1 - innate immunity...1 innate immunity lora bankova, nora barrett 1 section a asic sciences...

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Innate ImmunityLora Bankova, Nora Barrett

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SECTION A Basic Sciences Underlying Allergy and Immunology

INTRODUCTIONThe innate immune system has a long evolutionary heritage, with ele-ments shared by most vertebrates, plants, and insects. This group of conserved receptors, barrier and immune cells, and antimicrobial molecules are required to provide immediate protection from potential pathogens. An effective innate immune response must include immune recognition of pathogens by discriminating “self” from “nonself,” rapid induction of effector mechanisms for pathogen containment and clear-ance, stimulation of long-term adaptive immunity so that subsequent exposures are handled more efficiently, and regulation of the response to prevent damage to the host.

Innate immune responses are initiated by recognition of molecular components of microorganisms that are foreign to the host, so-called

pathogen-associated molecular patterns (PAMPs). They can also be acti-vated by host molecules that are released by damaged cells or produced by host cells during an inflammatory response, so-called damage-associated molecular patterns (DAMPs). The sensing receptors activated by PAMPs and DAMPs are termed pattern recognition receptors (PRRs). These receptors are germline encoded and therefore present at birth; they are not generated, tailored, or expanded by clonal selection in response to antigen, as are the recognition receptors of T and B lym-phocytes in acquired immunity.

Because innate immunity is “inborn,” it plays a critical role early in the life of an organism when the adaptive repertoire has not yet been shaped and continues to provide immediate protection throughout the life of an organism, bridging the gap to adaptive immune responses, which require days to amplify and become effective. In addition to its sentinel detection and first-responder roles, the innate immune system activates and instructs adaptive immunity, regulates inflammation, and maintains homeostasis to allow the organism to develop, grow, and thrive in its environment. Unfortunately, allergic sensitization, inflam-mation, and disease may originate in aberrant innate immune develop-ment. The innate roots of allergy are considered in this chapter.

MICROBIAL PATTERN RECOGNITION BY THE INNATE IMMUNE SYSTEMThe germline-encoded PRRs (Table 1.1) of the innate immune system have genetically predetermined specificities for microbial constituents. Natural selection has formed and refined the repertoire of these receptors to recognize microbe-specific PAMPs. Although different PAMP structures are biochemically distinct from each other, they share common features:• PAMPsareproducedonlybymicrobes,notbytheirhosts.• PAMPsarecommonmolecularstructures,typicallysharedbyentire

classes of pathogens.• PAMPsareusuallyfundamentaltotheintegrity,survival,andpatho-

genicity of the microbe.

C O N T E N T SIntroduction, 1Microbial Pattern Recognition by the Innate Immune System, 1Resident Cellular Responses of Innate Immunity, 6Infiltrative Cellular Responses of Innate Immunity, 8

Innate Instruction of Adaptive Immune Responses, 10Homeostasis in the Innate Immune System, 10Innate Immunity and Allergy, 12Summary, 15

• The innate immune system is composed of receptors, cells, and antimicrobial molecules that provide rapid protection from potential pathogens before adaptive immunity is established.

• Pathogens are identified by a limited number of key conserved molecular components that are not made by the host. These pathogen-associated molecular patterns (PAMPs) are recognized by germline-encoded pattern recognition receptors (PRRs).

• In addition to its sentinel function, the innate immune system activates and instructs the adaptive immune system for antigen-specific T and B lymphocyte responses and the development of immunologic memory.

• Innate immune defenses are highly efficient and include homeostatic mecha-nisms that downregulate inflammation to optimize the health of the host.

• Like antimicrobial immunity, allergen recognition and uptake and allergic sensitization, inflammation, and disease originate in the innate immune system.

SUMMARY OF IMPORTANT CONCEPTS

SECTION A Basic Sciences Underlying Allergy and Immunology2

Pattern Recognition Receptors

Pathogen-Associated Molecular Pattern Structures Recognized Functions

SecretedAntimicrobial peptides α- and β-defensins Cathelicidin (LL-37) Dermcidin RegIIIγ

Microbial membranes (negatively charged)

Opsonization, microbial cell lysis, immune cell chemoattractant

Collectins Mannose-binding lectin Microbial mannan Opsonization, complement activation, microbial cell lysis, chemoattraction,

phagocytosis

Surfactant proteins A and D Bacterial cell wall lipids; viral coat proteins

Opsonization, killing, phagocytosis, proinflammatory and antiinflammatory mediator release

Pentraxins C-reactive protein Bacterial phospholipids

(phosphorylcholine)Opsonization, complement activation, microbial cell lysis, chemoattraction,

phagocytosis

Secreted and Membrane BoundCD14 Endotoxin TLR4 signaling

LPS binding protein Endotoxin TLR4 signaling

MD-2 Endotoxin TLR4 coreceptor

Membrane BoundToll-like receptorsa Microbial PAMPs Immune cell activation

C-type lectin receptors

Mannose receptor (CD206) Microbial mannan Cell activation, phagocytosis, proinflammatory mediator release

DECTIN-1 β-1,3-Glucan Cell activation, phagocytosis, proinflammatory mediator release

DECTIN-2 Fungal mannose Cell activation, phagocytosis, proinflammatory mediator release

DC-SIGN Microbial mannose, fucose Immunoregulation, IL-10 production

Siglecs Sialic acid containing glycans Cell inhibition, endocytosis

CytosolicNOD-like Receptors NOD-1 Peptidoglycans from gram-negative

bacteriaCell activation

NOD-2 Bacterial muramyl dipeptides Cell activation

NLRP1 Anthrax lethal toxin PAMP recognition in inflammasome

NLRP3 (cryopyrin) Microbial RNA PAMP recognition in inflammasome

NLRC4 Bacterial flagellin PAMP recognition in inflammasome

RIG-I and MDA5 Viral double-stranded RNA Type 1 IFN responses

Cyclic GMP-AMP synthase (cGAS) Cytosolic double-stranded DNA Type 1 IFN responses

TABLE 1.1 Innate Pattern Recognition Receptors in Humans

DC-SIGN, Dendritic cell–specific intracellular adhesion molecule 3 (ICAM-3)–grabbing nonintegrin; DECTIN, dendritic cell–specific receptor; IFN, interferon; IL, interleukin; LPS, lipopolysaccharide; MD-2, myeloid differentiation factor 2 (also called lymphocyte antigen 96 [LY98]); MDA5, melanoma differentiation–associated 5 (also called interferon induced with helicase domain 1 [IFIH1]); NLR, NOD-like receptor; NOD, nucleotide-binding oligomerization domain protein; PAMP, pathogen-associated molecular pattern; RegIIIγ, regenerating islet-derived 3 γ (REG3G); RIG-I, retinoic acid–inducible 1 (also called DDX58); Siglecs, sialic acid–binding immunoglobulin-like lectins; TLR, toll-like receptor.aSee Table 1.2.

For example, bacterial endotoxin is a lipopolysaccharide (LPS) PAMP that makes up most of the outer membrane layer of all gram-negative bacteria. Lipid A, a highly conserved component of LPS that confers much of endotoxin’s biologic activities, is the recognized target of toll-like receptor 4 (TLR4) (Fig. 1.1).1 Other PAMPs include common microbial cell membrane components and nucleic acids with molecular features distinct from those of animals or humans.

