MicroRNA-5112 Targets IKK? To Regulate NF-?B-mediated Inflammatory Response

Inflammation,the primary response of innate immunity,is a protective response against pathogens infections and tissue damages.The initiation of inflammatory response is mainly mediated by signaling pathways,most commonly including NF-?B,MAPK,and JAK-STAT pathways.Salmonella species,the important zoonotic pathogen,cause substantial morbidity,mortality and burden of disease globally both in animal and human.Central to the pathophysiology of salmonellosis is the induction of a strong inflammatory response.The NF-?B pathway plays an important role in host inflammatory response induced by Salmonella.Hosts detection of the invading Salmonella was based on the recognition of pathogen-associated molecular patterns(PAMPs)by pattern recognition receptors(PRRs).The toll-like receptors(TLRs)are the one of the best-characterized PRR families.TLRs can recognize various bacterial compoents,e.g.,lipoproteins,lipopolysaccharide(LPS),flagellin,and unmethylated CpG DNA byTLR2,TLR4,TLR5 and TLR9,respectively.These recognitions can activate NF-?B pathway,leading to the production of inflammatory cytokines.For example,the recognition of flagellin by TLR5 induced the signaling cascades and activated I?B kinase(IKK).IKK,is composed of two kinase subunits,IKKa and IKK?,and a regulatory subunit,IKKy.The activated IKK induced the phosphorylation of I?B,subsequently resulting in the activation of NF-?B.Finally,the activated NF-?B induced the production of inflammatory cytokines to defend against invading bacteria.Although inflammation can defend against invading bacteria,it is also a double-edged sword.Deregulated or excessive inflammation can cause tissue injury,and inflammatory diseases.For example,inflammatory bowel diseases(IBD)is chronic bowel diseases caused by excessive activation of inflammatory response,and it has become a global health burden with increasing incidence and prevalence.Strong evidence suggests the involvement of NF-?B in the pathogenesis of IBD.Excessive NF-?B signal activation has been found in inflamed colonic tissue of IBD patients.Therefore,the inflammatory response need to be strictly and precisely controlled and regulated.MicroRNAs(miRNAs)are a class of small(approximately 22 nucleotides in length)and highly conserved noncoding RNAs.They can post-transcriptionally regulate gene expression by binding to 3'-untranslated regions(UTRs),resulting in translation repression or mRNA degradation.miRNAs play key roles in many biological processes,including cell proliferation,differentiation,development,immune response.Recently,increasing evidences have shown that miRNAs play important regulatory roles in inflammatory response.Although there have been reports of miRNAs involved in the regulation of inflammatory responses,the function of most miRNAs remains unknown.Particularly,the regulatory role of miRNAs in TLR5/NF-?B inflammatory pathways induced by flagellin needs to be explored.Previous study in our lab found that mmu-miR-5112(miR-5112)was down-regulated in flagellin-treated BMDCs,and preliminarily found that miR-5112 could target IKKy to regulate TLR5/NF-?B inflammatory pathways.In this study,the expression pattern of miR-5112 in dendritic cells(DCs)was further determined.We evaluated the function and mechanism of miR-5112 in inflammation induced by flagellin in vitro and in vivo.The regulation of miR-5112 in inflammatory response induced by Salmonella was also evaluated in vitro and in vivo.Impact of miR-5112 on colitis induced by DSS was investigated in vivo.1.The regulatory role of miR-5112 in inflammatory response induced by flagellinThe miR-5112 and cytokines expression in spleen DCs induced by flagellin was determined in vitro and in vivo.The expression of IL-6 and IL-12 p40 was increased in cultural supernatant and the expression of miR-5112 was down-regulated in spleen DCs stimulated with flagellin in vitro.The expression of IL-6 was up-regulated and the expression of miR-5112 was decreased in spleen DCs from mice immunized with flagellin.These results suggested that flagellin could downregulate the expression of miR-5112 and upregulated inflammatory cytokines in spleen DCs both in vitro and in vivo.The expression of miR-5112 was further determined in BMDCs after flagellin stimulation at different time points.After flagellin treatment,the miR-5112 expression was down-regulated at early time,and then returned to normal levels.The miR-5112 expression was decreased more than 1.5 fold changes in BMDCs after treatment with Pam3CSK4(TLR2 ligand),Poly(I:C)(TLR3 ligand),LPS(TLR4),Flagellin(TLR5),and R848(TLR7)except for CpG-ODNs(TLR9).miR-5112 was expressed in all examined tissues,but the expression level was variable in different tissues.These results suggested the expression of miR-5112 was time and tissue-dependent,and might be correlated with its function.The expression of miR-5112 was also determined in BMDCs treated with different flagellin mutants.Flagellin mutants,with deletion of TLR5 or NLRC4 recognition sites,could not induce the expression change of miR-5112 in BMDCs.The miR-5112 was only down-regulated in BMDCs treated with wild type flagellin containing both TLR5 and NLRC4 recognition sites.In addition,the expression of miR-5112 was also unchanged in BMDCs derived from MyD88-/-mice treated with wild type flagellin.These results suggested that flagellin requires both TLR5 and NLRC4 activity to down-regulate the expression of miR-5112 in BMDCs.When BMDCs were transfected