Available online at www.sciencedirect.com

ScienceDirect Antimicrobial inflammasomes: unified signalling against diverse bacterial pathogens Matthew JG Eldridge and Avinash R Shenoy Inflammasomes — molecular platforms for caspase-1 activation — have emerged as common hubs for a number of pathways that detect and respond to bacterial pathogens. Caspase-1 activation results in the secretion of bioactive IL-1b and IL-18 and pyroptosis, and thus launches a systemic immune and inflammatory response. In this review we discuss signal transduction leading to ‘canonical’ and ‘non-canonical’ activation of caspase-1 through the involvement of upstream caspases. Recent studies have identified a growing number of regulatory networks involving guanylate binding proteins, protein kinases, ubiquitylation and necroptosis related pathways that modulate inflammasome responses and immunity to bacterial infection. By being able to respond to extracellular, vacuolar and cytosolic bacteria, their cytosolic toxins or ligands for cell surface receptors, inflammasomes have emerged as important sentinels of infection.

is a molecular scaffold that catalyses the auto-proteolytic activation of pro-caspase-1 into its active p20 and p10 subunits [2]. The convergence of pathogen sensing mechanisms on a post-translational event, i.e. caspase-1 activation, sets inflammasome signalling apart from other innate immune networks that control gene expression programmes in the host.

Edited by David Holden and Dana Philpott

Active caspase-1 controls three key events, first, proteolytic processing of pro-IL-1b and pro-IL-18 into their bioactive forms, second, unconventional secretion of mature IL-1b/IL-18, and in most cases also that of mature or pro-IL-1a, and third, cell death by pyroptosis. These cytokines, as well as cell death, promote robust antimicrobial immunity to bacterial infection. Inflammasomes also play key roles in antiviral immunity, microbiome maintenance and autoinflammation, which have been reviewed recently [3,4]. Here we discuss the increasing complexity in inflammasome signalling since the identification of additional caspases, such as murine caspase-11 (represented by two genes, caspase-4 and caspase-5 in humans) and caspase-8, as indispensable upstream mediators of caspase-1 activation in a limited, but notable, number of settings. We also discuss the growing set of accessory signalling networks that modulate inflammasome activation and antimicrobial responses to bacterial pathogens.

http://dx.doi.org/10.1016/j.mib.2014.10.008

Activation of caspase-1 by ‘canonical’ inflammasomes by prion-like polymerization

Addresses Section of Microbiology, Medical Research Council Centre for Molecular Bacteriology and Infection, Imperial College London, Armstrong Road, London SW7 2AZ, UK Corresponding author: Shenoy, Avinash R ([email protected])

Current Opinion in Microbiology 2015, 23:32–41 This review comes from a themed issue on Host-microbe interactions: bacteria

1369-5274/# 2014 Published by Elsevier Ltd.

Introduction Mammalian innate immunity has evolved a diverse repertoire of sensors that respond to microbial infection, such as the Toll-like receptors (TLRs), RIG-I-like receptors/ helicases (RLRs/RLHs), C-type lectin receptors (CLRs), NOD-leucine-rich repeat proteins (NLRs) and AIM2like receptors (ALRs) [1]. Collectively, these proteins orchestrate appropriate responses to bacteria in extracellular or intracellular (including subcellular organellar, vacuolar or cytoplasmic) milieus. In this review we discuss new developments in mechanisms by which the NLR and ALR family of cytosolic proteins sense bacterial infection and assemble a large, multimeric signalling complex called the inflammasome. The inflammasome Current Opinion in Microbiology 2015, 23:32–41