This approach to microbial recognition by PRRs in innate immunity is fundamentally different from the development of microbial recogni-tion in the adaptive immune system by T and B lymphocytes. Each T and B lymphocyte acquires a structurally unique receptor during somatic recombination, generating a very diverse and almost limitless repertoire of antigen specificities (approximately 1014 different immunoglobulin receptors and 1018 different T cell receptors), from which the useful

CHAPTER 1 Innate Immunity 3

LBP

CD14 TLR4

TIRAP

MYD88 TRIF

TRAM

LPS

MD-2

MYD88-dependentpathway

Proinflammatorycytokines

TRIF-dependentpathway

Type 1interferons

Fig. 1.1 Endotoxin recognition and cell activation through toll-like recep-tor 4 (TLR4). Lipopolysaccharide (LPS) (i.e., endotoxin) from the outer cell wall of gram-negative bacteria is a prototypical microbial pathogen-associated molecular pattern (PAMP) that is bound by soluble LPS-binding protein (LBP) and CD14 and transferred to myeloid differentiation factor 2 (MD-2). MD-2 specifically binds LPS and forms signal-transducing multimers with TLR4. Four signal-transducing adaptor proteins are recruited to the LPS–MD-2–TLR multimers: MYD88 and TIRAP of the MYD88-dependent pathway and TRIF and TRAM of the TRIF-dependent pathway. The MYD88-dependent pathway induces the expression of inflammatory cytokines (e.g., TNF-α, IL-1, IL-6, IL-8) and costimulatory molecules (e.g., CD80). The TRIF-dependent pathway mediates the induction of type 1 interferons and interferon-inducible genes. IL, Inter-leukin; MD-2, myeloid differentiation factor 2 (also called lymphocyte antigen 96 [LY98]); MYD88, myeloid differentiation primary response gene 88; TIRAP, toll–interleukin-1 receptor (TIR) domain-containing adaptor protein; TNF, tumor necrosis factor; TRAM, TRIF-related adaptor mol-ecule; TRIF, TIR domain–containing adaptor protein inducing interferon β (also called toll-like receptor adaptor protein 1 [TICAM1]). (Adapted from Lu YC, Yeh WC, Ohashi PS. LPS/TLR4 signal transduction pathway. Cytokine 2008;42:145-51.)

receptors (e.g., those specific for microbial pathogens rather than self) are selected for clonal expansion. This process allows for greater diver-sification, specificity, and affinity of antibodies over the life of an organ-ism but cannot be passed on to progeny. By contrast, the innate immune system relies on evolutionarily conserved PRRs, which are not indi-vidualized for each host but which are passed on to progeny.2

Pattern Recognition ReceptorsPRRs of the innate immune system can be divided into two groups: secreted receptors and transmembrane signal-transducing receptors (Table 1.1). Secreted PRRs typically have multiple effects in innate immu-nity and host defense, including direct microbial killing, serving as helper proteins for transmembrane receptors, opsonization for phago-cytosis, and chemoattraction of innate and adaptive immune effector cells. Transmembrane PRRs such as TLRs are expressed on many innate immune cell types, including macrophages, dendritic cells (DCs),

monocytes, and B lymphocytes—the professional antigen-presenting cells (Fig. 1.2). Innate immune efficiency is achieved in part by the constitutive expression of some of these sentinel receptors and the rapid upregulation of others with innate immune activation. Notably, although most of the PRRs reviewed here are well-characterized, recent studies have demonstrated important antimicrobial functions of yet additional receptor classes, such as taste receptors,3 highlighting the emerging nature of this field and the additional work on PRRs to be done.

Antimicrobial PeptidesAntimicrobial peptides (AMPs) are highly diverse small cationic pep-tides with broad antimicrobial activity that are secreted by activated innate immune cells. A fundamental shared feature of all AMPs is their amphipathic structure: clustering hydrophobic and cationic amino acids in discrete regions of the molecule (Fig. 1.3). This structure allows AMPs to interact with negatively charged phospholipids in microbial cell membranes, integrate into the membranes, and disrupt them.4 Impor-tantly, while AMPs have antimicrobial activity against a broad range of bacteria, fungi, and enveloped viruses, they do not interact with the cell membranes of plants and animals, which lack polar phospholipids.4

AMPs are produced by hematopoietic cells and epithelial cells in the airway, skin, intestine, and urinary tract.5,6 In humans, the two main categories of AMPs are defensins (α and β classes) and the cathelicidin LL-37 (Table 1.1). There are six human α-defensins (HD1-6) and four well-characterized human β-defensins (HBD1-4), with computational genetic approaches predicting many more.4 Their production can be constitutive (e.g., HBD1) or inducible (e.g., HBD2, HBD3, HBD4) through multiple innate signaling pathways. For example, HBD2 expres-sion can be induced by bacterial PAMPs signaling through TLR2 or TLR4 or by innate inflammatory cytokines, including interleukin-1β (IL-1β) and tumor necrosis factor-α (TNF-α).4–6

Macrophages

Dendritic cells

C-reactive protein Mast cells

Neutrophils

Antimicrobial peptidesCollectins

Toll-likereceptors C-type lectin

receptors

Epithelialcells

NOD-likereceptors

Innate lymphoid type 2 cell

Fig. 1.2 Main categories of pattern recognition receptors and the innate immune cell types that express them. NOD, Nucleotide-binding oligomer-ization domain protein. (Adapted from Liu AH. Innate microbial sensors and their relevance to allergy. J Allergy Clin Immunol 2008;122:846-58.)


To summarize, when AMPs are induced at a site of injury, they act directly to destroy microbial invaders and to attract an array of defensive cells that provide backup support to defend the breached barrier. AMPs probably control commensal relationships to maintain health in the gut and maybe elsewhere. Inflammation results when the AMP-based defenses have proved inadequate and robust secondary defensive responses are mobilized.

CollectinsCollectins are secreted C-type lectin receptors (CLRs) that are structur-ally similar to the transmembrane CLRs (discussed later) and contain a collagenous domain. Mannose-binding lectin (MBL) is an acute-phase reactant that recognizes terminal mannose residues of carbohydrates on gram-positive and gram-negative bacteria, fungi, yeast, and some viruses and parasites.8 MBL is structurally similar to the complement component C1q, and, like C1q, it activates the classic complement cascade through MBL-associated serine proteases that are related to C1r and C1s and cleave C4, C2, and C3, leading to amplified opsonization, membrane pore formation, cell lysis, and neutrophil chemoattraction.9

Two of the four pulmonary surfactant proteins, SP-A and SP-D, are collectins with similar structures and multiple innate immune functions. They share carbohydrate-binding domains that bind oligosaccharides specific for a variety of microbes (e.g., gram-positive and gram-negative bacteria, viruses, fungi). They recognize a wide variety of PAMPs, such as bacterial LPS, mycobacterial lipoarabinomannan, other bacterial glycolipids, and common viral glycoproteins, such as influenza hemag-glutinin and neuraminidase envelope glycoproteins and respiratory

Human cathelicidin LL-37 is released from neutrophils, mast cells, epithelial cells, and keratinocytes, and it exhibits a broad range of anti-microbial activities. LL-37 is induced by vitamin D; the gene encoding LL-37 has a vitamin D receptor binding site. In keratinocytes and mac-rophages, stimulation of TLR2 results in the induction of CYP27B1, the cytochrome P-450 enzyme that converts 25-hydroxyvitamin D3 (25-OH-D) to the active form of 1,25-dihydroxyvitamin D3 (1,25-OH-D), which induces LL-37 expression. By this route, vitamin D can influ-ence microbicidal defenses of the skin and circulating phagocytic cells. It has been proposed that certain human infections, such as by Myco-bacterium tuberculosis, might be more prevalent among populations with inadequate plasma levels of vitamin D.5,7

Other types of AMPs include dermcidin in sweat and the lectin protein RegIIIγ (REG3G) in the intestine, which serves to both bind pathogens and alter the distribution of luminal mucus, segregating bacteria away from the epithelial cell surface.5

In addition to their direct bactericidal activities, AMPs also contribute to host defense through the control of cytokine/chemokine production, cell migration, and maintenance of skin barrier function. Both human α- and β-defensins act as chemoattractants for immature DCs and peripheral blood T cells, thereby enhancing antigen-specific adaptive immune responses. LL-37 attracts neutrophils, monocytes, mast cells, and T lymphocytes through formyl peptide-like receptor 1 (FPRL1), a PRR that also binds bacterial formyl peptides.5 Importantly, failure to upregulate AMP production has been linked to the increased Staphy-lococcus aureus colonization and susceptibility to viral skin infections in patients with atopic dermatitis.5

