Clinical Immunology_ Principles and Practice ( PDFDrive )

382 PARt tHREE Host Defenses to Infectious Agents



KEY CONCEPtS susceptibility to intracellular bacterial infection will continue
T Cell–Mediated Mechanisms to illuminate our understanding of how immunity is similarly
orchestrated across multiple infectious diseases.
Underlying Protection
Proinflammatory Cytokines and Phagocyte Attraction
IFN-γ- and TNF-α-mediated activation of phagocytes to kill bacteria by
means of: The recruitment of more phagocytes to the site of infection
Reactive oxygen intermediate (ROI) and reactive nitrogen intermediate represents a vital process in the resolution of infection. Phagocyte
(RNI) recruitment is achieved via the secretion by MPs and endothelial
Delivery of lysosomal hydrolytic enzymes and antimicrobial peptides cells of cytokines of the IL-1 family, TNF-α, IL-6, and chemokines.
to the bacteria-containing phagosome Signaling via IL-1 cognates is considered closely related to that
Autophagy
Formation and maintenance of granulomas of the TLRs because of the close homology of the cytoplasmic
T cell–mediated response controls but does not eradicate the domains of TLRs and IL-1 family receptors. The most studied
pathogen member is IL-1β, which, in synergy with chemokines and TNF-α,
increases the expression of adhesion molecules on the vascular
epithelium, thereby promoting extravasation of the inflammatory
cell infiltrate into infected tissues. Chemokines are a family of
listeriosis, TB, or typhoid in experimental animals. For macro- structurally related proteins. The positions of the first two cysteine
phages harboring intracellular bacteria, namely, M. tuberculosis, residues in the protein sequence have been used to divide che-
signaling with IFN-γ is a game-changer, summoning infected mokines into four subfamilies: CC (MIP-1β, MCP-1, MCP-2,
macrophages to escalate antimicrobial mechanisms. The action MCP-3), CXC (MIP-2, IL-8), C (lymphotactin), and CX3C
of TNF-α appears to augment IFN-γ and is also important in chemokines (fractalkine), where C represents cysteine and X
control of intracellular infection. This has been demonstrated represents any amino acid other than cysteine. These molecules
in humans through use of blocking of TNF-α by antibodies as are critical in controlling the migration of PMNs (IL-8) and
antiinflammatory therapy. Such treatments can activate TB in monocytes (MCP-1, also known as CCL2) from the bloodstream
24
individuals with LTBI. Despite this, these potent protective effects to infected tissue. Recently, the role of chemokines in intracellular
of IFN-γ and TNF-α come at a price. The need to kill intracel- infections has been increasingly appreciated, for example, with
lular bacteria often leads to death of the host cell as collateral mice lacking the receptor for CCL2 being deficient in their ability
damage. In part, the host manages this by controlling how the to clear listeria infection. It has been suggested that in the early
cell dies. Excessive TNF-α leads to less regulated necrotic cell stages of infection, M. tuberculosis exploits a delay in the mobiliza-
death, benefiting M. tuberculosis. For this reason elaborate host tion of T-cell immunity to recruit MPs to the site of infection,
mechanisms have evolved to maintain TNF-α at optimal levels which preferentially serve as habitat because of a lack of local
to control infection. The host enzyme leukotriene A4 hydrolase IFN-γ from T cells. Moreover, M. tuberculosis is thought to infect
(LT4H) catalyzes synthesis of a highly proinflammatory lipid the relatively sterile lower airways. The lack of commensal bacteria
leukotriene B4. In the event of enzyme deficiency, an antiinflam- could mean that M. tuberculosis uses cell surface phenolic gly-
matory lipid lipotoxin A4 accumulates that counteracts effects colipid (PGL) to signal epithelial cells to produce the chemokine
25
of TNF-α. Two common variant promoters control expression CCL2 in the absence of signaling via other PAMPs. This
of LT4H in humans, and homozygotes are associated with either mechanism then recruits MPs that are more permissive for
high or low inflammation. In contrast, the heterozygotes show a bacterial growth than those recruited by a more “global” MyD88-
balanced response to TNF-α associated with resistance against dependent signaling of TLRs, requiring coengagement of PAMPs
TB. Such a finding strongly suggests that genetic mechanisms on commensal bacteria that are more abundant in the upper
can maintain an optimal level of TNF-α responsiveness of cells airways. The initial macrophage infiltrates could play an important
harboring intracellular bacteria, namely, M. tuberculosis. A central role in early granuloma development.
antimicrobial mechanism stimulated by IFN-γ and TNF-α is
production of reactive nitrogen intermediates (RNIs) via the Cytokine-Induced Host-Protective Mechanisms
induction of nitric oxide synthase (NOS)2 and reactive oxygen Effector Molecules
intermediates (ROIs) via activation of nicotinamide adenine Activation of a membrane-bound NADPH oxidase by stimulation
dinucleotide phosphate (NADPH)–dependent oxidative burst. with IFN-γ or immunoglobulin G (IgG) initiates an oxidative
−
− 1
IFN-γ also promotes antimicrobial effects associated with vitamin burst that generates the ROIs O 2 , H 2 O 2 , OH , O 2 , and •OH
26
D and induces autophagy, a mechanism that plays an important radical (Table 26.3). In human PMNs and blood monocytes
role in host defense. It is now clear that the production of IFN-γ that possess myeloperoxidase, ROI activity is further augmented
depends on prior activation by IL-12 and/or IL-18. IL-12, in by the formation of hypochlorous acid. Oxidation and/or
concert with TNF-α, induces a cytokine loop resulting in the chlorination of bacterial lipids and proteins result in their
production of IFN-γ, which sustains the production of IL-12 inactivation and subsequent bacterial killing. The importance
and IL-18. These observations have been extended to humans, in of ROIs in antibacterial defense is underlined by recurrent
whom mutations that affect IFN-γ signaling cause susceptibility infections in patients whose phagocytes fail to generate an oxida-
to M. tuberculosis and salmonellae, as well as to BCG and com- tive burst. Nitric oxide synthase 2 (NOS2) is an inducible cytosolic
monly nonpathogenic mycobacteria, and are termed mendelian enzyme in professional phagocytes that delivers NO to the
susceptibility to mycobacterial disease (MSMD). The mutations are phagolysosome harboring bacteria while consuming O 2 and
−
−
located in genes that include IL12B and IL12RB, which encode L-arginine. NO is further oxidized to NO 2 and NO 3 . Nitrification
subunit β of the IL-12 cytokine and its receptor, respectively, and/or oxidation then inactivates bacterial molecules needed
27
and IFNGR1 and IFNGR2, which encode the IFN-γ receptor. for bacterial growth. The formation of •NO is catalyzed by
Further unraveling of the molecular basis of human genetic NOS2, which is promoted by both immunological stimuli, such

CHAPtER 26 Host Defenses to Intracellular Bacteria 383



TABLE 26.3 Antibacterial Effector Mechanisms of Activated Macrophages and Corresponding
Microbial Evasion Strategies
Macrophage Effector Mechanism Microbial Evasion Strategy
Production of ROIs Uptake via complement receptors; production of ROI detoxifying molecules
(superoxide dismutase, catalase); bacterial ROI scavengers (phenolic
glycolipids, sulfatides, lipoarabinomannans)
+
Production of RNIs Inhibition of phagosome maturation via blockage of H ATP pump, indirect
effect of ROI-detoxifying molecules
Autophagy, intraphagolysosomal killing Egression into cytoplasm;
Resistant cell wall
Phagosomal acidification, phagosome–lysosome fusion Inhibition of phagosome maturation
Defensins Modification of cell wall lipid A to resist defensins
Reduced iron supply (transferrin receptor downregulation, lipocalins) Expression of microbial siderophores to increase iron uptake
Tryptophan degradation Upregulation of bacterial tryptophan synthesis
ATP, adenosine triphosphate; ROI, reactive oxygen intermediate; RNI, reactive nitrogen intermediate.


as IFN-γ and TNF, and microbial products, such as LPS, lipo- Noninfected cells engulf bacterial antigens associated with vesicles
teichoic acid, and mycobacterial lipids. RNIs exert their bactericidal produced by apoptotic cells. Apoptosis as a prerequisite for this
activity by destroying iron-/sulfur-containing reactive centers pathway is induced by many intracellular bacteria, including
of bacterial enzymes and by synergizing with ROIs to form highly salmonellae, mycobacteria, and listeriae. This cross-presentation
−
reactive peroxynitrite (ONOO ). Despite being highly effective pathway in infections with intracellular bacteria adds an essential
in killing intracellular bacteria, NO production relies on a continu- function to the physiological role of apoptosis in the maintenance
ous supply of L-arginine, which becomes limited because of of tissue integrity and growth.
competition with another macrophage enzyme, Arginase-1 Upon signaling via IFN-γ, autophagy, a process common to
(Arg-1). Arg-1 metabolizes L-arginine to produce urea and all cells for removal of dysfunctional or damaged cellular
ornithine and demonstrates antiinflammatory activity. The organelles, can be harnessed to dispose of intracellular L. mono-
competitive function of Arg-1 likely regulates collateral tissue cytogenes and M. tuberculosis in a process termed xenophagy.
damage caused by overexuberant RNIs. The final downstream Signaling via members of the immunity-related guanosine tri-
product of NOS2 activity is citrulline, which is recycled to phosphatase (GTPase) family (IRG family) and the guanylate-
L-arginine by the enzymes argininosuccinate synthase (Ass1) binding protein family, TLR2 and TLR4 engagements and the
and argininosuccinate lyase (Asl). A mouse deficient in macro- active form of vitamin D 3 all act to augment xenophagy. Formation
phage Asl activity is unable to control mycobacterial infection, of double-membrane autophagosomes that mature analogously
highlighting the importance of this recycling pathway. The central to the phagosomal pathway and fuse with lysosomes that degrade
role of NOS2 in protection against intracellular bacteria is well bacteria contained within. The importance of this process is
established in murine models of infection. Whether NOS2 plays highlighted by polymorphisms in one of the three IRG families
a similarly central role in humans is still unclear. Defensins are of genes in humans, IRGM, being associated with susceptibility
small lysosomal polypeptides that are microbicidal at basic pH to TB. Recently, a host-encoded microRNA, miRNA-155, was
and are particularly abundant in phagocytes. These include shown to potentiate xenophagy during intracellular mycobacterial
granulysin, present in granules of human natural killer (NK) infection by targeting an endogenous inhibitor of autophagy,
28
and cytolytic T (CTLs) cells, and cathelicidin, which is regulated Ras homologue enriched in brain (Rheb) by suppressing Rheb
by vitamin D in a TLR-dependent manner and is converted by expression.
cleavage to the antimicrobial peptide LL-37.
Nutrient Deprivation
Apoptosis and Autophagy Deprivation of required nutrients to intracellular bacteria is also
Apoptosis is a highly regulated form of cell death that is critical a strategy employed by the host, markedly so within infected
29
for control of cell turnover, a vital process for tissue homeostasis. macrophages. Tryptophan degradation is achieved by the enzyme
Macrophage apoptosis also constitutes a defense mechanism, indoleamine 2,3-dioxygenase (IDO), which degrades tryptophan
allowing removal of phagocytes containing intracellular bacteria to kynurenine (see Table 26.3). This reaction is induced by IFN-γ
without the need to generate significant inflammation. Apoptosis, in both MPs and IFN-γ-responsive nonprofessional phagocytes
in contrast to cellular necrosis, results in cell death without and inhibits the growth of C. psittaci and C. trachomatis inside
permeabilization of the host cell membrane. The process can human macrophages and epithelial cells. Similarly, augmentation
be triggered by TNF-α signaling and augmented by IFN-γ, of NOS2 by IFN-γ and TNF-α depletes intracellular L-arginine,
resulting in activation of cellular caspases, mitochondrial mem- also required for growth of intracellular bacteria. 20
brane permeability, and cytochrome c release. These processes
result in cellular disintegration and generation of apoptotic bodies
that are engulfed and digested by neighboring phagocytic cells. EVASION FROM, INTERFERENCE WITH, AND
Apoptosis is protective against L. monocytogenes and Salmonella RESISTANCE TO MICROBIAL KILLING
spp. and is inhibited by M. tuberculosis, which promotes necrotic
cell death of infected cells to its benefit via mitochondrial Strategies Against Toxic Effector Molecules
membrane damage and by caspase-independent mechanisms Many intracellular bacteria have exploited successful strategies
during conditions of high bacterial burden in macrophages. against macrophage effector mechanisms (see Table 26.3). One

384 PARt tHREE Host Defenses to Infectious Agents


mechanism of evasion is determined by the receptor that is used are Rab3, -4, -5, -9, -7, -11, and -14. These proteins are associ-
for pathogen entry into the host cell. Internalization via CRs ated with different maturation stages of the phagosome and
inhibits the production of IL-12, a cytokine critical in facilitating chiefly orchestrate membrane fusion events to allow delivery of
macrophage activation. Engulfment by this receptor also bypasses vesicular protein cargo to the phagosomal compartment. The
activation of the oxidative burst, thereby avoiding ROI production. mycobacteria-containing phagosome acquires Rab5a but not the
Similarly, engaging mismatch repair (MMR) and DC-specific late endosomal marker Rab7a, which ultimately mediates fusion of
intercellular adhesion (DC-SIGN) molecules for uptake triggers the bacterial-containing phagosome with lysosomes that contain
secretion of the suppressive cytokines IL-10 and TGF-β. Several proteolytic enzymes active at low pH. By enabling arrest of this
intracellular bacteria also produce ROI detoxifiers, including maturation, M. tuberculosis maintains its compartment at an
superoxide dismutase and catalase, which nullify oxygen (O 2 ) early endosomal stage. This compartment does not acidify, partly
+
and hydrogen peroxide (H 2 O 2 ), respectively. Finally, a number because of a paucity of vacuolar H adenosine triphosphatase
of small bacterial products, such as the phenolic glycolipid and (ATPase); at the same time, it exchanges molecules with the
LAM of mycobacteria, scavenge ROIs. Many of the strategies plasma membrane, such as the transferrin receptor, to access
used to counteract the effects of ROIs also overlap in their effects iron. Activation of macrophages with IFN-γ restores the normal
on RNIs. A modification of lipid A renders gram-negative bacteria, maturation of the mycobacterial phagosome, resulting in a drop
including salmonellae, resistant to the effects of host antimicrobial in mycobacterial viability. Francisellae and brucellae are engulfed
peptides. by phagosomes that acquire the early endosomal markers EEA1
31
and Rab5a. The Francisella-containing vacuole acquires late
Intraphagosomal Survival endosomal markers, but the pathogen escapes into the cytosol
Inhibition of phagolysosome fusion represents a major intracel- by perforating the late endosomal membrane. After a transient
lular survival strategy for a number of intracellular bacteria, phagosomal stage, brucellae enter compartments enclosed by
including M. tuberculosis, Francisella spp., Brucella spp., and L. endoplasmic reticulum (ER) membranes to escape delivery to
monocytogenes (Fig. 26.2). After engulfment, these pathogens phagolysosomes.
manipulate the endocytic fate of the phagosome that contains
them. This is achieved in part by manipulation of Rab GTPases, Phenotypic Plasticity of the Infected Cell
proteins required for normal endocytic trafficking, positioned The ability of intracellular bacteria to influence the phenotypic fate
30
in the phagosome membrane. Rab GTPases associated with of both the cell in which it dwells and the cells within lesions that
phagosome maturation of the pathogen-containing phagosome form as a result of unresolved infection is becoming increasingly




Brucella
Francisella
Early endosome
Golgi Early BCV markers
EEA1 EEA1
Rab5 Rab5

Late endosome
ER Intermediate BCV markers Actin
Lamp-1, 2
Lamp-1 Rab5

ER
Replicative BCVs
Listeria


Rab5
Modified
phagosome
Mycobacterium
FIG 26.2 Inhibition of Phagolysosome Fusion Represents a Major Survival Strategy for a
Number of Intracellular Bacteria, Including Mycobacterium Tuberculosis, Francisella spp.,
Brucella spp., and Listeria Monocytogenes. The mycobacteria-containing phagosome acquires
Rab5 but not the late endosomal markers Lamp-1 and 2, enabling arrest of maturation of this
compartment at an early endosomal stage. Francisella spp. and Brucella spp. are engulfed by
phagosomes that acquire the early endosomal markers EEA1 and Rab5. The Francisella-containing
vacuole acquires late endosomal markers but escapes into the cytosol by perforating the late
endosomal membrane. A similar strategy is adopted by L. monocytogenes. After a transient
phagosomal stage, brucellae enter compartments enclosed by endoplasmic reticulum (ER)
membranes to escape delivery to phagolysosomes. BCV, Brucella-containing vacuole.

CHAPtER 26 Host Defenses to Intracellular Bacteria 385


appreciated. This has already been highlighted by the tendency to increased cell death and are not expressed by the vaccine BCG,
of M. tuberculosis to use its own membrane lipids to exploit which hence does not escape from the phagosome.
host chemotactic pathways to recruit bacterial growth-permissive
macrophages to the site of infection; this process subsequently T LYMPHOCYTES AS SPECIFIC MEDIATORS OF
allows a proliferative head-start before adaptive immunity kicks ACQUIRED RESISTANCE
in and amplifies intracellular defences by the action of cytokines,
such as IFN-γ and TNF-α. Moreover, bacterial killing must be Activated macrophages act as the nonspecific executors, whereas
tempered inside the granuloma to prevent destruction of host T lymphocytes are the specific mediators of acquired resistance
tissue. This is achieved by balancing macrophage phenotypes against intracellular bacteria. The dramatic increase in the
ranging from a phenotype highly bactericidal, termed “classically” incidence of TB and other intracellular bacterial infections in
activated, to a phenotype that is more suppressive of inflamma- patients with AIDS illustrate the central role of T lymphocytes
tion and is associated with wound healing and fibrosis, termed in protection. For instance, 15 million individuals are coinfected
“alternatively” activated. Tipping the balance one way or the with HIV and M. tuberculosis, and HIV increases the risk of
other is detrimental for the host in terms of disease. developing TB by several orders of magnitude resulting in more
Myeloid-derived suppressor cells (MDSCs) represent a certain than 1 million TB cases annually. At the site of microbial growth,
stage of development of myeloid cells (both of monocytic and T lymphocytes not only initiate the most potent defense mecha-
32
granulocytic lineage). Although most of our knowledge stems nisms available, they also focus this response to the site of
from their suppressive role in cancer, recent evidence suggests encounter, thus minimizing collateral damage to the host.
that they play a role in control of chronic infections, such as Although protective T-cell responses are multifactorial, they can
33
TB. They can be distinguished from canonical MPs and granu- be reduced to a few principal mechanisms (Fig. 26.3).
locytes by means of distinct surface markers. The granulocytic As previously mentioned, T cells inevitably also produce
+
int
hi
MDSCs are CD11b LY6G Gr1 , whereas monocytic MDSCs pathology through cytotoxic antimicrobial defense mechanisms.
+
+
hi
neg
are CD11b LY6G LY6C Gr1 . Moreover, pathogenesis of intracellular bacterial infection is
More recent findings point to host cell reprogramming result- highly influenced by T cells. It is therefore important that the
ing from intracellular infection. During intracellular infection of T-cell response be tightly controlled and downregulated, when
Schwann cells, M. leprae is able to downregulate genes active for necessary. Regulatory mechanisms, including regulatory T cells
the Schwann cell phenotype and upregulate genes that orchestrate (Tregs), are in place to limit immunopathology. 19
differentiation to a “stem cell–like” phenotype. This stem cell–like Protective immunity involves the so-called conventional T-cell
property allows the infected cell to differentiate further to multiple sets, CD4 αβ T cells, and CD8 αβ T cells, as well as unconventional
mesenchymal cell states, such as skeletal cells or smooth muscle T cells, such as γδ T cells, CD1-restricted αβ T cells, and T cells
34
cells. This ability to regress and then reprogram an infected cell that recognize antigen in the context of other nonclassic MHC
phenotype could play a role in spreading infection throughout class I molecules, such as mucosal-associated invariant T (MAIT;
the host during leprosy. Chapter 20) cells (see Fig. 26.3). Although these T-cell sets perform
Recently, mesenchymal stem cells (MSCs) were identified as different tasks, substantial redundancy exists. Furthermore, these
an intracellular niche of M. tuberculosis in mice, and the equivalent T-cell populations act in a coordinated way in close interaction
human MSC phenotype could be readily infected in vitro. Because with other leukocytes. Depending on the etiological agent and the
they reside in hypoxic niches and most antimycobacterial therapies stage of disease, the relative contribution of the different T-cell
are inactive in these conditions, it is feasible that MSCs could subsets to acquired resistance may vary. The conventional αβ T
maintain the bacteria during long-term infection and could cells make up more than 90% and γδ T cells less than 10% of all
represent a protective niche from drug therapy. Dormant M. lymphocytes in the blood and peripheral organs of humans and
tuberculosis has also been detected in hematopoietic stem cells mice. However, γδ T cells represent a significant proportion of
(HSCs) in mice and humans. HSCs are pluripotent, giving rise the intraepithelial lymphocytes in mucosal tissues, suggesting a
to both lymphoid and myeloid cell lineages in the blood. Clarifica- particular role at this important port of microbial entry.
tion of the pathophysiological context of carriage of M. tuber-
culosis by both HSCs and MSCs is an exciting prospect. CD4 T Cells
The CD4 T-cell population can be further subdivided into distinct
Escape Into Cytoplasm subsets, according to their pattern of cytokine production and
A successful strategy for survival inside activated macrophages expression of unique transcription factors that control patterns
is egression from the phagosome into the cytoplasm, which has of gene expression (Chapter 16). At least four major subsets
been exploited by L. monocytogenes and the various pathogenic exist, T-helper cell-1 (Th1), Th2, Th17, and Tregs. The first two
Rickettsia spp. (see Fig. 26.2). 35,36 This has the advantage of both subsets were discovered over 20 years ago and have been identified
avoiding the cellular defense mechanisms within the phagosome in both mice and humans: Th1 cells, which overwhelmingly
and providing the bacteria with a nutrient-rich environment. produce IFN-γ and IL-2, and Th2 cells, which produce IL-4,
L. monocytogenes possesses several virulence factors to facilitate -5, and -13. The Th1 subset can also be defined on the basis of
its escape from the phagolysosome, a pore-forming hemolysin the T-bet transcription factor and the signal transducer STAT4,
(listeriolysin [LLO]) that acts together with a metalloproteinase, whereas Th2 classification is consistent with expression of the
a lecithinase, and two phospholipases to efficiently promote the transcription factor GATA-3 and signal transducer STAT5.
rupture of the phagosomal membrane and to spread to other cells. Th17 cells express the retinoid orphan receptor γt (ROR-γt)
M. tuberculosis and M. leprae can also egress from the phagosome transcription factor and the signal transducer STAT3. They produce
into the cytoplasm of macrophages and DCs, a behavior that is the cytokines IL-17, IL-22, and granulocyte macrophage–colony-
37
mediated by a mycobacterial protein secretion system ESX-1. stimulating factor (GM-CSF). Cytokines of the IL-17 family are
Bacterial virulence factors secreted by ESX-1 may also contribute strong inducers of granulopoiesis; of proinflammatory mediators,

386 PARt tHREE Host Defenses to Infectious Agents


Unconventional T cells γδ IL-10
TGF-β

IL-1β
Phospholigand IL-6 IL-6
T P T reg IL-23 IL-17
IL-21
P IL-22
IL-27

T h17
Lipid TGF-β
Metabolite CD1, MR RA
CD8 Cytosol TGF-β IFN-γ
Conventional T cells Peptide MHC I Golgi 1 2 IL-21 (human) T h1 LT/TNF-α
IL-6 (mouse)
IL-2
GM-CSF
IL-12
IL-18
3 IFN-γ IL-4
IL-4
CD4 MHC II IL-25
Phagosome IL-33 T
h2
IL-4
IL-5
IL-13
IL-25


MHC B7.1
ICOS-L (B7.h) B7.2
B7.1 PD-L
B7.2
ICOS CD28 TCR PD-1 CTLA-4

T cell
Activation Inhibition
FIG 26.3 T-Cell Stimulation During Infection. (A) Recognition of bacterial antigen by T cells.
Antigen originating from intracellular bacteria is presented to conventional CD4 and CD8 T cells.
Unconventional T cells including γδ T cells, mucosa-associated invariant T (MAIT) cells, and
CD1-restricted T cells are also activated. Human γδ T cells recognize small molecules containing
pyrophosphate residues; MAIT cells recognize bacterial metabolites, such as vitamin B 2 derivatives,
in the context of major histocompatibility complex (MHC)–related (MR) gene products; CD1-
restricted T cells recognize glycolipids in the context of CD1 molecules. (B) CD4 T cells can be
subdivided into different T helper (Th) cells according to their cytokine expression pattern. Th1
cells are critical for protection against intracellular bacteria. They typically produce interferon-γ(IFN-γ),
tumor necrosis factor-α(TNF-α), lymphotoxin (LT), interleukin (IL)-2, and granulocyte macrophage–
colony-stimulating factor (GM-CSF). Th2 cells stimulate humoral immune responses via secretion
of IL-4 and IL-5. Other cytokines produced by Th2 cells include IL-13 and IL-25. Th17 cells produce
IL-6, -17, -21, and -22, which probably contribute to early protection. Regulatory T cells (Tregs)
produce transforming growth factor β(TGF-β) and IL-10, which suppress immune responses.
ICOS, inducible costimulatory molecule; PD-1, programmed death 1; PD-L, program death ligand;
RA, retinoic acid; TCR, T-cell receptor. See Chapters 9 and 12 for details. (Modified from Kaufmann
SHE, Parida SK. Tuberculosis in Africa: learning from pathogenesis for biomarker identification.
Cell Host Microbe 2008;4:219–28, with permission of Elsevier.)

