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Nod-like receptors in intestinal host defense: controlling pathogens, the microbiota, or both?

Robertson, Susan J.a; Girardin, Stephen E.b

Current Opinion in Gastroenterology: January 2013 - Volume 29 - Issue 1 - p 15–22
doi: 10.1097/MOG.0b013e32835a68ea
GASTROINTESTINAL INFECTIONS: Edited Mitchell B. Cohen
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Purpose of review Nod-like receptors (NLRs) are intracellular innate immune sensors of microbes and danger signals that control multiple aspects of inflammatory responses. We review the evidence that highlights the critical importance of NLRs in the host response to intestinal pathogens. Moreover, we discuss the potential roles played by NLRs in the dynamic control of the intestinal microbiota and how commensal microorganisms may affect host susceptibility to enteric bacterial pathogens through interactions with NLRs as well as with invading pathogens.

Recent findings Recent studies targeting the intestinal microbiota in the context of NLR deficiencies suggest inherent alterations in bacterial density or abundance may underlie the development of inflammatory diseases. As commensal microorganisms may also affect host susceptibility to enteric bacterial pathogens, NLRs might promote intestinal innate immune defense through mechanisms more complex than previously anticipated.

Summary The inclusion of the intestinal microbiota as a critical parameter in innate immunity represents an exciting new dimension for understanding NLR functioning and the clinical implications for human health.

aDepartment of Immunology

bDepartment of Laboratory Medicine and Pathobiology, University of Toronto, Toronto, Canada

Correspondence to Susan J. Robertson, University of Toronto, M5S 1A8 Toronto, Canada. Tel: +1 416 978 7527; e-mail: susan.robertson@utoronto.ca

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INTRODUCTION

The innate immune system is responsible for the detection and response to pathogens, resulting in inflammation, recruitment to the site of infection of professional phagocytes, and the production of vast repertoire of antimicrobial molecules, such as defensins, reactive oxygen species, and nitric oxide [1]. In addition, recent evidence showed that innate immune responses are tightly associated with alterations of cellular metabolic pathways [2▪–7▪], which in turn contribute to the cell autonomous defense against pathogens, particularly through the upregulation of autophagy [8]. Finally, in vertebrates, innate immunity is also critical for the induction of adaptive immune responses against pathogens, through the generation and presentation of antigens, and the secretion of mediators that in combination are required to activate T-cell and B-cell-dependent responses [9,10].

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Role of Nod-like receptors in host defense against enteric pathogens

Research from the past two decades has delineated the critical role played by specific families of host microbial sensors in the activation of innate immune defense in mammals [11]. Toll-like receptors (TLRs) are transmembrane pattern recognition molecules (PRMs) that detect microbe-associated molecular patterns (MAMPs) such as lipopolysaccharide, lipopeptides, CpG DNA, flagellin, and viral double stranded RNA, which are released in the extracellular milieu or the luminal side of intracellular endosomes [12]. Other families of PRMs have been characterized in the past decade, which detect MAMPs presented in the cytosol, and these include Nod-like receptors (NLRs) [13,14], RigI-like receptors (RLRs) [15], and Aim2-like receptors (ALRs) [16]. Whereas RLRs and ALRs specifically detect cytosolic nucleic acids, NLRs have evolved strategies to detect not only MAMPs (such as bacterial flagellin and peptidoglycan), but also host-derived molecules and cellular events that signal a stress or a danger. These danger-associated molecular patterns (DAMPs) detected by NLRs include molecular crystals, potassium efflux, ATP, reactive oxygen species, and possibly membrane damage [17]. Therefore, NLRs represent a fascinating point of convergence wherein microbes and aseptic cellular or tissue damage are detected, resulting in inflammation, thus providing remarkable support for the ‘danger model’ unifying concept of immune activation brought forward by Polly Matzinger [18] nearly two decades ago.

