We describe a novel regulatory sensing system in the colon dependent on the NLRP6 inflammasome. We show that genetic deletion of components of this sensing system has drastic consequences on the composition of the microbial communities leading to a shift towards a pro-inflammatory configuration that drives spontaneous and induced colitis.
On a molecular level, it appears unlikely that the evolutionarily conserved, innate mucosal immune arm possesses the ability to distinctly identify the myriad bacterial, archaeal and eukaryotic microbial phylotypes and virotypes that comprise the gut microbiota and differentiate autochthonous (entrenched) or allochthonous (transient/nomadic) components of this community that act as commensals or mutualists from those that act as pathogens. Rather, this function may be achieved by sensing signals related to tissue integrity, or factors released by tissue damage, that serve as “danger signals” promoting activation of an innate response (Matzinger, 2007
). Inflammasomes are capable of fulfilling this task as they can be activated by many microbial ligands, but also by host-derived factors released upon cell or tissue damage, such as uric acid, ATP, and hyaluronan (Schroder and Tschopp, 2010
). NLRP6 assembly in the colonic epithelial compartment may be driven by a low level of these substances or by yet unidentified molecules signaling tissue integrity, resulting in local production of IL-18. Interestingly, in the rat, NLRP6, caspase-1, ASC and pro-IL-18 are absent at embryonic day 16 (E16), and first appear at E20, with the processed form of IL-18 emerging in the gut during the early postnatal period (Kempster et al., 2011
), coinciding with the time of colonization of the gut ecosystem.
Dysbiosis may contribute to IBD by expansion of colitogenic strains such as enteroinvasive E.coli
(Darfeuille-Michaud et al., 2004
), by reduction of tolerogenic strains such as Faecalibacterium prausnitzi
(Sokol et al., 2008
), or through a combination of both mechanisms. In our study, a colitogenic microbiota with altered representation of distinct bacterial members formed in the intestines of NLRP6-deficient mice: this microbiota was transferred across generations within a kinship, and could displace the gut microbiota of cohoused immunocompetent mice. Once this community was horizontally transmitted to suckling or adult WT mice, it could persist. Compared to WT mice, NLRP6-inflammasome deficient mice exhibit both quantitative and qualitative changes in numerous taxa including increased representation of members of Prevotellaceae
and TM7, and reductions in members of genus Lactobacillus in the Firmicutes phylum.
There are several intriguing links between the abundance of Prevotellaceae
and TM7 and human diseases. Prevotellaceae
has been implicated in periodontal disease (Kumar et al., 2003
), and several reports have documented prominent representation of this group in samples from IBD patients (Kleessen et al., 2002
; Lucke et al., 2006
might disrupt the mucosal barrier function through production of sulfatases that actively degrade mucus oligosaccharides (Wright et al., 2000
); these enzymes are elevated in intestinal biopsies from IBD patients (Tsai et al., 1995
). While they have not been cultured, members of the TM7 phylum have been identified in 16S rRNA surveys of terrestrial and aquatic microbial communities as well in human periodontal disease (Brinig et al., 2003
; Marcy et al., 2007
; Ouverney et al., 2003
), and in IBD patients (Kuehbacher et al., 2008
). Defining the nature of the interactions of Prevotellaceae
and TM7 with the NLRP6 inflammasome may provide insights about probiotic interventions that may mitigate microbiota-mediated enhanced inflammatory responses.
Four previous reports indicated that caspase-1, ASC or NLRP3 deficiencies were associated with an increased severity of acute DSS colitis in mice, and suggested that exacerbated disease was mediated in part by a defect in repair of the intestinal mucosa (Allen et al., 2010
; Dupaul-Chicoine et al., 2010
; Hirota et al., 2010
; Zaki et al., 2010
). Opposing results were found in two other studies using the same colitis model. The first study to investigate the role of caspase-1 in intestinal auto-inflammation, even prior to the discovery of the inflammasome, found ameliorated acute and chronic colitis in caspase-1−/−
mice (Siegmund et al., 2001
). More recently, a second study demonstrated reduced severity of disease in NLRP3−/− mice that correlated with decreased levels of pro-inflammatory IL-1β (Bauer et al., 2010
). It has been hypothesized that these differences might be the result of distinct roles of inflammasomes in non-hematopoietic versus hematopoietic cells (Siegmund, 2010
). The proposed function in epithelial cells is to regulate secretion of IL-18 that stimulates epithelial cell barrier function and regeneration, while in hematopoietic cells inflammasome activation would have a pro-inflammatory effect. Varying degrees of tissue injury and subsequent inflammation may result in shifting the balance between protective and detrimental effects, depending on the experimental condition and the inflammatory context. However, we believe that inflammasome-driven effects on the colonic microbiota, as revealed in our study, add yet another layer of regulation that affects and effects initiation of auto-inflammation. As such, exacerbation in colitis severity in single-housed inflammasome-deficient mice may in fact involve defects in tissue regeneration, but this histopathological process may be dramatically influenced by the effects imposed by altered elements in the microbiota including, for example, the enhanced representation of Prevotellaceae
in the crypt. Thus, we propose that the fundamental role of the microbiota in shaping processes related to tissue damage, regeneration and stress response might offer an explanation for the opposing results between these studies. 16S rRNA enumeration studies combined with various permutated cohousing experiments of the type described in this report, coupled with mechanistic molecular studies, would allow this notion to be tested directly. Furthermore, our results suggest that prolonged cohousing (or littermate controls) should be used when NLRs and other innate receptors are studied: this would allow for equilibration of differences in gut microbial ecology that may exist between groups of mice, and allow investigators to determine which features of their phenotypes can be ascribed to the microbiota. Indeed, using co-housing conditions we were able to demonstrate that the NLRC4 inflammasome is a direct negative regulator of colonic epithelial cell tumorigenesis which is not driven by the microbiota (Hu et al., 2010
Our results show that the resultant aberrant microbiota promotes local epithelial induction of CCL5 transcription as a downstream mechanism, ultimately leading to an exaggerated auto-inflammatory response. CCL5 is potently induced by bacterial and viral infections, and in turn induces massive recruitment of a variety of innate and adaptive immune cells carrying CCR1, CCR3, CCR4 and CCR5 (Mantovani et al., 2004
). Interestingly, both NOD2 and TLRs have been shown to induce CCL5 transcription (Berube et al., 2009
; Werts et al., 2007
). It will be of interest to investigate the crosstalk between these immune recognition systems in future experiments.
Recent studies have highlighted the importance of the gut microbiota in the pathogenesis of various autoimmune disorders that manifest outside of the gastrointestinal tract. In some autoimmune models germ-free conditions or inoculation with a microbiota from healthy mice ameliorates disease (Lee et al., 2010
; Mazmanian et al., 2008
; Sinkorova et al., 2008
; Wu et al., 2010
). In contrast, rats with collagen-induced arthritis feature exacerbated disease when reared under germ-free conditions (Breban et al., 1993
) while germ-free NOD MyD88−/− mice fail to develop diabetes unlike their colonized counterparts (Wen et al., 2008
). In humans, epidemiological evidence points to possible links between dysbiosis and rheumatoid arthritis, asthma, and atopic dermatitis (Bjorksten, 1999
; Penders et al., 2007
; Vaahtovuo et al., 2008
). Our study indicates that deficiencies in the NLRP6 pathway should be added to the list of host genetic factors that may drive disease-specific alterations in the microbiota, which in turn may promote disease in these hosts or in individuals who have been exposed to these microbial communities and who have also experienced disruption in their gut epithelial barrier function due to a variety of insults.