Abstract:

Objective To investigate the promotion effect of Toll-like receptor 4（TLR4） adapters， myeloid differentiation factor 88（MyD88） and Toll/interleukin-1（IL-1） 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 interleukin-10 （IL-10） 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/Cas9 technique，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 IL-10 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 IL-10 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 IL-10 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.

Key words: Toll-like receptors, Myeloid differentiation factor 88, Toll/interleukin-1 receptor domain containing adaptor protein inducing interferon-β, Lactate, Macrophages