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.

Crohn disease (CD) is a chronic, relapsing inflammatory condition characterized by segmental, transmural inflammation that can affect any part of the gastrointestinal tract. A hallmark of CD is impaired intestinal barrier function, and intestinal fibrosis is a common complication. SHIP-deficient (SHIP-/-) mice spontaneously develop gut barrier dysfunction, ileal inflammation, and fibrotic pathology, recapitulating key features of CD-like ileitis. The SHIP-/- mice serve as a valuable model for testing anti-inflammatory and anti-fibrotic therapies in CD. Here, we describe methods to evaluate therapeutic interventions in this model, using dexamethasone as an example of an effective therapy. We provide a step-by-step protocol for characterizing disease and treatment response, including in vivo assessment of gut permeability measured by FITC-dextran, as well as gross pathological examination and detailed histological analyses. Histological assessments incorporate hematoxylin and eosin (H&E) staining for inflammation, Masson's trichrome staining for fibrosis, and Alcian blue/Periodic Acid-Schiff (PAS) staining for goblet cell hyperplasia and hypertrophy. Additionally, cytokines and inflammatory markers in ileal tissue are quantified to assess the inflammatory state. Together, these methods provide a comprehensive framework for evaluating treatment efficacy in the SHIP-/- mouse model of CD-like ileitis. This protocol is broadly applicable to investigators studying gut inflammation, mucosal healing, and intestinal fibrosis.

Crohn disease (CD) is a chronic, relapsing inflammatory condition characterized by segmental, transmural inflammation that can affect any part of the gastrointestinal tract. A hallmark of CD is impaired intestinal barrier function, and intestinal fibrosis is a common complication. SHIP-deficient (SHIP-/-) mice spontaneously develop gut barrier dysfunction, ileal inflammation, and fibrotic pathology, recapitulating key features of CD-like ileitis. The SHIP-/- mice serve as a valuable model for testing anti-inflammatory and anti-fibrotic therapies in CD. Here, we describe methods to evaluate therapeutic interventions in this model, using dexamethasone as an example of an effective therapy. We provide a step-by-step protocol for characterizing disease and treatment response, including in vivo assessment of gut permeability measured by FITC-dextran, as well as gross pathological examination and detailed histological analyses. Histological assessments incorporate hematoxylin and eosin (H&E) staining for inflammation, Masson's trichrome staining for fibrosis, and Alcian blue/Periodic Acid-Schiff (PAS) staining for goblet cell hyperplasia and hypertrophy. Additionally, cytokines and inflammatory markers in ileal tissue are quantified to assess the inflammatory state. Together, these methods provide a comprehensive framework for evaluating treatment efficacy in the SHIP-/- mouse model of CD-like ileitis. This protocol is broadly applicable to investigators studying gut inflammation, mucosal healing, and intestinal fibrosis.