Box 1

Box 1

Because NLRs detect cytosolic MAMPs and DAMPs while remaining insensitive to those molecular patterns presented to the extracellular milieu, it has been hypothesized that these sensors might play critical roles at mucosal surfaces, wherein overwhelming amounts of microbially derived molecules can be found in the extracellular milieu, released from either harmless microbes (such as the intestinal microbiota) or possibly by pathogens. Indeed, intracellular PRMs, such as NLRs, could, in theory, discriminate between pathogens that breach cellular and mucosal barriers and nonpathogenic microorganisms, thus providing a functional advantage over TLRs to serve as sentinels of the innate immune system at mucosal surfaces. In support of this concept, polymorphisms in the gene encoding Nod2, an NLR protein that detects the bacteria-derived peptidoglycan fragment muramyl dipeptide (MDP), have been shown to confer susceptibility to Crohn's disease, an inflammatory disorder that affects the gastrointestinal tract [19,20]. Importantly, although Crohn's disease etiology is complex and involves genetic and environmental factors, several lines of evidence have implicated a dysregulation of the control of the intestinal microbiota in the physiopathology of this disease [21]. However, because Nod2 is expressed in intestinal epithelial cells in inflammatory conditions [22,23], as well as in phagocytic cells, it remains unclear whether Nod2-associated Crohn's disease is caused by a dysfunctional control of microbes at the epithelial barrier, at the level of the phagocytes of the submucosa, or both.

Cellular as well as in-vivo models of infection have provided definitive evidence that NLRs contribute to host defense against enteric pathogens. In epithelial cells and macrophages, the peptidoglycan sensors Nod1 and Nod2 have been shown to detect enteric bacteria such as Shigella, Salmonella, Listeria, Yersinia, pathogenic Escherichia coli strains, and Mycobacterium species [24–27]. Following detection of peptidoglycan fragments, Nod1 and Nod2 trigger proinflammatory signaling dependent on nuclear factor-κB (NF-κB) and MAP kinases following the engagement of the adaptor protein Rip2 [28], and also induce autophagic responses through the recruitment of ATG16L1 [29]. Nod-dependent proinflammatory signaling induces the release of numerous cytokines and chemokines, and experiments in vivo using synthetic peptidoglycan fragments have demonstrated the critical importance of Nod1/2 in neutrophil recruitment [30,31] as well as in the Th2 polarization of adaptive immune responses in the absence of TLR ligands [32–34]. In in-vivo models of enteric infection, Nod2-deficient mice have been shown to be susceptible to several bacterial pathogens, including Listeria[35], Yersinia[36], adherent-invasive E. coli[37▪], and Citrobacter rodentium[38]. Similarly, Nod1-deficient mice have been shown to display enhanced susceptibility to Salmonella that enter the intestine through the dendritic cell pickup pathway [39]. There is also strong evidence that Nod1 and Nod2 may have partially overlapping functions in host defense against bacterial pathogens in the intestine, as Nod1/Nod2 double knockout (Nod DKO) mice display phenotypes that are not observed in Nod1 or Nod2 single knockout mice. In particular, Nod DKO mice cannot control Salmonella infection [40] and display a blunted capacity to mount a rapid and protective Th17 response to Salmonella and C. rodentium in the cecum of infected animals [41▪].

In addition to Nod1 and Nod2, other NLR proteins contribute to the host defense against enteric pathogens. NLRC4 detects flagellin from Salmonella, resulting in the upregulation of the caspase-1 inflammasome, which controls the generation of the mature forms of interleukin (IL)-1β and IL-18 [42–44]. Interesting evidence also shows that, in addition to flagellin, NLRC4 can detect membrane damage induced by the type three secretion system (TTSS) of Shigella[45], Salmonella[46] or pore-forming toxins from Legionella[47]. The differential formation of complexes composed of NLRC4 with either NAIP2 or NAIP5, two related NLR proteins encoded in the murine genome, has been shown to direct recognition of Salmonella TTSS proteins or flagellin, respectively [48▪,49▪]. In vivo, NLRC4-deficient mice were susceptible to Salmonella infection, and intestinal macrophages displayed an altered capacity to secrete IL-1β in response to this pathogen [50▪].

