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.

Jay Thiagarajah

Boston Children's Hospital

Appearances