分子生物学知识拓展 (17).pdf
Ma et al.,Sci.Adv.2020;6:eaaz6717 20 May 2020SCIENCE ADVANCES|RESEARCH ARTICLE1 of 14IMMUNOLOGYGasdermin D in macrophages restrains colitis by controlling cGAS-mediated inflammationChunmei Ma1*,Dongxue Yang1*,Bingwei Wang2*,Chunyan Wu1,Yuqing Wu1,Sheng Li1,Xue Liu1,Kara Lassen3,Lue Dai4,Shuo Yang1The functional relevance and mechanistic basis of the effects of the pyroptosis executioner Gasdermin D(GSDMD)on colitis remain unclear.In this study,we observed that GSDMD protein was activated during intestinal inflam-mation in a model of chemically induced colitis.GSDMD deficiency exacerbated experimental colitis independent of changes in the microbiota and without affecting the production of antimicrobial peptides.GSDMD deficiency in macrophages,but not epithelial cells,was sufficient to drive this exacerbated experimental colitis.We further demonstrate that GSDMD functions in macrophages as a negative regulator to control cyclic GMPAMP synthase(cGAS)dependent inflammation,thereby protecting against colitis.Moreover,the administration of cGAS inhibitor can rescue the colitogenic phenotype in GSDMD-deficient mice.Collectively,these findings provide the first demon-stration of GSDMDs role in controlling colitis and a detailed delineation of the underlying mechanism.INTRODUCTIONInflammatory bowel diseases(IBDs),including Crohns disease(CD)and ulcerative colitis,are chronic and complex disorders charac-terized by uncontrolled intestinal pathogenic inflammation and intestinal tissue injury(1).Incidences of IBD are increasing around world,and current therapies maintain remission in a subset of indi-viduals but cannot completely control inflammation and IBD re-lapse(2).Normally,the gut mucosal immune system coevolves with the intestinal bacteria,acquiring the capacity to tolerate the components of bacteria and maintaining the capacity to respond to invading pathogens(3).In addition,most intestinal bacteria are considered commensal to the host for contributing to the development and homeostasis of intestinal immune system(4).The intestinal mucosal immune system,which is mainly composed of the intestinal epithe-lium and lamina propria(LP),mediates the production of mucin and bactericidal molecules and induces immune response,thereby providing the effective defense line against invading bacteria(5).Dysfunction of the intestinal mucosal system exposes immune cells to excessive bacteria load,leading to an inflammatory state associated with violent responses to microbe-associated molecular patterns and a subsequent large amount of proinflammatory cytokines(6).The intestinal mucosal immune system uses several innate re-ceptors,including Toll-like receptors,nucleotide-binding oligom-erization domain protein-like receptors(NLRs),and C-type lectin receptors,to monitor bacterial colonization and regulate mucosal immune homeostasis(7).Among these receptors,NLR proteins,such as NLRP1,NLRP3,NLRC4,and NLRP6,are assembled into cytosolic multiprotein complexes termed as inflammasomes together with apoptosis-associated speck-like protein(ASC)and proinflammatory caspases(caspase-1 and caspase-11),which leads to caspase auto-activation that governs the cleavage of prointerleukin-1(IL-1)and proIL-18 precursors into their mature forms and also induces a type of proinflammatory programmed cell death termed as pyro-ptosis(8,9).The IL-1 family of cytokines has been reported to reg-ulate intestinal homeostasis,inflammation,and healthy microbiota(10,11).Specifically,NLRP3 inflammasome induces IL-1 in myeloid cells,which promotes intestinal inflammation with the accumulation of innate lymphoid cells(ILCs)and T helper 17(TH17)cells(12,13),whereas IL-18,processed by NLRP6 inflammasome,protects intestinal