Toll样受体4接头分子对乳酸诱导巨噬细胞M2极化的促进作用及其机制

Effect of adapters of Toll-like receptor 4 on M2 polarization of macrophages induced by lactate and its mechanism

目的:探讨Toll样受体4 (TLR4)接头分子髓样分化因子88 (MyD88)和诱导β干扰素的Toll/白细胞介素1 (IL)受体(TIR)结构域衔接蛋白(TRIF)基因敲除后对乳酸诱导巨噬细胞极化的促进作用,初步阐明乳酸免疫调控的可能机制。方法:体外培养小鼠巨噬细胞Raw264.7,采用慢病毒法构建CRISPR/Cas9基因编辑载体,定向敲除MyD88和TRIF基因(MyD88-KO组和TRIF-KO组),设未感染的Raw264.7细胞为对照组,实时荧光定量PCR (RT-qPCR)法和Western blotting法检测基因敲除效果。实验分为未处理Raw264.7细胞组、Raw264.7+乳酸组、MyD88-KO组、MyD88-KO+乳酸组、TRIF-KO组和TRIF-KO+乳酸组。15 mmol·L^(-1)乳酸诱导24 h后进行各指标检测。RT-qPCR法检测各组细胞中M2极化表型分子巨噬细胞甘露糖受体CD206和精氨酸酶1(Arg1) mRNA表达水平,光学显微镜下观察各组细胞形态表现,酶联免疫吸附测定(ELISA)法检测各组细胞上清中肿瘤坏死因子α (TNF-α)、γ干扰素(INF-γ)和白细胞介素10 (IL0)水平,Western blotting法检测各组细胞中核因子κB (NF-κB)信号通路相关蛋白表达水平。采用Autodock软件将乳酸分别与TLR4、MyD88和TRIF分子对接并观察其结合情况。结果:采用CRISPR/Cas9技术进行基因定向敲除后,与对照组比较,MyD88-KO组和TRIF-KO组细胞中MyD88和TRIF mRNA及蛋白表达水平明显降低(P<0.05或P<0.01)。乳酸作用24 h后,与未处理Raw264.7细胞组比较,Raw264.7+乳酸组细胞中M2极化标志物CD206和Arg1 mRNA表达水平明显升高(P<0.05);与MyD88-KO组比较,MyD88-KO+乳酸组细胞中CD206和Arg1 mRNA表达水平均升高(P<0.05);与TRIF-KO组比较,TRIF-KO+乳酸组细胞中CD206和Arg1 mRNA表达水平均降低(P<0.05)。细胞形态表现,Raw264.7+乳酸组细胞表现出M2极化形态,即体积增大、明显突起伪足和偏锥形细胞比例增加;MyD88-KO+乳酸组细胞同样表现出M2极化形态变化;TRIF-KO+乳酸组细胞形态变化不明显。ELISA法检测,与未处理Raw264.7细胞组比较,Raw264.7+乳酸组细胞中TNF-α水平明显降低(P<0.05),INF-γ和IL0水平差异无统计学意义(P>0.05);与MyD88-KO组比较,MyD88-KO+乳酸组细胞中TNF-α水平明显降低(P<0.05),INF-γ水平差异无统计学意义(P>0.05),IL0水平明显升高(P<0.05);与TRIF-KO组比较,TRIF-KO+乳酸组细胞中TNF-α水平明显升高(P<0.05),INF-γ和IL0水平差异无统计学意义(P>0.05)。Western blotting法检测,与未处理Raw264.7组比较,Raw264.7+乳酸组、MyD88-KO组、MyD88-KO+乳酸组、TRIF-KO组和TRIF-KO+乳酸组细胞中TLR4蛋白表达水平差异均无统计学意义(P>0.05),Raw264.7+乳酸组细胞中核因子κB抑制蛋白α (IκB-α)和p65蛋白表达水平明显降低(P<0.05);与MyD88-KO组比较,MyD88-KO+乳酸组细胞中IκB-α和p65蛋白表达水平明显降低(P<0.05);与TRIF-KO组比较,TRIF-KO+乳酸组细胞中IκB-α和p65蛋白表达水平差异无统计学意义(P>0.05)。分子对接,乳酸分别与TLR4、MyD88和TRIF形成氢键数为2、4和4,结合能分别为-2.72、-3.24和-3.66。结结论论:乳酸作用巨噬细胞后,可通过调控TRIF抑制IκB-α活性进而抑制NF-κB,促进M2极化。

