Chapter 20: Innate Immunity

In humans, as in all mammals, resistance to microbial infection is based partly upon lymphocytes, which yield highly specific responses to microbial antigens: either the production of antibodies or the expansion of T-cell cell clones that are directly cytotoxic to infected cells (Chaps. 75 and 76). This, the adaptive immune response, is a recent fixture in evolution, witnessed only in vertebrates and traceable to the development of a mechanism for recombination of genomic DNA that arose approximately 450 to 500 million years ago, operating on genes encoding proteins with immunoglobulin domains in some lineages and on genes encoding proteins with leucine-rich repeats in other lineages.^1,2 A more fundamental type of immunity, known as innate immunity, is represented in one form or another in all multicellular organisms. For this reason, a great deal of progress in the innate immunity field has come from the study of model animals such as Drosophila melanogaster, and model plants such as Arabidopsis thaliana. Despite the vast evolutionary divergence of these organisms from Homo sapiens, these species use defensive proteins and signaling pathways that are ancestrally related to those represented in humans.

Like the adaptive immune system, the innate immune system is endowed with a means of detecting microbes, destroying them, and at the same time, exercising self-tolerance. These mechanisms are far older than the analogous adaptive mechanisms and as a consequence are more refined. Although it is sometimes termed the “primitive” immune system, the innate immune system is both sophisticated and highly effective. Moreover, adaptive immunity is largely dependent upon innate immunity in the sense that antigen presentation and adaptive immune activation depend upon innate immune cells.

Innate immunity, which acts immediately to protect the host in the event of microbial inoculation, fills a temporal gap that would otherwise exist in the global immune response. Days or weeks are required for an effective adaptive immune response to develop when the naïve host encounters a new pathogen. During this time, innate immunity alone protects the host. Indeed, innate immunity is objectively more important than adaptive immunity. In a nonsterile environment, survival would be impossible without it (Table 20–1).

Innate immunity embraces a large number of host resistance mechanisms, which may be divided into cellular and noncellular components, and also into afferent and effector components. Noncellular components of innate immunity include antimicrobial peptides, which selectively disrupt microbial cell membranes, complement, components of which also disrupt cell membranes, and the proteins hemopexin and haptoglobin, which deny iron to invasive microbes. Cellular components include myeloid cells (granulocytes, monocyte/macrophages, mast cells, and dendritic cells) and lymphoid cells (natural killer [NK] cells and NKT cells). As such, it can be seen that despite their recent evolutionary origin, some lymphoid cells have been coopted to serve in the innate immune system rather than the adaptive immune system. Many other cells are also endowed with some degree of innate (often “cell-autonomous”) immune function. For example, fibroblasts can sense viral infection and respond with interferon (IFN) production.

Once initiated, the innate immune response runs its course in a preprogrammed fashion, proceeding from microbe sensing all the way through to microbial killing, making division into “afferent” and “effector” functions somewhat arbitrary. Nonetheless, the proteins responsible for microbial recognition, signaling, and the development of a transcriptional response within innate immune cells are generally considered “afferent” components; the cytokines that mediate the response and the cellular weaponry that is used to destroy viruses and bacteria may be considered “effector” components.

The remainder of this chapter emphasizes the afferent arm of cellular innate immunity, as the effector mechanisms (neutrophil-mediated killing, complement, and antimicrobial peptides) are covered in other chapters. Our understanding of innate immune responses has improved dramatically as forward and reverse genetic methods have been used to dissect the signaling pathways that permit host recognition of microbes. The initial interactions between molecules of microbes and molecules of the host that trigger an innate immune response have been studied in great detail over the past decade. The afferent pathways are each capable of activating responses that partly overlap with one another.

MICROBE RECOGNITION BY THE TOLL-LIKE RECEPTORS

Discovery of the Mammalian Toll Like Receptors as the Primary Sensors of the Innate Immune System

The Toll-like receptors (TLRs) collectively mediate the recognition of most microbes. Ten TLRs are encoded in the human genome. The molecular specificity of nine of these TLRs has been established, at least in part. Although publications can be found to suggest that some of the TLRs (notably TLRs 2 and 4) detect dozens of molecules, the evidence favoring most of these interactions is slender, and a conservative viewpoint is preferred; hence, Table 20–2 presents only those interactions that are deemed certain.

The microbe-sensing function of the mammalian TLR was discovered as a result of inquiry into the mechanism of endotoxin sensing. Endotoxin (later identified as lipopolysaccharide [LPS]) was first described by Pfeiffer as a toxic component of Vibrio cholerae more than 100 years ago.³ Its chemical structure was established many years later (reviewed in Ref. 4), and a toxic “lipid A” moiety of LPS was synthesized artificially in 1985 and found to have full biologic activity.⁵ The identity of the LPS receptor was established in 1998, through the positional cloning of Lps, a locus that was known to be required for all cellular responses to endotoxin, and for the effective clearance of Gram-negative bacterial infections⁶ in laboratory mice. In LPS-unresponsive mice, the Tlr4 locus was shown to be mutationally altered or deleted.⁷ It had previously been recognized that Toll, a Drosophila protein also known for its developmental effects,⁸ was required for the innate immune response to fungal infection in flies.⁹ Hence, the discovery of an LPS-sensing function for TLR4, a homologue of Toll, made evolutionary sense.

