THE DEVELOPMENT OF HUMAN PRIMARY CULTURE MODELS TO MODEL INFLAMMATORY SIGNALING IN CROHN’S DISEASE AND ULCERATIVE COLITIS

Abstract

Abstract Intestinal inflammation is a major pathological feature associated with gastrointestinal disorders such as Crohn’s disease and ulcerative colitis (UC). To unravel the complex mechanisms underlying these conditions and to develop effective therapeutic strategies, it is crucial to recapitulate intestinal inflammatory signaling using accurate and biologically relevant experimental systems. To establish primary cell-based models that reprise the inflammatory signaling in Crohn’s and UC, we developed the RepliGut platform. Briefly, we established a biobank of primary intestinal stem cells, derived from healthy full-length intestines from a range of donors of different sexes, ages, and ethnicities. From each donor, intestinal epithelial stem cells were isolated from each region of the small intestine and colon (duodenum, jejunum, ileum, ascending colon, transverse colon, and descending colon). The intestinal stem cells were passed to generate large and quality-controlled cell lots of “ready-to-use” vials which are cryopreserved and can be thawed directly onto 12- or 96-well Transwell inserts, generating the RepliGut platform. The RepliGut culture conditions allow for investigation into the physiology of intestinal epithelial cells as either proliferative stem or differentiated cell types and enable studies on focused regional differences in the intestinal tract, while the Transwell platform allows apical and basal media access that facilitates barrier function measures and convenient bi-lateral media sampling. Using this system, Crohn’s and UC-like inflammation can be produced via treatment with the most clinically and therapeutically validated cytokines associated with disease pathophysiology, TNF-α and IFN-γ. Circulating concentrations of both TNF-α and IFN-γ are observed to be elevated in patients with inflammatory bowel diseases and therapeutic antagonists including adalimumab (TNF-α) and JAK inhibitors such as tofacitinib (IFN-γ) represent the current standard of care. On the RepliGut platform, both TNF-α and IFN-γ produced a dose-dependent loss in epithelial barrier function, increased cytokine secretion, and caused cellular death, all of which were mitigated by adalimumab and tofacitinib, respectively. Moreover, at clinically relevant concentrations, combinations of TNF-α and IFN-γ produced synergistic inflammatory response on the intestinal epithelial cultures. In summary, the RepliGut platform provides a convenient and high-throughput platform that reproduces cytokine-induced intestinal inflammation for mechanistic studies and drug screening.