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^refHIO 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.

<b>Figure 1. DUOX2<sup>H678R;R701Q</sup> HIO Exhibit Reduced Mitochondrial Complex I Activity and Increased Collagen Content. </b>A) HIO mitochondrial complex I and II activity were measured using oroboros respirometry in HIO carrying the DUOX2 genotypes as shown, n=8-35 per group. B) LAP production was measured using ELISA, n=6 per group. C) ACAT2+ (red), VIM+ (green) and DAPI+ (blue) cells were quantified in ImageJ, n=8 per group. D) HIO collagen protein was detected using Sirius red staining and polarized light microscopy for type I and type III collagen bands (arrows), with HIO collagen content quantified using ImageJ. Data are shown as the mean (SEM), n=8 per group, **p<0.01, ***p<0.001, ****p<0.0001.

Figure 1. DUOX2^H678R;R701Q HIO Exhibit Reduced Mitochondrial Complex I Activity and Increased Collagen Content. A) HIO mitochondrial complex I and II activity were measured using oroboros respirometry in HIO carrying the DUOX2 genotypes as shown, n=8-35 per group. B) LAP production was measured using ELISA, n=6 per group. C) ACAT2+ (red), VIM+ (green) and DAPI+ (blue) cells were quantified in ImageJ, n=8 per group. D) HIO collagen protein was detected using Sirius red staining and polarized light microscopy for type I and type III collagen bands (arrows), with HIO collagen content quantified using ImageJ. Data are shown as the mean (SEM), n=8 per group, **p<0.01, ***p<0.001, ****p<0.0001.

<b>Figure 2. Sub-Mucosal Collagen Content in the CD Ileum and Transplanted Human Intestinal Organoids. </b>A) Overall tissue morphology was assessed using H&E staining, and percent collagen content (arrow) was measured using second harmonic imaging microscopy (SHIM) in Crohn’s Disease (CD) ileal resections and week 12 iPSC-derived HIO following kidney capsule transplantation. B) HIO gross morphology & total collagen content in DUOX2<sup>ref </sup>and DUOX2<sup>H678R;R701Q</sup> HIOs, and C) percent collagen content measured using SHIM is shown as the mean (SEM) for inflammatory (INFL) or fibrotic (FIBR) CD ileal resections, and transplanted (TRX) HIO, n=10-43, *p<0.05, ****p<0.001

Figure 2. Sub-Mucosal Collagen Content in the CD Ileum and Transplanted Human Intestinal Organoids. A) Overall tissue morphology was assessed using H&E staining, and percent collagen content (arrow) was measured using second harmonic imaging microscopy (SHIM) in Crohn’s Disease (CD) ileal resections and week 12 iPSC-derived HIO following kidney capsule transplantation. B) HIO gross morphology & total collagen content in DUOX2^refand DUOX2^H678R;R701Q HIOs, and C) percent collagen content measured using SHIM is shown as the mean (SEM) for inflammatory (INFL) or fibrotic (FIBR) CD ileal resections, and transplanted (TRX) HIO, n=10-43, *p<0.05, ****p<0.001

Patrizia Tornabene

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