Other molecules of microbial origin (for example, di- and tri-acylated lipopeptides and lipoproteins, lipoteichoic acid, unmethylated DNA bearing CpG dinucleotides in a particular context, flagellin, and double-stranded RNA [dsRNA]) were known to elicit responses qualitatively similar to those elicited by LPS. The other TLR paralogs seemed excellent candidate receptors for these molecules. Reverse genetic methods established that each of these molecules is indeed recognized by a particular TLR or heteromeric combination of TLRs.^{10,11,12,13,14} Moreover, genetic complementation analyses have shown that at least some microbial ligands directly engage the TLRs in order to elicit a signal.^15,16 On the other hand, other molecules enhance the signal, and also participate in ligand recognition. Dectin-1 is a type II transmembrane C-type lectin that recognizes glucans present in the cell walls of fungi, signals via spleen tyrosine kinase (Syk) and the Card9/Bcl-10/MALT1 complex to activate nuclear factor-κB (NF-κB),^17,18 and enhances TLR2/6 signaling.¹⁹ Similarly, proteinase-activated G-protein–coupled receptor (PAR-2) signaling enhances TLR4 responses to LPS.²⁰ Other examples include the binding of cluster of differentiation (CD) 14 to LPS²¹ which augments LPS responses,²² as well as the enhancement of responses to bacterial diacylglycerides by CD36.²³ It is likely that these accessory molecules form complexes with the TLRs, which are responsible for transducing the signal across the cell membrane. TLR4 exists in a tight complex with MD-2, a small secreted protein that is required for TLR4 to reach the cell surface and required for LPS sensing as well.²⁴

Structure of the Toll-Like Receptors

The TLRs are single-spanning transmembrane proteins with leucine-rich repeat (LRR) motifs in their extracellular domains and a characteristic TIR (Toll/interleukin [IL]-1 receptor) motif in their cytoplasmic domains. The TIR domain is based on an ancient protein fold²⁵ evident in cytosolic plant disease resistance proteins (where it often is represented together with a nucleotide binding sequence [NBS] and/or LRR motifs), in proteins of the IL-1 and IL-18 receptor family, in the adapter proteins that carry signals from TLRs, and in the TLRs themselves.

The structure of the TLR2/1, 2/6, 3, 4, 5, and 8 ectodomains has been determined by x-ray crystallography, which showed a horseshoe-shape characteristic of LRR-containing proteins. TLRs form homodimers or heterodimers induced by the simultaneous binding of ligands to LRRs of distinct receptor chains. The nature of the ligand-receptor interaction has also been determined for several of the above receptors, and appears to be different in each individual case (Fig. 20–1). To activate TLR4, LPS interacts with MD-2, which has a hydrophobic pocket that accommodates the lipid A moiety of LPS.^26,27 TLR2/1 heterodimers are “crosslinked” by the engagement of two acyl chains by TLR1 and a single acyl chain by TLR2.²⁸ TLR3 molecules bind a linear, negatively charged dsRNA oligonucleotide, which triggers activation.²⁹

TLRs 3, 7, 8, and 9 are believed to be intracellular. Little (TLR3) or no (TLRs 7 and 9) surface expression can be detected, and tagged versions of the molecules are found to reside within the interior of transfected cells.³⁰ The ectodomains of these TLRs project into endocytic vesicles and there detect foreign molecules rather than within the extracellular space. TLRs 3, 7, 8, and 9 are trafficked from the endoplasmic reticulum (ER) to endosomal compartments via the secretory pathway, and depend on the aid of chaperones to do so. For example, UNC93B1, a 12-transmembrane spanning ER protein, directly binds and is necessary for TLRs 3, 7, and 9 to gain access to the endosomal compartment.³¹ UNC93B1 is believed to escort these molecules, and perhaps others, to their destination in the cell.³² PRAT4A (encoded by TNRC5) serves a critical role in chaperoning multiple TLRs to their destination,³³ while the ER chaperone protein, gp96 (also called GRP94 or HSP90B1) is critical for all TLR maturation (Fig. 20–2).³⁴ Proteolysis of TLR7 and 9 is known to occur in the endolysosome, and at least for TLR9, this cleavage increases ligand binding and is necessary for activating downstream signaling pathways.^35,36 In plasmacytoid dendritic cells, TLR7 and TLR9 are further trafficked from endosomes to lysosome-related organelles; this trafficking is necessary for the abundant production of type I IFN for which these cells are specialized.^37,38 The adaptor protein complex 3 (AP-3), which directs subcellular trafficking through the secretory pathway, and the peptide/histidine transporter 1 (PHT1) are necessary for TLR7 and TLR9 trafficking to lysosome-related organelles.

