B7-H4对牙龈卟啉单胞菌感染食管癌小鼠免疫应答的调控作用

Mounting evidence suggests a causal relationship between Porphyromonas gingivalis (P. gingivalis, Pg) infection and the development of systemic diseases. Elucidation of the mechanisms by which P. gingivalis subvert the host immune response in ways that promote its colonization would facilitate the rational design of therapeutic interventions for immune clearance. B7-homolog 4 (B7-H4), a most recent addition to the B7 family, has been described to exert co-stimulatory and immune regulatory functions. However, the mechanisms responsible for B7-H4 enable cancer cells to escape local immune responses through interactions with the surrounding host community are currently poorly understood. Our findings for the first time establish an association between infection with P.gingivalis and the progression of ESCC, and have recently provided in vitro evidence that, expression of B7-H4, on ESCC cells after P.gingivalis exposure promotes development of CD4+CD25highFoxP3+regulatory T cells. In addition, the mucosal presence of B7-H4 in individuals with P. gingivalis infections in ESCC patients correlated with the infiltration of CD4+CD25highFoxP3+ cells. In this regard, it is logical to hypothesize that: the expression of B7-H4, on ESCC cells after P.gingivalis exposure interferes with host immunity and thereby facilitating the persistence of P. gingivalis infections and the promotion of ESCC progression. To prove this hypothesis, this project is to establish a model of long-term infection with P. gingivalis in a mouse model of human esophageal cancer and to investigate histopathological changes of esophageal mucosa during infection, explore the immunophenotype and functional status of each sub-population by flow cytometry analysis. In addition, we plan to compare the differences of mucosal Pg colonization by a quantitative bacterial culture and serum levels of cytokines and Pg-specific immunoglobulin by an enzyme-linked immunosorbent assay (ELISA) between mice with or without B7-H4 neutralizing antibodies after inoculation. In this proposal, we are going to provide insight into the mechanisms by which P.gingivalis evade host immunity and the experimental evidences for revealing the exact pathogenesis of Pg, and provide new possibilities for the tumor immune intervention strategy in ESCC.

牙龈卟啉单胞菌(Pg)可参与全身系统性疾病进程。探索Pg逃逸机制对其免疫清除有重要意义。B7-H4是新近发现的免疫检查点，然其如何与微环境中的其他信号形成网络参与免疫应答尚未明确。课题组前期发现Pg感染与食管癌密切相关，并从组织及细胞学水平证实B7-H4表达增加在Pg感染阳性患者中与调节性T细胞浸润程度正相关。因此设想慢性Pg感染触发B7-H4抑制性信号网络，下调局部黏膜免疫应答，Pg持续定植，协同肿瘤进展。为证明此设想，本项目拟进一步建立Pg长期感染食管癌转基因小鼠模型，从组织形态学水平研究小鼠粘膜病理学改变；运用流式细胞术精准分析各细胞群免疫表型变化及功能状态；并采用定量分离培养及ELISA对比模型中B7-H4单抗治疗前后食管粘膜细菌载量、Pg特异性抗体及炎症因子滴度水平变化。以期探明Pg躲避宿主免疫应答机制，为进一步研究Pg病理机制提供理论支持；为探索食管癌免疫干预治疗提供应用指导。

病原体能够劫持免疫防御系统机制，从而为感染部位高度突变细胞的癌变转化的发生提供了一个容许环境。免疫检查点导向的免疫治疗已表现出良好的前景。同样，免疫逃逸表型的表观遗传重编程以协同方式激活免疫系统可以改善免疫治疗效果。这些崭新的学术进展为表观遗传学和免疫检查点靶向的结合提供了重要启示及理论依据。我们团队先前报道已证实，从食管鳞状细胞癌中分离到的牙龈卟啉单胞菌是与这种致死性肿瘤密切相关的主要病原体。在本研究中，我们将继续探索牙龈卟啉单胞菌感染过程中宿主免疫应答的相关机制，并证明牙龈卟啉单胞菌通过上调食管癌细胞表面免疫检查点分子B7-H4和赖氨酸去甲基化酶5B的表达而削弱宿主的抗感染及抗肿瘤免疫。我们的上述研究发现，为联合使用B7-H4中和抗体及组蛋白去甲基化酶抑制剂调节慢性感染模型中的抗感染免疫及增强小鼠PDXs模型中抗肿瘤免疫奠定了重要理论基础。在我们采用的三个不同的前临床小鼠模型中，即口腔细菌灌注食管慢性感染模型、负荷PDXs人源化小鼠模型及T淋巴细胞过继治疗NSG小鼠模型中，我们发现，给予长时程、同步方案的双靶应用的小鼠模型对牙龈卟啉单胞菌感染和肿瘤负荷均具有较强的免疫应答。这是通过在肿瘤微环境中产生T细胞介导的免疫应答反应和形成免疫记忆来实现的。在食管癌患者肿瘤组织中，我们进一步发现，B7-H4和KDM5B的共同表达与细菌负荷的相关性比单独表达两分子更为显著。这些结果突出了牙龈卟啉单胞菌逃避免疫的独特能力，并探索了潜在的治疗靶点用于改善牙龈卟啉单胞菌感染的控制及与感染密切相关的恶性肿瘤的进展。