1 Recognition of extracellular bacteria by NLRs and its role in the 2 development of adaptive immunity 3 4 Running title 5 NLR sensing of extracellular bacteria 6 7 Authors and affiliations 8 Jonathan Ferrand, Richard Louis Ferrero* 9 Centre for Innate Immunity and Infectious Diseases, Monash Institute of Medical Research, 10 Monash University, Clayton, VIC, Australia 11 Correspondence: 12 Richard Ferrero 13 Centre for Innate Immunity and Infectious Diseases 14 Monash Institute of Medical Research 15 Monash University 16 27-31 Wright St, Clayton 17 Victoria 3168 18 Australia 19 [email protected] 20 21 Number of words: 11069 22 Number of figure: 1 23 24 Abstract 25 Innate immune recognition of bacteria is the first requirement for mounting an effective 26 immune response able to control infection. Over the previous decade, the general paradigm 27 was that extracellular bacteria were only sensed by cell surface-expressed Toll-like receptors 28 (TLRs), whereas cytoplasmic sensors, including members of the Nod-like receptor (NLR) 29 family, were specific to pathogens capable of breaching the host cell membrane. It has 30 become apparent, however, that intracellular innate immune molecules, such as the NLRs, 31 play key roles in the sensing of not only intracellular, but also extracellular bacterial 32 pathogens or their components. In this review, we will discuss the various mechanisms used 33 by bacteria to activate NLR signaling in host cells. These mechanisms include bacterial 34 secretion systems, pore-forming toxins and outer membrane vesicles. We will then focus on 35 the influence of NLR activation on the development of adaptive immune responses in 36 different cell types. 37 38 Keywords 39 NLRs, extracellular bacteria, OMVs, adaptive immunity, innate immunity 40

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42 1. Introduction 43 A balanced relationship between humans and their microbiota is required for a variety of 44 biological functions, including optimal protection against invasion by microbial pathogens, 45 development of the mucosal immune system and control of metabolic processes (reviewed in 46 Sommer and Backhed, 2013). The ability of the host immune system to distinguish between 47 commensals and pathogens is required to avoid the development of persistent immune 48 responses against the normal microbiota, yet maintain appropriate immune responses to 49 pathogens. However, bacterial pathogens are able to avoid or subvert the host immune system 50 to promote their survival and colonization. To this end, bacteria can either secrete different 51 components into the extracellular medium or inject molecules into the host cell cytoplasm. In 52 parallel, host cells have developed a wide range of pattern-recognition receptors (PRRs), 53 including Toll-like receptors (TLRs) and Nod-like receptors (NLRs), to detect 54 microorganism- and/or danger-associated molecular patterns (MAMPS and DAMPS, 55 respectively) present in the extracellular medium or in their cytoplasm. MAMPS include 56 virulence factors, but also essential components of both commensals and pathogens, e.g. 57 lipopolysaccharide (LPS), peptidoglycan or nucleic acids. Recent studies have shown that the 58 recognition of the microbiota that takes place in the gut is necessary for the development of a 59 normal epithelium, by controlling the balance of proliferation and differentiation, as well as 60 maintaining a properly functioning immune system (Rakoff-Nahoum et al., 2004; Sommer 61 and Backhed, 2013). 62 Over the previous decade, the general paradigm was that extracellular bacteria were only 63 sensed by cell surface-expressed TLRs, whereas cytoplasmic sensors, including members of 64 the NLR family, were specific to pathogens capable of breaching the host cell membrane. 65 Structurally, NLRs share a typical tripartite architecture with a conserved central nucleotide- 66 binding domain, which restrains the catalytic activity of NLR family proteins. This central 67 domain is named NACHT after the original proteins which defined the features of this 68 domain: Neuronal apoptosis inhibitory protein (NAIP), MHC class II transcription activator 69 (CIITA), incompatibility locus protein from Podospora anserine (HET-E) and a telomerase- 70 associated protein (TP1). At the C-terminal region of NLR proteins are a series of leucine- 71 rich repeats (LRRs) that are believed to initiate NLR activation after recognition of the 72 appropriate signal, although this mechanism is still unclear (Ting et al., 2008). The N- 73 terminal effector domain, which specifies the function of NLRs, is less conserved. Indeed, 74 NLRs may harbor either a pyrin domain (PYD), a caspase-activation and recruitment domain 75 (CARD), a baculovirus inhibitor of apoptosis domain (BIR) or an as yet characterized domain 76 (Table 1) (Kufer and Sansonetti, 2011). To date, 23 NLR family members have been 77 reported, each playing different roles in pathogen recognition, homeostasis, apoptosis or gut 78 development (reviewed in Kufer and Sansonetti, 2011). In the context of host-pathogen 79 responses, NLR activation has been shown to induce the production of pro-inflammatory 80 effectors through either nuclear translocation of Nuclear Factor-κB (NF-κB) or formation of 81 high-molecular-weight platforms, named inflammasomes, which activate caspase-1. The 82 main substrates of caspase-1 are cytokine pro-forms of IL-1β and IL-18, which are usually 83 expressed in an NF-κB-dependent manner. Hence, to be expressed in a fully mature form, 84 these cytokines require regulation at both transcriptional and post-transcriptional levels. 85 Several distinct inflammasomes have been described in the literature, consisting of different 86 scaffolding proteins of the NLR or the PYHIN (PYRIN and HIN-200) superfamilies 87 (reviewed in von Moltke et al., 2013).

2 88 Despite their intracellular localization, it has become apparent that NLRs play key roles in 89 the sensing of not only intracellular, but also extracellular bacterial pathogens or their 90 components. In this review, we will summarize the mechanisms used by extracellular 91 pathogens to deliver bacterial components into host cells and how infections by these 92 microorganisms are sensed via NLRs. Lastly, we will discuss the importance of the activation 93 of innate immunity receptors, such as NLRs, in tailoring an appropriate adaptive immune 94 response.