Toll/Interleukin-1 Receptor Adapter Signaling

The signaling events initiated by the TLRs are increasingly complex and have been studied in great detail [reviewed in Refs. 39 and 40]. Figure 20-3 illustrates the pathways as they are presently understood. It must be recognized that not all TLRs operate within the same cells, nor are all cells equivalent in their responses to TLR ligation. Notably, macrophages and conventional (myeloid) dendritic cells respond to different stimuli than do lymphoid cells, or plasmacytoid dendritic cells (which are specialized for type I IFN production). Moreover, some cells not usually regarded as “professional” components of the innate immune system are capable of responding to TLR ligands in one way or another.

A total of five TIR adapter proteins are encoded in the human genome. These adapters are MyD88 (myeloid differentiation primary response 88), MAL (MyD88 adaptor-like; also known as TIRAP), TRIF (Toll/interleukin-1 receptor domain-containing adaptor inducing IFN-β; also known as TICAM1 and first identified by a mutant allele known as Lps2), TRAM (TRIF-related adaptor molecule; also known as TICAM2), and SARM (sterile-α and armadillo motif). The function of SARM remains unknown, and it is the most distantly related paralog among the adaptors. However, the four remaining adapters have well-defined roles in signal transduction. All four of these adapters are required for normal signaling from the LPS receptor, TLR4; MyD88 and MAL act in concert with one another, and TRIF and TRAM act together, so that two primary branches of the LPS signaling pathway diverge at the level of the receptor.^41,42 In contrast, TRIF alone serves TLR3 signaling; MyD88 and MAL (but neither TRIF nor TRAM) serve TLR2; and MyD88 alone serves TLRs 7, 8, and 9. Mutational inactivation of MyD88 creates a severe immunodeficiency state in mice and humans,^43,44 and compound homozygosity for mutations affecting both MyD88 and TRIF causes immunodeficiency that is still more severe, in which animals are essentially unable to sense the presence of most microbes.⁴²

Two main branches of signaling, dependent on MyD88 or TRIF, mediate the effects of TLR activation in conventional dendritic cells, macrophages, and fibroblasts (see Fig. 20–3). The MyD88-dependent pathway is used by all TLRs except TLR3, as mentioned above. MyD88 is believed to assemble into a helical complex called the Myddosome upon receptor activation, engaging the serine kinases IRAK (interleukin-1 receptor-associated kinase) 4 and IRAK2 or IRAK1 through death domain interactions.⁴⁵ Signaling proceeds via phosphorylation of IRAK2 or IRAK1 by IRAK4. No comparable structural data illuminate the function of MAL, TRIF, or TRAM proteins, but it is clear that TRIF can directly engage TLR3.⁴⁶ The activated Myddosome recruits the E3 ubiquitin ligase tumor necrosis factor (TNF) receptor-associated factor (TRAF) 6, a cellular scaffold protein that coordinates the recruitment of several other protein kinases. MyD88 also interacts with TRAF3; however, degradative K48-linked ubiquitination of TRAF3 by cIAP1/2 during MyD88-dependent TLR signaling is necessary for the activation of mitogen-activated protein kinases (MAPKs) and production of inflammatory cytokines.⁴⁷ In conjunction with the E2 ubiquitin-conjugating enzyme 13 (Ubc13) and the Ubc-like protein Uev1a, TRAF6 adds chains of K63-linked polyubiquitin to itself, as well as inhibitor of κB (IκB) kinase γ (IKKγ; also called NEMO [NF-κB essential modulator]) and to TRAF2 (reviewed in Ref. 48). Transforming growth factor-β–activating kinase 1 (TAK-1) forms a complex with TAB1, TAB2, and TAB3, is recruited to the TRAF6 complex, and phosphorylates IKKβ, which in complex with IKKα and IKKγ phosphorylates IκB (an inhibitor of the p65 form of NF-κB), leading to its K48-ubiquitin–mediated degradation.⁴⁸ Nuclear translocation of homo- or heterodimers composed of p65 and/or p50 NF-κB ensues. NF-κB drives the transcription of hundreds of genes encoding proteins that form the inflammatory response. Mitochondrial reactive oxygen species (ROS) are also produced in macrophages as a result of TLR4, TLR2, and TLR1 activation; this antibacterial response depends on the translocation of TRAF6 to mitochondria to engage and ubiquitinate a protein called ECSIT, which functions in mitochondrial respiratory chain assembly.⁴⁹

At the same time, the IKK complex activated by TAK-1 phosphorylates the p105 form of NF-κB and MAP3K8 (also known as Tpl2), proteins that form a complex in which MAP3K8 is inactive under basal conditions. This leads to the degradation of p105 NF-κB, and to the activation of MAP3K8.^50,51 MAP3K8 phosphorylates and activates MEK1 and MEK2, while independently MEK3 and MEK6 are activated by TAK-1.⁴⁰ The MEKs activate MAPK family members, including extracellular signal-regulated kinase (ERK) 1 and ERK2, c-Jun N-terminal kinase (JNK), and p38 kinases. These kinases trigger the activation of other transcription factors, including c-Jun, which together with c-Fos forms the transcription factor AP1, and members of the cyclic adenosine monophosphate (AMP) response element-binding protein (CREB) family.

