Advances in Nod-like receptors (NLR) biology

Cytokine & Growth Factor Reviews xxx (2014) xxx–xxx

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CGFR-802; No. of Pages 17

Survey

Advances in Nod-like receptors (NLR) biology

Francois Barbe a, Todd Douglas a, Maya Saleh a,b,*a Department of Microbiology and Immunology, McGill University, Montreal, Quebec H3A 2B4, Canadab Department of Medicine, McGill University, Montreal, Quebec H3G 0B1, Canada

Contents

1. Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 000

2. Nlrs that do not assemble inflammasomes. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 000

2.1. NLRA (CIITA) . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 000

2.2. The NLRC sub-family . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 000

2.2.1. NOD1 and NOD2 . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 000

2.2.2. NLRC3. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 000

2.2.3. NLRC5. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 000

2.2.4. NLRX1 . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 000

3. Inflammasome-forming Nlrs . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 000

3.1. NLRP1 . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 000

3.2. NLRP3 . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 000

3.2.1. Priming. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 000

3.2.2. Post-translational modifications . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 000

3.2.3. Activation mechanisms . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 000

3.2.4. Lysosomal destabilization . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 000

3.2.5. Ion flux. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 000

3.2.6. Mitochondria . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 000

3.2.7. Binding partners of the NLRP3 inflammasome. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 000

3.3. NLRP6 . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 000

3.4. NLRP7 . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 000

3.5. NLRP10 . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 000

3.6. NLRP12 . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 000

3.7. NLRC4 and NLRB . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 000

A R T I C L E I N F O

Article history:

Available online xxx

Keywords:

NLR

Innate immunity

Inflammasome

Inflammation

Cell death

A B S T R A C T

The innate immune system is composed of a wide repertoire of conserved pattern recognition receptors

(PRRs) able to trigger inflammation and host defense mechanisms in response to endogenous or

exogenous pathogenic insults. Among these, nucleotide-binding and oligomerization domain (NOD)-like

receptors (NLRs) are intracellular sentinels of cytosolic sanctity capable of orchestrating innate

immunity and inflammatory responses following the perception of noxious signals within the cell. In this

review, we elaborate on recent advances in the signaling mechanisms of NLRs, operating within

inflammasomes or through alternative inflammatory pathways, and discuss the spectrum of their

effector functions in innate immunity. We describe the progressive characterization of each NLR with

associated controversies and cutting edge discoveries.

� 2014 Published by Elsevier Ltd.

Contents lists available at ScienceDirect

Cytokine & Growth Factor Reviews

jo ur n al ho mep ag e: www .e lsev ier . c om / loc ate /c yto g f r

* Corresponding author at: McGill Life Sciences Complex, Bellini Pavilion,

Rm.364, 3649 Promenade Sir-William Osler, Montreal, Quebec H3G0B1, Canada.

Tel.: +1 514 398 2065; fax: +1 514 398 2603.

E-mail address: [email protected] (M. Saleh).

Please cite this article in press as: Barbe F, et al. Advances in Nod-like receptors (NLR) biology. Cytokine Growth Factor Rev (2014),http://dx.doi.org/10.1016/j.cytogfr.2014.07.001

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4. Conclusion and future perspectives . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 000

Acknowledgements . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 000

References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 000

1. Introduction

The innate immune system is equipped with a set of receptors,termed pattern recognition receptors (PRRs), that detect imminentdangers such as microbial invasion, environmental or endogenousnoxious substances, and elicit protective responses to contain andeliminate these harmful triggers while providing the host withresistance mechanisms to tolerate damage and restore normalcy.PRRs can be classified into five main classes that include Toll-likereceptors (TLRs), nucleotide binding and oligomerization domain(NOD)-like receptors (NLRs), retinoic acid-inducible gene-I (RIG-I)-like receptors (RLRs), absent-in-melanoma (AIM)-like receptors(ALRs) and C-type lectins (CTLs). Membrane-bound receptorssurvey the extracellular environment, whereas intracellular PRRssuch as the NLRs ensure cytosolic sanctity by acting as criticalback-up defenses and providing synergistic responses in the face ofpersistent danger. The presence of NLRs across different speciesand kingdoms of life indicates that they are an essential product ofevolution, which is consistent with their conservation from plantsto humans [1]. NLRs are composed of three functional domains: anN-terminal protein–protein interaction domain required for signaltransduction, a central NACHT (or NBD) domain necessary foroligomerization, and a C-terminal leucine-rich repeat (LRR) thatconfers ligand recognition, but acts as a repressor of NLR signalingin the absence of ligand stimulation by masking the N-terminaldomain [2]. Mammalian NLRs are classified according to the type oftheir N-terminal domain: NLRA or Class II transactivator (CIITA)contains an acidic transactivation domain, NLRBs or neuronalapoptosis inhibitor proteins (NAIPs) have a baculovirus inhibitor ofapoptosis protein (IAP) repeat (BIR), NLRCs possess a caspase-activation and recruitment domain (CARD), and NLRPs a pyrindomain (PYD) [3]. NLRX1 contains a CARD-related X effectordomain of unknown function. Upon activation, NLRs scaffold largesignaling complexes to mediate innate immune responses such asthe induction of inflammation, autophagy or cell death. NLRP1,NLRP3, NLRP6, NLRP7, NLRP12, NLRC4 and NAIP operate throughthe formation of inflammasomes, signaling platforms dependenton the inflammatory protease caspase-1. On the other hand,NLRP10, NOD1, NOD2, NLRC3, NLRC5, NLRX1 and CIITA functionindependently of the inflammasome and mediate innate immunitythrough the regulation of nuclear factor-kB (NF-kB) and mitogen-activated protein kinases (MAPK) pathways, or by acting in thenucleus as transcriptional regulators.

2. Nlrs that do not assemble inflammasomes

2.1. NLRA (CIITA)

Class II transactivator (CIITA) is a key regulator of MHC class Iand II gene expression [4]. Its role as an MHC class II gene regulatorwas established when a 24 amino acid deletion splice mutant ofCIITA was identified in patients with bare lymphocyte syndrome(BLS) [5]. It is constitutively expressed in cells with high MHCclass II expression, such as dendritic cells (DCs) and macrophages,but is highly inducible by IFNg in many other cell types [6,7].CIITA consists of the conventional tripartite architecture of NLRs,but also comprises three additional N-terminal domains: anacidic domain (AD), a guanosine-binding domain (GBD) and aproline/serine/threonine (PST) domain. The AD and GBD mediate

Please cite this article in press as: Barbe F, et al. Advances in Nod-lihttp://dx.doi.org/10.1016/j.cytogfr.2014.07.001

interactions with general transcription factors, DNA-bindingtransactivators and chromatin-remodeling enzymes to form anenhanceosome [8]. The GBD also contains a nuclear localizationsignal (NLS) that permits trafficking between the cytosol and thenucleus [6]. The PST is essential for CIITA function but its preciserole is unclear. The class II transactivator function of CIITA isregulated by phosphorylation by a number of protein kinases suchas PKA, PKC, glycogen synthase kinase (GSK)3 and casein kinase(CK)II on various sites within the AD, PST and LRR domains [9].Within the enhanceosome, CIITA recruits histone acetyltrans-ferases, such as p300 and CBP, and methyltransferases, such asCARM1, to induce transcription, and histone deacetylases, such asHDAC1, HDAC2 and HDAC5 to repress it [10,11]. Beyond thisfunction, CIITA can also act as a general transcription factor in theTFIID basal transcription complex in response to IFNg [12]. Itinteracts with components of the TFIID machinery, includingTATA-binding protein (TBP), TAF6, TAF9, P-TEFb and TFIIB [13,14].Furthermore, it functionally replaces the TFIID complex compo-nent TAF1 (TATA-binding protein associated factor 1) by mediatingsimilar auto-phosphorylation events that promote the dissociationof the inhibitory factor TAF7, allowing transcriptional initiation[15]. Thus, CIITA possesses dual functions, acting as a co-activatorthat nucleates an enhanceosome and a general transcription factorsimilar to TAF1.

2.2. The NLRC sub-family

2.2.1. NOD1 and NOD2

Nunez et al. first discovered the NLRC family members NOD1(CARD4) and NOD2 (CARD15) in 1999 and 2001, respectively[16,17]. Two groups simultaneously reported that NOD2 isactivated by muramyl dipeptide (MDP), a bacterial cell wallmoiety derived from peptidoglycan [18,19]. NOD1 was shown tobind gamma-D-glutamyl-meso-diaminopimelic acid (iE-DAP), alsoderived from bacterial peptidoglycan [20]. Interestingly, the directbinding of these receptors by their respective ligands was onlyrecently proven. NOD1 was shown to directly interact with ie-DAPvia its LRR motif [21], and biochemical and in vitro analyses showedthat NOD2 directly bound to MDP [22,23]. Coulombe et al. recentlycharacterized N-glycolyl MDP as a more potent activator of NOD2[24].

