Epithelial permeability is often increased in inflammatory bowel diseases. We hypothesized that perturbed mitochondrial function would cause barrier dysfunction and hence epithelial mitochondria ...could be targeted to treat intestinal inflammation. Mitochondrial dysfunction was induced in human colon-derived epithelial cell lines or colonic biopsy specimens using dinitrophenol, and barrier function was assessed by transepithelial flux of Escherichia coli with or without mitochondria-targeted antioxidant (MTA) cotreatment. The impact of mitochondria-targeted antioxidants on gut permeability and dextran sodium sulfate (DSS)–induced colitis in mice was tested. Mitochondrial superoxide evoked by dinitrophenol elicited significant internalization and translocation of E. coli across epithelia and control colonic biopsy specimens, which was more striking in Crohn’s disease biopsy specimens; the mitochondria-targeted antioxidant, MitoTEMPO, inhibited these barrier defects. Increased gut permeability and reduced epithelial mitochondrial voltage-dependent anion channel expression were observed 3 days after DSS. These changes and the severity of DSS-colitis were reduced by MitoTEMPO treatment. In vitro DSS-stimulated IL-8 production by epithelia was reduced by MitoTEMPO. Metabolic stress evokes significant penetration of commensal bacteria across the epithelium, which is mediated by mitochondria-derived superoxide acting as a signaling, not a cytotoxic, molecule. MitoTEMPO inhibited this barrier dysfunction and suppressed colitis in DSS-colitis, likely via enhancing barrier function and inhibiting proinflammatory cytokine production. These novel findings support consideration of MTAs in the maintenance of epithelial barrier function and the management of inflammatory bowel diseases.
Background & Aims The 5-hydroxytryptamine receptor 4 (5-HT4R or HTR4) is expressed in the colonic epithelium but little is known about its functions there. We examined whether activation of colonic ...epithelial 5-HT4R protects colons of mice from inflammation. Methods The 5-HT4R agonist tegaserod (1 mg/kg), the 5-HT4R antagonist GR113808 (1 mg/kg), or vehicle (control) were delivered by enema to wild-type or 5-HT4R knockout mice at the onset of, or during, active colitis, induced by administration of dextran sodium sulfate or trinitrobenzene sulfonic acid. Inflammation was measured using the colitis disease activity index and by histologic analysis of intestinal tissues. Epithelial proliferation, wound healing, and resistance to oxidative stress-induced apoptosis were assessed, as was colonic motility. Results Rectal administration of tegaserod reduced the severity of colitis compared with mice given vehicle, and accelerated recovery from active colitis. Rectal tegaserod did not improve colitis in 5-HT4R knockout mice, and intraperitoneally administered tegaserod did not protect wild-type mice from colitis. Tegaserod increased proliferation of crypt epithelial cells. Stimulation of 5-HT4R increased Caco-2 cell migration and reduced oxidative stress-induced apoptosis; these actions were blocked by co-administration of the 5-HT4R antagonist GR113808. In noninflamed colons of wild-type mice not receiving tegaserod, inhibition of 5-HT4Rs resulted in signs of colitis within 3 days. In these mice, epithelial proliferation decreased and bacterial translocation to the liver and spleen was detected. Daily administration of tegaserod increased motility in inflamed colons of guinea pigs and mice, whereas administration of GR113808 disrupted motility in animals without colitis. Conclusions 5-HT4R activation maintains motility in healthy colons of mice and guinea pigs, and reduces inflammation in colons of mice with colitis. Agonists might be developed as treatments for patients with inflammatory bowel diseases.