Opsonization

Chemoattraction

Cell lysis

Outside

Inside

T lymphocyte Monocyte+ +Outer leaflet

Weak

Antimicrobialpeptide

Strong

Inner leafletBacterial cytoplasmic

membranePrototypic plasma membrane of a

multicellular animal

Hydrophobic interactions ++

+ +

Electrostatic andhydrophobicinteractions

Dendritic cellNeutrophil

Mast cell

– –

– –

– – –

– –

–

Fig. 1.3 Mechanism of antimicrobial peptide-mediated host defense. Antimicrobial peptides (AMPs) are amphipathic, containing a discrete cationic region of the molecule. They target the exposed outer membrane of bacteria that is dense with negatively charged phospholipid head groups. This is different from the cell membranes of plants and animals that are spared AMP binding, because their outer cell membrane lipids have no net charge. AMPs integrate into bacterial membranes and form holes that physically disrupt mem-brane integrity and lyse target cells. AMPs are chemoattractive for a variety of immune cells, while also carpeting and opsonizing bacterial targets for recognition and uptake by phagocytes bearing AMP receptors. (Adapted from Zasloff M. Antimicrobial peptides of multicellular organisms. Nature 2002;415:389-95.)


protein (LBP) and CD14 are soluble proteins that capture and transfer LPS to the MD-2/TLR4 complex. MD-2 specifically binds LPS and forms signal-transducing multimers with TLR4.1,13 Although LBP and CD14 are not classic PRRs in that their binding specificity is not limited to PAMPs, they improve cellular detection of and sensitivity to endo-toxin.14 Conversely, repeated, prolonged, or high-level endotoxin exposure induces cellular unresponsiveness or tolerance.15

Ligand-induced oligomerization of TLR4 induces the recruitment of four intracellular signal-transducing adaptor proteins through their toll/IL-1 receptor (TIR) domains: MYD88 and TIR domain-containing adaptor protein (TIRAP) of the MYD88-dependent pathway and TIR domain–containing adaptor protein–inducing interferon-β (TRIF) and TRIF-related adaptor molecule (TRAM) of the MYD88-independent or TRIF-dependent pathway.16 Different TLRs use different combina-tions of adaptor proteins for downstream signaling; TLR4 is the only known TLR that uses all four of these adaptor proteins. The MyD88-dependent pathway induces the expression of costimulatory molecules (e.g., CD80) and inflammatory cytokines (e.g., TNF-α, IL-1, IL-6, IL-8) through a series of signal-transducing intermediates that lead to the nuclear translocation of transcription factors NF-κB and activator protein 1 (AP-1). The TRIF-dependent pathway mediates the induction of type 1 interferons and interferon-inducible genes through activation of the transcription factor interferon regulatory factor 3 (IRF3).

Ten human TLRs have been identified (Table 1.2). They collectively recognize a diverse range of microbial cell wall components, proteins, and nucleic acids, the classic PAMPs.17 Unlike gram-negative bacteria, the cell walls or membranes of other bacteria (e.g., gram-positive bac-teria, mycoplasma) do not contain endotoxin, but they contain pepti-doglycan and lipoproteins that are recognized by TLR2, TLR1, TLR6, and possibly TLR10. TLR5 recognizes bacterial flagellin. The cytosine-phosphate-guanine (CpG) sequences of bacterial and viral DNA are unmethylated, distinguishing microbial DNA from mammalian DNA; microbial unmethylated CpG is recognized by TLR9. TLR7 and TLR8 are closely related to TLR9 and recognize virus-derived, single-stranded RNA. Double-stranded RNA, unique to certain viruses, is recognized by TLR3. TLR2 and TLR4 also bind members of the family of alarmins, proteins that are passively released from necrotic cells during infection or tissue injury, thereby eliciting inflammation.18

syncytial virus (RSV) G (attachment) and F (fusion) proteins.9 SP-A and SP-D mediate multiple antimicrobial functions. They aggregate and opsonize microbes for phagocytosis by alveolar macrophages, monocytes, neutrophils, and DCs. They also trigger nuclear factor-κB (NF-κB) activation and cytokine production through TLR4 and TLR2. SP-A induces the expression of scavenger and mannose receptors on phagocytes, thereby improving phagocytosis. SP-A and SP-D have direct bactericidal and fungicidal properties, and they help to dampen inflam-matory responses by enhancing the clearance of proinflammatory apoptotic cells by macrophages.10

PentraxinsPentraxins are acute-phase reactant PRRs that are secreted in response to TLR activation or proinflammatory cytokines.11 C-reactive protein (CRP) was the first PRR and the first pentraxin to be described. CRP specifically binds bacterial phospholipids (e.g., phosphorylcholine) and the complement factor C1q, thereby opsonizing bacteria and activating the classic complement cascade. CRP also directly binds Fcγ receptors on phagocytes, further promoting phagocytosis.

Toll-Like Receptors (Table 1.2)The immediate cellular responders of the innate immune system (e.g., epithelial cells, monocytes, macrophages, DCs, mast cells, neutrophils) and other cell types express a family of transmembrane PRRs with functional roots found in the toll receptor of Drosophila. These toll-like receptors (TLRs) are structurally similar, with large, leucine-rich extra-cellular domains and cytoplasmic domains that are similar to those of the mammalian IL-1 receptor (Table 1.2).12 The IL-1 receptor and TLRs share a signaling pathway that leads to NF-κB activation through the adaptor protein myeloid differentiation primary response protein 88 (MYD88), described further below.

TLR4 was the first human TLR identified, and it is specific for bacte-rial endotoxin. Endotoxin, a prototypical PAMP, is a gram-negative bacterial cell wall LPS with a highly conserved lipid A moiety. Very small amounts of endotoxin (i.e., picogram amounts, estimated to equal about 10 LPS molecules/cell) are immunostimulatory. This very high sensitivity for endotoxin-mediated cell activation can be attributed to the endotoxin receptor complex (Fig. 1.1). Lipopolysaccharide binding

Toll-like Receptors Cell Location Ligands Microbial Sources

TLR1 Surface Lipoproteins, lipoteichoic acid Gram-positive bacteria, mycoplasma

TLR2 Surface Lipoproteins, alarminsPeptidoglycan, lipoteichoic acidZymosanLipoarabinomannan

Bacterial cell walls and membranesGram-positive bacteria cell wallsFungi and mycobacteria cell walls

TLR3 Cytosol Double-stranded RNA Viral RNA

TLR4 Surface Endotoxin, alarmins, viral coat proteins Gram-negative bacteria cell wallsRespiratory syncytial virus

TLR5 Surface Flagellin Bacteria

TLR6 Surface Lipoproteins, lipoteichoic acid Gram-positive bacteria cell walls and membranes

TLR7 Cytosol Single-stranded RNA Viral RNA

TLR8 Cytosol Single-stranded RNA Viral RNA

TLR9 Cytosol Unmethylated CpG DNA Bacterial and viral DNA

TLR10 Surface Lipoproteins Bacterial cell walls and membranes

TABLE 1.2 Toll-Like Receptors in Humans

CpG, Cytosine-phosphate-guanine oligonucleotide.


toxin (NLRP1), bacterial flagellin (NLRC4), bacterial and viral RNA (NLRP3), and bacterial pore-forming toxins such as nigericin and mai-totoxin (NLRP3). NLRP3 is likely the most important among them, because it is activated in response to a broad array of diverse stimuli, perhaps through a common mediator of cellular stress.26 On activation, NLRP3 associates with several other proteins to form the inflammasome and activate the caspase-1-dependent cleavage of proIL-1β and proIL-18 to their mature active forms. Notably, caspase-1 also cleaves gasdermin D, leading to cell death after activation of the inflammasome.26

Additional Cytosolic Nucleic Acid ReceptorsThe RNA helicases retinoic acid–inducible protein 1 (RIG-I), melanoma differentiation–associated 5 (MDA5, now called interferon induced with helicase domain 1 [IFIH1]), and RIG-I-like receptor LGP227 are another class of cytosolic PRRs that recognize double-stranded RNA viruses and mediate type 1 interferon antiviral responses.28 Cytosolic double-stranded DNA also triggers the generation of type 1 interferons, and many DNA sensors have been proposed.29 GMP-AMP synthase (cGAS) is a double-stranded DNA sensor that is activated to generate a cyclic dinucleotide second messenger and, through a variety of signaling intermediates, cGAS activates the TBK1-IRF3-dependent production of interferons.30 The roles of other sensors and additional downstream signaling pathways are still emerging.