Activation of NLR/ALRs results in their assembly into a single inflammasome ‘speck’ which may act as a signalling hub containing multiple NLR/ALRs and caspases [2,5,6]. NLR/ALR oligomerization has emerged as a common mechanism for caspase-1 activation. Thus, NLRP3 oligomerises in response to K+ efflux, AIM2 by direct binding to double stranded DNA and NLRC4 through oligomeric NAIP (neuronal apoptosis inhibitor protein) proteins (Figure 1). Cutting-edge structural studies recently elucidated a ‘prion-like’ polymerization process nucleated by NLR/ALRs to generate star-like fibres of ASC (Apoptotic speck-associate protein) that form inflammasomes [7,8]. Clustering of pro-caspase-1 within these fibres leads to its activation. Interestingly, assembly driven activation of inflammasomes is analogous to polymerization of proteins that also contain domains of the death domain superfamily such as those present in NLRs/ALRs (Figure 1), for example the MyDDosomes, PIDDosomes, Fas/FADD-DISC and MAVS [9]. www.sciencedirect.com

Antimicrobial inflammasomes Eldridge and Shenoy 33

Figure 1

‘Non-canonical activation’

‘Canonical activation’ 1 PYD NOD

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Current Opinion in Microbiology

Canonical and non-canonical routes of activation of caspase-1 1 & 2 From among 22 NLRs and 4 ALRs in the human and 34 NLRs and 6 ALRs in the mouse, a handful can activate caspase-1 as shown in the schematic above [2]. Names of human proteins appear in capital letters; only the first letter of mouse proteins are capitalised. Canonical inflammasomes can be reconstituted in vitro (for example HEK293 cells) however, non-canonical signalling, as shown in 3 & 4 , requires additional as yet unknown proteins. ALRs have HIN200 domains that bind cytosolic DNA and assemble inflammasomes whereas NLRs shown are not receptors of microbial ligands [2]. Mouse Nlrp1b is activated by proteolysis and can induce cytokine maturation and pyroptosis by caspase-1 independently of Asc [58]. 2 NLRC4/Nlrc4 activation in human and mouse cells requires the NAIP proteins. A single NAIP in human (which detects the type 3 secretion system (T3SS) needle protein) and Naip1-7 in mouse (which detect cytosolic flagellins, T3SS rod or needle proteins) act upstream of NLRC4/Nlrc4. Ligand binding promotes activation and oligomeric assembly of NAIPs [59,60,61,62]. In mouse cells, Asc-independent caspase-1 activation is sufficient for pyroptosis and whether this is also true in human cells has not been tested (dotted line with question mark) [63]. 3 Detection of LPS in the cytosol activates caspase-11, 4 or 5 [14,15,16]. Caspase-11 promotes the assembly of Asc via Nlrp3 and this activates caspase-1. In the non-canonical pathway, caspase-11 controls pyroptosis and IL-1a release independently of Nlrp3, Asc and caspase-1. A lack of understanding of how caspase-11/4/5 activate Nlrp3 is indicated by (?). 4 Yersinia infection triggers caspase-1 activation via caspase-8, which itself is activated through proteins in necroptosis associated pathways — Ripk1 (receptor-interacting protein kinase-1), Ripk3 and Fadd (Fas associated protein with death domain) [17,18]. The mechanism of caspase-8-dependent caspase-1 activation and the role of Ripk3-dependent necroptosis are unclear and indicated by (?) marks. Domain compositions are colour coded and abbreviated as follows: BIR — baculovirus inhibitor of apoptosis domain; CARD — caspase-activation and recruitment domain; DD — death domain; DED — death effector domain, HIN200 — haematopoietic expression, interferon inducible, nuclear localized (HIN) DNA binding domain of 200 residues; NOD, nucleotide binding and oligomerization domain; LRR — leucine rich repeat, p20 & p10 — large and small catalytic subunits; PK — protein kinase; PYD — pyrin domain, RHIM — Receptor-interacting protein (RIP) homotypic interaction domain. CARD, PYD, DED and DD are related in structure but less so in sequence.

