1、Nlrp9b inflammasome restricts rotavirus infection in intestinal epithelial cellsShu Zhu1,*,Siyuan Ding2,3,*,Penghua Wang4,Zheng Wei1,Wen Pan5,Noah W Palm1,Yi Yang1,Hua Yu1,Hua-Bing Li1,Geng Wang1,Xuqiu Lei1,Marcel R.de Zoete1,Jun Zhao1,6,Yunjiang Zheng1,Haiwei Chen1,Yujiao Zhao4,Kellie A.Jurado7,Nin
2、gguo Feng2,Liang Shan1,Yuval Kluger6,Jun Lu5,Clara Abraham7,Erol Fikrig7,8,Harry B.Greenberg2,3,and Richard A.Flavell1,81Department of Immunobiology,Yale University School of Medicine,New Haven,Connecticut 06520,USA2Department of Medicine and Department of Microbiology&Immunology,Stanford University
3、 School of Medicine,Stanford,California 94305,USA3Palo Alto Veterans Institute of Research,VA Palo Alto Health Care System,Palo Alto,California 94304,USA4Department of Microbiology and Immunology,New York Medical College,Valhalla,New York 10595,USA5Department of Genetics,Yale University School of Me
4、dicine,New Haven,Connecticut 06520,USA6Department of Pathology,Yale University School of Medicine,New Haven,Connecticut 06520,USA7Department of Internal Medicine,Yale University School of Medicine,New Haven,Connecticut 06520,USA8Howard Hughes Medical Institute,Chevy Chase,Maryland 20815,USAReprints
5、and permissions information is available at and requests for materials should be addressed to R.A.F.(richard.flavellyale.edu).*These authors contributed equally to this work.Online Content:Methods,along with any additional Extended Data display items and Source Data,are available in the online versi
6、on of the paper;references unique to these sections appear only in the online paper.Supplementary Information is available in the online version of the paper.Author Contributions:S.Z.wrote the manuscript,designed,performed and interpreted experiments with technical assistance from S.D.and P.H.W;S.D.
7、and H.B.G.provided rotavirus related reagents,S.D.performed the rotavirus fecal shedding assay,qPCR and immunostaining experiments;P.H.W,W.P and Y.Z performed RNA binding experiments;Z.W.and H.C.performed infections in intestinal organoids;Y.Y.and H.Y performed TUNEL staining;H.-B.L.and K.A.J.helped
8、 with DHX9 functional studies;G.W.,X.L.and Y.Z.assisted in the characterization of Nlrp9b mice;N.P.,M.Z.,and J.Z.performed 16s sequencing and PcoA analysis;N.F.performed the human IEC organoid experiment;C.A.provided human gut biopsies;S.D.,P.H.W.,N.W.P.,W.P.,L.S.,Y.K.,J.L.,C.A.,E.F.,H.B.G.and R.A.F
9、.provided critical comments and suggestions;R.A.F.supervised the project.The authors declare no competing financial interests.Readers are welcome to comment on the online version of the paper.Publishers note:Springer Nature remains neutral with regard to jurisdictional claims in published maps and i
10、nstitutional affiliations.Reviewer Information:Nature thanks I.Goodfellow,C.Reis e Sousa and the other anonymous reviewer(s)for their contribution to the peer review of this work.HHS Public AccessAuthor manuscriptNature.Author manuscript;available in PMC 2018 January 28.Published in final edited for
11、m as:Nature.2017 June 29;546(7660):667670.doi:10.1038/nature22967.Author ManuscriptAuthor ManuscriptAuthor ManuscriptAuthor ManuscriptAbstractRotavirus,a leading cause of severe gastroenteritis and diarrhoea in young children,accounts for around 215,000 deaths annually worldwide1.Rotavirus specifica
12、lly infects the intestinal epithelial cells in the host small intestine and has evolved strategies to antagonize interferon and NF-B signalling25,raising the question as to whether other host factors participate in antiviral responses in intestinal mucosa.The mechanism by which enteric viruses are s
13、ensed 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,N
14、lrp9b 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 intestin
15、e 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 s
16、uggested that anti-rotavirus antibody isotype switching depends on Il-1 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 infection
17、s 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 addi
18、tion,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.1bd,Extended Data Fig.1a),suggesting that inf
19、lammasome 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 w
20、hich are known to activate inflammasomes in the intestine811.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 sy