RESIDENT CELLULAR RESPONSES OF INNATE IMMUNITYTissue-resident innate immune cells serve as critical first responders in host defense. This includes epithelial cells, DCs, macrophages, mast cells, and innate lymphoid cells (ILCs) (Fig. 1.4).

Epithelial cells provide a first line of host defense by maintaining a barrier function, trapping and killing potential pathogens, and activat-ing additional innate immune cells. Their physical barrier to the external environment is achieved through a network of junctional complexes including tight junctions and underlying junction adherens.31 An addi-tional layer of defense is provided by the mucociliary apparatus. Cell-associated and secreted mucins trap pathogens in the conducting airways and act in concert with antimicrobial peptides and the ciliary apparatus to clear pathogens. In addition to its barrier role, the homeostatic mucin MUC5B regulates alveolar macrophage function, indicating an unex-pected role for mucins in regulating innate immune responses.32 Epithelial cells secrete many of the AMPs reviewed previously and orchestrate innate immunity through inflammatory cytokine generation elicited by PRR signaling. Perhaps the best characterized are TLR4 and TLR2, which are expressed in epithelial cells from the lung, skin, and gut and which mediate NF-κB-dependent production of proinflammatory cyto-kines in response to both pathogens and commensal organisms.33,34

DCs are key sentinels of the innate immune system that link innate and adaptive immunity through their unique capacity to potently acti-vate naïve T cells. DCs can be subdivided into classic myeloid (mDC) and plasmacytoid (pDC) types, which are thought to originate from a common DC precursor in the bone marrow.35 The mDCs are recruited from the blood to histologic sites with high levels of antigen exposure (e.g., skin, mucosal surfaces, lymph nodes, spleen). With their long dendrites and their PRR-rich cell surfaces, mDCs form a subepithelial web that is sensitive to microbes, inflammation, and cellular stress. In the airways and intestine, antigens are immediately captured by mucosal mDCs that extend dendrites into the lumen for antigen sam-pling. After activation, mDCs quickly alert and instruct the immune system by secreting proinflammatory cytokines such as interferons and interleukin-12 and migrating to draining lymph nodes for T lymphocyte instruction.

C-Type Lectin ReceptorsTransmembrane C-type lectin receptors (CLRs) are defined by their lectin structure, although they bind additional moieties beyond glycans. They are expressed widely on innate immune cells and epithelial cells. They function as sensors for diverse microbes, but several play a par-ticularly important role in fungal recognition and immunity, including the mannose receptor, dendritic cell–specific ICAM-3–grabbing non-integrin (DC-SIGN), Mincle, Dectin-1, and Dectin-2.19 CLR activation elicits several antimicrobial functions. Some receptors such as the mannose receptor (CD206), DEC-205, and DC-SIGN function as endo-cytic receptors, facilitating DC and macrophage antigen uptake, recycling, and presentation. Many elicit the activation of NF-κB with attendant proinflammatory cytokine production. Importantly, several receptors including Dectin-1, Dectin-2, and Mincle elicit immunoreceptor tyrosine-based activating motif (ITAM)–based signaling to activate spleen tyrosine kinase, elicit a calcium flux, and trigger NFAT activation. This signaling pathway is not shared by TLRs and is critical for generation of IL-6, IL-10, and IL-23; shaping subsequent adaptive immunity with skewing of naïve T cells to Th17; and generation of proinflammatory lipid media-tors, the cysteinyl leukotrienes.20,21 CLR activation also plays a role in activating the inflammasome for the production of mature IL-1β.19 The structure of the inflammasome is reviewed below (see Nucleotide-Binding Oligomerization Domain–Like Receptors).

Sialic Acid–Binding Immunoglobulin-Like LectinsSialic acid–binding immunoglobulin-like lectins (Siglecs) are a family of receptors that bind sialic acid–containing glycans expressed on cell surfaces. Siglecs promote cell-cell interactions, regulate cell functions, and mediate microbial endocytosis.22

Different Siglecs are expressed by different immune cell types. For example, Siglec-1 (CD169 or sialoadhesin) is macrophage specific, Siglec-2 (CD22) is B lymphocyte specific, and the CD33-related Siglecs are specific for innate immune cells and resident macrophages, includ-ing microglia. Eosinophils, basophils, and mast cells express Siglec-8, whereas monocytes, dendritic cells, natural killer cells, and neutrophils express Siglec-9.22 Siglecs typically are inhibitory receptors. Some (e.g., sialoadhesin, CD33-related Siglecs) recognize sialic acid–expressing microbes, mediate their endocytosis, and dampen inflammatory and immune responses to these pathogens. Specific Siglecs have been impli-cated for their role in limiting tissue damage from activated granulocytes. Activation of Siglec-8 and Siglec-9 has differential effects on granulocytes leading to apoptosis of cytokine-primed eosinophils and neutrophils and inhibition of FcεR1-mediated activation of mast cells.23

Nucleotide-Binding Oligomerization Domain–Like ReceptorsNucleotide-binding oligomerization domain (NOD)–like receptors (NLRs) are cytosolic PRRs that are structurally similar and recognize microbial PAMPs that find their way into the cytoplasm. The human NLR family has 23 members that can be conceptually organized in two groups. The best characterized, NOD1 and NOD2, recognize different core motifs of bacterial peptidoglycans. NOD1 is specific for a core motif of peptidoglycans from primarily gram-negative bacteria. NOD2 detects the peptidoglycan muramyl dipeptide, present in all gram-positive and gram-negative bacteria.24 NOD2 has been of particular interest, because mutations in the human NOD2 gene are associated with an increased risk of Crohn disease.25

A different set of NLRs (including NLRP1, NLRP3, and NLRC4) are the sensing portion for the cytosolic protein complex termed the inflammasome. These NLRs recognize a diverse range of microbial PAMPs that find their way into cellular cytoplasm, including anthrax lethal


Macrophages share many features with DCs, including their enrich-ment in tissue areas with high environmental antigen exposure, cell surface expression of PRRs, capacity to phagocytose and digest organisms, and presentation of antigens to lymphocytes. However, macrophages have a limited capacity to migrate to regional lymph nodes and cannot stimulate naïve T cell proliferation as strongly as DCs.41,42 By contrast, macrophage phagolysosomes reach a lower pH than that of DCs, endow-ing them with increased killing capacity.43 Recent work in mouse and human cells has underscored the considerable heterogeneity of tissue-resident macrophages, likely reflecting their development from local tissue progenitors seeded in embryonic life from the fetal yolk sac.44 In addition to their phagocytic and killing capacity, macrophages secrete more than 100 proteins that mediate host defense and inflammation, and macrophages play important roles in removal of dead tissue and apoptotic cells, metabolism, tissue development, wound healing, and homeostasis (reviewed in reference 45) (see Homeostasis in the Innate Immune System).

