Society: AGA

A key feature in intestinal immunity is the dynamic intestinal barrier, which separates the host from resident and pathogenic microbiota through a mucus gel impregnated with antimicrobial peptides. The mechanisms underlying the maintenance and function of this intestinal barrier are not completely understood. Using a mouse forward genetic screen for defects of intestinal homeostasis, we have found a mutation in Tvp23b, which conferred susceptibility to chemically induced and infectious colitis. Golgi apparatus membrane protein TVP23 homolog B (TVP23B) is a transmembrane protein conserved from yeast to humans. The protein is localized to the intestinal epithelium and its deficiency in the hematopoietic extrinsic compartment was essential to protecting against colitis. We found that TVP23B controls the homeostasis of Paneth cells and function of goblet cells in vivo, leading to a decrease in antimicrobial peptides as well as a defective and more penetrable mucus layer. As a result, Tvp23b^-/- mice displayed decreased barrier function and a loss of host-microbe separation. Using unbiased glycomics, TVP23B-deficient colonocytes have a loss of core-3 O-glycosylation of colonic proteins, which is the major O-glycosylation present on gel forming mucins. TVP23B binds with another Golgi protein, YIPF6, which is similarly critical for intestinal homeostasis. The Golgi proteomes of YIPF6 and TVP23B-deficent colonocytes have a common deficiency of several critical glycosylation enzymes, including those necessary for core-3 glycosylation of mucins. TVP23B is necessary for the formation of the sterile mucin layer of the intestine and its absence disturbs the balance of host and microbe in vivo.

Background: Endoplasmic reticulum (ER) stress is critical in the pathogenesis of inflammatory bowel disease (IBD). ER stress is observed under various pathologic situations, including inflammation. Recent studies have revealed that chronic ER stress augments the suppressive phenotypes of immune cells. Existing data suggest that XBP1, a key component of ER stress pathways is a “synergistic” transcription factor for FoxP3 with critical involvement in Treg development. We hypothesize that ER stress during inflammation is required to activate Treg development and function via XBP1.

Methods: We aimed to validate this hypothesis using both in vivo and in vitro methods. In vivo we used a TNF-driven model of Crohn’s disease, the B6.129S-Tnf^tm2GKl/Jarn strain (TNF^Δ^ARE/+; MGI:3720980) mouse model, characterized by a spontaneous chronic murine ileitis which we crossed with the XBP1^loxpCD4^lckcre to assess XBP1 deletion in an inflamed mouse and confirmed the effects in an adoptive transfer (CD45RB^hi) model of colitis using XBP1^loxpCD4^lckcre CD45RB^hi cells. In vitro, Treg development was assessed in a Treg conversion assay using ER stress activators. Treg function was measured using a 3-day suppression assay and IL-10 output was measured by ELISA and by intracellular cytokine staining.

Results: Isolated CD4⁺CD25⁺FoxP3⁺ Tregs from TNF^Δ^ARE/+-Foxp3GFP mice expressed high levels of XBP1 (20-fold) compared to WT FoxP3-GFP mice, suggesting that the ER stress pathways are activated in FoxP3⁺ Treg cells with inflammation. This was recapitulated using in vitro conversion assays identifying an increase in the spliced (active) form of XBP1. We next demonstrated that glucose depletion and tunicamycin (ER stress activators) increased Treg development by 32% in vitro. With increased expression of XBP1 with inflammation we next evaluated conditional XBP1 knockout mice for development of Tregs but only identified a mild decrease in Treg development without inflammation. We thus hypothesized that inflammation would trigger a more significant effect, therefore we crossed the XBP1^loxpCD4^lckcre to the TNF^Δ^ARE/+. These mice had decreased Treg numbers and more severe inflammation compared to the TNF^Δ^ARE/+littermates. This was confirmed using the CD45RB^hi (XBP1^loxpCD4^lckcre) adoptive transfer model of colitis.

Conclusion: While ER stress has been shown to be important in intestinal inflammation, models have focused on the deleterious effects. Here we demonstrate that ER stress has beneficial anti-inflammatory effects as well and that XBP1 is required for Treg development in the inflamed intestine. Understanding ER stress in a critical cell type for intestinal homeostasis and disease pathogenesis is indispensable. elucidating these mechanisms will impact our understanding of IBD pathogenesis and will provide novel methods to expand more potent Tregs ex vivo or in vivo.

Tom Nguyen

Appearances

ER STRESS PROTEIN XBP1, ACTIVATED BY INFLAMMATION, STIMULATES INTESTINAL REGULATORY T CELL DEVELOPMENT

Speaker

Presenter