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IBD Basic Science

Introduction: Inflammatory bowel disease (IBD) is a chronic inflammatory condition of the gastrointestinal tract. Although the pathogenesis of IBD is not fully understood, it is believed to result from the interaction between various genetic and environmental factors, including the microbiome, resulting in inappropriate gut inflammation. Under homeostatic conditions, the gut immune system exists in a tolerogenic state with the microbiota. However, microbial dysbiosis and an imbalance of proand anti-inflammatory cytokines in the gut characterize IBD. In this study, we investigated the ability of anaerobic gut bacteria cultured from healthy human fecal samples to produce bioactives that suppress NF-κB activity. Methods: The colonic epithelial cell lines Caco-2 and LS174T were transduced with an NF-κB luciferase construct to generate stable reporter cell lines. These cells responded to stimulation with IL-1β and TNFα, respectively, with increased luciferase expression. We exposed the reporter cells to a 10% v/v solution of anaerobically produced bacterial culture supernatants (CS) diluted in tissue culture medium to investigate NF-κB suppressive effects from 23 different isolates, as well as Faecalibacterium prausnitzii A2-165, a bacterium known to produce NF-κB suppressive peptides. Suppressive isolates were treated with elevated temperature or proteinase K to assess their biochemical characteristics. Suppressive CSs were also tested on human-derived whole peripheral blood mononuclear cells (PBMCs) and human-derived colonic organoids from patients with IBD and healthy controls to measure IL-8 expression in response to TNFα and IL-1β stimulation, respectively. Results: LS174T and Caco-2 reporter cells stimulated with TNF-α and IL1β resulted in increased luciferase activity. Of the 23 isolates screened, CS from five isolates supressed NF-κB activation comparable to F. prausnitzii (z-score ≤ 3). Three classes of bioactives were identified: small (<3 kDa) bioactives that were heat and proteinase K labile; small bioactives that were both heat and proteinase K resistant; and large bioactives (>3 kDa). The selected CSs also suppressed the expression of the NF-κB-dependent cytokine IL-8 in PBMCs and gut organoids from patients with both ulcerative colitis and Crohn’s disease, as well as healthy controls, with individual variation. Conclusions: Our anaerobic culturing and NF-κB reporter assay system allowed for the rapid identification of bacteria producing immunomodulatory bioactives, which could lead to the future development of novel therapeutics, and demonstrated the potential of precision medicine using patient-derived organoids. NLRP3: A key player at the tipping point of colitis into colitis-associated colorectal cancer AP PERERA, R ERI School of Health Sciences, University of Tasmania, Hobart, Tasmania, Australia