Another NLR protein of particular importance in host response against intestinal pathogens is NLRP3. This NLR protein detects a very large spectrum of MAMPs and DAMPs, making this molecule a versatile and multifunctional sensor of bacteria, bacterial toxins, viruses, fungi, and aseptic tissue damage [17]. Following activation of this NLR protein, recruitment of the caspase-1 inflammasome results in NLRP3-dependent secretion of IL-1β and IL-18. However, despite the critical importance of NLRP3 in inflammasome activation, its implication in host defense against enteric pathogens in vivo remains poorly characterized, although a recent report identified a role for NLRP3 in the defense against C. rodentium[51]. In addition, NLRP3-dependent secretion of IL-1β in response to the intestinal microbiota has been shown to protect mice from Clostridium difficile infection [52].

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Nod-like receptor interactions with the resident microbiota

In the gut, the innate immune system controls, and is controlled by, the resident microbiota [53▪]. The microbiota is composed of complex communities of eukaryotes, bacteria, archaea, and viruses, but the term generally refers to bacteria, which have been the primary focus of recent research. As few bacteria have been isolated for study, current understanding is almost entirely based on analysis of the 16S ribosomal RNA gene, which provides insight into community structure (i.e., number, composition, and relative abundance) at various levels of phylogenetic resolution. These analyses have revealed extraordinarily high diversity at the genus and species levels, contained within a limited number of dominant phyla, including the Bacteroidetes, Firmicutes, Actinobacteria, and Proteobacteria [54]. Community structure varies along the length and breadth (from lumen to mucosa) of the gut [55,56], as dictated by the local intestinal ecosystem (e.g., metabolic substrates, colonization sites, other organisms, immune and physiological molecules) and stimuli from the external environment (e.g., pathogens, antibiotics, local environment). The role of the innate immune system in maintaining intestinal homeostasis is via chemical fortification of the epithelial barrier through secretion of antimicrobial peptides and other immune molecules. Immune functions rely on microbial stimulation, through MAMP and DAMP recognition, as well as via specific bacterial signals that are only now beginning to come to light [57,58]. Thus, a more detailed understanding of the gut microbiota is required to better understand its contributions to innate immune functioning and responses to pathogenic infection.

Dysbiosis, an imbalance in bacterial community structure leading to disease, has been associated with human inflammatory diseases linked to deficiencies in NLRs [59]. Only a few studies have explored the relationships between NLRs and the gut bacteria in animal models (Table 1) [60–66]. Results seem to support the hypothesis that NLR-deficient individuals host an altered microbiota compared to wild-type controls. However, conclusions should be considered preliminary due to a general lack of replication and differences in methodology (e.g., gut sampling location and intensity, mouse breeding/housing conditions, and DNA analysis method and depth) that likely impacted on individual findings. Importantly, the fundamental question of whether NLRs, in the absence of all other environmental variables, determine the composition of the microbiota remains inconclusive, as experiments would require analysis of multiple cohorts of F2 generation littermates, raised together in random mixtures of genotypes, and validation across multiple institutions. Although most studies took measures to standardize the microbiota in their mouse strains, replicated littermate studies are needed, especially in light of recent findings that the microbiota of MyD88-deficient and TLR-deficient (TLR2, TLR4, TLR5, TLR9) mouse colonies included distinct bacteria in the small and large intestine, representing divergence after extended isolation [67▪]. Here, we critically compare the NLR studies to reveal common trends and identify areas for future research.

Table 1

Table 1

In the terminal ileum of Nod1-deficient, Nod2-deficient, and Rip2-deficient mice, elevated bacterial density (assessed using qPCR and compared to wild-types) was correlated with reduced β-defensin secretion [60] and lower bactericidal capacity of crypt secretions [61], likely due to reduced α-defensin secretion [35]. These findings are consistent with a model in which failure of Nod1 or Nod2 to recognize bacterial peptidoglycan and recruit Rip2 for NF-κB activation allows for bacterial expansion. Similarly, higher bacterial density (compared to wild-type littermates) was reported from the mucosa-associated, but not luminal, ileum of MyD88-deficient mice, which lack the adaptor protein for NF-κB signaling through many TLRs [68]. In this case, greater density was related to decreased expression of the antimicrobial peptide, RegIIIγ, and its spatial limits of activity were reflected by differences between mucosal and luminal communities. The extent of bacterial expansion and its consequences for host immunity and health status remain unknown.

