摘要
目的尿素酶B(UreB)是幽门螺杆菌疫苗设计的重点候选抗原,本研究拟探究UreB对巨噬细胞的免疫调控作用及潜在机制。方法用重组UreB蛋白刺激小鼠骨髓来源M0型巨噬细胞,经流式细胞术和ELISA检测巨噬细胞凋亡、极化和抗原提呈分子表达;共培养试验、CFSE和流式细胞术检测CD4+T细胞增殖及IFN-γ表达水平。构建UreB截短体,NanoBiT和免疫共沉淀检测UreB与TLR2结合表位。结果UreB可诱导巨噬细胞凋亡并逆转脂多糖诱导的巨噬细胞M1型极化,促进巨噬细胞向M2型极化。同时,UreB也能抑制巨噬细胞抗原提呈分子MHCⅡ和CD86的表达,并进而抑制CD4+T细胞增殖和IFN-γ表达。分子机制研究表明UreB主要依赖于C端7个氨基酸残基与TLR2结合发挥以上调控作用。结论本研究证实UreB可通过C端7个氨基酸残基与TLR2结合发挥免疫负调控功能,有助于以UreB为基础的幽门螺杆菌疫苗设计及优化。
Objective To evaluate the regulatory role and potential mechanism of Urease B(UreB)on macrophages.Methods Bone marrow-derived macrophages(M0)were stimulated by recombinant UreB protein and then flowcytometry and ELISA were used to detect the apoptosis,polarization and antigen presentation-related biomarkers expression.CD4+T cell co-culture assay,CFSE stain and flowcytometry were used to evaluate the impacts of UreB on antigen presentation capacity of macrophages.Truncated UreB protein,NanoBiT assay and co-immunoprecipitation were used to identify the binding sites of UreB to TLR2.Results UreB promoted apoptosis and skewed macrophages from M1 to M2 in the presence of M1-inducer LPS.Moreover,UreB inhibited the expression of antigen presentation biomarkers,MHCⅡand CD86 on macrophages,and further inhibited the proliferation and IFN-γexpression of CD4+T cells.Molecular analyses revealed that the binding between seven carboxy-terminal amino acid residues of UreB and TLR2 were required for the UreB-mediated inhibitory effects.Conclusions The findings in this study demonstrate that UreB mainly depends on the binding between seven carboxy-terminal amino acid residues and TLR2 to perform immune-suppressive activities,and which may provide valuable information for the design and optimization of UreB-based vaccines against Helicobacter pylori infection.
作者
李智超
沈芯
袁纯辉
王军
向贇
蔡秦真
Li Zhichao;Shen Xin;Yuan Chunhui;Wang Jun;Xiang Yun;Cai Qinzhen(Department of Rheumatism Immunology,Wuhan Children′s Hospital,Huazhong University of Science and Technology,Wuhan 430016,China;Department of Laboratory Medicine,Wuhan Children′s Hospital,Huazhong University of Science and Technology,Wuhan 430016,China)
出处
《中华微生物学和免疫学杂志》
CAS
CSCD
北大核心
2021年第7期507-515,共9页
Chinese Journal of Microbiology and Immunology
基金
湖北省自然科学基金(2018CFB164)
武汉市科技局"黄鹤英才计划"项目(武人才办[2017]2号)。