Intestinal epithelial cell (IEC) junctions constitute a robust barrier to invasion by viruses, bacteria and exposure to ingested agents. Previous studies showed that microgravity compromises the human immune system and increases enteropathogen virulence. However, the effects of microgravity on epithelial barrier function are poorly understood. The aims of this study were to identify if simulated microgravity alters intestinal epithelial barrier function (permeability), and susceptibility to barrier-disrupting agents. IECs (HT-29.cl19a) were cultured on microcarrier beads in simulated microgravity using a rotating wall vessel (RWV) for 18 days prior to seeding on semipermeable supports to measure ion flux (transepithelial electrical resistance (TER)) and FITC-dextran (FD4) permeability over 14 days. RWV cells showed delayed apical junction localization of the tight junction proteins, occludin and ZO-1. The alcohol metabolite, acetaldehyde, significantly decreased TER and reduced junctional ZO-1 localization, while increasing FD4 permeability in RWV cells compared with static, motion and flask control cells. In conclusion, simulated microgravity induced an underlying and sustained susceptibility to epithelial barrier disruption upon removal from the microgravity environment. This has implications for gastrointestinal homeostasis of astronauts in space, as well as their capability to withstand the effects of agents that compromise intestinal epithelial barrier function following return to Earth.

Background

VSL#3 is a probiotic compound that has been used in the treatment of inflammatory bowel disease. T-cell protein tyrosine phosphatase (TCPTP) is the protein product of the inflammatory bowel disease candidate gene, PTPN2, and we have previously shown that it protects epithelial barrier function. The aim of this study was to investigate whether VSL#3 improves intestinal epithelial barrier function against the effects of the inflammatory bowel disease-associated proinflammatory cytokine, interferon-gamma (IFN-γ) through activation of TCPTP.

Methods

Polarized monolayers of T84 intestinal epithelial cells were treated with increasing concentrations of VSL#3 to determine effects on TCPTP expression and enzymatic activity. Therapeutic effects of VSL#3 against barrier disruption by IFN-γ were measured by transepithelial electrical resistance and fluorescein isothiocyanate-dextran permeability. A novel TCPTP-deficient HT-29 intestinal epithelial cell line was generated to study the role of TCPTP in mediating the effects of VSL#3. Tight junction protein distribution was assessed with confocal microscopy.

Results

VSL#3 increased TCPTP protein levels and enzymatic activity, correlating with a VSL#3-induced decrease in IFN-γ signaling. VSL#3 corrected the decrease in transepithelial electrical resistance and the increase in epithelial permeability induced by IFN-γ. Moreover, the restorative effect of VSL#3 against IFN-γ signaling, epithelial permeability defects, altered expression and localization of the tight junction proteins claudin-2, occludin, and zonula occludens-1, were not realized in stable TCPTP/(PTPN2)-deficient HT-29 intestinal epithelial cells.

Conclusions

VSL#3 reduces IFN-γ signaling and IFN-γ-induced epithelial barrier defects in a TCPTP-dependent manner. These data point to a key role for TCPTP as a therapeutic target for restoration of barrier function using probiotics.

Linaclotide, a synthetic guanylyl cyclase C (GC-C) agonist, and the prostone analog, Lubiprostone, are approved to manage chronic idiopathic constipation and constipation-predominant irritable bowel syndrome. Lubiprostone also protects intestinal mucosal barrier function in ischemia. GC-C signaling regulates local fluid balance and other components of intestinal mucosal homeostasis including epithelial barrier function. The aim of this study was to compare if select dosing regimens differentially affect linaclotide and lubiprostone modulation of ion transport and barrier properties of normal human colonic mucosa. Normal sigmoid colon biopsies from healthy subjects were mounted in Ussing chambers. Tissues were treated with linaclotide, lubiprostone, or vehicle to determine effects on short-circuit current (I sc). Subsequent I sc responses to the cAMP agonist, forskolin, and the calcium agonist, carbachol, were also measured to assess if either drug caused desensitization. Barrier properties were assessed by measuring transepithelial electrical resistance. I sc responses to linaclotide and lubiprostone were significantly higher than vehicle control when administered bilaterally or to the mucosal side only. Single versus cumulative concentrations of linaclotide showed differences in efficacy while cumulative but not single dosing caused desensitization to forskolin. Lubiprostone reduced forskolin responses under all conditions. Linaclotide and lubiprostone exerted a positive effect on TER that was dependent on the dosing regimen. Linaclotide and lubiprostone increase ion transport responses across normal human colon but linaclotide displays increased sensitivity to the dosing regimen used. These findings may have implications for dosing protocols of these agents in patients with constipation.

