Society: AGA

Background: RNA-binding motif protein 47 (RBM47) is a component of the APOBEC-1 dependent C-to-U RNA editosome required for RNA editing. However, the range of targets and functions of RBM47 in intestine is unknown. Aims: We studied spontaneous and induced tumor susceptibility in intestine-specific Rbm47 knockout mice (Rbm47^IKO) and human colorectal cancer (CRC). Results: We observed spontaneous intestinal and colonic adenomatous polyps in 92% (11/12) of 12-month-old chow-fed Rbm47^IKO mice, while only 8% (1/13) of aged Rbm47^flox controls developed polyps. High milk fat-fed Rbm47 ^IKO mice exhibited enhanced polyposis with a 3-fold and 2-fold increase in total small intestine (5.9 vs 2) and colon (1.9 vs 0.71) polyp count respectively compared to control mice (Fig 1A). In the Apc^Min/+background, total numbers of polyps were similar by genotype, but Rbm47 ^IKO Apc^Min/+mice developed significantly larger polyps (3.04 vs 1.82 mm²) in the small intestine compared to Rbm47^f/f Apc^Min/+mice. In contrast, colonic polyp burden was decreased in Rbm47 ^IKO Apc^Min/+mice (4.3 vs 7.4), with no change in polyp size (Fig 1B). We also observed upregulation of stem cell markers (Lgr5, Lrig 1, Atoh 1), WNT signaling (Wnt 11, Wnt7b, Fzd8, Fzd3, Ccnd1) pathway genes. Publicly available single cell RNA-seq data (retrieved from PMID 29144463) revealed increased expression of Rbm47 in mature intestinal enterocytes compared to stem and transit amplifying cells (Fig 2A), suggesting that RBM47 normally suppresses Wnt activity and the stem cell program. In addition, we observed increased expression of anti-oxidative pathway genes (Fndc5, Fndc4, Gsta4, Gstm1, Gsta3, Sod1, Nqo1) in Rbm47 ^IKO intestine. In order to examine the mechanisms involved in Rbm47-dependent regulation of mRNAs we undertook polysome profiling and mRNA stability assays on candidate target RNAs. We observed Fndc5 mRNA exhibited increased stability in Rbm47 ^IKO enteroids and colonoids (Fig 2B). We further extended these findings to humans where we observed decreased expression of Rbm47 mRNA (–ΔCT -2.53±3.12 vs 0.16±3.33, P<.0001) in CRC samples (n=24) vs paired uninvolved tissue. Tumors from CRC patients with higher N-stage exhibited lower expression of Rbm47 compared to patients with lower stage (Fig 2C). TCGA analysis showed Rbm47 overexpression (third quartile vs first quartile) is associated with improved overall (OS, HR=0.4, P=.01) and progression-free survival (PFS, HR =0.44; P=.001)) in CRC. Conclusion: Rbm47^IKO mice demonstrate increased susceptibility to both spontaneous and diet induced polyposis and exhibit accelerated polyposis in the Apc^Min/+background through upregulation of stem cell markers, Wnt signaling, and anti-oxidative pathways. RBM47 also exhibits a tumor suppressive role in human CRC as evidenced by findings of reduced expression of Rbm47 and association with OS and PFS.

Figure 1. A: Representative gross pictures of small intestine from <i>Rbm47<sup>f/f</sup></i><sup> </sup>and <i>Rbm47-IKO</i> mice fed with high milk fat diet for 6 months. B: Representative gross pictures of small intestine from <i>Apc<sup>min/+</sup></i> <i>Rbm47<sup>f/f</sup> </i>and <i>Apc<sup>min/+</sup>Rbm47-IKO</i> mice

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

$Figure 2. A. Top: <i>Rbm47</i> mRNA expression in small intestine epithelial cells with different levels of maturation (cell count: enterocyte 1041, EP (enterocyte progenitor) 1957, stem (3679), and TA (transit amplifying) 2311) derived from single cell RNA-seq data. Bottom: Three-dimensional exhibition of single cell RNA-seq survey for <i>Rbm 47</i> mRNA expression in murine intestinal epithelial cells. B. Relative <i>Fndc5</i> mRNA abundance in successive time-points after actinomycin treatment as a fraction of baseline <i>Fndc5</i> in enteroids (top) and colonoids (bottom). C. top:<i> Rbm47</i> mRNA expression in uninvolved and paired tumor tissue samples from colorectal cancer patients. Bottom: <i>Rbm47 </i>mRNA expressions in colorectal cancer patients with low (0) and high (1-2) N stage.$

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.

