Christina Hirota

Increased prevalence of circulating novel IL-17 secreting Foxp3 expressing CD4+ T cells and defective suppressive function of circulating Foxp3+ regulatory cells support plasticity between Th17 and regulatory T cells in inflammatory bowel disease patients.

Background:IL-17 and Foxp3 double-expressing (DE) CD4+ T lymphocytes are novel crossover immune cell population, but the presence and role of these cells in human intestinal inflammation is unclear. The aim of this study was to investigate the circulating IL-17 and Foxp3 DE CD4+ T lymphocytes in patients with inflammatory bowel disease (IBD). Methods:The entire cohort consisted of 79 subjects: 31 patients with Crohns disease, 28 patients with ulcerative colitis, and 20 healthy control subjects (HC). IBD patients with evidence of active disease at endoscopy were entered into the study. Peripheral blood mononuclear cells were used for ex vivo and in vitro studies to assess the characteristics and generation of these novel cells and the function of circulating Foxp3+ CD4+ regulatory T lymphocytes (Treg) in patients with IBD compared with HC. Results:Patients with IBD had significantly higher prevalence of IL-17 and Foxp3 DE CD4+ T lymphocytes compared with age- and gender-matched HC. These cells expressed ROR&ggr;t. The ability of Treg cells to suppress autologous T-cell proliferation was reduced by approximately 60% in patients with IBD compared with HC. Increased generation of these DE cells was demonstrated by the modulation of cytokine environment of CD4+ lymphocytes in vitro in patients with Crohns disease. Conclusions:Prevalence of circulating IL-17 and Foxp3 DE CD4+ T cells is increased in patients with IBD. Coexpression of ROR&ggr;t and Foxp3 in these cells implies conversion from Treg cells to Th17 cells. This is associated with a decreased suppressive function of Foxp3+ CD4+ T lymphocytes in patients with IBD.

Cholinergic regulation of epithelial ion transport in the mammalian intestine

Acetylcholine (ACh) is critical in controlling epithelial ion transport and hence water movements for gut hydration. Here we review the mechanism of cholinergic control of epithelial ion transport across the mammalian intestine. The cholinergic nervous system affects basal ion flux and can evoke increased active ion transport events. Most studies rely on measuring increases in short‐circuit current (ISC = active ion transport) evoked by adding ACh or cholinomimetics to intestinal tissue mounted in Ussing chambers. Despite subtle species and gut regional differences, most data indicate that, under normal circumstances, the effect of ACh on intestinal ion transport is mainly an increase in Cl‐ secretion due to interaction with epithelial M3 muscarinic ACh receptors (mAChRs) and, to a lesser extent, neuronal M1 mAChRs; however, AChR pharmacology has been plagued by a lack of good receptor subtype‐selective compounds. Mice lacking M3 mAChRs display intact cholinergically‐mediated intestinal ion transport, suggesting a possible compensatory mechanism. Inflamed tissues often display perturbations in the enteric cholinergic system and reduced intestinal ion transport responses to cholinomimetics. The mechanism(s) underlying this hyporesponsiveness are not fully defined. Inflammation‐evoked loss of mAChR‐mediated control of epithelial ion transport in the mouse reveals a role for neuronal nicotinic AChRs, representing a hitherto unappreciated braking system to limit ACh‐evoked Cl‐ secretion. We suggest that: i) pharmacological analyses should be supported by the use of more selective compounds and supplemented with molecular biology techniques targeting specific ACh receptors and signalling molecules, and ii) assessment of ion transport in normal tissue must be complemented with investigations of tissues from patients or animals with intestinal disease to reveal control mechanisms that may go undetected by focusing on healthy tissue only.