The TRIF-dependent TLR signaling pathway is activated by TLR3 and TLR4, and results in the induction of type I IFNs as well as inflammatory response genes (see Fig. 20–3). Upon receptor activation, TRIF interacts with TRAF3, which recruits TANK-binding kinase 1 (TBK1) and IKKε (both distantly homologous to the IKKs).^52,53 This complex engages and phosphorylates interferon response factor (IRF) 3, an interaction that may be mediated by phosphatidylinositol-5-phosphate generated by PIKfyve.⁵⁴ IRF3 dimerizes and translocates to the nucleus to activate transcription of type I IFN genes with the aid of deformed epidermal autoregulatory factor-1 (DEAF-1).⁵⁵ Two other IRF proteins, IRF1 and IRF7, also activate type I IFN genes, but in response to signaling from TLR7 and TLR9 particularly in plasmacytoid dendritic cells.^56,57 Activation of IRF3 and IRF1 can initiate expression of the IFN-β gene.^58,59 IFN-β mediates antiviral effects, and is also required for the upregulation of costimulatory proteins (e.g., CD40, CD80, and CD86) that enhance the activation of an adaptive immune response. Hence, the adjuvant effects of LPS and dsRNA are dependent upon the type I IFN receptor.⁶⁰ IRF7 induces the expression of the IFNα genes.^59,61 Both α and β IFNs bind to the type I IFN receptor rendering similar if not identical biological responses.

To induce inflammatory response genes, TRIF recruits receptor-interacting protein (RIP) 1 following its polyubiquitination by the E3 ligase Pellino.⁶² RIP1 interacts with the TRAF6/TAK-1 complex leading to NF-κB activation following the pathway described above for MyD88-dependent signaling. For reasons that remain unclear, the heteromeric MyD88/MAL complex is incapable of driving type I IFN gene expression.

Countervailing Influences in Toll/Interleukin-1 Receptor Adapter Signaling

IRAK-M, a homologue of IRAKs 1, 2, and 4, is an inhibitor of TIR domain signaling and may participate in feedback inhibition of signaling known as “endotoxin tolerance.”⁶³ In addition, suppressor of cytokine signaling 1 (SOCS-1) inhibits signal transduction from the Janus kinase (JAK)/signal transducer and activator of transcription (STAT) pathway (Chap. 17) activated by type I IFN, one of the key cytokines elicited in the course of an innate immune response.⁶⁴ A20 and CYLD, both deubiquitination enzymes, remove the K63 ubiquitin tails from TRAF6, NEMO, and RIP, inhibiting the activation cascade.⁴⁸ Still more distally, inhibition of signaling via antiinflammatory cytokines (such as IL-10 or transforming growth factor [TGF]-β) acts to limit responses initiated by the TLRs.

SENSORS OF THE NUCLEOTIDE-BINDING OLIGOMERIZATION DOMAIN-LIKE RECEPTOR FAMILY

An extensive family of proteins defined by their motif structure has recently been recognized for its participation in innate immune responses to intracellular microbes as well as noninfectious inflammatory stimuli, including, for example, uric acid crystals and aluminum hydroxide particles. Collectively called the nucleotide-binding oligomerization domain (NOD)-like receptors (NLRs), the proteins contain CARD (caspase activating and recruitment domain), Pyrin, or BIR (baculovirus inhibitor of apoptosis repeat) domains followed by nucleotide-binding NACHT domains and LRR domains arranged in tandem, and have been assigned to several subfamilies (Fig. 20–4).⁶⁵ Mutations within different representatives of the family produce dominant or semidominant inflammatory diseases. In some cases there is limited penetrance and strong dependence upon the presence of mutations in other genes. For example, NOD2 mutations have been clearly shown to enhance the likelihood of Crohn disease⁶⁶ and cause Blau syndrome,⁶⁷ while distinct NLPR3 mutations are the proximal cause of cold-induced autoinflammatory syndrome (CIAS1), chronic neurologic cutaneous and articular (CINCA) syndrome, or neonatal onset multisystem inflammatory disease (NOMID).^68,69,70 Mutations in the structurally related MEFV (pyrin-encoding) gene are responsible for familial Mediterranean fever.⁷¹ Pyrin has been shown to interact with the adaptor protein PSTPIP1 (proline serine threonine phosphatase-interacting protein 1). Mutations in the gene encoding this protein also cause an inflammatory disorder, pyogenic arthritis, pyoderma gangrenosum and acne (PAPA) syndrome.⁷²

The inflammatory potential of the NLR superfamily is exerted through two signaling pathways: the “inflammasome” pathway and the “NOD1/2” pathway. Each is less fully elucidated at present than the TLR signaling pathways. Moreover, each likely interacts with the TLR signaling pathways and in the case of the inflammasome, is dependent upon the TLR signaling for full expression of activity.

