Protease-Activated Receptor 2, Dipeptidyl Peptidase I, and Proteases Mediate Clostridium difficile Toxin A Enteritis

Harvard University, Cambridge, Massachusetts, United States
Gastroenterology (Impact Factor: 16.72). 06/2007; 132(7):2422-37. DOI: 10.1053/j.gastro.2007.03.101
Source: PubMed

ABSTRACT We studied the role of protease-activated receptor 2 (PAR(2)) and its activating enzymes, trypsins and tryptase, in Clostridium difficile toxin A (TxA)-induced enteritis.
We injected TxA into ileal loops in PAR(2) or dipeptidyl peptidase I (DPPI) knockout mice or in wild-type mice pretreated with tryptase inhibitors (FUT-175 or MPI-0442352) or soybean trypsin inhibitor. We examined the effect of TxA on expression and activity of PAR(2) and trypsin IV messenger RNA in the ileum and cultured colonocytes. We injected activating peptide (AP), trypsins, tryptase, and p23 in wild-type mice, some pretreated with the neurokinin 1 receptor antagonist SR140333.
TxA increased fluid secretion, myeloperoxidase activity in fluid and tissue, and histologic damage. PAR(2) deletion decreased TxA-induced ileitis, reduced luminal fluid secretion by 20%, decreased tissue and fluid myeloperoxidase by 50%, and diminished epithelial damage, edema, and neutrophil infiltration. DPPI deletion reduced secretion by 20% and fluid myeloperoxidase by 55%. In wild-type mice, FUT-175 or MPI-0442352 inhibited secretion by 24%-28% and tissue and fluid myeloperoxidase by 31%-71%. Soybean trypsin inhibitor reduced secretion to background levels and tissue myeloperoxidase by up to 50%. TxA increased expression of PAR(2) and trypsin IV in enterocytes and colonocytes and caused a 2-fold increase in Ca(2+) responses to PAR(2) AP. AP, tryptase, and trypsin isozymes (trypsin I/II, trypsin IV, p23) caused ileitis. SR140333 prevented AP-induced ileitis.
PAR(2) and its activators are proinflammatory in TxA-induced enteritis. TxA stimulates existing PAR(2) and up-regulates PAR(2) and activating proteases, and PAR(2) causes inflammation by neurogenic mechanisms.

