O026 Negative regulation of toll like receptor signaling by IL-10 dependent microRNAs

Abstract

Introduction MicroRNAs (miRs) have emerged as important controllers of Toll-like-Receptor (TLR) but their functional role in the inflammatory response remains incompletely understood. Methods Cells. Human monocytes obtained from healthy donor buffycoats and THP-1 monocytic cell lines were stimulated with 100 ng/ml LPS or Pam3CSK4. Constructs. 3′UTR of potential target genes were cloned in psiCHECK vector for the evaluation of miR activity by luciferase assay. For the transduction of THP-1 cell line we generated miRNA/lentiviral-based expression vectors (pRRL-cluster) and cytokines levels were evaluated by ELISA assay. Bioinformatics and statistical analysis. The IPA software has been used for the study of pathways associated to predicted miR-125a∼99b∼let-7e targets. Statistical evaluation was determined using the Student t -test or the One-way ANOVA. Results To identify candidate miRs potentially involved in the response of human monocytes to stimuli of bacterial origin, we previously analyzed miR expression profile of monocytes stimulated for 8 h with LPS [1] . Among the miRs strongly induced by LPS stimulation we identified the cluster of miR-125a, miR-99b and let-7e. We observed a higher expression of miR-125a, miR-99b and let-7e levels at later time points. As IL-10 is a cytokine late-induced during monocyte activation and exert its anti-inflammatory activity through modulation of gene expression program triggered by LPS, we measured the expression levels of these miRs in monocytes stimulated with IL-10, in the presence or not of LPS, showing a potentiating effect of IL-10 on LPS-induced cluster miR-125a∼99b∼let-7e expression. We identified a putative STAT3 binding site and demonstrated the binding of STAT3 to this site in the condition of IL-10 stimulation. These data were also consistent with the reduction of cluster miR-125a∼99b∼let-7e expression levels in the presence of blocking antibody against IL-10R and JAK-STAT inhibitors. An in silico analysis was performed to assess the potential role of cluster miR-125a∼99b∼let-7e in the context of inflammation and strikingly we found that the TLR pathway was potentially regulated by these miRs at multiple steps of the signaling cascade. We validated both the TLR4 and CD14 receptors as direct targets, as well as key adaptor/signalling proteins, in particular MyD88 and IRAK1 proteins and also most of the pro-inflammatory cytokines and chemokines expressed in THP-1 monocytic cells. Furthermore, the enforced expression of cluster miR-125a∼99b∼let-7e in LPS stimulated human monocytes led to an extensive down-regulation of pro-inflammatory cytokines. Conclusion Altogether, our results identify a class of IL-10-responsive miRs with anti-inflammatory activity in monocytes based on multiple targeting of components of the TLR4 pathway. We provide a mechanistic insight into the role of these miRs in the suppression of the inflammatory role and candidate them as new feedback modulators of LPS response involved in the resolution of inflammation. A broader and deeper understanding of these issues may well lead to novel therapeutic approaches to those diseases shown to be marked by dysregulated inflammatory responses.