Upon activation, NOD1 and NOD2 undergo oligomerizationthrough their central NOD domain, enabling the recruitment ofthe kinase RIP2 (RICK) through a CARD-CARD homotypicinteraction [25]. The essential role of RIP2 in NOD signalingwas illustrated by the abrogation of NOD-dependent NF-kBactivation in Rip2-deficient mice [26]. Rip2 engagement by theNOD receptors leads to its K63-linked ubiquitination by thecellular inhibitors of apoptosis proteins (cIAP)1 and cIAP2 [27],enabling the recruitment of the TAK1/TAB2/TAB3 kinase complexto RIP2. X-linked inhibitor of apoptosis protein (XIAP) similarlyinteracts with RIP2 and serves as a recruitment platform for thelinear ubiquitination assembly complex (LUBAC) that conjugateslinear ubiquitin chains to the IKK complex regulatory componentNEMO to mediate NF-kB activation [28]. NOD1 and NOD2stimulation also results in the activation of p38, c-JUN N-terminalkinase (JNK) and ERK MAPK pathways [26]. Together, thesesignaling pathways converge on the induction of pro-inflamma-tory cytokines, chemokines and antimicrobial peptides to elicitinnate immunity (Fig. 1a).

ke receptors (NLR) biology. Cytokine Growth Factor Rev (2014),


Fig. 1. Activation of the NOD1 and NOD2 pathways. (A) Bacterial peptidoglycan fragments can access the cytosol via the endosomal SLC15A channel, the hPepT1 plasma

membrane transporter or scavenger receptors (SR-A, MARCO). MDP and iE-DAP from peptidoglycan activates NOD1 and NOD2, respectively. Following direct or indirect

peptidoglycan sensing, NOD1/2 are relocalized to the plasma membrane in complex with ERBIN, FRMPD2 or DUOX2. Activation of NOD1 or NOD2 leads to their

oligomerization, promoting the recruitment of RIP2, TRAF2, TRAF5, TRAF6, cIAP1, cIAP2 and XIAP. The cIAPs conjugate K63-linked ubiquitin chains to RIP2, subsequently

recruiting the TAB-TAK complex. XIAP-generated polyubiquitination of RIP2 recruit the linear ubiquitin complex (LUBAC), which then targets NEMO in the IKK complex with

linear polyubiquitin that is required for maximal activation. IKKb is phosphorylated by TAK1 and in turn phosphorylates the NF-kB inhibitor IkB. Phosphorylated IkB is

targeted to the proteasome for degradation following ubiquitination. p50-p65 dimers are free to enter the nucleus to induce NF-kB-dependent gene expression. TAK1 also

activates the MAPK cascade, stimulating AP-1-dependent gene expression. Activation of the NODs also initiates autophagy. Several positive (green) and negative (red)

regulators of this pathway have been identified, which can act at the level of NOD1/2, RIP2 or elsewhere. (B) NOD1 and NOD2 possess antiviral functions. Viral RNA can

activate NOD2 and signal through the mitochondrial MAVS independently of RIP2, which induces the activation of IRF3, promoting its translocation to the nucleus to induce

type I IFN. Influenza viral RNA induces mitophagy following the phosphorylation of ULK1 by RIP2.

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2.2.1.1. Membrane recruitment. Even though the NOD receptorsare firmly established as cytosolic sensors of pathogenic insults,the membrane re-localization of these receptors upon activationwas reported as a required step in MDP-induced NF-kB activation.Barnich et al. generated NOD2 deletion and substitution mutantsthat were incapable of inducing NF-kB activation upon MDPstimulation in intestinal epithelial cells (IECs). These mutants, aswell as the Crohn’s disease (CD)-associated NOD2 3020insCmutant, lack the leucine- and tryptophan-containing motif atthe C-terminal end of the protein, which was proven to be requiredfor plasma membrane association [29]. It was later shown thatNOD2 is responsible for the membrane recruitment of RIP2 [30].FERM and PDZ domain containing 2 (FRMPD2), in complex withERBB2-interacting protein (ERBIN), were subsequently implicatedin NOD2 recruitment to the basolateral membrane of polarizedIECs [31,32]. NOD1 was also shown to localize to the membraneduring Shigella flexneri infection, where it interacts with the tightjunction-associated protein THO guanine nucleotide exchangefactor 2 (ARHGEF2) [33]. Additionally, CD147, a membrane-boundregulator of cellular migration and differentiation, was reported todirect NOD2 to sites of bacterial invasion [34]. Collectively, thesestudies suggest that guiding NOD receptors intracellular localiza-tion is a regulatory mechanism that might favor more efficientinteraction with cognate ligands.

2.2.1.2. Ligand internalization and transport. Vavricka et al. firstdiscovered the role of the intestinal tripeptide transporter hPepT1in MDP transport to the cytosol in a human colonic epithelial cell-line [35]. This transporter was later shown to be specific to MDP[36]. Inflammatory bowel disease (IBD) patients display abnormalhPepT1 expression, and IEC-specific transgenic overexpression ofhPept1 in mice exacerbated colitis induced by dextran sulfatesodium (DSS) treatment [37]. However, hPepT1-mediated MDPuptake appears to be specific to IECs, as macrophages deficient inhPept1 displayed normal MDP uptake and NOD2 activation[35–37]. Scavenger receptor A (SR-A) and Macrophage Receptorwith COllagenous structure (MARCO) were also linked to rapidNOD1 and NOD2 ligand internalization [38]. Further characteriza-tion of MDP uptake mechanisms pointed to a role of clathrin- anddynamin-mediated endocytosis in this process [39]. More recently,the endosomal peptide transporters SLC15A3 and SLIC15A4 wereshown to be key components of the MDP endosome-cytosol egressmachinery [40–42]. Highly expressed in DCs following TLRstimulation, these proteins mediate MDP transport and signalingthrough recruitment of NOD2 and RIP2 to endosomes [42].

2.2.1.3. Post-translational modifications. Post-translational modifi-cations of NOD2 and its downstream effectors constitute animportant mechanism in regulating NOD signaling and NOD-mediated innate immunity. Bertrand et al. reported that cIAP1 andcIAP2 were required for RIP2 K63-linked poly-ubiquitination andNOD signal transduction. cIAP1 and cIAP2-knockout mice failed tomount an immune response following administration of NODagonists [27]. XIAP is also required for NOD signaling byubiquitinating RIP2 and recruiting LUBAC to the NODosome[28]. Mutations in BIRC4, the gene encoding XIAP, are linked toX-linked lymphoproliferative syndrome type-2 (XLP2), a rareprimary immunodeficiency associated with fatal dysregulation ofthe immune system [43]. Notably, a subset of XLP2 patientsexhibited colitis-like symptoms [43]. Consistent with a role of XIAPin IBD, a recent exome sequencing and genotyping effort of IBDpatients reported frequent occurrence of BIRC4 mutations in �4%male pediatric-onset Crohn’s disease patients [44]. Ubiquitinationevents also negatively regulate the NOD signaling pathway.K63-linked polyubiquitination of RIP2 by the E3 ubiquitin ligaseITCH led to downregulation of NOD2-induced NF-kB activation


[45]. Furthermore, The E3 ubiquitin ligase TRIM27 was describedto negatively regulate NOD2 via K48-linked ubiquitination andsubsequent proteasomal degradation [46]. Recently, a group hasdemonstrated that direct binding of ubiquitin chains to the CARDdomains of NOD1 and NOD2 interfered with RIP2 association anddownstream signaling [47]. Thus, ubiquitination plays a diverserole in NOD signaling and mediates both activation and repressionvia distinct mechanisms.

2.2.1.4. Regulators. Production of various transcriptional spliceisoforms is a strategy adopted by the cell to intrinsically regulatethe NOD2 pathway. In 2006, Rosenstiel et al. identified NOD2-S, ashort isoform that encodes a protein truncated within the secondCARD domain. By binding to NOD2, NOD2-S acts as an endogenousinhibitor of NOD2 oligomerization and activation. Overexpressionof NOD2-S was shown to dampen NOD2-mediated signaling [48].Kramer et al. later reported an additional alternative splice variantof NOD2, NOD2-C2, composed of the two tandem CARD domains.Unlike NOD2-S, NOD2-C2 triggered NF-kB activation indepen-dently of MDP but competed with MDP-induced activation [49].

Beyond transcriptional and post-translational modifications,regulatory proteins can modulate signaling cascades via directbinding. For example, NOD2 was shown to interact with thechaperones Hsp90 and Hsp70, which confer stabilization andprotection from proteasomal degradation in the absence ofstimulus [50,51]. The proteasome subunit alpha type-7 (PSMA7)was also shown to regulate NOD1 levels in a proteasome-dependent manner [52].

