MTB Hsp16.3重组蛋白通过Fractalkine/CX3CR1信号轴调控小鼠肺泡巨噬细胞M2型极化

MTB Hsp16.3 recombinant protein regulates M2-type polarization in mouse alveolar macrophages via the Fractalkine/CX3CR1 signaling axis

目的初步探讨Fractalkine/CX3CR1信号轴在MTB Hsp16.3重组蛋白诱导小鼠肺泡巨噬细胞向M2型极化中的作用及机制。方法提取小鼠肺泡巨噬细胞,观察细胞形态后用其特异性标志CD68鉴定;MTB Hsp16.3(100 ng/mL)刺激肺泡巨噬细胞后,采用Real-time qPCR检测TNF-α、Arg-1等极化相关分子mRNA表达水平,Western blot检测Fractalkine、CX3CR1的表达情况。用慢病毒干扰肺泡巨噬细胞CX3CR1的表达后,荧光显微镜观察病毒感染情况;流式细胞术检测感染效率;Real-time qPCR和Western blot检测感染前后肺泡巨噬细胞CX3CR1的表达情况。采用MTB Hsp16.3刺激慢病毒感染过的肺泡巨噬细胞,Real-time qPCR检测各组极化相关分子mRNA表达水平;Western blot检测CD206、Arg-1的蛋白表达情况。利用Western blot检测各组中JNK、p38、ERK的磷酸化水平。结果显微镜下观察发现新分离的肺泡巨噬细胞为圆形,大小不等,约2 h后贴壁较牢,24 h后,肺泡巨噬细胞呈圆形、梭形等多种形态,且胞体更大,通过CD68鉴定为肺泡巨噬细胞;经MTB Hsp16.3处理后巨噬细胞TGF-β、IL-10、Arg-1、Fizz1的mRNA水平明显增高(P<0.05),巨噬细胞表面Fractalkine、CX3CR1的表达增高(P<0.05),与JNK、p38的磷酸化水平升高(P<0.05)相关。干扰肺泡巨噬细胞CX3CR1的表达后,慢病毒感染效率在72 h达到最高峰,并且可降低巨噬细胞表面CX3CR1的表达。经MTB Hsp16.3刺激慢病毒感染过的肺泡巨噬细胞发现,TGF-β、IL-10、Arg-1、Fizz1 mRNA水平降低(P<0.05),CD206、Arg-1的表达水平降低(P<0.05),与JNK、p38磷酸化水平被抑制(P<0.05)相关。结论Fractalkine/CX3CR1信号轴可能参与调控MTB Hsp16.3重组蛋白诱导的肺泡巨噬细胞M2型极化,并且可能通过激活JNK、p38的磷酸化水平发挥作用。

Objective A preliminary investigation of the role and mechanism of the Fractalkine/CX3CR1 signaling axis in MTB Hsp16.3 recombinant protein-induced polarization of mouse alveolar macrophages toward the M2 type.Methods Mouse alveolar macrophages were extracted and characterized by their specific marker CD68 and cell morphology.After MTB Hsp16.3(100 ng/ml)stimulation of alveolar macrophages,the mRNA expression levels of polarization-related molecules(TNF-α,Arg-1)were detected by Real-time qPCR,and the expressions of Fractalkine and CX3CR1 were detected by Western blot.After interfering with the expression of CX3CR1 in alveolar macrophages,fluorescence microscopy was used to observe the viral infection;flow cytometry was used to detect the efficiency of infection;Real-time qPCR and Western blot were used to detect the expression of CX3CR1 in alveolar macrophages before and after infection.MTB Hsp16.3 was used to stimulate lentivirus-infected alveolar macrophages.Real-time qPCR was used to detect the mRNA expression levels of polarization-related molecules in each group;Western blot was used to detect the protein expression of CD206 and Arg-1.The phosphorylation levels of JNK,p38 and ERK in each group were detected by Western blot.Results Microscopic observation revealed that the newly isolated alveolar macrophages were round,unequal in size,and more firmly adhered to the wall after 2 h.After 24 h,the alveolar macrophages showed a variety of morphologies,such as round and spindle shaped,and the cytosol was larger,which was identified as alveolar macrophages by CD68;The mRNA levels of TGF-β,IL-10,Arg-1 and Fizz1 in macrophages treated with MTB Hsp16.3 were significantly increased(P<0.05),and the expression of Fractalkine and CX3CR1 on the surface of macrophages was increased(P<0.05),which might be related to the increased phosphorylation levels of JNK and p38(P<0.05).Lentiviral infection efficiency peaked at 72 h after interfering with CX3CR1 expression in alveolar macrophages and reduced macrophage surface CX3CR1 expression.Stimulation of lentivirus-infected alveolar macrophages by MTB Hsp16.3 revealed a decrease in the levels of TGF-β,IL-10,Arg-1 and Fizz1 mRNA(P<0.05),and a decrease in the expression levels of CD206,and Arg-1(P<0.05),which were associated with inhibition of JNK and p38 phosphorylation levels(P<0.05).Conclusion The Fractalkine/CX3CR1 signaling axis may be involved in regulating MTB Hsp16.3 recombinant protein-induced M2-type polarization in alveolar macrophages,and may play a role by activating the phosphorylation levels of JNK and p38.