Background

Gastroenteritis is the most common manifestation of nontyphoidal Salmonella enterica infections, but little is known about the pathogenesis of diarrhea in this infection

Methods

To determine whether polymorphonuclear neutrophils (PMNs) are required for diarrhea for Salmonella colitis, we infected kanamycin-pretreated interleukin 8R (IL-8R) mutant mice and controls, both with nonmutant Slc11a1 (Nramp1, ItyR). We compared the 2 mouse strains for increases in fecal water content (diarrhea) 3 days after infection, changes in expression of ion transporters in colonic epithelial cells, proliferation of epithelial cells, and severity of infection as measured by colony-forming units (CFUs).

Results

The IL-8R knockout mice had fewer PMNs in the colon but the other variables we measured were unaffected except for an increase in CFUs in the colon. The pathologic changes in the cecum were similar in both groups except for the lack of PMNs in the IL-8R knockout mice. There was minimal damage to the colon more distally.

Conclusions

In the early stage of Salmonella colitis, PMNs are not required for diarrhea or for the decrease in expression of colonic epithelial cell apical ion transporters. They contribute to defense against infection in the cecum but not extracolonically at this stage of Salmonella colitis.

Congenital tufting enteropathy (CTE) is a rare chronic diarrheal disease of infancy caused by mutations in epithelial cell adhesion molecule (EpCAM). Previously, a murine CTE model showed mis-localization of EpCAM away from the basolateral cell surface in the intestine. Here we demonstrate that mutant EpCAM accumulated in the endoplasmic reticulum (ER) where it co-localized with ER chaperone, GRP78/BiP, revealing potential involvement of ER stress-induced unfolded protein response (UPR) pathway in CTE. To investigate the significance of ER-localized mutant EpCAM in CTE, activation of the three UPR signaling branches initiated by the ER transmembrane protein components IRE1, PERK, and ATF6 was tested. A significant reduction in BLOS1 and SCARA3 mRNA levels in EpCAM mutant intestinal cells demonstrated that regulated IRE1-dependent decay (RIDD) was activated. However, IRE1 dependent XBP1 mRNA splicing was not induced. Furthermore, an increase in nuclear-localized ATF6 in mutant intestinal tissues revealed activation of the ATF6-signaling arm. Finally, an increase in both the phosphorylated form of the translation initiation factor, eIF2α, and ATF4 expression in the mutant intestine provided support for activation of the PERK-mediated pathway. Our results are consistent with a significant role for UPR in gastrointestinal homeostasis and provide a working model for CTE pathophysiology.

Unlabelled

Congenital tufting enteropathy (CTE) is a devastating diarrheal disease seen in infancy that is typically associated with villous changes and the appearance of epithelial tufts. We previously found mutations in epithelial cell adhesion molecule (EpCAM) to be causative in CTE. We developed a knock-down cell model of CTE through transfection of an EpCAM shRNA construct into T84 colonic epithelial cells to elucidate the in vitro role of EpCAM in barrier function and ion transport. Cells with EpCAM deficiency exhibited decreased electrical resistance, increased permeability, and decreased ion transport. Based on mutations in CTE patients, an in vivo mouse model was developed, with tamoxifen-inducible deletion of exon 4 in Epcam resulting in mutant protein with decreased expression. Tamoxifen treatment of Epcam (Δ4/Δ4) mice resulted in pathological features of villous atrophy and epithelial tufts, similar to those in human CTE patients, within 4 days post induction. Epcam (Δ4/Δ4) mice also showed decreased expression of tight junctional proteins, increased permeability, and decreased ion transport in the intestines. Taken together, these findings reveal mechanisms that may underlie disease in CTE.

Key messages

Knock-down EpCAM cell model of congenital tufting enteropathy was developed. In vivo inducible mouse model was developed resulting in mutant EpCAM protein. Cells with EpCAM deficiency demonstrated barrier and ion transport dysfunction. Tamoxifen-treated Epcam (Δ4/Δ4) mice demonstrated pathological features. Epcam (Δ4/Δ4) mice showed improper barrier function and ion transport.