21、stemically examined the transcription levels of all NLRs by RNA sequencing of ileum intestinal epithelial cells(IECs).Interestingly,as well as Nlrp6,Nlrc4 and Naips9,1315,we discovered a novel NLR termed Nlrp9b that has not yet been characterized(Fig.2a).Nlrp9b,but not the closely related Nlrp9a and
22、 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
23、Nature.Author manuscript;available in PMC 2018 January 28.Author ManuscriptAuthor ManuscriptAuthor ManuscriptAuthor Manuscriptalso exhibited elevated viral load in IECs,fecal antigen shedding,diarrhoea occurrence and more severe pathology compared to wild-type pups(Fig.2be).Notably,loss of Nlrp9b di
24、d not affect normal gut homeostasis(Extended data Fig.2e,f).16S ribosomal DNA sequencing further indicated that the microbiota composition in Nlrp9b/mice was not different from that in wild-type mice(Extended data Fig.2f).Taken together,these findings strongly suggest that Nlrp9b,along with other in
25、flammasome components,is involved in the host innate immune defence against rotavirus infection,independent of microbiota effects.We next isolated both IECs and intraepithelial lymphocytes(IELs)from lamina propria and found Nlrp9b expression only in the EDTA-dissociated fraction,consisting of 8090%I
26、ECs and 20%IELs(Extended Data Fig.3a).A further separation using Villin-Cre-tomato+reporter mice(Extended Data Fig.3b)revealed that Nlrp9b and Il-18 were exclusively expressed in Villin-Cre-tomato+IECs(Fig.2f,Extended Data Fig.3c).To definitively confirm an IEC-specific role of Nlrp9b,we generated c
27、onditional knockout mice with specific depletion of Nlrp9b in IECs(Nlrp9bIEC)(Extended Data Fig.3d,e).Paralleling whole-body Nlrp9b/mice,Nlrp9bIEC mice were more susceptible to rotavirus infection compared to control(Nlrp9bfl/fl)mice(Fig.2g,h).Likewise,Casp1IEC mice with Casp1 deletion in IECs also
28、showed enhanced susceptibility to rotavirus infection compared to control(Casp1fl/fl)mice(Fig.2i,j).Taken together,these data strongly suggest that the Nlrp9b inflammasome functions only in IECs and restricts rotavirus infection.To further dissect the molecular mechanisms underlying Nlrp9b protectio
29、n against rotavirus infection,we examined the two downstream signals of inflammasome activation,the induction of pyroptosis and the secretion of Il-1 and Il-18.Consistent with previous publications17,18,we found disruption of the IEC monolayer and induction of cell death(Extended Data Fig.4).Notably
30、,rotavirus-induced IEC death at an early stage was largely dependent on Nlrp9b and Casp1(Fig.3a),whereas Aim2 was critical for radiation-induced death11.Using a mouse intestinal organoid model,we also found that Il-18 secretion from rotavirus infection was noticeably reduced in the absence of Nlrp9b
31、,Casp1/11 or Asc(Fig.3b,Extended Data Fig.5),suggesting that Nlrp9b accounts for both downstream events.By contrast,interferons(IFNs)and other pro-inflammatory cytokines were comparably expressed in wild-type and Nlrp9b/mice after infection(Extended Data Fig.5b,c).To assess the relative contribution
32、 of pyroptosis versus Il-18,we generated mice lacking gasdermin D(Gsdmd)(Extended Data Fig.6ad),a central mediator of pyroptosis19,20,and highly expressed in the small intestine(Extended Data Fig.6e,f).Notably,mice deficient in Gsdmd but not Il-18 displayed marked susceptibility to rotavirus infecti
33、on compared to wild-type and Ifnar1/mice(Fig.3c,d),previously shown to minimally restrict homologous rotavirus infection21.Moreover,we observed more robust rotavirus replication and reduced IEC death from Nlrp9b-,Casp1/11-and Gsdmd-deficient intestinal organoids(Fig.3e,f).In contrast,Nlrp9b deficien
34、cy had no effect on Salmonella infection(Fig.3g),highlighting its specific role in RNA virus infections.Collectively,our results support that Nlrp9b partners with adaptors Asc and Casp1 to assemble the inflammasome and mediate IEC pyroptosis to confine rotavirus replication.Zhu et al.Page 3Nature.Au
35、thor manuscript;available in PMC 2018 January 28.Author ManuscriptAuthor ManuscriptAuthor ManuscriptAuthor ManuscriptWe next performed in vitro experiments to examine how rotavirus infection triggers the Nlrp9b inflammasome.Using HEK293T cells reconstituted with Flag-tagged human NLRP9 and HA-tagged
36、 ASC,we observed an interaction between the two only during rotavirus infection(Fig.4a).Such binding was not detected for mouse Nlrp9b,probably owing to low expression.We hypothesized that rotavirus segmented double-stranded RNA(dsRNA)genome or other RNA byproducts during replication may activate ce