The Inflammasome Pathway

The “inflammasome” pathway (Fig. 20–5) is induced by at least three proteins, and possibly others. Ice-protease activating factor (IPAF/NLRC4; encoded by CARD12), NACHT domain-, LRR-, and pyrin domain (PYD) containing protein 1 (NLRP1, also known as CARD7), and NLRP3 (also known as cryopyrin) each trigger the inflammasome response. Diverse cellular perturbations probably lead to activation of IPAF, NLRP1, and NLRP3. Cytosolic flagellin introduced via type III or type IV bacterial secretion systems activates IPAF.⁷³ Anthrax lethal factor and muramyl dipeptide (MDP, a product of bacterial cell walls) introduced via pore-forming toxins activate NLRP1.^74,75 Peptidoglycan (PGN), MDP, lipopeptides, nucleic acids, uric acid crystals, alum, and other foreign substances activate NLRP3.^{76,77,78,79,80} Full activation of NLRP3 depends upon a drop in cytosolic potassium concentration, mediated in part by the potassium exporting channel P2X7. Activation of P2X7 recruits the gap junction channel Pannexin-1 (Panx-1), allowing entry of bacterial products and other molecules into the cell.⁸¹ Although it is not clear that the inducers have direct contact with the NLRPs or IPAF, the latter undergo oligomerization (mediated by the NACHT domain). They then signal either directly (in the case of IPAF) or via adapter proteins (ASC in the case of NLRP1, and both ASC and CARDINAL in the case of NLRP3) to activate the cytosolic cysteine proteases caspase-1 and/or caspase-5. Activation occurs through CARD interactions. Homodimeric caspase-1 and caspase-5 act to convert the inflammatory cytokine pro–IL-1β into its active form. Importantly, inflammasome signaling does not initially activate expression of the IL-1β encoding gene. However, TLR signaling, which activates NF-κB, or signaling by IL-1β itself, can do so.⁶⁵

IL-1β signals via its receptor to activate a signaling pathway very similar to those used by the TLRs, dependent upon MyD88 and the downstream signaling cascade components described earlier in this chapter in the section “Toll/Interleukin-1 Receptor Adapter Signaling”. As such, IL-1β may be viewed as an endogenous ligand that elicits a response similar to those elicited by microbial ligands. This signal may initially be induced by a focal infection operating in conjunction with a noninfectious inflammatory stimulus.

The Nucleotide-Binding Oligomerization Domain Pathway

NOD1 (CARD4) and NOD2 (CARD15) proteins have been mentioned as sensors of γ-d-glutamyldiaminopimelic acid (DAP) and MDP, respectively, both components of microbial cell walls. They are believed to detect intracellular bacteria or fragments thereof.^82,83,84 NOD2 has been strongly implicated in the pathogenesis of Crohn disease through linkage disequilibrium mapping and sequence analysis,⁶⁶ but mutations of NOD2 cause disease with low penetrance, suggesting the importance of other genetic and environmental factors. No clear disease association has been defined for NOD1. The NOD proteins do not form the core of inflammasomes, although recent data suggests that NOD2 is able to associate with NLRP1 and caspase-1 upon stimulation with MDP.⁸⁵ In response to microbial stimuli, the NOD proteins oligomerize and signal via TRAF2, TRAF5, and TRAF6 to cause K63 ubiquitination of RICK2 (also known as RIP2, or CARDIAK), a protein with a domain structure similar to receptor interacting protein (RIP), known for its involvement in TNF signal transduction. RICK2 activates TAK-1, and by way of TAK-1, elicits the activation of both NF-κB and the mitogen-activated protein (MAP) kinase cascade, leading to activation of the transcription factor AP1 (Fig. 20–6).