‘Non canonical’ activation of caspase-1 and the involvement of upstream caspases

An indispensable upstream role for capsase-11 in activating Nlrp3-Asc-caspase-1 was first identified in cells infected with non-pathogenic E. coli or treated with a combination of LPS and cholera toxin B (which delivers www.sciencedirect.com

LPS into the cytosol; Figure 1) [10,11,12]. How caspase-11 controls the assembly of Asc specks by Nlrp3 in this scenario is not yet understood. LPS is detected independently of Tlr4 when present in the cytosol by the activation of caspase-11 or human caspase-4 and caspase-5 [13,14,15]. However, all other Nlrp3 activating Current Opinion in Microbiology 2015, 23:32–41

34 Host-microbe interactions: bacteria

Figure 2

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8 Current Opinion in Microbiology

Inflammasome activating signals during bacterial infections Commonly activated inflammasomes during bacterial infection are illustrated and numbered. 1 Human NAIP and mNaip1 bind needle proteins from Gram negative, vacuolar, cytosolic or surface adherent bacteria and activate NLRC4. 2 Mouse Naip2 analogously detects indicated T3SS rod proteins. 3 Mouse Naip5 and Naip6 detect bacterial flagellins and are differentially expressed in different cell types. 4 Modification of Rho GTPases by indicated toxins activates human and mouse Pyrin. Glucosylation (TcdB), adenylylation (VopS and IbpA), ADP-ribosylation (C3) and asparagine deamidation within the switch I region of Rho GTPases are sensed by Pyrin [64]. ?(Bc); unknown T6SS effector; blue ?, unknown signalling protein. 5 Cytosolic DNA from some bacteria can be detected by Aim2. The related IFI16 is a sensor for viral infection in humans and whether it can detect bacterial DNA remains to be tested. 6 Mouse Nlrp6 acts as a negative regulator of inflammatory pathways, helps maintain commensal flora, prevents colonic tumorigenesis and may have inflammasome related roles [65]. The Nlrp6 activating signal remains unknown and indicated by a blue ?. 7 Human NLRP7 detects acylated lipoproteins and restricts bacterial replication [66]. 8 Anthrax lethal toxin induces proteolytic cleavage within the N-terminal region of Nlrp1b which activates it [67]. 9 Yersinia expressing YopJ trigger caspase-8-dependent activation of caspase-1 [17,18]. 10 Y. pestis is detected by Nlrp12 in the mouse [24]. 11 Indicated pore forming toxins stimulate K+ efflux from cells and activate NLRP3 [2]. 12 ATP is a major extracellular ligand that activates inflammasomes, in addition to others such as Tim3-Gal9 and the Complement system [31,32]. 13 Unknown sensors detect RNA:DNA hybrids and activate caspase-1 via Nlrp3 [68]. Salmonella lacking or poorly expressing SPI-1 T3SS (stationary phase bacteria) are detected by both Nlrc4 and Nlrp3 inflammasomes when SPI-2 T3SS is expressed later in infection [20,69]. 14 Transfection of purified LPS from Salmonella, Legionella, Escherichia, Shigella, Burkholderia, Pseudomonas activates caspase-11 [10,11,14,15]. However, Helicobacter, Rhizobium, Francisella and Yersinia (grown at 37 8C) evade detection by this pathway as a consequence of modifications of their LPS [14]. Stationary phase Salmonella DSPI-2 or Dflagellin and Legionella Dflagellin mutant strains are sensed by Nlrp3, most likely by detection of their LPS in the cytosol [19,20]. Abbreviations: Ah — Aeromonas hydrophila; Ba — Bacillus anthracis; Bc — Burkholderia cenocepacia; Bt — B. thailandensis; Cv — Chromobacterium violaceum; Cb — Clostridium botulinum; Cd — C. difficile; EHEC — Enterohaemorrhagic E. coli;

Current Opinion in Microbiology 2015, 23:32–41

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Antimicrobial inflammasomes Eldridge and Shenoy 35

