| Summary: | Interleukin-10 (IL-10) is an anti-inflammatory cytokine essential for maintaining immune homeostasis. One of its major targets is the macrophage where it inhibits production of pro-inflammatory cytokines, chemokines and other soluble mediators. However, the intracellular signaling mechanisms by which IL-10 achieves macrophage deactivation remain under intense investigation. Our studies suggest that in addition to canonical STAT3 signaling, IL-10 mediates its early phase anti-inflammatory response through SHIP1 in a STAT3-independent manner. Upon macrophage activation by bacterial lipopolysaccharide, the phosphoinositide 3′ kinase (PI-3 kinase) pathway is activated to produce cytokines such as tumor necrosis factor α (TNFα). SHIP1 is a negative regulator of the PI-3 kinase pathway and its activation downstream of the IL-10 receptor suppresses PI-3 kinase-initiated signals that trigger transcriptional elongation of TNFα and other pro-inflammatory related genes. We next investigated whether SHIP1 activation could mimic the anti-inflammatory actions of IL-10. We screened for small-molecule activators of SHIP1 and isolated the meroterpenoid compound Pelorol. Pelorol and its derivatives specifically enhanced SHIP1’s phosphatase activity and thus suppressed inflammation in macrophage cultures, and in murine models of endotoxic shock, acute anaphylaxis, and inflammatory bowel disease. Closer examination of SHIP1’s enzyme kinetics indicated that SHIP1 is subject to allosteric activation by its product phosphatidylinositol-3,4-bisphosphate (PI-3,4-P₂). We subsequently identified a previously unrecognized C2 domain residing C-terminal of SHIP1’s phosphatase domain which is required for its allosteric activation and is the binding site for both PI-3,4-P₂ and the small-molecule SHIP1 agonists. Bioinformatic and structural analyses also revealed another previously unappreciated domain located N-terminal of SHIP1’s catalytic domain. Using NMR spectroscopy, we characterized this domain as having pleckstrin homology (PH) domain-like topology. We demonstrate that SHIP1’s PH-related (PH-R) domain participates in recruiting SHIP1 to the plasma membrane upon cell stimulation via direct interactions with phosphatidylinositol-3,4,5-trisphosphate. The PH-R domain is essential for SHIP1 inhibition of FcR-dependent phagocytosis and represents another target to which to develop modulators of SHIP1 function. Together, this work suggests that IL-10 activation of SHIP1 is important in its inhibition of macrophage activation, and that mimicking IL-10 with small-molecule SHIP1 agonists could be an effective and viable approach to treating various inflammatory and autoimmune conditions.
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