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RESCUE OF EPITHELIAL STRUCTURE AND FUNCTION IN MICROVILLUS INCLUSION DISEASE PATIENT-DERIVED ENTEROIDS BY NOTCH INHIBITION

Figure 1. A: Representative gross pictures of small intestine from Rbm47f/f and Rbm47-IKO mice fed with high milk fat diet for 6 months. B: Representative gross pictures of small intestine from Apcmin/+ Rbm47f/f and Apcmin/+Rbm47-IKO mice

Figure 2. A. Top: Rbm47 mRNA expression in small intestine epithelial cells with different levels of maturation (cell count: enterocyte 1041, EP (enterocyte progenitor) 1957, stem (3679), and TA (transit amplifying) 2311) derived from single cell RNA-seq data. Bottom: Three-dimensional exhibition of single cell RNA-seq survey for Rbm 47 mRNA expression in murine intestinal epithelial cells. B. Relative Fndc5 mRNA abundance in successive time-points after actinomycin treatment as a fraction of baseline Fndc5 in enteroids (top) and colonoids (bottom). C. top: Rbm47 mRNA expression in uninvolved and paired tumor tissue samples from colorectal cancer patients. Bottom: Rbm47 mRNA expressions in colorectal cancer patients with low (0) and high (1-2) N stage.
Methods: Tamoxifen-inducible, intestinal epithelial cell-specific knockout mice (Ptpn2dIEC). Ptpn2dIEC and control littermates (Ptpn2fl/fl) were infected with either PBS, non-invasive E. coli K12, or fluorescent-tagged mAIEC (mAIECred) for 4 consecutive days. After sacrifice, bacterial colonies were enumerated in mouse tissues. mRNA and protein expression was assayed in intestinal epithelial cells (IECs) or whole tissue lysates by PCR and Western blot.
Results: Ptpn2dIEC mice exhibited intestinal region-specific higher mAIECred - but not K12 - bacterial load in distal colon tissue compared to Ptpn2fl/fl mice (P=0.038; n=9-12). To identify if the higher susceptibility to mAIECred infection was associated with altered host defenses, we measured levels of anti-microbial peptide (AMPs) in these mice. Ileal RNA expression of the alpha-defensins, Defa5 and Defa6, were significantly lower (P=0.008, 0.0182 respectively; n=3-5) in Ptpn2dIEC vs. Ptpn2fl/fl mice, after mAIECred but not K12 infection. Expression of the protease matrilysin-7 (MMP7) - which is responsible for proteolytic cleavage of alpha-defensins – was significantly decreased in ileum (P=0.042) and distal colon (P=0.007) of Ptpn2dIEC mice post mAIECred infection compared to Ptpn2fl/fl littermates (n=4-6). Next, we investigated if expression levels of cytokines that stimulate AMP expression were lower in Ptpn2dIEC mice after infection. We found that mRNA and protein levels of the IL-22 cytokine were significantly lower in Ptpn2dIEC mice infected with mAIECred vs. floxed littermates (P=0.0234 and P=0.0436 respectively; n=8-10). Next, we investigated if the reduction of IL-22 was due to lower IL-22 producing immune cells in Ptpn2dIEC mice. Th17+ T-cells are the major producers of IL-22. Expression of the Th17 T cell marker RORgT was lower in the Ptpn2dIEC/mAIECred group (P= 0.0103), while protein expression of the mature T-cell marker, CD3, was significantly reduced of Ptpn2dIEC/mAIECred mice compared to Ptpn2fl/fl/mAIECred (P=0.0398; n=4) controls.
Conclusion: Intestinal epithelial PTPN2 plays an important role in maintaining intestinal homeostasis by mounting protective anti-microbial peptide and cytokine responses against IBD-associated pathobionts.
Methods: Enteroids were generated from duodenal biopsies from patients with pathogenic mutations (Q526K and P660L) in MYO5B, and age-matched healthy controls and imaged using confocal, light-sheet, multiplex immunofluorescence, and electron microscopy. Enteroids were grown in enteroid expansion media (high Wnt/Rspondin/Noggin/EGF), differentiation media (low Wnt/Rspondin/Noggin), and differentiation media with the γ-secretase inhibitor, DAPT, and assessed for expression of markers of epithelial cell differentiation. Enteroids were also grown as monolayers and NHE3 activity measured by live fluorescence imaging of intracellular pH changes with or without DAPT.
Results: Electron and super-resolution (STED) microscopy of MVID enteroids revealed abnormal microvilli and reduced NHE3 in the apical brush border compared to healthy controls. Brightfield imaging and transcriptional analysis of MVID enteroids showed defective differentiation of MVID enteroids that is partially rescued by DAPT treatment. Assessment of the brush border by electron microscopy and NHE3 localization by STED microscopy also indicated recovery of apical brush border in MVID (P660L and Q526K) epithelial cells following DAPT treatment. Functional assessment of NHE3 activity in MVID enteroids by live-cell fluorescence imaging indicated highly reduced NHE3-mediated transport in primary MVID epithelial cells (5-10% of control) that was significantly rescued by notch inhibition (75% of control).
Conclusion: MVID patient-derived primary cells show defective differentiation, a structurally abnormal brush border, and reduced NHE3 transport activity. We show for the first time that epithelial defects in human MVID can be functionally and structurally rescued by altering Wnt/Notch signaling, opening a novel therapeutic avenue for this severe genetic intestinal disorder.
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