Therapeutic Mechanisms Of Anti-mouse CD52 In Interleukin-10 Knockout Mice With Spontaneous Chronic Colitis Similar To Crohn's Disease

Crohn's disease(CD)is a segmental inflammatory disease of the intestine characterized by a massive infiltration of CD4+T cells and macrophages and by intestinal barrier dysfunction.The exact aetiology is still not completely understood,it has been proposed that the environmental factor,genetic factor,microbe and dysfunction of immune response contribute to the disease.CD is characterized by both helper type 1(Th1)and Th17 polarized inflammation in lamina propria.The successful application of anti-TNF-a antibody treatment signified a major breakthrough in the treatment of IBD.Despite the success of anti-TNF-a biological agents in IBD,approximately one-third of patients do not respond to anti-TNF-a treatment,and many others eventually lose responsiveness or become intolerant to these agents.In addition,patients treated with anti-TNF-a show an increased incidence of severe infections and malignancies.Taken together,these results underscore the urgent need to develop new biological agents,particularly for patients who do not respond,lose responsiveness or cannot receive TNFa blockers.Although the anti-IFN-? and anti-IL-17 treatment were not satisfactory,the targeting of T cell subsets themselves or the simultaneous targeting of multiple cytokines(rather than targeting a single effector cytokine)may hold promise for future therapy of IBD.CD52 is a cell-surface glycoprotein mainly expressed on mature T lymphocytes and B lymphocytes,and expressed in the monocytes,macrophages and neutrophils at low levels.Alemtuzumab(Campath-1H),a humanized monoclonal antibody(mAb),directly targets the cell surface CD52 and is effective in depleting mature lymphocytes through complement-dependent cytotoxicity(CDC),antibody-dependent Cell Cytotoxicity(ADCC),apoptosis and other pathways,leading to long-lasting changes in adaptive immunity(particularly the low levels of CD4+T cells).This antibody has also been used in the treatment of a wide range of diseases including rheumatoid arthritis,lymphocytic leukaemia and organ transplantation.In recent phase 3 clinical studies,alemtuzumab showed efficacy in the treatment of relapsing-remitting multiple sclerosis.Our previous studies demonstrated that treatment with 20 mg of antimouse CD52 mAb once per week for 2 weeks improved histological inflammation scores in interleukin-10 gene knockout mice(IL-10-/-)However,there are still many unknown aspects that need to be identified to make us understand the complex immunity and ecosystem of the intestine after CD52 mAb treatment.Much of our current understanding of the molecular mechanisms involved in inflammatory bowel diseases(IBD)has come from transgenic,knockout and chemically induced mouse models.Although mouse models of IBD do not fully recapitulate the human disease,IL-10-/-mice display similar characteristics to those of human CD.Because the anti-inflammatory effects of IL-10 are required to regulate Thl and Thl7 cytokine production and promote immune homeostasis,loss of IL-10 in mice results in colitis under specific pathogen-free conditions driven by an aberrant immunological response to luminal content,including pathogens,toxins and antigens.In the present study,IL-10 KO mice were used as animal model of CD and were given CD52 mAb treatment.After the administration,the mononuclear cells from peripheral blood were isolated for measuring CD4+T cells every week for 6 weeks after the final drug administration.CD4+T cells were evaluated for lymphocyte depletion and repopulation after CD52 mAb treatment.Percentage of CD4+T lymphocytes changes and mechanisms of CD4+T lymphocytes polarization in IL-10 KO mice were measured before CD4+T lymphocytes repopulation.Furthermore,the effect of CD52 mAb treatment on lymphocyte homing and related mechanisms,severity of colitis,epithelial expression of tight junction proteins,morphology of tight junctions,TNF-?/TNFR2 mRNA expression,myosin light chain kinase(MLCK)expression and activity,as well as epithelial apoptosis in the proximal colon were studied 4 weeks after the final drug administration.The results could indicate the therapeutic effects and the potential mechanism of the CD52 mAb in CD animal model,which provided theoretical support for the clinic usage of CD52 mAb treatment in CD patients.This thesis is divided into three studies in two parts?Part 1The influence of CD52 monoclonal antibody on the mucosal immunity and the related mechanism in IL-10 knockout miceStudy 1:The effect of CD52 mAb on depleting CD4+T lymphocytes in the lamina propria and the-mechanisms of CD4+ T lymphocytes differentiation in IL-10 knockout miceObjectives:The aim of this study was to investigate the effect of CD52 