MMP-7 Regulates Mucosal Barrier In Inflammatory Bowel Disease Through Degrading Tight Junction Protein Claudin-7

Background:Inflammatory bowel disease（IBD）is a group of chronic,non-specific inflammatory bowel diseases,including Crohn’s disease（CD）and ulcerative colitis（UC）,which are often recurrent and hard to get complete remission.The etiology and pathogenesis of IBD remain obscure.Many studies suggest that intestinal mucosal barrier injury is an important part of progression to IBD.It is of great importance to maintain the expression and function of tight junction proteins.Previous studies have shown that MMP-7 is highly expressed in IBD.It is also reported MMP-7 can not only degrade a variety of extracellular matrix components,but also regulate intestinal inflammation by activating pro-or anti-inflammatory factors.However,the molecular mechanisms underlying the effects of MMP-7 on tight junctions and the intestinal mucosal barrier are unclear,and we have therefore undertaken this mechanistic elucidation.Aims:The aims of this study were to investigate the effect of MMP-7 on intestinal mucosal barrier in IBD:to clarify the differences in intestinal MMP-7/Claudin-7 expression between controls and UC patients;to decipher the mechanisms by which MMP-7 regulates Claudin-7 and its effects on the intestinal mucosal barrier and intestinal inflammation;and to investigate the effect of MMP-7 antibody intervention on intestinal mucosal barrier and Claudin-7 expression in dextran sulfate sodium（DSS）mouse model and trinitrobenzene sulfonic acid（TNBS）chronic colitis rat model,in order to identify a novel strategy for the treatment of IBD.Methods:1.Colon biopsies were collected from UC patients and controls,and MMP-7 expression was detected by reverse transcriptase-quantitative polymerase chain reaction（RT-q PCR）and immunohistochemistry.In this study,C57BL mice and SD rats were given 3%DSS solution ad libitum for 7 consecutive days or TNBS（65mg/kg）enema to induce colitis in vivo.In addition,IL-10^-/-spontaneous colitis mice and T-cell transfer colitis mice were housed.RT-q PCR and immunohistochemistry were used to verify the expression of MMP-7 in these animal models of colitis.2.In order to investigate the cellular source of MMP-7,eleven-commonly found inflammatory factors in IBD and lipopolysaccharides were used to stimulate the major constituent cells of the intestine,including normal human colon epithelial cells FHC,primary rat colon smooth muscle cells,and macrophages THP-1.MMP-7 transcript levels of cells above were measured for screening.A concentration gradient test was then performed to detect MMP-7 expression by RT-q PCR after24 hours of intervention with IL-1β,TNF-αand LPS in FHC or THP-1and TNF-α,IL-4 and IL-13 in primary rat colon smooth muscle cells.3.In vitro,MMP-7 was used to treat FHC according to a concentration gradient,and western blot was applied to detect the expression of tight junction proteins including Claudin-1,Claudin-7,Claudin-8 and Claudin-15.Normal human colonic epithelial cells CCD841 Co N were transfected with pc DNA-MMP7 plasmid to detect Claudin-7 levels upon MMP-7 overexpression.To investigate the mechanism,we constructed a Claudin-7 prokaryotic expression purification system and incubated the obtained Claudin-7 protein with MMP-7 for proteolysis experiments before applying Western blot to detect Claudin-7.A Caco-2 intestinal cell barrier model was constructed with the following groups for intervention:control,MMP-7,MMP-7+Claudin-7 overexpression group,Claudin-7 silencing group,and the barrier function was assessed by measuring the absorbance value of the fluorescent dye Lucifer Yellow.In addition,immunohistochemistry was applied to detect Claudin-7 expression in the intestine of UC patients with high MMP-7 expression.4.C57BL wild-type mice and Mmp7^-/-mice were given 3%DSS solution for 7 consecutive days to induce colitis model,while the control group was their littermates with normal water intake.The pathological characteristics of the four groups of mice were assessed by HE staining;the inflammatory status was assessed by detecting MPO activity and RT-q PCR for IL-1β,TNF-α,IL-6 and Nlrp3;the effect on the intestinal mucosal barrier was assessed by RT-q PCR for tight junctions,adherens junctions and mucus-related proteins,and the expression of MMP-7 was