ICOS介导Tfh活化对自发干燥综合征小鼠模型唾液腺致病的作用及机制研究

Primary Sjögren’s syndrome (pSS) is a chronic autoimmune inflammatory disorder that primarily affects exocrine glands leading to their functional impairment. The costimulatory molecule ICOS on follicular help T cells (Tfh) mediates Tfh/B cell interactions and plays a pivotal role in pathological damages in salivary glands. Thus, interventional measures targeting Tfh and B cells are promising therapeutical options for future pSS treatment. However, the effects of ICOS blockade in Tfh/B cell activation and targeting glands protection in pSS has not been well clarified. In this study, we would apply blockade strategy to the spontaneous non-obese diabetic mouse model via in vivo intraperitoneal administration of anti-ICOS neutralizing antibody. The frequencies of Tfh/B cells in salivary glands would be detected through flow cytometry, while the functional alternations would be evaluated through ELISA analysis for inflammatory cytokines. Expressions of PI3K/AKT kinases and transcription factors FOXO1/3 and Bcl-6 would be detected by real-time quantitative PCR and western blotting, with tissue damages in targeted glands detected via immunohistochemical staining techniques, to determine the protective effects of ICOS blockade in pSS models. Meanwhile, using previously established in vitro Tfh/B cell co-culture system, by inducing ICOS knockdown via siRNA techniques, the mRNA and protein expression levels of downstream molecules including PI3K/AKT kinases and transcription factors FOXO1/3 and Bcl-6 would be detected, for a clarification of underlying molecular mechanisms involved in ICOS blockade mediated protection against salivary glands in pSS. Through this research, a further knowledge of ICOS-mediated signals in salivary glands damage would provide novel insights into future development of targeted therapeutical strategies for pSS

靶向干预Tfh及其活化的B细胞是前景看好的pSS治疗策略。ICOS是Tfh表面介导与B细胞相互作用的重要共刺激分子，其对pSS唾液腺致病的影响和机制尚未明晰。本研究采用自发型pSS小鼠模型，采取体内ICOS抗体阻断策略，通过FACS、ELISA等方法检测Tfh/B细胞数量和功能，唾液流率评价下颌下腺功能，免疫组化和免疫荧光方法检测靶腺体结构损伤，考察ICOS阻断对pSS腺体保护作用。并应用前期建立的Tfh/B细胞体外共培养体系，采用RNA干扰阻断ICOS表达，通过qRT-PCR、免疫印迹等考察ICOS下游PI3K/AKT激酶和转录因子FOXO1/3、Bcl-6的mRNA和蛋白质水平，研究ICOS介导Tfh活化诱发pSS唾液腺致病的新型分子机制。本研究将探索ICOS阻断策略对pSS腺体保护作用和免疫生物学机制，为靶向治疗提供实验依据。

滤泡辅助T细胞（Tfh）能够促进生发中心（GC）B细胞成熟和抗体生成，靶向干预Tfh活化是治疗抗体介导自身免疫病的治疗策略。已知Foxo家族参与调控了效应T细胞的分化和功能，Foxo1 通过负性调控Bcl-6抑制Tfh细胞分化，而Foxo3调控Tfh细胞分化和功能的内在机制尚未见报道。本研究发现，OVA蛋白免疫后的小鼠模型中，转录因子Foxo3表达明显上调，Foxo3基因敲除鼠中Tfh细胞和GC B细胞比例，以及IL-21和OVA特异性抗体水平明显降低。进而应用骨髓嵌合体小鼠模型，以及过继转移Rag-/-小鼠模型，发现Foxo3缺乏使OVA免疫后小鼠Tfh细胞和GC B细胞形成明显减少，IL-21分泌减少，抗体生成能力明显下降。此外，通过体外实验和共培养实验，进一步证实Foxo3缺乏抑制了体外转化Tfh细胞的比例及功能，并且阻断了ICOS诱导Tfh细胞分化的能力。应用免疫共沉淀技术，证实Foxo3通过直接结合IL-21启动子调控IL-21分泌。研究首次揭示了Foxo3对Tfh细胞分化和功能调控的内在机制，Foxo3促进了Tfh细胞分化及其诱导GC B细胞分化为浆细胞的能力，在体液免疫中发挥重要作用。靶向调控Foxo3活性对于治疗抗体介导自身免疫病有一定的临床应用前景。