epithelial cells(IECs)against colitis with enhanced intestinal epithelial integrity and secretion of antimicrobial peptides(AMPs)(14).Nevertheless,a recent study showed that IL-18 con-tributes to a pathologic breakdown of the intestinal barrier by inhibiting goblet cell maturation(15).In general,the inflammasome-related proteins,especially NLRP6,ASC,or caspase-1,are thought to play protective roles in experimental colitis in a microbiota-dependent manner(16).Consistent with this hypothesis,NLRP6 has been reported,together with ASC and caspase-1 or caspase-11,to regulate mucin granule exocytosis and maintain the mucosal integrity through auto-phagy in goblet cells(17).However,recent research has suggested that the NLRP6 inflammasome may not be required for the formation or function of baseline colonic inner mucus layer(18).As such,the functions of inflammasome in mucosal immunity and colitis seem complicated,and their underlying mechanisms remain not well un-derstood.Gasdermin D(GSDMD)is a newly identified pyroptosis execu-tioner that operates downstream of inflammasome activation(19).Upon activation,proinflammatory caspases(caspase-1 and caspase-4/5 in humans and caspase-1 and caspase-11in mice)cleave GSDMD in its central linker domain.Once cleaved,the N-terminal domain of GSDMD(GSDMD-N)is released from the autoinhibition by the C-terminal domain of GSDMD(GSDMD-C).Next,GSDMD-N fragments oligomerize and translocate to the plasma membrane and form pores,which induces cell lytic death and the secretion of IL-1 and IL-18(20).GSDMD has been linked to potential pathogenesis of some diseases such as septic shock and experimental autoimmune encephalomyelitis(EAE)(21,22).A more recent study has revealed how GSDMD restrains cGAS-dependent inflammation(23).Moreover,GSDMD mediates IEC pyroptosis,which may be required for removing pathogen-infected epithelial cells(24).However,the exact function of 1Department of Immunology,Key Laboratory of Immunological Environment and Disease,State Key Laboratory of Reproductive Medicine,Center for Global Health,Nanjing Medical University,Nanjing,China.2Department of Pharmacology,Nanjing University of Chinese Medicine,Nanjing,China.3Roche Innovation Center Basel,Basel,Switzerland.4Roche Innovation Center Shanghai,Shanghai,China.*These authors contributed equally to this work.Corresponding author.Email:(S.Y.);(L.D.);(B.W.)Copyright 2020 The Authors,some rights reserved;exclusive licensee American Association for the Advancement of Science.No claim to original U.S.Government Works.Distributed under a Creative Commons Attribution NonCommercial License 4.0(CC BY-NC).on May 19,2021http:/advances.sciencemag.org/Downloaded from Ma et al.,Sci.Adv.2020;6:eaaz6717 20 May 2020SCIENCE ADVANCES|RESEARCH ARTICLE2 of 14GSDMD in the context of colitis is largely unknown.In this study,we sought to understand the role of GSDMD in colitis and elucidate underlying mechanisms.RESULTSGSDMD is highly activated in the intestine of dextran sodium sulfatetreated miceGSDMD protein was found highly expressed in various tissues of mice,especially mesenteric lymph nodes(mLNs)and intestine(Fig.1A).Immunofluorescence analysis showed wide distribution of GSDMD in both LP Cx3cr1+myeloid cells(tdTomato reporter)and epithelial E-cadherin+cells(Fig.1B).To further investigate whether GSDMD has a possible role in intestinal inflammation,we used the dextran sodium sulfate(DSS)model of colitis.Briefly,mice were given 2.5%DSS in the drinking water for 6 days and then switched to normal drinking water until day 9.We tested GSDMD and caspase-11 expression on day 9in both control and DSS-treated mice.Immunoblotting analysis revealed the substantially increased expression and cleavage of GSDMD and caspase-11in