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Mutations in NOD2 — which encodes NOD-like receptor 2 — are a major risk factor for the inflammatory bowel disease (IBD) Crohn's disease, but it has been unclear how disrupted bacterial recognition can increase the risk of inflammation. Now, Ken Cadwell and colleagues show that NOD2 prevents intestinal inflammation in mice by controlling the growth of the commensal bacterium Bacteroides vulgatus.

Using microarray analysis, the authors found that transcripts associated with inflammation, fatty acid biosynthesis and host immune defence were enriched in small intestinal epithelial cells from Nod2−/− mice, and genes encoding antimicrobial molecules such as regenerating islet-derived protein 3β (which is encoded by Reg3β) were among the most highly expressed. Patients with IBD have increased expression of the human homologue of REG3β, and the authors showed that compared with controls, Nod2−/− mice have increased levels of REG3β in intestinal epithelial cells. Interestingly, treatment of Nod2−/− mice with antibiotics reduced the expression of REG3β to wild-type levels, which suggests that the production of REG3β is dependent on bacteria.

Next, the authors investigated whether Nod2−/− mice develop other abnormalities in the small intestinal epithelium. Compared with control mice, Nod2−/− mice had decreased levels of the mucus-producing protein mucin 2 (MUC2), and their goblet cells showed an abnormal appearance and were reduced in number. Of note, mucus production by goblet cells is an important barrier between intestinal bacteria and the epithelium, and patients with IBD have decreased MUC2 expression.

B. vulgatus cannot stably colonize wild-type mice

As treatment of Nod2−/− mice with antibiotics led to reduced expression of REG3β, the authors speculated that commensal bacteria could have a role in the generation of the intestinal abnormalities. Deep sequencing showed that the microbiota of Nod2−/− mice differed substantially from that of wild-type mice. Nod2−/− mice had an over-representation of B. vulgatus, which could not be detected in controls. To investigate whether this accumulation was specific to Nod2−/− mice, or was owing to vertical transmission after exposure in a previous generation, wild-type mice were co-housed with Nod2−/− mice. In this setting, wild-type mice were readily colonized with B. vulgatus; however, when the mice were separated, B. vulgatus disappeared over time in wild-type mice but remained stable in Nod2−/− mice. Thus, B. vulgatus cannot stably colonize wild-type mice, but a deficiency of Nod2 results in the overgrowth of Bacteroides species.

So, is B. vulgatus responsible for the epithelial abnormalities in Nod2−/− mice? Treatment of Nod2−/− mice with metronidazole — which targets anaerobic bacteria — led to decreased levels of epithelial REG3β and restored normal goblet cell morphology. However, this was not observed when mice were treated with an antibiotic that does not kill B. vulgatus. Furthermore, when B. vulgatus (either live or heat-killed) was introduced by oral gavage to metronidazole-treated wild-type and Nod2−/− mice, the authors found that only Nod2−/− mice receiving live B. vulgatus displayed intestinal epithelial abnormalities. Hence, B. vulgatus seems to be responsible for the intestinal abnormalities observed in Nod2−/− mice.

Finally, the authors connected their findings to IBD by investigating the role of B. vulgatus in Nod2−/− mice treated with piroxicam, which induces small-intestinal injury. Compared with controls, piroxicam-treated Nod2−/− mice displayed increased focal ulceration, epithelial hyperplasia and blood in the small intestinal lumen. Of note, Nod2−/− mice receiving metronidazole did not show this response to piroxicam. These results suggest that the intestinal microbiota exacerbates inflammation induced by piroxicam.

In summary, this study indicates that NOD2 prevents harmful inflammatory immune responses by controlling the expansion of commensal bacteria in the small intestine.