Inflammatory bowel diseases are associated with dysregulated electrolyte and water transport and resultant diarrhea. Aquaporins are transmembrane proteins that function as water channels in ...intestinal epithelial cells. We investigated the effect of the inflammatory cytokine, interferon-γ, which is a major player in inflammatory bowel diseases, on aquaporin-1 expression in a mouse colonic epithelial cell line, CMT93. CMT93 monolayers were exposed to 10 ng/mL interferon-γ and aquaporin-1 mRNA and protein expressions were measured by real-time PCR and western blot, respectively. In other experiments, CMT93 cells were pretreated with inhibitors or were transfected with siRNA to block the effects of Janus kinases, STATs 1 and 3, or interferon regulatory factor 2, prior to treatment with interferon-γ. Interferon-γ decreased aquaporin-1 expression in mouse intestinal epithelial cells in a manner that did not depend on the classical STAT1/JAK2/IRF-1 pathway, but rather, on an alternate Janus kinase (likely JAK1) as well as on STAT3. The pro-inflammatory cytokine, interferon-γ may contribute to diarrhea associated with intestinal inflammation in part through regulation of the epithelial aquaporin-1 water channel via a non-classical JAK/STAT receptor signalling pathway.
Cancer cell lines have been the mainstay of intestinal epithelial experimentation for decades, due primarily to their immortality and ease of culture. However, because of the inherent biological ...abnormalities of cancer cell lines, many cellular biologists are currently transitioning away from these models and toward more representative primary cells. This has been particularly challenging, but recent advances in the generation of intestinal organoids have brought the routine use of primary cells within reach of most epithelial biologists. Nevertheless, even with the proliferation of publications that use primary intestinal epithelial cells, there is still a considerable amount of trial and error required for laboratories to establish a consistent and reliable method to culture three-dimensional (3D) intestinal organoids and primary epithelial monolayers. We aim to minimize the time other laboratories spend troubleshooting the technique and present a standard method for culturing primary epithelial cells. Therefore, we have described our optimized, high-yield, cost-effective protocol to grow 3D murine colonoids for more than 20 passages and our detailed methods to culture these cells as confluent monolayers for at least 14 days, enabling a wide variety of potential future experiments. By supporting and expanding on the current literature of primary epithelial culture optimization and detailed use in experiments, we hope to help enable the widespread adoption of these innovative methods and allow consistency of results obtained across laboratories and institutions.
Primary intestinal epithelial monolayers are notoriously difficult to maintain culture, even with the recent advances in the field. We describe, in detail, the protocols required to maintain three-dimensional cultures of murine colonoids and passage these primary epithelial cells to confluent monolayers in a standardized, high-yield and cost-effective manner.
The roles of proteinase-activated receptors (PARs) in platelet functions other than aggregation are not well understood. Among these is the release of factors that regulate the process of ...angiogenesis, such as endostain and VEGF, which, respectively, inhibit and promote angiogenesis. PAR1 and PAR4 are expressed on the surface of human platelets and can be activated by thrombin. In the present study, we have attempted to determine the roles of PAR1 and PAR4 in regulating release of endostatin and VEGF from human platelets. Aggregation and endostatin release could be elicited by a specific PAR4 agonist ( AYPGKF- NH2). The PAR4 agonist concentration dependently suppressed VEGF release. A selective PAR1 agonist ( TFLLR- NH2) induced platelet aggregation and VEGF release but suppressed endostatin release. Thrombin did not affect endostatin or VEGF release. However, in the presence of a selective PAR1 antagonist (SCH79797), thrombin stimulated endostatin release and suppressed VEGF release. Conversely, in the presence of a selective PAR4 antagonist (transcinnamoyl- YPGKF- NH2), thrombin stimulated VEGF release. In vivo, treatment of rats with established gastric ulcers with a PAR1 antagonist each day for 1 wk resulted in a significant retardation of healing. We conclude that PAR1 and PAR4 counter-regulate the release of endostatin and VEGF from platelets. These protease-activated receptors could therefore play a crucial role in regulating angiogenesis and in turn could regulate the processes of wound healing and tumor growth.
