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