Ronald R. Marchelletta

Spermidine stimulates T-cell protein tyrosine phosphatase-mediated protection of intestinal epithelial barrier function

Background: TCPTP is a negative regulator of proinflammatory cytokine signaling and may be a therapeutic target for IBD. Results: Administration of spermidine to intestinal epithelial cells reduced proinflammatory cytokine signaling and subsequent barrier defects in a TCPTP-dependent manner. Conclusion: Activation of TCPTP by spermidine attenuates inflammatory responses in intestinal epithelial monolayers. Significance: Protection of epithelial barrier integrity by spermidine in vitro suggests its potential importance for barrier protection in vivo. The gene locus encoding protein-tyrosine phosphatase non-receptor type 2 (PTPN2) has been associated with inflammatory bowel disease. Expression of the PTPN2 gene product, T cell protein-tyrosine phosphatase (TCPTP), in intestinal epithelial cells has been shown to play an important role in the protection of epithelial barrier function during periods of inflammation by acting as a negative regulator of the proinflammatory cytokine IFN-γ. Therefore, agents that increase the activity of TCPTP are of general interest as modifiers of inflammatory signaling events. A previous study demonstrated that the small molecule spermidine is a selective activator of TCPTP in vitro. The aim of this study was to investigate whether activation of TCPTP by spermidine was capable of alleviating IFN-γ-induced, proinflammatory signaling and barrier dysfunction in human intestinal epithelial cells. Studies revealed that treatment of T84 and HT29/cl.19A colonocytes with spermidine increased both TCPTP protein levels and enzymatic activity, correlating with a decrease in the phosphorylation of the signal transducers and activators of transcription 1 and 3, downstream mediators of IFN-γ signaling, upon coadministration of spermidine to IFN-γ-treated cells. On a functional level, spermidine protected barrier function in the setting of inflammation, restricting the decrease in transepithelial electrical resistance and the increase in epithelial permeability induced by IFN-γ in coincubation experiments. These data implicate spermidine as a potential therapeutic agent to treat conditions associated with elevated IFN-γ signaling and a faulty mucosal barrier.

Interferon-γ Alters Downstream Signaling Originating from Epidermal Growth Factor Receptor in Intestinal Epithelial Cells FUNCTIONAL CONSEQUENCES FOR ION TRANSPORT

Background: Inflammation alters epithelial signaling. We studied how IFN-γ regulates EGFr signaling. Results: IFN-γ regulated EGFr expression, membrane localization, tyrosine residue phosphorylation, and linkage to MAPK. EGF no longer inhibited ion transport in IFN-γ-treated cells. Conclusion: EGFr signaling outcomes are altered by inflammation. Significance: These effects may contribute to diarrheal and other symptoms of inflammatory bowel diseases. The epidermal growth factor receptor (EGFr) regulates many cellular functions, such as proliferation, apoptosis, and ion transport. Our aim was to investigate whether long term treatment with interferon-γ (IFN-γ) modulates EGF activation of downstream signaling pathways in intestinal epithelial cells and if this contributes to dysregulation of epithelial ion transport in inflammation. Polarized monolayers of T84 and HT29/cl.19A colonocytes were preincubated with IFN-γ prior to stimulation with EGF. Basolateral potassium transport was studied in Ussing chambers. We also studied inflamed colonic mucosae from C57BL/6 mice treated with dextran sulfate sodium or mdr1a knock-out mice and controls. IFN-γ increased intestinal epithelial EGFr expression without increasing its phosphorylation. Conversely, IFN-γ caused a significant decrease in EGF-stimulated phosphorylation of specific EGFr tyrosine residues and activation of ERK but not Akt-1. In IFNγ-pretreated cells, the inhibitory effect of EGF on carbachol-stimulated K+ channel activity was lost. In inflamed colonic tissues, EGFr expression was significantly increased, whereas ERK phosphorylation was reduced. Thus, although it up-regulates EGFr expression, IFN-γ causes defective EGFr activation in colonic epithelial cells via reduced phosphorylation of specific EGFr tyrosine residues. This probably accounts for altered downstream signaling consequences. These observations were corroborated in the setting of colitis. IFN-γ also abrogates the ability of EGF to inhibit carbachol-stimulated basolateral K+ currents. Our data suggest that, in the setting of inflammation, the biological effect of EGF, including the inhibitory effect of EGF on Ca2+-dependent ion transport, is altered, perhaps contributing to diarrheal and other symptoms in vivo.

