在脓毒症中"整合素β3-CD14通路"正向调节巨噬细胞TLRs功能的机制研究

A new way to regulate Toll-like receptors (TLRs) activation effectively will advance sepsie treatment. Recently, we found that TLR4 activation up-regulated integrinβ3 expression in peritoneal macrophages. Integrinβ3-/- alleviated the severity of organ injury and repressed the elevation of CD14.Integrin inhibitor RGDs pretreatment improved survival rate in septic mice. Thus, we hypothesize that “Integrinβ3-CD14 pathway” might modulate TLRs activation (the project will focus on TLR2、3、4) of macrophages positively in septic mice. In the current study: first, we will observe the protective effect of integrinβ3 inhibtion in tissue injury in cecal ligation and puncture induced sepsis and the modulated effect on CD14 expression. Second, we will verify the effect of CD14 in macrophages during sepsis which will be measured using the flow cytometry analysis after deletion or retransfusion of macrophage. Third, we will investigate the mechanism of integrinβ3 on regulation of CD14 through three aspects : membrane receptors,adaptor proteins and downstream signaling proteins by mice with related gene knocked out and by precondition of indicated protein inhibitor, respectively. Last, we will elucidate the effect of “Integrinβ3-CD14 pathway”regulates TLRs activation positively by blocking/restoring the pathway. Taken together, our aims are to verify the protective effect of integrinβ3 inhibition and to elucidate the mechanism of CD14 expression in sepsis. The results of this study might contribute to the immunoloregulation treatment of sepsis through blocking “Integrinβ3-CD14 positive feedback loop”.

调控TLRs激活的新方法将推动脓毒症治疗。我们发现:巨噬细胞TLR4通路的激活上调整合素β3表达；β3-/-减轻脓毒症小鼠炎症反应并抑制CD14表达；整合素抑制剂RGDs预处理可提高脓毒症小鼠生存率。因而 “整合素β3-CD14”通路可能存在，并通过正向调节TLRs功能（本项目以TLR2、3、4为例）在脓毒症中发挥重要作用。本研究拟通过：1）采用CLP脓毒症模型，明确抑制β3对器官功能保护作用及对CD14表达影响；2）采用巨噬细胞耗竭与补偿实验（包括CD14-/-）和流式细胞仪分析技术，明确巨噬细胞上CD14在脓毒症中的重要作用；3）采用基因敲除小鼠的巨噬细胞，从膜受体、衔接蛋白、下游信号通路蛋白三个层面，研究β3调控CD14表达的机制；4）采用阻断/恢复“整合素β3-CD14”通路，明确该通路对TLRs炎症激活的正向作用。本项目拟补充与完善TLRs激活传统途径，为脓毒症诊治提供新支点。

近来研究发现整合素β3参与 TLR 通路的激活。我们前期研究发现整合素抑制剂预处理减轻小鼠脓毒症引起的肺损伤，提高小鼠生存率。但整合素β3与 TLRs激活引起的炎症反应之间的相互作用及机制目前尚不清楚。本研究通过小鼠脓毒症在体模型与 TLR 激动剂刺激的巨噬细胞体外模型，从细胞膜、衔接蛋白及下游信号通路水平研究整合素β3与 CD14 通路激活在脓毒症炎症反应中的作用机制。通过研究发现在小鼠脓毒症模型中，整合素β3 -/-抑制TLR激活引起的细胞因子分泌，减轻脓毒症引起的ALT/AST、Cre/BUN、LDH，抑制组织中CD14表达增加，CO-IP检测发现整合素β3与TLR4存在相互作用。LPS刺激整合素β3敲除巨噬细胞，炎症因子分泌明显减少，CD14表达降低；补充rCD14,炎症因子分泌增加。Myd88-/-小鼠抑制TLRs激活引起的细胞因子分泌，减轻脓毒症引起的组织损伤，抑制组织中CD14表达增加，但TRIF-/-小鼠与野生型小鼠在细胞因子分泌、组织损伤程度及CD14表达方面无明显差异。另外LPS 刺激野生型小鼠 PMø后，p-ERK、p-JNK、 p-p38MAPK 水平明显增加，具有时间依赖性。整合素β3敲除后，p-ERK、 p-JNK、 p-p38MAPK水平在 30min 和 60min 时明显降低。U0126、 SP600125、 SB203580、BMS-345541、QNZ 预处理野生型小鼠 PMø后， LPS 诱导 CD14 的表达明显降低。我们认为：LPS 刺激整合素β3与 TLR4 形成复合物招募 MyD88 依赖型途径 TRIF 非依赖型衔接蛋白，激活 MAPK 和 NF-κB 通路参与 CD14 表达。CD14 可进一步与 TLR4 结合，增强TLR4 引起的炎症反应，形成正反馈环路，补充了经典 TLR 通路的激活途径，深化脓毒症机制研究。干预“整合素β3 -CD14”通路，可减轻炎症反应和组织损伤为脓毒症治疗提供新的靶点。