Mast cells are evolutionarily ancient immune cells and the only granulocyte that resides primarily in peripheral tissues. With granules containing preformed mediators including proteases (tryptase, chymase), heparin, histamine, platelet activating factor, AMPs (cathelicidin), defen-sins, and some cytokines (TNF-α), they are poised to rapidly neutralize microbial invaders. While the best-characterized pathway for mast cell activation is the IgE-dependent crosslinking of FcεRI, highlighting its effector function in amplifying adaptive immunity, mast cells are also activated through a wide variety of classical PRRs, including TLR1,

The functions of mDCs are developmentally related.36 They migrate from the bone marrow to peripheral tissues in an immature form, at which stage their role is primarily sentinel detection. They readily sense, sample, and process incoming antigen through dense PRR expression (i.e., TLR1 through TLR6), but they have a poor ability to stimulate T lymphocytes. After sensing environmental microbial PAMPs or inflam-matory stress, mDCs become activated scavengers of antigen, and they subsequently return to draining lymph nodes. They mature during this migration. As mature mDCs, their antigen uptake and processing func-tions are shut down, and large amounts of processed antigen are displayed in cell surface major histocompatibility complex (MHC) molecules with a battery of costimulatory factors for T lymphocyte education. The central role of DCs in directing T lymphocyte development in health (see Innate Instruction of Adaptive Immune Responses) and in allergic and asthmatic disease (see Innate Immunity and Allergy) is addressed later in this chapter. The mDCs also can be superior stimula-tors of natural killer (NK) and natural killer T (NKT) cells by virtue of their robust IL-12 production.36

Compared with mDCs, pDCs are sentinel antiviral responders, expressing TLR7 and TLR9 for recognizing viral infections37 and releas-ing large amounts of interferon α (IFN-α) to limit viral replication.38 They can also act as antigen-presenting cells and control T lymphocyte responses.39 Langerhans cells, although similar to DCs in their function, seem to originate from an embryonic precursor that populates the epidermis before birth, differentiates and self-renews in situ, and pro-liferates during inflammation.40

In�ltrative• Neutrophils• Monocytes• Dendritic cells• NK cells• Eosinophils• Basophils• NKT , MAIT

Innate immunity

IFNsChemoattraction

CytokinesILs

Antigen presentation instruction

Homeostatic• Macrophages• Monocytes• Dendritic cells• Epithelial cells• Regulatory lymphocytes

Immediate(resident)• AMPs• Phagocytes:– Macrophages– Dendritic cells• Epithelial cells• ILC2s• Mast cells

Microbes Acquired (adaptive)• T lymphocytes• B lymphocytes– antibody

Fig. 1.4 Innate immune responses to microbes can be broadly characterized as antimicrobial or homeostatic. Antimicrobial responses begin with protective layers of antimicrobial peptides and detection by immune cells residing at the epithelial interface. Often, these immediate responses sufficiently protect the host. If this first layer of host defense is inadequate, the frontline responders attract infiltrative innate immune cells that are activated as they approach the source of inflammation. Immediate and infiltrating immune cells stimulate adaptive immune responses and educate lymphocytes through antigen presentation and costimulation. Homeostatic responses by innate immune cells downregulate inflammatory and antimicrobial immune responses when they are no longer needed to optimize the use of resources and well-being of the host. AMP, Antimi-crobial peptide; IFN, interferon; IL, interleukin; NK, natural killer.


inflammatory without the extraordinarily efficient uptake and processing of apoptotic neutrophils by macrophages and DCs to prevent release of toxic constituents, a process called efferocytosis.58

In response to tissue infection, circulating neutrophils adhere to adjacent vascular endothelium, extravasate across it, and migrate to the site. Neutrophils have a number of receptors for diverse chemoattrac-tants, including bacterially derived N-formyl oligopeptides and host-derived C5a, IL-8, and leukotriene B4, secreted by activated innate immune cells. These neutrophil chemoattractants diffuse from the site of infection to provide a chemotactic gradient for neutrophil migration and further neutrophil activation.59,60

Neutrophils have several modes of killing. On reaching the infected site, they can phagocytose invading microorganisms that are opsonized by complement C3 fragments (e.g., C3b, iC3b) and immunoglobu-lin G (IgG).61 After phagocytosis, ingested microbes are killed almost immediately through several mechanisms. Microbicidal products such as α-defensins (HD 1-4) are released into the phagosome from intracellular granules. Additionally, highly reactive oxidizing agents (e.g., O2−, H2O2, hypochlorous acid) are generated by myeloperoxidase and membrane NADPH oxidase,62 which has an essential role in killing and preventing infection with certain common organisms (e.g., Staphylococcus aureus, Serratia, enteric bacteria, Aspergillus).63 An increased role for neutrophil granule proteases (i.e., neutrophil elastase and cathepsin G) has been recognized. These cationic proteases are released and activated with alkalinization and K+ ion fluxes into phagocytic vacuoles. These pH and potassium requirements for protease solubilization and activity restrict their toxicity to phagocytic vacuoles and limit damage to host tissues.62 Finally, neutrophils can form extracellular traps (NETs) in response to gram-positive and gram-negative bacteria and upon stimulation with LPS or interleukin-8. NETs consist of extruded DNA of either nuclear or mitochondrial origin, histones, neutrophil granule proteins, and antimicrobial peptides.64,65 They have direct bactericidal activity and trap bacteria in the extracellular space, preventing their spread.66

NK cells are an innate immune cell type with unique features. Similar to ILCs, they are lymphoid cells that are not activated through antigen-specific receptors such as the T cell receptor or surface immunoglobulin. Although NK cells express PRRs such as TLR2, TLR3, TLR4, TLR5, TLR7, and TLR8 and recognize and respond to the respective TLR ligands directly,67,68 they are best known for responding in an antigen-independent manner to help contain viral infections (especially herpes-virus infections) and malignant tumors by recognizing aberrant host cells for elimination. NK cells distinguish healthy host cells through inhibitory receptors such as the killer cell immunoglobulin-like receptor (KIR) and CD94/NKG2A receptors that recognize MHC class I molecules expressed on healthy cells (Fig. 1.5).69,70 Binding of these receptors inhibits NK cell–mediated lysis and cytokine secretion. Virus-infected and malignant cells often downregulate MHC class I molecules, rendering them susceptible to attack by NK cells.71 These inhibitory receptors on NK cells are counterbalanced by activating receptors, such as the NKG2D receptor that recognizes stress ligands expressed on cell surfaces in response to intracellular DNA damage.72

Recruited and activated NK cells mediate antimicrobial activities by induction of apoptosis in target cells and cytokine secretion, which promotes innate immune functions and contributes to adaptive immune responses. Target cell apoptosis results from granule exocytosis and death-receptor engagement. NK cell granules contain perforins and granzymes that are released on activation into the synapse between target and effector cell, disrupting target cell membranes and inducing apoptosis (Fig. 1.5).73 NK cells also mediate apoptosis by expressing FAS ligand (FASLG) and TNF-related apoptosis-inducing ligand (TRAIL, now called tumor necrosis factor superfamily member 10 [TNFSF10]), which bind the FAS and TNFRSF10 receptors, respectively, on target cells.74

TLR2, TLR4, and TLR6; and complement receptors for C3a and C5a.46 Furthermore, mast cells can sense small molecules like substance 48/80 through the Mas-related G protein–coupled receptor MRGPRX247 and can sense some allergens in an FcεR1-independent fashion.48 On activa-tion, mast cells can rapidly degranulate, generate lipid mediators such as cysteinyl leukotrienes and prostaglandin D2, and synthesize numerous proinflammatory cytokines. They are key sources of immediate release of TNF-α and IL-8, which are uniquely preformed in mast cells, and their immediate release of TNF-α may have a central role in effective antimicrobial responses to infections.46 Additionally, mast cells play a role in intestinal parasite clearance, and their proteases play a role in reducing the systemic toxicity of some venoms.49,50

Innate lymphoid cells (ILCs) are a recently described family of lym-phoid cells derived from the common lymphoid progenitor. They lack the canonical T cell receptor and are instead activated by tissue-derived mediators in an antigen-independent manner.51 Largely following the nomenclature for T cells, ILCs have been subdivided into three subsets: ILC1, 2, and 3. Group 1 ILCs include NK cells and ILC1s; the transcrip-tion factors that regulate their terminal development are Eomes and T-bet, respectively. They are activated by IL-12, IL-15, and IL-18 secreted in response to intracellular pathogens and generate high amounts of interferon γ (IFN-γ). ILC2s, on the other hand, are regulated by Gata-3, ROR-α, Gfi1, and T cell factor 1 and respond to epithelial cytokines and products of the arachidonic acid pathway generated in the setting of cellular injury from helminths or allergens.52,53 Activation of ILC2s leads to the production of high amounts of IL-4, IL-5, and IL-13. They also play homeostatic roles in barrier repair responses through the production of amphiregulin and in regulating thermogenesis in adipose tissue.54 Lastly, ILC3s respond mainly to IL-1β and IL-23 produced by myeloid cells in response to bacterial and fungal infection and are tran-scriptionally controlled by RORγt.53