SENSORS OF THE RIG-I–LIKE HELICASE PATHWAYS

While TLRs are capable of detecting nucleic acids within the endosomal compartment and do make an essential contribution to the detection of some viruses (notably herpesviruses), other viruses are detected chiefly or entirely by cytosolic receptors. Among these, the RIG-I–like helicases (RLHs), including retinoic acid inducible gene I (RIG-I), melanoma differentiation-associated gene 5 (MDA5), and LGP2, are the best known sensors and are believed to undergo direct interaction with nucleic acids to initiate a response. RIG-I and MDA5 sense specific viruses; for example, viruses detected by RIG-I include influenza A, Sendai, and vesicular stomatitis viruses, and MDA5 detects encephalomyocarditis and murine hepatitis viruses.⁸⁶ RLH proteins have RNA helicase domains (involved in binding nucleic acids), as well as a regulatory domain (RD) that has been implicated in inhibiting downstream signaling,⁸⁷ but is also necessary for RNA sensing.^88,89 Both RIG-I and MDA5 have more proximal CARD domains involved in signaling, whereas LGP2 does not. On this basis it was initially believed that LGP2 might have an inhibitory function.⁹⁰ However, it appears to contribute to sensing in a positive manner, and may augment RIG-I and MDA5 signaling.^89,91,92

Although TLR3 can detect dsRNA and its synthetic analogue poly I:C, the dominant sensor of poly I:C (long polymers in particular) in vivo is MDA5⁹³; shorter poly I:C polymers are better detected by RIG-I. RIG-I is able to form stable complexes with dsRNA molecules containing blunt ends or 5′-overhangs, while dsRNA with 3′-overhangs are unwound by its helicase activity.⁸⁸ RIG-I additionally recognizes single-stranded RNA (ssRNA) molecules, distinguishing them from host RNA by detecting 5′-triphosphate structures such as are found in ssRNA from the influenza virus.^94,95 RIG-I must be activated by T-cell receptor interacting molecule 25 (TRIM25), a host resistance factor that ubiquitinates RIG-I (K63 linkages).

Upon virus recognition the RLHs initiate signaling, leading to type I IFN and inflammatory cytokine production dependent, respectively, upon IRF and NF-κB activation. RLHs signal by CARD domain-mediated interaction with mitochondrial antiviral signaling protein (MAVS; also known as IPS-1, VISA, or CARDIF), an integral protein of the mitochondrial outer membrane with a CARD domain that projects into the cytoplasm.^96,97,98,99 Upon activation by RIG-I interaction, MAVS forms prion-like polymeric fibers that induce the formation of similar aggregates by untouched MAVS molecules, which thereby gain competence to activate IRF3.¹⁰⁰ MAVS, in its active form, is capable of triggering three different signaling pathways. One pathway mimics the TNF signaling pathway, and includes the adaptor protein TRADD, Fas-associated death domain protein (FADD), RIP1, caspase-8 and caspase-10, and leads to IKK complex and NF-κB activation. A second pathway recruits TRAF6 and MEKK1, leading to activation of the MAP kinases and AP1. These two pathways are responsible for inflammatory cytokine production. The third pathway entails activation of TBK1 and IKKε, and leads to the activation of IRF3 and IRF7, with ensuing type I IFN production (Fig. 20–7).

A pathway for responses to cytoplasmic double-stranded DNA (dsDNA) has also been identified in mammalian cells (see Fig. 20–7).^101,102 Cyclic adenosine monophosphate (AMP)/guanosine monophosphate (GMP) synthetase (cGAS) is an enzyme allosterically activated by binding to dsDNA, whereon it synthesizes cyclic GMP:AMP (cGAMP). cGAMP activates stimulator of IFN genes (STING),¹⁰³ a penta-spanning ER membrane protein that undergoes a conformational change leading to the activation of TBK1 to phosphorylate IRF3, which dimerizes and translocates to the nucleus to induce type I IFNs. STING also activates the IKK complex, leading to degradation of IκB and release of NF-κB to enter the nucleus and induce other cytokines. A putative cytosolic DNA sensor DAI (for DNA-dependent activator of IRFs) has also been described. DAI contains DNA binding domains and enhances DNA-mediated induction of type I IFNs in vitro.¹⁰⁴ However, the role of DAI as a sensor of cytoplasmic DNA appears to be redundant.¹⁰⁵

Both the RLH-MAVS pathway and the cGAS-STING pathway become activated in B cells when they are stimulated by type 2 T-cell–independent antigens, such as pneumococcal vaccine (PPSV23).¹⁰⁶ These pathways sense the induction of endogenous retroviruses, leading to sustained B-cell activation and an immunoglobulin (Ig) M response.

KEY EFFECTOR CYTOKINES IN THE INNATE IMMUNE RESPONSE

Cells of the innate immune system exhibit a measure of autonomy (e.g., neutrophils directly engulf and destroy pathogens), but also initiate the adaptive immune response to microbes and summon “reinforcements” to the site of infection. These functions depend upon the production of cytokines, too numerous to describe in this chapter. However, a few of the key mediators are listed here.