mAb on depleting CD4+T lymphocytes in the lamina propria and the mechanisms of CD4+T lymphocytes differentiation in IL-10 knockout mice.Methods:Interleukin-10 knockout mice(IL-10 KO)of 16 weeks on a C57BL/6 background were divided into IL-10 KO group and CD52 mAb group,and C57BL/6 background wild-type mice were served as WT group.The IL-10-/-mice in the CD52 mAb group received CD52mAb(20 ug diluted in PBS)treatment once a week for 2 weeks,whereas the mice in WT and IL-10 KO groups received the same volume of vehicle(IgG).The mononuclear cells from peripheral blood were isolated for measuring CD4+T cells every week for 6 weeks after the final drug administration.CD4+T cells were evaluated for lymphocyte depletion and repopulation after CD52 mAb treatment.Percentage of CD4+T lymphocytes changes and mechanisms of CD4+T lymphocytes polarization in IL-10 KO mice were measured before CD4+T lymphocytes repopulation.Results:CD52 mAb treatment effectively reduced the percentage of CD4+T for 4 weeks.Compared with mice in IL-10 KO group,CD52 mAb treatment significantly decreased the percentage of CD4+ CD45+ lymphocytes in peripheral blood,spleen,and lamina propria(P<0.05).Levels of CD4+CD8a+T cells,IL-17,IFN-? and IL-12/23 p40 in lamina propria were also significantly reduced(P<0.05).The Stat3,Stat4 and IL-21,which were required for CD4+T lymphocytes differentiating into Th17 and Th1,were also decreased after CD52 mAb treatment(P<0.05).Conclusion:The therapeutic mechanisms of CD52 mAb treatment in IL-10 KO mice may be associated with decreased percentage of CD4+T lymphocytes and proinflammatory cytokines,as well as inhibition of Thl and Th17 polarization.Study 2:The effect of CD52 mAb on migration of lymphocytes to lamina propria in IL-10 knockout miceObjectives:The aim of this study was to observe the effect of CD52 mAb treatment on migration of lymphocytes to lamina propria in IL-10 knockout mice.Methods:Interleukin-10 knockout mice(IL-10 KO)of 16 weeks on a C57BL/6 background were divided into IL-10 KO group and CD52 mAb group,and C57BL/6 background wild-type mice were divided into WT group.The IL-10-/-mice in the CD52 mAb group received anti-CD52mAb(20 ug diluted in PBS)treatment once a week for 2 weeks,whereas the mice in WT and IL-10 KO groups received the same volume of vehicle(IgG).The effect of CD52 mAb treatment on lymphocyte homing and relatively mechanisms were studied 4 weeks after the final drug administration.Results:Compared with mice in IL-10 KO group,CD52 mAb treatment significantly decreased the relative mRNA expression of ICAM-1,MAdCAM-1 and TNF-?(P<0.05),and the percentage of CD11b+F4/80+ macrophages(P<0.05).However,CD11b+F4/80+and F4/80+macrophages in spleen and lamina propria,as well as CD11b+F4/80+macrophages in spleen were reduced with no significantly statistical differences(P>0.05).Conclusion:The therapeutic mechanisms of CD52 mAb treatment in IL-10 KO mice may be associated with decreased lymphocyte homing to lamina propria.Furthermore,inhibition of F4/80+macrophages differentiating into CD11b+F4/80+macrophages may be another crucial mechanism of CD52 mAb treatment.Part 2CD52 mAb ameliorates intestinal epithelial barrier function in interleukin-10 knockout mice with spontaneous chronic colitisObjectives:The aim of this study was to investigate the therapeutic effect of CD52 mAb on epithelial barrier function in animal models of CD.Methods:Interleukin-10 knockout mice(IL-10 KO)of 16 weeks on a C57BL/6 background were divided into IL-10 KO group and CD52 mAb group,and C57BL/6 background wild-type mice were divided into WT group.The IL-10-/-mice in the CD52 mAb group received anti-CD52mAb(20 ug diluted in PBS)treatment once a week for 2 weeks,whereas the mice in WT and IL-10 KO groups received the same volume of vehicle(IgG).Severity of colitis,epithelial expression of tight junction proteins,morphology of tight junctions,TNF-?/TNFR2 mRNA expression,myosin light chain kinase(MLCK)expression and activity,as well as epithelial apoptosis in proximal colon were measured at the end of the experiment.Results:CD52 mAb treatment effectively attenuated colitis.After CD52 mAb treatment,attenuation of colonic permeability(P<0.05),increased epithelial expression(P<0.05)and correct localization of tight junction proteins(occludin and zona occludens protein-1),as well as ameliorated tight junction morphology were observed in IL-10 KO mice.CD52 mAb treatment also effectively suppressed the epithelial apoptosis(P<0.05),mucosa TNF-? mRNA expression(P<0.05),epithelial expression of long MLCK,TNFR2 and phosphorylation of MLC(P<0.05).Conclusion:Our results indicated that anti-CD52 therapy may inhibit TNF-?/TNFR2-mediated epithelial apoptosis and MLCK-dependent tight junction permeability by depleting activated T cells in the gut mucosa.