95 2. Mechanisms whereby bacterial components gain access to the cytoplasm 96 2.1 Bacterial secretion systems 97 Bacteria that need to deliver their effectors across both bacterial and cell membranes have 98 developed highly specialized secretion systems to reach their cytoplasmic targets. Among the 99 6 described secretion systems, the injection of bacterial components through either type-3 or 100 type-4 secretion systems (T3SS and T4SS, respectively) has been reported to result in the 101 activation of NLR signaling in host cells (Figure 1). 102 The T3SS, or “injectisome”, is a specialized molecular machine which is closely related 103 to the bacterial flagellar apparatus. T3SSs have been identified in numerous Gram-negative 104 bacteria, including pathogens, symbionts and commensals, suggesting that the T3SS is not a 105 hallmark of pathogenic microorganisms (reviewed in Cornelis, 2006 and Tseng et al., 2009). 106 The global architecture of injectisomes is conserved between bacteria and comprises a needle 107 complex, composed of two pairs of rings that are connected by a rod, spanning the inner and 108 outer bacterial membranes. This structure has at its end a hollow needle, a filament or a pilus. 109 The main function of T3SSs is to deliver effector proteins across the membranes of host cells, 110 in which these molecules are able to activate cell signaling pathways. 111 Similarly, T4SSs are specialized macromolecular machines that can deliver DNA and/or 112 proteins to host cells. In contrast to T3SSs, however, those of the type-4 family are believed 113 to be related to bacterial conjugation systems, rather than the flagellar apparatus. T4SSs have 114 been identified in many Gram-negative and -positive bacteria, as the complex can span both 115 types of membrane (reviewed in Tseng et al., 2009 and Voth et al., 2012). T4SSs are 116 classified as Type A or B, depending on structure composition, but both aim to deliver 117 bacterial effectors to host cells. The Type A T4SSs are defined by their homology with the 118 VirB/D4 system of the plant pathogen Agrobacterium tumefaciens, whereas the Type B 119 T4SSs are closer to the conjugal transfer systems of the self-transmissible IncI plasmid (Voth 120 et al., 2012). 121 The first study to report a mechanism for the recognition of extracellular bacteria by 122 intracellular receptors was described by Viala et al., who showed how virulent Helicobacter 123 pylori strains are able to activate the cytosolic NLR family member, nucleotide-binding 124 oligomerization domain-containing protein 1 (NOD1) (Viala et al., 2004). Specifically, the 125 authors showed that H. pylori strains with a functional T4SS were able to deliver degradation 126 products of Gram-negative cell wall peptidoglycan, identified as potent activators of NOD1 127 signaling (Girardin et al., 2003), to epithelial cells (Table 1). The mechanism of how 128 H. pylori peptidoglycan is delivered to cytosolic NOD1, however, is still unclear, even if it 129 was reported that depletion of cholesterol-rich domains, or lipid rafts, interferes with 130 peptidoglycan delivery into host cells (Hutton et al., 2010). In any case, H. pylori T4SS- 131 dependent induction of the NOD1 pathway was shown to result in the downstream activation 132 of NF-κB and MAP kinases, most likely through the recruitment of the serine-threonine 133 kinase adaptor molecule, RIP2 (Allison et al., 2009; Allison et al., 2013). These findings are 134 consistent with the general view that the NOD1 signaling pathway converges on the master 135 transcriptional regulator, NF-κB, leading to pro-inflammatory cytokine production (reviewed

3 136 in Correa et al., 2012). One group, however, suggested that NOD1 signaling is largely 137 independent of NF-κB and MAPK activation (Watanabe et al., 2010). Instead, these workers 138 presented data showing that the dominant response mediated by NOD1 activation involves 139 the formation of a complex known as IFN-stimulated gene factor 3 (ISGF3) and production 140 of CXCL10 and type I IFN by epithelial cells (Watanabe et al., 2010). The authors 141 demonstrated that stimulation of AGS gastric epithelial cells with either a synthetic NOD1 142 agonist or live H. pylori bacteria alone induced increased CXCL10 production. Nevertheless, 143 the potential link between NOD1 and type I IFN in epithelial cell responses, though 144 interesting, awaits confirmation by other researchers. 145 Since the work on the role of NOD1 in H. pylori sensing, other bacterial pathogens have 146 been identified as also being activators of this pathway e.g. Pseudomonas aeruginosa and 147 Campylobacter jejuni (Travassos et al., 2005; Zilbauer et al., 2007). These bacteria have 148 essentially extracellular lifestyles, although some data suggest that NOD1 activation in these 149 infection models may be due to the presence of intracellular bacteria. However, even if 150 invasive P. aeruginosa and C. jejuni have been shown to be present in defined intracellular 151 structures, peptidoglycan delivery and activation of cytoplasmic sensors, such as NOD1, must 152 still require an efficient secretion system (Angus et al., 2008; Bouwman et al., 2013). 153 Furthermore, a recent study has shown that Salmonella typhimurium activation of NOD1 and 154 NOD2, a related molecule that senses all forms of bacterial peptidoglycan, may be invasion- 155 independent but requires an intact T3SS for the injection into the cytoplasm of the bacterial 156 protein, SipA (Keestra et al., 2011). This work, identifying a novel agonist of NOD1- and 157 NOD2-signaling, suggests that these NLR family members may be activated through a 158 distinct pathway from that induced by bacterial peptidoglycan. The same group suggested 159 recently that NOD1 could sense other patterns of pathogenesis, such as modification of the 160 actin cytoskeleton. According to this intriguing new model, a T3SS-secreted protein of S. 161 typhimurium, SopE, activates the small Rho GTPases, which then triggers the NOD1 162 signaling pathway (Keestra et al., 2013). 163 Another major NLR family member that is activated by extracellular pathogens 164 through the action of bacterial secretion systems is the CARD-containing protein, NLRC4 165 (previously known as Ice Protease-Activating Factor, IPAF) (Table 1). The early paradigm 166 for NLRC4 activation was that this NLR senses bacterial flagellin within the cytoplasmic 167 compartment of cells. Specifically, it was shown that T3SS-dependent translocation of 168 S. typhimurium and P. aeruginosa flagellin into the host cell triggered NLRC4 activation in 169 macrophages (Franchi et al., 2006; Miao et al., 2006; Sun et al., 2007; Miao et al., 2008). 170 Interestingly, subsequent reports contradicted this hypothesis as both Shigella flexneri, an 171 aflagellated bacterium, and P. aeruginosa were also able to activate NLRC4 inflammasome 172 in a flagellin-independent, but T3SS-dependent mechanism, suggesting the existence of one 173 or several other ligands (Sutterwala et al., 2007; Suzuki et al., 2007). These findings have 174 since been confirmed in various bacterial species and the molecules responsible for NLRC4 175 activation have now been identified. Thus, it was shown that the basal body rod components 176 of T3SSs (rod protein) are detected during infection with either S. typhimurium (PrgJ), 177 Burkholderia pseudomallei (BsaK), Escherichia coli (EprJ and EscI), S. flexneri (MxiI) or P. 178 aeruginosa (PscI) (Miao et al., 2010). Interestingly, all these proteins appear to share a 179 sequence motif present in the carboxy terminal region that is conserved in the flagellar 180 protein, FliC, and detected by NLRC4 (Miao et al., 2010). The mechanism of how NLRC4 is 181 able to sense and respond to two distinct bacterial products, flagellin or PrgJ-like proteins, 182 has thus been deciphered. Concomitant animal infection studies from two different groups 183 showed that the specificity of the NLRC4 inflammasome is determined by different NAIP 184 paralogues (Kofoed and Vance, 2011; Zhao et al., 2011). In these new models, both flagellin 185 and rod protein are injected through the T3SS and are specifically recognized by NAIP5/6 or

