| Objective:To investigate the effects of MTB Hsp16.3 on the polarization of Bone marrow-derived macrophage(BMDM)and Alveolar macrophages(AM)in mice.To observe the expression of Fractalkine/CX3CR1 in macrophages derived from bone marrow and alveolar macrophages of mice treated with MTB Hsp16.3.To investigate the mechanism of Fractalkine/CX3CR1 in MTB Hsp16.3-induced M2 polarization of macrophages.Methods:1.Extract mouse bone marrow-derived macrophages and detect F4/80 and CD11 b double positive expression rates by flow cytometry;IFN-γ and IL-4 induce them respectively to be M1/M2 macrophages,and observe the morphological changes of BMDM after induction under light microscope;MTB Hsp16.3 stimulates BMDM at 0h,12 h,24h,36 h,48h,72 h,Real-time PCR detects TNF-α,i NOS,TGF-β,IL-10,Arg-1 and Fizz1 at different time points and other M1/M2 polarization-related molecules as well as Fractalkine and CX3CR1 m RNA expression levels;Using Western blot to detect TNF-α,Arg-1 and other M1/M2 polarization markers and Fractalkine and CX3CR1 protein expression levels;Fluorescence staining were used to observe the expression of Fractalkine and CX3CR1.2.By using alveolar lavage method to make isolation and extraction of mouse alveolar macrophages,observation of 0h,2h,24 h morphological changes of cells under light microscope;immunofluorescence staining method was used to identify the expression of AM specific phenotype marker CD68;Real-time PCR detects the m RNA expression levels of M1/M2 polarization-related molecules such as TNF-α,i NOS,TGF-β,IL-10,Arg-1,Fizz1,etc.The AM 24 h group induced by IFN-γ and the AM 24 h induced by IL-4Groups were used as the positive control group of M1 and M2 macrophages respectively;Western blot technology was used to detect the expression of Fractalkine and CX3CR1 after MTB Hsp16.3 stimulated AM at 0h,12 h,24h.3.Use lentiviral interference vector to down-regulate the expression of CX3CR1 in mouse alveolar macrophages.observe the infection efficiency under a fluorescence microscope;Flow cytometry was used to observe the infection efficiency at 0h,24 h,48h,72 h and 96h;Real-time PCR and Western blot to detect the expression of CX3CR1 after the lentiviral interference vector infects AM;the negative control vector LV-Cont and the interference vector LV-CX3CR1 were used to infect AM for 72 h,and then the cells were stimulated with MTB Hsp16.3 for 12 h.The experimental groups are: normal alveolar macrophage group(AM),negative control vector(LV-Cont),and then MTB Hsp16.3stimulation group(LV-Cont+Hsp16.3 and LV-CX3CR1+Hsp16.3).Real-time PCR detects the m RNA expression levels of TNF-α,i NOS,TGF-β,IL-10,Arg-1,Fizz1 and other M1/M2 polarization-related molecules in each group.Western blot detects M2 polarization in each group the protein expression levels of markers CD206 and Arg-1.4.Phosphorylation levels of JNK,p38 and ERK in AM group,LV-Cont group,LV-Cont+Hsp16.3 group and LV-CX3CR1+Hsp16.3 group were detected by Western blot.Results:1.After stimulated by MTB Hsp16.3,the expression of M2 polarization-related cytokines was increased and Fractalkine and CX3CR1 were highly expressed in BMDM.After induced by GM-CSF at a final concentration of 20ng/ml for 7 days,CD11 b and F4/80 of macrophages were detected as positive as 94.7%,indicating the successful culture of BMDM.Micromicroscope observation of BMDM morphology showed that IFN-γ induced cells presented long spindle type and elongated pseudopod,IL-4 induced cell overall morphology was round cells stretching out pseudopodia,MTB Hsp16.3induced cell morphology was more consistent with IL-4.Further stimulate BMDM with MTB Hsp16.3 0h,12 h,24h and 36 h,48h,72 h,Real-time PCR results show that the TGF-β,IL-10,Arg-1,Fizz1 M2 polarization related molecules at different time points of expression significantly higher in the untreated group and the difference was statistically significant(P<0.05).Western blot showed that the protein expression of M2 polarized molecular marker Arg-1 was higher than that of the untreated group,The difference was statistically significant(P<0.05).Real-time PCR and Western blot found that after MTB Hsp16.3 stimulated BMDM,the expression levels of Fractalkine and CX3CR1 were significantly higher than those in the untreated group at 0-72 h,and the difference was statistically significant(P<0.05).The results of immunofluorescence staining showed that the positive expression rate of Fractalkine and CX3CR1 was significantly higher than that of the group without MTB Hsp16.3 stimulation.2.After MTB Hsp16.3 stimulated AM,the expression of M2 polarization-related cytokines increased and Fractalkine and CX3CR1 were highly expressed.Observation under a microscope shows that the isolated alveolar macrophages appear round and vary in size.Approximately 2 hours later,the adherence is relatively firm.After24 hours of culture,alveolar macrophages were rich in cytoplasm,showing a variety of cell shapes such as round,oval,and spindle.The AM was identified by immunofluorescence staining.The results showed that the cytoplasm of mice alveolar macrophages treated with CD68 monoclonal antibody was red,and the nuclei were not stained,suggesting that AM was successfully cultured.Further stimulate AM with MTB Hsp16.3 0h,12 h,24h,Real-time PCR results show that the TGF-β,IL-10,Arg-1,Fizz1 M2 polarization related molecules at different time points of expression significantly higher in the untreated group,and the difference was statistically significant(P<0.05).Western blot results showed that the expression levels of Fractalkine and CX3CR1 after AM stimulation with MTB Hsp16.3 were significantly higher than those in the unstimulated group at 12 and 24h,with statistical significance(P<0.05).3.Down-regulating the expression of CX3CR1 hinders the M2 polarization of macrophages induced by MTB Hsp16.3.Lentiviral interference vector was used to down-regulate the expression of CX3CR1 in AM.The expression of green fluorescence was observed under a fluorescence microscope,and the infection efficiency was over 80%.Flow cytometry found that the infection efficiency reached the highest peak at 72 h.Real-time PCR detects the m RNA expression levels of M1 polarization-related molecules TNF-α,i NOS and M2polarization-related molecules TGF-β,IL-10,Arg-1,Fizz1.The results showed that interference with CX3CR1 expression in AM decreased the m RNA expression of M2 polarization related molecules(P<0.05),but did not affect the expression of M1 polarization related molecules(P>0.05).Western blot results showed that interfering with the expression of CX3CR1 in AM inhibited the expression of M2 polarization molecular marker CD206 and Arg-1 induced by MTB Hsp16.3,with statistical significance(P<0.05).4.CX3CR1 may participate in MTB Hsp16.3-induced macrophage M2 polarization through activation of JNK and p38 phosphorylation levels.Western blot results showed that after MTB Hsp16.3 stimulated AM,the phosphorylation levels of JNK and p38 were significantly higher than those in the normal alveolar macrophages,and the difference was statistically significant(P<0.05).The down-regulation of CX3CR1 expression in AM significantly inhibited the increase of JNK and p38 phosphorylation levels induced by MTB Hsp16.3,with statistical significance(P < 0.05).There was no significant difference in ERK phosphorylation level(P > 0.05).Conclusion:1.MTB Hsp16.3 can promote AM to M2 polarization.2.Fractalkine/CX3CR1 signal axis may be involved in the regulation of MTB Hsp16.3induced macrophage M2 polarization.3.CX3CR1 may participate in MTB Hsp16.3-induced macrophage M2 polarization through activation of JNK and p38 phosphorylation levels. |
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