The apoptotic protein BH3-interacting domain death agonist(BID) was identified in an siRNA screen as a potential positiveregulator of NOD signaling. Co-immunoprecipitation experimentsrevealed that BID acted as an adaptor protein between activatedRIP2 and the IKK complex [53]. Macrophages derived from Bid�/�

mice stimulated with Nod agonists displayed decreased activationof NF-kB and ERK1/2 but intact JNK and p38 signaling, suggestingthat additional factors regulated the latter MAPK pathwaysdownstream of Nod activation. Another group reported thatNod-induced responses were not strikingly different in Bid-deficient conditions [54], indicating that additional studies areneeded to define the determinants of Bid involvement in the NODpathway.

Yamamoto-Furusho et al. characterized Centaurin beta1(CENTB1), a GTPase-activating protein as a negative regulator ofNOD2-mediated NF-kB activation [55]. The MAP kinase kinasekinase (MAP3K) MEKK4 was also shown to bind to RIP2 andsequester it from the NOD2 machinery [56]. Another negativeregulator of NOD2 is c-Jun N-terminal kinase-binding protein 1(JNKBP1), which acts through its WD-40 domain to bind NOD2 andprevent its oligomerization in response to MDP [57]. The inositolphosphatase SHIP-1 similarly inhibited NOD2 signaling, bypreventing the association between RIP2 and XIAP [58]. Regardingthe antimicrobial activity of the NOD pathway, Lipinski et al.demonstrated that reactive oxygen species (ROS) are an integralpart of this process, and characterized the NADPH oxidase familymember DUOX2 as a key component of the NOD pathway. Theinteraction between NOD2 and DUOX2 was found to promoteprotection against Listeria monocytogenes infections [59].

2.2.1.5. Autophagy. Crosstalk between the NOD2 and autophagypathways was first put forth when genome-wide associationstudies identified polymorphisms in the gene encoding theautophagy regulator ATG16L1 to be strongly associated withsusceptibility to Crohn’s disease – a disease which has long beenlinked to deregulated NOD signaling [60,61]. Several studies havelater shown that NOD1 and NOD2 are able to induce autophagy.In one study, NOD1 and NOD2 were shown to direct autophagy




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to bacterial entry sites at the plasma membrane throughinteraction with ATGL16L1 [62]. This mechanism was reportedto be RIP2- and NF-kB-independent. In parallel, Cooney et al.reported a role of NOD2-ATG16L1-mediated autophagy inbacterial handling and antigen presentation in dendritic cells(DCs). Consistent with the results of Travassos et al., they showedthat DCs from CD patients expressing the NOD2 or ATG16L1 riskvariants were defective in autophagy. However, unlike Travassoset al., they demonstrated a requirement for RIP2 in this process[63]. Homer et al. similarly reported that the NOD2-ATG16L1interaction constituted part of an anti-bacterial mechanism butfound that the CD-associated ATG16L1 T300A variant was impairedin MDP-induced Salmonella killing only in epithelial cells but notmyeloid cells [64]. The contribution of RIP2 to NOD-inducedautophagy was later confirmed by Homer et al. who demonstratedthat RIP2 exerted a dual function; on one hand it stimulated p38activation, which was required for autophagy, and on the otherhand blocked the phosphatase PP2A, which acted as an inhibitor ofthis process [65]. More recently, the NOD2-RIP2 pathway wasshown to promote mitophagy (autophagy of mitochondria) duringinfluenza A virus infection as a means to blunt the NLRP3inflammasome, otherwise activated by damaged mitochondria[66]. Mechanistically, the kinase activity of RIP2 was required toactivate the mitophagy inducer ULK1. In addition to its role inautophagy, an autophagy-independent role for ATG16L1 in NOD-induced inflammation was recently reported. It was shown thatATG16L1 inhibited NOD1 and NOD2 signaling at the level of RIP2,specifically by interfering with its polyubiquitination [67]. It is thusclear that autophagy and NOD1/2 signaling exist in a dynamiccross-regulatory state, both of which are essential for optimalhomeostatic balance.

2.2.1.6. Anti-viral role of NOD2. In 2009, Sabbah et al. were first toshow that NOD2 recognizes viral ssRNA and triggers activation ofinterferon-regulatory factor 3 (IRF3) and production of interferon-beta (IFNb). This function was reported to be independent of RIP2.Instead, NOD2 interacted with the anti-viral signaling factormitochondrial adaptor protein (MAVS) to mediate signaling [68](Fig. 1b). Additionally, IFNb induction by viral RNA was shown toincrease the proinflammatory response to subsequent stimulationwith MDP, a finding with therapeutic relevance in the context ofthe severity of disease in patients with respiratory syncytial virus(RSV)-induced lower respiratory tract infections [69]. Morerecently, NOD2 was shown to play a key role in priming anoptimal CD8T cell response and adaptive immunity to influenza Avirus infection, by mediating the activation and survival of DCs[70]. Together with their earlier report that RIP2 triggersmitophagy by mediating the phosphorylation of ULK1 to limitinflammasome-mediated pulmonary tissue damage [66], theseresults implicate the NOD2-RIP2 pathway as an importantmodulator of the host response to influenza infection.

2.2.1.7. Role in T cells?. The contribution of the NOD receptors to Tcell function has been controversial. It was first shown that T-celldeficient mice reconstituted with Nod2-deficient T cells displayedan impaired Th-1 response following Toxoplasma gondii infection,and that Nod2 played a role in interleukin-2 (IL-2) production andCD4+ T cell proliferation and Th1 differentiation [71]. This T cell-intrinsic function of NOD2 was shown to be independent of RIP2[71]. However, these results were later disputed by Caetano et al.,who reported unimpeded Th1 response following T. gondii in Nod2deficient animals [72]. Recently, using a T cell transfer model ofcolitis, the expression of Nod2 in T cell was shown to bedispensable for the regulation of colitis. Although Nod2 expressionwas inducible following T cell receptor ligation and was increasedin activated/memory T cells, its deficiency in these cells did not


affect the onset or progression of colitis [73]. The same groupinterrogated the intrinsic function of NOD2 in regulatory T cells(Treg), and demonstrated that Nod2-deficient Foxp3+ Treg cellscould adequately proliferate and suppress effector T cell prolifera-tion and function [73]. T cell-intrinsic Nod2 expression wassimilarly not required for CD8+ T cell responses to OVA- orinfluenza antigens in vitro and in vivo, respectively. However, therewas a defect, albeit modest, in Nod2-deficient CD8+ T cellsaccumulation during infection [74].

2.2.2. NLRC3

Conti et al. were first to clone and characterize the CATERPILARgene CLR16.2 encoding NLRC3 (NOD3). NLRC3 was shown to be anegative regulator of T cell function potentially through itssuppressive effects on the NF-kB, NFAT and AP1 pathwaysfollowing T cell activation [75]. More recently, NLRC3 was alsoreported to blunt inflammation downstream of innate immunereceptors in myeloid cells. Nlrc3�/� mice injected with LPSdisplayed increased IL-6 levels and macrophage accumulation,which correlated with enhanced K63-ubiquitination of TRAF6 andan overt activation of NF-kB downstream of TLR signaling [76].However, the mechanism by which NLRC3 opposes inflammationstill remains obscure. Interestingly, a genetic screen in zebrafishalso revealed anti-inflammatory properties of NLRC3-like proteinin microglia. NLRC3-like mutants exhibited systemic inflammationand a defect in microglia development [77]. More recently, Zhanget al. described that NLRC3 blunted the activation of the cytosolicDNA sensor STING (stimulator of interferon genes) in response tointracellular DNA, cyclic di-GMP and DNA viruses (Fig. 2a). Herpes-simplex virus 1 (HSV1)-infected Nlrc3�/�mice displayed enhancedtype I interferon production, reduced viral loads and lethality [78].However, the precise mechanism of action of NLRC3 and theproperties of its ligand(s) still remain to be elucidated.

2.2.3. NLRC5

NLRC5 is the largest member of the NLR family, and structurallyresembles CIITA, notably in the NACHT and LRR motifs, suggestingfunctional similarity. NLRC5 contains the canonical NLR tripartitestructure, but has an intriguingly long C-terminal LRR motif, aswell as an N-terminal death domain (DD) [79]. Kuenzel et al.reported that human fibroblasts infected with cytomegalovirus(CMV) had increased expression of NLRC5, which positivelycorrelated with MHC I expression. This suggested a role of NLRC5in anti-viral immunity via MHC I transcriptional regulation [80].Indeed, NLRC5 was found to interact with the promoter of MHC Igenes, and IFNg upregulation of MHC I transcription was NLRC5-dependent [79].