signals, such as ATP or pore forming toxins (see next section and Figure 2), promote Nlrp3-driven Asc assembly independently of caspase-11. Therefore the involvement of caspase-11 is a special signalling modality of the Nlrp3Asc-caspase-1 axis when responding to cytosolic LPS — a ‘non-canonical’ mechanism of caspase-1 activation. This is unique to Nlrp3; all other inflammasome sensors, for example Aim2, Nlrp1 and Nlrc4, activate caspase-1 independently of caspase-11 (Figure 1). A recent study identified LPS binding sites within the CARD domains of caspase-11 and human caspase-4 and 5 [16]. LPS binding results in their oligomerization and activation [16]. An important feature is that during non-canonical activation caspase-11 directs pyroptotic cell death and IL-1a secretion, both of which are under the control of caspase-1 in canonical settings; IL-1b and IL-18 maturation is caspase-1-dependent in both pathways (Figure 1) [10,11,14,15]. The non-canonical activation of caspase-1 by caspase-4 or caspase-5 is yet to be demonstrated in human cells, however, caspase-4 and caspase-5 trigger pyroptosis in response to cytosolic LPS and are therefore functionally orthologous to murine caspase-11 [16]. Therefore, the term ‘non-canonical pathway’ should be preferred over ‘non-canonical inflammasomes’ as the latter gives the (incorrect) impression of the existence of an analogous signalling complex that has not yet been identified. In addition, some bacteria, such as Yersinia, trigger caspase-8-dependent activation of caspase-1 independently of Nlrp3, Nlrc4 and Asc and could therefore also be considered non-canonical (Figures 1 and 2) [17,18]. Thus, while not universally required, additional caspases acting upstream of caspase-1 may be critical in response to some infections.

There are conflicting reports on how Mtb elicits IL-1b maturation. However, the T7SS appears essential. Mtb infected human monocytes, macrophages and dendritic cells release IL-1b in a partially caspase-1-independent manner [21,22]. For example, Dectin1-mediated detection of Mtb in human dendritic cells can result in direct IL1b processing by caspase-8 independently of caspase-1 [23]. In the case of Yersinia, the T3SS effector YopJ supresses NF-kB and MAPK signalling and triggers the Ripk1(Receptor interacting kinase), Fadd- (Fas-associated death domain protein), and caspase-8-dependent pathway of caspase-1 activation, cytokine processing and cell death (Figures 1 and 2) [17,18]. Yersinia pestis is detected by Nlrp12 and Nlrp3 which together promote IL-18/ immunity in mice [24] caspase-1-dependent (Figure 2). The molecular determinants of Nlrp12 activation are presently unknown. Bacterial effectors may also subvert inflammasome signalling. For example, Yersinia which lack all known T3SS effectors but retain a functional injectisome are sensed solely by Nlrp3 [25]; however, this pathway is naturally suppressed by YopK which binds to the T3SS translocon and prevents its detection. Similarly, subversion of Nlrc4and Nlrp3-dependent detection of Salmonella requires bacterial oxidative phosphorylation genes such as aconitase (acnB), isocitrate dehydrogenase (icdA) and isocitrate lyase (aceA) [26]. Mtb subverts Aim2 and Nlrp3 inflammasomes in mouse cells in a T7SS dependent manner [27].

Cytosolic bacteria and toxins

Activation of inflammasomes during bacterial infections Vacuolar bacteria and the role of secretion systems

Specialised secretion systems, for example, the type 3 secretion system (T3SS), T4SS and T7SS in Salmonella, Legionella and Mycobacterium tuberculosis (Mtb) respectively, are critical for inflammasome activation by these vacuolar pathogens. In the special case of T3SS, structural needle and rod proteins of the injectisome are detected by the NAIP-NLRC4 pathway (Figures 1 and 2). In addition, flagellin can be translocated by both T3SS and T4SS and is detected through Naip5/Naip6-Nlrc4 in mice (Figures 1 and 2). Salmonella and Legionella lacking T3SS/T4SS/flagellins are detected by the non-canonical caspase-11 pathway presumably via their LPS (Figures 1 and 2) [19,20].