Greater bacterial density in the Nod-deficient ileum is the outcome of net increases in abundance within the Bacteroidetes and Firmicutes phyla [60,61]. Using community sequencing, Rehman et al. [62] further showed that altered abundance patterns occurred in the ileum of both 2-week old and adult Nod2-deficient mice and that community structure varied between the age groups. Further comparisons are impossible due to methodological differences. More research using standardized techniques is essential to define bacterial abundance patterns at these higher levels of resolution and to address questions concerning the individual and complementary functions of Nod1 and Nod2. For example, how do differences in bacterial abundance patterns reflect the spatial (i.e., location in epithelial cell types) and functional (i.e., recognition of different moieties of bacterial peptidoglycan) differences between the Nod1 and Nod2 receptors? How do Nod1 and Nod2 compensate for deficiency in one or the other and how do other parts of the immune system compensate for the total lack of Nod signaling in Rip2-deficient or Nod DKO mice? Finally, how do differences in bacterial abundance relate to the distinct diseases associated with Nod1 and Nod2 deficiency in humans?

Deep sequencing of the colon bacteria from Nod2-deficient and NLRP6-deficient (and apoptosis-associated speck-like protein, ASC-deficient) mice showed different abundance patterns for genera within the Bacteroidetes [62,63,65]. One possibility is that these abundance patterns reflect differences in the intestinal environment that are controlled through distinct functions of these NLRs, but the possibility that they are artefacts associated with different mouse strains and facilities must be eliminated. Shifts in abundance may also reflect a predisposition to inflammatory disease, as many Bacteroidetes are known to be opportunistic pathogens (pathobionts) under permissive environmental conditions [69]. Indeed, the Prevotella sp. present in NLRP6-deficient and ASC-deficient mice was transferred to wild-types upon cohousing and associated with greater susceptibility to dextran sulfate sodium (DSS)-induced colitis [65]. However, coligenicity is not necessarily predicted based on disease-associated shifts in Bacteroides abundance [70▪]. Experiments using relevant models of colitis (e.g., infection, T-cell transfer) are needed to address questions of disease predisposition associated with different NLR deficiencies. In contrast to the sequencing studies, no differences in Bacteroidetes abundance were revealed using DNA fingerprinting analysis of NLRP3-deficient [66] and Nod DKO [64] bacterial communities. Although TRFLP (terminal restriction fragment length polymorphism) and DGGE (denaturing gradient gel electrophoresis) may not be as sensitive as deep sequencing, the microbiota of the comparison groups was tightly standardized in both studies. It is also worth noting that the appropriate level of resolution for revealing meaningful patterns in bacterial community structure remains a subject of debate.

Finally, several of the NLR studies reported shifts in abundance within the Firmicutes, including Lactobacillaceae, Clostridaceae, and segmented filamentous bacteria (SFB) [60,65,66]. Members of these groups are included on a rapidly growing list of gut bacteria that have been shown to exhibit immunomodulatory activities that may directly or indirectly impact on NLR functions. Several Clostridium species (clusters IV and XIVa) promote accumulation of CD4+ T regulatory cells in the colon [71] and the closely related SFB induce intestinal T helper 17 cells in the ileum [72,73]. Many lactic acid bacteria have been shown to act directly on the immune system by acting on dendritic cells or altering cytokine profiles [74]. Recently, a strain of Lactobacillus paracasei was shown to produce lactocepin, a serine protease that selectively degrades the proinflammatory chemokine, IP-10 [75]. The potential for individual microbe–host interactions is staggering and these are completely unknown in the context of NLR functioning and disease predisposition. Unraveling the molecular mechanisms defining this complex web of interactions will require continued advances in sequencing technologies and metabolic profiling techniques that address functional aspects of bacterial community structure.

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Microbe–microbe interactions

Interactions between the microbiota and organisms entering the gastrointestinal tract from the external environment impact on immune response and ultimately determine outcomes for health and disease. Therefore, if NLR expression in the intestine could impact on the microbiota (see section above), it is likely that NLR-dependent control of intestinal pathogens would depend, at least in part, on the dynamic structure of the microbiota (Fig. 1). This section reviews some of the aspects of the microbiota–pathogen interplay in the intestine.

