activated by Gram-negative bacteria and through Relish induces the production of a number of different AMPs, including Diptericin. Whereas the Toll pathway shares significant homology with the TLR and interleukin (IL)-1R pathways in mammals, the Imd pathway is related to the mammalian tumor necrosis factor receptor (TNFR) and NOD pathways (Figure 32-6). This striking conservation between the fly and mammalian pathways points to a common ancestry of these immune mechanisms.

3.2. PRRs and inflammation

3.2.1. TLRs

The innate immune system was extensively studied in Drosophila with the aim of elucidating how fruit flies that lack adaptive immunity responded to infectious pathogens. The discovery of Drosophila Toll was followed by the cloning of a human homolog and its characterization as a PRR able to stimulate the NF- κB pathway.24 So far, 12 members of the Toll family have been identified in mammals and are referred to as the Toll-like receptors (TLRs). All known TLRs are type I transmembrane proteins consisting of an extracellular domain with leucine-rich repeats (LRRs) responsible for ligand detection and a cytoplasmic Toll/IL1R homology (TIR) domain essential for initiating signaling25 (Figure 32-7). TLRs function as homoor heterodimers and recognize a broad range of microbial components.26 Mouse genetics studies and investigation of TLR knockout mouse phenotypes demonstrated the essential role of these receptors in pathogen recognition. Mice with a point mutation in Tlr4 are hyporesponsive to LPS.27 TLR3−/– mice fail to recognize viral doublestranded RNA (dsRNA) and are defective in stimulating inflammatory and type I interferon (IFN) responses.28 By associating with other TLRs, TLR2 responds to a variety of bacterial molecules that include peptidoglycan, lipoproteins, and lipopeptides. TLR5 recognizes bacterial flagellin,8 TLR7 responds to synthetic compounds with potent antiviral and antitumor activities such as resiquimod (R848), and TLR9 senses CpG motifs in bacterial and viral DNAs.29 TLRs share common signaling determinants with IL-1R family members; their TIR domains interact with TIR-containing adaptors, including MyD88, TIRAP/MAL, TRIF, and TRAM.30 With the exception of TLR3, all TLRs signal through MyD88. TIRAP/MAL, TRIF, and TRAM are more specialized. TRIF is engaged in response to viral PAMPs downstream of TLR3 and TLR4, MAL interacts with TLR2 and TLR4, and

TRAM binds TRIF in the TLR4 complex. The multistep signaling cascade induced by TLR activation results in the production of antimicrobial effectors by means of the NF-κB, mitogen-activated protein kinases (MAPKs), and interferon regulatory factor IRF 5/3/7 pathways31 (Figure 32-7).

3.2.2. NLRs

NLRs are evolutionarily related to disease resistance or “R” proteins in plants. These are cytosolic proteins that contain a nucleotide-binding site (NBS) and leucinerich repeats (LRRs) and are often involved in the recognition of PAMPs and pathogen-induced host danger signals. Upon activation, R proteins elicit a hypersensitive or guard response, which induces antimicrobial proteins, cell wall modification, and programmed cell death.32 Although the defense systems in animals and plants evolved under selective pressure imposed by distinct infectious pathogens, they stayed remarkably conserved across the two kingdoms. Another level of similarity between the two systems is the role that Hsp90 and SGT1 play in stabilizing the NBS-LRR/NLR proteins and maintaining their conformation in an auto-repressed but activation-competent state.33

The first described mammalian NLR, Nod1, was identified in a screen aimed at finding Apaf-1–related proteins.34 Similar to Apaf-1, Nod1 possesses an N- terminal CARD domain, a central nucleotide binding and oligomerization domain (Nod), and a C-terminal agonist-binding domain. The latter is a WD-40 repeat domain in Apaf-1 that binds cytochrome c, whereas it is an LRR domain in Nod1 that senses bacterial peptidoglycan derivatives.35 In response to agonist sensing, NLRs oligomerize and activate inflammatory effectors. To date, there are 22 NLRs, including 14 Nlrp proteins (Nlrp1–14), 5 Nods, Ipaf, Naip5, and CIITA. Comparison of the NLRs reveals both structural and functional differences. The N-terminal effector domain is variable among NLRs; it is a CARD in Nod1 and Ipaf, two CARDs in Nod2, three BIRs in Naip, and a pyrin domain (PYD) in Nlrp1-14 (Figure 32-8).

