脓毒症免疫抑制期单核巨噬细胞Tim4表达上调对Nalp3炎性体活化的影响

During the stage of sepsis-induced immunosuppression, the ability of monocyte/macrophage to produce cytokines was suppressed. Our preliminary study demonstrated that Tim-4 ,a newly identified type I membrane protein, was upregualted on monocyte/macrophage in septic patients. The expression of Tim-4 was associated with the severity of sepsis-induced immunosuppression. In vitro Tim-4 blockade can restore proinflammatory cytokines release from monocyte/macrophage.The Nod-like receptor (NLR) family of innate immune cell sensors, such as Nalp3 inflammasome, are implicted in recognizing certain antigen in sepsis. Nalp3 inflammasome is a key molecule in monocyte/macrophage to modulate cytokine synthesis, leading to caspase-1 activation and subsequent release of mature IL-1β and IL-18. We hypothesize that Nalp3 inflammasome in monocyte/macrophage was invoved in Tim-4 mediated immune and inflammatory response during sepsis. In current research, we will determine the association among Tim-4 expression and Nalp3 inflammasome in monocyte/macrophage as well as the severity of sepsis-induced immunosuppression in septic patients. The role of Tim-4 blockade on Nalp3 inflammasome activation and subsquent cytokine release will be determined in vitro.The effect of different level of Tim-4 on Nalp3 inflammasome and underlying mechanisms will be investigated. In animal studies, the effect of Tim-4 upregulation or blockade on activation of Nalp3, organ injury, and survival rate will be determined. Our research will provide new insight into the mechanisms of sepsis, which will be helful for the prevention and therapy of sepsis in future.

在脓毒症免疫抑制期，单核巨噬细胞分泌细胞因子能力下降。我们前期研究证实脓毒症患者单核巨噬细胞表面新型免疫分子Tim-4表达上调并与免疫抑制状态相关，体外阻断Tim-4可恢复单核巨噬细胞分泌促炎细胞因子能力。Nalp3炎性体是单核巨噬细胞分泌细胞因子的关键分子。我们推测，Tim-4表达上调可能抑制Nalp3炎性体活化进而导致单核巨噬细胞分泌细胞因子能力下降。本研究拟首先明确脓毒症患者单核巨噬细胞Tim-4表达与炎性体活化和免疫抑制状态的相关性；观察阻断Tim-4对脓毒症患者单核巨噬细胞Nalp3炎性体和分泌细胞因子能力的影响；研究不同表达水平Tim-4对单核巨噬细胞Nalp3炎性体活化的影响并探讨其分子机制；动物模型上观察干预Tim-4对脓毒症小鼠Nalp3炎性体活化、组织损伤和生存率的影响。本项目的实施，将丰富脓毒症免疫炎症反应的机制，为探索脓毒症免疫调控新策略提供理论与实验依据。

研究背景：免疫抑制是脓毒症患者死亡的重要原因。脓毒症是严重感染时发生的全身炎症反应综合征，随着现有治疗水平的提升，脓毒症患者大多在短暂的促炎反应期后，进入持久的免疫抑制期，临床表现为继发条件致病菌感染、体温下降、白细胞数量减少等。单核巨噬细胞是脓毒症使的主要炎症反应细胞和细胞因子的来源之一，巨噬细胞通过产生IL-1β、和IL-18 等炎症因子参与脓毒症各个阶段的病情演变。 IL-1β、和IL-18的产生依赖于炎性体激活。炎性体在2002年第一次发现NLRP1炎性体以来，陆续发现NLRP3(或NALP3)、NLRC4、AIM2、RIG-1炎性体等，其中，NLRP3炎性体在最受关注，其中NLRP3分子通过接头蛋白ASC招募Caspase-1,从而组成多蛋白复合物即NLRP3炎性体。Tim4是T细胞免疫球蛋白域黏蛋白样蛋白（T-cell immunoglobulin domain and mucin domain,Tim）成员，Tim-4分子在免疫调节及维持机体稳态中具有重要的作用。主要表达在活化的抗原提呈细胞表面，能维持腹腔巨噬细胞平衡。最新研究表明，Tim4可以作为磷脂酰丝氨酸的受体，通过与凋亡细胞表面的磷脂酰丝氨酸结合，促进单核巨噬细胞或DC吞噬凋亡细胞。故脓毒症时单核巨噬细胞TIM4的表达和Nalp3炎性体活化的相关性值得深入探讨。主要研究结果：1）脓毒症免疫抑制期患者与脓毒症早期患者相比，外周巨噬细胞HLA-DR表达显著降低；同时HLA-DR表达下降的患者，巨噬细胞TIM4表达显著抑制，外周血中被释放的IL-1β 也降低。.2）在小鼠腹腔巨噬细胞系，shRNA抑制TIM4表达显著抑制Nalp3、Caspase-1、ACS 表达和细胞因子的分泌（IL-1β 表达下降，IL-18表达下降）。.3）在小鼠腹腔巨噬细胞系，shRNA抑制TIM4表达，严重抑制巨噬细胞吞噬凋亡细胞的能力。4）在shRNA抑制TIM4表达的小鼠腹腔巨噬细胞系，和C57BL/6小鼠盲肠 结扎穿孔（CLP）脓毒症模型中，P2X7R的下降，都无显著统计学差异。5）C57BL/6小鼠盲肠结扎穿孔（CLP）脓毒症模型，盲肠结扎72h后腹腔巨噬细胞表面TIM4表达增加，NALP3 和Caspase-1表达升高，外周血中IL-1β释放显著增高。ACS和IL-18改变无显著统计学差异