4 186 NAIP2 respectively, confirming previous studies showing a physical association between 187 NLRC4 and NAIP5 (Figure 1) (Zamboni et al., 2006; Kofoed and Vance, 2011; Zhao et al., 188 2011). Interestingly, humans possess only one Naip gene compared with the 6 Naip genomic 189 loci in mice, and the specificity of human NAIP appears to be different to the murine NAIPs 190 as it is unresponsive to intracellular delivery of flagellin or PrgJ-like rod proteins (Zhao et al., 191 2011). Although it has been shown that a PrgJ homologue, from the bacterium 192 Chromobacterium violaceum, is specifically recognized by human NAIP (Zhao et al., 2011), 193 further studies are required to determine the importance of human NAIP during infection 194 with common bacterial pathogens. 195 T4SS and flagellin have also both been suggested to play roles in NLRC4 activation 196 during Legionella replication within macrophages, since the presence of all three elements 197 (i.e. flagellin, a functional T4SS and NLRC4) is required to activate caspase-1 in these cells. 198 Consistent with these findings, in vivo studies confirmed that clearance of Legionella 199 pneumophila required the presence of both flagellin and NLRC4 (Amer et al., 2006). 200 Finally, bacterial secretion systems have recently been implicated in the activation of the 201 NLR pyrin domain-containing protein 12 (NLRP12) by the pathogen, Yersinia pestis (Table 202 1) (Vladimer et al., 2012). The nature of the NLRP12 ligand is still unknown, however, by 203 using a Y. pestis strain which lacks the virulence plasmid necessary for the formation of a 204 T3SS, the authors were able to show that this secretion system was required for 205 inflammasome activation and IL-1β release in bone marrow-derived macrophages (BMDMs) 206 (Vladimer et al., 2012). The precise mechanism by which the Y. pestis T3SS mediates 207 NLRP12 inflammasome activation remains to be elucidated. 208

209 2.2 Outer membrane vesicles 210 In addition to bacterial secretion systems, in which individual proteins or macromolecules 211 are secreted, bacteria have developed other mechanisms to transfer a wide variety of 212 components within the host cells. The release of outer membrane vesicles (OMVs) is one 213 such strategy developed by Gram-negative bacteria to secrete toxins, enzymes, DNA, 214 adhesins or other periplasmic constituents into the extracellular medium (reviewed in Ellis 215 and Kuehn, 2010). It is also noteworthy that commensal bacterium, Bacteroides fragilis, 216 releases a capsular polysaccharide in its OMVs that has immunomodulatory effects and can 217 prevent inflammation in an experimental colitis model (Shen et al., 2012). OMVs are released 218 by virtually all Gram-negative bacteria, whereas the level of expression differs between 219 bacterial species (Ellis and Kuehn, 2010). More recent reports also suggest that Gram- 220 positive bacteria can secrete membrane vesicles, however, these are less well studied than 221 those of Gram-negative organisms (Lee et al., 2009a; Rivera et al., 2010). OMVs can be 222 released under different conditions in vitro and in vivo from free-living cells, biofilms or by 223 internalized bacteria (Garcia-del Portillo et al., 1997; Schooling and Beveridge, 2006; 224 Toyofuku et al., 2012). 225 The primary roles of OMVs are believed to be the delivery of toxins or bacterial 226 components into host cells and the evasion of host immune responses (Tan et al., 2007). On 227 the other hand, several studies have revealed that OMVs are able to induce inflammatory 228 responses that may protect the host from infection. Indeed, OMVs contain different MAMPs, 229 including LPS, flagellin or DNA that could be recognized by TLR and NLR family members 230 (Ellis et al., 2010; Sharpe et al., 2011; Shen et al., 2012). Traditionally, many studies in the 231 literature have focused on OMV-associated LPS, however, it is now becoming apparent that 232 peptidoglycan represents a major promoter of the inflammatory responses induced by OMVs. 233 An initial clue to the potential role of OMV-associated peptidoglycan in innate immunity 234 arose from the observation that S. flexneri culture supernatants, when microinjected into