2.2.3.1. Inflammation and anti-viral responses. NLRC5 was initiallydescribed in vitro to inhibit NF-kB and type I interferon pathways,as siRNA-mediated depletion of Nlrc5 in RAW264.7 cells causedincreased TNFa, IL-6 and type I interferon production, which hasbeen corroborated in a recent report [81,82]. Moreover, it wasshown that LPS-induced IL-10 secretion by macrophages waspromoted by NLRC5 [83], further suggesting that NLRC5 is anegative modulator of inflammation. The antiviral and anti-inflammatory role of NLRC5, however, may be cell type-dependent.While bone marrow-derived macrophages and dendritic cellsderived from Nlrc5�/� mice produced normal levels of IFNb, IL-6and TNFa when challenged with bacteria, RNA or DNA viruses [84],mouse embryonic fibroblasts (MEFs) and peripheral macrophagesfrom Nlrc5�/� mice displayed increased IL-6 and IFNb productionfollowing LPS treatment or vesicular stomatitis virus (VSV)infection, respectively [85]. This was confirmed in vivo asNlrc5�/� mice injected with either LPS or VSV had increasedserum IL-6 and IFNb [85].



Fig. 2. NLRC3 and NLRX1 signaling cascades. (A) NLRC3 acts as an inhibitor of TCR signaling by dampening AP-1-, NF-kB- and NFAT-dependent gene transcription. HSV-1, viral

DNA and cyclic di-GMP trigger a STING-dependent type I IFN response from the endoplasmic reticulum that is blunted following NLRC3 binding to STING. (B) NLRX1 is

activated during viral infection or during stimulation with poly I:C and has been shown to directly bind viral RNA. The subcellular localization of NLRX1 is disputed. Some

groups have found it embedded in the outer mitochondrial membrane, where it interacts with MAVS to inhibit MAVS-dependent type I IFN induction, while others have found

it inside the mitochondrial matrix. NLRX1 interacts with the mitochondrial TUFM to mediate ATG5-ATG12-dependent autophagy during viral infection, subsequently

dampening the type I IFN response. Ubiquitination of NLRX1 downstream of TLR4 activation leads to its dissociation from TRAF6 and its binding to NEMO, which dampens the

NF-kB response.


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Besides this discrepancy, NLRC5 has been linked to the NLRP3inflammasome. First, it was shown that overexpression of NLRC5 inHEK293T cells induced increased secretion of IL-1b [84]. Second,Davis et al. substantiated this link by showing that RNAi-mediateddepletion of NLRC5 in human monocytic cells blunted caspase-1activation and IL-1b and IL-18 maturation in response to a panel ofNLRP3 agonists [86]. Thus, NLRC5 clearly has a diverse role in theantiviral and inflammatory responses as it seems to blunt theantiviral response while boosting inflammasome activation.

2.2.3.2. Class I transactivation. Like CIITA, NLRC5 is stronglyinducible by IFNg in a STAT1-dependent manner, and is able totravel from the cytosol to the nucleus to carry out its effectorfunction through the action of an enhanceosome [87,88]. The MHCI transactivator property of NLRC5 was reported to be dependenton an intact NBD, which supports both nuclear localization andtransactivation [89]. In the nucleus, NLRC5 was seen to interactwith a sequence of the MHC I promoter termed the X1 box, throughits association with the transcription factors RFX5, RFXAP andRFXANK/B [87,90]. Biswas et al. recently reported that NLRC5 isnecessary for both constitutive and inducible expression of MHCclass I genes [91]. Consistent with this function, Nlrc5�/� miceinfected with the intracellular pathogen L. monocytogenes hadincreased bacterial loads and a defect in CD8+ T cell activation,consolidating the critical function of NLRC5 as important regulatorof adaptive immunity [91].

2.2.4. NLRX1

The role of NLRX1 is a subject of controversy. NLRX1 (alsoknown as CLR11.2 and NOD9) was first identified to play a role inanti-viral response [92]. Through its unique association with MAVSat the outer mitochondrial membrane, which interacts with the


RIG-I-like helicase (RLH) family of cytosolic viral nucleic acidsensors, NLRX1 acted as a negative regulator of virus-induced typeI interferon production in vitro [92]. Soon after, these findings werechallenged, when a later report localized NLRX1 to the mitochon-drial matrix, not the outer membrane [93] (Fig. 2b). Allen et al.maintained that NLRX1 is a negative regulator of type I IFNproduction and showed increased IFNb, STAT2 and OAS1expression following influenza A virus infection in Nlrx1�/� mice[94]. However, Nlrx1�/� mice generated by two other groupsexhibited normal IFNb levels following poly I:C injection [95] orinfluenza A virus infection [96]. A recent crystallographiccharacterization of NLRX1 supported a role of NLRX1 in anti-viralimmunity by revealing direct interaction between the C-terminusof NLRX1 and RNA [97]. As of yet, the source of the discrepancybetween laboratories in relation to NLRX1 function remainsunclear.

2.2.4.1. NF-kB, JNK signaling and virus-induced autophagy. NLRX1was also shown to negatively regulate inflammation by dampeningToll-like receptor (TLR)-mediated activation of NF-kB and JNKsignaling pathways [94,98]. NLRX1 associated with TRAF6 or theIKK complex. Upon LPS stimulation, NLRX1 got rapidly ubiquiti-nated, dissociated from TRAF6, and interacted with the IKKcomplex inhibiting canonical NF-kB activation [98]. In vivo, Nlrx1�/

� mice challenged with LPS displayed increased plasma IL-6 levelsand increased susceptibility to septic shock [94,98]. NLRX1 wasalso reported to act as a positive regulator of virus-inducedautophagy. Following viral infection, NLRX1 associated with themitochondrial Tu translation elongation factor (TUFM) and theautophagy-related proteins Atg5-Atg12 and Atg16L1 to form themitochondria-immune signaling complex (MISC) [99]. NLRX1 andTUFM worked in concert to reduce cytokine production and to




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promote virus-induced autophagy that dampens type I interferonproduction [99]. This suggests an alternative route by whichNLRX1 dampens type I interferon production, further supportingits link to mitochondria-associated anti-viral responses.

3. Inflammasome-forming Nlrs

In 2002, Martinon et al. characterized a high-molecular weightcytosolic complex that served as a platform for the recruitment ofthe inflammatory caspases, notably caspase-1 and caspase-5, thatthey dubbed the inflammasome [2]. Nlrp1 was a key component ofthis newly defined complex, and was the first characterized PYD-containing NLR that could aggregate and induce IL-1b and IL-18production. Inflammasomes are multimeric structures that are inmost part composed of three main components: a sensor protein(an NLR, AIM2 or RIG-I), an adaptor (generally ASC (or PYCARD)),and caspase-1. NLR proteins that are capable of forming aninflammasome include NLRP1, NLRP3, NLRP6, NLRP7, NLRP12,NLRC4 and NAIP proteins. Stimulation of the LRR domain bycognate agonists triggers a conformational remodeling leading tothe oligomerization of the sensing receptor and caspase-1recruitment and activation.

3.1. NLRP1

NLRP1 was a key constituent of the first characterizedinflammasome [2]. Mice harbor three Nlrp1 gene paralogs Nlrp1a,Nlrp1b and Nlrp1c. In 2005, Boyden and Dietriech mapped asusceptibility locus on mouse chromosome 11 to Bacillus

anthracis infection. They reported that the Nlrp1b gene washighly polymorphic and was the primary mediator of mousemacrophage susceptibility to B. anthracis lethal toxin (LeTx)-induced cell death [100]. Indeed, it was shown that LeTx-infectedmacrophages underwent a rapid highly inflammatory form of celldeath dependent on caspase-1 termed pyroptosis, suggesting forthe first time that Nlrp1b could activate caspase-1 in response toLeTx. This finding was confirmed in vivo recently, by thegeneration of Nlrp1b-deficient mice [101]. LeTx is composed oftwo sub-units: protective antigen (PA) and lethal factor (LF). PA isa pore-forming protein that enables the translocation of LF intothe cytosol [102]. LF is a zinc metalloprotease and its catalyticactivity is required for the activation of Nlrp1b. Consistently,protease-mutants of LF failed to induce pyroptosis [103]. Thissuggested that Nlrp1b does not directly bind to LeTx, but rathersenses an event that depends on the catalytic activity of LF. Aliet al. proposed a mechanism by which LeTx-dependent inhibitionof p38 and AKT signaling resulted in inflammasome activation. Itinvolved the leakage of ATP from infected macrophages throughconnexin channels and subsequent stimulation of the ATP-responsive purinergic P2X7 receptor leading to inflammasomeactivation [104]. Levinsohn et al. demonstrated that the ratortholog of Nlrp1 was directly cleaved by LF at the N-terminus,and that this cleavage was required for NLRP1 activation andpyroptosis [105]. The direct cleavage of murine Nlrp1b by LF wasalso reported to be required and sufficient for Nlrp1b activation[106] (Fig. 3). This group emitted the hypothesis that Nlrp1b,alongside other intracellular receptors, are capable of sensing‘‘patterns of pathogenesis’’, in this context the protease activity ofLeTx LF, allowing the distinction between pathogenic andharmless microbes.

