The role of SHIP in regulating IL-1β production and RIPK2 signalling in macrophages

Inflammatory bowel disease is characterized by chronic intestinal inflammation driven in part by dysregulated immune responses to microbial products. These responses are mediated by pattern recognition receptors such as NOD2, which detects muramyl dipeptide (MDP), and TLR4, which detects lipopolysaccharide (LPS). NOD2 and TLR4 synergize to increase cytokine production in macrophages, and while this synergy can support host defense, it must be tightly regulated to prevent excessive inflammation. The SH2 domain-containing inositol 5'-phosphatase (SHIP) is a negative regulator of PI3K signalling that plays an important role in regulating immune homeostasis. SHIP-deficient (SHIP-/-) mice develop spontaneous Crohn's disease-like ileitis in which macrophage-derived IL-1β is a key driver of intestinal inflammation. SHIP has been reported to reduce NOD2 signalling by disrupting downstream interactions between RIPK2 and XIAP, raising the possibility that RIPK2 inhibition could ameliorate IL-1β-driven inflammation in SHIP-/- mice. However, SHIP's role during NOD2–TLR4 co-stimulation remains undefined. I found that the RIPK2 inhibitor GSK2983559 reduced IL-1β production in SHIP-/- bone marrow-derived macrophages co-stimulated with MDP and LPS in vitro. However, in vivo GSK2983559 treatment unexpectedly exacerbated intestinal inflammation in SHIP-/- mice, increasing ileal IL-1β. In an acute peritonitis model, RIPK2 inhibition reduced cytokine production in SHIP+/+ peritoneal cells but had no effect in SHIP-/- peritoneal cells or peritoneal macrophages of either genotype. SHIP protein abundance inversely associated with NOD2 and LPS synergy for IL-1β production. Reducing SHIP protein concentrations by either differentiating cells in media containing different growth factors, treating cells with IL-4, or using SHIP-targeting siRNA, enabled synergy. This regulation did not require SHIP phosphatase iv activity and was independent of PI3K activity. NOD2 inhibition abolished synergy in SHIP-/- macrophages, and sequential stimulation experiments demonstrated that MDP priming was required for enhanced responses to subsequent LPS stimulation. Together, these findings support a non-catalytic role for SHIP in limiting synergy for IL-1β production during NOD2–TLR4 co-stimulation.