VSL#3 Probiotic Stimulates T-cell Protein Tyrosine Phosphatase–mediated Recovery of IFN-γ–induced Intestinal Epithelial Barrier Defects

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-&ggr;) 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-&ggr; 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-&ggr; signaling. VSL#3 corrected the decrease in transepithelial electrical resistance and the increase in epithelial permeability induced by IFN-&ggr;. Moreover, the restorative effect of VSL#3 against IFN-&ggr; 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-&ggr; signaling and IFN-&ggr;-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.

Salmonella-induced diarrhea occurs in the absence of IL-8 receptor (CXCR2)-dependent neutrophilic inflammation

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.

A comparison of linaclotide and lubiprostone dosing regimens on ion transport responses in human colonic mucosa

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 (Isc). Subsequent Isc 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. Isc 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.

Human evolutionary loss of epithelial Neu5Gc expression and species-specific susceptibility to cholera

While infectious agents have typical host preferences, the noninvasive enteric bacterium Vibrio cholerae is remarkable for its ability to survive in many environments, yet cause diarrheal disease (cholera) only in humans. One key V. cholerae virulence factor is its neuraminidase (VcN), which releases host intestinal epithelial sialic acids as a nutrition source and simultaneously remodels intestinal polysialylated gangliosides into monosialoganglioside GM1. GM1 is the optimal binding target for the B subunit of a second virulence factor, the AB5 cholera toxin (Ctx). This coordinated process delivers the CtxA subunit into host epithelia, triggering fluid loss via cAMP-mediated activation of anion secretion and inhibition of electroneutral NaCl absorption. We hypothesized that human-specific and human-universal evolutionary loss of the sialic acid N-glycolylneuraminic acid (Neu5Gc) and the consequent excess of N-acetylneuraminic acid (Neu5Ac) contributes to specificity at one or more steps in pathogenesis. Indeed, VcN was less efficient in releasing Neu5Gc than Neu5Ac. We show enhanced binding of Ctx to sections of small intestine and isolated polysialogangliosides from human-like Neu5Gc-deficient Cmah-/- mice compared to wild-type, suggesting that Neu5Gc impeded generation of the GM1 target. Human epithelial cells artificially expressing Neu5Gc were also less susceptible to Ctx binding and CtxA intoxication following VcN treatment. Finally, we found increased fluid secretion into loops of Cmah-/- mouse small intestine injected with Ctx, indicating an additional direct effect on ion transport. Thus, V. cholerae evolved into a human-specific pathogen partly by adapting to the human evolutionary loss of Neu5Gc, optimizing multiple steps in cholera pathogenesis.

Sa1775 Salmonella Typhimurium Infection Alters Cell Fate During Differentiation of Intestinal Epithelium

could be attributed to specific donors. Results: A total of 34 patients (mean age 68.8 years, 67.6% female) received at least 1 dose of RBX2660. Nineteen patients received 1 dose and 15 patients received 2 doses. The individual donor or the order of donors made no difference in the success or failure of treatment with RBX2660. This was especially clear in the case of 1 patient who received 2 doses of the exact same product (both doses from the same donor and same batch). The first dose failed and the second was a success. The donor to patient results for the cohort receiving 2 doses is shown in Figure 2 as an example. Similar trends were observed for patients receiving a single dose. Conclusion: Based on an analysis of small numbers, it appears that the specific donor does not affect the outcomes of RBX2660 for recurrent CDI. Additional research with a larger patient cohort and comparative analysis of donor and patient microbiota communities is needed to provide more details on patientspecific factors predisposing to success or failure with this therapy. RBX2660 Outcomes by Donor

570 In Vivo PTPN2-Deficiency and a Dominant-Negative PTPN2 Mutation Cause Increased Intestinal Permeability and Alter Tight Junction Composition

