PRO-FIBROTIC DUOX2 GENE VARIANTS IMPAIR CELL METABOLISM AND INCREASE TISSUE COLLAGEN CONTENT IN AN INDUCED PLURIPOTENT STEM CELL DERIVED HUMAN INTESTINAL ORGANOID MODEL OF ILEAL CROHN’S DISEASE

Introduction: Missense mutations in the epithelial NADPH oxidase DUOX2 are associated with strictures in children with Crohn’s Disease (CD). Mitochondrial dysfunction and extra-cellular matrix production are potential mediators of this genetic association and have been previously linked to ileal fibrostenosis.

Hypothesis: We hypothesized that DUOX2 genetic variation would be associated with decreased mitochondrial activity and increased collagen content in a Human Intestinal Organoid (HIO) model system.

Methods: Induced pluripotent stem cell (iPSC) lines were derived from pediatric CD patients carrying the DUOX2 reference genotype (DUOX2^ref). DUOX2^H678R;R701Q iPSC lines were generated using CRISPER-Cas9 gene editing to introduce missense mutations associated with strictures (H678R and R701Q). iPSC lines were differentiated into HIO and studied in either tissue culture or following kidney capsule transplantation into NSG^TM immunodeficient mice. Oroboros respirometry measured mitochondrial complex I and complex II activity. Pro-fibrotic TGFB1 gene expression was measured by RT-PCR, and secretion of the LAP complex containing TGFB1 was measured by ELISA. ACTA2+ myofibroblasts were detected using immunofluorescence. HIO collagen content was measured using Sirius Red staining with polarized light microscopy, and second harmonic imaging microscopy (SHIM).

Results: HIO mitochondrial complex I activity was reduced in HIO carrying the pro-fibrotic DUOX2 genotypes under basal conditions. This was specific, as mitochondrial complex II activity did not vary with DUOX2 genotype (Fig. 1A). TGFB1 expression and LAP secretion were increased in DUOX2^H678R;R701Q HIO under basal and TGFB1 stimulated conditions (Fig. 1B). Consistent with this, we observed a trend towards an increased frequency of ACTA2+ myofibroblasts in DUOX2^H678R;R701Q HIO (Fig. 1C), which reached significance for both Type I and Type III collagen content (Fig. 1D). Following murine kidney capsule transplantation, the HIO sub-mucosal collagen architecture and smooth muscle layers matched CD ileal stricture resections (Fig. 2A). We observed increased growth of DUOX2^H678R;R701Q HIO relative to DUOX2^ref HIO following transplantation (Fig. 2B), with quantification of HIO percent collagen content by SHIM demonstrating comparable levels to fibrotic CD ileum, increased relative to inflammatory CD ileum (Fig. 2C).

Conclusions: The pro-fibrotic DUOX2 genotype is associated with reduced mitochondrial function and increased collagen content in the tissue culture HIO model system, replicating pathways observed in refractory ileal CD patients. Following murine transplantation, DUOX2^H678R;R701Q HIO exhibit expansion of the submucosal collagen architecture and smooth muscle layers, replicating CD ileal resections. These data provide insight into DUOX2 regulation of barrier function and wound healing in ileal CD.