Two additional subsets of lymphoid cells sit at the interface of innate and adaptive immunity. Invariant natural killer T (iNKT) cells and mucosal-associated invariant T (MAIT) cells express invariant T cell receptors with highly restricted diversity of the T cell receptor alpha chain.55,56 They are unique because of their ability to recognize non-peptide antigens: iNKT cells respond to glycolipids, and mucosal-associated invariant T cells (MAIT cells) recognize microbial metabolites. These antigens are presented to them by nonclassical antigen-presenting molecules: CD1d for iNKT cells and MR1 for MAIT cells.55 In addition to activation through the T cell receptor, MAIT and iNKT cells can respond rapidly to cytokine stimulation, similarly to ILCs.56 Activated MAIT cells produce IFN-γ, whereas iNKT cells produce high levels of many cytokines including IFN-γ, TNF-α, IL-2, IL-3, IL-4, IL-5, IL-9, IL-10, IL-13, IL-17, IL-21, and GM-CSF. Although much less is known about MAIT cells, studies in mice suggest that both MAIT and iNKT cells can be powerful modulators of the immune system, supplying critical cytokines before the generation of adaptive immunity.

INFILTRATIVE CELLULAR RESPONSES OF INNATE IMMUNITYInfiltrative cellular responses are potent antimicrobial effectors that usually are recruited by an innate immune intermediary to induce the full weight of their response, but they can respond directly to microbial stimuli through their own surface-expressed PRRs (Fig. 1.4).

Neutrophils, the most abundant circulating phagocytes in the human host, are recruited to sites of infection and inflammation where they are activated to degranulate, phagocytose, and release neutrophil extra-cellular traps (NETs), to kill microorganisms. Circulating neutrophils are short-lived (approximately 24 hours), and about 1011 cells die each day.57 This constant stream of neutrophil death would be potently


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Fig. 1.5 Natural killer (NK) cells recognize and target infected or malignant cells in an antigen-independent manner. They distinguish healthy host cells by receptors on NK cells (e.g., KIR, NKG2A/CD94) that interact with MHC class I molecules on host cells to inhibit NK cell activation. Pathogen-infected or malignant cells typically downregulate MHC class I molecule expression while concurrently expressing stress ligands that are recognized by activating receptors on NK cells (e.g., NKG2D). These changes in the balance of activation-to-inhibition receptor engagement lead to NK cell activation and targeting for killing. NK cells induce target cell apoptosis by the release of toxic granules containing granzymes and perforins that disrupt cell membranes. The cells also express apoptosis-inducing FAS ligand (FASLG) and TRAIL that interact with their counterparts on target cells (FAS and TRAILR, respectively). Activated NK cells produce stimulatory cytokines and che-mokines and are a rich source of IFN γ that augment innate and adaptive cytotoxic T lymphocyte and type 1 helper T lymphocyte (Th1) immune responses. IFN, Interferon; KIR, killer cell immunoglobulin-like receptor; MHC, major histocompatibility complex; NKG2A, natural killer cell receptor (also called 159A); NKG2D, natural killer receptor (now called killer cell lectin-like receptor subfamily member 1 [KLRK1]); TRAIL, tumor necrosis factor–related apoptosis-inducing ligand (now called TNFSF10); TRAILR, TRAIL receptor. (Adapted from Orange JS, Ballas ZK. Natural killer cells in human health and disease. Clin Immunol 2006;118:1-10.)


Activated NK cells are known for their secretion of IFN-γ in par-ticular, but they also secrete TNF-α, growth factors, IL-5, IL-10, IL-13, and chemokines.75 DCs recruit, interact with, and activate NK cells through cytokines (e.g., type I interferons, IL-12, IL-18) and cell-to-cell surface interactions.75 NK cells can activate bystander immature DCs by producing TNF-α and IFN-γ along with cell-cell contact.76 Reciprocal NK-DC interactions occur in secondary lymphoid organs, where NK cells respond to IL-12 produced by mature DCs by producing IFN-γ and promoting the development of helper T cell type 1 (Th1) and cytotoxic T lymphocytes.77–79

Eosinophils are terminally differentiated circulating granulocytes derived from the common granulocyte-monocyte precursor (GMP). The eosinophil precursor was recently identified as Gata1+GMP distinct from the Gata1+GMP that gives rise to neutrophils, monocytes, and macrophages.80 Human eosinophil granules are rich in four cationic proteins: major basic protein 1 (MBP1; also known as MBP and PRG2), eosinophil cationic protein (ECP; also known as RNase3), eosinophil-derived neurotoxin (EDN; also known as RNase2) and eosinophil peroxidase (EPX; also known as EPO).81 In addition, mature eosino-phil granules contain preformed stores of cytokines and chemokines including IFN-γ, IL-4, IL-6, TNF, IL-10, IL-12, and IL-13.82 Notably, rather than receptor-mediated activation, the most common path-ways for eosinophil granule extrusion are cytolysis and/or piecemeal degranulation.

Eosinophils and their extruded granules increase in number in patients with atopic disease or helminth infection, likely because of increased levels of IL-5 and GM-CSF/IL-3 that expand and activate the population, respectively.83 This observation, and data demonstrating that eosinophils can kill helminths in vitro, led to speculation that eosinophils are important in antihelminth immunity. Although murine models have not born this out, they have demonstrated roles for them in regulating mucosal barrier integrity and the intestinal microbiome through regulation of secretory IgA production. Additional studies have demonstrated largely homeostatic roles for IL-4-secreting eosinophils in beige fat biogenesis and thermoregulation83 (see Homeostasis in the Innate Immune System).

Basophils are circulating granulocytes derived from a common GMP shared with mast cells. Unlike mast cells, they lose surface expression of the kit receptor as they differentiate in the bone marrow under the direction of IL-3 and are released in the circulation as mature granu-locytes.84 Basophil granules contain histamine, serine proteases including cathepsin G, granzyme B, and a basophil-specific mediator, basogranulin. Upon activation, basophils generate cysteinyl leukotrienes and high levels of IL-4 and IL-13. In murine models, basophils are an important source of serine protease-elicited IL-4,85 leading to a polarization of the immune response to type 2 immunity. Because serine proteases are shared by multiple allergens and some helminths, basophils may play a role in initiating and amplifying type 2 immunity, but a definitive role in humans has not been established.85

INNATE INSTRUCTION OF ADAPTIVE IMMUNE RESPONSESThe immediate and infiltrative responses of innate immunity set the stage for their instruction of adaptive immunity and the maintenance of immunologic memory—long-lived memory T lymphocytes and a persistent antibody response. Because the adaptive immune system has a near limitless antigen receptor repertoire, instruction is necessary to guide adaptive antimicrobial immune responses toward pathogens and not self-antigens or harmless environmental antigens. Microbial pattern recognition by innate immune cells controls the activation of adaptive immune responses by directing microbial antigens linked to TLRs through

the cellular processes leading to antigen presentation and the expression of costimulatory molecules (e.g., CD80 with CD86).

A legacy of research on the prototypical PAMP endotoxin is helpful in understanding PAMP control of adaptive immunity. Endotoxin can be used as an essential adjuvant in the induction of antigen-specific T cell memory. Although T cells mount a short-lived proliferative response to protein antigens alone, classic immunologic memory depends on immunization with an adjuvant such as endotoxin. Endotoxin potently induces IL-12 and IFN-γ secretion, which are key regulators of memory Th1-type immune development.86 LPS strongly influences innate antigen-presenting cells (especially DCs) to produce IL-12 and to costimulate naïve T lymphocytes to become effector T lymphocytes that primarily secrete IFN-γ.87–89 In a reciprocal manner, IFN-γ primes innate immune cells to produce greater amounts of IL-12 in response to stimulation, fostering a positive feedback relationship between the innate and adap-tive immune compartments for Th1-type immune development.

