Declan F. McCole

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.

Activation of protein tyrosine phosphatase non-receptor type 2 by spermidine exerts anti-inflammatory effects in human THP-1 monocytes and in a mouse model of acute colitis.

Background Spermidine is a dietary polyamine that is able to activate protein tyrosine phosphatase non-receptor type 2 (PTPN2). As PTPN2 is known to be a negative regulator of interferon-gamma (IFN-γ)-induced responses, and IFN-γ stimulation of immune cells is a critical process in the immunopathology of inflammatory bowel disease (IBD), we wished to explore the potential of spermidine for reducing pro-inflammatory effects in vitro and in vivo. Methods Human THP-1 monocytes were treated with IFN-γ and/or spermidine. Protein expression and phosphorylation were analyzed by Western blot, cytokine expression by quantitative-PCR, and cytokine secretion by ELISA. Colitis was induced in mice by dextran sodium sulfate (DSS) administration. Disease severity was assessed by recording body weight, colonoscopy and histology. Results Spermidine increased expression and activity of PTPN2 in THP-1 monocytes and reduced IFN-γ-induced phosphorylation of signal transducer and activator of transcription (STAT) 1 and 3, as well as p38 mitogen-activated protein kinase (MAPK) in a PTPN2 dependent manner. Subsequently, IFN-γ-induced expression/secretion of intracellular cell adhesion molecule (ICAM)-1 mRNA, monocyte chemoattractant protein (MCP)-1, and interleukin (IL)-6 was reduced in spermidine-treated cells. The latter effects were absent in PTPN2-knockdown cells. In mice with DSS-induced colitis, spermidine treatment resulted in ameliorated weight loss and decreased mucosal damage indicating reduced disease severity. Conclusions Activation of PTPN2 by spermidine ameliorates IFN-γ-induced inflammatory responses in THP-1 cells. Furthermore, spermidine treatment significantly reduces disease severity in mice with DSS-induced colitis; hence, spermidine supplementation and subsequent PTPN2 activation may be helpful in the treatment of chronic intestinal inflammation such as IBD.

Mechanisms of Intestinal Epithelial Barrier Dysfunction by Adherent-Invasive Escherichia coli

Pathobiont expansion, such as that of adherent-invasive Escherichia coli (AIEC), is an emerging factor associated with inflammatory bowel disease. The intestinal epithelial barrier is the first line of defense against these pathogens. Inflammation plays a critical role in altering the epithelial barrier and is a major factor involved in promoting the expansion and pathogenesis of AIEC. AIEC in turn can exacerbate intestinal epithelial barrier dysfunction by targeting multiple elements of the barrier. One critical element of the epithelial barrier is the tight junction. Increasing evidence suggests that AIEC may selectively target protein components of tight junctions, leading to increased barrier permeability. This may represent one mechanism by which AIEC could contribute to the development of inflammatory bowel disease. This review article discusses potential mechanisms by which AIEC can disrupt epithelial tight junction function and intestinal barrier function.

Role of protein tyrosine phosphatases in regulating the immune system: implications for chronic intestinal inflammation.

Abstract:Current hypothesis suggests that genetic, immunological, and bacterial factors contribute essentially to the pathogenesis of inflammatory bowel disease. Variations within the gene loci encoding protein tyrosine phosphatases (PTPs) have been associated with the onset of inflammatory bowel disease. PTPs modulate the activity of their substrates by dephosphorylation of tyrosine residues and are critical for the regulation of fundamental cellular signaling processes. Evidence emerges that expression levels of PTPN2, PTPN11, and PTPN22 are altered in actively inflamed intestinal tissue. PTPN2 seems to be critical for protecting intestinal epithelial barrier function, regulating innate and adaptive immune responses and finally for maintaining intestinal homeostasis. These observations have been confirmed in PTPN2 knockout mice in vivo. Those animals are clearly more susceptible to intestinal and systemic inflammation and feature alterations in innate and adaptive immune responses. PTPN22 controls inflammatory signaling in lymphocytes and mononuclear cells resulting in aberrant cytokine secretion pattern and autophagosome formation. PTPN22 deficiency in vivo results in more severe colitis demonstrating the relevance of PTPN22 for intestinal homeostasis in vivo. Of note, loss of PTPN22 promotes mitogen-activated protein kinase–induced cytokine secretion but limits secretion of nuclear factor &kgr;B–associated cytokines and autophagy in mononuclear cells. Loss of PTPN11 is also associated with increased colitis severity in vivo. In summary, dysfunction of those PTPs results in aberrant and uncontrolled immune responses that result in chronic inflammatory conditions. This way, it becomes more and more evident that dysfunction of PTPs displays an important factor in the pathogenesis of chronic intestinal inflammation, in particular inflammatory bowel disease.