5 235 epithelial cells, induced the activation of the NF-κB signaling pathway (Philpott et al., 2000; 236 Girardin et al., 2003). Indeed, it was shown that the OMVs normally present in such 237 supernatants are able to enter non-phagocytic cells and deliver peptidoglycan directly to 238 cytoplasmic NOD1, resulting in the up-regulation of NF-κB and IL-8 responses in cells 239 (Figure 1) (Kaparakis et al., 2010). This OMV-dependent mechanism of NOD1 activation 240 was demonstrated for three extracellular pathogens: H. pylori, P. aeruginosa, and Neisseria 241 gonorrhoeae (Table 1) (Kaparakis et al., 2010). Moreover, it was shown that Nod1-deficient 242 mice intragastrically fed with H. pylori OMVs failed to mount local Cxcl2 and systemic 243 antibody responses, when compared with their wild-type littermates (Kaparakis et al., 2010). 244 Subsequent studies reported that Vibrio cholerae strains, which also produce large 245 amounts of OMVs, can promote immune responses in the host via a NOD1- and NOD2- 246 dependent mechanism (Bielig et al., 2011; Chatterjee and Chaudhuri, 2013). OMVs isolated 247 from Moraxella catarrhalis have also been shown to induce IL-8 production through TLR2- 248 initiated NF-κB activation, but a role for NOD1 could not been excluded, as cellular 249 responses to whole bacteria involved both TLR2 and NOD1 (Slevogt et al., 2007; Schaar et 250 al., 2011). Pro-inflammatory responses to OMVs were also observed for several other 251 bacteria (reviewed in Ellis and Kuehn, 2010), however, the role of peptidoglycan in these 252 responses was not assessed. 253 Besides peptidoglycan, OMVs contain other molecules able to activate the innate immune 254 system. For example, N. gonorrhoeae OMVs contain lipooligosaccharide (LOS) which has 255 been shown to activate NLRP3-induced IL-1β secretion and pyronecrosis in monocytes and 256 macrophages (Figure 1) (Fisseha et al., 2005; Duncan et al., 2009). This LOS-mediated 257 activation of the NLRP3 inflammasome is believed to be triggered by the release of cathepsin 258 B (Duncan et al., 2009). 259 Mechanistic data on OMV uptake are scarce, but recent findings suggest that disruption 260 of lipid rafts by treatment with Fumonisin B1, an inhibitor of sphingomyelin incorporation 261 into lipid rafts, or methyl-β-cyclodextrin, a cholesterol-depleting agent, abrogates both 262 internalization and NOD1-dependent immunostimulatory capacity of OMVs (Kaparakis et 263 al., 2010). The mechanism and intracellular compartment(s) involved in NOD1 sensing of 264 OMV-associated peptidoglycan, however, have yet to be determined. 265 The findings concerning the role of peptidoglycan delivery by OMVs provide a new 266 mechanism to understand how extracellular bacteria, which are unable to invade cells or to 267 inject components through a secretion system, may be able to initiate innate immune 268 signaling in non-phagocytic cells, such as epithelial cells. 269

270 2.3 Pore-forming toxins 271 In addition to OMVs, bacteria may secrete toxins to alter host cell integrity distant to the 272 original point of invasion. Among these proteins are the pore-forming toxins (PFTs), which 273 are produced by a wide range of pathogens. PFTs are secreted in a soluble form and are 274 subsequently multimerized into a transmembrane channel that perforates the plasma 275 membrane of host cells. Pore formation may be an entry door for bacterial molecules to 276 penetrate into host cells or lead to cellular ion imbalance (Gonzalez et al., 2008). Efforts 277 during the last decade have been focused on determining how cells are able to mount a 278 response against pore formation, thereby contrasting with the existing paradigm, which 279 suggested that cells possessed no defenses against these toxins and that the only outcome was 280 cell death (Aroian and van der Goot, 2007; Gonzalez et al., 2008). Numerous studies, indeed, 281 found that stimulation of immune cells with different PFTs activates pro-inflammatory 282 signaling or vacuolation in response to treatment (Ratner et al., 2006; and reviewed in Aroian 283 and van der Goot, 2007). It is also noteworthy that the concentrations of PFTs during in vivo

6 284 infection could be sublytic, thus allowing cells to mount an antibacterial response (Spreer et 285 al., 2003), capable of controlling the infection. 286 Recent intensive studies on PFTs and cellular responses have revealed a major role for 287 NLRP3 in the sensing of pore formation. Mariathasan et al. demonstrated a role for 288 listeriolysin O from Listeria monocytogenes, as well as for an unknown Staphylococcus 289 aureus toxin, in inflammasome activation and IL-1β production (Table 1). Furthemore, this 290 study speculated that the observed effects were dependent on intracellular potassium levels 291 (Figure 1) (Mariathasan et al., 2006). Concerning S. aureus, recent studies showed that 292 caspase-1 activation requires the presence of all three of its α-, β- and γ-hemolysins and the 293 release of bacterial lipoproteins (Munoz-Planillo et al., 2009; Kebaier et al., 2012). In 294 addition, a small percentage of S. aureus isolates also produce another toxin, named Panton- 295 Valentine leukocidin (PVL), which is able to trigger NLRP3 inflammasome activation 296 (Holzinger et al., 2012). These results were confirmed using aerolysin from Aeromonas 297 hydrophila which was shown to mediate the efflux of intracellular potassium ions and 298 activation of caspase-1 through the assembly of NLRC4 and NLRP3 inflammasomes (Gurcel 299 et al., 2006). Subsequent studies suggested that aerolysin from either A. veronii or 300 A. hydrophila activates only the NLRP3 inflammasome, through potassium efflux, whereas 301 NLRC4 activation was T3SS-dependent but potassium-independent (McCoy et al., 2010a; 302 McCoy et al., 2010b). During the last decade, a large number of bacterial PFTs have been 303 shown to be able to activate NLRP3 via the same molecular mechanism of potassium efflux. 304 These PFTs include the adenylate cyclase toxin (CyaA) from Bordetella pertussis (Dunne et 305 al., 2010), pneumolysin from Streptococcus pneumoniae (McNeela et al., 2010), HlyA 306 hemolysin and MARTX from Vibrio vulnificus and V. cholerae (McNeela et al., 2010), 307 streptolysin O from Streptococcus pyogenes (Harder et al., 2009). Even if the molecular 308 mechanism is unclear, NLRP3 inflammasome assembly occurs spontaneously at low 309 potassium concentrations and is prevented at higher concentrations, thus confirming its role 310 in the detection of DAMPs (Petrilli et al., 2007). In a recent study, potassium efflux was 311 shown to be the minimal membrane permeabilization event triggering NLRP3 inflammasome 312 activation by PFTs and particulate matter (Munoz-Planillo et al., 2013). 313 In contrast to our understanding of NLRP3 biology, far less is known about activation of 314 the NLRP1 inflammasome and its activation by PFTs. The human Nlrp1 gene has 3 murine 315 paralogues, which encode proteins lacking the N-terminal pyrin domain sequence found in 316 human NLRP1 (Moayeri et al., 2012). Sensitivity of mice to the effects of anthrax lethal toxin 317 from Bacillus anthracis has been correlated with a polymorphism in the Nalp1b gene, 318 encoding Nlrp1 (Boyden and Dietrich, 2006). Due to the absence of a PYD domain in the 319 mouse sequence of Nlrp1, it is not clear whether caspase-1 recruitment requires ASC or 320 dimerization with another NLRP. However, recent data suggest that upon stimulation with 321 anthrax lethal toxin, NLRP1 undergoes autoproteolysis to form an inflammasome (Frew et 322 al., 2013). Interestingly, inflammasome formation and caspase-1 recruitment are inhibited by 323 high levels of potassium (Wickliffe et al., 2008). Hsu et al. demonstrated a role for NOD2 in 324 lethal toxin-induced IL-1β production and suggested the formation of a complex between 325 NLRP1 and NOD2 (Hsu et al., 2008). This association has since been confirmed by other 326 authors (Wagner et al., 2009). Interestingly, NOD2-recognition of S. aureus is facilitated by 327 the presence of its hemolysin, probably by promoting cytoplasmic access of NOD2 ligand 328 (Hruz et al., 2009). 329 Synergistic responses from the activation of multiple NLR pathways have been observed 330 following co-stimulation with two different pathogens. Indeed, it was shown that 331 Haemophilus influenzae peptidoglycan enters epithelial cells more efficiently in the presence 332 of S. pneumoniae pneumolysin, suggesting the ability of intracellular NLRs to sense 333 extracellular bacteria that do not encode secretion systems or express OMVs or PFTs (Ratner

