Role of SHIP1 in the interleukin 10 signaling pathway

Background: Inflammation is a major immune response that protects the host from infections. Upon pathogen encounter, immune cells are activated and produce pro-inflammatory mediators. Although inflammation is a critical step in both innate and acquired immune response, excessive inflammation can damage tissues and lead to inflammatory diseases such as rheumatoid arthritis and inflammatory bowel disease. Interleukin 10 (IL10) is a key anti-inflammatory cytokine that deactivates various immune cells including macrophages. IL10 signaling has classically been described to include ligand stimulated phosphorylation of the IL10 receptor (IL10R), recruitment of the signal transducer and activator of transcription 3 (STAT3) and the subsequent phosphorylation, dimerization and translocation of STAT3 to the nucleus, where it stimulates transcription of anti-inflammatory genes. Our studies have shown that in addition to the canonical STAT3 signaling, IL10 mediates its anti-inflammatory effect through SHIP1 inositol phosphatase. We hypothesized that IL10 signaling leads to the recruitment and activation of a SHIP1-STAT3 complex that then mediates the anti-inflammatory effects of IL10. Our SHIP1 pulldown studies have revealed that SHIP1 and STAT3 form a complex in response to IL10. In addition, we observed the AMP-activated protein kinase (AMPK), which has been previously reported to play a role in IL10 signaling, to be part of this complex. The pro-inflammatory counterpart of IL10, IL6 is unable to initiate the formation of such complex although both IL10 and IL6 induce tyrosine phosphorylation of STAT3. In addition to IL10, our small molecule SHIP1 agonist, AQX-151, was also found to induce the formation of the SHIP1-STAT3-AMPK complex. Our experiments in STAT3 knock-out bone marrow derived macrophages suggested that STAT3 mediates the association of SHIP1 and AMPK. We also mutated a number of phospho-Tyr residues in SHIP1 and assessed IL10 signaling. Tyr190 was identified to play an important role in both complex assembly and cellular response to IL10 (evaluated by inhibition of TNFα secretion). These studies showed for the first time the importance of SHIP1, STAT3 and AMPK complex in IL10 signaling. Understanding the mechanisms by which IL10 down regulates immune cell function will give insight into development of novel therapeutics that mimic the beneficial effects of IL10.Medicine, Faculty ofExperimental Medicine, Division ofMedicine, Department ofGraduat