A large array of cytosolic toxins can be sensed by Nlrp1b, Nlrp3 and Pyrin, which reveals the versatility of inflammasomes in detecting changes to host cytosolic components (Figure 2). Infection by wild-type strains of cytosolic Gram negative bacteria such as Burkholderia, Vibrio, Proteus and Haemophilus predominantly trigger the non-canonical caspase-11 pathway [11]. Other bacteria, for example Salmonella, Legionella, Pseudomonas, Shigella or pathogenic E. coli, also have LPS that is detectable by caspase-11 when transfected directly into the cytosol. However, they express more potent activators of the NAIPNLRC4 axis (such as the secretion systems discussed above; see Figure 2) which override the caspase-11 pathway. Thus, in the case of some Gram negative bacteria non-canonical activation via caspase-11 is triggered only in experimental conditions using strains that lack

( Figure 2 Legend Continued ) Fn — Francisella novocida; Ft — F. tularensis; Hs — Histophilus somni; KSHV — Kasposi’s sarcoma-associated herpes virus; Lm — Listeria monocytogenes; Lp — Legionella pneumophila; Mt — Mycobacterium tuberculosis; Pa — Pseudomonas aeruginosa; Pl — Photorhabdus luminescence; Sa — Staphylococcus aureus; Sf — Shigella flexnerii; Sp — Streptococcus pneumoniae; St — Salmonella enterica Typhimurium; Td — Treponema denticola; Vc — Vibrio cholerae; Vp — V. parahemolyticus; Ye — Yersinia enterocolitica; Yp — Y. pseudotuberculosis; Ype — Y. pestis. www.sciencedirect.com

Current Opinion in Microbiology 2015, 23:32–41

36 Host-microbe interactions: bacteria

other activators, for example Salmonella [20] or Legionella [19] lacking flagellins and/or secretion systems. Signalling by host–cell surface receptors

Binding of ATP to its P2X7 receptor triggers K+ efflux and activates Nlrp3 mediated cytokine and caspase-1 processing, secretion and cell death (Figure 2) [28,29]. More broadly, during infection dying cells are major sources of extracellular ATP in vivo [30]. In addition, Td92 expressed by Treponema denticola binds integrin a5b1 on macrophages and modulates ATP release [28]. Similarly, the C3a-receptor can stimulate ATP release from cells [31]. In a poorly understood pathway, Tim3 expressed on T-helper cells binds galectin9 which decorates Mtb infected macrophages and activates caspase-1 in the macrophage [32]. This promotes cell-autonomous immunity in Mtb infected macrophages. Thus, secretion systems, secreted effectors and toxins, as well as host pathways can direct caspase-1 activation. Furthermore, the deployment of inflammasomes as common effectors of multiple sensory pathways may provide fail-safe mechanisms for the host to counter bacterial evasive strategies.

Modulators of inflammasome signalling During infection, expression of NLRs/ALRs, caspases and pro-IL-1b is transcriptionally induced by upstream sensory pathways [1]; thus, lack of ‘priming’ of cells results in low expression of ‘core’ signalling components and blunted inflammasome responses [2]. Below, we discuss newly identified pathways that affect post-translational inflammasome activation (Figure 3). Guanylate binding proteins (GBPs)

GBPs are a family of interferon-inducible, large (65– 72 kDa) antimicrobial GTPases which include 7 members encoded at a single locus on human chromosome (Chr) 1. In the mouse, 11 Gbps are encoded at two loci on Chr3 (5 Gbps) and Chr5 (6 Gbps) [33]. Gbp1, Gbp2, Gbp6/10 and Gbp7 provide robust cell-autonomous immunity by trafficking to Listeria and Mycobacterium containing vacuoles and restricting bacterial growth [34]. Gbp1 / , Gbp2 / , Gbp5 / and GbpChr3 (lacking all Chr3 GBPs i.e. Gbp1, Gbp2t1 (a trans-spliced variant), Gbp2, Gbp3, Gbp5 and Gbp7) mice are thus susceptible to bacterial or protozoal infections [34,35,36]. However, unlike mammalian GBPs, some lower vertebrates such as zebra fish have GBPs that contain NLR-like CARD domains which first hinted at their involvement in inflammasome signalling [36]. GBP5 assists the oligomerization of ASC by NLRP3 in IFN-g activated macrophages exposed to canonical signals such as K+ efflux, live Salmonella or Listeria (Figure 3). Gbp5 / mice show reduced caspase-1 activation and IL-1b levels in vivo and are susceptible to these bacteria. GBP5 is unique in being dispensable for activation of NLRP3 by sterile particulates (for Current Opinion in Microbiology 2015, 23:32–41