7 334 et al., 2007). One hypothesis from the authors is that host organisms have evolved to detect a 335 combination of pathogens in order to mount optimal responses in ways that are different to 336 the responses induced by a single infection. It is now recognized that co-infection plays a 337 previously unappreciated yet important role in the development of mucosal immunity and 338 disease progression (Lijek and Weiser, 2012). 339 In conclusion, increasing numbers of studies suggest that osmotic changes induced by 340 pore formation may be sensed by intracellular NLRs as an early warning system (Munoz- 341 Planillo et al., 2013). Furthermore, these signals can cooperate with other signaling pathways, 342 thus leading to the generation of antibacterial responses before the concentration of PFTs 343 reaches a lytic concentration. 344

345 2.4 Endocytosis 346 Asides from the active processes described above, intracellular NLRs may be activated by 347 passive mechanisms. One such mechanism involves cellular entry by the peptidoglycan 348 fragments that are released during bacterial growth or are degraded by host enzymes. Bacteria 349 express peptidoglycan-degrading enzymes, necessary for maintaining functional growth, 350 division and development (reviewed in Cloud-Hansen et al., 2006; Park and Uehara, 2008 351 and Uehara and Bernhardt, 2011). In addition to their role in shaping bacterial membranes, 352 these enzymes are responsible for the release of free peptidoglycan fragments in the 353 extracellular compartment. It is thus possible that these fragments interact with surrounding 354 organisms, mediating pathogenic effects on host cells, as mutations in peptidoglycan- 355 recycling proteins result in decreased pathogenesis. Conversely, released peptidoglycan 356 fragments can also play a role in symbiotic relationships, such as is the case with Vibrio 357 fischeri, which induces developmental changes in its squid host (reviewed in Cloud-Hansen 358 et al., 2006). Moreover, peptidoglycan can have effects on host cells distant to the point of its 359 release. For example, peptidoglycan released in the gut was shown to circulate and play a 360 major role in the priming of neutrophils in the bone marrow (Clarke et al., 2010). 361 Interestingly, a study by Hasegawa et al., characterizing NOD1- and NOD2-stimulatory 362 activities in different bacterial preparations, showed that the highest levels of NOD1- 363 stimulatory activity were found predominantly in culture supernatants, whereas NOD2 364 activity was associated with extracts from whole bacterial cells (Hasegawa et al., 2006). This 365 study further underscores the likely important role of released peptidoglycan during infection 366 with extracellular bacteria. 367 On the other hand, host cells have also developed some mechanisms to degrade bacterial 368 peptidoglycan in order to kill invading pathogens and provide ligands to host receptors 369 (reviewed in Davis and Weiser, 2011). Enzymes such as lysozyme or peptidoglycan 370 recognition protein family members generate fragments small enough to be sensed by NOD1 371 and NOD2 (Clarke and Weiser, 2011; Davis and Weiser, 2011). 372 In the context of extracellular pathogens, we can then wonder how these peptidoglycan 373 fragments are processed to be presented to intracellular innate immune sensors. It is now 374 established that different internalization mechanisms can be used by the host cell and these 375 are probably cell-type dependent. In the case of epithelial cells, it has been reported that 376 NOD1 and NOD2 ligands are likely to be internalized by clathrin-mediated endocytosis (Lee 377 et al., 2009b; Marina-Garcia et al., 2009). In immune cells, it is more likely that 378 internalization occurs through phagocytosis. Interestingly, it has been observed that Nod1- or 379 Nod2-deficient mice have decreased phagocytic abilities (Deshmukh et al., 2009; Dharancy 380 et al., 2010). 381 Once the fragments have been internalized in vesicles by clathrin-mediated endocytosis 382 or phagocytosis, they have to be delivered across membranes and be presented to the