Among other NLRP1 activators, MDP was reported to inducecaspase-1-dependent IL-1b secretion in vitro via direct binding ofMDP to the LRR of NLRP1, triggering the conformational changerequired to recruit caspase-1 to the inflammasome [107]. NOD2association with NLRP1 was later shown to be required forthe MDP-induced activation of caspase-1 [108]. Interestingly, the


anti-apoptotic proteins Bcl-2 and Bcl-Xl were reported as negativeregulators of MDP-induced IL-1b production, as Bcl-2 deficientmacrophages stimulated with MDP displayed robust increase in IL-1b production [109]. Concordantly, Gerlic et al. identified avaccinia virus Bcl-2 homolog, F1L, which bound to and inhibitedNLRP1 activation [110]. Macrophages infected with a non-functional F1L-bearing vaccinia virus strain displayed increasedcaspase-1 activation during infection [110]. The link between Bcl-2family members and NLR regulation highlights the co-evolution ofthe cell death machinery and the NLR-mediated innate immunitypathways.

Decrease in cellular ATP was also reported as an activatingmechanism for NLRP1 [104,111]. In contrast to the ATPase domainof NLRP3, that of NLRP1 was suggested to inhibit inflammasomeassembly [111]. The ability of Nlrp1 to detect cellular energy levelsmight provide a link between metabolism and immunity.

A novel role for NLRP1 in sensing Toxoplasma infection hasbeen recently unraveled [112,113]. Ewald et al. observed thatT. gondii-induced IL-1b production in macrophages requiredcaspase-1, the adaptor Asc and Nlrp1b [113]. Activation of Nlrp1bin this case did not require its N-terminal processing [106,113].T. gondii-infected Nlrp1b�/� mice displayed increased mortality[112]. These recent findings depict a novel function of NLRP1 incontrolling parasitic infections, by a mechanism that seemsdistinct from Anthrax LeTx-induced inflammasome activation(Fig. 3).

NLRP1 protein structure is distinct from that of other NLRs. Itcontains a pyrin domain (PYD) on the N-terminus and a CARD onthe C-terminus, as well as ZU5 and UPA internal domains (or FIIND)enabling autoproteolytic activity at a conserved SF/S motif withinthe FIIND [114]. This unique ability of NLRP1 to autoprocess wasreported to be required for its assembly and activation [115,116].Martinon et al. established that ASC was required for NLRP1inflammasome assembly and function, by demonstrating that ASCdepletion in LPS-primed THP1 cells abrogated caspase-1 andcaspase-5 activation and IL-1b processing [2]. Similarly, ratsinjected with anti-ASC neutralizing antibodies in a study oftraumatic brain injury showed reduced activation and processingof caspase-1 [117]. However, accumulating evidence suggests thatASC may be dispensable for the initial steps of NLRP1 inflamma-some activation. Faustin et al. reconstituted the NLRP1 inflamma-some, and showed that ASC enhanced but was not mandatory forNLRP1-mediated caspase-1 activation in vitro [107]. A more recentreport confirmed these findings in vivo by demonstrating thatASC was required for murine NLRP1b inflammasome-mediatedcaspase-1 autoproteolysis, but was dispensable for Anthrax lethaltoxin (LeTx)-induced IL-1b secretion and pyroptotic cell death[118]. Indeed, LPS-primed LeTx-treated macrophages derived fromAsc�/�mice showed normal IL-1b secretion compared to cells fromheterozygous mice or wild-type controls [118].

In contrast to the highly polymorphic Nlrp1b gene, Nlrp1a isconserved among mouse strains [119]. Nlrp1a expression wasproposed to be under the control of the lipogenic transcriptionfactor SREB1a [120]. However, this finding was recently contested[121]. Interestingly, using an N-ethyl-N-nitrosourea (ENU) randommutagenesis approach in mice, Masters et al. identified an Nlrp1a

mutant mouse that exhibited inflammasome hyperactivation,pyroptosis of hematopoietic progenitor cells and systemic lethalinflammation [122]. These results suggest that NLRP1a might be akey sensor of hematopoietic stress and an important regulator ofinflammation.

3.2. NLRP3

The NLRP3 inflammasome is arguably the most well describedinflammasome to date. In 2002, gain-of-function mutations in



Fig. 3. Agonists of the NLRP inflammasomes. Several of the NLRP family members have been shown to assemble into distinct inflammasomes. Anthrax lethal toxin from

Bacilllus anthracis cleaves the N-terminus of NLRP1b and mediates its activation. Low cellular ATP, Toxoplasma gondii and MDP via NOD2 have also been shown to engage

NLRP1 to form an inflammasome. NLRP6 has been shown to signal through an inflammasome in the context of colitis in mouse models, yet its ligand is currently unclear, but is

potentially a derivative of the intestinal microbiota. Acylated lipopeptides from several bacteria engage NLRP7, and a yet unknown virulence factor from Yersinia pestis

activates NLRP12. Following direct or indirect sensing of these agonists, the NLRP proteins oligomerize with ASC and pro-caspase-1, leading to activation of caspase-1 and

initiation of downstream signaling. NLRP10 has been shown to potentially inhibit inflammasome activation.


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the NLRP3 gene were discovered to be associated with autoin-flammatory cryopirin-associated syndromes [123,124]. NLRP3 iscomposed of a central NACHT domain, a C-terminal LRR and an N-terminal pyrin domain (PYD) allowing it to interact with theadaptor protein ASC to assemble an inflammasome that recruitsand activates caspase-1.

3.2.1. Priming

Activation of the NLRP3 inflammasome requires an initialpriming step (signal 1) that upregulates NLRP3 levels, followed byagonist sensing (signal 2) that induces its oligomerization andcomplex assembly. The priming signal was thought to rely on NF-kB-mediated induction of NLRP3 gene expression downstream ofTLRs, NLRs and cytokine receptors. However, increasing evidencesuggest that priming can also occur independently of transcrip-tional upregulation of inflammasome components. For instance, inmacrophages, acute stimulation of TLR4 by LPS triggers rapidpriming of NLRP3 through its deubiquitination [125]. IRAK1, IRAK4and ERK1 were implicated in such rapid non-transcriptionalpriming of NLRP3 [126,127]. Thus, early priming is independent ofprotein synthesis but requires TLR-IRAK1 signaling, followed by a


late phase that involves transcriptional induction of NLRP3 by NF-kB [128]. The role of reactive oxygen species (ROS) in NLRP3priming is a matter of debate [129,130]. Whereas Zhou et al.initially proposed that ROS induced NLRP3 inflammasome activa-tion [129], Bauernfeind et al. later showed that ROS were requiredfor priming but dispensable for activation [130].

3.2.2. Post-translational modifications

The observation that priming can rapidly occur independentlyof de novo protein synthesis suggests that more dynamic layers ofcontrol regulate inflammasome activation. Post-translationalmodifications represent such events that have been shown toenable inflammasome function. For instance, NLRP3 is ubiquiti-nated at its LRR domain in resting conditions, which prevents itsoligomerization. Recently, the deubiquitinase BRCC3 was reportedto be required for NLRP3 deubiquitination and subsequentactivation, which is thus far the only post-translational modifica-tion reported to activate NLRP3 [131]. However, ubiquitination is adynamic process and both NLRP3 and AIM2 inflammasomes werepreviously reported to be downregulated by ubiquitination-induced autophagy and destruction [132]. Nitrosylation of NLRP3




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was also reported as an endogenous mechanism that bluntsinflammation during persistent infection [133,134]. Phosphoryla-tion of ASC by the kinases Syk and Jnk is an additional layer ofcontrol required for the formation of ASC specks and subsequentcaspase-1 activation [135].

3.2.3. Activation mechanisms

A wide variety of unrelated ligands from endogenous andexogenous sources trigger NLRP3 activation. These includemonosodium urate crystals [136], elevated ATP levels whichengage the cell surface receptor P2X7R [137], asbestos, silica andother particulate matter [138,139], amyloid b aggregates [140]and pore-forming toxins [137,141] to name a few. Because theseagonists are structurally diverse, it is likely that they converge onselect pathways to mediate activation. Recent studies have focused

Fig. 4. Signaling mechanisms of the NLRP3 inflammasome. The NLRP3 inflammasome is

converge on only a handful of pathways: potassium efflux, induced by ATP binding to

following rupture caused by particulate matter; and reactive oxygen species (ROS). Vario

extracellular calcium signals through CASR and GPCR6A cause a release of endoplasmic c

ion channels. Cytosolic calcium inhibits cAMP, which itself inhibits NLRP3, and can indu

leading to the release of large amounts of ROS, oxidized mitochondrial DNA (mtDNA) and

mitophagy. ROS causes the dissociation of TXNIP from TRX, allowing TXNIP to directly b

localization of NLRP3 is currently under debate, but it has been shown to localize to the

following activation, in a manner that may be mediated through MAVS. Post-translati

undefined deubiquitinases mediates NLRP3 activation, and phosphorylation of ASC

inflammasome activation.


on unraveling common pathways triggered by the above-mentioned molecules in finding a mechanism for canonical NLRP3activation (Fig. 4).

