Nlrp9b inflammasome restricts rotavirus infection in intestinal epithelial cells

Rotavirus, a leading cause of severe gastroenteritis and diarrhoea in young children, accounts for around 215,000 deaths annually worldwide1. Rotavirus specifically infects the intestinal epithelial cells in the host small intestine and has evolved strategies to antagonize interferon and NF-κB signalling2–5, raising the question as to whether other host factors participate in antiviral responses in intestinal mucosa. The mechanism by which enteric viruses are sensed and restricted in vivo, especially by NOD-like receptor (NLR) inflammasomes, is largely unknown. Here we uncover and mechanistically characterize the NLR Nlrp9b that is specifically expressed in intestinal epithelial cells and restricts rotavirus infection. Our data show that, via RNA helicase Dhx9, Nlrp9b recognizes short double-stranded RNA stretches and forms inflammasome complexes with the adaptor proteins Asc and caspase-1 to promote the maturation of interleukin (Il)-18 and gasdermin D (Gsdmd)-induced pyroptosis. Conditional depletion of Nlrp9b or other inflammasome components in the intestine in vivo resulted in enhanced susceptibility of mice to rotavirus replication. Our study highlights an important innate immune signalling pathway that functions in intestinal epithelial cells and may present useful targets in the modulation of host defences against viral pathogens. Previous studies suggested that anti-rotavirus antibody isotype switching depends on Il-1β and Il-18 (ref.6), and that recombinant Il-18 prevents and resolves rotavirus infection7. However, the precise role of inflammasome signalling that mediates the maturation of these cytokines in the context of enteric virus infections is largely unknown. To directly interrogate whether inflammasomes participate in the restriction of enteric viruses, we orally inoculated suckling pups with mouse rotavirus. Notably, rotavirus infection potently induced caspase-1 (Casp1) p10 cleavage, indicative of inflammasome activation (Fig. 1a). In addition, mice deficient in Asc or Casp1, two universal inflammasome components, exhibited higher viral loads in the small intestine, increased fecal shedding of viral antigens, and more frequent incidences of diarrhoea compared to their wild-type littermates (Fig. 1b– d, Extended Data Fig. 1a), suggesting that inflammasome signalling protects against rotavirus infection. The heightened susceptibility of Asc−/− (also known as Pycard−/−) mice to rotavirus infection implies a role of certain NLR(s) in this antiviral response. Therefore, we examined a panel of knockout mice deficient in Nlrp3, Nlrp6, Nlrc4 and Aim2, all of which are known to activate inflammasomes in the intestine8–11. With the exception of Nlrp6, none of these strains had increased susceptibility to rotavirus infection comparable to Asc−/− or Casp1−/− mice (Extended Data Fig. 1b, c). Given that mouse rotavirus has a specific tropism for the small intestine12, we systemically examined the transcription levels of all NLRs by RNA sequencing of ileum intestinal epithelial cells (IECs). Interestingly, as well as Nlrp6, Nlrc4 and Naips9,13–15, we discovered a novel NLR termed Nlrp9b that has not yet been characterized (Fig. 2a). Nlrp9b, but not the closely related Nlrp9a and Nlrp9c, was detected in different sections of the small and large intestines, highlighting its unique expression pattern16. To functionally examine the potential anti-rotavirus role of Nlrp9b, we generated Nlrp9b−/− mice (Extended data Fig. 2c, d). Nlrp9b−/− mice resembled Asc−/− and Caspi−/− mice and Zhu et al. Page 2 Nature. Author manuscript; available in PMC 2018 January 28. A uhor M anscript