免疫检查点蛋白VISTA调控FcγRⅡb抑制牙周破骨细胞分化的作用和机制

The abnormal differentiation of osteoclasts in inflammatory microenvironment is an important pathological basis for the occurrence and development of periodontitis. Previously, our team found that VISTA can inhibit the differentiation of hematopoietic progenitor cells into macrophages, but whether VISTA mediates the abnormal differentiation of osteoclasts caused by inflammation is still unknown. Preliminary results showed that the absence of VISTA signal promoted the osteoclast differentiation of macrophages, and the expression of immune complex receptor protein FcγRⅡb, which inhibited osteoclast differentiation, decreased on the surface of osteoclast precursors. Therefore, we propose a scientific hypothesis that pathogenic bacteria in periodontitis microenvironment can inhibit the expression of VISTA in osteoclast precursors, thereby reducing the inhibitory effect of FcγRⅡb on osteoclast differentiation. In this study, we will analyze the direct regulation of VISTA by epigenetics of periodontal pathogens and the indirect regulation of ligand VSIG-3 to clarify the reasons for the decline of VISTA expression in microenvironment, and elucidate the new function of VISTA in regulating the differentiation of osteoclasts by means of VISTA, FcγRⅡb gene knockout mice, in vivo and in vitro intervention. This project is expected to propose a new molecular mechanism for increasing the number of osteoclasts in periodontitis, and provide a new idea for clinical use of immunotherapy in the treatment of periodontitis.

炎症微环境中破骨细胞的分化异常是牙周炎发生发展的重要病理基础。课题组前期发现免疫检查点蛋白VISTA能抑制造血祖细胞向巨噬细胞分化，但VISTA是否介导了炎症导致的破骨细胞分化异常仍然未知。预实验结果显示VISTA信号缺失促进巨噬细胞破骨向分化；且破骨细胞前体表面具有抑制破骨向分化功能的免疫复合物受体蛋白FcγRⅡb表达下降。由此我们提出科学假说：牙周炎微环境中致病菌能够抑制破骨细胞前体表达VISTA，进而降低FcγRⅡb对破骨细胞分化的抑制作用。本课题拟对VISTA受到牙周致病菌表观遗传学的直接调控和配体VSIG-3的间接调控两方面进行分析，明确微环境中VISTA表达下降的原因；并利用VISTA、FcγRⅡb基因敲除鼠，体内外干预等多种手段阐明VISTA调控FcγRⅡb抑制破骨细胞分化的新功能。本项目有望提出牙周炎破骨细胞分化异常的分子新机制，为临床利用免疫疗法治疗牙周炎提供新的思路。

在以往的研究中，新型免疫共抑制分子VISTA因其抑制T细胞活化的特点在获得性免疫相关的研究中被广泛研究。而其在天然免疫过程中是否存在具体的功能尚未明确。前期研究结果显示，VISTA在破骨细胞的前体细胞表面高表达，且Vsir-/-小鼠长骨骨量较野生型小鼠显著下降。于是本项目针对这一表型，首先在小鼠体内体外对VISTA基因缺失而导致的破骨细胞的分化，功能异常进行了全面的分析，结果发现VISTA基因缺失主要影响了破骨细胞前体向破骨细胞分化的能力。为了进一步明确其作用机制，通过对破骨分化相关信号通路的删选，发现VISTA信号缺失显著影响破骨细胞分化过程中经典NF-κB信号的传递。进一步通过高通量基因删选后发现VISTA能够与NF-κB信号传递过程中的STAT3基因相结合，影响该蛋白Ser705的磷酸化。利用VISTA激动抗体能够显著缓解绝经后骨质疏松模型和破骨细胞异常激活模式动物的骨量下降现象，并利用单细胞测序技术进一步证明VISTA对STAT3-NF-κB介导的破骨细胞分化过程的影响。本项目通过完整的实验从多角度对VISTA结合STAT3影响NF-κB信号传导的机制进行了阐述，并对使用VISTA激动抗体作为治疗骨质疏松的临床应用提出了理论依据。在接下来的研究中，我们将继续探究VISTA配体在破骨细胞异常分化过程中是否发挥作用，为进一步阐明VISTA在天然免疫细胞中的生物学功能提供理论支持。