3.2.4. Lysosomal destabilization

The NLRP3 inflammasome is activated by several particulateand crystalline compounds, including the vaccine adjuvantaluminum hydroxide [142], cholesterol crystals [143] and uricacid crystals [136]. One mechanism posited for unifying theactivation of NLRP3 by these compounds is the rupture of thelysosome following phagocytosis and the release of cathepsin B.

3.2.5. Ion flux

Potassium and calcium fluxes across the cell membrane havebeen shown to enable the activation of the NLRP3 inflammasome.

activated by a vast array of diverse stimuli; however, classically these were seen to

P2X7R or bacterial pore-forming toxins, release of cathepsin B from the lysosome

us other activating mechanisms have been described in recent years. High levels of

alcium stores into the cytosol, and can enter the cell via TRPM2, TRPM7 and TRPV2

ce mitochondrial damage. Most NLRP3 agonists induce mitochondrial dysfunction,

cardiolipin, other direct NLRP3 agonists. Damaged mitochondria can be cleared via

ind and activate NLRP3, or by inducing the deubiquitination of NLRP3. Subcellular

endoplasmic reticulum at rest and relocalize to the mitochondria where ASC exists

onal modifications regulate NLRP3 activity: deubiquitination by BRCC3 and other

by Syk and Jnk promotes inflammasome assembly, while nitrosylation inhibits




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Petrilli et al. initially depicted a role for potassium efflux as acommon trigger of both NLRP1 and NLRP3 inflammasomeactivation, where extracellular ATP sensing by the P2X7 receptorfacilitated potassium efflux [144]. This mechanism was confirmedwhen a subsequent study demonstrated that blocking potassiumchannels with glyburide abrogated NLRP3 inflammasome activa-tion in vitro [145]. Munoz-Planillo et al. similarly showed that adrop in cellular potassium levels was the unifying mechanism forNLRP3 activation by a number of triggers including particulatematter or bacterial toxins [146]. Increased extracellular calciumwas also shown to play a role in NLRP3 activation, a processmediated by plasma membrane G-protein coupled receptorGPCR6A and calcium sensor CASR [147]. Extracellular calciumsensing triggered the release of cytosolic endoplasmic reticulumcalcium stores, leading to increased intracellular calcium levels,which facilitated inflammasome assembly, in part throughsuppression of the NLRP3 inhibitor cyclic AMP (cAMP)[147,148]. Moreover, calcium entry through the cation channelsTRPM7 and TRPV2 was suggested to regulate cytosolic calciumlevels during cell volume re-adjustment following cellular swelling[149]. A recent report showed that the ROS-sensitive cationchannel TRPM2 equally contributed to increased intracellularcalcium levels required for NLRP3 inflammasome activation, asTrpm2�/� mice were resistant to liposome-induced IL-1b produc-tion [150]. Finally, Triantafilou et al. reported that the membraneattack complex (MAC) can mediate increased cytosolic calciumleading to calcium accumulation in the mitochondrial matrix via

mitochondrial calcium uniporters, and subsequently cause loss ofmitochondrial transmembrane potential and NLRP3 inflamma-some activation [151]. While ion flux is correlated with activationof the inflammasome, the underlying mechanism still remainsunclear. One possibility could be that NLRP3 is sensitive to shifts incellular ion concentrations, which could result in conformationalchange of the protein and exposure of the pyrin domain todownstream inflammasome effectors. Further investigation on thestructure of NLRP3 during ionic imbalance would greatly contrib-ute to the understanding of how NLRP3 is activated.

3.2.6. Mitochondria

Many studies have implicated the mitochondria in NLRP3inflammasome activation. The respiring organelle plausiblyconstitutes an adequate recruitment platform for NLRP3 oligo-merization, as well as a source of various mitochondria-derivedagonists that can be released if the mitochondria were compro-mised. A number of NLRP3 agonists were reported to trigger ROSproduction, suggesting that ROS might be a second messengerupstream of the NLRP3 inflammasome (reviewed in [152]). Thesignificance of ROS in NLRP3 activation was supported by theobservation that mitochondrial ROS induces the oxidation ofthioredoxin, triggering its dissociation from thioredoxin-interact-ing partner (TXNIP) [153]. The released TXNIP directly interactswith and induces NLRP3 inflammasome activation [153]. TXNIPthereby acts as an indirect NLRP3 sensing adaptor, bridging theconserved ROS response following infection or tissue damage toactivation of the inflammasome. Besides ROS, decreased mito-chondrial transmembrane potential (DCM) and mitochondrialdysfunction were correlated with inflammasome activation [129].Consistently, disrupting the mitochondrial membrane potential byblocking voltage dependent anion channels (VDAC) inhibitedNLRP3 inflammasome activation [154]. Similar alterations ofmitochondrial membrane potential and NLRP3 inflammasomeactivation were displayed by the overexpression of uncouplingprotein 2 (UCP-2) in HEK293T cells, a mitochondrial innermembrane protein that mediates mitochondrial proton leakage[155]. Interestingly, a recent group identified that the microRNAmiR-133a-1 targets UCP-2 [156]. miR-133a-1 overexpression in


the THP1 monocytic cell-line resulted in increased NLRP3inflammasome activation and IL-1b production, which rendersmiR-133a-1 an endogenous positive regulator of the inflamma-some by acting at the level of the mitochondria [156]. Release ofmitochondrial damage-derived factors was reported to promoteinflammasome activation. Indeed, released oxidized mitochondrialDNA (mtDNA) during apoptosis was shown to directly bind to andactivate NLRP3 [157]. Mitophagy, a form of autophagy of damagedor dysfunctional mitochondria, was shown to be critical incontrolling inflammasome activation [157]. A recent studydemonstrated that linezolid, an oxazolidinone antibiotitic, couldinduce NLRP3 inflammasome activation by causing mitochondrialdamage and the release of cardiolipin, which interacted with andactivated NLRP3. Consistently, inhibition of cardiolipin synthesissignificantly reduced NLRP3 inflammasome activation [158].

In support of a role of the mitochondria as a recruitmentplatform for the inflammasome, NLRP3 has been demonstrated tophysically associate with respiring mitochondria through themitochondrial adaptor MAVS [159,160]. At rest, NLRP3 is localizedto the ER, while the adaptor ASC is localized to the mitochondria[161]. In response to mitochondrial damage, inactivation of sirtuin2 enhances the levels of acetylated a-tubulin, which promotesmicrotubule-dependent mobilization of mitochondria to theperinuclear space bringing NLRP3 and ASC in close proximity[161]. Some reports however refuted the mitochondrial localiza-tion of NLRP3 upon activation. One study reported that activatedNLRP3 remained cytosolic, with no association with any organelle[162]. Munoz-Planillo et al. similarly showed that NLRP3 did notassociate with mitochondria following activation, and that ROS or achange in cell volume was not required for NLRP3 activation [146].Promyelocytic leukemia protein (PML), which is required for IP3R-mediated ER calcium release, was required for NLRP3 activation. Loet al. showed that PML deficiency in macrophages resulted indecreased release of mitochondrial DNA and ROS and impaired IL-1b production [163]. In contrast, Dowling et al. recently reportedthat PML limited inflammasome activation, by interacting withASC and retaining it inside the nucleus [164]. PML-deficientmacrophages had increased ASC dimer formation in the cytosoland increased IL-1b production in response to NLRP3 activation inmacrophages infected with herpes simplex virus-1 (HSV-1) orSalmonella [164]. Further investigation is thus required to resolvethese contradictory results.

3.2.7. Binding partners of the NLRP3 inflammasome

A wide array of interacting proteins that differentially regulateactivation of the inflammasome has been identified. Stronginflammatory responses to pathogenic insults provide hostprotection, however sustained and uncontrolled inflammationmay cause detrimental effects in the host. Endogenous repressorsof the inflammasome thus represent important regulators ofinflammation that prevent immunopathology. The myeloid-specific microRNA miR-223 and the Epstein–Barr virus (EBV)-encoded microRNA miR-BART15 were reported to target NLRP3

mRNA causing its degradation [165,166]. CARD8 is anotherinhibitor of the NLPR3 pathway [167,168]. Mutant forms of NLRP3linked to cryopyrin-associated periodic fever syndromes (CAPS)have impaired CARD8 binding, suggesting that absence of CARD8-mediated modulation of NLRP3 activation might underlie theimmunopathology in CAPS [168]. An additional suppressor of theNLRP3 pathway is Leucine-rich repeat Fli-I-interacting protein 2(LRRFIP2). It suppresses caspase-1 activation through associationwith Flightless-1 that acts as a caspase-1 pseudosubstrate [169].

