The gut microbiota of mammals possess distinctive metabolic pathways with untapped therapeutic potential. Using molecular insights into dietary fiber metabolism by the human gut microbiota, we designed a targeted drug delivery system, called GlycoCaging, that is based on bespoke glycoconjugates of a complex plant oligosaccharide. GlycoCaging of exemplar anti-inflammatory drugs enabled release of active molecules triggered by specific glycosidases of autochthonous gut bacteria. GlycoCaging ensured that drug efficacy was potentiated, and off-target effects were eliminated in murine models of inflammatory bowel disease. Biochemical and metagenomic analyses of gut microbiota of individual humans confirmed the broad applicability of this strategy.

Macrophages coordinate cytokine responses by integrating signals from pattern recognition receptors including NOD2 and TLR4. NOD2 detects muramyl dipeptide derived from bacterial peptidoglycan, and TLR4 recognizes lipopolysaccharide in the outer membrane of Gram-negative bacteria. NOD2 and TLR4 signals synergize to enhance cytokine production in myeloid cells. While synergy can support host defense, it must be regulated to minimize the risk of excessive inflammation. The lipid phosphatase SHIP, a negative regulator of Class I PI3Ks, reduces inflammatory signaling, but its role during NOD2-TLR4 co-stimulation remains undefined. We found that SHIP limits IL-1β production by blocking synergy in bone marrow-derived macrophages co-stimulated with muramyl dipeptide and lipopolysaccharide. SHIP−/− macrophages showed a synergistic increase in IL-1β that was not evident in SHIP+/+ macrophages. Moreover, reducing SHIP protein concentrations by differentiation in different growth factors, IL-4 treatment, or siRNAs enabled synergy for IL-1β production in SHIP+/+ macrophages. Pharmacologic inhibition of SHIP's catalytic activity did not promote synergy, and similarly, blocking PI3K had no effect, suggesting that the response is independent of SHIP's phosphatase activity. Synergy for IL-1β production was dependent on NOD2 signaling despite NOD2 stimulation alone resulting in little to no IL-1β. Moreover, IL-1β was selectively enhanced in SHIP−/− macrophages during co-stimulation with MDP and LPS, or when MDP stimulation preceded LPS. These findings identify SHIP's adaptor function as a negative regulator of IL-1β induced by NOD2-TLR4 co-stimulation in macrophages, and suggest that SHIP acts as a gatekeeper for macrophage IL-1β during early innate immune activation.

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.