detected by immunofluorescence;tight junction proteins and inflammatory status were assessed by Western blot for Claudin-7,IL-1β,andβ-catenin.5.3%DSS wild type mouse with colitis was constructed and treated with MMP-7 antibody in one group of DSS mice.The mice classified as the control group,the DSS group and the DSS+MMP-7antibody group.The intestinal permeability of each group was assessed by measuring the serum FITC-dextran-4000 concentration,and the intestinal inflammation was assessed by measuring MPO activity and IL-1βby RT-q PCR.The chronic TNBS model was established by administration of TNBS（50 mg/kg）to SD rats four times at 3-week intervals.The control group was treated with saline and one TNBS group was treated with MMP-7 antibody.As a whole,the rats were divided into following groups:control group,TNBS group and TNBS+MMP-7 antibody group.Result:1.RT-q PCR showed that MMP-7 m RNA expression was significantly upregulated in the intestinal tissues of UC patients compared to controls.Immunohistochemistry revealed a large number of brownish-yellow areas in UC colonic epithelial cells and inflammatory cells,indicating an increased expression of MMP-7 protein.The results were validated in various animal models of colitis,including DSS rats,TNBS rats,DSS mice,TNBS mice,IL10^-/-mice and T-cell transfer of colitis mice.2.RT-q PCR results showed that IL-1βand TNF-αconcentration-dependently stimulated MMP-7 expression in FHC intestinal epithelial cells;IL-4 and IL-13 concentration-dependently up-regulated MMP-7expression in primary rat colon smooth muscle cells;while IL-1β,TNF-αand LPS concentration-dependently increased MMP-7 expression in THP-1-derived macrophages.3.Western blot showed that MMP-7 treatment of intestinal epithelial cells or overexpressing MMP-7 in epithelial cells decreased Claduin-7 expression,but did not affect Claudin-1,Claudin-8 and Claudin-15 expression.Proteolysis assay showed that MMP-7 was able to degrade Claduin-7,and we innovatively identified Claudin-7 as a substrate for MMP-7.Caco-2 intestinal barrier permeability assay showed that MMP-7 significantly increased intestinal epithelial permeability,and overexpression of Claudin-7 significantly alleviated the disruption of the intestinal epithelial barrier by MMP-7.In addition,knockdown of Claudin-7 increased intercellular permeability in Caco-2.Immunohistochemistry showed that intestinal tissue Claudin-7expression was reduced in UC patients with high MMP-7 expression.4.MMP7 knockout was found to alleviate colonic inflammation in Mmp7^-/-mice compared to wild-type mice,as indicated by milder intestinal pathological damage with no obvious ulcerative erosion,significantly lower MPO activity,and significantly lower expression of IL-1β,TNFα,IL-6,Nlrp3,MMP-3 and MMP-9.The expression of E-cadherin and Muc-2 was decreased in wild-type mice after DSS,suggesting that epithelial cell adhesion junctions and the mucus barrier were impaired,but MMP-7 knockdown had a protective effect on this.We found that tight junction protein transcript levels were not downregulated after DSS intervention,suggesting that its regulation of tight junction proteins is post-transcriptional.5.MMP-7 antibody treatment in DSS mice and TNBS rats with chronic colitis resulted in alleviation of pathological damage,significant improvement in intestinal mucosal permeability,significant reduction in MPO activity,and significant reduction in IL-1βand inflammatory cells（neutrophil,mast cell,eosinophil）infiltration.Immunofluorescence showed significant loss of Claudin-7 from the apical part of the crypt in the TNBS chronic colitis group,whereas Claudin-7 was protected and not reduced in the MMP-7 antibody treated group.Conclusion:MMP-7 expression was significantly increased in UC patients and in various murine models of colitis,whereas Claudin-7 expression was significantly down-regulated.This study demonstrates for the first time that MMP-7 disrupts tight junctions between intestinal epithelial cells by degrading Claudin-7,leading to increased intestinal permeability and thus exacerbating intestinal inflammation.This study reveals that MMP-7 regulates intestinal barrier and demonstrates that targeted blockade of MMP-7 in intestinal inflammation can maintain the intestinal barrier and alleviate intestinal inflammation by protecting Claudin-7 protein.