Loss of PTPN2 in Intestinal Epithelial Cells Increases Invasion of Human Intestinal Epithelial Cells and Intestinal Permeability in Mice by Adherent‐invasive E. coli

Abstract

Background : Alterations in epithelial tight junction proteins can compromise its ability to act as a barrier between luminal contents and underlying tissues, thereby increasing intestinal permeability, an early event in inflammatory bowel disease (IBD). Additionally, shifts in the gut microbiota and expansion of pathobionts such as adherent-invasive Escherichia coli (AIEC) are associated with the pathogenesis of IBD. Mice deficient in expression of the IBD gene candidate, Ptpn2,exhibit increased intestinal permeability and a pronounced expansion of a novel murine adherent-invasive Escherichia coli (mAIEC). This study aimed to investigate how PTPN2 expression in intestinal epithelial cells modifies mAIEC invasiveness and host-barrier properties after infection using in vitro and in vivo models. Methods First, we generated a fluorescent-tagged mAIEC strain — mAIECred. Caco-2BBe intestinal epithelial cells (IEC), stably expressing either scrambled shRNA or shRNA against Ptpn2, were infected with mAIECred. Tamoxifen-inducible, intestinal epithelial cell-specific knockout mice (Ptpn2∆IEC) and control littermates (Ptpn2fl/fl) were infected with either PBS, E. coli K12 or mAIECred. FITC-Dextran (FD4) and Rhodamine-Dextran (RD70) permeability probes were administered by oral-gavage. Post-necropsy, bacterial colonies were enumerated from mouse tissues. Results First we tested if PTPN2 deficiency in human IEC altered mAIECred invasion. Immunofluorescence showed that PTPN2 knockdown (KD) IECs exhibited increased bacterial invasion compared to control IECs (n=3). To confirm our results in an in vivo model, we infected Ptpn2∆IEC mice with either E. coli K12 or mAIECred or administered PBS as vehicle control. E. coli K12 increased FD4 but not RD70 permeability in Ptpn2∆IEC mice compared with Ptpn2fl/fl mice (P=0.0025; n=5-9) while mAIECred caused an even greater increase in FD4 permeability in Ptpn2∆IEC vs. Ptpn2fl/fl mice (P< 0.0001; n=6-9). Ptpn2∆IEC mice also exhibited higher mAIECred - but not K12 - bacterial load in distal colon tissue compared to Ptpn2fl/fl mice (P=0.038; n=9-12). As FD4 permeability was increased in Ptpn2∆IEC mice infected with K12 and mAIECred, we wanted to determine if this corresponded with changes in expression of relevant tight junction proteins. Western blot analysis from isolated IEC's revealed Ptpn2∆IEC mice infected with mAIECred had decreased E-cadherin (P=0.0142; n=4) and Occludin (P=0.0030; n=4). Conversely, we observed increased Claudin-2 in PBS and K12-infected Ptpn2∆IEC vs. Ptpn2fl/fl mice (P=0.015; n=4). mAIECred increased Claudin-2 levels above control(s) to comparable levels in Ptpn2fl/fl and Ptpn2∆IEC mice (P= 0.025; n=4). Conclusion Intestinal epithelial PTPN2 is critical in maintaining intestinal homeostasis by restricting bacterial invasion and preserving intestinal barrier function. Given that loss of PTPN2 in vivo also provokes expansion of AIEC, these studies indicate that PTPN2 is an important mediator of host-microbiome crosstalk.