CHAPtER 26 Host Defenses to Intracellular Bacteria 387


such as IL-6; and of the chemokines CXCL1, CXCL8, and CXCL6, are rapid producers of IL-17 at sites of bacterial implantation.
which attract neutrophilic and eosinophilic granulocytes and Transient participation of γδ T cells in protection and a unique
38
prolong their survival. Th17 cells, too, have limited importance requirement for γδ T cells in granuloma formation have been
for protection in murine models against primary infection with described for murine listeriosis and TB. Murine γδ T cells appear
mycobacteria, salmonellae, and listeriae. However, Th17 cells to recognize peptides presented by nonpolymorphic MHC class
can drive more rapid Th1 responses against pulmonary TB in I molecules, whereas human γδ T cells respond to nonpeptidic
mice after vaccination, resulting in enhanced protection. IL-17 phosphorylated metabolites, notably from the isoprenoid pathway
is also required for optimally protective Th1 responses during of bacterial and host origin. 43
murine F. tularensis infection. 39 MAIT cells are primarily localized at mucosal sites, and current
Despite the convenience of defining T-cell populations in evidence suggests that they play a role in control of bacterial
terms of subsets, recent evidence suggests considerable plasticity infections in mucosal tissues, such as lung (M. tuberculosis) and
in cytokine production by T cells. This was first suggested by gut (gram-negative bacteria) tissues. Antigenic ligands include
demonstration that all subsets could produce IL-10, which derivatives of vitamin B 2 (riboflavin), produced by many intracel-
regulates potency of T-cell responses to limit host collateral lular bacteria, including salmonellae and mycobacteria. 44-46
damage during immune responses. IL-10 expression might be CD1 comprises a group of nonpolymorphic MHC-related
an intrinsic control mechanism common to all T cells. However, molecules that can present glycolipid antigens to unconventional
reduction in T-cell potency also favors chronic intracellular T cells. In humans, group 1 CD1-restricted T cells respond to a
bacterial infection. T-cell subsets may acquire the ability to variety of microbial glycolipids, including LAM, PIMs, mycolic
47
produce additional cytokines by expression of additional tran- acids, sulfatides, sulfoglycolipids, and lipopeptides. Group I
40
scription factors or by remodeling chromatin structure. Future CD1 molecules are absent in mice. The group II CD1 molecule
research will redefine T-cell behavior during intracellular bacterial CD1d is present in both humans and mice and controls develop-
infection. ment of natural killer T cells (NKT cells) that express the NK
cell marker NK1.1. Upon antigen activation, these T cells rapidly
CD8 T Cells produce cytokines and are capable of producing both IL-4 and
Infection of mice deficient in specific T-cell subsets has conclu- IFN-γ. Bacterial antigens recognized by NKT cells, PIMs from
sively demonstrated a role for CD8 T cells during listeriosis and mycobacteria, and glycosphingolipids from Ehrlichia and Sphin-
41
TB. Furthermore, CD8 effector T cells have been identified in gomonas spp. have been identified. NKT cells also respond to
granulomas of patients with tuberculoid leprosy with low numbers host endogenous lysosomal lipids loaded onto CD1d. 48
of bacteria. The cytolytic potential of these T cells can serve two In summary, unconventional T cells often recognize nonpep-
roles in infection with intracellular bacteria, namely, target cell tidic ligands of bacterial origin, emphasizing that they play a
killing or lysis of cells that are unable to control the infection, particular role in immunity against bacteria, including intracel-
thus releasing the bacteria for phagocytosis by more activated lular bacteria. Because of the highly skewed T cell receptor, these
cells. In humans, CD8 T cell–mediated killing is cell contact T cells are specific for a limited variety of bacterial ligands. Because
dependent and based on production of perforin, granzymes, of their less demanding antigen recognition and activation
42
and granulysin. Finally, CD8 T cells are also a potent source requirements, unconventional T cells may fill a gap between
of IFN-γ and TNF-α, thus contributing to direct activation of prompt innate resistance and the delayed conventional T-cell
infected macrophages to enhance protective mechanisms (see response.
Fig. 26.3).
CD8 T cells recognize antigenic peptides in the context of T-Cell Memory and Regulation of Immune Responses
MHC class I gene products, which are responsible for presentation Long-term protective immunity against infectious agents relies
of antigens residing in the cytosol. Initially, therefore, it was on immune memory, which forms the basis for the success of
mysterious how CD8 T cells were stimulated by intracellular all vaccines. Memory T cells can be divided into central memory
bacteria, which were thought to have a uniquely restricted T (T CM ) cells and effector memory T (T EM ) cells, based on dif-
49
phagosomal residence. However, with the knowledge that many ferential surface phenotypic and tissue migration patterns.
intracellular bacteria egress into the cytoplasm, one major T EM cells accumulate in peripheral tissues where they express
mechanism for MHC class I processing became obvious: proteins effector functions, whereas T CM cells persist in lymph nodes,
secreted by bacteria in the cytoplasm undergo antigen processing where they rapidly develop into T EM cells after secondary antigen
and presentation similarly to newly synthesized proteins of viral encounter. The tissue-resident memory T (T RM ) cells are local-
35
or host origin. Yet, alternative contact points for MHC class I ized to mucosal sites, where they provide efficient protection
50
molecules and bacterial peptides exist. Cross-presentation by against invading pathogens. Although it is generally accepted
noninfected antigen-presenting cells (APCs) of antigens engulfed that memory T cells can survive in the absence of persistent
within apoptotic blebs from infected cells represents a critical antigen, little is known about the induction and maintenance of
pathway to induce CD8 T cells by phagosomal bacteria (see long-lasting T-cell memory in chronic infections with intracellular
below). One major advantage of CD8 T cells over CD4 T cells bacteria. 51
is their recognition of antigen bound by MHC class I gene
products, which are expressed by almost all host cells. Thus CD8 B Cells
T cells recognize professional and nonprofessional phagocytes Although antibodies produced by B cells appear to play a minor
equally well. biological role in infections with intracellular bacteria, many
facultative intracellular bacteria spend some time outside their
Unconventional T Cells host cells, where they are accessible to antibodies. Thus antibod-
52
The relevance of the γδ T cells to antibacterial immunity is ies contribute to protection against salmonellae. Besides antibody
incompletely understood. Several studies indicate that γδ T cells production, B cells are potent APCs for soluble antigens, including

388 PARt tHREE Host Defenses to Infectious Agents


lipids presented by CD1c, and secrete many cytokines otherwise Our deepening understanding of the molecular events governing
associated with T cells, DCs, and macrophages. B-cell signaling via intracellular bacterial infections is allowing development of novel
MyD88 during S. typhimurium infection has been associated with therapeutic and preventive approaches. The need for such
B-cell production of IL-10, and mice with B-cell-specific deficiency interventions is becoming all the more pronounced in the face
in MyD88 were more resistant to infection demonstrating that, of increasing levels of antibiotic resistance of bacteria, such as
like T cells, B cells can perform regulatory functions. 53 M. tuberculosis, which render canonical drugs that specifically
KEY CONCEPtS target bacterial molecular processes ineffective. A new approach,
termed host-directed therapy (HDT) aims to develop new drugs
How Might a Vaccine Work? or repurpose previously approved ones that are directed at host
55
molecular processes. Such approaches include monoclonal
Activation of innate immunity for instruction of appropriate acquired antibodies (mAbs) to neutralize cytokines, such as TNF-α or
immune responses (PRRs)
Activation of the appropriate array of T-cell populations IL-6, to abrogate tissue destructive inflammation, repurposed
T-cell secretion of appropriate cytokine combination use of licensed drugs, such as ibuprofen and verapamil, to
Efficient development of T-cell memory modulate inflammation and enhance antibiotic effectiveness,
Efficient activation of antibacterial effector mechanisms respectively, and use of immunostimulatory natural molecules,
Best case scenario: sterile pathogen eradication such as vitamin D 3 to enhance bacterial killing by xenophagy.
Second best case scenario: driving the infection “deeper” into latency, These approaches range from preclinical and early-stage clinical
thus efficiently preventing reactivation of disease
development to late-stage clinical development and offer promise
to shorten the traditional duration of therapy.
Regulatory T Cells It is being increasingly recognized that the interaction between
Intracellular bacteria can cause detrimental inflammation and intracellular bacteria and the immune system is not of the “all
tissue damage, for example, as a result of IFN-γ- and TNF- or nothing” type but is instead a “continuous struggle.” This
α-mediated Th1 responses. Under normal circumstances, control realization has far-reaching implications for preventive and
mechanisms are in place to limit immunopathology. Such therapeutic strategies against intracellular bacterial infections.
countermeasures are elicited as part of the ongoing immune First, vaccination against intracellular bacteria has not yet been
response during infection. The main cytokines that limit inflam- effected satisfactorily because of the involvement of several distinct
mation and control IFN-γ production are IL-10 and TGF-β. T-cell subsets with different modes of stimulation and activity
Although macrophages and DCs produce these cytokines, their profiles. Second, chemotherapy has frequently proven to be
main producers are Tregs, which are the prime cells involved in suboptimal for the sterile eradication of bacteria hidden in cellular
immune regulation. Natural Tregs are responsive to IL-2 because niches. A better understanding of the complex crosstalk between
of high constitutive CD25 expression and are characterized by cytokines, T lymphocytes, macrophages, and infected host cells
54
expression of the transcription factor FOXP3. Expansion of will no doubt directly promote the development of improved
Tregs appears to be both antigen dependent and antigen inde- control measures. 55
pendent. In addition, Tregs selectively express TLRs and can be
activated, for example, by LPS and possibly other TLR ligands. ACKNOWLEDGMENTS
This makes their immediate activation during bacterial infection
a probable scenario. Although Tregs limit CD8 T-cell responses We are grateful to Mary Louise Grossman for excellent assistance
in experimental listeriosis, their general role in infections with and Diane Schad for the figures.
intracellular bacteria has not been fully elucidated. Suppression
of T-cell responses and anergy have been clinically documented Please check your eBook at https://expertconsult.inkling.com/
for TB and leprosy. Although Treg functions can limit detrimental for self-assessment questions. See inside cover for registration
T-cell responses and immunopathology, they can also prevent details.
elimination of bacteria, and hence are likely a key factor in
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CHAPtER 26 Host Defenses to Intracellular Bacteria 389.e1


MUL t IPLE-CHOICE QUES t IONS

1. Which disease caused by intracellular bacterial infection results 3. Which of these host cell processes is NOT harnessed for
in the HIGHEST level of global mortality? protection against intracellular bacterial infection?
A. Typhoid A. Autophagy
B. Tuberculosis B. Necrosis
C. Legionnaires’ disease C. Apoptosis
2. Which host cytokine molecule is the MOST important in 4. Which bacterial-derived ligand is presented to T cells via major
augmenting host cell defense mechanisms against intracellular histocompatibility complex (MHC) class I?
bacteria? A. Lipopolysaccharide
A. Interleukin (IL)-4 B. Polypeptide
B. IL-17 C. RNA
C. Interferon (IFN)-γ

27









Host Defenses to Extracellular Bacteria



Marcos C. Schechter, Sarah W. Satola, David S. Stephens







The human host has developed protective mechanisms to interact Conversely, bacteria typically classified as “intracellular” can have
with the multitude of bacterial species encountered in nature. an extracellular component to their lifecycle (e.g., Mycobacterium
These host defenses include nonspecific mechanisms of clearance, tuberculosis in cavitary lesions).
as well as innate and specific adaptive immune responses. Partly Host defense mechanisms against extracellular bacteria are
because of these mechanisms, the vast majority of bacterial species a continuum. The innate and adaptive immune systems cooperate
do not cause human disease. Many bacterial species have estab- to protect the host from extracellular bacterial infections. The
lished symbiotic or commensal relationships with the human innate immune system senses bacteria through pattern recognition
host and colonize skin and mucosal surfaces. These commensals receptors (PRRs; Chapter 3). These receptors activate antimicrobial
are generally of low virulence except in individuals whose host defenses and stimulate the adaptive response, all while balancing
defenses are compromised. Given the diversity of the microbial excessive immune and inflammatory responses with the need
world, a relatively few pathogenic bacterial species or subpopula- to protect against the infecting pathogen. 2
tions of those species have evolved virulence factors or strategies
that can overcome or circumvent intact human host defense CLINICAL PEARLS
mechanisms to cause localized or systemic disease. Distinguishing Clinical Characteristics of
Important bacterial pathogens of clinical importance reside Infections With Extracellular Bacteria
mostly extracellularly (Table 27.1). Examples are bacterial
pathogens typically labeled extracellular, such as Streptococcus • Sterilizing immunity
pneumoniae, Streptococcus pyogenes, Haemophilus influenzae, • Colonization of mucosal surfaces often precedes disease
Neisseria meningitidis, Neisseria gonorrhoeae, and Bordetella • Causes of pyogenic infections
pertussis, which are transmitted from one individual to another • T-helper (Th)17 response critical in generating a neutrophilic response
by close contact. Other “extracellular” bacterial pathogens, such • Antibodies are protective for some of the major pathogens
• Effective vaccines available for many of the major pathogens
as Clostridium spp. Vibrio cholerae, Shigella dysenteriae, entero-
pathogenic Escherichia coli, and Bacillus anthracis, are transmitted
through food, water, animal, or other environmental contact. CLEARANCE AND NONSPECIFIC HOST DEFENSES
Staphylococcus aureus is an important extracellular pathogen for AT MUCOSAL EPITHELIAL SURFACES
humans and can be acquired from other humans, from animals,
or through environmental contact. Acquisition of these pathogenic Bacteria first encounter a physical barrier, which comprises skin,
bacteria may be transient, result in variable intervals of coloniza- mucus and mucosal surfaces, and the normal microbiota, as well
tion, or rapidly cause localized or systemic disease. as nonspecific factors, such as nutrient limitation (e.g., iron),
Extracellular bacterial pathogens can produce acute inflam- and antimicrobial proteins or peptides. Intact skin and mucosal
matory and purulent infectious diseases, such as meningitis, surfaces provide complex chemical and biological obstacles to
septicemia, pneumonia, urethritis, pharyngitis, inflammatory extracellular bacteria and are an important line of defense prevent-
3
diarrhea, cellulitis, and abscesses and/or produce disease by the ing the invasion of these pathogens and their products. Humans
release of toxins. Disease associated with some extracellular commonly carry extracellular pathogens asymptomatically on
bacteria (e.g., Helicobacter pylori) results from chronic coloniza- skin and at respiratory and gastrointestinal (GI) mucosal surfaces.
tion. Susceptibility to extracellular bacterial pathogens is enhanced To cause disease, pathogens breach or disrupt epithelial barriers.
by hereditary, acquired, or age-related defects in innate or adaptive Damage to epithelial barriers as a result of trauma; coinfections;
host defenses. Resistance to extracellular bacterial pathogens or drugs, such as those used in chemotherapy; environmental factors,
their toxins can be accentuated by chemoprophylaxis, by vaccines, such as smoking, allergies, or low humidity; and catheterization
and by other immune modulation processes (e.g., passive immune and intubation circumvent these barriers and allow bacteria
globulin administration). Caution is urged in the interpretation access to subcutaneous tissues, blood vessels, and other normally
of the term “extracellular.” The classification of bacteria as sterile sites. Additionally, acquired and genetic diseases affecting
“extracellular” and “intracellular” is primarily based on observa- epithelial barriers are also linked to increase in infections.
tions in vitro and has been challenged by some authors, as some The human epithelium has evolved to prevent colonization and
3
”extracellular” bacterial species invade host cells as a part of invasion by pathogenic organisms. Skin is a relatively dry, acidic
their normal lifecycle and during steps in the disease process (pH 5–6) barrier that contains growth-inhibiting fatty acids and
1
(e.g., S. aureus, S. pneumoniae, S. pyogenes, N. meningitidis). antimicrobial peptides (AMPs) (see below), characteristics that
391

392 PARt tHREE Host Defenses to Infectious Agents



TABLE 27.1 Examples of Clinically Relevant Pathogenic Extracellular Bacteria
Examples of Human Selected Mechanisms of Special Features Key Examples of Susceptible
Species Disease Pathogenesis to Host Infection Populations/Risk Factors
Staphylococcus Cellulitis, abscesses, Protein A: promotes fibronectin binding Asymptomatic Injection drug users, patients on
aureus bacteremia, PVL: cytotoxic colonization, resistant to hemodialysis, defects on Th17
endocarditis, toxic α-toxin: membrane damage dehydration response (Job’s syndrome),
shock syndrome, TSST-1: superantigen Surgical procedures and skin
osteomyelitis, trauma
pneumonia, wound
infections
Streptococcus Pneumonia, otitis media, Capsule: prevents phagocytosis, Asymptomatic Smokers, cerebrospinal fluid leak,
pneumoniae meningitis antigenic variation colonization, readily asplenia, hypogammaglobulinemia,
(Pneumococcus) Pneumolysin: cytotoxic acquires new genes human immunodeficiency virus/
PspA & C: inhibition of complement through transformation acquired immunodeficiency
Neuraminidase, hyaluronidase: spread syndrome (HIV/AIDS),
and colonization unvaccinated children
IgA 1 protease
Streptococcus Pharyngitis, cellulitis, Hyaluronic acid capsule, M-protein: High diversity of M- School-age children, crowded-
pyogenes (group erysipelas, toxic shock prevents phagocytosis proteins, molecular conditions (e.g., military barracks),
A Streptococcus) syndrome, necrotizing Streptolysin O & S: cytotoxic mimicry of human injury to lymphatic system (e.g.,
fasciitis, scarlet fever, Streptococcal pyrogenic exotoxins antigens surgical harvest of saphenous
rheumatic fever C5a peptidase vein)
Streptococcus Neonatal sepsis, FbsA: fibrinogen receptor, promotes Asymptomatic Neonates and infants (immunity
agalactiae (group pneumonia and adherence colonization, acquisition dependent on passive transfer of
B Streptococcus) meningitis, perinatal Capsule by infants during birth maternal antibodies), diabetes
infections, bacteremia β-hemolysin mellitus
C5a peptidase
β protein: downregulates complement
Neisseria Meningitis, bacteremia Capsular polysaccharide: promotes Molecular mimicry of Terminal complement deficiencies,
meningitidis (purpura fulminans) adherence and prevents phagocytosis human antigens, phase hypogammaglobulinemia
(Meningococcus) Type IV pili: promote attachment to host and antigenic variation,
cells asymptomatic carriage
LOS: analogues to LPS, activates TLR4
pathway
IgA 1 protease
fHbp: downregulates the host alternative
complement pathway
Neisseria Urogenital infections, Type IV pili: promote attachment to host Phase and antigenic Terminal complement deficiencies,
gonorrhoeae disseminated cells variation, molecular women during menstrual period
(Gonococcus) gonococcal infection, Opa protein adhesion mimicry of human (increases risk of dissemination)
pharyngitis IgA 1 protease antigens
LOS: analogs to LPS, activates TLR4
pathway
Escherichia coli Urinary tract infections, Capsular polysaccharide Antigenic heterogeneity of Bladder instrumentation, pregnancy
gastroenteritis, sepsis, Tissue specific fimbriae LPS and capsule
neonatal meningitis Heat-labile enterotoxins: increases
intestinal chloride secretion
LPS: activation of TLR4
Pseudomonas Ventilator-associated Pili and flagella: attachment to the host Considerable adaptability Orotracheal intubation, cystic
aeruginosa pneumonia, and formation of biofilms to changes in fibrosis
bronchiectasis LPS environment, large
Exotoxin A genome size, biofilms
Lipases, lecithinases, elastase
Clostridium difficile Colitis Toxin B: cytotoxic Endospore formation, Antibiotics and other disruptions of
Flagella asymptomatic carriage the microbiotia
Haemophilus Otitis media, pneumonia, LPS with phosphorylcholine Phase variation of pili, Unvaccinated children,
influenzae epiglottitis, bacteremia, Pili: adherence asymptomatic carriage immunocompromised, sickle cell
meningitis Capsule disease, smoking
High-molecular weight adhesins
IgA 1 protease
Helicobacter pylori Peptic ulcer disease Urease: colonization of gastric mucosa Polymorphism of CagA Crowded living conditions, unreliable
Flagella: motile in gastric mucus source of clean water, living with
CagA; bacteria-derived carcinogen someone who has H. pylori
Bordetella pertussis Whopping cough Pertussis toxin: inhibits neutrophils, Antigenic variation of Nonvaccinated adults and children,
(children), chronic macrophages, lymphocytes adhesins infants who have not completed
cough (adults) Pertactin & filamentous hemagglutinin; vaccine series, adults and
mediates attachment adolescents whose immunity has
diminished
TSST-1, toxic shock syndrome toxin 1; Psp, pneumococcal surface protein; LOS, lipooligosaccharide; fHbp, Factor H-binding protein; LPS, lipopolysaccharide; CapA, cytotoxin-
associated gene A; Ig, immunoglobulin; TLR, Toll-like receptor; PVL, Panton-Valentine leukocidin.

CHAPtER 27 Host Defenses to Extracellular Bacteria 393


present in secretions, such as ABO blood group antigens. Cell
adhesion and extracellular matrix molecules, such as fibronectin
and proteoglycans, can also inhibit or enhance bacterial binding
to epithelial surfaces. The Tamm-Horsfall glycoprotein, found
in urine, can bind avidly to a variety of bacteria and facilitate
clearance. Proteins, such as lactoferrin (Lf), present at mucosal
surfaces, bind iron, an important requirement for bacterial growth.
This action may reduce microbial proliferation, but some mucosal
pathogens bind Lf and remove iron from the molecule for growth.
Normal Microbiota as Host Defense
The human microbiome is now recognized as a major host defense
against bacterial pathogens by providing “colonization resistance,”
maintaining a balance of commensals to pathogens, and by
5
priming the immune system (Chapter 14). Altering or disrupting
the normal microbiota by antibiotics facilitates the expansion
of enteric pathogens as Clostridium difficile and Salmonella
typhimurium or selection of antibiotic-resistant members of the
microbiome. Similarly, changes in human physiology, for example,
exposure of skin to elevated temperatures and humidity, chronic
stress, host immune suppression, or active behavioral changes,
such as smoking, can cause a commensal-to-pathogen switch.
Recent studies have demonstrated that certain resident microbiota
can resist pathogen colonization and infection. For example,
FIG 27.1 Mucociliary Host Defense. Scanning electron micro- matched volunteers were inoculated with Haemophilus ducreyi
graph of human upper respiratory mucosa showing the ciliated into the arms, and the subsequent infection either resolved or
and nonciliated epithelial surface (×16000). resulted in formation of abscesses; characterization of the skin
41
microbiome before, during, and after the experimental inoculation
showed that the microbiomes of those with pustule formation
are detrimental to many bacteria. The constant desquamation and of those with resolved infection were distinct and influenced
of stratified epithelial surface of skin helps in the removal of the course of the H. ducreyi infection. 6
4
microorganisms. Nevertheless, a complex skin microbiome that The interaction of the microbiome with the immune system
can include bacterial pathogens has been identified, and this differs is also important for defense against extracellular pathogens.
remarkably, depending on location in the body. Disruption of Normal microbiota facilitate a high level of priming of the
these physical barriers can augment pathogen tissue colonization immune system by maintaining high levels of major histocompat-
and invasion. Infections by S. aureus and S. pyogenes, bacteria that ibility complex (MHC) class II molecule expression on macro-
can colonize skin, are often preceded by skin damage. Repeated phages and other antigen-presenting cells (APCs). PRRs (see
trauma to skin (e.g., dialysis and intravenous drug use) also below) are traditionally known to recognize microbial molecules
enhances skin colonization with pathogens, including that by during infection; however, ligands for PRRs are abundantly
S. aureus. produced by the resident microbiota during normal colonization.
Mucosal surfaces have additional nonspecific antibacterial The integrity of the intestinal epithelial layer is dependent on
defenses. The mucociliary blanket of the respiratory tract (Fig. activation of Toll-like receptors (TLRs; see below) by normal
4
27.1) and the female urogenital tract (fallopian tube) move microbiota. Stimulation of TLR-5 has been shown to increase
bacteria away from epithelial surfaces, as does the flushing of resistance to E. faecium infection in a murine model. Activation
the urinary tract with urine, intestinal peristalsis, and the bathing of nucleotide oligomerization domain 1 (NOD1) receptors by
of the conjunctiva with tears. Lysozyme is found in most mucosal gut resident microbiota is necessary for priming of the innate
secretions and lyses bacterial cell walls by splitting muramic acid immune system. Additionally, resident microbiota produce such
β(1–4)-N-acetyl-glucosamine linkages. The acid pH of the factors as bacteriocins, lantibiotics, and phenol-soluble modulin
stomach, intestinal peristalsis, and the antibacterial effect of (PSM), which function in a similar manner to that of host-derived
proteolytic enzymes present in intestinal secretions are important antimicrobial proteins and peptides (APPs; see below), suggesting
7
GI tract host defenses against many pathogenic bacteria. The an important host defense strategy against pathogen colonization.
GI mucosa has a layer of mucus that acts as a physical shield to Importantly, members of the resident microbiota can cause
bacteria. Mucus is rich in mucin, glycoproteins that limit pathogen disease, particularly with loss of epithelial integrity and transloca-
binding to other host molecules necessary for mucosal adhesion. tion to a different host tissue.
Additionally, the mucus layer may function more than as a physical
barrier by acting as a diffusion barrier to concentrate antimicrobial Antimicrobial Peptides and Antimicrobial Proteins
proteins at the appropriate epithelial cell surface. The glycocalyx, Pathogens colonizing or invading epithelial surfaces are confronted
an extracellular layer of the apical surface of mucosal cells with APPs, which can be produced both by the host and the
7
composed of carbohydrates, also protects cells against bacterial microbiota (Table 27.2). In addition to pathogen killing, APPs
attachment. control host physiological functions, such as inflammation,
Bacterial attachment and colonization of mucosal surfaces angiogenesis, and wound healing. They also limit pathogen
can be inhibited by bacterial binding to human cellular antigens colonization and shape the composition of the host microbiome

394 PARt tHREE Host Defenses to Infectious Agents



TABLE 27.2 Antimicrobial Proteins (AMPs) Against Extracellular Bacteria
AMP tissue/Cell Sources Mechanism of Action target Organisms
α-defensins Small intestines, Paneth cells Membrane disruption; inhibits complement Gram-positive bacteria
activation; chemoattracts dendritic cells Gram-negative bacteria
β-defensins Large intestines, skin, respiratory Membrane disruption; lipid II binding Gram-positive bacteria
tract epithelial cells Gram-negative bacteria
Cathelicidin (LL37) Large intestine, skin, lung, Membrane disruption Gram-positive bacteria
urogenital tract Gram-negative bacteria
RNases Skin, intestine, respiratory Unknown Gram-positive bacteria
epithelia, placenta Gram-negative bacteria
Psoriasin (S100A7) Skin, urogenital tract Unknown Escherichia coli
Calprotectin (S100A8-A9) Abscesses/neutrophils Metal Chelation Staphylococcus aureus
C-type lectins Small intestines Peptidoglycan recognition Gram-positive bacteria
Bactericidal/permeability- Neutrophils, epithelial cells Neutralizes lipopolysaccharide Gram-negative bacteria
increasing protein (BPI)
Lysozyme Skin, body fluids, tears, intestinal Degrades peptidoglycan Gram-positive bacteria
Paneth cells Some gram-negative bacteria activity
Dermcidin Sweat glands Membrane disruption Gram-positive bacteria
Gram-negative bacteria
Peptidoglycan recognition Liver, intestines, skin, neutrophils Activates bacterial two-component systems; Especially active against gram-
proteins targets peptidoglycan positive bacteria
Gram-negative bacteria
Phospholipase A2 Tears, intestines Hydrolysis of bacterial phospholipids Gram-positive bacteria




TABLE 27.3 Immune Microbial Pattern discovery and characterization of specific pattern recognition
Recognition Molecules molecules (see below) has revolutionized our understanding of
the initial specific events occurring between microbes and human
Toll-like receptors (TLRs) cells. The identification and function of these molecules is rapidly
Nucleotide-binding oligomerization domain (NOD), caterpillar proteins, expanding and include TLRs, NOD and caterpillar proteins, RNA
peptidoglycan recognition proteins (PGRPs) helicases, complement proteins, antimicrobial peptides, collectins
RNA helicases/PkR
Complement proteins: C1q, C1 inhibitor and surfactants, C-lectins, and S-lectins, such as mannose-binding
Antimicrobial peptides lectin and L-ficolin.
Collectins and surfactants
C- and S-lectins: mannose-binding lectin, L-ficolin Pattern Recognition Receptors
Innate immune recognition relies on the detection of unique
8,9
molecular structures found on microorganisms by host PRRs.
and are both constitutively present and inducible by infection TLRs and NOD-like receptors (NLRs) are the best studied PRRs
7
and injury. Humans produce two main classes of APPs: defen- (Table 27.4). TLRs (TLR1–11) (Chapter 3) are found on macro-
sins and cathelicidins. Defensins, for example, are expressed in phages, neutrophils, and other host cells. These receptors recognize
skin, intestines, and the respiratory tract and have activity against a variety of microbial ligands or pathogen-associated molecular
gram-positive and gram-negative bacteria. Interestingly, APPs patterns (PAMPs), including lipoproteins, lipopolysaccharide
expression can be altered by epithelial injury. Keratinocytes of (LPS), flagellin, and nucleic acids produced by gram-negative
inflamed psoriatic lesions produce increased levels of certain and/or gram-positive bacteria. Alterations (polymorphisms) in
APPs, and patients with such lesions rarely have secondary TLRs (e.g., TLR4) and other pattern recognition molecules are
bacterial infections. In contrast, keratinocytes from patients with associated with susceptibility or severity of specific infections (e.g.,
10
atopic dermatitis have dampened APP production in response sepsis). TLR expression can be regulated by type I interferons
to inflammation, and colonization and superinfections of the (IFNs) and by microRNAs (miRNAs), and the dysregulation
skin by S. aureus are common. Not surprisingly, successful of TLRs can be involved in acute and chronic inflammatory
pathogens have developed several mechanisms to counteract diseases and cancer. 11
APPs. Gonococci, for example, counteract APPs with efflux The NLRs are a family of intracellular receptors, some of
8
pumps. which function as PRRs. NOD1 and NOD2 are well characterized
as PRRs to extracellular pathogens, such as S. flexneri. Importantly,
in concert with TLR signaling, NLR can respond to a variety of
RECOGNITION OF EXTRACELLULAR BACTERIA AND PAMPs by forming the inflammasome complex. Inflammasome
ACTIVATION OF THE IMMUNE SYSTEM activation generates interleukin-18 (IL-18) and the active form
of IL-1, an important step in the immune response to many
Immune pattern recognition molecules (Table 27.3) are a major bacteria. In addition to PAMPs, danger-associated molecular
arm of the innate immune system and are released or expressed patterns (DAMPs), typically associated with activation of the
by a range of host cells, including lymphocytes, macrophages, innate immune system upon injury by noninfectious mechanisms,
or tissue histiocytes, dendritic cells (DCs), polymorphonuclear can be released both by the host and bacteria and result in
leukocytes or neutrophils (PMNs), and epithelial cells. The amplification of the inflammatory response.

