NLRP6 and Dysbiosis: Avoiding the Luring Attraction of Over-Simplification
2018; Cell Press; Volume: 48; Issue: 4 Linguagem: Inglês
10.1016/j.immuni.2018.04.002
ISSN1097-4180
AutoresEran Elinav, Jorge Henao‐Mejia, Till Strowig, Richard A. Flavell,
Tópico(s)Tryptophan and brain disorders
ResumoIdentification of the individual factors contributing to the formation of "healthy" or disease-modulating "dysbiotic" microbiome communities is an important aspect of microbiome research. In light of the high individuality of human and animal microbiomes across geography and time, the reproducibility of findings between different labs is increasingly recognized as a challenge to this exponentially expanding field. The NLRP6 inflammasome was one the first host factors suggested to regulate the composition and function of the intestinal microbiome. Early cross-sectional studies of wild-type (WT) and gene-deficient mice suggested that a deficiency in the NLRP6 inflammasome was associated with a state of dysbiosis, leading to aggravation of intestinal inflammation and metabolic dysfunction (Elinav et al., 2011Elinav E. Strowig T. Kau A.L. Henao-Mejia J. Thaiss C.A. Booth C.J. Peaper D.R. Bertin J. Eisenbarth S.C. Gordon J.I. Flavell R.A. NLRP6 inflammasome regulates colonic microbial ecology and risk for colitis.Cell. 2011; 145: 745-757Abstract Full Text Full Text PDF PubMed Scopus (1451) Google Scholar, Henao-Mejia et al., 2012Henao-Mejia J. Elinav E. Jin C. Hao L. Mehal W.Z. Strowig T. Thaiss C.A. Kau A.L. Eisenbarth S.C. Jurczak M.J. et al.Inflammasome-mediated dysbiosis regulates progression of NAFLD and obesity.Nature. 2012; 482: 179-185Crossref PubMed Scopus (1687) Google Scholar). These findings, of significant conceptual importance, encouraged further studies to explore the nature and degree of the contribution of NLRP6-deficiency versus husbandry effects on gut microbiome community structure. The first step to assess the contribution of NLRP6 to the regulation of the microbiota, while controlling for husbandry effects, included experimentation under littermate-controlled conditions. Indeed, two labs reported that such F1 to F1 (i.e., +/− x +/−) breeding led to the development of distinct microbiome compositions in NLRP6 inflammasome-deficient mice housed in two different vivaria (Levy et al., 2017bLevy M. Shapiro H. Thaiss C.A. Elinav E. NLRP6: A Multifaceted Innate Immune Sensor.Trends Immunol. 2017; 38: 248-260Abstract Full Text Full Text PDF PubMed Scopus (79) Google Scholar, Seregin et al., 2017Seregin S.S. Golovchenko N. Schaf B. Chen J. Pudlo N.A. Mitchell J. Baxter N.T. Zhao L. Schloss P.D. Martens E.C. et al.NLRP6 Protects Il10-/-Mice from Colitis by Limiting Colonization of Akkermansia muciniphila.Cell Rep. 2017; 19: 733-745Abstract Full Text Full Text PDF PubMed Scopus (181) Google Scholar). In contrast, a recent publication by Wullaert and colleagues (Mamantopoulos et al., 2017Mamantopoulos M. Ronchi F. Van Hauwermeiren F. Vieira-Silva S. Yilmaz B. Martens L. Saeys Y. Drexler S.K. Yazdi A.S. Raes J. et al.Nlrp6- and ASC-Dependent Inflammasomes Do Not Shape the Commensal Gut Microbiota Composition.Immunity. 2017; 47: 339-348 e334Abstract Full Text Full Text PDF PubMed Scopus (110) Google Scholar) and another by Robertson and colleagues (Lemire et al., 2017Lemire P. Robertson S.J. Maughan H. Tattoli I. Streutker C.J. Platnich J.M. Muruve D.A. Philpott D.J. Girardin S.E. The NLR Protein NLRP6 Does Not Impact Gut Microbiota Composition.Cell Rep. 2017; 21: 3653-3661Abstract Full Text Full Text PDF PubMed Scopus (63) Google Scholar) did not observe such genotype-dependent effects on microbiome composition. Could these conflicting results be explained by differences in microbiome compositions between different facilities? To illustrate this possibility, one may envision a hypothetical simplified scenario. Assuming a mouse deficient in a bona-fide microbiome-modulating factor is mono-inoculated with a single commensal, such a hypothetical animal would not develop dysbiosis, as the microbial spectrum of both case and control does not allow the host factor under examination to exert its effect. In a more complex scenario, an enhanced SPF barrier facility may likewise vary in the abundance of organisms capable of mediating