example, alum and uric acid crystals), NLRC4 or AIM2 inflammasomes [36]. Thus, GBP5 acts as an inducible modulator of selective inflammasome-dependent host defence pathways. The mechanisms for its selectivity and the role of its GTPase activity are presently unclear but are likely to involve differential vacuolar trafficking. Two recent studies have offered conflicting evidence on the role of GBPs in non-canonical signalling [37,38]. These papers showed that GbpChr3 cells are resistant to pyroptosis when infected with Salmonella, Legionella, Enterobacter, Vibrio and Citrobacter. Therefore one or more Chr3 GBPs may be involved in caspase-11-dependent pyroptotic cell death. However, the two reports differ in the mechanisms they propose (Figure 3) [37,38]. Pilla et al. [38] tested whether less cell death was because of reduced capase-11 activation as a result of an inability of phagocytosed bacteria to escape from vacuoles. However, SalmonellaDsifA or LegionellaDsdhA mutant strains, which readily escape vacuoles and activate caspase-11 in wild-type macrophages, failed to do so in GbpChr3 cells despite comparable efficiency in escape from vacuoles in these cells. In agreement with these observations, caspase-11 activation, pyroptosis and IL-1b maturation in response to transfected LPS was also greatly reduced in GbpChr3 cells. This led the authors to conclude that Chr3 GBPs are required for ‘downstream’ caspase-11 activation rather than destabilising bacterial vacuoles (Figure 3) [38]. On the other hand, Meunier et al. [37] found slightly fewer cytosolic Salmonella in GbpChr3 cells which suggested a role for Chr3GBPs in the rupture of bacterial vacuoles. Further, Meunier et al. found normal caspase-11 activation in GbpChr3 cells when transfected with LPS and therefore ascribed a role for Chr3GBPs in promoting vacuole disruption rather than caspase-11 activation (Figure 3) [37]. The use of different backgrounds of bacterial strains, priming conditions (for example, use of naı¨ve, LPS or IFN-g primed) may explain some of these discrepancies. Moreover, Pilla et al. [38] used Galectin3YFP whereas Meunier et al. [37] used endogenous Galectin8 as markers of damaged phagosomes to enumerate ‘cytosolic’ bacteria indirectly. Clearly, a number of questions remain unanswered, such as which GBP(s) on Chr3 are involved in pyroptosis and what their roles are in human inflammasome signalling. It is tempting to speculate that different GBP GTPases may affect inflammasome signalling at different steps in the pathway. Caspase-8 and necroptosis related pathways

Caspase-8 triggers classical apoptosis in response to Fasligand [39]. However, it also acts as a negative regulator of necroptotic cell death triggered by TNF-a via Ripk1 or Ripk3; therefore Casp8 can be deleted only when Ripk1 or Ripk3 are also deleted to prevent embryonic lethality in mice [39]. Ripk1 and Ripk3 mediated necroptosis is www.sciencedirect.com

Antimicrobial inflammasomes Eldridge and Shenoy 37

Figure 3

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IL-18 Current Opinion in Microbiology

Regulatory networks that modulate inflammasome signalling 1 Nlrp3 activation by canonical signals can be regulated by nitrosylation [48], ubiquitylation [46] and other proteins such as GBP5 [36], BRCC3 [47] and MAVS [50–53]. Syk and Jnk phosphorylate Asc (Tyr144 in mAsc) which promotes its assembly by Nlrp3 and Aim2 (not depicted) [41]. 2 Activation of Nlrc4 by Salmonella or Legionella requires its phosphorylation on Ser353 by Pkcd [42]. 3 The Tlr4-typeI IFN signalling axis triggered by LPS from Gram-negative bacteria, such as Citrobacter, promotes caspase-8 activation via Fadd [40]. How caspase-8 controls caspase-11 or caspase-1 activation is presently unclear (? marks). 4 Both Fadd and caspase-8 are required for the transcriptional upregulation of Nlrp3 and pro-IL-1b expression (purple arrows) [40]. 5 The interferon inducible GBPs GTPases encoded at the locus on mouse Chr3 (Chr3GBPs) are required for caspase-11-dependent pyroptosis triggered by Gram negative bacteria such as Salmonella and Legionella [37,38]. There are conflicting reports on the mechanisms by which they promote caspase-11 activation. Chr3GBPs may promote the destabilization of bacteria containing vacuoles and bacterial release into the cytosol to allow detection of LPS by caspase-11 [37]. Alternatively, they might act at a step more proximal to caspase-11 without affecting vacuolar integrity [38]. (?) marks indicate a need for further clarification of the mechanisms involved.