CHAPtER 27 Host Defenses to Extracellular Bacteria 395


4
TABLE 27.4 Pattern Recognition immunoglobulins. In most tissues, DCs are at low level of
Receptors (PRRs) Recognize Pathogen- activation and are immature, but upon activation, they take up
Associated Molecular Patterns (PAMPs) and process antigen. DCs are rich in PRRs (e.g., TLRs), and
microbe–PRR interaction has a key role in shaping the T-cell
From Various Bacteria response. For example, TLR5 stimulation by bacterial flagellin
14
PRRs PAMPs Microbes can induce a Th17 response and B-cell immunoglobulin A (IgA)
Toll-like receptor Triacyl lipoproteins Bacteria production. Skin contains a major supply of tissue DCs (Lang-
(TLR)2/1 erhans cells), and their involvement in combating skin and soft
TLR2/6 Diacyl lipoproteins Mycoplasma tissue infections must be considered along with their function
Lipoteichoic acid Gram-positive bacteria and contribution to stimulating immunity during vaccination.
TLR2 Peptidoglycan Gram-positive bacteria Limited information is available regarding the role of DCs in
Porins Bacteria (Neisseria) host resistance to extracellular bacteria, but some studies have
TLR4 Lipopolysaccharide Gram-negative bacteria examined the interaction between bacteria and DCs. For instance,
(LPS)
TLR5 Flagellin Flagellated bacteria Unkmeir et al. studied the interaction of serogroup B menin-
12
(Helicobacter pylori, gococci with DCs. Infection of DCs by meningococci resulted
Salmonella) in a significant and rapid production of proinflammatory
TLR7/8 RNA Group B Streptococcus cytokines and chemokines, including TNF-α, IL-6, and IL-8
TLR9 CpG-DNA Bacteria (Salmonella) through a lipooligosaccharide (LOS)–dependent mechanism.
12
DNA Bacteria (Staphylococcus Murine studies provide some insight into the mechanisms
at low MOI)
TLR11 Not determined Uropathogenic bacteria extracellular bacteria use to avert phagocytosis by DCs. For
Nucleotide- Meso-diaminopimelic H. pylori, Bacillus spp., example, S. suis polysaccharide capsule reduces bacterial adhesion
15
binding acid Campylobacter jejuni, to the DC plasma membrane. Once bacteria are internalized,
oligomerization Pseudomonas they must resist degradation in the DC autophagolysosomes.
domain (NOD)1 aeruginosa
NOD2 Muramyl dipeptide Streptococcus KEY CONCEPtS
(MDP) pneumoniae, Host Defenses and Immune Response at
Staphylococcus aureus,
Salmonella typhimurium Epithelial Surfaces to Extracellular Bacteria
NOD-like Whole pathogens S. aureus • Clearance and nonspecific host defenses at skin and mucosal
receptor (NLR) Toxins, LPS, MDP, Bacteria
P3 and RNA surfaces
NLRP1 MDP Bacteria • Epithelial barriers
NLRP1b Microbial toxin Bacillus anthracis • Antibacterial factors (fatty acids, antimicrobial peptides, lysozyme,
phospholipase A 2 )
NLRC4 Flagellin P. aeruginosa
• Mucociliary activity
• Normal microbiota
MOI, multiplicity of infection.
• Adherence blocking molecules
• Specific immune defenses at mucosal surfaces
Complement • Innate immune mechanism
• Immunoglobulins
Complement, a series of more than 20 proteins, is activated by • Phagocytosis at mucosal surfaces
microbial surfaces (alternative complement [AP] cascade) or via • Mucosa-associated lymphoid tissue (MALT), gut-associated lympho-
antibody or by the mannose-binding lectin system (Chapter 21). reticular tissue (GALT), bronchus-associated lymphoid tissue (BALT)
Complement activation leads to microbial lysis and the release
13
of opsonins and chemoattractant molecules for phagocytic cells. Macrophages
The classical complement pathway (CP) can be initiated either Phagocytic cells, macrophages, and PMNs are also present at
by antibody binding to cell surface epitopes or by antibody- mucosal surfaces (Fig. 27.2). These cells express PRRs and migrate
independent autocatalytic activation of C1 to form C1q. Initiation to mucosal surfaces by chemotaxis and diapedesis between
of the APs by bacterial products or mannose-binding protein epithelial cells. Macrophages are also encountered after crossing
leads to the direct deposition of the C3b complex on the bacterial the epithelial barrier. Specialized epithelial M cells of mucosal
surface. Complement activation results in generation of opsonins surfaces are key sites for antigen sampling, including viruses,
16
(as C3b), anaphylatoxins (as C3a), and activation of the late and bacteria and macrophages surround these sites. However,
components of the complement pathway, which results in the enteroinvasive pathogens, such as Shigella, can resist macrophages.
formation of a membrane attack complex (MAC). Gram-positive Shigella induce macrophage apoptotic death by direct interaction
extracellular pathogens resist the bacteriolytic action of the MAC of the bacterial protein IpaB with IL-1β-converting enzyme.
as a result of a thick peptidoglycan layer, which impedes the
insertion of the MAC C5b-9 complex. Gram-negative bacteria Polymorphonuclear Leukocytes
can resist the MAC through structural alterations in their LPS In areas of epithelial inflammation, PMNs can be recruited to
(the possession of O antigen keeps the MAC at a distance from mucosal and skin surfaces. PMNs are more effective in the
the bacterial surface) or by masking or deleting the epitope(s) presence of specific immune defenses, such as antibody and
responsible for binding bactericidal antibody. complement components. PMNs express PRRs and have both
oxygen-dependent and oxygen-independent mechanisms of
Dendritic Cells killing (Chapter 3) (Fig. 27.3). Activated neutrophils can release
DCs sample live bacteria at the mucosal surface, traffic to mucosal granule proteins with direct antibacterial action (e.g., bactericidal/
lymphoid tissue, and induce B cells to produce bacteria-specific permeability-increasing [BPI]) or degradative activity (e.g.,

396 PARt tHREE Host Defenses to Infectious Agents


deficiency, to name a few). Bacterial infections associated with
phagocytic dysfunction are described elsewhere (Chapter 22).
Innate Lymphoid Cells
Innate lymphoid cells (ILCs), a heterogeneous group of cells of
the innate immune system, have lymphoid morphology but lack
the capacity for rearrangement of the antigen receptors, a cardinal
18
feature of the cells of the adaptive immune system. Conventional
natural killer (NK) cells, better known for generating inflam-
matory cytokines and cytotoxic activity against malignant cells
and cells infected by viruses, seem to have a role in the defense
against bacterial pathogens. Murine study data suggest that
conventional NK-cell activation by lung macrophages is protective
19
against S. aureus pneumonia. A subset of ILCs, known as NK-like
cells, produces IL-22 and has been found in mucosal sites, where
these ILCs appear to have a protective activity against bacterial
FIG 27.2 Bacterial Phagocytosis at Mucosal Surfaces. Transmis- pathogens. IL-22 derived from these cells modulates AMP expres-
sion electron micrograph of phagocyte engulfing Neisseria sion by epithelial cells. NK-like cells have minimal cytotoxic
meningitidis at a human respiratory epithelial mucosal surface activity and are not strong producers of IFN-γ, core characteristics
41
(×19 000). of conventional NK cells. 20
Lymphocytes
Carbohydrate Th1 cells are characterized by IFN-γ and function to activate
capsule
macrophages to phagocyte and kill pathogens. While this
Antiphagocytic mechanism of pathogen elimination is primarily directed against
structures pathogens with a predominant intracellular lifecycle, Th1 cells
are relevant for typical extracellular bacteria as pneumococcus
21
and S. aureus. The neutrophilic response to extracellular bacteria
Secreted Pilus is primarily coordinated by Th17 cells. Animal models have
22
bacterial Leukocidin
products C5a peptidase suggested that the Th17 response is central for protection against
that lyse a wide variety of gram-positive and gram-negative bacteria. For
phagocytes or example, Th17 response has been shown to induce nasopharyngeal
impede chemotaxis clearance of Pneumococcus in both animal models and in children.
Differentiation toward the Th17 subtype appears to be favored
by strong antigenic signals and broad activation of pathogen
recognition receptors. IL-17 and IL-22, the signature interleukins
(ILs) of the Th17 response, promote AMP secretion by epithelial
cells, neutrophil migration, and epithelial integrity. The increased
susceptibility of subjects with Job’s syndrome to S. aureus infec-
FIG 27.3 Bacterial Resistance to Polymorphonuclear Leuko- tions demonstrates the importance of the Th17 response in
cytes (PMNs) in the Extracellular Environment. The two humans.
principal mechanisms of bacterial resistance to PMN killing. One in five cells in the intestinal epithelium is a lymphocyte.
These consist of resistance to phagocytosis as a result of bacterial Mucosa-associated lymphoreticular tissue (MALT) comprises
surface components (e.g., capsule or pili) and the action of intraepithelial lymphocytes (IELs), lamina propria lymphocytes,
extracellular proteins that can lyse PMNs (e.g., leukocidins) or and lymphoid follicles (e.g., Peyer patches) and is sometimes
decrease chemotaxis (e.g., C5a peptidase). Bacteria growing in divided into gut-associated (GALT), bronchial-associated (respira-
biofilms may be more protected from PMNs than are bacteria tory tract) (BALT), and genitourinary tract lymphoid tissues
growing in the planktonic state. (Chapter 20). Lymphocytes are important for homeostatic
23
regulation and the maintenance of immune response against
elastase) and chromatin containing the antibacterial histone microbes at mucosal surfaces, including “extracellular” bacteria.
17
H2A. These released compounds work together to form extracel- These cells express PRRs (e.g., TLRs), have constitutive cytotoxic
lular fibers, termed neutrophil extracellular traps (NETs), which activity, secrete chemokines and cytokines important in regulation
can trap and kill gram-positive and gram-negative bacteria and and host defense, and act in concert with mucosal epithelial cells
degrade their virulence factors as well. NETs have been observed and exocrine glands.
in instances of acute inflammation (experimental dysentery and Innate T cells represent a heterogeneous group of cells that
spontaneous appendicitis) and provide a mechanism for reducing possess T-cell receptors (TCRs) and are restricted to MHC-like
bacterial spread at sites of acute infection. The importance of molecules. 24,25 Unlike other T cells, innate T cells gain effector
PMNs in host defense against extracellular pathogens can best capacity before exiting the thymus and therefore can respond
be highlighted by the increased frequency of bacteremias and more readily to stimuli, including infections. This has led to the
other life-threatening infections in patients with neutropenia or idea that innate T cells provide a bridge between the innate and
those individuals with neutrophil deficits (e.g., chronic granu- adaptive immune systems during infections. Invariant natural
lomatous disease, Chediak-Higashi syndrome, or specific granule killer T (iNKT) cells, a subset of innate T cells, are restricted to

CHAPtER 27 Host Defenses to Extracellular Bacteria 397


the MHC-like molecule CD1d. iNKT cells can be directly activated
by bacterial pathogens as a result of binding of cell wall com-
ponents of gram-negative bacteria to CD1d. Interestingly,
cytokines secreted by APCs that have encountered bacterial
pathogens can increase iNKT-cell accumulation of self-lipid
antigen for CD1d activation. Mucosa-associated invariant T
(MAIT) cells are restricted by MR1, a MHC-like receptor that
binds molecules derived from the microbial riboflavin synthesis
pathway. 24,26 Both gram-positive and gram-negative bacteria have
been shown to activate MAIT cells. Murine models of intraperi-
toneal inoculation of gram-negative bacteria suggest the MAIT
cells are important for the early clearance of pathogens. 25,26

Immunoglobulins
Immunoglobulins (Igs), principally secretory IgA and IgG, are
present at mucosal surfaces and in mucosal secretions. Important
in the generation of these immunoglobulins at mucosal surfaces
is the dissemination of IgA and IgG class–committed B- and
T-helper (Th) cells with specificity to an antigen encountered
and processed at one mucosal site to local and distant mucosal
sites. Protective mucosal antibodies against bacteria may be
derived from prior colonization, vaccines, or shared cross-reactive
antigens on normal flora. Mucosal Igs may neutralize bacterial
toxins, facilitate phagocytosis or bactericidal activity, inhibit
bacterial adherence ligands, or sterically hinder other events
necessary for bacterial colonization and invasion. Many extracel-
lular bacterial pathogens (N. meningitidis, N. gonorrhoeae, H. FIG 27.4 Colonization and Adherence of Extracellular Bacteria
influenzae, certain streptococci) colonize and/or infect mucosal at Mucosal Surfaces. Scanning electron micrograph of Neisseria
27
surfaces where protective IgA 1 antibodies could become available. meningitidis adherence and microcolony formation of a human
41
These pathogens secrete an IgA 1 protease that cleaves IgA 1 , thereby upper respiratory mucosa (×16 250).
inactivating the molecule. IgA 1 protease can also recognize other
substrates, notably lysosomal-associated membrane protein 1
(LAMP-1), which are important in host defense. Bacterial infec- provides initial attachment. The pneumococcal CbpA surface
tions associated with abnormal immunoglobulin production or protein promotes mucosal adhesion and dissemination. 29
function are summarized in Chapter 34. Bacteria utilize several mechanisms to avert the host immune
response to bacterial surface antigens (see Table 27.1). Phase
variation of adhesins is a mechanism of immune evasion common
MECHANISM OF IMMUNE EVASION AND DISEASE to pathogenic Neisseria spp. Meningococcus, for example, utilizes
BY EXTRACELLULAR BACTERIA phase variation of the adhesion protein Opa and type IV pili
during the process of colonization of human upper respiratory
31
To colonize human epithelial and mucosal surfaces, bacteria must mucosal surfaces. Sialylation of LPS, a potent inducer of host
overcome the local host defense mechanisms described above. inflammatory response, is an example of bacterial “hiding” of
After navigating these defenses, adhesion to host cells is usually surface antigens. For example, sialylation of lipooligosaccharide,
the first important step for bacterial pathogens (Fig. 27.4). Initial a molecule analogous to LPS, in meningococci has been shown
attachment of bacteria to human epithelial cells is, in part, to increase resistance to CP and AP complement-mediated killing
mediated by pili, fimbriae, or other bacteria ligands or adhesins, by decreasing the deposition of C3b and IgM on the cell surface,
and close adherence of bacteria to the human cell-surface receptors irrespective of capsular phenotype.
involves the cell wall, outer membrane proteins, LPS, and other Many pathogenic extracellular bacteria interact with compo-
bacterial surface structures. The attachment of bacteria to human nents of the complement system to induce negative regulation
29
epithelial cells prevents elimination of bacteria from the host. of the complement pathway. Binding of human factor H (hfH)
Attachment can also induce host cell pathways leading to cyto- by meningococci factor H-binding protein (fHbp) downregulates
skeletal rearrangements, such as elongation and branching of the host AP and helps the organism to evade host innate immunity
32
the microvilli, the accumulation of actin, and calcium efflux, and is now included in the new serogroup B vaccines. Proteolytic
which facilitates close adherence and invasion of epithelial cells degradation of IgA 1 present in the urogenital and respiratory
33
by normally “extracellular” bacteria, especially at sites with fluid tracts is used to avert the action of the humoral system. The
movement. Strains of E. coli that successfully colonize the bladder elaboration of superoxide dismutase and catalase can reduce
and cause renal infection possess pili that allow adhesion to the the efficacy of oxygen (O 2 )–dependent killing of bacteria, but
renal epithelium. 28,29 Type IV pili are fundamental for attachment the high levels of O 2 radicals that accumulate in PMNs probably
of gonococci to the male reproductive tract and play a role on overcome these bacterial enzymes, as evidenced by the susceptibil-
the attachment of meningococci to vascular endothelial cells. 28,30 ity of S. aureus to intraleukocytic killing. Several extracellular
Meningococcal pili also facilitate twitching motility and micro- bacteria possess polysaccharide-rich capsules that resist phago-
colony formation, which allows the penetration of mucus and cytosis. Polysaccharide capsule antigens can mimic human

398 PARt tHREE Host Defenses to Infectious Agents


antigens. Antigen mimicry can lead to autoantibodies, as in the LPS, PG monomers, DNA repeats
case of rheumatic fever and glomerulonephritis after S. pyogenes Lipoproteins, Teichoic acid
infection. Antigen mimicry can also dampen the immune response Microbial toxins, other microbial components
to bacterial antigens, as in the case of serogroup B Meningococcus. (Superantigens)
Other microbial surface structures, such as the pili of the gonococ-
cus, can “stiff-arm” neutrophils, keeping them at a distance. A Pattern recognition receptors (e.g., TLRs)
number of pyogenic bacteria (e.g., S. aureus) secrete leukocidins,
which lyse phagocytes. Other pathogens (e.g., group A strepto-
cocci) inhibit chemotaxis of neutrophils through the elaboration Cytokine stimulation
of enzymes (e.g., C5a peptidase) that proteolytically cleave
chemotactic signals. Some bacteria possess mechanisms to prevent Coagulopathy TNF-a Complement activation
30
opsonization by changing surface antigens. Many bacteria form Kinin stimulation IL-1 C5a
biofilms, which shield these microorganisms from host defense Prostaglandins INF-γ C3a
34
molecules and antibiotics. Leukocytes that invade S. aureus Leukotrienes IL-6, IL-8 Leukocyte chemotaxis
biofilms exhibit impaired phagocytosis and decreased ability to PAF IL-10 Inflammation
kill bacteria. In addition, biofilm matrices can protect bacteria
from antibody-mediated phagocytosis. Fibrin deposition Nitric oxide
As previously noted, many “extracellular” bacteria have an DIC
intracellular component to their lifecycle. The intracellular
environment provides protection from proteins of the comple- Generalized endothelial damage
ment system, Igs, and nonspecific barriers to infection present Vascular leak
28
in the epithelia. The entry of bacteria into epithelial cells provides Tissue edema
access to nutrients and protection from host defenses, allows Vasodilation
protected multiplication, and leads to shedding of organisms Leukocyte activation
Bleeding
back to the mucosal surface, to facilitate transmission and further Temperature dysregulation (e.g. fever)
spread of the infection on the epithelium. Attachment can also
initiate epithelial cell apoptosis or toxin-mediated cell death and Tachycardia, hyperventilation
lead to the breakdown of the epithelial barrier. Hypotension (↑CO, ↓SVR)
Pallor, peripheral vasoconstriction
Cutaneous signs
HOST RISK FACTORS FOR LOCAL AND SYSTEMIC Multiorgan failure
INVASION BY EXTRACELLULAR PATHOGENS (ARDS, renal failure)
Altered mental status
Bacteria that breach mucosal and skin barriers and reach sub- Shock
Death
mucosal tissues of sites, such as pulmonary alveoli or the middle
ear and/or the bloodstream, induce immune responses, including FIG 27.5 Inflammatory cascade initiated during sepsis.
cytokine release, phagocytosis, complement activation, antibody
release or production, and other local or systemic induction of
the inflammatory cascade (Fig. 27.5). The survival of bacteria invasive bloodstream meningococcal and gonococcal infections,
following colonization of the epithelium and access to the indicating an important role for insertion of the complement
bloodstream depends on the integrity of the host immune MAC in the bactericidal activity of human serum against
response (including variability caused by genetic polymorphisms) pathogenic Neisseria. In adults, 10–20% of invasive meningococcal
and on the ability of the bacteria to resist this host immune disease has been associated with a defect in the complement
response. Host factors that increase the risk for the development system.
of systemic disease as a result of extracellular bacteria include In infants, antibacterial activity wanes as levels of passively
polymorphisms in innate immune mechanisms, the absence of transferred maternal antibody fall. This waning of antibody is
bactericidal or opsonizing antibodies, deficiencies in the comple- correlated with the highest incidence of several “extracellular”
ment pathways, and an absence of or reduction in neutrophil pyogenic bacterial diseases (caused by S. pneumoniae, N. men-
function or levels (see Table 27.1). ingitidis, H. influenzae type b) in young children. During child-
Complement deficiencies, either congenital or acquired, hood and adolescence, levels of bactericidal antibodies rise and
increase the risk for invasive bacterial diseases (Chapter 21). rates of these diseases decline. Specific antibodies are acquired
Because C3 plays a critical role in the complement cascade, through carriage and through cross-reacting epitopes on other
congenital C3 deficiency or conditions that reduce C3 (e.g., commensal species. For example, cross-reactive antibodies to N.
systemic lupus erythematosus, cirrhosis, nephritis, C3 nephritic meningitidis are acquired by colonization with commensal Neis-
factor) increase the risk for invasive disease due to pyogenic seria spp. (e.g., Neisseria lactamica) and unrelated bacteria (e.g.,
bacteria, such as S. pneumoniae and N. meningitidis. Mannose- Enterococcus faecium, Bacillus pumilus, and E. coli). The lack of
binding lectin (MBL) is a plasma opsonin that initiates comple- bactericidal antibodies against a strain recently acquired in the
ment activation. MBL gene polymorphisms are found in children upper respiratory tract is an important risk factor for invasive
with meningococcal and pneumococcal sepsis. Properdin defi- meningococcal disease.
ciency, leading to defective AP killing, is also associated with In addition to defects in innate immunity, Igs, and complement
severe and recurrent meningococcal infections. Terminal comple- deficiencies, human genetic polymorphisms are associated with
ment deficiencies (C5–C8) are also associated with recurrent an increased risk or severity of bacterial diseases. For example,