Background: The intestinal epithelium provides the first line of defense against luminal stimuli and the underlying immune cells. Expansion of intestinal pathobionts, such as adherent-invasive E. coli (AIEC), are strongly implicated in the pathogenesis of IBD. Our previous work showed, mice deficient for the expression of the IBD risk gene, Ptpn2, exhibit pronounced expansion of a novel murine adherent-invasive Escherichia coli (mAIEC) strain. This study aimed to investigate how PTPN2 expression in intestinal epithelial cells restricts mAIEC colonization in vivo.
Methods: Tamoxifen-inducible, intestinal epithelial cell-specific knockout mice (Ptpn2^d^IEC). Ptpn2^d^IEC and control littermates (Ptpn2^fl/fl) were infected with either PBS, non-invasive E. coli K12, or fluorescent-tagged mAIEC (mAIEC^red) 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: Ptpn2^d^IEC mice exhibited intestinal region-specific higher mAIEC^red - but not K12 - bacterial load in distal colon tissue compared to Ptpn2^fl/fl mice (P=0.038; n=9-12). To identify if the higher susceptibility to mAIEC^red 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 Ptpn2^d^IEC vs. Ptpn2^fl/fl mice, after mAIEC^red 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 Ptpn2^d^IEC mice post mAIEC^red infection compared to Ptpn2^fl/fllittermates (n=4-6). Next, we investigated if expression levels of cytokines that stimulate AMP expression were lower in Ptpn2^d^IEC mice after infection. We found that mRNA and protein levels of the IL-22 cytokine were significantly lower in Ptpn2^d^IECmice infected with mAIEC^red 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 Ptpn2^d^IEC mice. Th17⁺ T-cells are the major producers of IL-22. Expression of the Th17 T cell marker RORgT was lower in the Ptpn2^d^IEC/mAIEC^redgroup (P= 0.0103), while protein expression of the mature T-cell marker, CD3, was significantly reduced of Ptpn2^d^IEC/mAIEC^redmice compared to Ptpn2^fl/fl/mAIEC^red(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.

Background: Congenital Diarrheas and Enteropathies (CoDEs) are rare monogenic disorders that result in dehydrating diarrhea and malnutrition. Mutations in the gene myosin VB (MYO5B) result in a severe CoDE disorder, microvillus inclusion disease (MVID). Patients with MVID have severe fluid and nutrient malabsorption secondary to loss or malformation of epithelial microvilli and loss of apical electrolyte transporters. Clinical management of MVID is currently only supportive with no known disease modifying strategies. Electroneutral sodium transport via the intestinal epithelial Na^+/H⁺exchanger, NHE3, is critical for normal intestinal fluid absorption and has been proposed to be decreased in MVID. Previous data from mouse models of MVID have suggested that altering Wnt/Notch signaling can recover apical proteins in epithelial cells. Here we investigate epithelial structure and NHE3 activity in patient-derived enteroids with two different loss of function mutations of MYO5B(Q526K and P660L) and the effects of Notch inhibition.
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.