3.2.3. The Nod signalosome

Despite the presence of a CARD in Nod1 and Nod2, these proteins do not engage caspases directly. Instead, they interact with a CARD-containing kinase known as RIP2. This assembles a Nod signalosome that recruits TRAF proteins, cellular inhibitor of apoptosis proteins cIAP1 and cIAP2, TAK1, TAB1 and TAB2, needed to activate the

DAP-type

Peptidoglycan

derivatives

PGRP

-LC

IMD

DD

BIR BIR BIRCARD RING

DIAP2

TAB2

TAK1

IKK Ird5

Kenny

Rel

Gram-negative

bacteria

dFADD

p20

Dredd

p10

Relish

ANK

Rel

Mammalian

Gram-positive and Gram-negative

bacteria

Insect

derivatives

TRAF6

TAB2

TAK1

TAB1

JNK

p38

Fungi

Endosome

p50 RelA

Induction of survival and inflammatory genes

Rel Rel

Induction of Drosomycin and other antimicrobial genes

proinflammatory NF-κB and MAPK pathways. Recently, a protein termed CARD9 has been reported as a new addition to the Nod signalosome. CARD9 interacts with the Nod-Rip2 complex and selectively mediates activation of the MAPK pathway downstream of Nod stimulation (36 and references therein). Similar to RIP1’s

activation at the TNFR level, polyubiquitination of RIP2 by cIAP1 and cIAP2 is key to the transduction of the signal from Nod proteins to NF-κB and MAPK.37 The Nod signalosome is negatively regulated by caspase-12, which was shown to interact with RIP2 and inhibit Nod signaling by blocking RIP2 ubiquitination.38

3.2.4. The inflammasome

Unlike Nod proteins that signal through RIP2, NlRp proteins, Ipaf, and Naip recruit and activate inflammatory caspases, predominantly caspase-1, in a multiprotein complex known as the inflammasome.39,40 The activation of caspase-1 results in the processing and maturation of the cytokines IL-1β and IL-18. IL-1β and IL-18 exert various effects on different tissues, which result in the induction of fever, anorexia, fatigue, fat catabolism, secretion of acute-phase proteins, and activation of immune cells, leading to the release of other cytokines and chemokines.41 The recruitment of caspase-1 and its activation in the inflammasome mirrors that of caspase-9 in the apoptosome during mitochondrial apoptosis (Figure 32-9). Structurally, the NlRp1 inflammasome observed by cryoelectron microscopy also seems to share with the apoptosome its oligomeric nature.42 Binding of caspases to NLRs occurs through a CARD–CARD interaction, which is either direct, as in the case of Ipaf binding to caspase-143

and NlRp1 binding to caspase-5,44 or indirect, as in the case of PYD-containing NlRp proteins.44 Adaptor molecules mediate the association between caspases and NlRp proteins.40 Two adaptors have been identified, namely a PYD-CARD–containing adaptor termed Apoptosis-associated Speck-like protein containing a Caspase-recruitment domain (ASC) (also known as PYCARD), and Cardinal, a protein with homology to the C-terminal region of NlRp1. Among the inflammatory caspases, caspase-1 is universal to all inflammasomes and is the sole “effector” of cytokine processing. Caspase-5 is recruited to the NlRp1 inflammasome,44 where it acts as a caspase-1 cofactor, but does not appear to be involved in the NlRp3, Ipaf, and Naip5 complexes. Caspase-11, the murine ortholog of caspase-5, has also been suggested to act in vivo as an essential activator of caspase-1. In support of this, caspase-11–deficient mice were shown to be incapable of producing IL-1β and IL-18 in response to LPS stimulation.45 Nonetheless, it seems that the requirement for caspase-11 is not absolute but is restricted to certain stimuli, as caspase-1

could be normally activated in the absence of caspase-11 in response to Listeria infection.46 Caspase-12 has been recently shown to act as an inhibitor of caspase-1.47 The extent of caspase-1 activation appears to vary among inflammasomes. Ipaf, as compared with NlRp3, activates caspase-1 more robustly. This results in rapid caspase- 1–dependent cell death, or pyroptosis (discussed below in 3.3.2), in addition to cytokine maturation and release. It has been recently proposed that, in response to K+ efflux, cells assemble one large ASC oligomer, or speck, per cell. Termed the pyroptosome, this platform is hypothesized to recruit most of the cellular caspase-1

off NLRs, leading to increased caspase-1 activation and cell death.48 In vivo findings, however, do not implicate ASC in pathogen-induced pyroptosis, as cell death proceeds normally in ASC-deficient cells, whereas it is inhibited in caspase-1−/– or Ipaf−/– cells. Both Ipaf and ASC are fully required for IL-1β production in response to Salmonella, Pseudomonas, or Legionella infection, yet only Ipaf-deficient macrophages are fully resistant to pyroptosis induced by these pathogens, whereas ASCdeficient cells are only partially protected.43,49,50,51

The presence of LRRs in NLR proteins is thought to mediate recognition of PAMPs by these cytosolic