CHAPtER 27 Host Defenses to Extracellular Bacteria 399


FcγIIa (CD32) receptor polymorphisms, Fcγ-receptor III (CD16), TABLE 27.5 Examples of Extracellular
MBL, TLR4, tumor necrosis factor (TNF) promoter region Bacteria Causing Sepsis by Presumed Site
polymorphisms, plasminogen activator and inhibitor expression, of Infection
and hereditary differences in cytokine induction influence
susceptibility to meningococcemia. Each of these polymorphisms Lung Streptococcus pneumoniae, Haemophilus
can influence the course of invasive bacterial infection by influenc- influenzae, Pseudomonas aeruginosa
ing the response of the inflammatory cascade. Abdomen Escherichia coli, mixed anaerobic infections
Urinary tract E. coli, Klebsiella spp., Enterobacter spp.,
Enterococcus spp.
tHERAPEUtIC PRINCIPLES Soft tissue Streptococcus pyogenes, Staphylococcus
Sepsis aureus, polymicrobial
Intravenous line S. aureus, Candida spp.
• Early effective antibiotic therapy associated with improved Other Neisseria meningitides
outcomes
• Lack of “source control” (e.g., removal of infected lines, drainage of
abscesses) linked to poor outcomes even in the presence of effective
antibiotics (e.g., LPS) and intracellular environments (e.g., DNA fragments)
• Intensive and supportive care, management of fluid, electrolytes, and (see Table 27.3) The severity of sepsis is also influenced by
respiratory function polymorphic alleles of genes involved in the inflammatory
• Insulin for glucose control—unknown mechanism of protection; cascade. 35
neutrophils have impaired function in the presence of hyperglycemia,
insulin can have antiapoptotic effects
KEY CONCEPtS
DELETERIOUS HOST RESPONSES Definitions of Sepsis and Septic Shock (Third
International Consensus Definitions for Sepsis
Inflammation and Autoimmunity and Septic Shock) 42
The host immune response can be the leading cause of tissue
injury in the acute phase of an infection. Brain edema and infarcts, • Sepsis—life-threatening organ dysfunction causes by dysregulated
host response to infections (including nonbacterial pathogens)
which are devastating consequences of pyogenic meningitis, occur • Septic shock—subset of patients with sepsis with increased risk
as a result of the host inflammatory response. Corticosteroids of death due to profound circulatory, cellular, and metabolic
are currently recommended as an adjunctive therapy to pneu- abnormalities
mococcal meningitis, an acute pyogenic infection. The use of • SIRS (systemic inflammatory response syndrome)—nonspecific
small molecules targeting specific immunological pathways is response to infectious and noninfectious insults; current guidelines
an area of ongoing research. As acute infections are initially recommend use of qSOFA (Quick Sequential Organ Failure Assessment
Score) score in lieu of SIRS criteria, given its higher predictive value
characterized by an inflammatory response followed by an for sepsis
antiinflammatory response, the timing of use of these compounds
is of the utmost importance. 35,36 Infections can lead to autoimmune
diseases. Molecular mimicry between a bacterial antigen and a The morbidity and mortality of bacteremia and sepsis have
host protein is a mechanism of generation of autoantibodies. been directly correlated with the initial levels of proinflammatory
Examples include rheumatic fever and glomerulonephritis after cytokines and the amount of circulating bacterial components.
S. pyogenes infections, reactive arthritis following Chlamydia Indeed, the severity of gram-negative sepsis has been equated
trachomatis urethritis, and the Guillain-Barré syndrome following with high levels of endotoxin, increased levels of cytokines, and
Campylobacter jejuni enteritis. Molecular mimicry can also limit excessive activation of the AP. Disseminated intravascular coagula-
selection of epitopes for vaccine development. tion, which often accompanies gram-negative sepsis, is caused
by excessive activation of the coagulation cascade and downregula-
Sepsis tion of the fibrinolytic system associated with high levels of LPS.
Septicemia remains a leading cause of death in the United States Levels of natural anticoagulants in the vasculature, such as
and accounts for several billion dollars in health care expendi- antithrombin and protein C, are often low in gram-negative
ture. 35,36 Both gram-negative and gram-positive bacteria can sepsis. The onset and severity of disseminated intravascular
rapidly multiply in the bloodstream and trigger sepsis and septic coagulation may be influenced by genetic polymorphisms in
shock (Table 27.5). Septic shock is a result of an initial and plasminogen activation or inhibition. The generalized, altered
widespread systemic proinflammatory response, resulting in vascular endothelial lining facilitates thrombosis and thrombo-
hypotension, organ failure, and death. The later phase of sepsis cytosis. Although much remains to be learned about the mecha-
is also characterized by an antiinflammatory response. Although nisms by which gram-negative and gram-positive bacteria and
survival of patients with the acute phase of sepsis has improved, microbial products trigger sepsis, significant advances have been
advances on treatment and prevention of death, which can be made recently, particularly with endotoxin-mediated sepsis.
secondary to nosocomial infections, have been slower. These Advances during the past decade include the identification of
secondary infections are often caused by less virulent organisms, certain LPS–host protein interactions that result in delivery of
likely as a result of “immunoparalysis” (as a result of an exag- LPS to host cell receptors and gene activation events that result
gerated antiinflammatory reaction) and also breaching of the in elevated expression of a diverse array of proinflammatory
physical barriers to infection as a result of invasive medical and antiinflammatory mediators (Fig. 27.6).
procedures (e.g., intravenous lines, intubation, and bladder For example, TLR4 signaling requires an accessory protein,
catheterization). The systemic inflammatory cascade of sepsis myeloid differentiation protein-2 (MD-2), which binds directly to
37
is initiated by recognition of PAMPs by PRR, both in extracellular endotoxin. The key point, however, is that the LPS engagement

400 PARt tHREE Host Defenses to Infectious Agents



Gram-negative bacteria Macrophage

Release of
cytokines
Cytokine
LPS gene
expression

Nucleus
Release of LPS in vesicles or by mCD14
the action of bacteriolytic agents Mobilization of
transcription factors
LPS oligosaccharide
mCD14 Signal
MD-2 transduction
Lipid A

LBP
sCD14 TLR-4
LBP sCD14

LPS Lipopolysaccharide mCD14 Membrane-bound CD14
LBP LPS-binding protein TLR-4 Toll-like receptor 4
sCD14 Soluble CD14
FIG 27.6 Lipopolysaccharide (LPS) Triggering of Cytokine Production by Macrophages.
Steps known or presumed to be necessary for LPS triggering of proinflammatory cytokines.


of MD-2/TLR4 on host cells, particularly macrophages, triggers
intracellular signaling events that ultimately, through NF-κB and CLINICAL RELEVANCE
other pathways, result in cytokine gene activation and production Signs of Septicemia
of cytokines (TNF-α, IL-1, IL-6, IL-8, IFNs). Other TLRs (e.g.,
TLR2) play a critical role in the recognition of lipoproteins, and • Shaking chills, spiking fevers, or hypothermia (<36°C)
• Tachycardia, hyperventilation
the recognition of these components is a likely a key determinant • Pallor (peripheral vasoconstriction) and acrocyanosis, but 10–20% are
in the development of septic shock seen with gram-positive infec- flushed “warm shock”
tions. TLR5 recognizes bacterial flagella, and such recognition • Nausea/vomiting, diarrhea
is of importance in the host response to motile bacteria. Some • Hypotension <90 mm Hg or >40 mm Hg decrease from baseline, in
human pathogens (e.g., H. pylori) produce flagellin molecules 20–35% of cases
that do not engage TLR5. TLR9 has been shown to recognize • Cardiac output, decreased systemic vascular resistance (SVR)
• Cutaneous signs: purpura fulminans, petechiae, palpable purpura,
14
bacterial DNA CpG dinucleotides. Taken together, the clinical ecthyma gangrenosum
syndrome of septic shock represents a series of interactions of • Change in mental status
bacterial products in the vascular space with pattern recogni- • Signs may be more subtle in elderly and uremic (renal failure)
tion molecules on serum proteins (Lipopolysaccharide-binding patients
3
protein [LBP] and soluble CD14) and with host cell receptors • White blood cell (WBC) count >12 000 cells/mm or <4000 cells/
3
(MD-2/TLR4 and TLR2, other TLRs) leading to signaling events mm with >10% immature (band) forms
and release of transcriptional factors that modulate cytokine • Oliguria (<20 mL/h of urine)
gene expression. These events also trigger other events in the
inflammatory cascade, leading to activation of the coagulation,
complement, and kinin pathways. Superantigens can activate ENHANCEMENT OF IMMUNE RESPONSES
large pools of non–antigen-specific T cells by binding to MHC TO EXTRACELLULAR BACTERIA (VACCINES
class II molecules and the TCR Vβ region outside the peptide- AND IMMUNOMODULATION)
binding domain. The result is a cytokine storm with clinical
manifestations of sepsis. Vaccines are the most affordable and cost-effective health interven-
Early and effective antimicrobial therapy is the primary goal tion for enhancing the immune response to extracellular bacterial
in the treatment of sepsis. In contrast to the initial phase of and other microbial pathogens (Chapter 90). The use of vaccines
sepsis characterized by the release of TNF-α, IL-1, IL-6, and to prevent diphtheria, pertussis, tetanus, and infections by N.
IFN-γ, an antiinflammatory response may predominate during meningitidis, H. influenzae b (Hib), and S. pneumoniae is a major
the latter phase. The clinical failures of antiinflammatory thera- public health success. Vaccines are also used for prevention of
peutic mediators (antiendotoxin antibodies, TNF-α, antagonists disease caused by Salmonella typhi, Vibrio cholera, and Bacillus
of IL-1, or platelet-activating factor) in sepsis suggests that this anthracis. The efficacy of vaccines to extracellular bacteria is most
hypoinflammatory state encountered in many patients at presenta- often correlated with enhanced bactericidal antibodies, opsonic
tion could be an additional target of immune modulation. antibodies, and/or neutralizing antibodies, both systemically and

CHAPtER 27 Host Defenses to Extracellular Bacteria 401


at mucosal surfaces. Enhancement of these immune mechanisms squalene. Cytokines, such as IL-1, IL-2, IL-12, IL-18, and
can provide protection even in immunocompromised individuals granulocyte macrophage–colony-stimulating factor (GM-CSF),
(although immune responses are diminished in these individuals). also modify and enhance immune responses to vaccines. IL-12,
For example, vaccination with meningococcal capsular vaccines for example, induces strong Th1 shifts, and GM-CSF is a comigrat-
protect patients with hereditary complement deficiencies by ing signal for DCs and stimulates antigen processing and presenta-
enhancing opsonophagocytic activity. However, vaccines have tion. Antigen recognition and processing in macrophages is critical
limitations in terms of long-lived immune responses, safety issues, to determining T-cell responses and can be manipulated by
and poor responses in certain populations (extremes of age) selected adjuvants. Immune modulation is being evaluated not
when infections with extracellular bacteria are most common. only for the enhancement of bacterial vaccines but also as adjunct
Advances in genetic engineering, immunology, molecular patho- therapy for serious bacterial infections, such as sepsis. Vaccines
genesis, vaccine adjuvants, and delivery systems are resulting and specific immunotherapeutic approaches, such as cytokines,
in the development of new vaccines and vaccine approaches may also find use against chronic tissue-damaging inflammatory
that enhance the immune response to extracellular bacterial reactions created by persistent extracellular bacteria (e.g., Heli-
pathogens (e.g., protein–polysaccharide conjugate vaccines for cobacter) and autoimmune reactions that may be induced by
Hib, S. pneumoniae, and N. meningitidis). cross-reactive bacterial antigens (e.g., C. jejuni and Guillain-Barré
The conjugation of bacterial polysaccharides to carrier proteins, syndrome).
such as diphtheria or tetanus toxins, has been a major advance
in stimulating immune responses to saccharide bacterial antigens. TRANSLATIONAL RESEARCH OPPORTUNITIES
Polysaccharide capsules, for example, when used alone, are
“T-independent” antigens; they do not require the presence of An important challenge for the next decade will be to take the
T cells to induce an immune response, and generate IgM as the rapidly expanding basic discoveries in innate immunity, systems
dominant antibody produced (Chapter 6). Failure to induce biology, and response to bacterial antigens into clinical applica-
memory and failure of affinity maturation follow polysaccharide tions. The design and use of bacterial vaccines through the
immunization. Thus polysaccharides are poorly immunogenic assessment of innate immune molecular signatures after vaccina-
in infants, older adults, and those with impaired antibody tion, both for general use and for subpopulations of nonre-
production—groups most susceptible to encapsulated bacterial sponders, is one example. A second is the continued development
pathogens. Covalent linkage of the polysaccharide to a carrier of small-molecule inhibitors or enhancers that specifically target
protein converts the polysaccharide to a thymus-dependent innate immune pathways to modulate bacterial immune responses.
antigen generating IgG anticapsular antibodies and memory B A third is the control of mucosal immune responses to prevent
cells. Because these vaccines induce vigorous mucosal immune or eliminate colonization by bacterial pathogens. A fourth is the
responses, they also provide “herd” protection. A major (and understanding of role of the microbiome in shaping the immune
unanticipated) result of vaccination with the Hib, meningococcal, response to pathogens and vaccines and its therapeutic potential
and pneumococcal conjugate vaccines is the interruption of for both infections and noninfectious diseases. Fecal microbiota
mucosal carriage, decreased transmission, and herd protection. transplantation for C. difficile colitis is an early example of
These vaccines are now used as part of the routine immunization therapeutic use of the microbiome. Finally, the development of
series in all age groups. new therapies for acute bacterial sepsis may be based on improved
The presentation to CD4 T cells of an antigen by MHC class understanding and control of the immune responses in sepsis. 36
II molecules is critical for an immune response and influences
the amount of antibody, the affinity of that antibody, and the
duration of response. CD4 T cell subsets influence the qualitative ON tHE HORIZON
and quantitative features of an immune response to vaccines • Tailoring vaccine design based on assessment of innate immune
and bacterial antigens. As new subsets of CD4 T cells are described, molecular signatures
research is necessary to determine their role in existing vaccines • Small molecule inhibitors or enhancers specifically targeting innate
and strategies to exploit them in new preparations (Chapter immune pathways
38
16). Mucosal vaccination is an attractive strategy for protection • Identification of immune responses that prevent or eliminate mucosal
bacterial pathogen colonization
against extracellular bacteria as mucosal colonization often • Development of new therapies modulating immune response in sepsis
precedes invasive disease. The hope is to generate effective immune • Defining microbial community and metagenome changes after antibiotic
responses at the mucosa and avoid the need for parenteral treatment
39
injection. In addition, recent advances in the application of • Managing disease based on the human microbiome
systems biology to define molecular signatures that correlate
with and predict vaccine immunity have greatly enhanced our
understanding of immune responses to vaccination. 40 Please check your eBook at https://expertconsult.inkling.com/
Considerable progress is being made in the development of for self-assessment questions. See inside cover for registration
new vaccine adjuvants and immune modulators (see Chapter details.
90 for updated information). Aluminum salts, used since the
1930s in many vaccines against bacteria, induce a >90% Th2
response. As noted above, bacterial toxins as conjugates can be REFERENCES
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6. van Rensburg JJ, Lin H, Gao X, et al. The human skin microbiome 26. Napier RJ, Adams EJ, Gold MC, et al. The role of mucosal associated
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MBio 2015;6:e01315–15. 27. Mulks MH, Plaut AG. IgA protease production as a characteristic
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2006;24:353–89. 31. Rouphael NG, Stephens DS. Neisseria meningitidis: biology, microbiology,
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CHAPtER 27 Host Defenses to Extracellular Bacteria 402.e1


MUL t IPLE-CHOICE QUES t IONS

1. With regard to the use of corticosteroids as adjunctive therapy C. This patient should have diagnostic workup for terminal
for acute infections: complement deficiencies. Meningococcal vaccination should
A. Corticosteroids are immunosuppressive and should be be offered.
avoided during acute infections. D. This patient should have diagnostic workup for terminal
B. Corticosteroids are generally recommended for the treat- complement deficiencies. If confirmed, stem cell trans-
ment of sepsis. plantation can be offered.
C. Corticosteroids are never contraindicated during acute 3. The normal microbiota maintains a balance with the host
infections. by avoiding activation of pathogen recognition receptors.
D. Corticosteroids are beneficial in certain types of acute Pathogen recognition receptors are activated by pathogenic
infection, presumably because of their antiinflammatory bacteria only.
effect.
A. False. Colonizers and pathogens have pathogen-associated
2. A 18-year-old college freshman presents to the hospital with molecular patterns, and both can activate pattern recogni-
meningococcal meningitis. Upon review of his medical history, tion receptors.
you learn that he had disseminated gonococcal infection at B. True. Colonizers are devoid of pathogen-associated
the age of 16 years. Should you consider investigation for an molecular patterns.
immunodeficiency? If so, which one? Should you offer any C. True. Colonizers have pathogen-associated molecular
prophylactic therapies for this patient? patterns but do not activate pathogen recognition
A. No investigation for an immunodeficiency is warranted. receptors.
Meningococcal vaccination should be offered. D. False. Colonizers and pathogens have pathogen-associated
B. These infections suggest a T-helper (Th)17 deficiency, molecular patterns, and both can activate pattern recogni-
and there is no approved prophylactic therapy for this tion receptors, but colonizers only do so when invading
disorder. host tissues.

28









Host Defenses to Spirochetes



Nicolás Navasa, Erol Fikrig, Juan Anguita








Spirochetes constitute a unique group of bacteria that inhabit are the major immunogens of B. burgdorferi and most likely T.
many different environments, such as soil, arthropods, and pallidum, and thus they are their dominant proinflammatory
mammals. These microorganisms cause numerous human ill- agonists. Five percent of the chromosomal ORFs and 14.5–17%
nesses, including syphilis and Lyme disease. Spirochetes share of the functionally complete ORFs contained in the plasmids of
a typical spiral shape and a distinctive flat-wave morphology. B. burgdorferi encode putative lipoproteins. Interestingly, only
Cellular dimensions vary over a wide range, with a diameter 2.1% of the T. pallidum ORFs encode putative lipoproteins. The
between 0.09 to 0.75 µm and lengths that range from 3 to 500 µm. abundant lipoprotein coding potential of B. burgdorferi suggests
They are motile organisms with a multilayered outer membrane that these molecules may be important for the survival of the
that encapsulates a peptidoglycan layer surrounding their inner spirochete. In fact, as a result of environmental changes, expres-
membrane. Motility is enabled by the presence of endoflagella sion of several B. burgdorferi lipoproteins changes throughout
5
located in the periplasmic space. These axial filaments are arranged the lifecycle of the spirochete. For instance, the lipoprotein
in a bipolar fashion and extend toward the opposite end of the outer surface protein (Osp) A is expressed at high levels in the
cell (Fig. 28.1). The viability of the organism is dependent on gut of the unfed tick, but upon feeding OspA expression is
5
an intact outer membrane, which can be damaged by variations downregulated, and the expression of OspC increases 90-fold.
in osmolarity, antibodies, or complement resulting in the loss of It is thought that many Osps have adhesive functions, and it
intracellular components and ultimately death of the bacterium. has been shown that OspA is involved in the attachment of B.
Several spirochetal species are able to induce disease in burgdorferi to the gut of the tick through an interaction with the
6
mammals (Table 28.1). Lyme disease was first discovered in 1976 tick receptor TROSPA. Furthermore, OspC may be necessary
as an illness affecting a cluster of children in Lyme, Connecticut. for the migration of B. burgdorferi from the gut of the tick to
Several manifestations of the disease, however, had been known the salivary glands and for its survival in the mammalian host.
for over a century in parts of Europe. In 1982, the agent of Lyme Changes in lipoprotein expression are primarily regulated by the
7
1
disease was identified as Borrelia burgdorferi. In contrast, alternative sigma factors, RpoS and RpoN and other unknown
Treponema pallidum subspecies pallidum is the causative agent mechanisms.
of venereal syphilis, a disease that has been recognized for over
5 centuries, although its agent was not identified until 1905. 2 CLINICAL MANIFESTATIONS
The genomes of B. burgdorferi and T. pallidum have been
3,4
sequenced. Despite similar ancestry and morphological features, Lyme Disease
these spirochetes have striking differences at the genetic level, B. burgdorferi sensu stricto (B. burgdorferi) is the etiological agent
which may account for the differences in their lifecycles, envi- of Lyme disease in the United States and other parts of the world,
ronmental adaptations, and the diseases they cause. Both B. whereas B. afzelii and B. garinii are agents of Lyme disease that
burgdorferi and T. pallidum have relatively small genomes are restricted to Europe and Asia. Infection with Lyme disease–
compared with other microorganisms. B. burgdorferi has one of causing spirochetes has also been observed in Japan, Russia, and
the most complex genomes known among prokaryotes, with a China. In the United States, transmission of the spirochete is
single linear chromosome and 21 plasmids, the largest number through the hard-bodied ticks of the Ixodes complex, mainly I.
of plasmids of any characterized prokaryote. Of the 21 plasmids, scapularis and I. pacificus, whereas I. ricinus is responsible for
nine are circular and 12 are linear. Furthermore, less than 10% the transmission of the spirochete in much of Eurasia. According
of B. burgdorferi plasmid-coding regions are found in other to the Centers for Disease Control and Prevention (CDC), Lyme
microorganisms, including spirochetes, which underscore the disease is the most common tickborne disease in the United
8
uniqueness of this spirochete among microorganisms. Unlike States and the fifth most common nationally notifiable disease.
B. burgdorferi, T. pallidum contains a single circular chromosome The disease is concentrated in the North Eastern seaboard, with
with no plasmids. Yet, 476 open reading frames (ORFs) in T. further presence in the northern Midwest and the West of the
pallidum have orthologous genes in B. burgdorferi, and almost Continental United States. In Europe, diagnosed Lyme disease
60 of these orthologous genes encode proteins of unknown accounts for more than 65 000 cases annually, which tend to
biological function that are specific to spirochetes. concentrate in countries of Central and Eastern Europe. However,
In contrast to other spirochetes, B. burgdorferi and T. pal- the disease is present to different degrees throughout much of
lidum do not contain lipopolysaccharide (LPS). Lipoproteins Europe. In both the United States and Europe, it is widely believed

403

404 Part tHrEE Host Defenses to Infectious Agents


that the disease is underreported and that the suspected real
number of cases may be much higher.
20-30µm An early hallmark of infection is the appearance of a skin
0.2-0.3µm rash, known as erythema migrans (Fig. 28.2), which appears at
the inoculation site, often during the first week of infection
(stage I Lyme disease), as a result of local inflammatory responses.
A Other symptoms secondary to the local inflammatory responses
Outer membrane occurring during stage I can affect more distal sites and may
include fever, headache, malaise, myalgia, and/or arthralgia.
Periplasmic flagella Inner membrane Hematogenous dissemination of the spirochete is stage II Lyme
disease and results in colonization of different tissues and/or
Protoplasm organs and presentation of a range of symptoms, such as conduc-
tion system abnormalities, meningitis, and acute arthritis. Joint
inflammation appears in 60% of untreated individuals in the
United States and predominately affects large joints, especially
the synovium of the knee. Some untreated individuals develop
B stage III Lyme disease, which is generally characterized by
FIG 28.1 Borrelia burgdorferi structure is characterized by a prolonged infection with the spirochete. Late-stage symptoms
distinctive flat-wave morphology consisting of approximately 18 may include chronic arthritis, neuroborreliosis, or cutaneous
bends and a length of 20–30 µm (A). A cross-section of this lesions, such as acrodermatitis chronica atrophicans. The range
spirochete reveals the endoflagella, which are responsible for of symptoms that appear upon infection in Europe and the
the unique morphology and motility of this organism (B). United States vary in relative terms: arthritis and carditis are



TABLE 28.1 Major Diseases Caused by Spirochetes*
Disease agents Distribution transmission Symptoms
Lyme disease Borrelia burgdorferi North America, Europe Tick engorgement Development of a skin rash known as erythema
B. garinii Asia, Europe migrans, accompanied by other symptoms, such as
B. afzelii Asia, Europe malaise, myalgia, and/or arthralgia. Symptoms can
B. andersonii North America progress to include carditis and arthritis. Persistent
B. japonica Japan infection can result in chronic arthritis,
B. lusitaniae Southern Europe neuroborreliosis, or cutaneous symptoms
B. valaisiana Europe, Ireland, UK (acrodermatitis chronica atrophicans).
B. mayonii North America
B. miyamotoi North America
Relapsing fever B. hermsii Western USA Tick engorgement Clinical manifestations of infection include high-
B. turicatae Southwestern USA, Mexico density spirochetemia, high fever, myalgias, and
B. parkeri Western USA arthralgias and can even include cerebral
B. mazzotti Central America hemorrhage and fatality.
B. venezuelensis Central America
B. duttonii Sub-Saharan Africa
B. crocidurae North Africa, Middle East
B. persica Middle East, Central Asia
B. hispanica Iberian peninsula, North Africa
B. latyschewii Iran, Iraq, Eastern Europe
B. caucasia Iraq, Eastern Europe
Venereal syphilis Treponema pallidum Worldwide Sexual contact Disease progresses from a primary lesion (chancre)
pallidum to a secondary eruption and then to a latent period,
and if left untreated, tertiary symptoms may
appear.
Endemic syphilis or T. pallidum Eastern Mediterranean region, Nonsexual skin Symptoms begin with a slimy patch inside the
Bejel syphilis endemicum West Africa contact mouth, followed by blisters on the trunk and limbs.
Bone infection in the legs soon develops, and in
the later stages, lumps may appear in the nose and
on the soft palate of the mouth.
Yaws T. pertenue Humid equatorial countries Nonsexual skin Destructive lesions of the skin and bones, which is
contact rarely fatal but can be debilitating.
Pinta T. carateum Mexico, Central America, Nonsexual skin Dark-colored skin lesions found on those areas of the
South America contact body that are exposed to sunlight. Eventually, the
skin lesions become discolored.
Leptospirosis Leptospira Worldwide Urine from an Symptoms include fever, headache, chills, nausea
interrogans infected animal and vomiting, eye inflammation, and muscle aches.
In more severe cases, the illness can result in liver
damage and kidney failure.
*Spirochetes are the causative agents of many diseases, which can have social as well as lasting health-related consequences.

