Background: IL-6 is the most important cytokine that plays a central role in the development of chronic inflammation. Recently, non-immune cells like fibroblast have been postulated to mediate chronic allograft rejection via activation of the IL-6 amplifier loop (IL-6+IL-17) via NFκB and STAT3 signaling pathways. We evaluated IL-6 amplifier loop activation by IL-6 and Il-17 in chronic antibody-mediated rejection (CABMR) in renal transplant recipients.

Methodology: Fibroblasts from grafted kidneys from CABMR patients (n=6) were cultured and stimulated with IL-6 (20ng/ µl), IL-17(50ng/ µl), IL-6 plus IL-17 for 24 hours. Levels of IL-6, MCP-1, and CCL20 were estimated in culture supernatants by ELISA as markers of IL-6 amplifier loop activation. mRNA expression of IL-6, MCP1, CCL20, and SOCS3 genes were measured in the stimulated fibroblasts. Human Renal fibroblast cells from CABMR patients were lysed with Lysis buffer and subjected to SDS–PAGE and western blotting with anti-STAT3, anti-phospho-STAT3, anti-NFκB p65, and anti-phospho-NFκB p65. Additionally, IL-6, MCP1, and CCL20 levels were measured in Healthy control (n=10), CABMR (n=20), and non-CABMR (n=30) patients.

Results: IL-6 and IL-17 synergistically induced more IL-6, CCL-20 & MCP-1 production from fibroblasts.

Gene expression analysis of IL-6, MCP1, and CCL20 was significantly higher with synergistic activation of IL-6 and IL-17 as compared to either IL-6 or IL-17 alone, while SOCS3 gene expression was downregulated. Our results also suggested that IL-6 Amplifier loop activation induces the NFκB and STAT3 signaling pathway activation in the non-immune cells like fibroblast derived from CABMR patients. Additionally, concentrations of IL-6, CCL-20 & MCP-1 in sera were significantly higher in CABMR patients compared to non-rejection patients (p<0.001). There was a significant reduction in IL-6 concentration in culture supernatant with IL-6 and IL-17 inhibitor together and mRNA expression of IL-6 and MCP-1 were significantly reduced.

Conclusion: In humans after kidney transplantation, IL-6 amplifier activation plays an active role in chronic rejection responses. Inhibition of IL-6 with Anti-IL-6 (Tocilizumab) and inhibition of IL-17 with Anti-IL-17 together reduces markers of tissue injury (IL-6, MCP1, CCL20) and rejection of allografts. so, the IL-6 amplifier may be a therapeutic target for chronic transplant rejection.