Tumor Necrosis Factor-α

A homotrimeric cytokine that is made by many cells, TNF is synthesized in greatest amounts by mononuclear phagocytes that have been exposed to LPS or other TLR-activating stimuli. It was recognized as a key endogenous mediator of endotoxicity,¹⁰⁷ and later, as a mediator of other forms of inflammation (including sterile inflammation, as observed in rheumatoid arthritis and cancer, Crohn disease, ankylosing spondylitis, and psoriasis). The TNF signaling pathway depends upon two receptors, involves NF-κB activation, and is ancestrally related to the Drosophila Imd (immunodeficiency) pathway for recognition of Gram-negative bacteria.¹⁰⁸ The ancient phylogenetic origins of TNF signaling, its large representation in distant species, the therapeutic efficacy of TNF neutralization in the diseases just mentioned, and the immunocompromising effects of TNF and TNF receptor mutations in animals all suggest that TNF is one of the most important of the cytokines utilized by the innate immune system for effective containment of infection.

Interleukin-1α and β

Once known as pleiotropic inflammatory cytokines, IL-1α and IL-1β, two distantly related ligands that share the same set of receptors, are produced in response to innate immune stimuli and evoke fever, swelling, and neutrophil adhesion in the region of an infectious nidus. The type I IL-1 receptor, responsible for most or all of the agonist activity of the IL-1 proteins, has two chains, each of which is endowed with a cytoplasmic TIR domain. The receptor complex signals via MyD88 and no other adapters are known to be required. IL-1 signaling may act as an amplification mechanism that augments the primary infectious signal, and transmits awareness of infection to cells that lack the innate immune sensors required for detection of microbes.

Interleukin-6

Signaling via a receptor that uses the JAK/STAT pathway, IL-6 activates many elements of the “acute phase response”; that is, hepatic production of fibrinogen, serum amyloid A protein, and C-reactive protein. It also has thrombopoietic activity, both directly (minor effect) and through its stimulation of thrombopoietin production (major effect) (Chap. 111), which stimulates platelet production, often consumed in the course of a serious infection.

Interleukin-12

A cytokine made in abundance by dendritic and other cells in response to TLR stimulation, IL-12 activates the production of IFN-γ by lymphoid cells, which, in turn, increases the microbicidal activity of mononuclear phagocytes. Unlike most cytokines, IL-12 is a heterodimeric protein, and the IL-12 p40 subunit is subject to induction, whereas the p35 subunit is synthesized constitutively. Mutations of the genes encoding IL-12 or its receptor, or IFN-γ or its receptor, are known to cause relatively severe susceptibility to infection by mycobacteria and other intracellular infections. Hence the IL-12/IFN-γ feedback loop is considered one of the most important innate/adaptive immune interactions.

Chemokines

A family of small proteins, highly redundant in receptor specificity and organized into CC and CXC subfamilies, the chemokines are induced by primary microbial stimuli and by TNF and IL-1. Binding to G-protein–coupled receptors, they exhibit phagocyte chemotactic activity (Chaps. 61 and 68), and are believed to contribute to the egress of neutrophils from blood into infected tissue.

Granulocyte Colony-Stimulating Factor and Granulocyte-Macrophage Colony-Stimulating Factor

The central hematopoietic response is attuned to events in the peripheral tissues, and granulocyte colony-stimulating factor (G-CSF) and granulocyte-macrophage colony-stimulating factor (GM-CSF) promote the production and release of granulocytes and monocytes to cope with an infectious challenge. These cytokines are produced by macrophages, endothelial cells and fibroblasts in direct response to TLR signaling (Chap. 61), and also in response to secondary cytokines such as TNF. They signal via JAK/STAT-coupled receptors.

Interferons

Type I IFNs (IFN-α and IFN-β) are expressed immediately in response to LPS, dsRNA, or unmethylated DNA, and have broad activity in the containment of viral infections. LPS-induced type I IFN production depends upon TLR4 and the adapters TRIF and TRAM. dsRNA-induced type I IFN depends upon TLR3 and TRIF (but not TRAM). Unmethylated CpG motifs in DNA stimulate type I IFN production that depends upon MyD88. Although many cells are induced into an antiviral state as the result of IFN stimulation, NK cells, which are specialized for the elimination of virus-infected targets, are particularly dependent upon type I IFN signaling (Chap. 77),¹⁰⁹ and require it for the elimination of specific pathogens such as cytomegalovirus.¹¹⁰ The type I IFNs are also involved in protection against bacterial infection,¹¹¹ and type I IFN signaling has been shown to be important to the development of endotoxic shock.¹¹² Plasmacytoid dendritic cells are a particularly important source of type I IFN.¹¹³

Type II IFN (IFN-γ) has less antiviral activity than type I IFN, is produced by T cells in response to IL-12 receptor stimulation, and is crucial for the elimination of intracellular pathogens such as mycobacteria, which reside within macrophages of the infected host.