8 383 cytosolic molecules, NOD1 and NOD2. This mechanism implies the presence of specific 384 transporters, with the transporter family SLC15 having been proposed to play a role. This 385 transporter family comprises membrane proteins controlling the cellular uptake of 386 di/tripeptides and peptide-like drugs (reviewed in Smith et al., 2013). Roles for SLC15A4 387 (PHT1) and SLC15A2 (PepT2) have indeed been identified for the delivery of NOD1-ligands 388 (Figure 1) (Swaan et al., 2008; Lee et al., 2009b; Sasawatari et al., 2011). Another member of 389 this family, SLC15A1 (PepT1), is believed to play a role in NOD2 ligand transport (Vavricka 390 et al., 2004; Marina-Garcia et al., 2009). Nevertheless, the specificity of each of these 391 transporters is still unclear, as both the minimal motif recognized by NOD1, iE-DAP (γ-D- 392 Glu-mDAP), and the NOD2 agonist, MDP (MurNAc-L-Ala-D-isoGln, also known as 393 muramyl dipeptide) may be delivered through SLC15A2 (Swaan et al., 2008; Charriere et al., 394 2010). Interestingly, there are higher expression levels of SLC15A1, SLC15A2 and 395 SLC15A4 in the small intestine, with lower bacterial loads of 103 organisms per gram, than in 396 the colon, where microbial densities reach 1012 organisms per gram. This may suggest a role 397 for such transporter proteins in MAMP uptake (reviewed in Ingersoll et al., 2012, and 398 Sommer and Backhed, 2013). 399

400 3. Control of adaptive immunity 401 Activation of NLRs has been shown to prime T and B cells, suggesting the contribution 402 of these innate immune molecules in the development of adaptive immune responses. 403 Interestingly, many of the known NLRs activators (e.g. MDP, flagellin, alum) play roles as 404 adjuvants during vaccination, suggesting a role for these molecules in tailoring the adaptive 405 immune response (reviewed in Coffman et al., 2010). 406 The most striking example is the role played by NOD2 in the mediation of the adjuvant 407 effect of complete Freund’s adjuvant, first described in 1937 (Freund et al., 1937). The 408 adjuvant activity of this compound, which is composed of paraffin oil and Mycobacterium 409 tuberculosis extract, is believed to be attributed to its ability to prolong antigen release, to 410 promote the recruitment of immune cells and antigen presentation by inducing expression of 411 cytokines and chemokines (reviewed in Foss and Murtaugh, 2000). The minimal component 412 of complete Freund’s adjuvant was subsequently identified to be MDP, as this molecule 413 provided the same level of adjuvant activity as whole killed M. tuberculosis (Adam et al., 414 1981). The mechanism responsible for the activity of Freund’s adjuvant was determined less 415 than ten years ago by Kobayashi et al., who showed that NOD2 was required for the 416 development of protective immunity mediated by the adjuvant effects of MDP (Kobayashi et 417 al., 2005). During immunization assays in Nod2-/- animals, a severe deficiency was observed 418 in the production of antigen specific immunoglobulins, specifically in those of the IgG1 419 subclass, suggesting that NOD2 is able to activate the adaptive immune system and promote 420 the production of antibodies to T cell-dependent antigens (Kobayashi et al., 2005). These 421 results were confirmed subsequently, when it was shown that MDP injection in mice 422 triggered Th2 polarized responses in a NOD2-dependent manner (Magalhaes et al., 2008). 423 Specifically, it was shown that Nod2-deficient mice displayed impaired chemokine and Th2 424 responses with low numbers of splenic IL-4- and IL-5-producing T cells, as well as the loss 425 of antigen-specific T and B cell responses (Magalhaes et al., 2008). Interestingly, NOD2 can 426 also cooperate with TLRs to generate Th1-polarized responses to co-stimulation with MDP 427 and TLR2 or TLR4 ligands, suggesting the importance of complementary effects between 428 TLRs and NLRs in the balance of immune effector responses (Magalhaes et al., 2008). In 429 addition to the recognition of MDP by NOD2, dual recognition of a mycobacterial glycolipid, 430 also known as cord factor, and peptidoglycan is essential for Th17-differentiation in an

