The Role And Possible Mechanisms Of Th17/IL-17 Pathway In Atherogenesis Of ApoE^-/- Mice

Objective It is now generally accepted that atherosclerosis is an inflammatory disease and is driven by innate and adaptive immune responses. IL-17-producing T helper cells (Th17 cells) are currently considered playing an essential role in various autoimmunity and inflammatory diseases. We and others have reported that the Th17/IL-17 pathway (Th17 cells, IL-17 and RORγt ) were up-regulated in patients with coronary heart diseases. But the exact role of Th17/IL-17 pathway in atherogenesis remains unclear. The purpose of this study was to clarify whether or not there were more Th17 cells, relative cytokines and transcriptional factor in atherosclerotic mice.Methods Th17 function at different levels including cell frequencies (in periaortic lymph nodes), related cytokine secretion (IL-17 and IL-6) and key transcription factors(RORγt) were investigated comparatively in 8, 16, 24 and 32 weeks old ApoE-/- mice and their age-matched C57BL/6J mice.Results Our results demonstrated that there is functional upregulation of Th17 cells ratio, Th17 relative cytokines (IL-17 and IL-6) and specific transcription factor (RORγt) in ApoE-/- mice compared with age-matched C57BL/6J mice at all four time-points. Th17 related mediators reached their maximum expression values at the early-intermediate stage (8-16 weeks of age) in ApoE-/- mice, and then followed by continuous degression of their expression.Conclusions There is functional upregulation of Th17/IL-17 pathway during atherogenesis in ApoE-/- mice, suggesting a potential role of Th17/IL-17 pathway in the formation and progression of atherosclerosis. Objective Our experiments of PartⅠhave showed that there are more Th17 cells, relative cytokines(IL-17 and IL-6) and transcriptional factor RORγt in atherosclerotic mice, which suggested the potentially regulatory role of Th17/IL-17 pathway in atherosclerosis. This study is to clarify whether or not IL-17 is pro-atherogenic by inhibition of IL-17 in ApoE-/- mice.Methods Eight-week-old male ApoE-/- mice were treated with neutralized anti-IL-17 antibodies or isotype controls for 12 weeks (n=10 per group) weekly for 12 weeks, respectively. The plaque area was evaluated by Oil-red stain. Meanwhile, we analyzed the composition of AS including three major types of atheroma-associated cell (macrophages, T lymphocytes and SMCs) and collagen respectively by immunofluorescence and Masson's trichrome stain. Then the protein expression of VCAM-1 and MMP-9 in plaques was detected by immunohistochemistry stain. We also detected the mRNA expression of various cytokines ( IFN-γ, IL-17, IL-6, TNF-α, and IL-4 and IL-10) and MMP-9 in the aorta by real-time qPCR. The flow cytometry were used to measure the ratio of Th1, Th2, Treg and Th17 cells in total CD4+ cells.Results We found that inhibition of IL-17 in ApoE-/- mice markedly reduced plaque size by 47% and induced biologic features of stable plaque (more SMCs and collagen, and fewer inflammaory mediators VCAM-1 and MMP-9 in plaques), meanwhile decreased the expression of inflammatory mediators IL-6,TNF-α,VCAM-1 and MMP-9 in the aorta. And treatment with anti-IL-17 antibodies had not affected the ratios of Th1, Th2, Treg and Th17 cells in total CD4+ cells.Conclusions IL-17 may play important role in atherogenesis, and inhibition of IL-17 can induce features of plaque stability that is low in inflammation and high in fibrosis. Objective Our experiments of PartⅠhave demonstrated that there are more Th17 cells, relative cytokines and transcriptional factor in atherosclerotic mice, and then the experiments of PartⅡdemonstrated that inhibition of IL-17 reduced obviously the size and increased SMCs in plaque of in ApoE-/- mice. To explore further the role mechanisms of IL-17 in atherosclerosis, we use mrIL-17 cytokine to stimulate SMCs in vitro and observe the biological changes of SMCs.Methods SMCs were cultured with mrIL-17 in different concentration (0, 50, 100 and 200 ng/ml) and for 12h or 24h. Annexin/PI double staining was performed and flow cytometry was used to detect the apoptosis of SMCs. Meanwhile, different concentration of mrIL-17 (0, 50, 100 and 200 ng/ml) was used to stimulate SMCs for definite periods (1, 2, 4 and 8 h) and then the mRNA expression of IL-17, IFN-γ, VCAM-1, ICAM-1, MCP-1, IL-6, MMP-2 and MMP-9 in SMCs was measured by real-time qPCR.Results IL-17 can induce SMCs apoptosis in dose-dependent and time-dependent way in vitro, and mostly obviously at the time-point of 24h. In addition, the mRNA expression of VCAM-1, IL-6 and MMP-9 in SMCs was up-regulated by IL-17 time-dependently, and peaked at early periods of stimulation (respectively at 2h, 1h and 4h).Conclusions IL-17 can induce apoptosis and inflammatory mediators expression of SMCs dose-dependently and time-dependently alone in vitro.