TRPM8 activation attenuates inflammatory responses in mouse models of colitis

Transient Receptor Potential Melastatin–8 (TRPM8), a recently identified member of the transient receptor potential (TRP) family of ion channels, is activated by mild cooling and by chemical compounds such as the supercooling agent, icilin. Since cooling, possibly involving TRPM8 stimulation, diminishes injury-induced peripheral inflammation, we hypothesized that TRPM8 activation may also attenuate systemic inflammation. We thus studied the involvement of TRPM8 in regulating colonic inflammation using two mouse models of chemically induced colitis. TRPM8 expression, localized immunohistochemically in transgenic TRPM8GFP mouse colon, was up-regulated in both human- and murine-inflamed colon samples, as measured by real-time PCR. Wild-type mice (but not TRPM8-nulls) treated systemically with the TRPM8 agonist, icilin showed an attenuation of chemically induced colitis, as reflected by a decrease in macroscopic and microscopic damage scores, bowel thickness, and myeloperoxidase activity compared with untreated animals. Furthermore, icilin treatment reduced the 2,4,6-trinitrobenzenesulfonic acid–induced increase in levels of inflammatory cytokines and chemokines in the colon. In comparison with wild-type mice, Dextran Sodium Sulfate (DSS)-treated TRPM8 knockout mice showed elevated colonic levels of the inflammatory neuropeptide calcitonin-gene–related peptide, although inflammatory indices were equivalent for both groups. Further, TRPM8 activation by icilin blocked capsaicin-triggered calcitonin-gene–related peptide release from colon tissue ex vivo and blocked capsaicin-triggered calcium signaling in Transient Receptor Potential Vaniloid-1 (TRPV1) and TRPM8 transfected HEK cells. Our data document an anti-inflammatory role for TRPM8 activation, in part due to an inhibiton of neuropeptide release, pointing to a novel therapeutic target for colitis and other inflammatory diseases.

Proteinase-activated receptors induce interleukin-8 expression by intestinal epithelial cells through ERK/RSK90 activation and histone acetylation

Proteinase‐activated receptors (PARs) are involved in both inflammation and tumorigenesis in epithelial cells. Interleukin (IL)‐8 is a potent chemoattractant and is also involved in angiogenesis. The molecular mechanism whereby PARs induce epithelial IL‐8 expression is not known. In HT‐29 colonic epithelial cells, PAR1 or PAR2 agonists stimulated the expression of IL‐8 through a NF‐κB‐dependent pathway without inducing IκB degradation and disassociation of IκB from NF‐κB. Further studies revealed that PAR activation induced the phosphorylation of p65 at Ser‐276 in the nucleus, which increased the recruitment of histone acetyltransferase (HAT) p300 to p50. Inhibition of ERK activation completely blocked PAR‐induced IL‐8 expression, phosphorylation of p65 and HAT activity. We also demonstrated that RSK p90 was the downstream kinase that mediated ERK‐induced nuclear p65 phosphorylation. In conclusion, activation of either PAR1 or PAR2 stimulated the transcriptional up‐regulation of IL‐8 in HT‐29 colonic epithelial cells through a pathway that involved ERK/RSK p90, NF‐κB phosphorylation, and HAT activity. These studies provide evidence of a new role for serine proteinases and PARs in the regulation of gene expression in colonic inflammation and tumorigenesis.—Wang, H, Moreau, F., Hirota, C. L., MacNaughton, W. K. Proteinase‐activated receptors induce interleukin‐8 expression by intestinal epithelial cells through ERK/RSK90 activation and histone acetylation. FASEB J. 24, 1971–1980 (2010). www.fasebj.org

The EGF Receptor and HER2 Participate in TNF-α-Dependent MAPK Activation and IL-8 Secretion in Intestinal Epithelial Cells

TNF-α activates multiple mitogen-activated protein kinase (MAPK) cascades in intestinal epithelial cells (IECs) leading to the secretion of interleukin 8 (IL-8), a neutrophil chemoattractant and an angiogenic factor with tumor promoting properties. As the epidermal growth factor receptor (EGFR) is a known transducer of proliferative signals and a potent activator of MAPKs, we hypothesized that the EGFR participates in TNF-dependent MAPK activation and IL-8 secretion by intestinal epithelial cells (IECs). We show that the EGFR is tyrosine-phosphorylated following treatment of IECs (HT-29 and IEC-6) with TNF-α. This requires EGFR autophosphorylation as it was blocked by the EGFR kinase inhibitor AG1478. Autophosphorylation was also inhibited by both a Src-kinase inhibitor and the metalloproteinase inhibitor batimastat. TNF treatment of IECs resulted in the accumulation of soluble TGF-α; treatment of IECs with batimastat suppressed TGF-α release and immunoneutralization of TGF-α resulted in decreased EGFR and ERK phosphorylations. TNF-α treatment of IECs resulted in an association between EGFR and HER2 and inhibition of HER2 using a specific inhibitor AG879 in combination with AG1478-suppressed TNF-α-dependent ERK phosphorylation and IL-8 release. Downregulation of HER2 via siRNA resulted in a significant decrease in ERK phosphorylation and a 50% reduction in IL-8 secretion.

Epidermal growth factor receptor transactivation is required for proteinase-activated receptor-2-induced COX-2 expression in intestinal epithelial cells

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.

