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Abstract

Crohn’s disease is a chronic, idiopathic inflammatory bowel disease characterized by patchy, transmural inflammation that is influenced by genetic, environmental, and microbial factors. The NOD2 pathway mediates NFκB activation and pro-inflammatory cytokine production. In the SHIP–/– murine model of Crohn’s disease-like ileitis, macrophage-derived IL-1β production drives intestinal inflammation. SHIP reduces NOD2 signaling by preventing downstream interaction between RIPK2 and XIAP, leading us to hypothesize that blocking RIPK2 in SHIP–/– mice would ameliorate intestinal inflammation. We examined the effects of RIPK2 inhibition on pro-inflammatory cytokine production in SHIP+/+ and SHIP–/– macrophages and in mice, using the RIPK2 inhibitor, GSK2983559. We found that GSK2983559 blocked RIPK2 activation in SHIP+/+ and SHIP–/– bone marrow-derived macrophages (BMDMs), and reduced Il1b transcription and IL-1β production in (MDP+LPS)-stimulated SHIP–/– BMDMs. Despite the reduction of IL-1β production in BMDMs, in vivo treatment with GSK2983559 worsened intestinal inflammation and increased IL-1β concentrations in the ileal tissues of SHIP–/– mice. GSK2983559 only modestly reduced IL-1β in (MDP+LPS)-stimulated SHIP–/– peritoneal macrophages, and did not suppress pro-inflammatory cytokine production in response to TLR ligands in peritoneal macrophages from either SHIP+/+ or SHIP–/– mice. Taken together, our data suggest that although RIPK2 inhibition can block IL-1β production by (MDP+LPS)-stimulated macrophages in vitro, it is not an effective anti-inflammatory strategy in vivo, highlighting the limitations of targeting RIPK2 to treat intestinal inflammation in the context of SHIP deficiency.