Introduction: Microfold (M) cells are specialized epithelial cells that play an integral role in immune surveillance of the gut lumen. They act as gatekeepers within the follicle-associated epithelium (FAE) of Peyer’s patches to regulate antigen delivery to immune cells of gut-associated lymphoid tissue. Inflammation in the FAE is associated with Crohn’s Disease; however, studies designed to understand how human M cells contribute to disease onset and/or pathogenesis remain incomplete.
Aims: Since M cells differentiate from Lgr5+ stem cells, we utilized human enteroids to identify factors that regulate human M cell expression and function. Proteomics of M cell-expressing enteroids revealed enrichment of coronin 1A (CORO1A), a phagocytosis protein expressed in immune cells. Since M cells engulf luminal gut antigens via phagocytosis, we hypothesized that CORO1A is uniquely expressed in the human FAE and plays a role in gut antigen uptake and transcytosis in human M cells.
Methods: Human ileal enteroids were grown as monolayers and differentiated (no Wnt3a, R-spondin-1) for at least 5 days. To express M cells, monolayers were exposed to RANKL and TNF during differentiation. Human expression of CORO1A was validated by immunoblot and confocal microscopy of ileal biopsies from healthy donors. M cell monolayers, expressing or lacking CORO1A (via shRNA knockdown (KD)), were exposed to commensal and pathogenic strains of E. coli and Candida albicans to define the contribution of gut microbes to stimulate M cell transcytosis, as measured by uptake of fluorescent beads and immunoglobulins.
Results: CORO1A was expressed in human enteroid monolayers differentiated to express M cells, but not in monolayers differentiated to mimic the villus epithelium. Similarly, CORO1A was detected in the FAE, but not in crypt or villus epithelia in ileal biopsies. This staining was unique for human, as CORO1A expression in mouse ileum was confined to lamina propria and lymphoid follicle immune cells. CORO1A expression in human FAE was also confirmed by single cell RNAseq. CORO1A KD decreased the expression of glycoprotein 2 (mature M cell marker), while other markers such as CD14, ICAM1, Spi-B, SOX8, and FcRN were unchanged. CORO1A KD also impaired uptake/transcytosis of IgG (via FcRN), enteric pathogens, as well as pathogen-stimulated uptake/transcytosis of beads by human M cells.
Conclusions: The studies show: (1) human enteroids can be differentiated to model CORO1A expression in the intact human FAE; (2) CORO1A is necessary for terminal differentiation and function of human M cells; and (3) CORO1A is required for pathogen-stimulated uptake and transcytosis of luminal gut cargo. We conclude that M cell-expressing enteroids are a relevant human model to interrogate tropism for host-pathogen interactions, mechanisms of luminal surveillance, and response to oral vaccine therapies.

Background and aims: Wnt2b is a canonical Wnt ligand which was previously thought to be fully redundant with other Wnts in the intestinal epithelium. Humans with Wnt2b deficiency have severe intestinal disease, however, highlighting the critical importance of Wnt2b. Therefore, we sought to understand why Wnt2b is critical for intestinal health.

Methods: We investigated the intestinal health Wnt2b knock out (KO) mice at baseline. We also assessed the impacts of inflammatory challenge to the small intestine with anti-CD3ε antibody and to the colon with dextran sodium sulfate (DSS) administration. In addition, we generated human intestinal organoids (HIOs) and enteroids from Wnt2b deficient patients for transcriptional and histologic analyses.

Results: In contrast to humans, Wnt2b KO mice were healthy and had normal small intestinal and colonic histology at baseline but decreased intestinal stem cells. Small intestinal response to anti-CD3ε was similar to controls. The colons of Wnt2b KO mice were severely affected by DSS compared to controls (Figure 1), and this was due to increased inflammation and immune infiltrates. In contrast, HIOs generated from Wnt2b-deficient patients showed a developmental phenotype with abnormal epithelial organization and enriched mesenchymal signature (Fig. 2), and epithelial enteroids showed restricted growth.

Conclusion: Wnt2b deficiency in mice more severely impacts the colon than the small intestine and results in reduced intestinal stem cells. In human Wnt2b deficiency the epithelium is severely disorganized from the early stages of intestinal lineage specification in addition to having diminished intestinal stem cells and proliferation.

Figure 1. DSS colitis in control versus Wnt2b KO mice. Mice were treated with 2.5% dextran sodium sulfate for 4 days and then euthanized and the colons were assessed using periodic acid Schiff (upper panel) or hematoxylin & eosin (lower panel). e - epithelium, s - submucosa, m – mesenchyme.

Figure 1. DSS colitis in control versus Wnt2b KO mice. Mice were treated with 2.5% dextran sodium sulfate for 4 days and then euthanized and the colons were assessed using periodic acid Schiff (upper panel) or hematoxylin & eosin (lower panel). e - epithelium, s - submucosa, m – mesenchyme.

<b>Figure 2.</b> <b>Wnt2b Deficient Human Intestinal Organoids (HIOs) Have Altered Epithelial-Mesenchymal Organization. </b>Confocal microscopy for indicated proteins by immunofluorescence detailing abnormal organization of epithelial tissue component.

Figure 2. Wnt2b Deficient Human Intestinal Organoids (HIOs) Have Altered Epithelial-Mesenchymal Organization. Confocal microscopy for indicated proteins by immunofluorescence detailing abnormal organization of epithelial tissue component.

Michael Helmrath

Cincinnati Children's Hospital

Appearances