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Available from: Graeme Cottrell, May 08, 2014
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    • "This is supported by our observations of colocalization of PAR2 and tryptase expression in and around goblet cells, the induced mucin depletion within these cells, and the blockade of inflammation , mucin depletion, and mastocytosis by the PAR2 antagonist GB88. Various serine proteases and their precursors [e.g., trypsinogens (Hansen et al., 2005; Cottrell et al., 2007) and tryptases (He and Xie, 2004; He et al., 2004)] released by cells resident in the colon may have antibacterial roles (Huang et al., 2001; Thakurdas et al., 2007). These endogenous antibiotics could contribute to the primary barrier protection of the gut. "
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    ABSTRACT: Many trypsin-like serine proteases such as β-tryptase are involved in the pathogenesis of colitis and inflammatory bowel diseases. Inhibitors of individual proteases show limited efficacy in treating such conditions, but also probably disrupt digestive and defensive functions of proteases. Here, we investigate whether masking their common target, protease-activated receptor 2 (PAR2), is an effective therapeutic strategy for treating acute and chronic experimental colitis in rats. A novel PAR2 antagonist (5-isoxazoyl-Cha-Ile-spiro[indene-1,4'-piperidine]; GB88) was evaluated for the blockade of intracellular calcium release in colonocytes and anti-inflammatory activity in acute (PAR2 agonist-induced) versus chronic [2,4,6-trinitrobenzenesulfonic acid (TNBS)-induced] models of colitis in Wistar rats. Disease progression (disease activity index, weight loss, and mortality) and postmortem colonic histopathology (inflammation, bowel wall thickness, and myeloperoxidase) were measured. PAR2 and tryptase colocalization were investigated by using immunohistochemistry. GB88 was a more potent antagonist of PAR2 activation in colonocytes than another reported compound, N¹-3-methylbutyryl-N⁴-6-aminohexanoyl-piperazine (ENMD-1068) (IC₅₀ 8 μM versus 5 mM). Acute colonic inflammation induced in rats by the PAR2 agonist SLIGRL-NH₂ was inhibited by oral administration of GB88 (10 mg/kg) with markedly reduced edema, mucin depletion, PAR2 receptor internalization, and mastocytosis. Chronic TNBS-induced colitis in rats was ameliorated by GB88 (10 mg/kg/day p.o.), which reduced mortality and pathology (including colon obstruction, ulceration, wall thickness, and myeloperoxidase release) more effectively than the clinically used drug sulfasalazine (100 mg/kg/day p.o.). These disease-modifying properties for the PAR2 antagonist in both acute and chronic experimental colitis strongly support a pathogenic role for PAR2 and PAR2-activating proteases and therapeutic potential for PAR2 antagonism in inflammatory diseases of the colon.
    Journal of Pharmacology and Experimental Therapeutics 02/2012; 340(2):256-65. DOI:10.1124/jpet.111.187062 · 3.97 Impact Factor
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    • "PAR2 has been proposed to be important in inflammation (Niu et al., 2008; Shin et al., 2009) and proliferation (Nishibori et al., 2005; Arisawa et al., 2007; Matej et al., 2007; Iwaki et al., 2008). Based mainly on the properties of these exogenous peptide agonists, together with some knockout mouse studies, activation of PAR2 has been purported to be proinflammatory in arthritis (Ferrell et al., 2003; Kelso et al., 2007; Lam, 2007; McIntosh et al., 2007), pancreatitis (Maeda et al., 2005; Hirota et al., 2006), gastric (Hansen et al., 2005; Cottrell et al., 2007; Kawabata et al., 2008) and kidney (Moussa et al., 2007; Vesey et al., 2007a,b) cells, but antiinflammatory in airways inflammation (Cocks et al., 1999; D'Agostino et al., 2007). Increased expression of PAR2 associated with proliferation has been consistently found in many types of cancer cells, with reports on PAR2-induced cancer progression in breast (Matej et al., 2007), colon (Nishibori et al., 2005), gastric (Arisawa et al., 2007) and pancreatic cancers (Iwaki et al., 2008). "
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    ABSTRACT: Many cells express proteinase activated receptor 2 (PAR2) on their plasma membrane. PAR2 is activated by proteolytic enzymes, such as trypsin and tryptase that cleave the receptor N-terminus, inititating signalling to intracellular G proteins. Studies on PAR2 have relied heavily upon activating effects of proteases and peptide agonists that lack stability and bioavailability in vivo. A novel small molecule agonist GB110 and an antagonist GB88 were characterized in vitro against trypsin, peptide agonists, PAR2 antibody, PAR1 agonists and flow cytometry,in seven cell lines using intracellular Ca(2+) mobilization and examined in vivo against PAR2- and PAR1-induced rat paw oedema. GB110 is a potent non-peptidic agonist activating PAR2-mediated Ca(2+) release in HT29 cells (EC(50) ∼200 nM) and six other human cell lines, inducing PAR2 internalization. GB88 is a unique PAR2 antagonist, inhibiting PAR2 activated Ca(2+) release (IC(50) ∼2 µM) induced by native (trypsin) or synthetic peptide and non-peptide agonists. GB88 was a competitive and surmountable antagonist of agonist 2f-LIGRLO-NH(2), a competitive but insurmountable antagonist of agonist GB110, and a non-competitive insurmountable antagonist of trypsin. GB88 was orally active and anti-inflammatory in vivo, inhibiting acute rat paw oedema elicited by agonist GB110 and proteolytic or peptide agonists of PAR2 but not by corresponding agonists of PAR1 or PAR4. The novel PAR2 agonist and antagonist modulate intracellular Ca(2+) and rat paw oedema, providing novel molecular tools for examining PAR2-mediated diseases.
    British Journal of Pharmacology 08/2011; 165(5):1413-23. DOI:10.1111/j.1476-5381.2011.01610.x · 4.84 Impact Factor
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    • "One explanation for these findings is that C. difficile toxins cause direct injury to the intestinal epithelium, which is associated with a robust host inflammatory response with neutrophil activation and recruitment leading in turn to greatly amplified intestinal injury. In fact, a wide range of anti-inflammatory agents can reduce intestinal injury in animal models of CDI (Anton et al., 2004; Chen et al., 2006; Cottrell et al., 2007; Kim et al., 2005, 2007; Kokkotou et al., 2009; Pothoulakis et al., 1993; Warny et al., 2000). The use of combinations of antibiotic and anti-inflammatory agents to treat severe CDI in humans warrants greater attention and investigation, especially given the rising incidence of severe and fatal disease (McDonald et al., 2006; Redelings et al., 2007). "
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    ABSTRACT: Clostridium difficile is the most common cause of nosocomial bacterial diarrhoea in the Western world. Diarrhoea and colitis are caused by the actions of toxins A and B released by pathogenic strains of C. difficile. Adaptive immune responses to these toxins influence the outcomes of C. difficile infection (CDI). Symptomless carriers of toxinogenic C. difficile and those with a single episode of CDI without recurrence show more robust antitoxin immune responses than those with symptomatic and recurrent disease. Immune-based approaches to CDI therapy and prevention have been developed using active vaccination or passive immunotherapy targeting C. difficile toxins. Innate immune responses to C. difficile and its toxins are also central to the pathophysiology of CDI. An acute intestinal inflammatory response with prominent neutrophil infiltration and associated tissue injury is characteristic of CDI. Furthermore, inhibiting this acute inflammatory response can protect against the intestinal injury that results from exposure to C. difficile toxins in animal models. Studies examining host risk factors for CDI have led to validated clinical prediction tools for risk of primary and of recurrent disease. Risk factors associated with severe CDI with poor clinical outcomes have also been identified and include marked elevation of the peripheral white blood cell count and elevated creatinine. However, further work is needed in this area to guide the clinical application of new approaches to disease prevention and treatment including new antimicrobials as well as passive and active immunization.
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