CHaPtEr 28 Host Defenses to Spirochetes 405



Treatment
During early stages of Lyme disease, such as that during which
erythema migrans is present, oral administration of doxycycline
(100 mg twice daily) or amoxicillin (500 mg three times a day)
for approximately 2 weeks is recommended. Doxycycline has
the advantage of being effective against Anaplasma phagocyto-
philum, which may also be transmitted by ticks. In areas where
B. burgdorferi infection is prevalent, some experts recommend
antibiotic therapy for individuals who served as hosts to I.
scapularis ticks that were attached longer than 40–48 hours—the
time required for transmission of the spirochete. It is extremely
difficult, however, to consistently make accurate determinations
of the species of tick and the degree of engorgement. Furthermore,
randomized double-blind clinical trials involving individuals
who were bitten by I. scapularis ticks led to the conclusion that
antibiotic treatment of all individuals who had vector ticks
FIG 28.2 Erythema migrans caused by infection with Borrelia removed is probably not warranted.
burgdorferi, the Lyme disease agent. (Courtesy of Gary Wormser,
MD.) Venereal Syphilis
Infection with the agent of syphilis, T. pallidum subspecies pal-
lidum, occurs worldwide. T. pallidum is an obligate human parasite
that is almost exclusively transmitted when contact with infectious
exudates from lesions of the skin and mucous membranes of
more commonly found in the United States, whereas patients infected individuals occurs. Clinically, this treponemal infection
in Europe tend to show higher incidence of skin and nervous is first characterized by the formation of a hardened and painless
system involvement. This may be attributed to the heterogeneity ulcer at the initial site of infection. This primary lesion, called
of the B. burgdorferi sensu lato genospecies that cause the disease. a chancre, forms after invasion of the bloodstream by the spi-
In the United States, in the large majority of cases, B. burgdorferi rochete. Four to 6 weeks after infection, the edges of the chancre
sensu stricto is involved, whereas in Europe infections by B. afzelii roll inward and upward and, in most cases, a secondary eruption
and B. garinii predominate. appears, often accompanied by a rash on the palms of the hands
and the soles of the feet. The secondary manifestations resolve
Diagnosis within weeks to a year after the development of a vigorous
A detailed clinical history and comprehensive physical examina- cell-mediated immune response. Long periods of latency followed
tion are critical for the accurate diagnosis of Lyme disease. by late lesions of skin, bone, and viscera, as well as the cardio-
Appropriate laboratory testing, however, is a valuable diagnostic vascular system and the CNS, can occur despite clearance of the
aid. A two-tiered approach is standard for the serodiagnosis of majority of the treponemes, which coincides with the resolution
Lyme disease. Using this approach, serum is first tested for the of the primary syphilitic lesion.
presence of B. burgdorferi–specific antibodies by using a sensitive
method, such as enzyme-linked immunosorbent assay (ELISA) Diagnosis
or immunofluorescent assay (IFA). Sera testing negative for Much like Lyme disease, the diagnosis for syphilis is based on
antibodies generally need not be tested further; however, those the clinical presentations of the disease and serological tests. In
found to be positive or equivocal are further evaluated by the addition, dark-field microscopy can be used for the identification
more specific immunoblotting for immunoglobulin M (IgM) of T. pallidum in the serous exudates of the chancre. This
and IgG antibodies. The detection of at least two of three specific approach, however, is limited by the number of live treponemes
bands in IgM or five of 10 specific bands in IgG is considered in the exudates and by the presence of nonpathological trepo-
positive. Patients with early Lyme disease often report to a nemes in oral and anal lesions; as such, negative examinations
physician during the first few days of infection, at which time a on three independent days are required before a lesion is con-
detectable humoral response may not have developed. Conse- sidered negative for T. pallidum.
quently, the two-tiered approach is considered highly sensitive Infection with T. pallidum leads to the production of non-
during the later stages of the disease (>90%) and less sensitive specific antibodies, which is the basis for other diagnostic tests,
during very early infection. Furthermore, a positive serological such as the traditional nontreponemal serological tests, including
test, particularly IgG, is evidence of exposure to B. burgdorferi, the Venereal Disease Research Laboratory (VDRL) and rapid
but it does not necessarily indicate active infection. All serological plasma reagin (RPR) tests. Because these tests are nonspecific,
tests must then be evaluated in conjunction with a clinical false-positive reactions can occur as a result of pregnancy,
assessment by the attending physician. Other diagnostic methods, autoimmune disorders, or infections. Therefore treponemal-
such as culture and polymerase chain reaction (PCR) detection specific tests, which detect antibodies to various antigens of T.
of B. burgdorferi, may be very useful to detect active infection, pallidum, are often used to confirm the results of a nonspecific
particularly of skin, joints, and the central nervous system (CNS). test. Interestingly, treponemal-specific tests are just as sensitive
Culture, however, is generally limited to research laboratories, as nontreponemal tests; however, they are much more difficult
and the sensitivity and specificity of PCR can vary greatly among and expensive to perform, which limits their use. These tests
testing centers. include, but are not limited to, the enzyme immunoassay (EIA)

406 Part tHrEE Host Defenses to Infectious Agents


test for T. pallidum-specific IgG, T. pallidum hemagglutination or epithelial cells, resulting in the recruitment of different types
test (TPHA), fluorescent treponemal antibody-absorption test of innate immune cells and their activation. Each PRR recognizes
(FTA-ABS), and ELISA. a specific structure that is present in a group or groups of
microorganisms and that distinguishes them from the more
Treatment specific recognition of antigens by the T- and B-cell receptors.
Susceptibility to infection with T. pallidum is universal, although The recognition of patterns instead of specific antigens provides
only 30% of exposures with lesions teeming with the spirochete the innate immune system with a rapid way to respond to infecting
result in infection. Infection results in gradual development of organisms until the more specific response mediated by T and
immunity against T. pallidum and often against heterologous B cells develops.
treponemes as well. However, treatment with long-acting penicillin A distinct innate cellular infiltration profile has been observed
subverts the development of immunity against T. pallidum. in B. burgdorferi–infected tissues. Based on studies using mice,
Administration of 2.4 million units in a single intramuscular the inflamed heart appears to predominately comprise macro-
dose the day that the primary, secondary, or latent syphilis is phages, with smaller amounts of T and invariant natural killer
diagnosed is effective at killing the spirochetes. For people who T cells (iNKT cells). However, neutrophils are the primary innate
are allergic to penicillin, there are alternative treatments, such immune cell found in the joints. In this tissue, resident myeloid
as doxycycline. cells act as the initiators of type I interferon (IFN) production
upon encounter with B. burgdorferi, whereas endothelial cells
HOST DEFENSES TO B. burgdorferi and joint fibroblasts expressing adhesion/ activation markers
amplify the response and serve as the major source of disease-
KEY CONCEPtS promoting chemokines. This tissue-specific tropism complicates
our understanding of the host defense against the spirochete,
Protective Versus Pathological Responses to but this suggests that macrophages are more efficient at preventing
Borrelia burgdorferi successful colonization of B. burgdorferi because carditis is a less
frequent manifestation of Lyme disease. These early defense
The early immune response to B. burgdorferi is necessary to control
spirochetal burden; however, by itself, it is not sufficient to resolve responses also have a role in determining the type, strength, and
infection. duration of the more specific adaptive immune response, beyond
Phagocytosis is a key element of the innate immune response, which their direct bacterial-killing capability.
is involved in the elimination of the bacteria while also contributing
to the proinflammatory output of macrophages Early Pathogen Recognition
The T cell–mediated response appears to be involved in pathology arising The interaction of B. burgdorferi lipoproteins with complexes
from infection.
A T cell–independent B-cell response is sufficient to resolve infection formed by TLRs 1 and 2 initiates a series of signaling cascades
with B. burgdorferi. that results in the production of proinflammatory cytokines
(IL-1β, tumor necrosis factor [TNF], interleukin [IL]-12, and
IL-18, among others), chemokines (IL-8, monocyte chemoat-
B. burgdorferi virulence is attributed, in part, to the evolution tractant protein [MCP]-1, keratinocyte chemoattractant [KC]),
of the spirochete’s sophisticated tactics to evade killing mecha- metalloproteinases, adhesion molecules (E-selectin, vascular cell
nisms during all stages of the immune response: the first stage, adhesion molecule-1 [VCAM-1] and intercellular adhesion
9
beginning with transmission into the host via tick engorgement, molecule-1 [ICAM-1]), and type I IFNs. These interactions are
at which time the spirochete is exposed to serum complement critical for generating an effective spirochete-clearing inflam-
and cellular immunity; and the second and third stages, that is, matory response, as demonstrated by experiments using mice
hematogenous dissemination to and colonization of peripheral deficient for TLR1, TLR2, or their adaptor molecule, MyD88.
sites, at which time the host is producing specific antibodies. These mice had significant increases in B. burgdorferi burdens
Both the innate and adaptive immune responses elicited by B. after infection. Moreover, humans with decreased TLR1 expression
burgdorferi are discussed, with the supposition that these responses are hyporesponsive to the original OspA-based Lyme disease
are required for efficient bacterial clearance, while acknowledging vaccine, and macrophages from these subjects also have dimin-
that unnecessarily prolonged or intense responses may contribute ished inflammatory responses to the lipoprotein. Besides TLR1/
to pathology arising from infection. Indeed, predisposition to TLR2 complexes, other members of this family of receptors
infection could be the result of one or more monogenic traits participate in, and amplify, the inflammatory response to B.
that confer primary immunodeficiencies; whether or not this is burgdorferi. These include TLR5, TLR7, TLR8, and TLR9, which
the case remains to be determined, but human studies have recognize different bacterial constituents. Importantly, the
shown that responses to B. burgdorferi are diminished in individu- development of inflammatory arthritis in the absence of TLRs
als with specific mutations in or diminished expression of innate or MyD88 was the first suggestion that alternative pathways
immune cell receptors (nucleotide-binding oligomerization trigger the inflammatory response to the spirochete. Other PRRs
domain-2 [NOD2] and Toll-like receptor-1 [TLR1]). that have been shown to be involved in the response to the
spirochete include NOD-like receptors. For the full inflammatory
Innate Immune Responses response to take shape upon recognition of B. burgdorferi, most
The initial recognition of pathogens with cells of the host relies of the interactions of B. burgdorferi constituents and PRRs occur
on a complex interplay between pathogen recognition receptors within the phagolysosome. Therefore phagocytosis is a hallmark
(PRRs) and bacterial constituents, and this initiates a cascade of the innate immune cell recognition of the spirochete.
of responses leading to the upregulation of chemokines and The interaction of several cell types with the spirochetes also
cytokines, adhesion molecules, and other effector molecules involves several integrins belonging to the β 1 , β 2 , and β 3 groups.
(Chapter 3). This response is often initiated by endothelial and/ Integrins are involved in the adhesion of cells to a variety of

CHaPtEr 28 Host Defenses to Spirochetes 407


Professional antigen-presenting cell Little is known about the molecular events or receptors that
mediate B. burgdorferi phagocytosis despite the importance of
Phagocytic receptor Endosomal PRRs this cardinal mechanism of pathogen clearance. MyD88-mediated
signals substantially mediate the phagocytosis of B. burgdorferi.
IL-1β IFNγ However, MyD88-mediated phagocytosis occurs independently
IL-12
TLR 1/2 IL-18 of any known B. burgdorferi-recognizing TLRs. The mechanism
MyD88 of MyD88-mediated phagocytic uptake is therefore unclear.
Furthermore, the analysis of MyD88-deficient macrophages shows
CD4 + that although reduced, phagocytosis is not absent in these cells.
TCR T cell Therefore the uptake of B. burgdorferi by phagocytic cells seems
Signaling to be mediated by more than one receptor with MyD88-dependent
and MyD88-independent components. Complement receptor 3
(CR3) is a β 2 integrin, which, in cooperation with the GPI-
anchored CD14, acts as a phagocytic receptor for B. burgdorferi
Type I IFN independently of TLR or MyD88-induced signals. Phagocytosis
10
Surface PRRs (i.e., integins) plays a major role in the pathogenesis of Lyme disease, not only
TNF
through the control of bacterial numbers but also through
Endothelial cell modulation of the potency and quality of proinflammatory
cytokine induction. Indeed, as opposed to MyD88-mediated
phagocytosis, which is proinflammatory, the internalization of
B. burgdorferi by CR3 tempers the inflammatory response of
macrophages. Therefore the presence of alternative phagocytic
IL-8, MCP-1, KC mechanisms has nonredundant physiological consequences during
FIG 28.3 The interaction of Borrelia burgdorferi with pattern infection with the spirochete.
recognition receptors (PRRs) in innate immune cells and endo-
thelial cells mediates the inflammatory response to the spirochete. Complement
Phagocytosis induces Toll-like receptor (TLR)–driven proinflam- The complement system is a key component of the innate immune
matory cytokine production as well as antigen presentation by system (Chapter 21). It comprises a collection of serum proteins
professional antigen-presenting cells (APCs), which leads to the and cell surface receptors that are involved in the early response
11
activation of CD4 effector T cells, marked by the production of to pathogens, including B. burgdorferi. Destruction of micro-
IFN-γ. Likewise, PRR- and tumor necrosis factor (TNF) receptor organisms via complement involves the formation of a pore in
signaling lead to the upregulation of chemokines by endothelial the microbial cell membrane by the membrane attack complex
cells. Overall, these responses lead to increased activation and (MAC), which results in the lysis of the organism. Three different
recruitment of innate immune cells in sites of infection. pathways elicit complement activation: the classical (antigen/
antibody–mediated) pathway (CP), the lectin pathway, and the
alternative (pathogen surface) pathway (AP). These pathways
converge at the level of C3 convertase, a protease that cleaves
ligands and mediate essential cellular processes, including attach- complement component C3 into C3a and C3b. As a result, C3b
ment and cell migration. Some integrins have also been associated can (i) bind to the surface of the bacteria and facilitate internaliza-
with the phagocytosis of microorganisms. For the most part, tion of the spirochete via opsonization; or (ii) it can bind C3
study of the interaction between the spirochete and integrins convertase and facilitate the deposition of downstream compo-
has focused on their role aiding the adhesion of B. burgdorferi nents onto the surface of the spirochete resulting in the formation
to cells and the colonization of tissues and as receptors that of MAC and lysis of the cell.
contribute to signals that induce the production of proinflam- B. burgdorferi activates the CP and AP of the complement
12
matory factors (Fig. 28.3). cascade. Moreover, the activation of complement has been
associated with dramatic decreases in spirochetal numbers in
Phagocytic Cell Recruitment and Spirochetal Clearance different tissues of infected mice, indicating the importance of
The recruitment of phagocytic cells and other cell types into the complement system early in B. burgdorferi infection.
sites of infection is mediated by the production of chemokines, The members of the B. burgdorferi sensu lato group, which
increased vascular permeability, and upregulated expression of includes B. burgdorferi sensu stricto, B. garinii, and B. afzelii, have
cell adhesion molecules in endothelial cells. B. burgdorferi induces evolved a variety of mechanisms enabling them to escape
the upregulation of these factors in different cell types. Chemokine complement-mediated lysis, including the expression of comple-
production at sites of pathology in disease-susceptible C3H/HeJ ment regulator-acquiring surface proteins (CRASPs). Of these
mice and disease-resistant C57BL/6 J mice shows that inflam- CRASPs, the Erp (OspEF-related protein) family of outer
mation is related to increased production of neutrophil and membrane proteins serve as binding sites for the complement
13
monocyte–macrophage chemokines, KC and MCP-1, respectively. inhibitor factor H and factor H–like protein 1 (FHL-1). The
In patients, the production of chemokines, especially IL-8, during interaction of factor H with these proteins recruits a protease
the initial response to B. burgdorferi correlates well with the (factor I) that cleaves and inactivates the complement serum
onset of symptoms known to occur during the early stages of proteins, C3b and C4b. Cleavage of these two complement proteins
infection, and this suggests that their production is increased prevents the deposition of downstream components onto the
during the beginning of the infection to recruit phagocytic cells, surface of the spirochete, thereby halting the formation of the
which are involved in the initial clearance of the spirochete. membrane attack complex. B. burgdorferi also express a CD59-like

408 Part tHrEE Host Defenses to Infectious Agents


molecule on the outer membrane that can inactivate MAC and with a ligand-binding lipoprotein known as decorin-binding
14
prevent complement-mediated lysis. It has been speculated that protein A (DbpA). A deficiency in decorin reduces the incidence
most borreliae are able to evade complement-mediated lysis, of Lyme arthritis in mice, suggesting that the interaction of DbpA
and recently, a novel protein expressed by B. hermsii (a relapsing with the extracellular matrix provides a protective niche for B.
fever spirochete) has been discovered that affords protection to burgdorferi, preventing humoral-mediated bacterial killing. Once
15
spirochete by inactivating C3b. More recently, it has been shown the spirochete is in the joints and the heart, its clearance may
that B. burgdorferi expresses a protein, BBK32, on its surface, be more dependent on cellular responses (e.g., macrophages in
which prevents activation of the CP by blocking activation of the heart) than on antibodies. In fact, the lack of IFNγ-mediated
the C1 complement complex. 16 activation of macrophages has profound consequences on murine
cardiac inflammation, even in the presence of strong antibody
Adaptive Immune Responses responses. Furthermore, the bacterial clearance potential of
T Cell–Mediated Responses infected mouse sera administered in newly infected mice is lost
Upon antigen presentation by macrophages, dendritic cells (DCs), when administered 4–8 days after infection, potentially the result
or B cells, naïve CD4 T cells are activated and differentiate into of the colonization of tissues into which antibodies are less able
effector T cells. Effector CD4 T cells are classified on the basis to penetrate.
of their cytokine production profile, which determines their B. burgdorferi can also avoid clearance by antibodies through
mode of action and downstream effects, and include Th1 (IFN- antigenic diversity. B. burgdorferi differentially expresses outer
γ-producing), T-helper [Th]2 (IL-4, IL-5, IL-13), Th17 (IL-17), membrane antigens under pressure from the immune response,
or regulatory T cells (Tregs; IL-10) (Chapter 16). Interaction of which might contribute to the ability of the spirochetes to persist
B. burgdorferi antigen with TLRs induces the production of IL-12, in the host. A mechanism that is potentially essential for spiro-
which drives the differentiation of CD4 T cells into Th1 effector chetal immune escape is the recombination that takes place at
18
cells. Th1 cells are regulators of the cell-mediated inflammatory the vls locus, located near the right telomere of the linear plasmid
reactions, which are characterized by macrophage activation, lp28-1. B. burgdorferi is also able to evade antibody responses
including phagocytosis, or the induction of opsonizing IgG during transmission from the tick by attachment to the tick
antibodies. Although IFNγ and Th1 CD4 T cells have been shown salivary protein, Salp15, which interacts with the lipoprotein,
to be protective during cardiac inflammation with B. burgdorferi, OspC, and protects the spirochete from antibody-mediated
joint inflammation is independent of this effector cell type in killing. 19
mice. However, in patients with Lyme disease, Th1 cells dominate
in the synovial fluid, and the severity of arthritis directly correlates HOST DEFENSES TO T. pallidum
17
with increased levels of Th1 cells in the synovium. Whether
neutrophilic infiltration during joint infection with the spirochete KEY CONCEPtS
is influenced by Th effector cells that more directly affect this
cell type, such as Th17 cells (through the production of IL-17) Protective Versus Pathological Responses to
remains to be elucidated. treponema pallidum
B Cell–Mediated Responses The role of the early innate response to T. pallidum is still poorly understood
because immunogenic cell surface proteins able to activate pattern
Antibodies are specific and powerful effector molecules of the recognition receptors remain to be found.
adaptive immune response (Chapter 15). Once antibodies bind It is likely that the cell-mediated immune response to T. pallidum is
to their specific foreign antigen, they confer protection to the involved in the development of pathology following infection with the
host by using a variety of effector mechanisms: antibodies spirochete. Likewise, this response seems to be involved in the resolu-
tion of infection with T. pallidum.
contribute to adaptive immunity by neutralizing microbes or The protection that the humoral response affords to the host is unclear.
their products, activation of complement, and opsonization, Furthermore, no definite antigens have been isolated from the spiro-
which leads to phagocytosis of microorganisms. The activation chete because of the inability to cultivate this organism in vitro.
of B cells and their differentiation into antibody-secreting plasma
cells often requires an interaction with Th cells, and this interac-
tion controls isotype switching as well as somatic hypermutation. T. pallidum is known colloquially as the “stealth pathogen”
However, in response to B. burgdorferi, T cell–independent because of its denuded outer membrane, which comprises mostly
humoral responses also confer protection to the host. Thus mice nonimmunogenic transmembrane proteins, whereas the highly
deficient in T cells, CD40L, and MHC class II infected with B. immunogenic lipoproteins are contained within the periplasmic
20
burgdorferi mount a protective antibody response, which, upon space. This molecular architecture, coupled with the ability to
passive serum transfer, affords protection to severe combined generate antigenic variants, is responsible for the treponeme’s
immune deficient (SCID) mice from homologous challenge. remarkable ability to cause persistent infection with relatively
21
However, mice that lack both B and T cells developed severe few organisms. We have yet to elucidate many of the specific
arthritis and carditis in response to infection with B. immunological phenomena that occur as a result of infection with
burgdorferi. the treponeme and that are inherent to the clinical manifestations
The role of antibodies in controlling B. burgdorferi infection of the disease. Our limited understanding of immunological
may be more important during the hematogenous dissemination phenomena during infection mainly results from our inability
phase, when they are easily accessible, than after the spirochetes to culture these organisms in vitro and to reproducibly infect
have colonized tissues. B. burgdorferi is able to evade the humoral an animal model. Consequently, our understanding of the
response by interacting with the extracellular matrix of the immune responses to this pathogen is not nearly as detailed as
mammalian host via attachment to decorin, a major component our knowledge of those elicited in response to infection with
of the extracellular matrix. B. burgdorferi attaches to decorin B. burgdorferi.

CHaPtEr 28 Host Defenses to Spirochetes 409


bactericidal monoclonal antibody (mAb), M131, which provides
Innate Immune Responses partial protection to experimental syphilis. M131 binds to a
Similar to B. burgdorferi lipoproteins, treponemal lipoproteins phosphorylcholine surface epitope of T. pallidum and is the
appear to be the major proinflammatory agonists during trepo- first demonstration of such an antigen on the surface of the
nemal infection through engagement with their cognate receptors, spirochete. 24
TLR1/2 and CD14. The inflammatory milieu established by
treponemal lipoproteins is a principal driving force for immune Adaptive Immune Responses
cell recruitment to T. pallidum–infected tissues. The importance T Cell–Mediated Responses
of this immune system compartment during the response to T. The infiltration of T cells (CD4 and CD8) and macrophages
pallidum is further supported by the systemic upregulation of into the primary and secondary syphilitic lesion facilitates local
innate immune cells during treponemal dissemination and by clearance of the majority of treponemes and lesion resolution
demonstration that macrophages are principal effectors of via a vigorous cell-mediated immune response characteristic of
treponemal clearance during infection. 22 a delayed-type hypersensitivity or Th1 response. 25,26 CD4 T cells
are the principal T-cell subset found in the lesions and are believed
Early Pathogen Recognition to promote macrophage activation and subsequent treponeme
Because of their location in the skin, DCs (i.e., Langerhans cells), clearance through IFN-γ secretion. The role of CD8 cells is not
likely mediate the initial responses to treponemes at the primary clear, but throughout the course of primary lesion development
syphilitic lesion. Because of the denuded outer membrane of T. during experimental syphilis, their proportion related to CD4
pallidum, extremely high treponeme to DC ratios (500 : 1 and cells increases. Once the treponemes are cleared and the lesion
higher) are required for observable levels of phagocytosis in heals, a latency stage ensues, which is characterized by long-lasting,
vitro. Thus unhindered replication of spirochetes at the initial protective T-cell memory to T. pallidum antigens.
site of infection probably underlies the vigorous cell-mediated
immune response, as well as chancre appearance, which follows B Cell–Mediated Responses
antigen presentation to and differentiation and activation of Many functional activities of human syphilitic serum originate
CD4 T cells. from B-cell responses to T. pallidum. Infection with T. pallidum
invokes a humoral immune response early in the course of infec-
Phagocytic Cell Recruitment and Spirochetal Clearance tion, which strengthens as the number of recognizable antigens
It is not until after the recruitment of macrophages and their increases during the progression of infection. A polymorphic
activation via CD4 T cell–derived IFN-γ, that the majority of gene family of T. pallidum, T. pallidum repeat (tpr), has been
spirochetes are killed and resolution of syphilitic lesions occurs. recently identified as related to the major surface protein (msp)
27
Activated macrophages readily phagocytose antibody-opsonized genes of T. denticola. A single member of the tpr family, tprK,
treponemes through Fc receptor–mediated uptake. Recently, it serves as an antigen for opsonizing antibodies, suggesting
has been shown that MyD88 mediates the opsonophagocytosis that this protein is a surface antigen of T. pallidum. However,
of T. pallidum by macrophages without affecting the production there are only inferential indications that surface antigens of
of opsonic antibodies. As result, MyD88-deficent mice infected T. pallidum exist. So, although candidate T. pallidum surface
with T. pallidum showed a defect in bacterial clearance that proteins have been advanced on the basis of porin activity or
resulted in worse inflammation. These results reveal a previously homology with a surface protein of T. denticola, there is no direct
unsuspected link between MyD88 signaling and FcR-mediated evidence identifying specific surface antigens on T. pallidum.
phagocytosis. However, as with DCs, uptake via a direct PAMP– Furthermore, like most pathogenic organisms, T. pallidum has
PRR interaction does not occur readily, and because of treponemal evolved various mechanisms to escape host killing. TprK is an
antigenic variation, not all spirochetes will be eliminated via immunogen with seven discrete variable regions differing among
27
opsonophagocytosis despite the vigorous inflammatory response. isolates of the spirochete. In fact, the antibody response to T.
The relatively few remaining spirochetes are able to cause per- pallidum is directed against these variable regions, which leads to
sistent infection. immune selection of new TprK variants; thus antigenic variation
is involved in the reinfection of hosts despite robust immune
Complement responses. 27,28
The immunoprotection afforded by human syphilitic serum is,
in large part, a result of the activation of the complement cascade Translational Research
by bactericidal antibodies and spontaneous hydrolysis of C3. ON tHE HOrIZON
Thus like B. burgdorferi, T. pallidum activates the CP and AP of
the complement cascade, and there is a considerable amount of Efficient diagnostic methods that are required to rapidly identify infection
evidence for an important role of these pathways in syphilitic and procure antimicrobial treatments
lesion resolution during human infection with treponemes. In Development of new generation vaccines for Lyme disease, including
contrast to B. burgdorferi, there is no evidence indicating that those that target the tick vector and that can prevent other tick-borne
infections
T. pallidum has evolved mechanisms to evade complement- Attention to communities at risk for contracting syphilis with investments
dependent killing, suggesting that these complement pathways in prophylaxis and efficient treatments
have a larger role in controlling treponemal infection than in
the case of B. burgdorferi.
During experimental syphilis, immunization with purified The challenge in the next 5–10 years is to develop effective
outer membrane vesicles (OMVs) isolated from T. pallidum diagnostic methods for both Lyme disease and syphilis and to
23
results in complement-dependent bactericidal activity. More devise new preventive measures. For Lyme disease, advances in
recently, immunization with OMVs led to the isolation of a genetic manipulation, as well as other means to study structure/

410 Part tHrEE Host Defenses to Infectious Agents


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22. Salazar JC, Cruz AR, Pope CD, et al. Treponema pallidum elicits innate
Please check your eBook at https://expertconsult.inkling.com/ and adaptive cellular immune responses in skin and blood during
for self-assessment questions. See inside cover for registration secondary syphilis: a flow-cytometric analysis. J Infect Dis
details. 2007;195:879–87.
23. Blanco DR, Champion CI, Lewinski MA, et al. Immunization with
REFERENCES Treponema pallidum outer membrane vesicles induces high-titer
complement-dependent treponemicidal activity and aggregation of T.
1. Burgdorfer W, Barbour AG, Hayes SF, et al. Lyme disease-a tick-borne pallidum rare outer membrane proteins (TROMPs). J Immunol
spirochetosis? Science 1982;216:1317–19. 1999;163(5):2741–6.

CHaPtEr 28 Host Defenses to Spirochetes 411


24. Blanco DR, Champion CI, Dooley A, et al. A monoclonal antibody that 27. Centurion-Lara A, Castro C, Barrett L, et al. Treponema pallidum major
conveys in vitro killing and partial protection in experimental syphilis sheath protein homologue Tpr K is a target of opsonic antibody and the
binds a phosphorylcholine surface epitope of Treponema pallidum. Infect protective immune response. J Exp Med 1999;189:647–56.
Immun 2005;73:3083–95. 28. Giacani L, Molini BJ, Kim EY, et al. Antigenic variation in Treponema
25. McBroom RL, Styles AR, Chiu MJ, et al. Secondary syphilis in persons pallidum: TprK sequence diversity accumulates in response to immune
infected with and not infected with HIV-1: a comparative pressure during experimental syphilis. J Immunol 2010;184:3822–9.
immunohistologic study. Am J Dermatopathol 1999;21:432–41.
26. Van Voorhis WC, Barrett LK, Koelle DM, et al. Primary and secondary
syphilis lesions contain mRNA for Th1 cytokines. J Infect Dis
1996;173:491–5.