Positive regulators of the inflammasome are equally importantin modulating inflammasome activation in the case of attenuatedor circumvented inflammatory responses. The E3 ubiquitin ligasescIAP1 and cIAP2 were demonstrated to be required for efficient




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inflammasome activation [170]. In contrast, XIAP indirectlyblunted the inflammasome response downstream of TNF in ayet undefined mechanism involving the kinase RIP3 [171]. A recentpaper found that NLRP3 is activated in response to double strandedRNA via association with the RNA helicase DHX33. siRNA depletionof DHX33 in human macrophages challenged with poly I:C,reoviral or bacterial RNA displayed decreased caspase-1 activation[172]. Protein kinase R (PKR), an RNA dependent kinase involved inantiviral defenses, was initially proposed to mediate inflamma-some activation [173], but this function is controversial. While achemical biology screen implicated PKR in pyroptosis [174], Heet al. later showed that PKR was dispensable for NLRP3 activation[175].

3.3. NLRP6

Reports on the role of NLRP6 function hitherto are now well inagreement with its role as a guardian of intestinal mucosalintegrity. Initially described to have the capacity to interact withASC and caspase-1 to form an inflammasome [176], the generationof Nlrp6-deficient mice have allowed for the characterization of thephysiological relevance of NLRP6. Nlrp6�/� mice are moresusceptible to DSS-induced colitis, which displayed decreasedserum IL-18, a key cytokine involved in intestinal wound healingfollowing inflammasome activation [177]. Cohousing of wild-typemice with Nlrp6�/�mice transferred the disease to wild-type mice,indicative of the involvement of a dysbiotic and transmissiblemicrobiota. The expansion of certain bacterial communities inNlrp6�/�mice intestinal flora was also seen in Asc�/� and Casp1�/�

mice, which displayed similar susceptibility to colitis [178,179],thus supporting the role for NLRP6 in inflammasome formation[177]. Nlrp6�/� mice displayed increased colonic CCL5 (RANTES)production, a chemokine that promotes the recruitment ofinflammatory leukocytes and indirectly exacerbates inflammation.Genetic ablation of Ccl5 in Nlrp6�/� mice conferred resistance toDSS-induced colitis [177]. Colonic IL-18 production by the NLRP6inflammasome was subsequently described to participate in theregulation of the intestinal microbiota and intestinal barrierintegrity. Il18-deficient mice displayed expansion of colitogenicbacterial communities and increased CCL5 production in the colon,thereby demonstrating the role of IL-18 in maintaining a healthymicrobiota [177]. Moreover, Nlrp6�/� mice treated with azox-ymethane (AOM) and DSS displayed increased colitis-relatedtumorigenesis, which was associated with decreased IL-18production in the colon and in circulation. Normand et al.subsequently demonstrated that NLRP6 ablation lead to impairedintestinal tissue repair, linking deficiency in inflammasome-dependent IL-18 production to a dysregulation of the mechanismsthat govern intestinal cell proliferation [180]. Recently, the role ofNLRP6 in intestinal and microbial homeostasis has been substan-tiated in its capacity to regulate goblet cell mucus production inthe gut. Indeed, Nlrp6-, Asc- and caspase-1/11-deficient mice wereunable to clear enteric pathogens from the colonic mucosa, due toimpaired mucus production in the large intestine [181]. Mecha-nistically, NLRP6 deficiency caused disruption of autophagy inintestinal goblet cells, which prevented the release of mucus-containing granules in the lumen of the colon, therefore abrogatingthe formation of a protective mucus layer [181]. Together, thesefindings demonstrate the pivotal role of NLRP6 in the host–microbiota interface, by controlling both the composition of themicrobiota and maintaining intestinal homeostasis. Aside of itsimplication in intestinal physiology, recent evidence havedemonstrated an anti-inflammatory role for NLRP6 in responseto Gram-positive and Gram-negative bacteria. Nlrp6�/� mice wereresistant to infection with Salmonella typhimurium, L. monocyto-

genes and Escherichia coli [182]. Listeria infection of macrophages


from Nlrp6�/�mice produced aberrant levels of TNFa, IL-6 and KC,due to de-repression of MAPK and NF-kB pathways downstream ofTLR stimulation [182]. Interestingly, the negative role of NLRP6 inNF-kB activation did not affect NOD1 and NOD2 signaling,suggesting an upstream role for NLRP6 in the TLR signalingpathway [182]. Taken together, NLRP6 thus appears as a negativeregulator of inflammation in the context of pathogenic infection,while modulating intestinal and microbial homeostasis in theintestine. The exact activation mechanism of NLRP6 remains to bediscovered (Fig. 3).

3.4. NLRP7

In 2005, NLRP7 was described as an inhibitor of the inflamma-some pathway [183]. Its overexpression in HEK293T cells resultedin impaired caspase-1-mediated IL-1b production, with noapparent effect on NF-kB activation. Moreover, the N-terminalfragment of NLRP7 was sufficient to inhibit LPS-induced IL-1bproduction in THP1 cells, suggesting that a functional pyrindomain (PYD) was required for inhibition [183]. However, morerecent evidence suggests that NLRP7 can assemble an inflamma-some in response to microbial acylated lipopeptides [184] (Fig. 3).Indeed, siRNA-mediated depletion of NLRP7 or ASC in humanmacrophages resulted in impaired IL-1b secretion when exposedto heat-killed Mycoplasma spp., Staphylococcus aureus or Legionella

pneumophilia [184]. NLRP7 overexpression resulted in the forma-tion of a high molecular-weight complex composed of ASC andcaspase-1, indicative of an NLRP7-induced inflammasome. NLRP7and NLRP3 inflammasome-mediated production of IL-1b and IL-18was required in blocking intracellular bacterial replication ininfected THP1 cells independently of NF-kB [184]. Mutations inNLRP7 have been linked to hydatidiform moles (HMs), anabnormal pregnancy characterized by impaired embryonic devel-opment and placental hyperproliferation [185]. As of yet, it isunclear whether aberrant NLRP7 inflammasome activation isimplicated in disease, though a role for NLRP7 in coordinating thesecretion of cytokines via interaction with the Golgi apparatus hasbeen suggested as a potential mechanism of pathogenesis [186].

3.5. NLRP10

Human NLRP10 (PYNOD) was first described by Wang et al. as anegative regulator of inflammation and apoptosis. Structurally, it iscomposed of a NOD domain, a pyrin domain, but devoid of C-terminal LRRs [187]. The role of NLRP10 in repressing inflamma-tion, however, has been reconsidered due to conflicting reports.Human NLRP10 inhibited caspase-1 auto-processing and ASCaggregation in HEK293T cells and overexpression of murineNLRP10 in macrophages led to decreased IL-1b productionindependently of these processes (Fig. 3). Moreover, NLRP10transgenic mice were resistant to endotoxic shock [188]. However,NLRP10 was recently shown to promote IL-6 and IL-8 production inepithelial cells and primary fibroblasts infected with S. flexneri

[189]. In vitro, NLRP10 interacted with multiple components of theNOD1 signaling pathway, and was proposed to act as a scaffold forthe assembly of the nodosome, which potentiated the activation ofNF-kB and p38 [189] (Fig. 1a). These findings suggested thatNLRP10 both negatively regulates ASC-dependent inflammation,while amplifying NF-kB-derived pro-inflammatory cytokineproduction in response to bacterial insults. Alternatively, a rolefor NLRP10 in initiating adaptive immunity was also proposed.Eisenbarth et al. showed that Nlrp10�/� mice displayed impairedT-cell-mediated immune responses to a variety of adjuvants [190].Whereas Nlrp10�/� mice-derived B and T cell function remainedintact in vitro, impaired Th1 and Th2 responses in these mice wasdue to a migration defect of circulating DCs, which were unable to



Fig. 5. Sensing of bacterial infection by NAIPs and the NLRC4 inflammasome. The

NLRC4 inflammasome is activated by bacterial flagellin and the rod proteins of

bacterial type III and IV secretion systems (T3SS, T4SS) within the cytosol. Direct

recognition of flagellin by NAIP5 and NAIP6, and rod proteins, such as Mxil, by

NAIP2 initiate the activation of NLRC4 inflammasome. While ASC has been shown to

partially promote the NLCR4 inflammasome, it is not absolutely required.

Phsophorylation of NLRC4 by PKCd has been suggested to be essential for its

activation, although this remains to be fully characterized.


