NKT细胞及其LIGHT信号调节H7N9感染的肺损伤机制研究

A novel reassortant avian-origin influenza A virus of the H7N9 subtype caused severe viral pneumonia and fatal acute respiratory distress syndrome in humans, and leading to 118 death of 368 infected humans to date. Similar to other avian influenza viruses, H7N9 induces much more immunological activity than is necessary to clear the virus and it is the cytokine storm that cause most of the damage in the lung. The mechanisms of such immunopathogenesis are still remain poorly understood. Our previous studies showed that αβTCR expressing but not CD4+ or CD8+ T cells, which are NKT cells, are involved in the immunopathology. In addition, LIGHT-/- mice were protected from H7N9 induced death, suggesting a potential involvement of LIGHT in inflammation and pathogenesis in H7N9 infection. Thus, we hypothesized that NKT cells, which recruited to lung after H7N9 infection and activated with increased expression of LIGHT, promote pulmonary immune injury via promoting cytokine storm. The interaction between the LIGHT, which expressed on NKT cells, and LTβR and HVEM expressed on monocytes/macrophages, neutrophils and NK cells might promote excessive activation and proinflammatory cytokines production of these cells, increased NETs generation by neutrophils and enhanced cytotoxic effect of NK cells, and then cause deleterious pulmonary immunopathology. LIGHT expressed on NKT cell might also activate receptor LTβR expressed on airway epithelial cell, resulting in lung injury directly. This project is expected to clarify the mechanism that NKT cell promote immunopathology via LIGHT in H7N9 infection mouse model, and to seek new methods to intervene immune injury caused by novel avian-origin influenza A virus.

新发甲型禽流感病毒H7N9感染导致病毒性肺炎和呼吸窘迫综合症，致死率高，目前尚无有效药物和疫苗。与其他甲型禽流感病毒相似，H7N9感染引起的细胞因子风暴是导致呼吸道免疫病理损伤和急性呼吸窘迫综合症的主要致病原因，表现为炎性因子大量产生和炎性细胞大量浸润，但分子与细胞机制仍不详。TNF超家族分子LIGHT可通过其受体（HVEM和LTβR）激活下游STAT3和NF-κB，启动和促进炎性因子产生，参与多种疾病的炎症反应。NKT细胞是LIGHT的重要来源。本申请前期研究发现H7N9感染引起的炎性反应依赖NKT细胞和LIGHT的存在。本申请拟进一步研究H7N9感染中NKT细胞通过LIGHT促进细胞因子风暴的作用和机制，揭示LIGHT作用的细胞类型(表达HVEM和LTβR)、相关炎性病理反应特征，阐明H7N9感染所致肺损伤的分子机制，并为探寻针对新发甲型流感病毒的免疫干预策略提供实验依据。

严重的甲型流感病毒（influenza A virus，IAV）感染常常会导致急性呼吸窘迫综合症（acute respiratory distress syndrome，ARDS），这也是甲流引起严重后果甚至病人死亡的重要原因。其中，除了病毒本身的复制之外，由IAV引起机体产生的过激炎症反应也是甲流致病的非常重要原因，但其中的分子和细胞学机制仍然有待研究。本项目通过生物信息学分析、RT-PCR以及ELISA等多种方法证明，TNF超家族分子LIGHT（TNFSF14）在高致病性IAV感染的肺组织中表达显著升高，且其表达水平与肺炎的严重程度呈显著的正相关。在IAV感染小鼠的模型发现，与野生型小鼠（WT）相比，LIGHT缺陷小鼠（LIGHT-/-）表现出更低的发病率、严重程度以及死亡率，且肺组织的损伤程度也明显降低。此外，本项目发现，IAV感染可引起肺泡巨噬细胞（alveolar macrophages，AM）的大量死亡，LIGHT的缺陷则显著提高AM的存活。通过机制性的研究我们发现，LIGHT引起死亡的AM为 LTbR+细胞而非HVEM+细胞，同时体内给予WT小鼠LTbR刺激性抗体或是HVEM封闭性抗体都会加重IAV感染引起的疾病进展，证明LIGHT可通过LTbR介导AM的死亡而引起IAV感染后的疾病加重。最后，我们发现在小鼠体内，NKT细胞是LIGHT的主要来源之一，通过过继转输NKT细胞证明NKT细胞是IAV感染引起的肺炎的充分必要条件。综上，本项目证明了NKT细胞来源的LIGHT通过引起组织保护性的AM的死亡，从而加重肺组织的损伤和疾病的进展。后续我们将会研究通过封闭LIGHT的相关信号通路是否能治疗流感相关的呼吸窘迫综合症。