INTESTINAL EPITHELIAL PTPN2 RESTRICTS ADHERENT-INVASIVE <i>E. COLI </i>COLONIZATION AND PROMOTES IL-22 PRODUCTION IN MICE

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.

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^dIEC). Ptpn2^dIEC 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^dIEC 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^dIEC 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^dIEC mice post mAIEC^red infection compared to Ptpn2^fl/fl littermates (n=4-6). Next, we investigated if expression levels of cytokines that stimulate AMP expression were lower in Ptpn2^dIEC mice after infection. We found that mRNA and protein levels of the IL-22 cytokine were significantly lower in Ptpn2^dIEC mice 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^dIEC mice. Th17⁺ T-cells are the major producers of IL-22. Expression of the Th17 T cell marker RORgT was lower in the Ptpn2^dIEC/mAIEC^red group (P= 0.0103), while protein expression of the mature T-cell marker, CD3, was significantly reduced of Ptpn2^dIEC/mAIEC^red mice 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.