Background & aims

Salmonella enterica serovar Typhimurium is an enteropathogen that causes self-limiting diarrhea in healthy individuals, but poses a significant health threat to vulnerable populations. Our understanding of the pathogenesis of Salmonella-induced diarrhea has been hampered by the lack of a suitable mouse model. After a dose of oral kanamycin, Salmonella-infected congenic BALB/c.D2(NrampG169) mice, which carry a wild-type Nramp1 gene, develop clear manifestations of diarrhea. We used this model to elucidate the pathophysiology of Salmonella-induced diarrhea.

Methods

BALB /c.D2(NrampG169) mice were treated with kanamycin and then infected with wild-type or mutant Salmonella by oral gavage. Colon tissues were isolated and Ussing chambers, quantitative polymerase chain reaction, immunoblot, and confocal microscopy analyses were used to study function and expression of ion transporters and cell proliferation.

Results

Studies with Ussing chambers demonstrated reduced basal and/or adenosine 3',5'-cyclic monophosphate-mediated electrogenic ion transport in infected colonic tissues, attributable to changes in chloride or sodium transport, depending on the segment studied. The effects of infection were mediated, at least in part, by effector proteins secreted by the bacterial Salmonella pathogenicity island 1- and Salmonella pathogenicity island-2-encoded virulence systems. Infected tissue showed reduced expression of the chloride-bicarbonate exchanger down-regulated in adenoma in surface colonic epithelial cells. Cystic fibrosis transmembrane conductance regulator was internalized in colonic crypt epithelial cells without a change in overall expression levels. Confocal analyses, densitometry, and quantitative polymerase chain reaction revealed that expression of epithelial sodium channel β was reduced in distal colons of Salmonella-infected mice. The changes in transporter expression, localization, and/or function were accompanied by crypt hyperplasia in Salmonella-infected mice.

Conclusions

Salmonella infection induces diarrhea by altering expression and/or function of transporters that mediate water absorption in the colon, likely reflecting the fact that epithelial cells have less time to differentiate into surface cells when proliferation rates are increased by infection.

The prostone analog, lubiprostone, is approved to manage constipation-predominant irritable bowel syndrome. Lubiprostone also protects intestinal mucosal barrier function in animal models of colitis. The aim of this study was to determine if lubiprostone improves barrier properties in isolated colonic biopsies from Crohn's disease (CD) and ulcerative colitis (UC) patients. Sigmoid colon biopsies from healthy subjects, CD and UC patients in remission, and CD patients with active disease were mounted in Ussing chambers. Tissues were treated with lubiprostone or vehicle to determine the effects on transepithelial electrical resistance (TER), FITC-dextran 4kD (FD4) permeability, and electrogenic ion transport responses to forskolin and carbachol. Localization of the tight junction protein, occludin, was determined by immunofluorescence. Lubiprostone significantly increased ion transport across control, CD and UC remission biopsies but not active CD. Lubiprostone selectively improved TER in both CD remission and active disease biopsies but not in control or UC biopsies. The improved TER was associated with increased membrane localization of occludin. Lubiprostone selectively improved barrier properties of biopsies from CD patients vs. UC and independent of an ion transport response. These data indicate that lubiprostone has potential efficacy in improving mucosal integrity in Crohn's disease.

Genome-wide association studies revealed that loss-of-function mutations in protein tyrosine phosphatase non-receptor type 2 (PTPN2) increase the risk of developing chronic immune diseases, such as inflammatory bowel disease (IBD) and celiac disease. These conditions are associated with increased intestinal permeability as an early etiological event. The aim of this study was to examine the consequences of deficient activity of the PTPN2 gene product, T cell protein tyrosine phosphatase (TCPTP), on intestinal barrier function and tight junction organization in vivo and in vitro. Here, we demonstrate that TCPTP protected against intestinal barrier dysfunction induced by the inflammatory cytokine IFN-γ by 2 mechanisms: it maintained localization of zonula occludens 1 and occludin at apical tight junctions and restricted both expression and insertion of the cation pore-forming transmembrane protein, claudin-2, at tight junctions through upregulation of the inhibitory cysteine protease, matriptase. We also confirmed that the loss-of-function PTPN2 rs1893217 SNP was associated with increased intestinal claudin-2 expression in patients with IBD. Moreover, elevated claudin-2 levels and paracellular electrolyte flux in TCPTP-deficient intestinal epithelial cells were normalized by recombinant matriptase. Our findings uncover distinct and critical roles for epithelial TCPTP in preserving intestinal barrier integrity, thereby proposing a mechanism by which PTPN2 mutations contribute to IBD.