“WISP1与TLR4正反馈促炎通路”介导机械通气肺损伤的机制

Recent studies show Toll-like receptors (TLR) in innate immunity play an important role in Ventilator-induced lung injury (VILI). Our previous work had showed TLR4/MyD88 pathway contributed to VILI. We recently using Genome-wide association study (GWAS) with 23 inbred mouse strains in VILI model identified that WNT1 inducible signaling pathway protein 1 (Wisp1) gene as one of significant single nucleotide polymorphisms associated with VILI. We verified the synergistic effect between extracelluar matrix protein WISP1 and TLR signaling in macrophages. Further, we found TLR4 signaling pathway is involved in the production and effect of WISP1. The current study focuses on the role of " WISP1-TLR4 Positive feedback Loop" in VILI: First, we will observe the differences of WISP1 and TLR expression in mechanical ventilation model using VILI-sensitive and resistent mice. Second, we will verify the relationship between the production and effect of WISP1 and TLR4 associated pathway (including CD14, TLR4, MyD88 and TRIF) in vivo and in vitro. Third, we need to further confirm the interplay between WISP1 and TLR by co-immunoprecipitation, TLR transfection test in HEK293 cells and confocal microscopy. Last, we use macrophage-TLR4 knock out mice and WISP1 protein inhalation to clarify the mechanism of WISP1 in VILI. The project will help understand and explore the novel treatment for acute lung injury through blocking the " WISP1-TLR4 Positive feedback Loop" .

Toll样受体（TLR）在机械通气肺损伤（VILI）中发挥重要作用。我们前期研究证明了VILI的产生有赖于TLR4/MyD88通路，新近通过全基因组关联分析发现WISP1是VILI的易感基因。在巨噬细胞中我们证明：一方面WISP1能显著放大TLR通路的促炎效应，另一方面TLR4介导了WISP1的表达/作用。因此本研究聚焦"WISP1-TLR4正反馈促炎通路"：首先比较VILI敏感鼠和耐受鼠机械通气后WISP1与TLRx表达水平差别；其次从动物和细胞水平证明WISP1表达/作用与TLR4及相关（CD14、MyD88或TRIF）通路的关系；再采用免疫共沉淀、293细胞TLR转染测试和激光共聚焦筛选和证实WISP1与TLRx的分子作用机制。最后使用巨噬细胞TLR4基因敲除小鼠和WISP1干预实验，阐明WISP1在VILI中作用途径。本研究有利于理解和开发通过阻断"正反馈"环节的肺损伤防治新策略。

研究背景：.Toll 样受体（TLR）在机械通气肺损伤（VILI）中发挥重要作用。我们前期通过全基因组关联分析发现 WISP1 是VILI 的易感基因。在巨噬细胞中WISP1放大TLR激动剂引起的炎症反应。因此本研究聚焦“WISP1-TLR4 正反馈促炎通路”的作用机制研究。.研究方法：.1）比较VILI对不同敏感株小鼠肺组织中 WISP1与 TLR表达差异以及与TLR4之间的相互作用。2）通过分离野生型小鼠及Toll相关基因敲除小鼠腹腔巨噬细胞，给予WISP1与TLR激动剂刺激，检测WISP1放大TLR激动剂引起的炎症反应的机制。3）采用293细胞过表达的方法通过免疫共沉淀检测WISP1与TLR4的相互作用。4）采用野生型与LyzTLR4-/-小鼠检测VILI后WISP1及炎症因子分泌。气管内给予重组WISP1蛋白后观察小鼠肺损伤。.研究结果：.1）VILI后A/J小鼠肺组织中WISP1，TLR4表达明显增加，C57BL/6小鼠也明显增加，CBA/J小鼠无明显增加。各品系小鼠肺组织中TLR3表达均无明显改变。免疫共沉淀结果显示VILI后WISP1与TLR4结合增加。 2）在小鼠原代腹腔巨噬细胞中，WISP1通过ERK通路的激活放大TLR激动剂LPS、PolyI:C引起的TNF-α分泌。WISP1的协同作用依赖于TLR4通路的完整性。3）293细胞过表达TLR4和WISP1后，LPS刺激，TLR4和WISP1相互作用增加。4）VILI后FloxTLR4小鼠炎症反应增加，肺组织损伤，组织中WISP1的表达明显升高，而LyzTLR4-/-小鼠肺损伤明显减轻，组织中WISP1表达降低。给予外源性重组WISP1加重LyzTLR4-/-小鼠肺损伤。.结论：.巨噬细胞TLR4信号通路激活参与上调WISP1表达，WISP1通过与细胞表面TLR4结合增强ERK通路的激活，继而放大TLR激活引起的炎症因子分泌，形成正反馈。本研究有利于理解和开发通过阻断“正反馈”环节的肺损伤防治新策略。