9 431 inflammasome-dependent manner (Shenderov et al., 2013). It was shown that recognition of 432 both the mycobacterial cord factor by the CARD9-dependent C-type lectin receptor mincle 433 and peptidoglycan, via a NOD1- and NOD2-independent mechanism, induces inflammasome 434 activation and IL-1β secretion and thus drives skewed Th17 responses. 435 NOD1 stimulation has also been shown to be sufficient to drive antigen-specific 436 immunity with a predominant Th2 polarization profile and to play a role in the onset of Th1, 437 Th2 and Th17 immune pathways in conjunction with TLR stimulation (Fritz et al., 2007). 438 Thus, depending on the presence of different MAMPS and the co-stimulation of TLRs, NOD 439 receptors can initiate different arms of the adaptive response. Although data are still scarce 440 concerning the role of NOD1 and NOD2 in the onset of adaptive immunity during microbial 441 infection, H. pylori-infected Nod1-deficient mice exhibited reduced Th1 immune responses 442 compared with their WT littermates (Fritz et al., 2007). Similar results were obtained in M. 443 tuberculosis or S. pneumoniae-infected Nod2-deficient mice, with lower titers of pathogen- 444 specific serum IgG and diminished antigen-specific T cell responses (Divangahi et al., 2008; 445 Davis et al., 2011). Consistent with these data, Citrobacter and Salmonella infections 446 triggered Th17 responses that were dependent on NOD1 and NOD2 (Geddes et al., 2012). 447 Injection of bone marrow reconstituted mice with NOD1 or NOD2 agonists and 448 ovalbumin allowed the group of Philpott and collaborators to determine the importance of 449 stromal factors, versus hematopoietic cells, in the initiation of Th2 immune responses (Fritz 450 et al., 2007; Magalhaes et al., 2011). In addition, that group showed that the capacity of 451 NOD1 ligand to cooperate with TLR agonists was completely abolished in Nod1-deficient 452 bone marrow-derived dendritic cells (BMDCs) (Fritz et al., 2007). Similarly, Nod2- 453 deficiency in BMDCs abolished pro-inflammatory cytokine production upon stimulation with 454 MDP alone, whereas synergy between MDP and TLR ligands was lost in Nod2-deficient 455 BMDMs (Kobayashi et al., 2005). Additionally, full responses required sensing within the 456 hematopoietic compartment, with a major role for dendritic cells, consistent with the well- 457 established role of dendritic cells in the onset of adaptive immunity (Le Bourhis et al., 2009; 458 Magalhaes et al., 2011). Furthermore, co-stimulation with NOD1 or NOD2 agonists in 459 combination with TLR agonist induces a synergistic production of Th1-associated cytokines 460 IFN-γ and IL-12 (Tada et al., 2005). 461 In contrast to the now well-defined role of NOD proteins in tailoring adaptive immune 462 responses, the role of the NOD1/2 adaptor protein, RIP2, is less well-defined. Several early 463 works in in vivo or in vitro RIP2-deficient models demonstrated impairment in the 464 development of anti-infectious responses, NF-κB signaling or T cell proliferation and 465 differentiation (Chin et al., 2002; Kobayashi et al., 2002; Ruefli-Brasse et al., 2004). On the 466 other hand, more recent papers, all of them using the same mouse model but different to those 467 used previously, claimed an absence of effect of RIP2 in T cell proliferation and T helper 468 differentiation (Hall et al., 2008; Nembrini et al., 2008). A comparison of the different RIP2- 469 knockout mouse lines may help to resolve these differences. 470 Recent studies have shown that non-hematopoietic cells can also be of importance during 471 the development of adaptive immune responses. Indeed, Watanabe et al. proposed that 472 activation of NOD1 and NOD2 in gastrointestinal epithelial cell lines induces production of 473 cytokines associated with a Th1 response (Watanabe et al., 2010). They also proposed that 474 NOD1 signaling, through ISGF3 activation and type I IFN responses, may lead to Th1 475 differentiation and Th1-dependent inflammation (Watanabe et al., 2010). 476 Concerning the other NLR family members, further studies will be needed to help to 477 understand their roles in adaptive immunity. It has been shown using the Listeria infection 478 mouse model that strains that activated the inflammasome generated significantly less 479 protective immunity, a phenotype that correlated with decreased induction of antigen-specific 480 T cells (Sauer et al., 2011). It is noteworthy that IL-1 family cytokines, including IL-1β and

10 481 IL-18, have adjuvant properties, as they can induce antigen-specific immune responses 482 against infection (Kayamuro et al., 2010). For example, CyaA, a pore forming toxin from B. 483 pertussis, activates the NLRP3 inflammasome and induces IL-1β expression, thereby playing 484 a critical role in promoting antigen-specific Th17 cells and in generating protective immunity 485 against B. pertussis infection (Dunne et al., 2010). Interestingly, a recent report suggested that 486 IL-1β production in trophoblasts after Chlamydia trachomatis infection may also be mediated 487 by NOD1, but the signaling pathway involved remains unclear (Kavathas et al., 2013). In 488 addition, IL-1β may be secreted after non-canonical inflammasome activation, where an 489 intracellular lipid A moiety of LPS has been showed to play major roles in the induction of 490 TLR4-independent inflammatory responses (Kayagaki et al., 2013). Although the receptor 491 has as yet to be characterized, these results suggest a new mechanism of intracellular sensing 492 in the mounting of innate immune responses against microbial infection. 493

494 4. Concluding remarks 495 As discussed above, various NLR family members have evolved to detect infection and 496 mount effective immune responses mediated by both innate and adaptive arms of the immune 497 system. Asides from these NLR family members, it is possible that other family members 498 could play roles during infection with extracellular bacteria. Indeed, NLRP6-deficiency in 499 mice was shown to result in increased inflammation, to alter the colonic microbial ecology 500 and was associated with susceptibility to colorectal tumorigenesis (Chen et al., 2011; Elinav 501 et al., 2011). More recently, NLRP6 has been shown to inhibit NF-κB translocation and 502 MAPK activation, with NLRP6 activation leading to increased susceptibility to both 503 intracellular and extracellular bacteria (Anand et al., 2012). Thus, a second subclass of NLR 504 family members, such as NLRP6 or NLRC5, may act as molecular switches to dampen host 505 responses induced by extracellular bacteria. For example, NLRC5 has been suggested to 506 interact with NF-κB regulators, IKKα and IKKβ, and to block their phosphorylation so as to 507 modulate inflammatory signaling to bacterial pathogens (Cui et al., 2010). Discordant results 508 were, however, obtained in different studies (Benko et al., 2010; Neerincx et al., 2010; Tong 509 et al., 2012), suggesting that further investigations are required to fully elucidate the role(s) of 510 NLRC5 in host responses to microbial pathogens. Besides the NLR family members 511 described in this review, other intracellular molecules, such as certain TLRs (i.e. TLR3, 512 TLR9) or Absent In Melanoma 2 (AIM2), are able to detect nucleic acids from extracellular 513 bacteria, allowing a wide range of MAMPs to be sensed. 514 The different examples of infection sensing described above highlight the existence of 515 dual systems of recognition for MAMPs from extracellular bacteria. First, conserved 516 molecular patterns in bacteria may be recognized by extracellular receptors. For instance, 517 sensing of bacterial lipoproteins by TLR2, LPS by TLR4 or flagellin by TLR5 have been 518 relatively well described. Activation of these extracellular receptors leads to a transcriptional 519 inflammatory response with production of type I IFNs or pro-inflammatory cytokines, such 520 as TNF-α or IL-12. However, particular cytokines, including IL-1β or IL-18, require an 521 additional post-transcriptional step to be fully functional. A growing amount of evidence 522 suggests that the signaling involved in this post-transcriptional response is due to activation 523 of inflammasome complexes, after sensing of microbes or danger signals by intracellular 524 molecules, including the NLRs. However, some intracellular sensors of extracellular bacteria, 525 such as NOD1 and NOD2, do not induce inflammasome formation and are generally thought 526 to activate NF-κB signaling instead. Nevertheless, we can reasonably hypothesize that host 527 cells are able to distinguish between the signals originating from extracellular and 528 intracellular pathogens, through the intensity, kinetics or cell-specific nature of the signal.