dysbiosis. In such case, the inability to detect differences in the microbiota between littermate WT and gene-deficient mice would simply reflect the absence of dysbiosis-triggering microbes in a given housing environment, thereby leading to over-simplified conclusions. Indeed, in the case of NLRP6, marked compositional microbiome differences existed between involved vivaria (Figure S1A, including reanalyzed data from Mamantopoulos et al., 2017Mamantopoulos M. Ronchi F. Van Hauwermeiren F. Vieira-Silva S. Yilmaz B. Martens L. Saeys Y. Drexler S.K. Yazdi A.S. Raes J. et al.Nlrp6- and ASC-Dependent Inflammasomes Do Not Shape the Commensal Gut Microbiota Composition.Immunity. 2017; 47: 339-348 e334Abstract Full Text Full Text PDF PubMed Scopus (110) Google Scholar). In light of this inter-facility microbiome variability, littermate approaches must be coupled to a second important step involving generation of germ-free NLRP6-deficient and wild-type (WT) mice (or alternatively mice re-derived into enhanced specific-pathogen-free conditions), followed by spontaneous or fecal microbiome transplantation-induced microbiome reconstitution in these mice under identical conditions (Figure S1B). Using these approaches, if an identical microbiome composition develops in reconstituted genetically-altered and WT recipient mice, a genetic contribution of the host factor in question to community structure becomes unlikely. If, however, de novo dysbiosis does develop in reconstituted germ-free mice, then the opposite conclusion may be reached, namely of a host-derived effect on microbiome community structure that becomes apparent only in specific microbiome contexts. Indeed, in the case of NLRP6, spontaneous recolonization of germ-free NLRP6-deficient mice yielded de-novo dysbiosis as compared to simultaneously recolonizing germ-free WT mice (Levy et al., 2015Levy M. Thaiss C.A. Zeevi D. Dohnalová L. Zilberman-Schapira G. Mahdi J.A. David E. Savidor A. Korem T. Herzig Y. et al.Microbiota-Modulated Metabolites Shape the Intestinal Microenvironment by Regulating NLRP6 Inflammasome Signaling.Cell. 2015; 163: 1428-1443Abstract Full Text Full Text PDF PubMed Scopus (573) Google Scholar). In addition, three independent labs (spanning 7,000 km and three different continents) have recently reported on studies using the fecal transfer strategy, i.e., diverse microbiomes of different origins were concomitantly transferred into germ-free NLRP6-deficient and WT recipient mice or into WT and NLRP6-deficient mice harboring a standardized microbiota (Gálvez et al., 2017Gálvez E.J.C. Iljazovic A. Gronow A. Flavell R. Strowig T. Shaping of Intestinal Microbiota in Nlrp6- and Rag2-Deficient Mice Depends on Community Structure.Cell Rep. 2017; 21: 3914-3926Abstract Full Text Full Text PDF PubMed Scopus (59) Google Scholar, Levy et al., 2017bLevy M. Shapiro H. Thaiss C.A. Elinav E. NLRP6: A Multifaceted Innate Immune Sensor.Trends Immunol. 2017; 38: 248-260Abstract Full Text Full Text PDF PubMed Scopus (79) Google Scholar, Seregin et al., 2017Seregin S.S. Golovchenko N. Schaf B. Chen J. Pudlo N.A. Mitchell J. Baxter N.T. Zhao L. Schloss P.D. Martens E.C. et al.NLRP6 Protects Il10-/-Mice from Colitis by Limiting Colonization of Akkermansia muciniphila.Cell Rep. 2017; 19: 733-745Abstract Full Text Full Text PDF PubMed Scopus (181) Google Scholar). Importantly, all three labs independently reported the post-transfer de novo development of dysbiosis in mice lacking NLRP6. Galvez et al. further showed that, while dysbiosis was not observed in the context of a low-diversity microbiome, it reemerged upon colonization with a complex microbial community, indicating that the impact of NLRP6 on microbiome structure requires the presence of certain taxonomic elements. Furthermore, two of the studies (Levy et al., 2015Levy M. Thaiss C.A. Zeevi D. Dohnalová L. Zilberman-Schapira G. Mahdi J.A. David E. Savidor A. Korem T. Herzig Y. et al.Microbiota-Modulated Metabolites Shape the Intestinal Microenvironment by Regulating NLRP6 Inflammasome Signaling.Cell. 2015; 163: 1428-1443Abstract Full Text Full Text PDF PubMed Scopus (573) Google Scholar, Seregin et al., 2017Seregin S.S. Golovchenko N. Schaf B. Chen J. Pudlo N.A. Mitchell J. Baxter N.T. Zhao L. Schloss P.D. Martens E.C. et al.NLRP6 