with miR-5112 mimics,secretion of IL-12 p40 was significantly decreased upon flagellin stimulation,suggesting that exogenous miR-5112 could inhibit the flagellin-mediated inflammatory response in vitro.Western Blotting analysis showed that the expression of IKK? was inhibited by miR-5112 mimics in BMDCs induced with flagellin.At the same time,the expression of p-P65 was also decreased in BMDCs that were transfected with miR-5112 mimics and induced by flagellin.These results suggested that miR-5112 can target the IKKy to regulate the expression of TLR5/NF-kB inflammatory pathways upon flagellin treatment.To elucidate the role of miR-5112 in vivo,we intraperitoneally administered mice with agomiR-5112 or antagomiR-5112 to overexpress or inhibit the miR-5112.Subsequently,the mice were treated with flagellin,and cytokines were measured.The result showed that the flagellin-induced the inflammatory cytokines IL-6,IL-12 p40,TNF-a were significantly decreased by administration of agomiR-5112 and increased by administration of antagomiR-5112.Production of the chemokine MCP-1 induced by flagellin was also significantly decreased by agomiR-5112.In addition,agomiR-5112 could also decrease the production of anti-inflammatory cytokine IL-10 induced by flagellin,while antagomiR-5112 enhanced IL-10 production.These results suggested that miR-5112 could regulate flagellin-mediated inflammatory response in vivo.2.The regulatory role of miR-5112 in inflammatory response induced by Salmonella infectionThe regulatory role of miR-5112 in inflammatory response was further evaluated during Salmonella infection both in in vitro and in vivo.In vitro experiment showed that transfection agomiR-5112 into peritoneal macrophages could decrease the IL-6 production triggered by infection with Salmonella enterica serovar enteritidis(S.enteritidis).The result suggested that miR-5112 could regulate the inflammatory response induced by Salmonella in vitro.A streptomycin-treated mouse model was used to evaluate miR-5112 regulatory role in inflammatory response induced by S.enteritidis in vivo.The inflammatory cytokines could not be detected in serum at 8 h post Salmonella-infection.Cytokines including IL-1?,IL-6,IL-12 p4O,IP-10,IFN-y,and TNF-a were increased in S.enteritidis-infected mice at 4 d post-infection.Importantly,the agomiR-5112 could markedly inhibit the production of these cytokines,including IL-1?,IL-6,IL-12 p40,IP-10 and TNF-?,in S.enteritidis-infected mice.Histopathological analysis showed that at 8 h and 4 d post-infection,the liver and cecum of the Salmonella-infected mice exhibited inflammatory cell infiltration,while miR-5112 agomiR administration dramatically reduced the accumulation of inflammatory cells compared with the administration of PBS.These results indicated that agomiR-5112-treated mice showed a reduced inflammatory response compared with PBS-treated mice during Salmonella infection,which suggested that miR-5112 could inhibit the inflammatory response induced by Salmonella.Furthermore,agomiR-5112-treated mice had significantly reduced Salmonella loads in ileum and colon tissues compared with PBS-treated mice at 8 h post-infection.Finally,administration of the agomiR-5112 alleviated the loss of body weight and delayed the death of mice infected with Salmonella.In summary,our results suggested that miR-5112 is an important regulator in inflammatory response and defending against Salmonella infection.3.The regulatory role of miR-5112 in colitis induced by dextran sulfate sodium(DSS)The colitis model was developed by treating the mice with 4%DSS.The effect of miR-5112 on colitis induced by DSS was evaluated.After drinking 4%DSS,the mice developed symptoms including anorexia,energielos,loss of weight,lack of gloss on the fur.As time goes on,the mice began to show watery feces and bloody stools,which are typical symptoms of colitis.These results suggested that the colitis model was successfully developed.Systemic and local inflammation were evaluated by measuring the cytokines and MPO(an indicator of neutrophil influx and acute inflammation)in serum and colon tissues.The levels of IL-6 in serum were significantly increased in DSS group at days 3 and 6 after DSS treatment.However,the increased IL-6 in serum could be dramatically suppressed by administration of agomiR-5112.Additionally,the inflammatory cytokines IL-6,MCP-1,TNF-?,and MPO induced by DSS in colon tissues were also reduced by agomiR-5112.We further performed a histological analysis of the colon to assess the severity of colitis.Compared with the colon in the control group,the DSS group showed massive epithelial destruction,submucosa oedema,and inflammatory cell infiltration in the submucosa and muscular layer.In contrast,administration of agomiR-5112 significantly alleviated these deteriorating pathological alternations.DSS typically caused colonic shortening,while administration of agomiR-5112 significantly reduced the DSS-induced colon shortening.These results suggested that miR-5112 could reduce the inflammation induced by DSS.Disease activity index(DAI)scores,an indicator of colitis,were significantly increased in the DSS group compared with the control group.Administration of agomiR-5112 significantly reduced the DAI scores compared with the DSS group.These results demonstrated that miR-5112 could regulate the DSS-induced colitis,dampen the inflammatory response,and alleviate DSS-induced colitis.These results suggested that miR-5112 could be a novel therapeutic target for inflammatory bowel disease.