Prostaglandin E2 derived from cyclooxygenases 1 and 2 mediates intestinal epithelial ion transport stimulated by the activation of protease-activated receptor 2.

Proteinase-activated receptor (PAR)2 is activated by trypsin-like serine proteinases and has been implicated in intestinal inflammation. However, its role in the regulation of intestinal mucosal function remains unclear. Using the intestinal epithelial cell line, SCBN, we have studied the stimulus-secretion coupling mechanisms of PAR2-induced epithelial chloride transport, focusing on cyclooxygenase (COX)-1 and COX-2 activities and prostaglandin (PG) E2 secretion. SCBN monolayers were grown on Snapwell supports, mounted in modified Ussing chambers, and exposed to the activating peptide, SLIGRL-NH2 (50 μM), to activate PAR2. Pretreatment with inhibitors of cytosolic PLA2 (cPLA2) (AACOCF3, arachidonyltrifluoromethyl ketone), COX-1 [SC560, 5-(4-chlorophenyl)-1-(4-methoxyphenyl)-3-(trifluoromethyl)-1H-pyrazole], and COX-2 (celecoxib) resulted in a significant concentration-dependent attenuation of PAR2-induced changes in short-circuit current. Immunoblot analysis showed a PAR2-induced increase in cPLA2 phosphorylation that was blocked by the mitogen-activated protein kinase kinase inhibitor, PD98059 [2-(2-amino-3methoxyphenyl)-4H-1benzopyran-4-one, C16H13NO3], and the pan-protein kinase C inhibitor, GFX (bisindolylmaleimide). PAR2 stimulation also resulted in a large increase in the production of PGE2 as determined by enzyme-linked immunosorbent assay and was also blocked by PD98059 and GFX. Immunofluorescence and immunoblot analysis determined that EP2 and EP4 are expressed at the basolateral membrane of SCBN cells. Through the use of selective inhibitors (EP2, AH6809 [6-isopropoxy-9-oxoxanthene-2-carboxylic acid]; EP4, GW627368X [N-{2[4,9-diethoxy-1-oxo-1,3-dihydro-2H-benzo[f]isoindol-2-yl)phenyl] acetyl}benzene sulphonamide]), it was found that both EP2 and EP4 were involved in mediating the PAR2-induced chloride secretory response. We conclude that basolateral PAR2 activation induces epithelial chloride secretion that is mediated by cPLA2, COX-1, COX-2, and the subsequent release of PGE2. The production of PGE2 results in an autocrine secretory response that is dependent on basolateral EP2 and EP4 receptors.

Profiles of Lamina Propria T Helper Cell Subsets Discriminate Between Ulcerative Colitis and Crohn's Disease.

Background:Distinction between 2 forms of inflammatory bowel disease (IBD), ulcerative colitis (UC) and Crohns disease (CD), can be challenging. Aberrant mucosal immunity suggests that CD is a T helper type 1 cell (Th1)-driven disease, whereas UC as Th2-driven response. However, whether this paradigm truly distinguishes CD from UC is controversial. We aimed to clarify the discriminating potential of lamina propria Th subsets in patients with IBD. Methods:Biopsies from 79 patients with IBD and 20 healthy controls were collected for Th subsets analysis (Th1:interferon &ggr; [IFN-&ggr;], T-bet; Th2:interleukin 13 [IL-13], Gata3; Th17:IL-17, ROR&ggr;t; Treg:FoxP3). The receiver-operating characteristic curves were constructed to assess the discriminating ability by calculating the area under the receiver-operating characteristic curve. The equation with the highest area under the receiver-operating characteristic curve was applied to newly diagnosed patients to evaluate discriminating ability. Results:Patients with CD showed increased IFN-&ggr;+ or T-bet+ cells and decreased IL-13+ or Gata3+ cells compared with UC. A discriminant equation composed of 4 markers (IFN-&ggr;+, T-bet+, IL-13+, and Gata3+) yielded the highest area under the receiver-operating characteristic curve. In 36 established CD or UC, the sensitivity, specificity, positive and negative predictive probabilities were 92.6%, 55.6%, 86.2%, and 71.4% and in 14 newly diagnosed patients were 100.0%, 42.9%, 63.6%, and 100.0%. Furthermore, Gata3+ cells were increased in tumor necrosis factor inhibitor therapy nonresponders compared with responders in CD. IFN-&ggr;+ cells were directly and inversely proportional to disease activity in patients with CD and UC, respectively. Conclusions:The Th1/Th2 paradigm can distinguish CD from UC and may be further associated with response to tumor necrosis factor inhibitor in CD and disease activity in patients with IBD.