Protects Il10-/-Mice from Colitis by Limiting Colonization of Akkermansia muciniphila.Cell Rep. 2017; 19: 733-745Abstract Full Text Full Text PDF PubMed Scopus (181) Google Scholar) exogenously replenished systemic IL-18 levels in NLPR6-deficient mice, which led to reversal of NLRP6 deficiency-associated dysbiosis. Additionally, a fourth recent study (Radulovic et al., 2017Radulovic K. Normand S. Rehman A. Delanoye-Crespin A. Chatagnon J. Delacre M. Waldschmitt N. Poulin L.F. Iovanna J. Ryffel B. et al.A dietary flavone confers communicable protection against colitis through NLRP6 signaling independently of inflammasome activation.Mucosal Immunol. 2017; (November 15, 2017)https://doi.org/10.1038/mi.2017.87Crossref PubMed Scopus (45) Google Scholar) identified apigenin as an NLRP6 regulator, thereby enabling to demonstrate NLRP6-dependent microbiome modulation even in WT mice, further ruling out confounding husbandry-related effects. Collectively, these results support the notion that NLRP6 contributes to regulation of microbiome community structure upon exposure to a sufficiently diversified microbiome. Moreover, as reported both by Flavell and colleagues (Elinav et al., 2011Elinav E. Strowig T. Kau A.L. Henao-Mejia J. Thaiss C.A. Booth C.J. Peaper D.R. Bertin J. Eisenbarth S.C. Gordon J.I. Flavell R.A. NLRP6 inflammasome regulates colonic microbial ecology and risk for colitis.Cell. 2011; 145: 745-757Abstract Full Text Full Text PDF PubMed Scopus (1451) Google Scholar), other laboratories (Levy et al., 2015Levy M. Thaiss C.A. Zeevi D. Dohnalová L. Zilberman-Schapira G. Mahdi J.A. David E. Savidor A. Korem T. Herzig Y. et al.Microbiota-Modulated Metabolites Shape the Intestinal Microenvironment by Regulating NLRP6 Inflammasome Signaling.Cell. 2015; 163: 1428-1443Abstract Full Text Full Text PDF PubMed Scopus (573) Google Scholar, Seregin et al., 2017Seregin S.S. Golovchenko N. Schaf B. Chen J. Pudlo N.A. Mitchell J. Baxter N.T. Zhao L. Schloss P.D. Martens E.C. et al.NLRP6 Protects Il10-/-Mice from Colitis by Limiting Colonization of Akkermansia muciniphila.Cell Rep. 2017; 19: 733-745Abstract Full Text Full Text PDF PubMed Scopus (181) Google Scholar) and by Wullaert and colleagues themselves (Mamantopoulos et al., 2017Mamantopoulos M. Ronchi F. Van Hauwermeiren F. Vieira-Silva S. Yilmaz B. Martens L. Saeys Y. Drexler S.K. Yazdi A.S. Raes J. et al.Nlrp6- and ASC-Dependent Inflammasomes Do Not Shape the Commensal Gut Microbiota Composition.Immunity. 2017; 47: 339-348 e334Abstract Full Text Full Text PDF PubMed Scopus (110) Google Scholar), colitis susceptibility was noted only when NLRP6 deficiency induced dysbiosis, while susceptibility was absent when dysbiosis was not present, thereby supporting the notion that the effect of NLRP6 on regulating susceptibility to intestinal inflammation is mainly mediated through its microbiome-modulatory activity. We hope that the collective knowledge gained by the above studies will increase the awareness of the importance in rigorously integrating multiple complementary experimental modalities into microbiome research. These should include both littermate breeding strategies and fecal microbiome transplantations of diverse sources, but never solely rely on only one or the other (Figure S1B). In generalizing beyond NLRP6, utilization of such a comprehensive stepwise approach may enable a conclusive verification or falsification of the microbiome modulatory roles suggested for other innate immune receptors, including NLPR3, NOD2, and TLR5. To the best of our knowledge, contributions by these receptors were thus far disputed only by littermate experimentation (such as in Robertson et al., 2013Robertson S.J. Zhou J.Y. Geddes K. Rubino S.J. Cho J.H. Girardin S.E. Philpott D.J. Nod1 and Nod2 signaling does not alter the composition of intestinal bacterial communities at homeostasis.Gut Microbes. 2013; 4: 222-231Crossref PubMed Scopus (110) Google Scholar), but not with the necessary complementary fecal transfer strategies outlined above. As in the case of NLRP6, only such an integrative approach will enable correct interpretations of host effects on microbiome community structure, while avoiding partial and non-generalizable conclusions. 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