ReIA/NF-kB and STAT3 transcription factors cooperate in trans-activating the human IL-17A proximal promoter in response to CD28 individual stimulation

Introduction CD28 is an important costimulatory receptor for T lymphocytes that, in humans, may delivers TCR-independent signal leading to the up-regulation of pro-inflammatory cytokines. We have recently reported that CD28 autonomous signalling induces the expression of IL-17A in peripheral CD4+ T lymphocytes from healthy donors, Multiple Sclerosis and type 1 diabetes patients. Objectives Due to the relevance of IL-17A in the pathophysiology of several inflammatory and autoimmune diseases, the aim of this work was to characterize the mechanisms and signalling mediators responsible for CD28-induced IL-17A expression. Patients & methods Primary CD4+ T cells isolated from the peripheral blood of healthy donor (HD) were stimulated with agonistic anti-CD28 antibodies and the expression of IL-6 and IL-17A (Real-time PCR, ELISA), the nuclear translocation of tyrosine phosphorylated STAT3 (pSTAT3) and RelA/NF-B (western blotting) as well as their specific recruitment on the human IL-17A proximal promoter (chromatin immunoprecipitation, ChIP assays) were analysed. Results: CD28-mediated up-regulation of IL-17A gene expression depends on RelA/NF-B and IL-6-associated STAT3 transcriptions factors. In particular, we found that CD28-activated RelA/NF-B induces the expression of IL-6 that, in a positive feedback loop, mediates the activation and nuclear translocation of pSTAT3. pSTAT3 in turn cooperates with RelA/NF-B by binding specific sequences within the proximal promoter of human IL-17A gene, thus inducing its expression. Finally, by using specific inhibitory drugs, we also identified class 1A phosphatidylinositol 3-kinase (PI3K) as a critical upstream regulator of CD28-mediated RelA/NF-B and STAT3 recruitments and trans-activation of IL-17A promoter. Conclusion: Our findings reveal a novel mechanism by which human CD28 may amplify IL-17A expression in human T lymphocytes and provide biological bases for immunotherapeutic approaches targeting CD28-associated class 1A PI3K to dampen IL-17A- mediated inflammatory response in autoimmune/inflammatory disorders.