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.

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.

PTPN2 Regulates Inflammasome Activation and Controls Onset of Intestinal Inflammation and Colon Cancer

Variants in the gene locus encoding protein tyrosine phosphatase non-receptor type 2 (PTPN2) are associated with inflammatory disorders, including inflammatory bowel diseases, rheumatoid arthritis, and type 1 diabetes. The anti-inflammatory role of PTPN2 is highlighted by the fact that PTPN2-deficient mice die a few weeks after birth because of systemic inflammation and severe colitis. However, the tissues, cells, and molecular mechanisms that contribute to this phenotype remain unclear. Here, we demonstrate that myeloid cell-specific deletion of PTPN2 in mice (PTPN2-LysMCre) promotes intestinal inflammation but protects from colitis-associated tumor formation in an IL-1β-dependent manner. Elevated levels of mature IL-1β production in PTPN2-LysMCre mice are a consequence of increased inflammasome assembly due to elevated phosphorylation of the inflammasome adaptor molecule ASC. Thus, we have identified a dual role for myeloid PTPN2 in directly regulating inflammasome activation and IL-1β production to suppress pro-inflammatory responses during colitis but promote intestinal tumor development.

The Hydrogen Peroxide Scavenger, Catalase, Alleviates Ion Transport Dysfunction in Murine Colitis

Reactive oxygen species (ROS) such as hydrogen peroxide (H2O2) contribute to epithelial damage and ion transport dysfunction (key events in inflammatory diarrhoea) in inflammatory bowel disease (IBD). The aim of this study was to identify if H2O2 mediates suppression of colonic ion transport function in the murine dextran sulfate sodium (DSS) colitis model by using the H2O2 degrading enzyme, catalase. Colitis was induced by administering DSS (4%) in drinking water for 5 days followed by 3 days on normal H2O. Mice were administered either pegylated catalase or saline at day −1, 0 and +1 of DSS treatment. Ion transport responses to the Ca2+‐dependent agonist, carbachol (CCh), or the cAMP‐dependent agonist, forskolin, were measured across distal colonic mucosa mounted in Ussing chambers. Parameters of DSS‐induced inflammation (loss in body weight, decreased colon length, altered stool consistency), were only partially alleviated by catalase while histology was only minimally improved. However, catalase significantly reversed the DSS‐induced reduction in baseline ion transport as well as colonic Isc responses to CCh. However, ion transport responses to forskolin were not significantly restored. Catalase also reduced activation of ERK MAP kinase in the setting of colitis, and increased expression of the Na+‐K+‐2Cl− cotransporter, NKCC1, consistent with restoration of ion transport function. Ex vivo treatment of inflamed colonic mucosae with catalase also partially restored ion transport function. Therefore, catalase partially prevents, and rescues, the loss of ion transport properties in DSS colitis even in the setting of unresolved tissue inflammation. These findings indicate a prominent role for ROS in ion transport dysfunction in colitis and may suggest novel strategies for the treatment of inflammatory diarrhoea.

All Hands on Deck: Commensals, Th17 Cells, and Neutrophils Provide Short-term Compensation of Constitutive Permeability Defects Against Acute Infection

