Role of toll-like receptor 4-dependent signal pathways in bone marrow-derived macrophage activation induced by high glucose

Chronic microinflammatory state plays an important role in occurrence and development of diabetic nephropathy. Macrophages are the main regulatory cells in the inflammatory response. Toll-like receptors (TLRs) play an important role in innate immune response and inflammation. In the present study, bone marrow-derived macrophages (BMDMs) were separated from C57BL/6J and B10ScNNju (TLR4 knockout mice). These were divided into a normal control group (LG), high glucose group (HG), TLR4 knockout group (TLR4-/-), and high glucose stimulated TLR4 knockout BMDM group (TLR4-/-+HG). The M1 phenotype of macrophages was detected by flow cytometry and co-expression of TLR4 while macrophage activation marker inducible nitric oxide synthase (iNOS) was observed by immunofluorescence. Tumor necrosis factor-alpha (TNF-alpha), monocyte chemoattractant protein-1 (MCP-1), and interleukin- 1 (IL-1 beta) were assessed by RT-PCR and ELISA, together with mRNA levels of iNOS. Western blot was performed to analyze protein levels of TLR4, myeloid differentiation primary response gene 88 (MyD88), TIR-domain-containing adapter-inducing interferon-b (Trif), p-IRAK-1, p-IRF3, IRF3, NF-kappa B p65, NF-kappa B p-p65, and iNOS. Compared to the LG group, high glucose increased the percentage of M1 macrophages and mRNA levels of TNF-alpha, MCP-1, IL-1 beta, and iNOS. In addition, expression of TLR4, MyD88, Trif, p-IRAK-1, p-IRF3, IRF3, NF-kappa B p65, NF-kappa B p-p65, and iNOS proteins were enhanced. Knockout of TLR4 genes eliminated the effects of macrophage activation induced by high glucose. The present study suggests that high glucose can promote BMDM to M1 phenotype polarization and knockout of TLR4 genes can inhibit the M1 phenotype of macrophage activation and production of inflammatory cytokines induced by high glucose.