CHaPtEr 28 Host Defenses to Spirochetes 411.e1


MUL t IPLE-CHOICE QUES t IONS

1. The major immunogens of Borrelia burgdorferi and Treponema 3. The generation of a protective antibody response against B.
pallidum are: burgdorferi:
A. Lipopolysaccharide molecules present on the outer A. Occurs even in the absence of T-cell responses
membrane B. Is targeted exclusively to variable regions of the vlsE protein
B. Sugar coated-proteins that decorate their surface C. Is always accompanied by a T-helper type 2 response
C. A group of lipoproteins that constitute Toll-like receptor D. Occurs within the first 3 days of infection and is the basis
(TLR)1/TLR2 ligands for the successful diagnosis of patients
D. Both spirochetes, which are considered “stealth pathogens” 4. T-cell responses against T. pallidum:
because of their lack of immunogens
A. Are always weak because of the low level of infection in
2. The phagocytosis of spirochetes: patients
A. Allows the elimination of the bacteria before they have a B. Induce a type I delayed hypersensitivity response that helps
chance to induce a proinflammatory response the elimination of the bacteria
B. Allows the interaction of pathogen recognition receptors C. Are always composed of CD4 T cells that exclusively aid
with their ligands within the phagolysosome t modulate the development of antibody responses
the inflammatory response D. Can be used as diagnostic and prognostic tools for the
C. Depends exclusively on the presence of opsonins that target disease
the bacteria to professional antigen-presenting cells
D. Occurs only on tissues where the pathogens reside, exclu-
sively upon activation of the adaptive immune system

29









Host Defenses to Fungal Pathogens



Allison K. Lord, Jatin M. Vyas








Advances in modern medicine have significantly improved the is an urgent need to define the mechanisms of host defense
prognosis and quality of life for patients with a wide spectrum against fungal pathogens with the goal of developing novel
of diseases, including many forms of cancer. However, oppor- therapeutics as well as improving diagnostic and preventative
tunistic fungal pathogens have exploited these modern immu- procedures.
nosuppressive and invasive medical interventions. Invasive fungal One of the greatest limiting factors in the treatment of IFIs
infections (IFIs) represent a significant cause of morbidity and is lack of effective and prompt diagnostic tools. Although Candida
mortality among immunocompromised patients. Patients at is capable of growing in conventional blood cultures, most other
highest risk for IFIs include solid organ or hematopoietic stem invasive fungal organisms, including Cryptococcus, Aspergillus,
cell transplant (HSCT) recipients on intensive immunosuppressive and Histoplasma, are typically not recovered by adding blood
regimens, patients with hematological malignancies, and other to growth media. There has been a heavy reliance on clinical
patients that are immunocompromised as a result of various diagnosis and a few biomarkers. Fungal cell wall carbohydrates
clinical conditions and treatments (e.g., human immunodeficiency can be detected in the bloodstream of some patients. β-1,3 glucan,
virus/acquired immunodeficiency syndrome [HIV/AIDS], galactomannan, and glucuronoxylomannan (GXM) are three
1,2
advanced age, recent surgery, etc.) (Fig. 29.1). The clinical carbohydrates that can be analyzed clinically. β-1,3 glucan is a
consequences of these pathogenic fungi include superficial disease, common fungal cell wall carbohydrate found in Candida albicans
allergic disease, and IFIs. and many other fungi (with the notable exception of Cryptococcus
neoformans). Galactomannan is typically associated with Aspergil-
lus fumigatus. This fungal biomarker can be measured in blood
KEY CONCEPTS and bronchoalveolar lavage fluid. GXM is an abundant fungal-
Invasive Fungal Infections derived carbohydrate produced by C. neoformans. GXM can also
be found on the surface of Candida gattii, a closely related fungal
• Invasive fungal infections are systemic life-threatening infections that organism that can cause disease in immunocompetent individuals.
are estimated to cause 1.5 million deaths annually. Although GXM appears to be a useful biomarker for Cryptococcus,
• Aspergillus, Candida, Cryptococcus, and Pneumocystis are opportunistic
fungi that cause the majority of invasive fungal infections. its widespread use is limited by technical and economic issues.
• Risk factors for invasive fungal infections include bone marrow or solid Finally, a urine test for histoplasmosis has been established. Again,
organ transplantation, intensive immunosuppressive regimens, hema- the antigen for this test is a polysaccharide derived from the cell
tological malignancies, HIV/AIDS, advanced age, recent surgery, and wall of this fungal organism. Many of these biomarkers suffer
other clinical conditions and treatments that cause immunosuppression. from poor sensitivity (galactomannan) and, in some cases, poor
specificity (e.g., testing urine for Histoplasma antigen).
Current therapeutic approaches for IFIs include the prompt
Superficial fungal infections of the skin and nails affect 25% institution of antifungal agents (including polyenes, azoles, and
of the global population (≈1.7 billion people) and give rise to echinocandins) and even more important, the reversal of underly-
various conditions, including athlete’s foot and ringworm of the ing host immune defects, such as neutropenia or high doses of
scalp. Fungi also commonly cause mucosal infections of the oral immunosuppressive therapy. The first-line therapy for the
and genital tracts; vulvovaginal candidiasis occurs at least once treatment of invasive aspergillosis (IA) is voriconazole, an
3,4
in 50–75% of women in their childbearing years. Superficial extended-spectrum azole. Despite its in vitro efficacy, voriconazole
fungal infections of the skin and mucosa can become chronic, only demonstrated a survival rate at 12 weeks of 70.8% compared
5
but they are rarely life threatening. with 57.9% in the amphotericin B group. Although voriconazole
IFIs occur when fungal pathogens invade the bloodstream, has become the gold standard for treatment, its high mortality
resulting in a systemic life-threatening infection that affects rate indicates that the host immune response plays a critical role
multiple organs. IFIs are estimated to cause 1.5 million deaths in determining host outcome. Despite clinical vigilance, preven-
annually, with four genera accounting for most of the infections: tion, diagnosis, and treatment of IFIs remain a significant
Cryptococcus, Candida, Aspergillus, and Pneumocystis. Mortality challenge because of lack of rapid and reliable diagnostic methods
resulting from infections with these fungal organisms exceeds and limited treatment options. Discovery of novel diagnostic
4
that caused by tuberculosis or malaria. As a result, IFIs have tools and antifungal therapies is crucial to ameliorating this
emerged as an escalating and worrisome clinical problem. There public health burden.
413

414 ParT ThrEE Host Defenses to Infectious Agents



Immunotherapy for Immunotherapy Cancer and
autoimmune for HIV / AIDS chemotherapy
diseases cancer


Parenteral Intensive
nutrition care unit
treatment
Catheterization Immunosuppressive
therapy
Neonates
and elderly Broad-spectrum
antibiotics
Organ/bone marrow Invasive
transplant fungal
infections Surgery

Hematologic Inherited
malignancy immunodeficiencies
FIG 29.1 Major risk factors for developing invasive fungal infections. (Adapted from Karkowska-
Kuleta J, Kozik A. Cell wall proteome of pathogenic fungi. Acta Biochim Pol 2015; 62: 339–51.)





CLINICALLY RELEVANT FUNGAL ORGANISMS Candida albicans
Candida species are ubiquitous commensal fungi that normally
Although it is estimated that there are in excess of 5 million colonize human mucosa and skin. There are over 165 species of
distinct species of fungi, only a handful are considered sig- Candida, but only a few are known to cause human disease. C.
nificant to human health: Aspergillus spp., Candida spp., and albicans accounts for the majority of infections, followed by C.
Cryptococcus spp. cause the majority of IFIs in the United States glabrata. Less frequently, C. parapsilosis, C. tropicalis, C. krusei,
7
and Europe. Invasive infections caused by these pathogens and others cause invasive candidiasis. In immunocompromised
in immunocompromised patients are serious and often life individuals, Candida can become pathogenic and infect host
threatening. tissues. For example, Candida is commonly associated with skin
infections, as well as oral and vaginal thrush. However, Candida
Aspergillus fumigatus can become invasive (invasive candidiasis), entering the blood-
Aspergillus fumigatus is a ubiquitous airborne mold found in soil, stream (candidemia) and other organs (e.g., bladder/kidneys,
air, food, and decaying organic material. Humans routinely inhale liver, spleen, gut, etc.), resulting in a life-threatening infection.
A. fumigatus conidia, but the microorganism is rapidly eliminated Despite the availability of antifungal therapy, the crude mortality
by the innate immune system in immunocompetent individuals. rate for candidemia exceeds 50%, indicating that the immune
In immunocompromised patients, A. fumigatus can cause IA, a system is a necessary partner for successful treatment of this
8
severe and usually fatal infection. It is estimated that IA occurs in infection. Every year, it is estimated that invasive candidiasis
4
200 000 patients annually. In cases of widespread infection, IA affects 250 000 people worldwide and causes more than 50 000
9
mortality rates approach 90%, with favorable responses to anti- deaths. As with other opportunistic fungal infections, patients
5
fungal therapy observed in <30% of patients. In particular, IA on immunosuppressive regimens (i.e., transplant recipients) and
has emerged as a significant cause of mortality among patients patients with hematological cancers are at high risk for invasive
who have undergone hematopoietic stem cell transplantation and candidiasis. Other major risk factors include recent surgery,
solid organ transplantations. Advanced age, respiratory viruses, broad-spectrum antibiotic therapy, and central vascular catheters.
graft-versus-host disease (GvHD), prolonged glucocorticosteroid Disruptions to the balance of C. albicans in the gut are associated
use, cytomegalovirus (CMV) infection, and iron overload are also with increasing severity of Crohn disease and ulcerative colitis
6
common risk factors for IA. Given that A. fumigatus spores are (UC). Moreover, disseminated candidiasis may originate in the
inhaled, IA most frequently manifests as an invasive pulmonary gut.
disease, but the pathogen can enter the bloodstream and infect
multiple organs. Less frequently, IA affects skin, sinuses, and the Cryptococcus neoformans
central nervous system (CNS). Clinical characteristics of IA vary, During the 1980s to the 1990s, cryptococcosis emerged as a
depending on the organ(s) affected. major cause of morbidity and mortality among persons with
Aspergillus can cause a condition called chronic pulmonary HIV/AIDS. The majority of cryptococcosis cases are caused by
aspergillosis (CPA), which causes progressive destruction of lung C. neoformans, a fungus that is widely distributed in soil. The
tissue. CPA is most common in patients with underlying lung major route of exposure to C. neoformans is inhalation of airborne
disease (e.g., asthma) and affects 3 million people worldwide. organisms into the lungs. In healthy individuals, the immune
CPA is estimated to have 15% mortality, often as a result of a system is effective at clearing the pathogen, but in immuno-
massive pulmonary hemorrhage within the first 6 months of compromised individuals (especially those with HIV/AIDS),
diagnosis. 4 C. neoformans can cause an IFI. Interestingly, C. neoformans

ChaPTEr 29 Host Defenses to Fungal Pathogens 415


targets the CNS, causing meningoencephalitis, a life-threatening to withstand diverse environmental conditions but also maintains
condition. plasticity to permit cell growth and division and formation of
different cell types throughout the life cycle of a fungal organism.
HOST DEFENSE AGAINST FUNGI BY Availability of nutrients, stress, hypoxia, and hypercarbia are
EPITHELIAL CELLS environmental cues to the modification of the cell wall. The
major constituents of the fungal cell wall are chitin, glucans,
Given the ubiquity of many fungal organisms, humans inhale and glycoproteins. Chitin is a structurally important component
a diverse species of fungi regularly. Moreover, the microbiota of the fungal cell wall located closest to the plasma membrane.
has a profound influence on human health. Early work on this The composition of the outer layer varies, depending on the
complex collection of microorganisms focused on bacteria, but fungal species, morphotype, and growth stage. Branched β-1,3
increasing evidence indicates that the mycobiome plays an glucan cross-links to chitin and is covalently linked to other
important role in human health. Indeed, sequencing data from polysaccharides (e.g., galactomannan and β-1,6 glucan). The
the respiratory sputum of normal volunteers showed over 40 fungal cell wall composition differs dramatically between conidia
10
distinct species of fungi. Interestingly, sputum from patients and hyphae, which leads to differential recognition and response
with allergies demonstrated a distinct mycobiome. Although by the immune system. Many fungal species produce melanin,
there is an association, it remains unclear as to whether specific a natural pigment found on the cell wall and is associated with
species of fungi are causal for allergic responses. Growth of the enhanced virulence of many pathogenic fungi. Melanin
commensal fungi is limited by release of antimicrobial peptides protects the organism from both environmental stressors and
and colonization by other microbial flora that compete for interferes with host defense mechanisms. Specifically, fungal
nutrients. Disruptions to this delicate balance can have serious melanin has been associated with decreased phagocytosis, an
pathological consequences. altered cytokine response, and reduces susceptibility to antifungal
16
Skin and the epithelial mucosa of the respiratory, gastro- drugs. In some organisms, such as A. fumigatus, a hydrophobic
intestinal (GI), and genitourinary tracts represent the first line rodlet layer composed of hydrophobins makes the conidia water-
of defense against fungal pathogens, acting as a physical barrier proof and shields underlying carbohydrates that may induce
against infection. There is evidence that epithelial cells provide inflammation.
more than just barrier function by triggering an immune response. Fungal dimorphism is a critical feature of virulence for both
Several groups have demonstrated that oral epithelial cells infected yeast and molds. C. albicans exist in three forms that have distinct
with fungi produce proinflammatory cytokines and chemokines shapes: blastospores (also known as yeast cells), pseudohyphal
in vitro. 11,12 Similarly, stimulation of human corneal epithelial cells, and true hyphal cells. Yeast cells are typically 5 µm in
cells with A. fumigatus hyphae activates Syk signaling and leads diameter with a round to ovoid shape. Pseudohyphae resemble
to production of inflammatory cytokines (interleukin-1β [IL-1β] elongated yeast cells that remain attached to one another. These
13
and IL-6) and chemokines (IL-8 and CXCL1). Hyphal formation fungi usually grow in a branching pattern that may facilitate
14
is critical for inducing a strong response by epithelial cells. scavenging for nutrients at distal sites. True hyphal cells are long
Recently, a fungal toxin termed candidalysin has been identified and highly polarized, with parallel sides and no obvious constric-
that is a cell permeable peptide that induces a strong inflammatory tions between cells. Similarly, A. fumigatus conidia are waxy
response within epithelial cells. Production of candidalysin by hydrophobic ovoid-shaped cells of approximately 3–5 µm in
C. albicans is required for T-helper 17 (Th17) responses. diameter. A. fumigatus can filament to generate hyphae that are
C. albicans is a commensal fungus in the human GI tract, but >40 µm in length. For both C. albicans and A. fumigatus, this
its abundance is limited by intestinal bacteria. Antibiotics that dimorphism is a virulence factor as organisms that fail to do so
disrupt the balance of bacterial microbiota favor overgrowth of are avirulent.
Candida species. It is thought that patients with fever and
neutropenia who develop invasive candidemia result from THE INNATE IMMUNE RESPONSE TO
translocation of fungi from the GI tract. Emerging data connect FUNGAL PATHOGENS
15
gut homeostasis and immune responses to fungi in the lung.
Many questions remain about how epithelial cells discriminate Once a fungal pathogen has invaded the host system, innate
fungal morphology and which proteins mediate epithelial recogni- immune cells, such as macrophages/monocytes, neutrophils,
tion and activation in response to pathogenic fungi. Compared and dendritic cells (DCs), phagocytose and degrade the organ-
with host defense mechanisms exerted by myeloid cells, much ism (Chapter 3). In immunocompetent individuals, physical
less is known about host–pathogen interactions in epithelial barriers (e.g., epithelial cells, mucous, skin) are effective at
mucosa. preventing infection. In the respiratory tract, macrophages
routinely neutralize fungal pathogens that make their way to the
THE FUNGAL CELL WALL alveoli. Understanding the rules that govern the recognition and
response to these fungal pathogens provide important insights
The fungal cell wall contains many of the relevant pathogen- into how these organisms cause disease in immunocompromised
associated molecular patterns (PAMPs) and epitopes for the individuals.
immune response. PAMPs are recognized by cells of the host Recognition of fungal pathogens by innate immune cells is
innate immune system and are often targets for antifungal agents. mediated by pattern recognition receptors (PRRs). Engagement
The ultrastructure of fungal organisms is similar to mammalian of PRRs and PAMPs triggers a cascade of molecular events that
cells. This feature has thwarted the development of a broad coordinates phagocytosis and degradation of the pathogen.
armamentarium of antifungal agents. However, fungi also possess Additionally, release of cytokines, reactive oxygen species (ROS)
a cell wall, a structure not found in mammalian cells. The fungal production, and presentation of fungal antigens to the adaptive
cell wall not only provides the organism with mechanical strength immune system aids in controlling infection.

416 ParT ThrEE Host Defenses to Infectious Agents



KEY CONCEPTS neutrophils. Macrophages are recruited to the site of infection by
Innate Immunity to Fungal Pathogens chemotaxis. They are also responsible for patrolling interfaces with
the environment, including the lung and the GI tract. In contrast
• Fungi express highly conserved pathogen-associated molecular patterns to neutrophils, macrophages express class II MHC and can activate
(PAMPs) that are recognized by pathogen recognition receptors (PRRs) T cells. Upon activation by fungi through PRRs and PAMPs,
expressed on host phagocytes. macrophages produce potent inflammatory cytokines, including
• Dectin-1 is a C-type lectin receptor (CLR) that acts as a PRR to recognize tumor necrosis factor α (TNF)-α and IL-6. Engagement of the
β-1,3 glucan expressed on the cell walls of Candida, Aspergillus, and inflammasome within these cells leads to copious production
other fungal pathogens.
• Toll-like receptors (TLRs) recognize fungal cell wall components and of IL-1β. Macrophages receive assistance from activated T cells
nucleic acids. that secrete interferon-γ (IFN-γ), which can activate a host of
• Engagement of PRRs and PAMPs initiates signaling that coordinates genes to improve the antifungal response from macrophages.
secretion of cytokines, reactive oxygen species (ROS) production,
and presentation of fungal antigens to the adaptive immune system Role of Dendritic Cells
to facilitate elimination of the pathogen. In contrast to macrophages, DCs can stimulate naïve T cells.
Moreover, DCs are capable of taking up both yeast and conidia.
DC subsets are characterized by expression of specific surface
Recently engulfed pathogens become enclosed in membrane markers. Plasmacytoid DCs (pDCs) expressing specific surface
delineated compartments called phagosomes, which traffic toward markers phagocytose A. fumigatus conidia and spread over hyphae.
the lysosome, as directed by modifications to the phagosomal pDCs incite an immune response by release of proinflammatory
membrane proteins and changes to the intraphagosomal environ- cytokines, including IFN-α and TNF-α. pDCs are typically found
ment. Proteins recruited to the phagosomal membrane are specific in the spleen but will migrate to the lungs following challenge
to its contents. Phagolysosomes intersect with class II major by A. fumigatus conidia. Depletion of pDCs has been shown to
histocompatibility complex (MHC) molecules and permit loading result in increased mortality, indicating a nonredundant role for
of pathogen-specific peptides. T cells are then activated in an pDCs in host defense against A. fumigatus.
antigen-specific manner to augment the immune response and C. albicans epithelial infections appear to recruit pDCs. These
generate long-term immunity. cells reorganize and become more concentrated in the T-cell
zones of lymph nodes. Draining lymph nodes of mice infected
Role of Neutrophils with Candida versus control mice showed that pDCs were the
Neutrophils are the most critical cell in the host defense against predominant DC subset after vaginal infection. pDCs are involved
fungal pathogens. Patients with neutropenia or acquired defects in the induction of Th1 responses to C. albicans vaginal infection.
in neutrophil-mediated killing are at higher risk of developing
IFIs, including IA. Although phagocytosis is a critical feature in PATTERN RECOGNITION RECEPTORS
controlling infection, other mechanisms also limit the spread of
infection and serve to kill invading organisms. Neutrophils rely Recognition of fungal pathogens is mediated by PRRs expressed
on multiple mechanisms for killing, including granule-dependent by innate immune cells, including DCs and myeloid cells (e.g.,
killing, nicotinamide adenine dinucleotide phosphate (NADPH) macrophages, monocytes, and neutrophils). The interaction
oxidase–dependent killing, and neutrophil extracellular trap between PRRs and PAMPs expressed on the fungal cell wall
(NET) formation (Chapter 22). triggers downstream signaling, which elaborates the host immune
Phagocytosis of fungal organisms by neutrophils triggers response and facilitates elimination of the pathogen. The major
production of antimicrobial ROS. Generation of ROS by the PRRs involved in antifungal immunity are Toll-like receptors
NADPH oxidase complex is a result of engagement of surface (TLRs) and C-type lectin receptors (CLRs). Other relevant PRRs
receptors, including dectin-1, and signaling adaptors, including include nucleotide-binding oligomerization domain (NOD)–like
caspase recruitment domain family member 9 (CARD9) and receptors (NLRs), and retinoic acid-inducible gene I (RIG-I)–like
Syk. Loss of ROS production has a clear phenotype in Aspergillus receptors (RLRs).
infections. Patients with chronic granulomatous disease (CGD)
fail to control Aspergillus hyphae, which leads to invasion and Toll-Like Receptor
metastatic spread of disease. In contrast, conidial forms of Toll-like receptors (TLRs) are transmembrane receptors that
Aspergillus do not require ROS. The data for Candida infections recognize a broad range of microbial ligands, including fungal and
are less compelling. There appears to be modest reduction in bacterial cell wall components, bacterial and viral nucleic acids,
killing of serum-opsonized C. albicans, but killing of unopsonized and bacterial lipoproteins (Fig. 29.2) (Chapter 3). TLR1, -2, -4, -5,
organisms is unimpaired. -6, and -10 are expressed on the cell surface, whereas TLR3, -7,
In addition to cytotoxic granules and ROS production, -8, and -9 are expressed on intracellular membranes. Intracellular
neutrophils are capable of NET formation. This relatively newly TLRs recognize nucleic acids derived from fungi, bacteria, and
discovered cytotoxic mechanism delivers a web-like structure viruses and signal through MyD88 or TRIF. MyD88 signaling
composed of chromatin and histones. For Candida, it appears triggered by TLRs is essential for host fungal defense; mice lacking
17
that NET formation contributes to fungal killing. However, in MyD88 are more susceptible to IFIs. However, humans with
Aspergillus, data from studies do not support a clear role for MyD88 signaling defects do not have increased incidence of fungal
NETs. infections. To date, several TLRs have been implicated in fungal
18
immunity in mice, including TLR2, TLR4, TLR7, and TLR9. The
Role of Macrophages importance of TLRs has been demonstrated in human biology.
Macrophages also play a critical role in neutralizing fungal In humans, polymorphisms in TLR1, TLR3, TLR4, and TLR6 are
19
organisms. These phagocytic cells are much longer lived than associated with increased susceptibility to IFIs (especially IA).

ChaPTEr 29 Host Defenses to Fungal Pathogens 417


Fungi C. albicans C. albicans A. fumigatus Candida spp. Candida spp.
A. fumigatus A. fumigatus RNA A. fumigatus RNA
C. neoformans C. neoformans C. neoformans
DNA

PAMPs GXM GXM
Mannan (O-linked) PLM (phospholipomannans)


TLR2 TLR6

Plasma membrane
TLR TLR4








Endosome
IFNδ
IL-12
TNFα TLR3
IFNγ TGF-β
Gene expression IL-12 IL-10 TLR9
IL-23
output
TLR7


IFNβ

IL-6
IL-10
IFNβ IFNβ
IL-12 IL-12



FIG 29.2 Toll-Like Receptors (TLRs) and Fungal Immunity. Surface and endosomal TLRs
recognize fungal pathogen-associated molecular patterns (PAMPs) resulting in downstream signaling
that promotes production of cytokines. (Adapted from Bourgeois C, Kuchler K. Fungal pathogens-a
sweet and sour treat for Toll-like receptors. Front Cell Infect Microbiol 2012;2:142.)


In contrast, TLR9 polymorphisms are more tightly associated phagosomal acidification, which permits cleaved TLR9 to traffic
with allergic bronchopulmonary aspergillosis. to the phagosomes. Moreover, dectin-1 regulates TLR9-dependent
20
Surface-disposed TLR2 recognizes β-glucans of several fungal changes in gene expression. Interestingly, TLR9 deficient
species (C. albicans, A. fumigatus, and C. neoformans). TLR2 also macrophages have increased fungicidal activity and production
interacts with phospolipomannans and linear β-1,2-oligomannoside of antiinflammatory cytokines. Thus TLR9 appears to modulate
structures found on the cell wall of C. albicans. TLR2 can het- the inflammatory response by downregulating cytokine
erodimerize with TLR1 or TLR6 to recognize GXM, a cell wall production. 21
component of C. neoformans. TLR2 is critical for early recruitment
19
and the killing abilities of neutrophils. TLR2-deficient mice C-Type Lectin Receptor
infected with Pneumocystis jiroveci display more severe symptoms, C-type lectin receptors (CLRs) comprise a diverse family of
increased fungal burden, and less TNF-α production. proteins characterized by a conserved C-type lectin domain
TLR4 recognizes O-linked mannan expressed on the cell wall (CTLD) that recognizes a variety of ligands. CLRs that are
of C. albicans and through downstream signaling, stimulates recognized to play a role in fungal immunity include dectin-1,
production of TNF-α. Mice deficient in TLR4 do not appear dectin-2, mannose receptor, Mincle, and dendritic cell–specific
to have increased susceptibility to disseminated candidiasis intercellular adhesion molecule–grabbing nonintegrin (DC-SIGN)
compared with wild-type mice. In contrast, killing of Aspergillus (Fig. 29.3). However, dectin-1 is the best-characterized CLR
−/−
is impaired in TLR4 mice in a corneal inflammation model, associated with fungal immunity. Dectin-1 is a type II trans-
although recruitment of immune cells to the infection, remains membrane protein expressed primarily on myeloid cells. It is
unaffected. considered the major mammalian cell surface receptor for β-1,3
TLR9 recognizes intracellular microbial ligands, such as glucan and β-1,6 glucan, carbohydrates widely expressed on the
CpG-rich fungal DNA. Dectin-1–dependent Syk activation triggers cell wall of many fungal organisms. Furthermore, dectin-1

418 ParT ThrEE Host Defenses to Infectious Agents


Fungi *C. albicans *C. albicans *C. albicans *Candida spp. *Candida spp.
*C. glabrata *C. glabrata *P. brasiliensis *Malassezia spp. *A. fumigatus
*Coccidioides spp. *Paracoccidioides *Saccharomyces spp. *Chrysosporium
*H. capsulatum brasiliensis tropicum
*P. jirovecii
PAMP β-glucan α-mannan β-mannan ? N-mannan

CLR Dectin-1 Dectin-2 Galectin-3 MINCLE DC-SIGN








Plasma membrane












TNFα TNFα IL-1β TNFα IL-6
Gene expression IL-1β IL-1β IL-6 IL-6 IL-10
output IL-23 IL-23 IL-10 IL-10 IL-12
IL-1β
IL-2
IL-6
TNFα
IL-10 IL-10 TGFβ IL-23
IL-6
IL-12



FIG 29.3 C-Type Lectin Receptors (CLRs) and Fungal Immunity. CLRs recognize fungal PAMPs
and trigger downstream signaling that leads to cytokine production.