suppressed by a signalling complex containing Fadd, FLIP (also called Cflar, caspase-8 and Fadd-like apoptosis regulator) and caspase-8 [39]. The role of these proteins in inflammasome activation was recently assessed. Defects in NF-kB signalling in Casp8 / cells result in lower caspase-1 activation and IL-1b levels due to poor expression of Nlrp3 and pro-IL-1b [6,40]. However, caspase-8 also has a post-translational role in caspase-1/IL-1b activation during Yersinia and Citrobacter rodentium infection (Figures 1 and 3). Interestingly, caspase-8 acts upstream of caspase-11 during C. rodentium infection (Figure 3). The contribution of caspase-8 in celldeath dependent immunity to infection is unclear at present. However, the interplay of necroptosis and pyroptosis are important findings that should clarify www.sciencedirect.com

mechanisms of these new forms of programmed cell death during infection. Protein kinases PKCd, Syk, Jnk and PKR

A chemical screen identified Syk and Jnk dependent regulatory phosphorylation of ASC in speck formation by NLRP3 and AIM2; however, these kinases are dispensable for ASC assembly by NLRC4 [41]. On the other hand, mNlrc4 undergoes an essential phosphorylation at a conserved Ser533 residue (also present in hNLRC4) upon Salmonella or Legionella infection in a protein kinase delta (PKCd)-dependent manner (Figure 3) [42]. Protein kinase R (PKR) has been implicated in inflammasome activation [43,44]; however, this was not seen in a later study [45]. Current Opinion in Microbiology 2015, 23:32–41

38 Host-microbe interactions: bacteria

Post-translational modifications of NLRP3

NLRP3 is rapidly deubiquitylated by BRCC3 upon exposure of cells to LPS and this is required for its full activation [46,47]. The ubiquitin ligase that ubiquitylates NLRP3 and whether this may be exploited by bacterial pathogens remains to be investigated. During Mtb infection high nitric oxide levels induced by IFN-g result in nitrosylation of Nlrp3 leading to suppression of its activity and inflammation in vivo (Figure 3) [48]. However, caspase-1- and Nlrp3-independent IL-1b production appears to be protective against Mtb infection [21,49]. Interestingly, nitrosylation-dependent inhibition of Nlrp3 is observed after a short (2–4 h) IFN-g priming [48], however, longer priming (12–18 h) promotes activation of human and mouse Nlrp3 by Gbp5 [36].

IL-18 secretion; both these cytokines are leaderless proteins that exit cells by unconventional methods. Research in this direction recently identified a membrane channel that negatively regulates IL-1b secretion, TRPC1, which is proteolytically cleaved and inactivated by caspase-11 [57]. The search for other such mediators should provide a better understanding of how the inflammasomes act as effector mechanisms in immunity against bacterial infections.

Acknowledgements We would like to apologise to colleagues whose work we could not cite due to space limitations. This work was supported by funds from Imperial College London and the Royal Society grant RG130811 to AS. AS would like to thank Sandhya Visweswariah for critical reading of the manuscript.

References and recommended reading Mitochondrial antiviral signalling protein (MAVS)

MAVS is a CARD containing protein involved in type I interferon induction by RLRs in promoting antiviral immunity. It has dual localization on mitochondria and peroxisomes from where it signals different transcriptional programmes. Recent reports implicated MAVSNLRP3 interaction in localizing NLRP3 to mitochondria for full activation of ASC assembly [50,51]; however, two subsequent reports have been unable to confirm this and further studies are required to clarify its role [52,53]. Autophagy suppresses inflammasome activity [37,54,55]; however, bacterial interaction with autophagy pathways is particularly complex [56] and beyond the scope of this review. Thus, multi-layered regulatory circuits involving post-translational modifications, trafficking and caspasecascades modulate inflammasome signalling and immunity during bacterial infection.