EDITORIAL All Hands on Deck: Commensals, Th17 Cells, and Neutrophils Provide Short-term Compensation of Constitutive Permeability Defects Against Acute Infection A lthough ample evidence has been generated that increased intestinal permeability contributes to many inflammatory and disease phenotypes, both in the intestines and in extra-intestinal sites such as the liver, it also has been shown in several model systems that a barrier defect alone generally is insufficient to induce chronic in- flammatory conditions such as ulcerative colitis and Crohn’s disease. This is reflected in clinical data showing that a subset of healthy first-degree relatives of Crohn’s disease patients show increased intestinal permeability but do not go on to develop full-blown disease, even though increased permeability can serve as a predictor of disease relapse. 1,2 This conundrum has perplexed investigators for many years as to how some people with a compromised intestinal barrier evade being condemned to a fate of chronic in- flammatory disease that many others endure. Clues to this puzzle have been gleaned from several studies using genetically manipulated rodent models that identified that a permeability defect precedes intestinal inflammation but required a second-hit to manifest disease. The consistency of these observations indicated that a safety mechanism existed to prevent random breaches of epithelial junctional integrity from precipitating chronic autoimmune disease. The assortment of genes studied that give rise to a perme- ability defect range from specific tight junction proteins junctional adhesion molecule A to anti-inflammatory cyto- kines (interleukin 10) to a number of inflammatory bowel disease candidate genes 3,4 (reviewed previously 5 ). Although the capacity of the epithelium itself to rapidly repair discreet disruptions, such as occurs during cell shedding, are well documented, the consequences of targeted disruption of tight junction permeability in the absence of epithelial cell loss have been somewhat unpredictable, with several models yielding conflicting results. Adding to the complexity of this system is the well-recognized dual role of innate and adaptive immune cells in both aiding and compromising epithelial barrier function in a context- dependent manner. Moreover, our increasing understand- ing of the intricate and essential interactions of the host mucosa with the intestinal microbiome for overall intestinal homeostasis also underlines how dynamic and interdepen- dent this relationship is. In this issue of Cellular and Molecular Gastroenterology and Hepatology, Edelblum et al 6 have identified a novel mechanism that may explain some of the key questions in this field. The investigators used a constitutively active myosin light-chain kinase (CA-MLCK) transgenic mouse that shows increased macromolecule permeability without evidence of epithelial damage or hyperproliferation. This group previously showed that this mouse shows subclinical inflammation and is more susceptible to experimental colitis (CD4 þ CD45 þ Rb(hi) T-cell transfer) than control mice. 7 Here, the investigators made the surprising finding that the presence of an underlying barrier defect did not lead to an immediate increase in susceptibility to infection with protozoan or bacterial pathogens, as predicted previously. On the contrary, CA-MLCK mice showed an immune response—mediated by T-helper 17 CD4 þ T cells and sub- sequent recruitment of neutrophils—that reduced acute Toxoplasma gondii and Salmonella typhimurium trans- location across the epithelial barrier. The protective response in CA-MLCK mice was the result of enhanced mucosal immune activation that required CD4 þ T cells and interleukin 17A, with no apparent requirement for increased overall or microbe-specific IgA production. This indicates a key mechanistic difference between the mode of immune compensation in this model and a previous study showing that mice lacking expression of the tight junction protein junctional adhesion molecule A also showed a compensatory adaptive CD4 þ T-cell immune response to an underlying permeability defect. This response, which pro- tected against injury-induced colitis, required IgA secretion driven by increased transforming growth factor-b produc- tion. 3 The protective response in CA-MLCK mice was lost in germ-free conditions but was restored by introduction of a fecal microbe community rich in segmented filamentous bacteria. Therefore, commensal microbes were able to pro- mote a mild Th17 response that limits early pathogen translocation. A key finding of the study was that this pro- tective immune response was effective only against acute, but not chronic, infection because chronic S typhimurium infec- tion resulted in a more severe outcome in CA-MLCK mice, suggesting that despite activation of protective mucosal im- munity, barrier defects ultimately resulted in enhanced dis- ease progression after a more sustained challenge. The study further underlined the differing and context-dependent re- sponses of T cells to pathogen infection in conditions of an intact vs a compromised epithelial barrier. These data pro- vide new insight into the mechanisms by which mucosal immunity adapts to epithelial tight junction defects, and how this compensation eventually fails in the context of chronic inflammatory disease. As concluded by the investigators, these mechanisms may explain both the absence of disease in relatives of Crohn’s disease patients, despite barrier loss, as well as the increased disease susceptibility induced by bar- rier dysfunction and the potential link between enteric Cellular and Molecular Gastroenterology and Hepatology 2017;4:326–327

T cell protein tyrosine phosphatase prevents STAT1 induction of claudin‐2 expression in intestinal epithelial cells

T cell protein tyrosine phosphatase (TCPTP) dephosphorylates a number of substrates, including JAK–STAT (signal transducer and activator of transcription) signaling proteins, which are activated by interferon (IFN)‐γ, a major proinflammatory cytokine involved in conditions such as inflammatory bowel disease. A critical function of the intestinal epithelium is formation of a selective barrier to luminal contents. The structural units of the epithelium that regulate barrier function are the tight junctions (TJs), and the protein composition of the TJ determines the tightness of the barrier. Claudin‐2 is a TJ protein that increases permeability to cations and reduces transepithelial electrical resistance (TER). We previously showed that transient knockdown (KD) of TCPTP permits increased expression of claudin‐2 by IFN‐γ. Here, we demonstrate that the decreased TER in TCPTP‐deficient epithelial cells is alleviated by STAT1 KD. Moreover, increased claudin‐2 in TCPTP‐deficient cells requires enhanced STAT1 activation and STAT1 binding to the CLDN2 promoter. We also show that mutation of this STAT‐binding site prevents elevated CLDN2 promoter activity in TCPTP‐deficient epithelial cells. In summary, we demonstrate that TCPTP protects the intestinal epithelial barrier by restricting STAT‐induced claudin‐2 expression. This is a potential mechanism by which loss‐of‐function mutations in the gene encoding TCPTP may contribute to barrier defects in chronic intestinal inflammatory disease.