the colon of colitis mice relative to untreated mice(Fig.1C).We next isolated IECs and myeloid cells from colons of colitis mice to investigate GSDMD activation in these cells.We found that the more notable GSDMD activation induced by colitis in myeloid cells was mainly characterized by a 30-kDa cleaved fragment,whereas both p30 and p47 fragments are the major cleaved bands in IECs(Fig.1D).Col-lectively,these results suggest the involvement of GSDMD-mediated pyroptosis in this colitis model.GSDMD deficiency exacerbates DSS-induced colitisTo directly evaluate the role of GSDMD in colitis,we treated age-matched GSDMD knockout(GSDMD/)or wild-type(WT)mice with DSS to compare their colitis phenotype.The GSDMD/mice exhibited normal growth and survival,and GSDMD deficiency did not affect the development and maturation of myeloid and lymphoid cells in central and peripheral lymphoid organs under homeostatic conditions,as previously reported by our laboratory(21).Notably,a more severe colitis on day 9 after DSS administration was observed in GSDMD/mice than in WT controls,as characterized by significantly greater body weight loss,higher disease activity index(DAI)score,and shorter colons in DSS-treated GSDMD/mice(Fig.1,EtoG).Consistently,histopathological analysis hematoxylin and eosin(H&E)staining showed that GSDMD deficiency led to increased inflamma-tory cell infiltration with a more severe disruption of the mucosal epi-thelium in response to DSS treatment(Fig.1H).In addition,we used ASC knockout(ASC/)mice as a control and confirmed the protective effect of inflammasome on colitis,as previously reported(Fig.1,ItoL)(16).Together,these data suggest that similar to other inflammasome proteins,GSDMD plays a protective role in DSS-induced colitis.GSDMD deficiency drives severe DSS colitis in a microbiota-independent mannerLoss of inflammasome components,such as NLRP6 and ASC,led to the shift of microbial communities toward a transmissible and more colitogenic microbiota that drives DSS-induced colitis(16).To evaluate whether the increased severity in colitis observed in GSDMD/mice relative to WTs is also driven by colitogenic microbiota,we cohoused littermate WT mice WT(GSDMD/)and GSDMD/mice GSDMD/(WT)for 6 weeks to equalize bacterial community before administration of DSS.Analysis of bacterial 16S ribosomal RNA(rRNA)sequencing of fecal pellets demonstrates similar bacterial composition including comparable bacterial diversity and abundance in family levels(including the colitogenic Prevotellaceae and Bacteroidales)between the cohoused WT and GSDMD/mice(fig.S1,A to C).Cohousing breeding did not change the develop-ment of more severe DSS-induced colitis in GSDMD/(WT)mice as compared with the cohoused WT(GSDMD/)controls(fig.S2,A to D).While cohousing of littermate WT mice WT(ASC/)and ASC/ASC/(WT)also led to similar bacterial diversity and abundance(fig.S1,D to F),this now almost equalized the severity of DSS-induced colitis(fig.S2,E to H).Therefore,unlike ASC,GSDMD protects against colitis independent of gut microbiota changes.GSDMD deficiency exacerbates colonic inflammation but does not impair the production of AMPs and mucus secretionTo further characterize the role of GSDMD in colitis as a negative regulator of intestinal inflammation,we analyzed the expression of proinflammatory cytokines and chemokines in colon explants of WT and GSDMD/colitis mice by real-time quantitative polymerase chain reaction(RT-qPCR)and enzyme-linked immunosorbent assay(ELISA).GSDMD deficiency significantly increased the production of proinflammatory cytokines and chemokine including IL-6,tumor necrosis factor(TNF-),interferon-,and Ccl2,and as expected,the secretion of IL-1 and IL-18 was impaired in the absence of