Phosphatidylcholine and the intestinal mucus layer: in vitro efficacy against Clostridium difficile-associated polymorphonuclear neutrophil activation.

BACKGROUND: Phosphatidylcholine (PC), an important component of intestinal mucus, protects against Clostridium difficile toxin-induced intestinal barrier injury in vitro. Polymorphonuclear neutrophil (PMN) activation may contribute to intestinal injury and systemic toxicity in patients with C. difficile-associated disease. We therefore hypothesized that the intestinal barrier function against C. difficile toxin by exogenous PC would ameliorate PMN activation. METHODS: Intestinal epithelial cell (IEC) monolayers were cocultured with C. difficile toxin A and/or exogenous PC. Naïve PMNs were cocultured with IEC culture supernatants and PMN activation, and chemotactic potential determined. RESULTS: PC treatment of IEC abrogated the enhanced PMN activation and chemotactic potential following toxin A exposure (P < .001). CONCLUSIONS: Exogenous PC ameliorated PMN activation from IECs exposed to C. difficile toxin. Administration of exogenous PC may be a useful adjunctive treatment in severely ill or immunocompromised patients with C. difficile-associated disease.

Exogenous phosphatidylcholine supplementation improves intestinal barrier defense against Clostridium difficile toxin.

BACKGROUND: The incidence and severity of Clostridium difficile colitis have increased dramatically in the last decade. Disease severity is related to C. difficile virulence factors, including toxins A and B, as well as the patient's immune status. The intestinal mucus is an important component of innate barrier function in the intestine. Phosphatidylcholine (PC) is a key constituent of the intestinal mucus barrier, and exogenous PC administration has had therapeutic efficacy in patients with ulcerative colitis. We studied the protective function of exogenous PC on C. difficile toxin effects on the intestinal barrier in vitro. METHODS: Mucus-producing (HT29-MTX strain) and non-mucus-producing (HT29 strain) intestinal epithelial monolayers were cocultured with PC and C. difficile toxin A added to the apical media. Basal chamber culture supernatants were subsequently obtained, and tumor necrosis factor and interleukin 6 were quantitated by enzyme-linked immunosorbent assay. In other experiments, HT29 toxin A uptake, intestinal monolayer permeability, necrosis, and actin microfilament disruption were determined. RESULTS: There was a threefold to fourfold decrease in tumor necrosis factor and interleukin 6 levels and similar decreases in toxin A uptake and permeability changes in intestinal epithelial cells with mucus or PC versus control. Intestinal epithelial cell necrosis was reduced by more than 50% with either mucus or PC versus control. The integrity of HT29 cell cytoskeleton was demonstrated by both the mucus layer of the HT29-MTX strain and by exogenous PC administration by phalloidin staining of actin microfilaments. CONCLUSION: PC supplementation was effective in improving intestinal barrier defense against C. difficile toxin A challenge. PC administration may be a useful therapeutic adjunct in severe cases of C. difficile colitis or in patients who do not improve with conventional treatment.

Effect of host defenses on Clostridium difficile toxin-induced intestinal barrier injury.

INTRODUCTION: The severity of Clostridium difficile-associated infection depends on the virulence factors of the organism and host factors, including intestinal barrier function. The intestinal mucus layer has recently been recognized as the first line of defense against enteric pathogens. Its interaction with mucosal humoral immunity provided by secretory immunoglobulin A (SIgA) is unknown as it relates to C. difficile disease severity. This was studied in vitro. METHODS: Confluent HT29 (non-mucus-producing) and HT29-MTX (mucus-producing) intestinal epithelial cells (IECs) with and without transcytosed SIgA were exposed to C. difficile toxin A (6 hours), and IEC toxin internalization, permeability (fluorescein isothiocyanate dextran), and necrosis (propidium iodide staining) were determined. In other experiments, colostral SIgA was added to the apical surface of IEC, and cleavage was determined by measurement of intact SIgA and secretory component fractions by enzyme-linked immunosorbent assay. Tumor necrosis factor α and interleukin 6 were measured from basal chamber culture supernatants by enzyme-linked immunosorbent assay. RESULTS: Toxin A uptake and subsequent enterotoxic effects on IEC were decreased by both the mucus layer and SIgA. Similar findings were noted with the effects of toxin A on IEC monolayer permeability, cytoskeleton changes, and proinflammatory cytokine release. The combination of the mucus layer and SIgA afforded the best protection against the adverse effects on IEC by toxin A. It seems that the mucus layer was also protective against SIgA cleavage, resulting in reduced protease activity by HT29 cells exposed to toxin A. CONCLUSION: Both intestinal mucus and SIgA were important in limiting C. difficile-associated disease severity in this model. A synergistic effect of mucus and IgA was also noted and may be due to protection of SIgA from proteolytic cleavage.