Establishment And Characterization Of A Novel Mouse Model Of Systemic Sclerosis Induced By Immunization With AT1R Peptide

Objective:Systemic scleroderma（SSc）is an autoimmune disease characterized by microvascular injury,autoimmunity,vasculopathy and fibrosis in multiple organ,with a largely unknown pathogenesis.It has been shown that autoantibodies against angiotensin type Ⅱ receptor（AT1R）and endothelin type Ⅰ A receptor（ETAR）are significantly increased in patients with SSc,and these autoantibodies are associated with clinical manifestations and mortality of the disease.Recently,our group established novel mouse models for SSc by immunizing mice with mice with human AT1R or ETAR protein.After the immunization,C57BL/6 mice develop some SSc-like clinical symptoms,including skin and lung inflammation as well as skin fibrosis.The aim of this study is two folds.On the one hand,we determined whether it is possible to generate SSc mouse model by immunizing AT1R peptide which contains a T cells epitope,aiming to investigate the role of cellular autoimmunity against AT1R in pathogenesis of the disease.On the other hand,we aimed to investigate the role of CD4+T cells and CXCR-1 in the development of disease in the mouse model of SSc.Methods:To predict T cell epitopes,NetMHCIIpan 3.2 software was used to analyze the protein sequence of AT1R and ETAR to search for peptides that could be bound by MHC Ⅱ molecules of C57BL/6 strain.To determine the pathogenicity of the T cell epitope,C57BL/6 mice were immunized with the AT1R₁49 peptide which contains a strong T cell epitope emulsified in CFA via subcutaneous（s.c.）injection or footpad（f.p.）injection and boosted two weeks later with the peptide emulsified in IFA.Three weeks after the immunization,mice were sacrificed and tissues including the lung,skin,kidney and heart were collected and further evaluated.To optimize the AT1R peptideinduced mouse model,repetitive immunization（four times with an interval of 2 weeks）with the AT1R₁49 peptide emulsified in CFA was performed.Eight weeks after the first immunization,mice were sacrificed and blood and tissues were collected for evaluation.Finally,to determine the role of CD4+T cells and CXCR1 in the pathogenesis of the mouse model for SSc,we immunized CD4+T cell-deficient mice and CXCR-1 knockout mice as well as their controls with the AT1R₁49 peptide.Eight weeks after the first immunization,mice were sacrificed and the lung samples were collected and analyzed.Results:The NetMHCIIpan 3.2 predicted that AT1R contain a strong T cell epitope at the amino acid residue of 149,and the corresponding position of ETAR also contains a strong T cell epitope.Therefore,we synthesized the AT1R₁49 peptide and utilized it for the immunization.In vivo results showed that immunization with the AT1R₁49 peptide via f.p.injection induced inflammation in the lung but not other organs of C57BL/6 mice,while control immunization did not.In addition,repetitive immunization with the AT1R₁49 peptide induced stable and severe pulmonary inflammation in C57BL/6 mice,providing an optimized AT1R peptide-induced mouse model for SSc.Moreover,the analysis of the serum antibodies revealed that both mice immunized with the AT1R₁49 peptide and the control mice dose not produce specific antibodies against AT1R,indicating that immunization with the AT1R₁49 peptide did not induce antibody responses to AT1R protein.Furthermore,immunization with the AT1R₁49 peptide induced strong pulmonary inflammation in wildtype C56BL/7 mice but not in CD4+T cell-deficient mice.As compared to wildtype C57BL/6 mice,CXCR1 knockout mice developed a significantly less severe inflammation in the lung after the immunization with AT1R₁49 peptide.Conclusion:In conclusion,immunization with the AT1R₁49 peptide is able to induce pulmonary inflammation in mice,suggesting a role of T cell responses against AT1R in the pathogenesis of SSc.Furthermore,both CD4+T cell and CXCR1 play an essential role in the AT1R peptide-induced mouse model for SSc.