GSDMD(Fig.2,AandB,and fig.S3,A and B).More-over,fluorescence-activated cell sorting(FACS)revealed a significant increase in the frequencies and absolute numbers of colon-infiltrating immune cells(CD45+),CD4+and CD8+T cells,macrophages(CD11b+F4/80+),monocytes(CD11b+Ly6c+),and neutrophils(CD11b+Ly6g+)in GSDMD/mice relative to those in WT mice on day 5 after DSS treatment(Fig.2,CandD).Moreover,we observed a reduction in PI+cells gated on the CD45+CD11b+F4/80+(colonic macrophages)in the colon of GSDMD/mice during colitis compared with control mice(Fig.2E).Thus,GSDMD deficiency promotes colonic inflammation and protects colonic macrophages from death during colitis.Inflammasome is expected to shape the composition of micro-biome to avoid the outgrowth of colitogenic bacteria by regulating the production of AMPs(14).Next,we investigated whether GSDMD affects the expression of AMPs and its inducing cytokine IL-22 but found no significant difference in the expression of Il22 or AMPs,such as Reg3b,Reg3g,Defn5,and Lysozyme1,between the colons of untreated(fig.S3C)and DSS-treated(Fig.2F)WT and GSDMD/mice.Because intestinal mucosal barrier integrity is also important in host defense against colitis(25)and NLRP6 inflammasome and IL-18 can regulate mucin granule exocytosis and goblet cell matura-tion to affect intestinal barrier function(15,17),we further assessed whether GSDMD affects the expression or secretion of mucus pro-teins in goblet cells.We found no difference in the expression of mature goblet cell mucin MUC2in colons from WT and GSDMD/mice untreated(fig.S3C)or treated with DSS(Fig.2F).Moreover,Periodic acidSchiff(PAS)and MUC2 staining showed the compa-rable abundance and configuration of mature PAS+and MUC2+goblet cells in untreated(fig.S3,D and E)or DSS-treated(Fig.2,GandH)WT and GSDMD/mice.Together,these data suggest that GSDMD has no impact on the production of AMPs and mucus but is indis-pensable in controlling colonic inflammation.on May 19,2021http:/advances.sciencemag.org/Downloaded from Ma et al.,Sci.Adv.2020;6:eaaz6717 20 May 2020SCIENCE ADVANCES|RESEARCH ARTICLE3 of 14ACBGSDMDMergeE-cadherinCx3cr1MergeGSDMDGEFH100200WTGSDMD/JKIL100 200WTDGSDMD/WT-TubulinGSDMD5555(kDa)45553545IECsMyeloid cellsFL-GSDMDCleaved-GSDMD(kDa)-Actin455535-ActinCtrlDSSFL-GSDMDPro-caspase-11Cleaved-caspase-11Cleaved-GSDMD(kDa)454525WTGSDMD/WTGSDMD/WTGSDMD/WTASC/246810Histology score*WTASC/Colon length(cm)WTASC/Weight change(%)WTGSDMD/2.5%DSSWeight change(%)WTASC/2.5%DSS1010864210864864Colon length(cm)Histology score200101234Days*864*DAI5678910234Days567891086420DAI10234Days567892030100101234Days*567892030Fig.1.GSDMD is activated in the intestine of DSS-induced colitis mice and protects against DSS-induced colitis.(A)Immunoblot analysis of GSDMD expression in various organs,including heart,liver,kidney,lung,mLNs,spleen,small intestine(SI),colon,cortex,midbrain,and cerebellum from GSDMD/and wild-type(WT)mice.(B)Immunofluorescence labeling of GSDMD(red),4,6-diamidino-2-phenylindole(DAPI)(blue)in colon sections from WT mice,and labeling of GSDMD(green),Cx3cr1(red),and DAPI(blue)in colon sections from Cx3cr1 reporter mice.The merging of GSDMD with E-cadherin or Cx3cr1 is indicated by arrows.Scale bar,200 m.(C)Immuno-blot analysis of full-length and cleaved GSDMD and caspase-11 in colon from control or DSS-treated WT mice on day 9.(D)Immunoblot analysis of full-length and cleaved GSDMD in IECs and myeloid cells sorted from colon of DSS-treated WT mice on day 9.(E and F)Age-matched male WT(n=12)and GSDMD/(n=12)mice were given 2.5%DSS in their drinking water for 6 days and then normal water for three further days.Weight changes(E)and DAI(F)were monitored daily.(G)Gross morphology images of colons and colon lengths of