Objective:To investigate the promotion effect of Toll-like receptor 4(TLR4) adapters,myeloid differentiation factor 88(MyD88)and Toll/interleukin(IL)receptor(TIR)domain containing adaptor protein inducing interferon-β(TRIF) after gene knowout in M2 polarization of macrophages induced by lactate, and to elucidate the regulation mechanism of immune responses by lactate.Methods:The murine macrophage cell line Raw264. 7 were cultured in vitro,CRISPR/Cas9 gene editing system was constructed by lentivirus method,MyD88 and TRIF genes were knocked out(Myd88-KO group and TRIF-KO group),and the untransfection Raw264. 7 cells were used as control group. The knockout effects of gene knowout were detected by real-time fluorescence quantitative PCR(RT-qPCR)and Western blotting methods. The experiment was divided into untreated Raw264. 7 cell group,Raw264. 7 + lactate group,MyD88-KO group,MyD88-KO + lactate group,TRIF-KO group and TRIF-KO+lactate group. The indexes were determined after 15 mmol·L^(-1)lactate were induction for 24 h.The expression levels of M2 polarization phenotype molecules macrophage mannose receptor CD206 and arginase 1(Arg1) mRNA in the cells in various groups were detected by RT-qPCR method, the morphology of cells in various groups was observed under light microscope,and the levels of tumor necrosis factor-α(TNF-α),interferon-γ(INF-γ) and interleukin0(IL0) in the cell supernatant in various groups were detected by enzyme linked immunosorbent assay(ELISA)method. The expression levels of nuclear factor-κB(NF-κB)signaling pathway-related proteins were detected by Western blotting method.The molecular docking simulation of lactate with TLR4,MyD88 and TRIF were carried out by Autodock software,and the binding was observed. Results:After targeted gene was knocked out using CRISPR/Cas9technique,compared with control group,the expression levels of MyD88 and TRIF mRNA and proteins in the cells in MyD88-KO group and TRIF-KO group were significantly reduced(P<0. 05 or P<0. 01).After 24 h of lactate treatment,compared with untreated Raw264. 7 cell group,the expression levels of markers of M2 polarization CD206 and Arg1 mRNA in the cells in Raw264. 7+lactate group were significantly increased(P<0. 05);compared with MyD88-KO group,the expression levels of CD206 and Arg1 mRNA in the cells in MyD88-KO+lactate group were increased(P<0. 05);compared with TRIF-KO group,the expression levels of CD206 and Arg1 mRNA in the cells in TRIF-KO+lactate group were decreased(P<0. 05). The cells in Raw264. 7+lactate group showed M2-polarized morphology,such as increased volume,obvious protruding pseudopods and increased proportion of conical cells;the cells in MyD88-KO+lactate group also showed M2-polarized morphological changes;the cells in TRIF-KO+lactate group showed no significant morphological changes. The ELISA results showed that compared with untreated Raw264. 7 cell group,the TNF-α level in the cells in Raw264. 7+lactate group was significantly decreased(P<0. 05),and the differences in INF-γ and IL0 levels were not statistically significant(P>0. 05);compared with MyD88-KO group,the level of TNF-α in the cells in MyD88-KO+lactate group was significantly decreased(P<0. 05),the difference in INF-γ level was not statistically significant(P>0. 05),and the IL0 level was significantly increased(P<0. 05);compared with TRIF-KO group,the TNF-α level in the cells in TRIF-KO+lactate group was significantly increased(P<0. 05),and the differences in the INF-γ and IL0 levels were not statistically significant(P>0. 05). The Western blotting results showed that compared with untreated Raw264. 7 cell group,the expression levels of TLR4 protein in the cells in Raw264. 7+lactate group,MyD88-KO group,MyD88-KO+lactate group,TRIF-KO group and TRIF-KO+lactate group had no statistically significant differences(P>0. 05);the expression levels of nuclear factor-κB inhibitor protein-α(IκB-α)and p65 protein in the cells in Raw264. 7+lactate group were significantly decreased(P<0. 05);compared with MyD88-KO group,the expression levels of IκB-α and p65 proteins in the cells in MyD88-KO+lactate group were decreased(P<0. 05);compared with TRIF-KO group,the expression levels of IκB-α and p65 proteins in the cells in TRIF-KO+lactate group had no statistically significant differences(P>0. 05). The number of hydrogen bonds formed by molecular docking of lactate with TLR4,Myd88 and TRIF was 2,4 and 4,and the binding energies were-2. 72,-3. 24 and-3. 66. Conclusion:Lactate can inhibit the activity of IκB-α,and further inhibit NF-κB and thus promote M2 polarization through regulation of TRIF after treatment in the macrophages.