22
promotes acidification and maturation of phagosomes. Patients
with mutations in dectin-1 are more susceptible to IFIs, which Collaboration Between TLRs and CLRs
demonstrates the essential role of dectin-1 in mediating fungal Although most cognate ligands found on the fungal cell wall are
immunity. 23 carbohydrates that preferentially trigger CLRs, experimental
Upon dectin-1 binding to β-1,3 glucan, a tyrosine residue evidence indicates that CLRs can collaborate with TLRs on the
within the cytoplasmic hemi–immunoreceptor tyrosine-based cell surface to coordinate cytokine production. Dectin-1 can
activation motif (ITAM) domain of dectin-1 is phosphorylated collaborate with TLR2 and TLR4 to induce synergistically
by Src family kinases, which leads to the recruitment and activa- cytokines, including TNF-α, IL-10, and IL-23. This collaboration
tion of Syk. Syk triggers the activation of the caspase recruitment has been used clinically for management of clinical disease.
domain family member 9–Bcl-10– mucosa-associated lympho- Fonsecaea pedrosoi is the fungus that causes chromoblastomycosis,
reticular tissue 1 (MALT1) complex, inducing nuclear factor a skin infection that is difficult to treat. The CLR Mincle recognizes
(NF)-κB and production of cytokines, including IL-1β, IL-6, this pathogen. If costimulation of both CLR and TLR pathways
IL-10, IL-112, IL-23, and TNF-α. Dectin-1 can signal through are not engaged, the inflammatory response is defective. Exog-
a Syk-independent pathway via activation of the serine/threonine enous administration of imiquimod, purified TLR7 ligand,
24
kinase, Raf-1, that phosphorylates p65, a subunit of NF-κB. facilitated pathogen clearance in mouse models and humans.
Cytokines produced in response to canonical (via Syk) and This signaling requires both Syk/CARD9 and MyD88. Imiquimod
noncanonical (via Raf-1) NF-κB signaling are essential for the is a synthetic compound with potent antiviral and antitumor
activation of Th1 and Th17 cells (Fig. 29.4). activity that stimulates the innate immune system through TLR7
Syk also triggers recruitment and activation of NADPH oxidase, activation. When applied to the lesions of four patients with
which stimulates production of ROS, elaboration of proinflam- chromoblastomycosis, there was a rapid resolution of the infec-
25
matory cytokines, and ultimately fungal killing. Syk appears to tion. This provides proof of concept that multiple signaling
be a master controller for phagosomal maturation and recruitment pathways are necessary for optimal activation of the innate
of light chain 3 (LC3), a protein associated with autophagy. immune system against fungal pathogens.

ChaPTEr 29 Host Defenses to Fungal Pathogens 419



Fungus


Dectin-1 Dectin-1
Neutrophil
recruitment

Plasma membrane
IL-17A
IL-17F
IL-2 2


Syk
Th17

CARD9
Raf-1 NFAT NADPH IL-6, TGFβ
BCl-10 MALT1 oxidase
LC3 Th2
ROS recruitment
to phagosome
NF-κβ IL-4
IL-4
NRLP3 IL-5
IL-13
etc.
caspase1
pro-IL1β IL1β

NFKβ IL-1β IL-10 IL-12
Cytokines IL-23 IL-12 Th1
IL-6 TNFα
NFAT IL-2
Cytokines IL-10 IFNδ
etc.
Nucleus
Macrophage
activation
FIG 29.4 Dectin-1 Signaling in Response to Fungal Pathogens. Upon recognition of fungal
pathogens, dectin-1 signaling triggers secretion of cytokines, production of reactive oxygen species
(ROS), and activation of the adaptive immune system to facilitate elimination of the pathogen.


CARD9 THE INFLAMMASOME
Mutations in the CARD9 adaptor molecule, downstream of IL-1β activation occurs when its precursor pro–IL-1β is cleaved
dectin-1, are associated with increased susceptibility to invasive by caspase-1. IL-1β is critical for neutrophil recruitment and
Candida infections. Patients with CARD9 deficiencies have for induction of the Th17 response. Activation of the inflam-
decreased Th17 cells, reduced chemokine/cytokine production, masome regulates IL-1β, IL-18, IL-17, and IFN-γ. NLR family
26
and impaired neutrophil activation. Numerous patients have domain containing protein 3 (NLRP3) is required for activation
been identified to have mutations in CARD9 that lead to fungal of caspase 1. Caspase 8 may also play a role in regulating activation
26
infections. The vast majority of inborn errors in immunity that of IL-1β. Syk-dependent production of ROS activates the NLRP3
predispose to fungal disease typically also lead to development inflammasome, which coordinates with NF-κB to promote
27
of nonfungal infections. Patients with gain-of-function (GOF) production of IL-1β. Mice lacking the inflammasome or IL-1β
28
signal transducer and activator of transcription 1 (STAT1) are highly susceptible to disseminated candidiasis. In addition
or autosomal dominant STAT3 also suffer from mycobacte- to ROS, lysosomal rupture, release of cathepsins, and efflux of
rial and other bacterial infections. In contrast, mutations of potassium can lead to activation of the inflammasome. Many
CARD9 appear to affect susceptibility to fungal infections components of the fungal cell wall can trigger inflammasome
exclusively. This observation makes CARD9 a key regulator in activation in vitro. However, in these experiments, cells were
antifungal immunity. Other genes associated with increased risk pretreated with adenosine triphosphate (ATP) or lipopolysac-
of fungal infections typically predispose patients to increased charide (LPS) to provide an extra signal. When whole live fungal
mucocutaneous or invasive disease. In contrast, CARD9 organisms are used, pretreatment with ATP or LPS is not required.
deficiency predisposes to both mucosal and systemic fungal Mice lacking NLRP3, apoptosis-associated speck-like protein
diseases. containing a C-terminal caspase recruitment domain (ASC), or

420 ParT ThrEE Host Defenses to Infectious Agents


caspase 1 demonstrate increased susceptibility to C. albicans has been demonstrated. Extracellular H. capsulatum are taken
infection. The mechanism appears to be a reduction in neutrophil up by DCs and are then capable of triggering CD8 T cells through
influx. However, in these mouse models, early neutrophil influx antigen-specific peptide-loaded class I MHC molecules.
does not appear to be dependent on an intact inflammasome; Monocyte-derived DCs have a nonredundant role in antifungal
−/−
rather, it is required for sustained neutrophil influx. immunity, through the induction of Th1 cells. CCR2 mice
show skewed Th2 responses and poorly controlled H. capsulatum
MEMORY OF INNATE IMMUNE CELLS infection compared with wild-type control mice. Priming of
+
Th1 lymphocytes require CCR2 monocyte-derived inflammatory
Historically, the innate immune response was considered a rapid DCs in mouse models of A. fumigatus or C. neoformans infection.
and nonspecific attack against invading pathogens. The widely These data indicate a critical role for DCs to connect the innate
accepted dogma was that innate immune cells confer no immu- immune response to drive T-cell responses and coordinate the
nological memory, unlike adaptive immune cells that respond adaptive immune response.
more slowly but execute a specific attack based on memory. An
increasing body of evidence suggests that the innate immune THE ADAPTIVE IMMUNE RESPONSE TO
system can, in fact, form memory, challenging the historical FUNGAL PATHOGENS
dogma. For example, innate immune memory has been dem-
onstrated in organisms that lack the adaptive immune system, The primary function of the adaptive immune system is to launch
29
such as plants and invertebrates. Several studies have demon- a highly specific attack to destroy invading pathogens. The adaptive
strated increased responsiveness of innate immune cells to second- immune system also provides long-term protection by creating
30
ary encounters with pathogens. Immunological memory of “immunological memory” following an initial response to a
innate immune cells has been described in the context of C. pathogen. The two major branches of the adaptive immune
albicans infection; in mice, preexposure to a nonlethal infection system are cell-mediated immunity (CMI) and humoral immunity.
or purified β-1,3 glucan confers protection from a secondary B cells and T cells carry out this adaptive immune response.
lethal infection in the absence of functional B cells and T cells. CMI by T cells is critical for the host response to fungal pathogens.
Epigenetic modifications serve as the mechanism by which innate There are two major classes of T cells that orchestrate CMI in
31
immune cells generate immunological memory. There are response to invading pathogens: CD4 T cells and CD8 T cells.
numerous lingering questions about innate immune memory, Antigen presentation by DCs activates T cells. T-cell activation
including the duration of this response, its ability to provide is also driven by cytokines secreted from macrophages and DCs.
protection from other microbes, and whether this type of response In turn, activated T cells secrete cytokines that elaborate the
can be elicited by vaccination. immune response.

LINKING THE INNATE IMMUNE RESPONSE TO
ADAPTIVE IMMUNE RESPONSE KEY CONCEPTS
Adaptive Immunity to Fungal Pathogens
A secondary, but critical, role of the innate immune system is
to amplify the immune response by activating and recruiting • T-cell immunity is critical for fungal host defenses. Patient with advanced
cells of the adaptive immune system, including T and B lym- HIV infection and AIDS are at heightened risk for C. neoformans
infection.
phocytes. The innate immune signaling events that trigger • T-helper 1 (Th1) and Th17 T cell subsets are the most important T
activation of adaptive immunity are complex and may be pathogen cells for control of fungal infection
dependent. This amplification of the immune response is partially • Th2 cells mediate fungal asthma and hyperreactivity.
achieved by secretion of cytokines and ROS that recruit immune • Dectin-1 signaling is required for Th17 immunity.
cells to the site of infection. Additionally, antigen-presenting • Humoral immunity may play a role in antifungal defense but appears
cells (APCs) of the innate immune system use class II MHC to play a supportive role.
molecules on their surface to present pathogen-derived antigens
to I CD4 T cells, resulting in differentiation into effector or
regulatory T cells (Tregs). CD4 T Cells
DCs provide the critical link between the innate and adaptive CD4 T cells are major players in host fungal immunity. The
arms of the immune system. DCs are capable of recognizing critical role for CD4 T cells became overwhelmingly apparent
fungal pathogens similar to macrophages. Endowed with both during the AIDS epidemic. Patients with HIV/AIDS experience
TLRs and CLRs, DCs are activated by components of the fungal loss of CD4 T cells and are extremely susceptible to opportunistic
cell wall. Subsets of DCs provide specialized function. pDCs fungal pathogens, especially C. neoformans infection. Additionally,
produce IFN-α in response to ligands that engage endosomal subsets of CD4 T cells, including Th1, Th2, and Th17 cells, have
TLRs. Although pDCs play a key role in viral infections, their specialized roles in fungal defense (Chapter 16).
role in fungal infections is not fully understood. pDCs are able
to recognize A. fumigatus through engagement of TLR9. When Th1 Cells
A. fumigatus and pDCs are coincubated in vitro, growth of the Th1 cells are a subset of CD4 T cells that play a critical role in
fungus is inhibited, indicating that these cells are capable of controlling fungal infections. After exposure to fungal pathogens,
controlling growth of this mold. A subset of pDCs secretes IL-6 APCs secrete IL-12, which is critical to the maintenance and
and IL-23, the latter of which can serve to prime antigen-specific expansion of Th1 lymphocytes. Mutations in the IL-12 signaling
Th17 lymphocytes. pathway predispose mammals to Candida, Cryptococcus, and
+
CD8 DCs possess the unique capacity to cross-present Coccidiomycosis infections. Th1 lymphocytes produce copious
antigens. Cross-presentation of Histoplasma capsulatum antigens amounts of IFN-γ, TNF-α, and granulocyte macrophage–colony

ChaPTEr 29 Host Defenses to Fungal Pathogens 421


stimulating factor (GM-CSF), all of which play a critical role in These lymphocytes play a critical and nonredundant role in the
antifungal defense. Although IFN-γ has multiple effects on control of IFIs. Th17 cells are characterized by production of
different cell types, this cytokine induces several genes in mac- cytokines, IL-17A, IL-17F, and IL-22. Differentiation of this T-cell
rophages that potently modify the distribution of proteins on lineage requires stage-specific stimulation by cytokines and
the phagosome. The net result of these effects is enhanced activation of specific transcription factors. Transforming growth
intracellular killing. IFN-γ–treated macrophages have enhanced factor-β (TGF-β) and IL-6 prime the initial differentiation of
phagocytosis and are more efficient in antigen presentation. CD4 T cells to Th17 cells. IL-23 is required for both maintenance
32
TNF-α has similar effects similar to those of IFN-γ and enhances and expansion of these cells. Activation of STAT3 by IL-6
the function of macrophages. Patients with mutations in the regulates transcription of retinoid orphan receptor-γt (ROR-γt),
TNF-α receptor or those who are treated with TNF-α are at the master transcription factor controlling Th17 lineage com-
heightened risk for fungal infections, including A. fumigatus. mitment. Several studies have confirmed the importance of Th17
33
GM-CSF appears to increase ROS production in neutrophils cells and IL-17/IL-23 signaling for antifungal immunity. Mice
and macrophages. Two lines of evidence in humans suggest an deficient in IL-17 receptor fail to generate functional Th17 cells
important role for Th1 cells in antifungal defense. Patients with and are highly susceptible to both systemic and mucocutaneous
fever and neutropenia with documented IFIs, including candi- candidiasis. Additionally, IL-23–deficient animals develop progres-
demia, have improved resolution of infection independent of sively worse mucocutaneous disease. Likewise, in mouse models
the effect of GM-CSF on neutrophil mobilization. Patients with of vulvovaginal C. albicans infection, impairment of Th17 cell
protein alveolar proteinosis, which is a disease related to anti– function led to increased fungal burden. Interestingly, fungal
GM-CSF autoantibodies, have increased risk of IFIs. pathogens have devised mechanisms to subvert this component
of the immune system. Cell surface receptors found on Candida
CLINICaL rELEVaNCE and Aspergillus bind IL-17 and cause increased hyphal growth
Opportunistic Fungal Pathogens and transcriptional changes within these fungal organisms that
are thought to combat the immune response.
Cause Invasive Fungal Infections in
Immunocompromised Patients CD8 T Cells
• Advances in clinical medicine provide treatment modalities that bring Although CD8 T cells are critical for immune defense against
new promise to once-deadly diseases. During the course of these viral pathogens and tumors, their contribution to fungal immunity
treatments, many patients develop significant deficiencies in their is incompletely understood. They may provide functional
immune system that permit the development of opportunistic redundancy to confer protection in the absence of CD4 T cells.
infections. These cells will be important in the development of fungal
• Invasive fungal infections (IFIs) caused by Aspergillus fumigatus, vaccines, but further studies are required to delineate if they
Candida albicans, or Cryptococcus neoformans are considered the have any nonredundant role in antifungal immunity.
most serious infections that affect immunocompromised patients.
Despite effective fungicidal therapies, such as voriconazole and B Cells
amphotericin, mortality in these patients exceeds 25%.
• These data indicate that a robust immune response is required for Early data dismissed the role of humoral immunity during fungal
clinical efficacy. Patients with defects in innate immunity are at highest infection. However, clinical observations have suggested that
risk despite the fact that they typically have functional circulating antibodies contribute to fungal host defense. Patients with B-cell
lymphocytes. defects, including X-linked hyper-IgM syndrome and hypogam-
• There is a dire clinical need to develop novel therapeutics for IFIs to
address the significant public health burden caused by opportunistic maglobulinemia, demonstrate increased susceptibility to cryp-
fungal pathogens. tococcosis. Additionally, pediatric patients with selective IgG2
deficiency are at heightened risk for IFIs and other bacterial
infections that rely on antipolysaccharide antibody formation.
Th2 Cells Antibodies observed in normal individuals are typically
In contrast to Th1 cells, Th2 lymphocytes appear to be defense against fungal cell wall components and have the capacity to
against most fungal infections. Th2 lymphocytes secrete IL-4, inflict negative changes within the fungal cell. These include
IL-5, and IL-13. The effects of these cytokines on macrophages inducing transcriptional changes that are deleterious to the
diminish the antifungal response. IL-4 drives macrophage dif- microbe. Antibodies can work in conjunction with complement
ferentiation toward the alternatively activated phenotype. In mice and phagocytic cells to facilitate damage of the cell wall and
exposed to either Aspergillus or Cryptococcus in the respiratory trigger uptake by cells, respectively. Antibodies also have the
tract, Th2 skewing drives an allergic phenotype with airway capacity to trigger transcriptional changes that increase pathogen
hyperresponsiveness (AHR) and goblet hyperplasia. There is one virulence, thereby exacerbating disease. Pleiotropic effects have
notable exception to the deleterious effect of Th2 cells on invasive made it difficult to resolve the net value of humoral immunity
fungal disease—P. jiroveci. Th2 responses seem to be protective to IFIs.
in this infection. Mouse studies suggest that alternatively activated
macrophages are critical for clearance of this fungal pathogen. Natural Killer Cells
Recent studies have identified a nonredundant role for Th17 Natural killer (NK) cells exert an antifungal response, but
lymphocytes, suggesting that Th2 responses are necessary, but the rules that govern this response have only been elucidated
not sufficient, for clearance of P. jiroveci. recently. NK cell–mediated killing requires direct contact with the
fungus, which leads to release of perforin. The NK cell receptor
Th17 Cells p30 is required for NK cell-fungal conjugate formation, pI3K
Th17 cells are a subset of CD4 T cells that have emerged as signaling, and perforin release. p30 was previously identified
major contributors to host defense against fungal pathogens. as an activating receptor against tumor cells. In patients with

422 ParT ThrEE Host Defenses to Infectious Agents


AIDS, who are highly susceptible to C. neoformans infection, p30 Chronic granulomatous disease (CGD) is a well-known
expression is decreased on NK cells leading to defective perforin example of a genetic disorder associated with increased susceptibil-
release and reduced microbicidal activity. IL-12 restored p30 ity to invasive bacterial and fungal infections, especially invasive
expression and fungal killing in a mouse model of C. neoformans aspergillosis. CGD is caused by mutations in genes encoding
infection by NK cells. These data indicate a direct role for NK subunits of the NADPH complex (e.g., CYBA, CYBB NCF1, NCF2,
cells against Cryptococcus through direct recognition by a cell NCF4), which results in defective ROS production, a critical step
surface receptor. Evidence for the role of NK cells against invasive in the microbial killing process. 34,35 Recent work has shown that
fungal pathogens has been expanded to show that they play a deficiencies in ROS production resulting from a mutation in a
nonredundant role in clearing C. glabrata infections. The human subunit of the NADPH complex also result in defective autophagy.
NK receptor p46 and its mouse orthologue natural cytotoxic- These defects prevent autophagy-dependent inhibition of IL-1β
ity triggering receptor 1 (NCR1) bind C. glabrata. In vitro, NK and increased inflammasome activation. Blocking of IL-1 protects
cells lacking NCR1 show reduced capacity to kill C. glabrata. mice with CGD from invasive aspergillosis and colitis and restores
−/−
Indeed, NCR1 mice have increased susceptibility to C. glabrata autophagic function in monocytes derived from patients with
infection. The ligands for p46/NCR1 are discrete members of CGD. 36
a family of proteins called EPA (epithelial adhesion), which are Chronic mucocutaneous candidiasis (CMC) is an immune
glycan-binding lectins and permit attachment of fungal cells to disorder characterized by persistent or recurrent candidal infec-
host cells. tions of the skin, nails, and mucous membranes. Primarily, CMC
appears to be associated with defects in IL-17 signaling. A direct
GENETIC SUSCEPTIBILITIES TO INVASIVE role for IL-17 signaling in the protection against CMC was
FUNGAL INFECTIONS demonstrated in two families with deficiencies in the IL-17
37
receptor A (IL-17RA) and the cytokine IL-17F. As mentioned
Although certain clinical diagnoses and medical interventions previously, mutations in dectin-1 cause increased susceptibility
are associated with susceptibility to fungal infections, not all to CMC as a result of impaired IL-1β production and a reduced
38
patients in these high-risk groups become infected. Moreover, frequency of Th17 cells. Similarly, patients with CARD9 deficien-
individuals lacking traditional risk factors (e.g., immunosup- cies have increased susceptibility to invasive fungal infections
pression, HIV, etc.) can develop chronic or invasive fungal resulting from decreased Th17 cells, reduced chemokine/cytokine
infections, suggesting that additional factors confer susceptibility. production, and impaired neutrophil killing. 39
Primary immunodeficiencies (PIDs) include a group of hereditary CMC is also common in patients with autoimmune polyen-
immune disorders that render patients more susceptible to docrinopathy syndrome type 1 (APS-1), an autosomal recessive
infection. Recent studies of PIDs have provided the opportunity disorder caused by defects in the autoimmune regulator (AIRE)
to define some of the genetic and molecular mechanisms that gene. In these patients, CMC results from the generation of
40
contribute to infection. A list of genes associated with susceptibility autoantibodies against IL-17 and IL-22. Hyper-IgE syndrome
to fungal infection are summarized in Table 29.1. (HIES) is an autosomal dominant or recessive immune disorder


TABLE 29.1 Genes associated With Immunity to Fungi in humans
Gene Product Disease Fungal Pathogen Immunological Phenotype
ACT1 CMC a Candida Impaired interleukin-17 (IL-17) signaling.
AIRE CMC; Autoimmune Polyendocrinopathy Syndrome-1 Candida Autoantibodies against IL-17 and IL-22
CARD9 CMC; disseminated candidiasis; Candida Candida, Trichophyton, Defective dectin-1 signaling and reduced
meningoencephalitis; deep dermatophytosis; Phialophora, Exophiala frequency of T-helper 17 (Th17) cells
subcutaneous phaeohyphomycosis; invasive Impaired neutrophil killing
Exophiala infection
CYBA CGD b Candida, Aspergillus Nicotinamide adenine dinucleotide
CYBB phosphate (NADPH) complex
NCF1 deficiency causing impaired reactive
NCF2 oxygen species (ROS) production
NCF4
Dectin-1 CMC Candida Impaired IL-1β and IL-17 production and
reduced frequency of Th17 cells
DOCK8 CMC; HIES c Candida Defective activation of Th17 cells
IL-17F CMC Candida Defective IL-17 signaling
IL-17RA CMC Candida Defective IL-17 signaling
RORC Candidiasis Candida Absent IL-17A/-17F–producing T cells
STAT1 CMC, cutaneous fusariosis, disseminated Candida, Fusarium, Coccidioides, Defective production of IL-17, IL-22, and
coccidioidomycosis, histoplasmosis, Histoplasma, Penicillium interferon-γ (IFN-γ) and reduced
mucormycosis, and Penicillium marneffei infection marneffei, Apophysomyces frequency of Th17 cells
STAT3 CMC; HIES Candida Impaired Th17 differentiation and
reduced frequency of Th17 cells
TYK2 CMC; HIES Candida Defective IL-23 signaling and reduced
frequency of Th17 cells
a Chronic mucocutaneous candidiasis.
b Chronic granulomatous disease.
c Hyper–immunoglobulin E syndrome.

ChaPTEr 29 Host Defenses to Fungal Pathogens 423


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32. Zuniga LA, Jain R, Haines C, et al. Th17 cell development: from the 43. Liu L, Okada S, Kong XF, et al. Gain-of-function human STAT1
cradle to the grave. Immunol Rev 2013;252(1):78–88. mutations impair IL-17 immunity and underlie chronic mucocutaneous
33. Hamad M. Innate and adaptive antifungal immune responses: partners candidiasis. J Exp Med 2011;208(8):1635–48.
on an equal footing. Mycoses 2012;55(3):205–17. 44. van de Veerdonk FL, Plantinga TS, Hoischen A, et al. STAT1 mutations in
34. Clark RA, Malech HL, Gallin JI, et al. Genetic variants of chronic autosomal dominant chronic mucocutaneous candidiasis. N Engl J Med
granulomatous disease: prevalence of deficiencies of two cytosolic 2011;365(1):54–61.
components of the NADPH oxidase system. N Engl J Med 1989; 45. Depner M, Fuchs S, Raabe J, et al. The extended clinical phenotype of 26
321(10):647–52. patients with chronic mucocutaneous candidiasis due to gain-of-function
35. Wang X, van de Veerdonk FL. When the Fight against Fungi Goes Wrong. mutations in STAT1. J Clin Immunol 2016;36(1):73–84.
PLoS Pathog 2016;12(2):e1005400. 46. Okada S, Markle JG, Deenick EK, et al. Immunodeficiencies. Impairment
36. de Luca A, Smeekens SP, Casagrande A, et al. IL-1 receptor blockade of immunity to Candida and Mycobacterium in humans with bi-allelic
restores autophagy and reduces inflammation in chronic granulomatous RORC mutations. Science 2015;349(6248):606–13.
disease in mice and in humans. Proc Natl Acad Sci USA 2014;111(9):
3526–31.

