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

Abstract

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. 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. 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. 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.