The Mechanism Of Phosphatase Wip1 In Regulating Septic Peritonitis And The Exploration In The Induction Of Transplantation Tolerance

Part ⅠThe mechanism of phosphatase Wipl in regulating septic peritonitisObjective:To explore the cellular and molecular mechanism of phosphatase Wip1 in the reulation of septic peritonitis.Methods:Through establishing cecal ligation and puncture model,survival rate and outcome was assessed between wild type and Wipl knock out mice.With the depletion strategy and adoptive transfer experiment,the importance of neutrophil in Wipl regulating septic peritonitis outcome was explored.With the use of flow cytometry and other experiments,the molecular mechanism of Wipl in regulating neutrophil chemotaxis was further identified.Results:Wip1 deficiency led to a better outcome with an increased survival rate,less bacterial burden in the peritoneal cavity and peripheral blood,and increased infiltration of neutrophils in the peritoneal cavity.Depletion of neutrophils reversed the protective function induced by Wip1 deficiency,indicating the critical role of neutrophils in Wipl deficiency mediating better outcome.Further mechanism studies showed Wipl deficiency prevented the internalization of CXCR2 on neutrophils and increased the anti-bacterial activity of neutrophils.Pharmacological inhibition of Wipl also prevented the downregulation of CXCR2 on human neutrophils under the stimulation of LPS.Conclusions:Phosphatase Wipl regulates septic peritonitis outcome through manipulating neutrophil chemotaxis and anti-bacterial function,and inhibition of Wipl might be a promising therapeutic strategy for septic peritonitis in the future.Part ⅡRegulatory T cells promote the formation of mixed chimera and induce immune tolerance for small bowel trasnlantationObjective:To explore the strategy for inducing small bowel transplantation tolerance based on manipulating regulatory T cells(Treg).Methods:Antigen-specific Tregs were obtained from the spleen of tolerant recipient mice which received heart transplantation,and were further expanded with the use of IL-2,rapamycin,and dendritic cells in vitro.The immunosuppressive function and amplification efficiency of expanded Tregs were examined.Adoptive trasnfer of these expanded antigen-specific Tregs or normal Treg isolated from normal mice,combined with rapamycin,CTLA-4 blocker,and donor bone marrow cell transfusion,was used to establish mixed chimeras.Small bowel transplantation was further performed in these mixed chimeras.Allograft survival rate was assessed and the mechanism accounting for immune tolerance was explored.Results:After in vitro expansion,the immunosuppressive function of antigen-specific Tregs was significantly enhanced and can induce the formation of mixed chimeras stably.In addition,the induced mixed chimeras could significantly prolong small bowel allograft survival.The molecular mechanisms of the induction of tolerance included central immnne tolerance as shown by the depletion of alloreactive T cells in the thymus and increased proportion of peripheral Treg in the graft and peripheral blood.Conclusions:In vitro expanded antigen-specific Treg can potently induce the formation of mexed chimeras,and further prolong the survival of small bowel allograft,which may provide promising prospects for the induction of immune tolerance in the field of organ transplantation.PartⅢDexamethasone potentiates the differentiation and immunosuppressive function of myeloid derived suppressor cells which faciliates cardiac allograft survivalObjective:To explore the inducntion of immune tolerance through the use of myeloid derived suppressor cells(MDSC)favoured by dexamethasone.Methods:In vitro,MDSC were induced from bone marrow cells with the use of GM-CSF and different concentrations of dexamethasone.The immunosuppressive function of induced MDSCs was assessed.With the use of iNOS knock out mice and other techniques,the mechanism of immunosuppressive function of MDSCs induced by dexamethasone was analysed.Adoptive transfer of dexamethasone-induced MDSC into recipients which received cardiac allograft transplantation was performed and survival rate of allograft,as well as the mechanism of prolonging allograft survival,was examined.Results:Dexamethasone treatment promoted the differentiation of MDSC and enhanced its immunosuppressive function.The function of dexamethasone-induced MDSC was dependent on the iNOS signaling pathway.Adoptive transfer of dexamethasone-induced MDSC prolonged the survival of cardiac allograft with increased infiltration of MDSC in allograft.Upregulation of CXCR2 on dexamethasone-induced MDSC was identified which was mainly mediated through the GR receptor signaling-dependent pathway.Furthermore,dexamethasone-induced MDSC also favoured the expansion of Treg in the peripheral,which may also promote the survival of allograft survival.Conclusions:Dexamethasone-induced MDSC promote cardiac allograft survival in an iNOS-and GR receptor signalling-dependent manner,and can be a potential strategy for the induction of immune tolerance in the future.Part ⅣGroup 2 innate lymphoid cells produce VEGFA and its biological significance in mucosal immunityObjective:To explore new molecule from group 2 innate lymphoid cells(ILC2)which plays a critical role in mucosal immunity.Methods:Human peripheral blood ILC2 was sorted and further cultured with IL-33,IL-2 and IL-25 in vitro.The production of VEGFA and other VEGF family members was assessed through RT-PCR and ELISA.Mouse model was established to detect the production of VEGFA from ILC2 in the lung.With the use of VEGFA receptor inhibitor SU1498 and adoptive transfer experiments,the role of VEGFA derived from ILC2 during mucosal immunity was further examined.In addition,the expression of VEGFA receptor on ILC2 was analysed,and was further examined for its involvement in ILC2 activation and function.Results:Both human peripheral blood ILC2 and mouse lung ILC2 had the ability to express VEGFA mRNA and protein,and the expression of VEGFA in asthmatic patients was significantly up-regulated.Blockade of VEGFA pathway significantly reduced airway inflammation and airway hyperresponsiveness.ILC2 from the lung expressed VEGFA receptor 2 after activation which indicated the possibility of ILC2 responding to VEGFA stimulation produced by themselves.With the use of SU1498,we confirmed and identified VEGFA signalling was required for ILC2 activation and function during mucosal immunity.Conclusions:ILC2-derived VEGFA not only promotes the development of mucosal disease such as asthma,but also is a novel mechanism in regulating the activation and function of ILC2.