ChaPTEr 29 Host Defenses to Fungal Pathogens 424.e1


MULTIPLE-C h OICE QUESTIONS

1. Loss of dectin-1 in patients leads to: C. Glucuronoxylomannan
A. Chronic mucocutaneous candidiasis D. Peptidoglycan
B. Candidemia 3. Which T-cell response typically makes fungal infection worse?
C. No clinical disease unless patient has neutropenia A. T-helper 1 (Th1)
D. Increased risk of Cryptococcus neoformans disease
B. Th2
2. Which of the following carbohydrates is NOT found on the C. Th17
fungal cell wall? D. CD8
A. β-1,3 glucan
B. Galactomannan

30









Host Defenses to Protozoa



Peter C. Melby, Robin Stephens, Sara M. Dann







Protozoal infections are an important cause of morbidity and T cells can be critically important through cytokine production
mortality worldwide (Table 30.1). Protozoan pathogens exact (e.g., Plasmodium spp., T. cruzi, T. gondii) or direct cytotoxic
their major toll in the tropics, but infection by these parasites activity (e.g., Cryptosporidium). For the parasites that have an
remains a significant problem in developed countries because extracellular stage (e.g., Plasmodium spp., Trypanosoma spp.,
of travel to and emigration from developing countries, the Giardia, and Trichomonas), specific antibodies mediate acquired
susceptibility of patients with acquired immunodeficiency immunity.
syndrome (AIDS) to opportunistic protozoans, and episodic Intensive effort has been dedicated to the development of
transmission within communities. effective vaccines for protozoal diseases, but as of 2016, only the
malaria circumsporozoite vaccine (RTS,S) has reached the stage
of clinical use. The reader is referred to a number of excellent
KEY CONCEPTS reviews of the potential vaccine candidates. A discussion of
1-4
Host Defense Against Protozoa the immune responses to some of the individual protozoal
pathogens follows.
• Interaction of the parasite with host cells induces an array of cytokines
that stimulate the innate and adaptive immune responses to eliminate
the pathogen, and/or cytokines that inhibit or downregulate the Plasmodium spp.
antiparasitical responses to enable the initiation of tissue parasitism.
• The outcome of infection is determined by the balance between the Pathogenesis
infection-promoting and the host-protective cytokines and effector Soon after Plasmodium spp. sporozoites are injected into the
cells. Often there is a mixed response, resulting in a persistent bloodstream by the Anopheles mosquito, they invade hepatocytes
infection. and undergo schizogony (asexual reproduction). A dormant form
• A persistently infected host may develop clinical disease if there is a of P. vivax and P. ovale (hypnozoites) can reside within hepatocytes
waning of the immune mechanisms (e.g., in acquired immunodeficiency
syndrome [AIDS]) that are critical to the control of infection. for months and then cause clinical bloodstream infection. Fol-
lowing schizogony, merozoites are released from hepatocytes
into the bloodstream in a membrane-bound structure, known
Protozoan pathogens make up a group of highly diverse as merosomes. The merosomes rupture in blood, and free mero-
organisms that utilize a wide array of mechanisms for pathogenesis zoites invade red blood cells (RBCs) to produce ring-stage para-
and immune evasion. There are numerous host targets for the sites. These parasites mature into trophozoites, which again
intracellular protozoan parasites, including erythrocytes (Plas- undergo schizogony, leading to rupture of the erythrocyte and
modium and Babesia), macrophages (Leishmania and Toxoplasma the release of new invasive merozoites. Merozoites can also develop
gondii), or multiple cell types (Trypanosoma cruzi). The luminal into sexual-stage gametocytes, which can be ingested by a feeding
parasitical protozoan may be extracellular, such as amebae and mosquito to continue the transmission cycle.
the flagellates (Giardia and Trichomonas), or primarily intracel- The clinicopathological features of malaria are caused by
lular, such as the coccidian parasite Cryptosporidium. intraerythrocytic infection and the associated immune response.
The innate and adaptive immune systems respond in diverse The cyclical rupture of erythrocytes is associated with fever. The
ways to the blood and tissue and intestinal protozoan pathogens. induction of a proinflammatory cytokine cascade plays a central
Neutrophils, macrophages, and natural killer (NK) cells are the role in the pathogenesis of P. falciparum malaria and its complica-
effector cells that mediate the innate response against the extracel- tions. Parasite antigens, particularly those having glycophospha-
lular protozoan parasites. The NK cell–activated macrophage tidyl inositol (GPI) membrane anchors, released during the
system is central to the innate response to intracellular parasites rupture and reinvasion of RBCs, activate the innate immune
(Fig. 30.1) (Chapters 3, 17). The innate cytokine response activates response. The production of proinflammatory cytokines (IL-1,
phagocytes and is critical to the induction of the adaptive immune TNF, lymphotoxin, IL-12, and IFN-γ) leads to fever, expression
response via antigen presentation by dendritic cells (DCs). For of endothelial adhesion molecules, and cytoadherence. It is
5
the intracellular pathogens (e.g., Leishmania spp., T. cruzi, T. mediated, in part, by TLR2 and is MyD88-dependent. NK cells
gondii), the early production of interleukin-12 (IL-12) and and memory T cells produce early IFN-γ, which contributes to
interferon-γ (IFN-γ) drives the differentiation of T cells to a the production of a pathologically high level of TNF. IL-10- and
protective T-helper 1 (Th1) phenotype. In most cases CD4 T transforming growth factor-β (TGF-β)–mediated downregulation
cells play a primary role in adaptive cellular immunity, but CD8 of the Th1 immune response and leukotriene (LT)/tumor necrosis

425

426 ParT THrEE Host Defenses to Infectious Agents



TABLE 30.1 Worldwide Significance of the Major Protozoal Infections
Parasite Estimated Worldwide Cases (annual Mortality) Clinical Manifestations
Plasmodium spp. 400–490 million (P. falciparum: >2 million deaths/year, Fever with potential complications of severe hemolysis, renal
primarily children) failure, pulmonary edema, cerebral involvement
Leishmania spp. 10–50 million people infected, 1.2 million new cases per Asymptomatic infection; skin ulcers or nodules; destructive
year oropharyngeal lesions; visceral disease with fever,
hepatosplenomegaly, cachexia, pancytopenia
Trypanosoma cruzi 24 million (60 000 deaths) Asymptomatic infection; dysrhythmias or chronic heart failure;
hypertrophy and dilation of the esophagus, colon
Toxoplasma gondii Several hundred million people infected worldwide. Self-limited fever, hepatosplenomegaly; lymphadenopathy and
5–9% of healthy US adults are seropositive encephalitis (reactivation in patients with acquired
immunodeficiency syndrome [AIDS]); congenital infection,
with fetal death, chorioretinitis, meningoencephalitis
Entamoeba histolytica 50 million (100 000 deaths) Asymptomatic infection, diarrhea, dysentery, or liver abscess
Giardia lamblia 200 million (most common in young children and Asymptomatic infection, chronic diarrhea
immunocompromised persons)
Cryptosporidium parvum Prevalence 3–10% in patients with diarrhea in developing Self-limited diarrhea in immunocompetent persons, severe
and C. hominis countries intestinal and biliary disease in patients with AIDS
Trichomonas vaginalis 170 million/year Asymptomatic infection, vaginal discharge, urethritis

Pathogen or KEY CONCEPTS
soluble products
IFN-α/β Immunopathogenesis of Severe Plasmodium
falciparum Malaria
IL-10
TGF-β (-) (+) • Release of malarial antigens stimulates tumor necrosis factor (TNF),
PGE 2 NK cell interleukin (IL)-1, and lymphotoxin production from innate immune
RNI cells.
IL-12 ROI • TNF/leukotriene (LT) induce vascular leakage, hemorrhage, endothelial
IFN-γ
TNF-α (+) TNF-α cell activation with expression of endothelial adhesion molecules,
IL-18 platelet activation and adhesion, and coagulation.
(+) IL-1β (+) NO Macrophage • Inflammatory cytokines amplify severe anemia caused by loss of
Macrophage activated infected red blood cells (RBCs) by inducing dyserythropoiesis and
or dendritic cell for intracellular phagocytosis of uninfected RBCs, which can be coated with parasite
IFN-γ killing antigens.
TNF-α • Lethal outcomes for the fetus of infected women during pregnancy
FIG 30.1 Macrophage, Natural Killer (NK) Cell, and Cytokine are caused by the local immune response induced by placenta-specific
Interactions in the Innate Immune Response to Intracellular parasites.
Protozoa. Exposure of macrophages or dendritic cells to a
pathogen or microbial product can result in the release of
cytokines and inflammatory mediators that may stimulate (+) or Innate Immunity
suppress (−) NK cell activation. Activated NK cells produce Complement-mediated lysis can occur at the sporozoite and
cytokines that can then activate macrophages for intracellular merozoite stages, though parasites have evolved evasive mecha-
killing. It must be recognized that this diagram is oversimplified nisms. While sporozoites rapidly transit from the skin to the
and that these cytokines, most notably interferon (IFN)-α/β, liver, they can activate DCs at the site of inoculation or in the
interleukin (IL)-10, transforming growth factor (TGF)-β, and IL-12, regional lymph node. Early activation of NK cells and IFN-γ
may be produced by other types of cells, such as epithelial cells are associated with better outcomes of infection. Although high
or enterocytes. NO, nitric oxide; RNI, reactive nitrogen intermedi- levels of TNF are associated with severe malaria, physiological
ates; ROI, reactive oxygen intermediates. levels are protective through the activation of macrophages. γδ
+
T-cell receptor (TCR) T cells that respond to phosphorylated
nonpeptide antigens on live parasites have been demonstrated.
factor (TNF) production, which have a role in severe and cerebral DCs initially induce a strong adaptive immune response to the
malaria, act to limit the inflammatory response. erythrocytic stage, which is tempered during prolonged infection.
Severe malaria includes severe anemia, respiratory distress,
placental malaria, and cerebral malaria, with the latter causing Adaptive Immunity
up to 90% of deaths. In cerebral malaria, a combination of Partial immunity to Plasmodium spp. infection is acquired slowly
6
inflammation, cytoadhesion of parasites, and leukocytes, and following repeated exposure in endemic areas. In areas of intense
vascular pathology leads to coma. Cytokines and endothelial cell perennial P. falciparum transmission, the density of parasitemia,
production of nitric oxide (NO) contribute to inflammatory morbidity, and the incidence of cerebral malaria and malaria-
lesions in the brain. Patients may die as a result of severe edema related deaths are highest in the early childhood years, declining
and swelling of the brainstem. Severe anemia is the result of a thereafter. Naturally acquired immunity to repeated infections
combination of destruction of infected and uninfected RBCs develops in young adults, with the exception of the morbidity
and dyserythropoiesis. Both destruction of uninfected RBCs and associated with placental infections.
malformation of RBCs in bone marrow are increased by inflam- Adaptive immunity to the preerythrocytic stage is primarily
matory cytokines, as is the damage in placental malaria. mediated through major histocompatibility complex (MHC)

CHaPTEr 30 Host Defenses to Protozoa 427


class I-restricted parasite-specific CD8 T cells and, to a lesser fly becomes infected by ingesting amastigotes during a blood
extent, CD4 T cells, via IFN-γ-induced NO-dependent killing meal. In the sand fly gut, the amastigotes differentiate into
7
of intrahepatocyte parasites. Potentially protective immune infectious metacyclic promastigotes that infect the vertebrate
mechanisms against the preerythrocytic stage have largely been host during the next blood meal. The surface lipophospho-
identified by study of mice vaccinated with irradiated sporozoites glycan (LPG) plays a central role in the parasite’s entry and
and challenged with murine Plasmodium spp. Irradiated sporo- survival in host cells. Immunomodulatory factors present in
zoite challenge also protects humans and is being scaled up for the sand fly saliva may enhance the infectivity of the parasite.
vaccine trials. Antisporozoite immunity requires the presence Once introduced into skin, the promastigotes are phagocytosed
of high antibody titers and high numbers of T cells to block (through complement–complement receptor–mediated coiling
sporozoite invasion of the hepatocyte, which occurs in just a phagocytosis) by neutrophils, DCs, and macrophages, where
few cells within minutes of inoculation. they transform to amastigotes and replicate within the acidic
Both antibody-dependent and cell-mediated immune mecha- and hostile environment of the phagolysosome. Eventually,
nisms are active against the erythrocytic stage of infection. Both the phagocytes rupture and release amastigotes to infect other
B cells and CD4 T cells are required for complete clearance of macrophages or a sand fly.
8
parasites. Adoptive transfer of human immune serum is protective
for naïve individuals. Antibodies directed against merozoite Innate Immunity
surface proteins can inhibit invasion. A significant proportion Much of what we know of immunity in leishmaniasis comes
of antibodies to infected erythrocytes are directed toward variant from studies of inbred mouse strains, which demonstrate a
antigens, such as PfEMP-1 on the surface of the RBC. Immu- genetically determined spectrum of innate and adaptive immune
noglobulin G1 (IgG1) and IgG3 isotype antibodies specific for responses that shape the outcome of infection. The innate immune
parasite antigens exported to the surface of the RBC play a role response to Leishmania is mediated by complement, NK cells,
11
in naturally acquired immunity by opsonizing infected cells for cytokines, and phagocytes. The production of IL-12 early in
phagocytosis in the spleen. the course of infection by DCs leads to the early activation of
CD4 T cells are implicated in protection by experiments NK cells and the production of IFN-γ. Chemokines (IP-10, MCP-1,
showing that MHC class II–restricted antigen presentation is and lymphotactin), as well as LPG–TLR interaction, can also
required for reduction of parasitemia and pathology. Protective promote the early NK-cell activation. Activated NK cells have
cellular immune responses (CD4 and CD8 T-cell proliferation, been shown to be cytolytic for Leishmania-infected macrophages,
IFN-γ production, and NO synthesis) in the absence of detectable but NK cell–derived IFN-γ plays a more prominent role in host
antibody responses were identified in naïve volunteers, who were defense by activating macrophages to kill the intracellular parasite
protected by repeated exposure to low doses of blood-stage through the generation of reactive oxygen intermediates (ROIs)
parasites. In addition to promoting phagocytosis, CD4 T cells or reactive nitrogen intermediates (RNIs). Parasite-induced,
are required to help B cells, especially by the production of IL-21. MyD88-dependent signaling through TLR2, TLR3, and TLR4
One of the most important roles for CD4 T cells is the regulation contributes to macrophage activation and NO production.
of the intense inflammatory response through production of Activated polymorphonuclear leukocytes (PMNs) can kill parasites
antiinflammatory cytokines IL-10 and TGF-β. through oxidative mechanisms, but the role of neutrophils in
vivo depends on the timing of their recruitment and their
Evasion of Host Immunity interaction with other immune cells. An important study
12
The malaria parasite uses several different mechanisms to evade demonstrated that infiltrating neutrophils promote sand fly–
9,10
the host immune response. Sporozoites and merozoites evade transmitted infection, probably through modulation of macro-
circulating antibody by rapidly entering hepatocytes or RBCs, phage function following engulfment of apoptotic parasitized
13
respectively. Some sporozoite proteins enter the hepatocyte nucleus neutrophils. Type 1 IFNs participate in the early induction of
and influence the expression of a number of host genes, thereby NO and control of parasite replication early in infection.
favoring parasite survival. Mature RBCs do not express MHC
molecules on their surface and so avoid recognition by T cells. Adaptive Immunity
The few parasite proteins that are expressed on the erythrocyte Within an endemic area there is acquisition of immunity in the
surface exist in multiple allelic forms to avoid quick recognition population over time. Retrospective epidemiological studies
by the adaptive immune system. Many of the immunodominant indicate that most individuals with prior (primary) infection
antigens in Plasmodium spp. are proteins having extensive repeat (subclinical or healed) are immune to a subsequent clinical
sequences that vary over time. The large number of bloodborne infection. Following primary infection, parasites persist for the
antigens induces a plasmablast response generating a short-lived life of the host and maintain long-term immunity.
burst of low-affinity antibodies. Although there is an increase There is extensive evidence from experimental models that
in atypical B cells, memory B cells and long-lived plasma cells cellular immune mechanisms mediate adaptive resistance to
are generated. It is unclear if poor immunity following a single Leishmania infection, and human studies have generally confirmed
infection is caused by ineffective, atypical B cells or misdirected this. Antileishmanial antibodies, which are produced at a low
antibody specificity. level in localized cutaneous leishmaniasis (LCL) and at a very
high level in visceral leishmaniasis (VL), play no role in protection.
Leishmania spp. The general mechanisms of cellular immunity in leishmaniasis
can be summarized (Fig. 30.2). Following infection in the skin,
Pathogenesis migratory dermal DCs phagocytose Leishmania and presumably
The intracellular Leishmania amastigote replicates within macro- transport the intracellular parasite to the regional lymph node,
phages in the vertebrate host, and the extracellular promastigote where they induce a T-cell response. Adaptive immunity is primar-
develops within the insect vector. The female phlebotomine sand ily mediated by parasite-induced production of IFN-γ by CD4

428 ParT THrEE Host Defenses to Infectious Agents


Leishmania promastigotes gain the capacity to produce IFN-γ, and then migrate to the site
of infection. Thus T cm cells act as a reserve of antigen-reactive
NK T cells that can expand and become effector T cells in response
IL-12 to secondary antigenic challenge.
DC
The generation of RNI by activated macrophages is the primary
IFN-γ mechanism of parasite killing in the murine model. Although
IL-12 IFN-γ-induced production of NO may not be detectable in human
IFN-γ IFN-γ macrophages, inhibition of nitric oxide synthase 2 (NOS2) was
IFN-γ shown to impair killing of intracellular Leishmania.
IFN-γ Th1 Several adaptive immune mechanisms promote parasite
15
IL-4 replication and disease. The progression of murine L. major
IL-10 IFN-γ infection has been correlated with the expansion of Th2 cells
Th2
IL-10 TNF-α and the production of IL-4, IL-5, and IL-10. In susceptible mice,
TGF-β IL-4 production within the first day of infection was shown to
IL-4 IL-10
IL-10 Activation TGF-β downregulate IL-12 receptor β-chain expression and drive the
RNI response to a Th2 phenotype. However, other nonsusceptible
ROI mouse strains appear to be able to overcome an early IL-4 response
IL-10
TGF-β Parasite killing and develop a resistant phenotype, and susceptibility to some
PGE 2 Deactivation L. major strains is not strictly mediated by IL-4 (IL-13 and/or
IL-10 may have a prominent role). The cytotoxic activity of CD8
Parasite Macrophage T cells may promote cutaneous inflammation and lesion pathol-
14
replication ogy. The macrophage production of immune suppressive
FIG 30.2 Immunity in Leishmaniasis. Exposure of dendritic molecules, such as TGF-β or prostaglandin E 2 (PGE 2 ), may also
cells to parasites or parasite antigens leads to the release of contribute to susceptibility.
interleukin (IL)-12, which induces natural killer (NK) cells to produce Peripheral blood mononuclear cells (PBMCs) isolated from
interferon (IFN)-γ and drives the adaptive immune response patients with localized cutaneous leishmaniasis demonstrate a
toward a protective T-helper 1 (Th1) phenotype. IL-12 production Th1 response to Leishmania antigens, and in the cutaneous lesion,
by dendritic cells and IFN-γ production by NK and Th1 cells there is an exuberant Th1 and granulomatous response that
negatively regulates the Th2 response. IFN-γ activates macro- mediates parasite killing and localized tissue damage, which
phages to kill the intracellular pathogen. In genetically susceptible usually leads to a scar. Patients with mucosal leishmaniasis (ML)
individuals, a counterregulatory Th2 cytokine response can exhibit vigorous cellular immune responses characterized by high
suppress the Th1 response and impair classic macrophage levels of TNF-α and Th1 and Th17 cytokines; it is postulated
activation, leading to parasite replication and uncontrolled infec- that this hyperresponsive state contributes to the prominent
tion. Counterprotective macrophage-derived cytokines can also tissue destruction of ML. Patients with diffuse cutaneous leish-
inhibit the Th1 response, stimulate the Th2 response, and impair maniasis (DCL) resemble the progressive infection caused by L.
classical activation through an autocrine loop. Activating stimuli major in BALB/c mice in that there are minimal or absent
are shown by solid arrows, and deactivating stimuli are shown Leishmania-specific lymphoproliferative responses, and predomi-
by dashed arrows. nant Th2 cytokine expression. During active VL in humans,
there is a marked depression of Leishmania-specific lympho-
proliferative and IFN-γ responses, contraction of circulating
memory T cells, and an absence of delayed-type hypersensitivity
T cells (Th1 subset). CD4 T cells are absolutely required, but (DTH) response to parasite antigens. This T-cell unresponsiveness
immunity to cutaneous disease is also mediated by CD8 T cells appears to be mediated, at least in part, by a suppressive effect
14
via production of IFN-γ. Both CD4 and CD8 T cells are required of IL-10 and low levels of IL-12. Successful treatment of active
for an effective defense against murine visceral L. donovani disease restores an antigen-specific Th1 response.
infection, but the precise role of CD8 T cells is unclear. The
generation of the Th1 response is critically dependent on CD40- Evasion of Host Immunity
CD40L-mediated IL-12 production and driven by NK cell–derived The Leishmania parasite has numerous ways in which it adapts
16
IFN-γ. IL-12 and STAT4 are required for the maintenance of to and survives within the vertebrate host. In skin, the pro-
immunity. Tumor necrosis factor-α (TNF-α) contributes to mastigotes may be phagocytosed by neutrophils and macrophages,
protective immunity by synergizing with IFN-γ to activate which, unlike DCs, do not actively participate in T-cell priming.
macrophages. Recently nuclear factor-kappa B (NF-κB) family Furthermore, the clearance of apoptotic neutrophils is likely to
13
members have been shown to regulate T-cell responses and make macrophages more permissive to infection. The parasite’s
immunity to L. major infection in mice. surface LPG (and, to a lesser extent, the surface protein gp63)
Two subpopulations of CD4 T cells mediate immunity induced plays an important role in the entry and survival of Leishmania
by primary infection. Effector memory cells, which are short-lived in the mammalian host by conferring complement resistance
and dependent on the persistence of antigen, rapidly respond and by facilitating the entry of complement-opsonized parasites
to secondary infection by migrating to the infected tissue and into the macrophage without triggering a respiratory burst.
generating effector cytokines. Central memory T (T cm ) cells, Macrophage phagosome–endosome fusion and phagolysosomal
which can be maintained in the absence of persistent antigen, biogenesis are also inhibited by parasite LPG.
circulate throughout the lymphatic system and upon secondary Leishmania-infected macrophages have diminished capacity
challenge migrate to and proliferate in the draining lymph node, to initiate and respond to a T-cell response, and the impaired

CHaPTEr 30 Host Defenses to Protozoa 429



TABLE 30.2 Evidence for autoimmune and Parasite-Induced Inflammatory Mechanisms in
Chronic Chagas Disease
Evidence for autoimmune-Mediated Disease Evidence for Parasite-Induced Inflammatory Disease
Inflammatory disease presents in tissues with few or no parasites on routine Sensitive parasite detection techniques (polymerase chain reaction
histopathology studies [PCR], immunohistochemistry) correlate with the presence of
parasites (or parasite material) and severity of inflammatory disease
Peculiar pattern of organ involvement (heart and gastrointestinal tract) in Organs free of parasites (by sensitive parasite detection techniques)
patients with chronic disease are also free of disease
Long delay in the onset of chronic disease following infection; only a minority Absence of effective cellular immune response (in mice or humans)
of infected persons develops disease almost invariably exacerbates rather than reduces the parasite
burden and disease
Wide variability in the expression of disease among infected people In chronically infected mice, the destruction of a transplanted heart is
dependent on parasite infiltrating the transplanted tissue
Self-reactive antibodies and T cells demonstrable in infected people and in Degree of disease in hearts transplanted into chronically infected
experimental animals. Level of antibodies to the ribosomal P protein (R13 mice correlates with level of parasite burden in transplanted tissue
peptide) and cardiac myosin (B13 antigen) correlate with cardiac disease
Transient or limited disease reported in experimental models following Reduction of parasite burden by chemotherapy usually leads to
lymphocyte transfer decreased tissue inflammation and disease




antimicrobial effector activity provides a safe haven for the tissue, but an intense chronic inflammatory infiltrate with fibrosis
16
intracellular parasite. Infected macrophages have decreased and loss of muscle fibers is evident. In the digestive tract, there
synthesis of IL-1 and IL-12, and blunted IFN-γ-mediated activa- is lymphohistiocytic infiltration of the myenteric plexuses, with
tion through the disruption of signal transduction pathways reduction in the number of ganglion cells.
involving JAK/STAT, protein kinase C, p38 MAPK, ERK, AP-1, The tissue damage of acute T. cruzi infection is the result of
and NF-κB. Signaling mediated by tyrosine phosphorylation is a direct effect of the parasite and an indirect effect of the acute
decreased by the rapid induction of the host phosphotyrosine inflammatory response. In chronic infection, the balance between
phosphatase SHP-1. Conversely, there is increased synthesis of immune-mediated parasite containment and host-damaging
the immunosuppressive molecules IL-10, TGF-β, and PGE 2 . inflammation determines the course of disease. The pathological
Parasite factors and IL-4/IL-13 enhance expression of arginase mechanisms related to chronic Chagas disease are controversial
by infected macrophages. Arginase promotes infection through and certain to be multifactorial. Trypanosomal clonal variation
depletion of l-arginine, enhanced production of polyamines, and host genetic polymorphisms both contribute to tissue tropism,
+
+
and reduced NO production. IL-10 produced by CD4 CD25 T parasite persistence, and disease severity. Whether tissue damage
regulatory cells (Tregs) has an essential role in parasite persistence. is caused directly by parasites or indirectly through parasite-driven
Recently, it was shown that metastasizing parasites that cause inflammatory or autoimmune mechanisms, parasite persistence
mucosal disease harbor a high burden of Leishmania RNA virus, is a significant driver of disease (Table 30.2). 18,19 Autoimmunity
which subverts the host immune response and promotes parasite could arise from molecular mimicry of self by parasite antigens
persistence through activation of Toll-like receptor 3 (TLR3). 17 or by the release of self molecules from damaged or dying host
cells within the environment of an activated immune response.
Trypanosoma cruzi There is evidence for both of these autoimmune mechanisms.
The production of IL-10 by T. cruzi–infected cells may down-
Pathogenesis regulate the pathological cellular immune response.
T. cruzi is transmitted to the mammalian host when the infectious
metacyclic trypomastigote, which is deposited on skin in the Innate Immunity
feces of the reduviid insect vector while it takes a blood meal, The early innate immune response to T. cruzi infection is mediated
20
is scratched into the wound or transferred to a mucous membrane by NK cells, DCs, and macrophages. Macrophages and DCs
(e.g., the eyes). The trypomastigotes can infect almost any cell exposed to T. cruzi trypomastigote antigens produce IL-12 and
type and replicate as amastigotes in the cytoplasm. Eventually, TNF, through a MyD88-dependent mechanism. MyD88-deficient
the amastigotes transform back into trypomastigotes and rupture mice had impaired inflammatory responses and host defense
the cell to enter the bloodstream, from where they invade other against T. cruzi. Immune activation through another protein
cells or are picked up by another insect vector. involved in TLR2 signaling, the Toll/IL1R domain-containing
Following primary infection, the parasites replicate locally adapter protein– inducing IFN-β (TRIF), promotes resistance
and then disseminate through the bloodstream to a variety of through production of IFN-β and downstream expression of
tissues. Muscle and glial cells are the most frequently infected IFN-β inducible genes, such as the p47 guanosine triphosphatases
cells, and acute myocarditis or meningoencephalitis can develop. (GTPase) IRG47. IL-12 activates NK cells to secrete IFN-γ, which
In most cases, however, primary infection occurs without clinical synergizes with TNF to activate macrophages to control parasite
symptoms, and the infected individual may enter an indeterminate replication. The generation of NO is the primary trypanocidal
phase of asymptomatic seropositivity. Only 10–30% of chronically mechanism in murine macrophages. A number of trypomastigote
infected individuals will ultimately develop symptomatic Chagas antigens, including free glycosyl-phosphatidylinositol (GPI)
disease, usually involving the heart or the gastrointestinal (GI) anchors, glycoinositol phospholipids (GIPLs), GPI-linked gly-
tract. Pathologically, there are few parasites observed in cardiac coproteins, and GPI-mucins activate the innate immune response,