The adjuvant effect of microbes has been known since the classic studies of Lewis and Loomis¹¹⁴ who coined the term “allergic irritability” to describe the augmented production of antibodies against a protein antigen in guinea pigs infected with Mycobacterium tuberculosis. Freund and McDermott¹¹⁵ demonstrated that heat-killed mycobacteria were capable of eliciting an exaggerated antibody response as well when coadministered with a protein antigen, indicating that molecular components of microbes (rather than infection per se) were responsible for adjuvanticity. LPS was shown to be endowed with adjuvant activity in 1955,¹¹⁶ and by 1975 the Lps locus was shown to be required for this effect of LPS (as it is required for all other cellular effects of LPS).¹¹⁷ By deduction, the positional cloning of Lps thus revealed the essential role of TLR4 in LPS-mediated adjuvanticity.⁷

Activation of an adaptive immune response to a specific antigen has long been known to depend upon two signals that occur in the course of antigen presentation. First, the T-cell receptor must be activated. In addition, costimulatory molecules upregulated on the antigen-presenting cell (e.g., CD40, CD69, CD80, and CD86) are known to interact with receptors (or in some cases ligands) on the T cell. An exchange of signals occurs over a period of approximately 12 hours,¹¹⁸ ultimately leading to autonomous expansion of the T-cell clone and, in turn, activation of specific B cells. Some of these signals are well characterized. For example, CD80 and CD86 both engage CD28 and cytotoxic T-lymphocyte antigen (CTLA) on the T-cell surface, and abrogation of signaling via these costimulatory receptors is known to substantially attenuate the adaptive immune response.¹¹⁹

Upregulation of costimulatory molecules (UCM) is therefore essential, although not by itself sufficient, for activation of the adaptive immune response. LPS depends upon TRIF (and specifically, upon TRIF-mediated type I IFN gene expression) to elicit UCM^42,60,120; absent TRIF, LPS cannot exert an adjuvant effect. TRAM is also required for UCM.⁴¹ Although MyD88 does not elicit UCM, it does contribute to LPS-induced adjuvanticity in an experimental setting.⁶⁰ It is likely that IL-12, a cytokine that is largely MyD88-dependent, also contributes to the adjuvant effect, along with other proteins yet to be identified.

Even though several publications initially suggested that TLR signaling is required for adaptive immune responses to occur, it has been observed that mice lacking all TLR signaling are quite capable of mounting adaptive immune responses, including antibody responses and recall responses to defined antigens administered with diverse adjuvants.¹²¹ It is now evident that there is much redundancy in adaptive immune responses, and several innate immune pathways can independently trigger such responses.

Premature death from infection is strongly heritable in humans.¹²² Defects of the innate immune sensing apparatus are expected to cause hypersusceptibility to infection in humans as they clearly do in mice, and specific examples of such mutations have recently come to light including the NLR disorders discussed above (see “Sensors of the NOD-Like Receptor Family”). Missense mutations of TLR4 that are rare among the normal white population are quite common in patients with systemic meningococcal disease, and have been assigned a role in susceptibility on this basis.¹²³ A nonsense mutation of TLR5 was found to be overrepresented in patients who developed Legionnaire disease as compared to a comparably exposed population that remained disease-free.¹²⁴ Mutations of IRAK4 and MyD88 create susceptibility to suppurative Gram-positive infections.^44,125 In humans, both TLR3¹²⁶ and UNC93B1¹²⁷ mutations cause susceptibility to recurrent Herpes simplex virus encephalitis, and presumably to other diseases as well.

On the effector side, examples of immunocompromise from innate immune defects are far better known, and include diseases caused by mutations affecting IFN-γ,¹²⁸ IL-12¹²⁹ and its receptor,^130,131 defects of granule formation,¹³² and defects of nicotinamide adenine dinucleotide phosphate (NADPH) oxidase.¹³³

Although the term “autoimmunity” is reserved for inappropriate adaptive immune responses that damage tissues of the host, the innate immune system may also cause injury or death, and typically does so when systemic activation occurs in the course of a serious infection. Innate immune responses, which entail cytokine-mediated inflammation and coagulation, evolved to contain small inoculates of microorganisms by encouraging the influx of granulocytes to engulf and destroy these pathogens, and by stimulating the development of an adaptive immune response. The mechanisms that are employed to these ends can be lethal if they are generalized rather than focal. In several examples, the importance of microbes as drivers of inflammation has been cited, and forward-feedback loops may perpetuate inflammation or autoimmunity. It has been reported, for example, that endogenous DNA, signaling via TLR9, is responsible for the generation and perpetuation of antinucleoprotein antibodies in a mouse model of systemic lupus erythematosus.¹³⁴ The involvement of TLRs 3 and 7 may also be important.

In hemophagocytic lymphohistiocytosis (HLH), an amplification loop involving a microbial driver, CTL expansion, and IFN-γ driven myeloid expansion has been well described in mice.¹³⁵ In SHP1 deficiency in mice, autoimmunity and inflammation also depend upon a microbial driver and activation of TIR domain signaling pathways.¹³⁶ NOMID and other mutations capable of activating the NLRP3 inflammasome have been mentioned earlier in this chapter. Beyond this, the innate immune system may also contribute to sterile inflammation (autoinflammatory disease), as witnessed in many human diseases that have so far eluded etiologic decipherment.