G Model


reach draining lymph nodes and prime naıve CD4+ T cells [190].Accordingly, Nlrp10�/� mice were highly susceptible to systemicinfection with Candida albicans, a fungal pathogen known to triggera Th17 response upon infection [191]. While NLRP3-derived IL-1band IL-18 levels were similar to those in infected WT mice, Nlrp10�/

�mice displayed a robust decrease in Th1 and Th17 responses, thusdepicting a role for NLRP10 in orchestrating an adaptive immuneresponse to fungal infection.

3.6. NLRP12

In 2002, Wang et al. reported a novel PYRIN-containing Apaf1-like protein NLRP12 (PYPAF7, Monarch-1), which was able topromote inflammation by inducing ASC and caspase-1-dependentIL-1b production in vitro [176]. Initial in vitro assays revealed thatNLRP12 acted as a positive regulator of canonical and non-canonical MHC class I expression. Accordingly, siRNA-mediateddepletion of NLRP12 greatly reduced basal expression of HLA-Band HLA-G genes [192]. An alternate role for NLRP12 wassubsequently defined by the same group, in which NLRP12 couldmodulate inflammation. Indeed, Williams et al. found that NLRP12was a negative regulator of the NF-kB signaling pathway.Monocytes depleted of NLRP12 displayed increased productionof pro-inflammatory cytokines when stimulated with agonists forTLR3 or TLR4, TNFa or infection with Mycobacterium tuberculosis.Mechanistically, it was demonstrated that NLRP12 bound to IRAK1and inhibited its auto-phosphorylation [193]. NLRP12 was alsoseen to attenuate the NF-kB alternative pathway triggered byCD40 by promoting proteasomal degradation of the NF-kB-inducing kinase (NIK) [194]. The ATP binding motif of NLRP12was described to allow its association with NIK and IRAK-1 [195].By a mechanism similar to R proteins in plants that necessitatestabilization by heat shock proteins to conduct inflammatorysignals, Arthur et al. described the required interaction betweenNLRP12 and HSP90 in order to dampen NF-kB-dependent pro-inflammatory cytokine production [196]. The relevance of NLRP12in modulating inflammation in vivo, however, was only recentlyuncovered. Nlrp12�/� mice were highly susceptible to DSS-induced colitis and colitis-associated colorectal cancer. The overtinflammation seen in the colon of Nlrp12�/� mice was due toregulatory defects in the activation of NF-kB and ERK signalingpathways in macrophages, which drove intestinal hyperplasia andsubsequent increased tumor burden [197]. Zaki et al. also reporteda novel role of NRLP12 in bacterial infection, in which S.

typhimurium utilizes NLRP12 to modulate intestinal inflammationallowing its persistence and survival in the host. Indeed, Nlrp12�/�

mice were highly resistant to S. typhimurium infection andmacrophages infected with S. typhimurium displayed markedlyenhanced production of IL-6, KC and TNFa compared to wild-typecells. Liver homogenates of Nlrp12�/� mice revealed enhancedphosphorylation of ERK and IkBa but not NF-kB p100, suggestingthat NLRP12 specifically affected the canonical route of activationof NF-kB during S. typhimurium infection. Even though theactivator of NLRP12 remains unknown, Salmonella LPS alonewas sufficient to induce NLRP12-mediated attenuation of NF-kB[198].

The pro-inflammatory capacity of NLRP12 was initiallyattributed to its ability to self-oligomerize and associate withASC and caspase-1 to promote IL-1b production in vitro [176]. Thephysiological relevance of NLRP12-dependent cytokine productionhas recently been shown to promote host protection duringbacterial infection. Vladimer et al. showed that the NLRP12inflammasome highly contributed to the production of IL-1b andIL-18 in response to Yersinia pestis infection (Fig. 3). Nlrp12�/�miceinfected with this bacterium exhibited increased mortality andenhanced bacterial burden, which was associated with decreased


serum IL-18 and IL-1b levels [199]. Interestingly, this defect wasnot accompanied by impaired production of NF-kB-dependentcytokines, suggesting that NLRP12 contributes to host protectionagainst Y. pestis in an inflammasome-dependent manner. Eventhough the exact nature of the ligand that bound NLRP12 was notelucidated, the activation of the NLRP12 inflammasome wasdependent on a functional bacterial type 3 secretion system (T3SS)[199].

3.7. NLRC4 and NLRB

NLRC4 (IPAF) contains an N-terminal CARD domain, enabling adirect CARD-CARD interaction with caspase-1 [200]. NLRC4 isactivated by bacterial flagellin [201,202] or the rod complex ofbacterial type III and IV secretion systems (T3SS, T4SS) [203]. Theability of NLRC4 to recognize flagellin and T3SS components waslinked to an interaction between NLRC4 and another class of NODproteins known as NAIPs (NLRB). Murine NAIP5 and NAIP6recognize flagellin and NAIP2 recognizes T3SS rod proteins suchas PrgJ and Mxil, respectively, which then activate NLRC4 [200,204](Fig. 5). Thethorey et al. recently found that ligand detection byNAIPs is mediated by an internal region composed of NBD-associated a-helical domains, and not by the LRR domain [205].NAIP recognition of bacterial moieties represents one of the firstinstances of direct ligand binding to an NLR protein and providesinsights into the mechanisms of inflammasome activation. A




G Model


recent report found that NAIP1 in mice recognizes the needlecomponent of some T3SS [206]. In humans, a single NAIP exists,which functionally resembles murine NAIP1 as it recognizes theneedle protein of T3SS of the bacterium Chromobacterium

violaceum [207]. PKCd phosphorylation of NLRC4 on Ser533 duringS. typhimurium infection of macrophages was reported to berequired for inflammasome activation, as macrophages expressinga NLRC4 Ser533 mutant did not display ASC specks, producedreduced IL-1b levels and failed to induce pyroptotic cell deathfollowing infection [208]. Interestingly, NLRC4 inflammasomeactivation through recognition of the Shigella T3SS inner rodprotein Mxil by NAIP2 did not require PKCd-mediated phopshor-ylation of NLRC4 [209]. These findings suggest that activation ofNLRC4 can occur independently of this phopshorylation event.

4. Conclusion and future perspectives

Collectively, a large number of studies have supported the roleof NLRs as pivotal drivers of innate immunity. These are cytosolicreceptors capable of regulating inflammation by triggering theassembly of inflammasomes and by modulating the NF-kB, MAPKand IRF signaling pathways. The capability of some NLRs tofunction independently of these inflammatory pathways, namelyby the transcriptional upregulation of MHC molecules, broadensthe implication of these receptors in innate immunity. However,mechanisms governing direct ligand binding and activation ofmost NLRs still remain elusive. In light of this, investigationscentered on the identification of interacting partners and upstreammodulators of NLRs are needed. Furthermore, tissue-specificfunctions of NLRs and their regulation by environmental signalsrequire further exploration using next-generation mouse modelsin which tissue-specific deletion can be achieved in definedenvironmental conditions. Mouse genetic background and micro-biota variability among different facilities might underlie some ofthe variability described in the field and need to be carefullycontrolled for in the future.

Acknowledgments

This work was supported by grants from the Canadian Institutesfor Health Research (MOP-82801) and the Burroughs WellcomeFund to M.S. who is a Fonds de Recherche en Sante du Quebec(FRSQ) Senior Investigator and a McGill University WilliamDawson Scholar.

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G Model


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Francois Barbe obtained a B.Sc. in Agricultural & Envi-ronmental Sciences at McGill University in 2013, fol-lowed by a graduate certificate in bioinformatics. Herecently joined Dr. Maya Saleh’s laboratory within theMcGill Complex Traits Group for a M.Sc. in Microbiology& Immunology. His project consists in the characteriza-tion of cell death modalities in inflammatory boweldiseases.


Todd Douglas completed his B.Sc. Honors in Microbiol-ogy and Immunology at McGill University in 2012. He iscurrently a Ph.D. student under the supervision of Dr.Maya Saleh. His research focus lies in understanding thebiochemical mechanisms that regulate the activation ofthe inflammasome, and their implication in complexinflammatory disorders.

Maya Saleh obtained her Ph.D. in 2001 from the De-partment of Biochemistry at McGill University studyingmechanisms of transcriptional regulation. In 2002, Dr.Saleh joined Merck Research Laboratories and in 2004moved to the La Jolla Institute for Allergy and Immu-nology in California where she investigated mecha-nisms of apoptosis and innate immunity in hostdefense. Dr. Saleh joined the Faculty of Medicine atMcGill University in 2005 and is currently AssociateProfessor in the Departments of Medicine and Biochem-istry and Director of the Inflammation and CancerProgram. She is a McGill University Dawson Scholar, aFRSQ Chercheur-Boursier Senior and a Burroughs Well-

come Fund Investigator in the Pathogenesis of Infectious Disease. Dr. Saleh’sresearch group investigates mechanisms of inflammation and immunity, and therole of cell death in host–pathogen interactions and complex diseases with a focuson inflammatory bowel diseases and colon cancer.





















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Advances in Nod-like receptors (NLR) biology

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