Conclusions and perspectives The inflammasomes are versatile signalling networks that enable host cells to respond rapidly by post-translational activation of caspase-1 and downstream events. Modular protein–protein interactions and regulated oligomerization and assembly are common features of all inflammasome pathways. Additionally, signalling is also controlled by post-translational events and actions of cytokines such as the interferons on host cells. Bacterial pathogens seem to have evolved mechanisms to promote or suppress inflammasomes, and on the other hand, multiple inflammasomes may be deployed to sense a particular pathogen. Many unanswered questions remain, related to the mechanisms of activation of inflammasomes through non-canonical routes and the roles of caspase-4 and 5 in human disease. How do non-pathogenic E. coli, which do not possess the machinery to breach phagosomal membranes, activate non-canonical signalling? What is the importance of this pathway during infection by wild-type strains of Gram negative bacterial human pathogens? Also poorly understood are the precise molecular events that follow caspase-1 activation leading to cell death, IL-1b and Current Opinion in Microbiology 2015, 23:32–41

Papers of particular interest, published within the period of review, have been highlighted as:  of special interest  of outstanding interest 1.

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2.

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Man SM, Hopkins LJ, Nugent E, Cox S, Gluck IM, Tourlomousis P, Wright JA, Cicuta P, Monie TP, Bryant CE: Inflammasome activation causes dual recruitment of NLRC4 and NLRP3 to the same macromolecular complex. Proc Natl Acad Sci USA 2014, 111:7403-7408. This study shows the recruitment of Nlrp3, Nlrc4, caspase-1 and caspase-8 to endogenous Asc foci in Salmonella infected human cells.

6. 

Man SM, Tourlomousis P, Hopkins L, Monie TP, Fitzgerald KA, Bryant CE: Salmonella infection induces recruitment of Caspase-8 to the inflammasome to modulate IL-1beta production. J Immunol 2013, 191:5239-5246. This is the first study to identify an inflammasome-priming role for caspase-8.

7. 

Lu A, Magupalli VG, Ruan J, Yin Q, Atianand MK, Vos MR, Schroder GF, Fitzgerald KA, Wu H, Egelman EH: Unified polymerization mechanism for the assembly of ASCdependent inflammasomes. Cell 2014, 156:1193-1206.

8. 

Cai X, Chen J, Xu H, Liu S, Jiang QX, Halfmann R, Chen ZJ: Prionlike polymerization underlies signal transduction in antiviral immune defense and inflammasome activation. Cell 2014, 156:1207-1222. Above two reports demonstrate a prion-like polymerization driven mechanism that generate inflammasome fibres within which caspase1 is activated.

9.

Kersse K, Verspurten J, Vanden Berghe T, Vandenabeele P: The death-fold superfamily of homotypic interaction motifs. Trends Biochem Sci 2011, 36:541-552.

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Antimicrobial inflammasomes Eldridge and Shenoy 39

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40 Host-microbe interactions: bacteria

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This study identified the internal proteolytic event within Nlrp1b catalysed by anthrax lethal factor as being sufficient for Nlrp1b inflammasome activation.

This interesting study identified bacteria RNA:DNA hybrids in the cytosol as activating ligands of an as yet unidentified inflammasome.

68. Kailasan Vanaja S, Rathinam VA, Atianand MK, Kalantari P,  Skehan B, Fitzgerald KA, Leong JM: Bacterial RNA:DNA hybrids are activators of the NLRP3 inflammasome. Proc Natl Acad Sci U S A 2014, 111:7765-7770.

69. Broz P, Newton K, Lamkanfi M, Mariathasan S, Dixit VM, Monack DM: Redundant roles for inflammasome receptors NLRP3 and NLRC4 in host defense against Salmonella. J Exp Med 2010, 207:1745-1755.

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Current Opinion in Microbiology 2015, 23:32–41