Corticotropin-releasing hormone (CRH) requirement inClostridium difficiletoxin A-mediated intestinal inflammation

Abstract

Clostridium difficile, the causative agent of antibiotic-associated colitis, mediates inflammatory diarrhea by releasing toxin A, a potent 308-kDa enterotoxin. Toxin A-induced inflammatory diarrhea involves many steps, including mucosal release of substance P (SP) corticotropin-releasing hormone (CRH) and neutrophil transmigration. Here we demonstrate that, compared with wild type, mice genetically deficient in CRH (Crh ؊/؊ ) have dramatically reduced ileal fluid secretion, epithelial cell damage, and neutrophil transmigration 4 h after intraluminal toxin A administration. This response is associated with diminished mucosal activity of the neutrophil enzyme myeloperoxidase compared with that of wildtype mice. In wild-type mice, toxin A stimulates an increase in intestinal SP content compared with buffer administration. In contrast, toxin A administration in Crh ؊/؊ mice fails to result in an increased SP content. Moreover, immunohistochemical experiments showed that CRH and SP are colocalized in some enteric nerves of wild-type mice, and this colocalization is more evident after toxin A administration. These results provide direct evidence for a major proinflammatory role for CRH in the pathophysiology of enterotoxin-mediated inflammatory diarrhea and indicate a SP-linked pathway.C orticotropin-releasing hormone (CRH), a 41-aa peptide, is a major peptide hormone that modulates the activity of the hypothalamic-pituitary-adrenal axis during stress, including inflammation (1-3). CRH mediates its effects by binding to its G protein-coupled receptor subtypes, CRH1 and CRH2 (4-6). CRH plays a dual role in the pathophysiology of inflammation. CRH secreted from the hypothalamus has indirect antiinflammatory effects via stimulation of glucocorticoid release, but CRH is also expressed peripherally, and its expression is increased in leukocytes, nerve fibers, and other cells involved in inflammatory reactions (7-12). Moreover, in vivo and in vitro studies indicate that CRH is a potent proinflammatory peptide (7,(12)(13)(14)(15).A limited number of studies indicate that CRH and its receptors play a role in the pathophysiology of intestinal inflammation. Thus, Van Tol et al. (11) found increased CRH expression in the rat cecum in colitis induced by injection of peptidoglycan-polysaccharide polymers. Increased expression of CRH-immunoreactive cells was also evident in the colonic mucosa of patients with inflammatory bowel disease (16). CRH gene expression is increased in hypothalamic neurons during colitis after intracolonic administration of 2,4,6-trinitrobenzenesulfonic acid (17) and appears to play a protective role in the worsening of experimental colitis induced by stress (18). We have recently shown that expression of CRH as well as CRH1 and CRH2 was dramatically increased in mouse ileum shortly after intraluminal administration of Clostridium difficile toxin A (19). Moreover, experiments with CRH receptor antagonists indicate that CRH plays a proinflammatory role in toxin A-induced intestinal secretion and inflammatio...

Application of Mutated Clostridium difficile Toxin A for Determination of Glucosyltransferase-Dependent Effects

“…In addition, the colonic cell line Caco-2 was used to investigate the cytopathic property of mutant toxins with respect to transepithelial electrical resistance (TER). The TER of confluent Caco-2 cell monolayers grown on filter inserts (Falcon; BD, Germany) was determined, starting with an initial value of at least 150 ⍀ · cm 2 (Fig. 1C).…”

“…Several reports of the in vivo effects of TcdA in animal models reflect efforts to understand the cellular mechanism leading to clinical symptoms as well as to the release of mediators that are involved in the inflammatory process (2,13,15,18,20). The inherent glucosyltransferase…”

Expression of corticotropin releasing factor (CRF) in the enteric nervous system of the jejunum of sheep

“…Th is is in contrast to a previous study of Liu et al (2006), who reported that in the small intestine of the guinea-pig 55% of CRF-expressing myenteric neurons were also SP-positive and no colocalization of CRF with SP was detected in submucous neurons. It is noteworthy that in the murine ileum, some CRF-IR/SP-IR enteric nerves were found (Anton et al, 2004). In the mammalian intestine SPexpressing myenteric neurons projecting into the circular smooth muscle layer are considered to be cholinergic excitatory motor neurons (Brookes, 2001).…”

“…Admittedly, Lazar et al (2003) suggested that capsaicin-sensitive neurons do not mediate the eff ect of exogenous CRF in the guinea-pig ileum but other reports have indicated that CRF is an important factor involved in visceral pain development during infl ammatory processes (for a review see Larauche et al, 2009). Since in the wild type mouse, the co-localization of CRF with SP in the ileal enteric nerves is more evident after Clostridium diffi cile toxin A administration it has been proposed that CRF secreted in response to toxin A is correlated with the pro-infl ammatory eff ect of SP (Anton et al, 2004).…”

Clostridium difficile-related postinfectious IBS: a case of enteroglial microbiological stalking and/or the solution of a conundrum?

“…A few studies on the effects of C. difficile toxins on enteric neurons showed that toxin A stimulates extrinsic sensory nerves and causes uncontrolled gut inflammation, likely mediated by substance P from primary afferent neurons [50]; this inflammation was prevented by extrinsic surgical denervation [51,52]. On the other hand, neuronal activation of VIP-positive pathways after exposure to toxin B has been hypothesized as an attempt of a protective adaptive response toward a pathogenic agent aggression of the intestinal mucosa [53].…”