Proteinase-activated Receptor 2 (PAR2) Decreases Apoptosis in Colonic Epithelial Cells

Background: Inflammatory bowel disease management lacks therapies that heal the epithelial barrier. Results: PAR2 activation increases activities of MEK1/2 and PI3K in intestinal epithelial cells, which blocks apoptosis. Conclusion: Cytokine-induced apoptosis in colonic epithelial cells is inhibited by PAR2 signaling. Significance: PAR2 is important in maintaining intestinal epithelial homeostasis. Mucosal biopsies from inflamed colon of inflammatory bowel disease patients exhibit elevated epithelial apoptosis compared with those from healthy individuals, disrupting mucosal homeostasis and perpetuating disease. Therapies that decrease intestinal epithelial apoptosis may, therefore, ameliorate inflammatory bowel disease, but treatments that specifically target apoptotic pathways are lacking. Proteinase-activated receptor-2 (PAR2), a G protein-coupled receptor activated by trypsin-like serine proteinases, is expressed on intestinal epithelial cells and stimulates mitogenic pathways upon activation. We sought to determine whether PAR2 activation and signaling could rescue colonic epithelial (HT-29) cells from apoptosis induced by proapoptotic cytokines that are increased during inflammatory bowel disease. The PAR2 agonists 2-furoyl-LIGRLO (2f-LI), SLIGKV and trypsin all significantly reduced cleavage of caspase-3, -8, and -9, poly(ADP-ribose) polymerase, and the externalization of phosphatidylserine after treatment of cells with IFN-γ and TNF-α. Knockdown of PAR2 with siRNA eliminated the anti-apoptotic effect of 2f-LI and increased the sensitivity of HT-29 cells to cytokine-induced apoptosis. Concurrent inhibition of both MEK1/2 and PI3K was necessary to inhibit PAR2-induced survival. 2f-LI was found to increase phosphorylation and inactivation of pro-apoptotic BAD at Ser112 and Ser136 by MEK1/2 and PI3K-dependent signaling, respectively. PAR2 activation also increased the expression of anti-apoptotic MCL-1. Simultaneous knockdown of both BAD and MCL-1 had minimal effects on PAR2-induced survival, whereas single knockdown had no effect. We conclude that PAR2 activation reduces cytokine-induced epithelial apoptosis via concurrent stimulation of MEK1/2 and PI3K but little involvement of MCL-1 and BAD. Our findings represent a novel mechanism whereby serine proteinases facilitate epithelial cell survival and may be important in the context of colonic healing.

Smooth Muscle Hyperplasia/Hypertrophy is the Most Prominent Histological Change in Crohn’s Fibrostenosing Bowel Strictures: A Semiquantitative Analysis by Using a Novel Histological Grading Scheme

Background: The simplistically and ambiguously termed ‘fibrostenosis’ of bowel is a hallmark of severe Crohn’s disease [CD] and a major contributor to medical treatment failure. Non-invasive imaging assessment and novel medical therapy targeting this condition are under investigation, which particularly requires a better understanding of the underlying histological basis. Methods: We analysed 48 patients with stricturing Crohn’s ileitis or/and colitis that required surgical resection. The most representative sections of the fibrostenotic, non-stenotic and uninvolved regions were reviewed for histological analysis. For each layer of bowel wall (mucosa including muscularis mucosae [MU], submucosa [SM], muscularis propria [MP], subserosal adventitia [SS]), histological abnormalities were evaluated individually, including active and chronic inflammation, fibrosis, smooth muscle hyperplasia or hypertrophy, neuronal hypertrophy and adipocyte proliferation. A novel semiquantitative histological grading scheme was created. Results: The most significant histopathological features characterizing the stricturing intestines were smooth muscle hyperplasia of SM, hypertrophy of MP and chronic inflammation. The muscular alteration was predominant in all layers. The overall muscular hyperplasia/hypertrophy was positively correlated with chronic inflammation and negatively correlated with fibrosis, whereas SM muscular hyperplasia was also associated with MU active inflammation. Similar changes, to a lesser extent, occurred in the adjacent non-stenotic inflamed bowel as well. Conclusions: In CD-associated ‘fibrostenosis’, it is the smooth muscle hyperplasia/hypertrophy that contributes most to the stricturing phenotype, whereas fibrosis is less significant. The ‘inflammation-smooth muscle hyperplasia axis’ may be the most important in the pathogenesis of Crohn’s strictures.