Abstract Aims Hydrogen sulphide (H2 S) exerts several anti-inflammatory effects, accelerates the healing of experimental gastric ulcers, and can stimulate intestinal secretion. Little is known about ...H2 S synthesis in the gastrointestinal tract. The aim of this study was to characterize H2 S synthesis throughout the gastrointestinal tract. Methods H2 S synthesis in various gastrointestinal tissues of rats and mice was determined. The effects and selectivity of inhibitors of two key enzymes for H2 S synthesis, cystathionine-γ-lyase and cystathionine-β-synthase, were examined. Cystathionine-γ-lyase and cystathionine-β-synthase expression was evaluated by Western blotting and immunohistochemistry. Cystathionine-γ-lyase and cystathionine-β-synthase expression in biopsies of human colon was also examined. Results H2 S synthesis was variable throughout the gastrointestinal tract in parallel with variations in cystathionine-γ-lyase and cystathionine-β-synthase expression. The efficacy of cystathionine-β-synthase and cystathionine-γ-lyase inhibitors to reduce H2 S synthesis in these tissues was also variable. Cystathionine-β-synthase is the predominant source of H2 S synthesis in the colon of rodents. Cystathionine-γ-lyase and cystathionine-β-synthase were also expressed in healthy human colon biopsies. Conclusions The capacity for H2 S synthesis varies throughout the rodent gastrointestinal tract, as does the distribution and contribution of the two key enzymes. Investigation of additional enzymatic sources of H2 S and the development of more selective inhibitors are suggested.
Proteinase-activated receptor (PAR)(2), a G protein-coupled receptor activated by serine proteinases, has been implicated in both intestinal inflammation and epithelial proliferation. Cyclooxygenase ...(COX)-2 is overexpressed in the gut during inflammation as well as in colon cancer. We hypothesized that PAR(2) drives COX-2 expression in intestinal epithelial cells. Treatment of Caco-2 colon cancer cells with the PAR(2)-activating peptide 2-furoyl-LIGRLO-NH(2) (2fLI), but not by its reverse-sequence PAR(2)-inactive peptide, for 3 h led to an increase in intracellular COX-2 protein expression accompanied by a COX-2-dependent increase in prostaglandin E(2) production. 2fLI treatment for 30 min significantly increased metalloproteinase activity in the culture supernatant. Increased epidermal growth factor receptor (EGFR) phosphorylation was observed in cell lysates following 40 min of treatment with 2fLI. The broad-spectrum metalloproteinase inhibitor marimastat inhibited both COX-2 expression and EGFR phosphorylation. The EGFR tyrosine kinase inhibitor PD153035 also abolished 2fLI-induced COX-2 expression. Although PAR(2) activation increased ERK MAPK phosphorylation, neither ERK pathway inhibitors nor a p38 MAPK inhibitor affected 2fLI-induced COX-2 expression. However, inhibition of either Src tyrosine kinase signaling by PP2, Rho kinase signaling by Y27632, or phosphatidylinositol 3 (PI3) kinase signaling by LY294002 prevented 2fLI-induced COX-2 expression. Trypsin increased COX-2 expression through PAR(2) in Caco-2 cells and in an EGFR-dependent manner in the noncancerous intestinal epithelial cell-6 cell line. In conclusion, PAR(2) activation drives COX-2 expression in Caco-2 cells via metalloproteinase-dependent EGFR transactivation and activation of Src, Rho, and PI3 kinase signaling. Our findings provide a mechanism whereby PAR(2) can participate in the progression from chronic inflammation to cancer in the intestine.
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Platelets contain an array of growth factors that can modulate healing processes, including both pro‐ (e.g., vascular endothelial growth factor (VEGF)) and antiangiogenic (e.g., endostatin) ...factors. Previous studies have shown that circulating platelets contribute significantly to gastric ulcer healing, acting as a delivery system for these growth factors to the site of injury. In this study, we examined the effects of orally administered human platelets on the healing of gastric ulcers in rats, and determined the contribution of VEGF and endostatin to healing in this model.
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Twice‐daily administration of human platelets significantly accelerated ulcer healing, but platelet‐poor plasma (PPP), lysed platelets and serum failed to produce this effect. There was no correlation between ulcer healing and the levels of VEGF or endostatin in serum, PPP or platelet‐rich plasma (PRP).