We have previously generated C57BL/6J-Tg(Car1-cre)5Flt transgenic mice (CAC) with large intestine, epithelial cell-specific expression of Cre recombinase and demonstrated that they can be used to study colorectal cancer. Here we report a novel observation with CAC mice that expands their utility as an experimental model for colitis and inflammation-induced colon cancer research. CAC mice were crossed to ROSA26R reporter mice (ROSA) that have a lox-STOP-lox site controlling β-galactosidase (β-gal) expression. In study 1, 8-wk-old CAC;ROSA mice were treated with 0, 0.65, 1.35, or 2% DSS in drinking water for 5 d. Proximal (CoP) and distal colon (CoD) segments were harvest 10 d after ending DSS treatment. The colon was fixed in 10% neutral buffered formalin, paraffin embedded, and processed for determination of an epithelial damage score (reflecting % surface ulceration and% regenerating epithelium) and for immunohistochemical detection of β-gal (an indicator of Cre-mediated recombination). In control mice (0% DSS) β-gal staining was superficial in CoP while 3% of CoD epithelium had β-gal staining from crypt base to luminal surface. DSS treatment caused a dose-dependent increase in damage score for the CoP (0% DSS = 0, 0.65%DSS = 30.5±4.3, 1.35% DSS = 53.6±4.1, 2% DSS = 58.5±4.9%) and CoD epithelium (0, 24.7±8.0, 46.7±9.8, 48.4±10.6%) and this was associated with increased β-gal staining (CoP = 0.7±0.2, 20.4±1.8, 41.0±2.9, 45.9±4.6%; CoD = 3.0±0.54, 20.4±5.2, 35.4±6.9, 42.2±9.4%). Interestingly, 60% regenerating epithelium was β-gal positive regardless of the DSS dose or the colon segment examined; this new staining was seen from crypt base to luminal surface in both CoP and CoD. In study 2, we examined whether β-gal induction persisted after the epithelium had healed, reflecting Cre mediated recombination in intestinal stem cells. 8-wk-old CAC;ROSA mice were treated with 1.35% DSS and β-gal staining was assessed in colon at 10 and 30 d post DSS treatment. Epithelial β-gal staining was low in the control (CoP = 0.8±0.2%; CoD = 6.6±0.8%) and increased with the epithelial damage score at 10 d (CoP damage = 18.7±3.4%, β-gal = 17.4±2.9%; CoD damage = 27.6±7.3%, β-gal = 33.6±6.8%). At 30 d, all ulcers had healed and the % regenerating epithelium was reduced by >45% in each segment compared to the 10 d timepoint. However, epithelial βgal staining remained high in both colon segments (CoP = 18.9±1.8%; CoD = 44.4±5.2%), especially in the epithelium of the healed ulcer (CoP=75.2±4.4%; CoD = 89.4±2.2%). These studies show that in CAC mice, DSS-mediated epithelial damage can be used to induce a persistent, Cre-mediated recombination of floxed alleles. This will permit examination of the function of genes in colon epithelium during experimental colitis and inflammationinduced colon cancer.

O-027 PTPN2 Deficiency Exacerbates Epithelial Barrier Dysfunction in vivo and in vitro via Redistribution of Occludin and ZO-1

BACKGROUND: Crohn’s disease (CD) and ulcerative colitis (UC), collectively known as inflammatory bowel disease (IBD), share many characteristics, one of which is a compromised intestinal epithelial barrier. Mutations in the protein tyrosine phosphatase non-receptor type 2 (PTPN2) gene are associated with IBD and have been linked to epithelial barrier dysfunction. The exact mechanism of barrier dysfunction remains unclear. PTPN2 knock-out (KO) mice exhibit severe inflammation and survive only 5–6 weeks after birth. Before death, these mice present with diarrhea. We performed barrier function studies on colonic tissues from KO mice at 3 weeks of age, prior to severe inflammation and diarrhea onset. METHODS: Segments of proximal colon from wild-type, heterozygote, and PTPN2 KO mice were mounted in Ussing chambers. To assess paracellular permeability to macromolecules, FITC-dextran (4 kD) was applied to the mucosal side and samples were taken at 0, 60 and 120 min from the serosal side, and after 10 min from the mucosal side. Paraffin embedded proximal colons were processed and immunostained for ZO-1 and occludin localization. In addition, a lentiviral vector encoding the C216S PTPN2 dominant-negative mutant was transduced into the HCA-7 colonic cell line and ZO-1 and occludin visualized by immunostaining and confocal microscopy. Statistical analysis was performed using ANOVA or Students t-test. RESULTS: In Ussing chambers studies, permeability of 4 KD FITC-Dextran (FD4) was increased in the proximal colon of PTPN2 KO mice (0.93% ± 0.5% of serosal FD4; P < 0.05; n = 7) compared to PTPN2 heterozygote (het) and wild-type (wt) mice (0.06% ± 0.01% and 0.05% ± 0.01% of serosal FD4 respectively; n = 7–8). FITC permeability studies were supported by confocal microscopy showing that occludin, was depleted at the apical membrane in the proximal colon of PTPN2 knock-out mice when compared to wt and het. Likewise, HCA7 cells overexpressing a PTPN2 active-site mutation (C216S) showed significant depletion of both occludin and ZO-1 (P < 0.05) from the apical tight-junctions (7.42 ± 0.44AU (Arbitrary Units)) when compared to control (9.68 ± 0.52AU) after both were challenged with IFNg (100 U/mL, 72 hours). CONCLUSIONS: In conclusion, these studies show that loss of functional PTPN2 in both mouse intestine and an epithelial cell line exacerbates inflammation-induced barrier defects. This dysfunction is likely due to an enhanced IFNg-driven depletion of occludin and ZO-1 from the apical tight-junctions.