37、ll-intrinsic innate immunity22.Indeed,despite the fact that the majority of rotavirus RNA is localized at the viroplasm23,we observed weak yet detectable levels of viral RNA in the cytoplasm during infection(Extended Data Fig.7a).This is consistent with previous observations24 and suggests that rota
38、virus dsRNA might be recognized by and activate NLRP9.To this end,we performed assays to determine the ability of different sensors to bind to rotavirus RNA and found that NLRP9 co-precipitated with viral RNA more strongly than the controls NLRP6 and RIG-I(Fig.4b).Interestingly,as opposed to NLRP6,w
39、hich preferably bound to high molecular weight poly(I:C),a long dsRNA mimetic25,and RIG-I,with an affinity for 5-triphosphate RNA26,NLRP9 preferentially bound to short dsRNA stretches,represented by low molecular weight poly(I:C)and weakly to 5-triphosphate RNA(Fig.4c).Intestinal organoid and in viv
40、o studies corroborated our conclusion that Nlrp9b is not involved in the control of encephalomyocarditis virus(EMCV)(Extended Data Fig.7b),which predominantly produces long dsRNA during infection27.We further tested whether Nlrp9b directly interacts with viral RNA.Although GST-tagged recombinant Dhx
41、15 co-precipitated with biotinylated poly(I:C)as previously reported25,we did not observe any interaction for Nlrp9b(Fig.4d),suggesting the potential involvement of other RNA binding proteins.To identify such factors in an unbiased manner,we performed immunoprecipitationmass spectrometry for Flag-ta
42、gged NLRP9 and biotin-labelled viral RNA,respectively,and identified RNA helicase Dhx9 as a common hit in both data sets.Importantly,NLRP9 co-precipitated with endogenous DHX9 upon rotavirus infection(Fig.4e)and the RNA-binding pattern of DHX9 also revealed a unique affinity for short dsRNA,similar
43、to that of NLRP9(Fig.4c).Functionally,small interfering RNA(siRNA)knockdown of DHX9 but not DHX15 or RIG-I impaired the RNA-binding ability of NLRP9(Fig.4g,Extended Data Fig.8a),and rotavirus-induced inflammasome formation(Extended Data Fig.8b),in support of the hypothesis that the host RNA helicase
44、 DHX9 serves as a direct RNA-binding protein that mediates NLRP9 recognition of viral RNA.Consistently,rotavirus-infected Dhx9-deficient intestinal organoids were defective in both pyroptosis and Il-18 production(Extended Data Fig.8cf).We next generated Flag-tag knock-in mice to study Nlrp9b regulat
45、ion better(Extended Data Fig.9a).Whereas Nlrp9b protein levels increased at 12 h.p.i(Extended Data Fig.9b),Nlrp9b mRNA and protein levels decreased at 36 h.p.i.(Extended Data Fig.9b,c),probably owing to negative feedback loop.We also measured Nlrp9b expression in ileum and colon at different days af
46、ter birth.Intriguingly,we observed an age-dependent increase in Nlrp9b and Asc expression,but not Nlrp3,Casp1 and Rig-I(Extended Data Fig.9d).Such a pattern was reported for Tlr3(ref.28)(Extended Data Fig.9d)and is particularly interesting as the upregulation of Nlrp9b and Asc levels coincides with
47、the decreased susceptibility to rotavirus infection with age29,30.Zhu et al.Page 4Nature.Author manuscript;available in PMC 2018 January 28.Author ManuscriptAuthor ManuscriptAuthor ManuscriptAuthor ManuscriptFinally,we sought to examine a physiological relevance of NLRP9 in the human intestine.Compa
48、red to peripheral blood mononuclear cells and lamina propria lymphocytes,NLRP9 was only detected in the IEC fraction,whereas ASC and CASP1 were expressed in both populations(Extended Data Fig.10ac).We further validated that both NLRP6 and NLRP9 are highly expressed exclusively in primary human IEC o
49、rganoids but not transformed intestinal epithelial cell lines(Extended Data Fig.10d).Using rotavirus infection as a model,our study comprehensively characterizes and functionally dissects the role of a novel NLR,Nlrp9b,with expression that is highly specific to IECs,in the host defence against enter
50、ic virus infection.Upon Dhx9 recognition of short dsRNA stretches,the Nlrp9b inflammasome is activated and promotes Il-18 release and Gsdmd-mediated pyroptosis.The latter is especially important for the host to restrict rotavirus replication by inducing premature death of infected IECs and maintaini
