The Role And Mechanisms Of MTORC1 In Flagellin-induced Inflammatory Cytokines Expression

Colitis is a common disease. Intestinal flora and increased opportunistic pathogens result in the production of bacterial bacterial lipopolysaccharide (LPS), peptidoglycan, and flagellin that lead to the colitis. The pathogen associated molecular patterns combine with pattern recognition receptors to activate colonic epithelial cells and immune cells to secret inflammatory factors, resulting in colitis. Flagellin is a major component of the bacterial flagellum and the ligand of TLR5 (toll like receptor 5) which recognized by TLR5 resulting in gathered and activation of MyD88 (myeloid differentiation 88) by its intracellular TIR domain, it can raise IRAK (IL-1-receptor kinase) and TRAF6 (TNF-receptor- associated factor 6) and activate nuclear transcription factors (transcription factor nuclear factor, NF-κB) to promote the expression of pro-inflammatory cytokines which finally cause inflammatory respose to clear the pathogen from body.Phosphatidylinositol 3-kinase (PI3K) is an intracellular lipid kinases and has the activity of serine/threonine kinase. It is already known that PI3K regulated the innate immune response induced by a variety of viruses and bacteria components via TLRs. lipopolysaccharide (LPS) activates PI3K and induces complex formation of PI3K and MyD88, which regulates the expression of cytokines. Flagellin activates PI3K rapidly through TLR5. mTOR is a very conserved serine/threonine kinases in evolution, it belongs to PIKK (phosphatidylinositol 3-kinase-related kinase) superfamily, forms two complexes with other proteins, mTORC1 and mTORC2, mTORC1 is sensitive to rapamycin. As PI3K downstream proteins, mTORC1 mediates upstream signals and activates downstream p70S6K (ribosomal protein p70S6 kinase) and 4EBP-1 (transcription initiation factor 4E binding protein 1) to regulate gene expression in a variety of physiological and pathological processes. mTOR has an important role in the innate immune response, such as effecting on the expression of bacteria or other stimulating factor-induced cytokines. However, it is unknown whether mTORC1 regulates flagellin-induced inflammation. In this study, we plan to explore the mechanism of mTORC1 in regulation of flagellin-induced inflammatory response using the mice macrophage cell line, mouse peritoneal macrophages and normal colon epithelial cell lines.In the present study, PI3K inhibitors LY294002 and wortmannin and rapamycin, mTORC1 inhibitor were used to block PI3K and mTORC1 signaling pathway in mouse macrophage cell line Ana-1, murine peritoneal macrophages and normal human colonic epithelial cell line NCM460 which stimulated by Salomonella typhimurium flagella protein. Inflammatory cytokines (TNF-α, IL-6 and IL-8) were detected by ELISA to conform whether PI3K and mTORCl regulate flagellin-induced inflammatory cytokine expression and cell proliferation. The results show that flagellin induces TNF-α and IL-8 expression in a time-dependent and dose-dependent manner. The optimal flagellin-stimulated doses and times were 100 ng/ml/24 h and 100 ng/ml/12 h in Ana-1 and NCM460 respectively. Flagellin plays different roles in different cell types respectively.100 ng/ml flagellin induces Ana-1 cell proliferation that can be inhibited by LY294002, wortmannin and rapamycin; however, flagellin has no effect on NCM460 cell proliferation. LY294002 and wortmannin attenuate flagellin-induced TNF-a, IL-6 and IL-8 expression, furthermore, rapamycin also decreases the secretion of flagellin-induced TNF-a, IL-6 and IL-8 expression. These results suggest PI3K/mTORC1 pathway plays an important role in the regulation of flagellin-induced inflammation.In order to investigate the molecular mechanisms of PI3K/mTORC1 pathway in flagellin-induced inflammatory response, we study the signaling pathways and transcription factors. Firstly, The expression and phosphorylation of Akt, mTOR, S6, and 4EBP1 were detected to confirm whether flagellin activates PI3K/Akt/mTOR signaling pathway; Cells were stimulated with flagellin after pretreated with LY294002 and rapamycin to inhibit PI3K and mTOR, then the activity of PI3K/Akt/mTOR pathway was measured by western blot and mRNA expression of TLR5 was detected by real time PCR; secondly, mTORC1 signal pathway and cytokines were detected after TLR5 was blocked through TLR5 antibody and TLR5 RNA interference vector respectively, using a TLR4 antibody as a control. Next, the level of protein expressions and phosphorylations of STAT3, NF-κB p65 were detected. Finaly, TLR5 mRNA expression was after Ana-1 and NCM460 were stimulated by flagellin. The data shows that flagellin activate rapidly PI3K/Akt, ERK1/2 and mTOR signaling pathway. Flagellin-actived PI3K/Akt and mTOR pathway were inhibited after PI3K was suppressed by LY294002, while the activity of ERK1/2 didn’t change; Flagellin activates PI3K/Akt, mTOR signaling pathway, STAT3 and inflammatory gene expression via a TLR5-dependent mechanism; Rapamycin impairs flagellin-induced NF-κB and STAT3 activation; Flagellin inhibits TLR5 mRNA expression, which can be inhibited by LY294002 and rapamycin in mouse macrophage; Flagellin induces TLR5 mRNA expression, which can be inhibited by LY294002 and rapamycin in human colonic epithelial cells. In summary, mTORC1 pathway plays an important role in regulating flagellin-induced inflammation. The possible mechanism is that flagellin activates mTORC1 to increase the product of pro-inflammatory cytokine via TLR5, which may be through activating NF-κB and STAT3.