11 529 This dual recognition of the pathogen itself, or of the consequences of the infection, may 530 be of importance to finely tune inflammatory responses in line with the threat. One 531 hypothesis is that non-pathogenic bacteria may be recognized by extracellular receptors only, 532 whereas pathogenic extracellular or invasive bacteria will be sensed by both families of 533 receptors, leading to more intense responses, suggesting that synergy between TLRs and 534 NLRs may be required for optimal responses. As evoked in this review, it was found that 535 NLR family members may synergize with TLR-dependent cytokine expression (Uehara et al., 536 2005). An interesting example of this possible dual recognition would be the gut, where there 537 is exposure to more than 500 species of commensal microorganisms. It has been shown that 538 TLR agonists induced tolerance to subsequent stimulation with the same agonist (Bagchi et 539 al., 2007). This process could thus play a role in the induction of tolerance to commensal 540 bacteria, whereas pathogenic microorganisms could then be sensed by NLR family members. 541 It is possible that, depending on the cell type, host cells may distinguish between the signal 542 originating from TLRs and NLRs. Gut immunology provides a good example of how this 543 might work. Indeed, the intestinal epithelium is composed of different layers allowing 544 discrimination between commensal and pathogenic bacteria. The outermost of these layers, 545 comprising the mucus, is a barrier surrounding intestinal epithelial cells. Some areas of the 546 intestinal epithelium, such as the Peyer’s patches, are devoid of mucus and serve as inductive 547 sites for the mucosal immune system. In addition, dendritic cells can extend pseudopodes 548 through the mucus and reach the lumen (reviewed in Rescigno, 2011). This multi-layer 549 system could allow the host to distinguish between a commensal, which should not progress 550 through the mucosa, and a pathogen, which could disseminate beyond this layer and/or 551 present bacterial components to the epithelium (Sansonetti, 2008). Hence, the ability of the 552 host to distinguish between commensals and pathogens and to mount efficient immune 553 responses could be dependent on how and where the MAMPs are sensed (Vance et al., 2009). 554 As discussed in this review, activation of NLR-dependent signaling pathways by 555 extracellular bacteria induces the direct production of pro-inflammatory molecules and also 556 tailors and drives adaptive immunity, suggesting that NLR family members are multifaceted 557 proteins. A comprehensive understanding of the functions of NLRs will help decipher their 558 roles in shaping both innate and adaptive immunity during infection with extracellular 559 pathogens. 560 561 Acknowledgments 562 This work was supported by the Victorian Government’s Operational Infrastructure Support 563 Program to MIMR and grant to R.L.F. from the National Health and Medical Research 564 Council of Australia (APP1030243). R.L.F. is supported by a Senior Research Fellowship, 565 awarded by the National Health and Medical Research Council.

566 Conflict of interest statement 567 The authors declare that the research was conducted in the absence of any commercial or 568 financial relationships that could be construed as a potential conflict of interest. 569

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977 Figure legends 978 Table 1. NLR family members and bacterial recognition. 979 NLR family members are classified depending on the function of the N-terminal effector 980 domain: CARD, caspase-activation and recruitment domain; PYD, pyrin domain; BIR, 981 baculovirus inhibitor domain. * indicates that proteins are expressed in mice only. The 982 ligands and the activation mechanisms are detailed in the text. 983 984 985 986 987 988 989 990 991 992 993 994 995 996 997 998 999 1000 1001 1002 1003 1004 1005 1006 1007 1008 1009 1010 1011 1012 1013 1014 1015 1016

21 Effector NLR family Activation Bacteria NLRs ligands References domain member mechanism (Viala et al., H. pylori Peptidoglycan T4SS 2004) (Kaparakis et al., H. pylori 2010; Bielig et al., P. aeruginosa NOD1 Peptidoglycan OMVs N. gonorrhoeae 2011; Chatterjee V. cholerae and Chaudhuri, 2013) (Keestra et al., S. typhimurium T3SS 2011) CARD S. typhimurium PrgJ B. pseudomallei BsaK (Kofoed and NLRC4/NAIP2* E. coli EprJ, EscI T3SS Vance, 2011; S. flexneri MxiI Zhao et al., 2011) P. aeruginosa PscI (Kofoed and NLRC4/NAIP5* S. typhimurium Flagellin T3SS Vance, 2011; Zhao et al., 2011) NLRC4/NAIP C. violaceum CprI (Zhao et al., 2011) (Vladimer et al., NLRP12 Y. pestis ? T3SS 2012) (Fisseha et al., N. gonorrhoeae Lipooligosaccharide OMVs, LOS 2005; Duncan et al., 2009) (Gurcel et al., 2006; Mariathasan et al., 2006; Harder et L. monocytogenes Listeriolysin O al., 2009; Munoz- S. aureus Hemolysins and PVL Planillo et al., PYR NLRP3 A. hydrophila Aerolysin A. veronii Aerolysin 2009; Dunne et B. pertussis CyaA al., 2010; McCoy S. pneumoniae Pneumolysin PFT et al., 2010a; S. pyogenes Streptolysin O McCoy et al., V. vulnificus HlyA V. cholerae MARTX 2010b; McNeela et al., 2010; Kebaier et al., 2012; Holzinger et al., 2012) (Boyden and BYR NLRP1 B. anthracis Anthrax lethal toxin Dietrich, 2006) 1017 1018 1019 1020 Figure 1. Mechanisms used by bacteria to activate NLR signaling in host cells. 1021 A schematic overview of the major NLR signaling pathways activated during bacterial 1022 infection, showing the mechanisms whereby extracellular MAMPs are sensed by intracellular 1023 NLRs. Upon detection of the appropriate signal, NLRs are believed to oligomerize and 1024 recruit adaptor proteins to transduce the signal to downstream effector proteins.

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