FIGURE 1

FIGURE 1

Microbe–microbe interactions are best understood in the context of probiotics, which are live organisms (often lactobacilli or bifidobacteria) that confer health benefits to the host when consumed in adequate amounts [76]. Probiotic bacteria are thought to enhance or maintain the beneficial components of the microbiota either indirectly, via regulation of host immune responses, or directly, through production of factors that enhance their competitive abilities in an intestinal niche [76–78]. In addition, many studies have shown that probiotic bacteria protect mice against pathogens. For example, Lactobacillus rhamnosus increases the survival of mice infected with Salmonella typhimurium[79], L. casei resists Listeria monocytogenes infection [80], and L. paracasei inhibits the inflammatory potential of pathogenic Salmonella and protects against experimental colitis [74]. There is currently much interest in identifying bacterially derived products such as bacteriocins that may facilitate colonization of probiotic bacteria, eliminate competing organisms and pathogens, and signal other bacteria or the immune system [76].

It is likely that the resident microbiota employ similar strategies when interacting with invading pathogens. The mouse pathogen, C. rodentium, colonizes the surface of the intestinal epithelium and its eradication requires the presence of the microbiota [81▪]. Ivanov et al. [73] showed that SFB, as a component of the microbiota, indirectly protect against C. rodentium infection, through induction of Th17 cytokines (especially IL-22), and limit C. rodentium growth in colonic tissue, at least partly via stimulation of host production of antimicrobial peptides such as RegIIIγ. Kamada et al.[81▪] found that C. rodentium-infected GF mice that were subsequently infected with Bacteroides thetaiotaomicron or B. vulgatus did not reduce C. rodentium loads in the feces. In contrast, secondary infection with E. coli did reduce fecal loads of the pathogen, which was directly related to competition for monosaccharides, their common preferred substrate. It has been previously reported that γ-Proteobacteria, such as E. coli, specifically accumulate after C. rodentium infection, likely because the inflammatory responses triggered by invading pathogens that function to enhance their own colonization also enhance microbiota able to occupy the same niche [82]. This change in the microbiota may actually benefit the host by increasing the number of resident bacteria that can outcompete the pathogen [81▪].

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CONCLUSION

It is well documented that NLRs are important contributors of host innate immune defense against microbial pathogens, in particular at the level of mucosal surfaces. In the intestine, these surveillance systems are complicated by the need for the host to discriminate between resident elements of the microbiota and potential pathogens. It is now recognized that the intestinal microbiota may critically impact on host defense against enteric pathogens and, furthermore, the innate immune sensors themselves might influence the composition and functioning of the microbiota. Inclusion of the microbiota as a key parameter in innate immunity has opened many new avenues of investigation. For these reasons, there is a crucial and urgent need to uniformize, between research groups worldwide, diet, housing conditions and surveillance, the use of littermate individuals, as well as techniques to monitor the dynamic composition of the microbiota along the intestinal tract. As exciting as the study of the intestinal microbiota could be, considerable resources and efforts will likely be wasted on inability to reproduce data from independent institutions if a collective effort is not made to apply standard housing and monitoring procedures. It might be advisable to advocate for the creation of an international label to minimize these issues. This is critical because of the multiple clinical implications of this research for human health.

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Acknowledgements

Research in the laboratory of S.E.G. is supported by grants from the Canadian Institutes of Health Research, Crohn's and Colitis Foundation of Canada, Burroughs Wellcome Fund, and Natural Sciences and Engineering Research Council of Canada and S.J.R. is supported through a fellowship from the Canadian Institutes of Health Research and Canadian Association of Gastroenterology.

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Conflicts of interest

The authors declare no conflict of interest with the content of this article.

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REFERENCES AND RECOMMENDED READING

Papers of particular interest, published within the annual period of review, have been highlighted as:

  • ▪ of special interest
  • ▪▪ of outstanding interest

Additional references related to this topic can also be found in the Current World Literature section in this issue (p. 93).

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Keywords:

enteric pathogens; innate immunity; intestinal microbiota; Nod-like receptors

© 2013 Lippincott Williams & Wilkins, Inc.