Among antigen-presenting cells, DCs are the most efficient educa-tors of T lymphocytes. Immature DCs are activated and recruited to epithelial surfaces, where they scavenge antigen and migrate to draining lymph nodes. During their migration, they mature and redirect their processes to MHC class II antigen presentation, and they express cyto-kines and cell surface molecules to attract antigen-specific T lymphocytes and direct their maturation and differentiation to helper T cell subsets (e.g., Th1, Th2, Th17) or regulatory T (Treg) cells (Fig. 1.6). The nature of DC instruction is affected by the dose and types of the PAMPs and PRRs involved, the duration of exposure, and the microenvironment in which the DCs are activated and located. For example, different cytokines influence TLR4-activated DCs to skew their subsequent instruc-tion of T lymphocyte differentiation as follows: TGF-β and IL-10 induce DCs to instruct Treg development; IL-12 promotes Th1 immune devel-opment; thymic stromal lymphopoietin (TSLP) and IL-33 promote Th2 immune responses; and IL-23, TGF-β, IL-6, and IL-1β induce Th17 development.90,91 These activated and differentiated T lymphocytes subsequently migrate to other lymph nodes and back to the mucosa, where they again interact with and are sustained by mature mDCs in the subepithelial periphery. This peripheral tissue-specific interaction between mature mDCs and progeny effector T lymphocytes may underlie the persistence of organ-specific immune memory.

Although immunologic memory is considered a function of long-lived T and B lymphocytes, innate immune cells also show altered responsiveness, termed trained immunity, after exposure to certain pathogens or cytokines.92 Notably, immunologic training in this setting does not involve genetic rearrangement but rather stable alterations in gene expression mediated by epigenetic changes. Moreover, this process is not antigen specific, such that exposure to one pathogen may alter subsequent responsiveness to an unrelated one. Although some innate immune cells have a short half-life, environmentally driven gene changes would seem likely to influence long-lived hematopoietic stem cells or stromal or hematopoietic populations that self-renew in peripheral tissues.

HOMEOSTASIS IN THE INNATE IMMUNE SYSTEMThe breadth and depth of innate immune activities that defend the host in its microbe-laden environment are silent from a clinical per-spective. In health, inflammation is the exception rather than the norm. This immune tranquility of the well-defended host is testament to the seamless efficiency of frontline defenses combined with active homeo-static processes that closely regulate inflammatory responses within the innate immune system.

Macrophages have an essential role in maintaining immune homeo-stasis (Fig. 1.7). Airway macrophages exemplify this antiinflammatory


actively maintains an asymmetric phospholipid distribution such that phosphatidylserine is kept on the inner side of the membrane bilayer. Apoptosis perturbs this asymmetry and exposes phosphatidylserine on the cell’s outer surface, leading to their recognition by macrophages bearing phosphatidylserine receptors.99,100 On recognition of apoptotic cells, macrophages release antiinflammatory IL-10, PGE2, and TGF-β to complete the task of maintaining immune homeostasis.

The complement component C1q and the collectins MBL and sur-factant proteins A and D bind to apoptotic cells and mediate their clearance.58 Components of the innate immune system accomplish this through PRRs that recognize both microbes and distinct apoptotic cell–associated molecular patterns (ACAMPs).101 Although macrophages have received most of the attention in mediating efferocytosis in the immune system, epithelial cells, endothelial cells, fibroblasts, and stromal cells can also contribute.

During resolution of acute inflammation, neutrophils switch their production of proinflammatory lipid mediators (e.g., leukotrienes, prostaglandins) to other molecular families of fatty acid–derived media-tors (e.g., lipoxins, resolvins, protectins) that are potently antiinflam-matory and injury resolving.102 They restore tissue homeostasis by stimulating efferocytosis of apoptotic or necrotic neutrophils, blocking neutrophil infiltration, and reducing vascular permeability.

role, actively suppressing DC maturation and antigen presentation in mouse airways, which is revealed when they are depleted.93,94 PAMP-PRR activation temporarily diverts alveolar macrophages from their antiinflammatory mode and primes them for antimicrobial functions.95,96 Classic activation of macrophages induces both proinflammatory (e.g. TNF-α) and antiinflammatory mediators (e.g., IL-10, TGF-β, and pros-taglandin E2 [PGE2]) that downregulate macrophage and DC functions (Fig. 1.7). Thus microbe-induced activation of the innate immune system is tightly linked to concurrent induction of downregulatory mechanisms to regain immune homeostasis. An alternative pathway of macrophage activation is mediated by IL-4 and IL-13. This modified response mod-estly downregulates macrophage and DC function and induces antiin-flammatory IL-10, IL-1 receptor antagonist (IL-1RA), and the decoy, nonsignaling, type II IL-1 receptor (IL1R2) while promoting MHC class II antigen presentation and antibody production.97

Macrophages also control inflammation through their constitutive ability to rapidly ingest and clear apoptotic cells (i.e., efferocytosis) (Fig. 1.7).58,98 The efficiency of this process is illustrated by the observa-tion that more than 1011 circulating neutrophils are eliminated each day without a trace of inflammation.58 Macrophages recognize apoptotic cells by molecular pattern recognition reminiscent of microbial recogni-tion by innate immune cells. The plasma membrane of viable cells

Ovalbumin

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Fig. 1.6 Innate immune instruction of adaptive immunity is exemplified by dendritic cells (DCs) in the lung. Microbial stimuli activate immature DCs in the periphery to take up and process antigen, migrate to draining lymph nodes, and mature as they migrate. On reaching lymph nodes, DCs secrete chemokines that attract T lymphocytes. The activities of mature DCs—presenting processed antigen, secreting cytokines, and express-ing costimulatory molecules—induce antigen-specific T lymphocyte activation, proliferation, and differentiation. The cytokine microenvironment of peripheral tissues from which DCs migrate biases them to direct naïve T cells in different directions (e.g., Th1, Th2, Th17, Treg). Memory T lymphocytes migrate to other lymph nodes, and effector T lymphocytes migrate to peripheral tissues. Mature DCs also migrate to peripheral tissues, where their interactions with effector T lymphocytes are thought to underlie tissue-specific immune memory. Ag, Antigen; DC, dendritic cell; MHC, major histocompatibility complex; PAMP, pathogen-associated molecular pattern; Th, helper T cell (types 1, 2, and 17); Treg, regulatory T cell. (Adapted from Lambrecht BN. Dendritic cells and the regulation of the allergic immune response. Allergy 2005;60:271-82; inset from Medzhitov R, Janeway C Jr. Innate immunity. N Engl J Med 2000;343:338-44. Copyright 2000 by Massa-chusetts Medical Society.)


INNATE IMMUNITY AND ALLERGYThe innate immune system of the airway, gastrointestinal tract, and skin is continuously exposed to potential allergens. Like microbial anti-gens, allergens can engage innate PRRs and be processed by innate immune cells. In the case of allergic disease, this recognition leads to generation of antigen-specific IgE; overexpression of type 2 cyto-kines (IL-4, IL-5, IL-13); and tissue injury, dysfunction, and aberrant remodeling. Although the circumstances leading to allergic immunity in humans are not clear, evidence suggests that allergic susceptibili-ties can originate in the innate immune system,111–113 with key roles for barrier epithelial cell programs that condition DCs for adaptive memory Th2 responses (Fig. 1.8). Indeed, GWAS studies in asthma114 and expression analyses in atopic patients115–118 point to key roles for epithelial-derived innate type 2 cytokines and DC activation pro-grams that underlie the atopic march. Notably, recent research has demonstrated that barrier epithelial cells may also activate innate cells such as ILC2s and mast cells to make substantial quantities of type 2 cytokines, indicating the possibility that type 2 inflammatory diseases could be generated or perpetuated in the absence of classical memory Th2 cells. The importance of these pathways in humans has yet to be clarified.

Allergen Recognition By the Innate Immune SystemBecause of the structural diversity of allergens that elicit IgE in humans, it is likely that type 2 immunity arises from several complex recognition programs, the subjects of ongoing study. Although some allergens contain structural motifs that are recognized by PRRs, others may have shared function such as the protease-mediated breakdown of tissue barriers and elicitation of aberrant tissue repair mechanisms.