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Accelerated ulcer healing could not be produced by oral administration of the angiogenic factors themselves, at concentrations matching those in PRP.
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The accelerated healing induced by platelets could be reversed by immuno‐neutralization of VEGF. In contrast, immuno‐neutralization of endostatin did not affect PRP‐induced ulcer healing.
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These studies indicate that VEGF released from platelets accounts for the accelerated healing of gastric ulcers. However, as intact (rather than lysed) platelets were required for the accelerated healing, the presentation of VEGF by the platelet at the site of injury appears to be crucial for enhancement of the healing process.
British Journal of Pharmacology (2006) 148, 274–278. doi:10.1038/sj.bjp.0706722
Patients with systemic inflammatory diseases (e.g., rheumatoid arthritis, inflammatory bowel disease, chronic liver disease) commonly develop debilitating symptoms (i.e., sickness behaviors) that ...arise from changes in brain function. The microbiota-gut-brain axis alters brain function and probiotic ingestion can influence behavior. However, how probiotics do this remains unclear. We have previously described a novel periphery-to-brain communication pathway in the setting of peripheral organ inflammation whereby monocytes are recruited to the brain in response to systemic TNF-α signaling, leading to microglial activation and subsequently driving sickness behavior development. Therefore, we investigated whether probiotic ingestion (i.e., probiotic mixture VSL#3) alters this periphery-to-brain communication pathway, thereby reducing subsequent sickness behavior development. Using a well characterized mouse model of liver inflammation, we now show that probiotic (VSL#3) treatment attenuates sickness behavior development in mice with liver inflammation without affecting disease severity, gut microbiota composition, or gut permeability. Attenuation of sickness behavior development was associated with reductions in microglial activation and cerebral monocyte infiltration. These events were paralleled by changes in markers of systemic immune activation, including decreased circulating TNF-α levels. Our observations highlight a novel pathway through which probiotics mediate cerebral changes and alter behavior. These findings allow for the potential development of novel therapeutic interventions targeted at the gut microbiome to treat inflammation-associated sickness behaviors in patients with systemic inflammatory diseases.
This research shows that probiotics, when eaten, can improve the abnormal behaviors (including social withdrawal and immobility) that are commonly associated with inflammation. Probiotics are able to cause this effect within the body by changing how the immune system signals the brain to alter brain function. These findings broaden our understanding of how probiotics may beneficially affect brain function in the context of inflammation occurring within the body and may open potential new therapeutic alternatives for the treatment of these alterations in behavior that can greatly affect patient quality of life.
Background & Aims Hydrogen sulfide (H2 S) is an endogenous gaseous mediator of mucosal defense with antiinflammatory effects that promote ulcer healing. The effects of H2 S during the pathogenesis of ...colitis have not been established. We analyzed the contribution of H2 S to inflammation and ulceration of the colon in a rat model of colitis. Methods Colitis was induced by intracolonic administration of trinitrobenzene sulfonic acid. The ability of the colon to synthesize H2 S was studied over the course of the resolution of the colitis. Expression of 2 enzymes involved in the synthesis of H2 S and the effects of inhibitors of these enzymes were examined. We also examined the effects of H2 S donors on the resolution of colitis. Results The capacity for the colon to produce H2 S increased markedly over the first days after induction of colitis and then declined toward control levels as the colitis was resolved. Inhibition of colonic H2 S synthesis markedly exacerbated the colitis, resulting in significant mortality. Inhibition of H2 S synthesis in healthy rats resulted in inflammation and mucosal injury in the small intestine and colon along with down-regulation of cyclooxygenase-2 messenger RNA expression and prostaglandin synthesis. Intracolonic administration of H2 S donors significantly reduced the severity of colitis and reduced colonic expression of messenger RNA for the proinflammatory cytokine tumor necrosis factor α. Conclusions In rats, H2 S modulates physiological inflammation and contributes to the resolution of colitis.
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