Role of the haem oxygenase/carbon monoxide pathway in Clostridium difficile toxin A-induced enteritis in mice

Abstract

Clostridium difficile is the major cause of antibiotic-associated colitis, a disease with significant morbidity and mortality. This study investigated the role of the haem oxygenase-1 (HO-1)/carbon monoxide (CO) pathway in C. difficile toxin A-induced enteritis in mice. The HO substrate haemin, zinc protoporphyrin IX (ZnPP IX), a specific HO-1 inhibitor, dimanganese decacarbonyl (DMDC), a CO donor, or an equivalent volume of their respective vehicles were injected subcutaneously 30 min prior to local challenge with toxin A (25 or 50 mg per ileal loop) or PBS. Intestinal ileal loop weight/length ratios were calculated 3 h later. Ileal tissues were collected for histological analysis and measurement of myeloperoxidase (MPO) activity, tumour necrosis factor alpha (TNF-a) and interleukin-1 beta (IL-1b) production by ELISA and immunohistochemistry for HO-1. Treatment of mice subjected to C. difficile toxin A (TcdA) with haemin or DMDC prevented oedema, mucosal disruption and neutrophil infiltration observed in histological analysis. It also decreased TcdAinduced MPO activity and TNF-a or IL-1b production. In contrast, the specific HO-1 inhibitor (ZnPP IX) exacerbated all these evaluated parameters. TcdA increased HO-1 expression as seen by immunohistochemistry. These results suggest that the HO-1/CO pathway exerts a protective role in TcdA-induced enteritis and that its pharmacological modulation might be important for the management of C. difficile-associated disease. INTRODUCTIONClostridium difficile is the major causative agent of antibiotic-associated diarrhoea. The main C. difficile virulence factors are two large exotoxins, toxin A (TcdA) and toxin B (TcdB), which show substantial sequence similarity and share common structural elements (Pothoulakis et al., 1986). A recent report demonstrated that, instead of TcdA, TcdB is actually the essential virulence factor for the development of C. difficileassociated disease (Lyras et al., 2009). However, by using a gene knockout system to inactivate the toxin genes permanently, Kuehne et al. (2010) found that C. difficile producing either one or both toxins showed cytotoxic activity in vitro that translated directly into virulence in vivo, re-establishing the importance of both toxins in the pathogenesis of C. difficile-associated disease. The mechanism of TcdA-induced enteritis involves toxin binding to enterocyte receptors, which leads to infiltration of the mucosa by neutrophils, degranulation of mast cells and activation of sensory and enteric nerves, resulting in enhanced intestinal secretion and motility (Brito et al., 2002b;Kelly et al., 1994a;Pothoulakis et al., 1998).Haem oxygenase (HO; EC 1.14.99.3) catalyses haem degradation to iron, carbon monoxide (CO) and biliverdin (BVD) (Abraham et al., 1988). Three isozymes have been identified: HO-2 and HO-3, which are constitutive forms (Maines et al., 1986;McCoubrey et al., 1997), and HO-1, which is induced by various stimuli such as haem, haemin, cytokines, mitogens, metals, reactive oxygen species, heat shock, UV ...

Transforming Growth Factor β1/SMAD Signaling Pathway Activation Protects the Intestinal Epithelium from Clostridium difficile Toxin A-Induced Damage

“…Accordingly, it was previously demonstrated that previous exposure to low concentrations of TcdA (Ͻ10 ng/ml) also induced expression of TGF-␤1 by T84 cells, a human colon adenocarcinoma cell line (24). The present report adds new insight about the intracellular mechanism of activation of the TGF-␤1 pathway in a nontransformed rat small intestinal epithelial cell line with characteristics of crypt epithelial cells and demonstrates that this cytokine is also expressed and produced in response to TcdA in vivo in a well-established TcdA-induced enteritis model in mice (27).…”

CO-releasing Metal Carbonyl Compounds as Antimicrobial Agents in the Post-antibiotic Era

“…Several bacteria also possess HO enzymes that function to degrade heme that is imported for use as an iron source ( 58 , 59 ). HO activity contributes to pathogenesis in certain bacteria by scavenging iron from heme ( 58 , 60 ).…”

“…CO is also implicated in the pathogenesis of Clostridium difficile . Inhibition of host HO activity by administering Zn protoporphyrin IX to mice exacerbated the histopathological alterations elicited by C. difficile toxin A; conversely, pretreatment of mice with a CO donor (dimanganese decacarbonyl) reduced the effect ( 60 ).…”

Carbon Monoxide (CO) Released from Tricarbonyldichlororuthenium (II) Dimer (CORM-2) in Gastroprotection against Experimental Ethanol-Induced Gastric Damage

“…It is nowadays accepted that CO exerts pleiotropic cellular effects by acting through a number of signaling pathways including those regulated by mitogen-activated protein kinases (MAPKs), peroxisome proliferator-activated receptor gamma (PPAR-γ), cellular ROS production, calcium-dependent potassium channels (Kca) and soluble guanylyl cyclase (sGC) [ 7 , 8 , 9 ]. Moreover, numerous studies have demonstrated that HO-1 and its metabolites, CO and biliverdin can modulate the inflammatory processes [ 10 , 11 ]. CO, acting via MAPKs activity, inhibits the production of pro-inflammatory cytokines (TNF-α, IL-1β) in vitro and in vivo and increases the expression of the anti-inflammatory cytokine IL-10.…”