The allergenicity of major allergens results in part from their recog-nition by PRRs. Allergen-elicited TLR4 signaling on airway epithelial cells plays a key role. In the case of house dust mite, activation of TLR4 elicits the generation of epithelial-derived innate type 2 cytokines such as GM-CSF and IL-33, promoting downstream allergic lung inflam-mation in mouse models.119 The major dust mite allergen, Der p 2, is a structural and functional homolog of MD-2, the TLR4 coreceptor responsible for binding LPS, and thereby augments TLR4 signaling.120 This facilitation is critical, because TLR4 signaling in human airway epithelial cells is limited by normally low MD-2 expression.121 Whether other major allergens can substitute for MD-2 is unknown, but like Der p 2, many are lipid-binding proteins (e.g., dust mite Der p 7, cat Fel d 1, lipocalins mouse Mus m 1, and horse Equ c 1). Finally, other allergens appear to facilitate epithelial cell TLR4 activation by binding to MD-2, including pollen allergens from diverse families of trees (Cottonwood, Walnut), grasses (Bermuda, Timothy, Rye), and weeds (Ragweed, Pigweed, Thistle).122 This again can drive TLR4-dependent allergic airway inflammation, highlighting the importance of TLR4 activation of airway epithelial cells in the allergic process.

Some CLRs recognize carbohydrate PAMPs common to fungi, pollens, and helminths but not mammals and mediate Th2 responses. DC-SIGN binds fucosylated glycan moieties on some major allergens (e.g., peanut Ara h 1, mite Der p 2, Bermuda grass BG60).123,124 The mannose recep-tor binds carbohydrate moieties on diverse major allergens (e.g., mite Der p 1 and Der p 2, dog Can f 1, cockroach Bla g 2, peanut Ara h 1).125 Of particular interest in allergen-induced disease mechanisms, Dectin-2 on DCs binds glycans in house dust mite and Aspergillus extracts, induc-ing cysteinyl leukotriene production and allergen-specific Th2 inflam-matory responses in the lung.20,126 The β glucan receptor Dectin-1, expressed on DCs, can also facilitate murine allergic airway inflamma-tion elicited by Aspergillus127 and by house-dust mite,128 but the ligand in dust mite extracts has not been characterized.

Epithelial cells also maintain homeostasis through several lines of defense. Poor expression of MD-2, a component of TLR4, on airway epithelial cells is likely an important adaptation to prevent excessive NF-κB-dependent airway inflammation. Furthermore, a recent study examining the protective effect of farm dust on type 2 inflammation (reviewed further in Environmental Determinants of Atopy: the Hygiene Hypothesis and the Microbiome) found a critical role for epithelial cell expression of A20, an inhibitor of NF-κB in preventing lung disease elicited by environmental antigen.103 Epithelial cell–derived cytokines also have a demonstrated role in restoration of tissue homeostasis. For example, IL-33–mediated activation of ILC2s leads to production of amphiregulin, an EGFR agonist that promotes restoration of epithelial integrity.104,105

Finally, an emerging theme from preclinical studies is the innate immune control of metabolic functions. For example, a well-studied murine model of homeostasis involving type 2 immunity is in visceral adipose tissue. In response to cold exposure, IL-33–mediated activation of ILC2s, eosinophils, and alternatively activated macrophages elicits transformation of white adipose tissue into metabolically active beige adipose tissue, essential for cold adaptation and thermogenesis.54,106 In mouse models of obesity-induced insulin resistance, eosinophils, alter-natively activated macrophages, ILC2s, and Foxp3+ T regulatory cells are protective in maintaining insulin sensitivity.107–110 Thus the function of innate type 2 immunity in maintaining homeostasis may be consid-erably more complex than we have understood.

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Fig. 1.7 Homeostasis in innate immunity. Macrophages have specialized regulatory functions that prevent inflammatory responses. Activation of macrophages induces antiinflammatory mediators such as interleukin-1 receptor antagonist (IL-1RA), interleukin-10 (IL-10), prostaglandin E2 (PGE2), and transforming growth factor β (TGF-β), which are thought to downregulate dendritic cell maturation and function. Macrophages also control inflammation by rapidly ingesting apoptotic cells to prevent their inflammatory rupture in the microenvironment, a process known as efferocytosis. The cell membranes of apoptotic cells have externalized phosphatidylserine (PS) that is actively maintained by healthy cells on the inner side of cell membranes. Macrophages have PS receptors (PS-R) that recognize apoptotic cells, triggering their ingestion and the release of antiinflammatory cytokines. During the resolution of inflam-mation, neutrophils switch their production of proinflammatory lipid mediators to antiinflammatory lipoxins, resolvins, and protectins. They help to restore tissue homeostasis by stimulating efferocytosis and blocking neutrophil infiltration.


DAMPs or “alarmins” released in the setting of cellular stress or non-programmed cell death such as uric acid, IL-1α, ATP, and High Mobility Group Box 1 (HMGB1) may play a key role in eliciting innate type 2 cytokines,143–147 but how this occurs in humans is not well substanti-ated, and what prevents this from happening in all people remains unknown.

Environmental Determinants of Atopy: The Hygiene Hypothesis and the MicrobiomeThe hygiene hypothesis was originally proposed to explain the inverse relationship between the development of hay fever and family size or birth order148 and suggested that the epidemic of atopic diseases noted in developed countries might be related to a decreased incidence of childhood infections. As further epidemiologic studies noted a reduced incidence of atopic diseases in children with early life exposures to farms,149,150 dogs,151,152 endotoxin,153 and pests,154 among other exposures, the hygiene hypothesis has been revised to propose that exposure to a diverse microbiome in neonatal life, and not specific infections per se, may mediate a protective effect. Support for a rich or diverse micro-biome mediating a protective effect has come from multiple studies. First, epidemiologic factors that protect against the development of atopy and atopic diseases profoundly alter the microbial composition

Many allergens, including mite, cockroach, fungi, and grass and weed pollen, have protease activity that is associated with their aller-genicity,129 and some can cleave and activate protease-activated receptors (PARs) expressed by innate immune cells.130 House dust mite proteases Der p 1, Der p 3, and Der p 9 stimulate proinflammatory cytokine release by airway epithelial cells through PAR2.131 In the case of house dust mite,132 cockroach,133 and the mold Alternaria,134 PAR2 mediates allergic airway inflammation, likely through the protease-mediated release of IL-33 from airway epithelial cells.134

Allergen-Elicited Innate InflammationThe barrier epithelial cell program activated in response to allergens is still emerging but includes the generation of canonical “innate type 2 cytokines” IL-33, IL-25, and thymic stromal lymphopoietin (TSLP). These cytokines activate mDCs to upregulate membrane-bound OX40L and generate CCL17 and CCL22 to induce allergen-specific Th2 dif-ferentiation.135–137 They also elicit expansion of cytokine-producing ILC2s138–140 and prime and activate innate effectors, such as mast cells.141,142 Although these epithelial cell cytokines are detected in response to protease-containing allergens, viruses, and impaired epithelial cell integrity, the mechanisms by which they are generated, released, and regulated remains poorly understood. Recent work suggests that

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Fig. 1.8 Epithelial control of type 2 immunity. After encounter with allergens, barrier epithelial cells generate the canonical innate type 2 cytokines, IL-33, IL-25, and thymic stromal lymphopoietin (TSLP). On the left, these cytokines can activate dendritic cells (DCs), conditioning them to upregulate cell surface expression of OX40L. On arrival in the tissue-draining lymph node, these DCs promote the skewing of T helper type 2 (Th2) cells from naïve (Th0) cells and the generation of type 2 cytokines. Additionally, B cells (B) are activated to undergo antibody class switching and the generation of IgE+ B cells. On the right, these same innate cytokines can directly activate tissue-resident and recruited innate effector cells such as mast cells (MCs), basophils (Ba), and group 2 innate lymphoid cells (ILC2s) to make type 2 cytokines.

1 - innate immunity...1 innate immunity lora bankova, nora barrett 1 section a asic sciences...

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