Effects of Toxin A fromClostridium difficileon Mast Cell Activation and Survival

Abstract

Toxins A and B from Clostridium difficile are the main cause of antibiotic-associated diarrhea and pseudomembranous colitis. They cause fluid accumulation, necrosis, and a strong inflammatory response when inoculated in intestinal loops. Since mast cells are a rich source of inflammatory mediators, abundant in the gut, and known to be involved in C. difficile-induced enteritis, we studied the in vitro effect of toxin A on isolated mast cells. Normal rats sensitized by infection with Nippostrongilus brasiliensis were used to isolate peritoneal mast cells (PMC). PMC from naive rats were stimulated with calcium ionophore A23187 as a model of antigen-independent activation, and PMC from sensitized rats were stimulated with N. brasiliensis antigens to study immunoglobulin E-dependent mast cell activation. After 4 h, toxin A did not induce release of nitric oxide or histamine in naive PMC. However, 10 ng of toxin per ml caused a significant release of tumor necrosis factor alpha (TNF-α). In contrast, 1 μg of toxin per ml inhibited antigen or A23187-induced histamine release by PMC. Toxin A at 1 μg/ml for 4 h caused disruption of actin which aggregated in the cytoplasm and around the nucleus. After 24 h, chromatin condensation, cytoplasmic blebbing, and apoptotic-like vesicles were observed; DNA fragmentation was documented also. These results suggest that mast cells may participate in the initial inflammatory response toC. difficile infection by releasing TNF-α upon interaction with toxin A. However, longer exposure to toxin A affects the release of inflammatory mediators, perhaps because of the alteration of the cytoskeleton and induction of apoptosis. The impaired functions and survival of mast cells by C. difficile toxin A could hamper the capacity of these cells to counteract the infection, thus prolonging the pathogenic effects of C. difficiletoxins.

Mast cells in innate immunity

“…In contrast, both cholera toxin and the toxin A from Clostridium difficile failed to induce histamine release from rat peritoneal mast cells while variably influencing the release of certain cytokines. Thus, 10 ng/ml of C. difficile toxin caused a significant release of TNF-a at 4 h of incubation, while the cholera toxin significantly enhanced IL-6 production by mast cells but inhibited TNF-a production (38,39). A differential effect on degranulation and cytokine release was also noticed when the effect of enterobacterial LPS on rat peritoneal mast cells was investigated.…”

The pathogenicity of Clostridium difficile

“…Neutrophil recruitment appears to be an essential step in the pathogenesis of C. difficile toxin-induced intestinal injury as biopsy specimens from patients with C. difficile colitis show marked vascular congestion, neutrophil infiltration of the lamina propria and inflammation. Although it was shown by Calderon et al [15] that toxin A was able to activate neutrophils, mast cells and macrophages in vitro, there is still some speculation as to how this works in vivo due to the large size of the toxin. Toxin A can cause detachment and apoptosis of enterocytes and so, in this disrupted epithelium, toxin A may diffuse and interact with the inflammatory cells in the lamina propria.…”

Modeling host–pathogen interactions: H. sapiens as a host and C. difficile as a pathogen

“…More reports are also available (Filho et al ., ; Rocha et al ., ), which show that both TcdA and TcdB induce an intense neutrophil migration that is mediated by macrophage‐derived TNF‐ α . Another study by (Calderon et al ., ) has also shown that mast cells release TNF‐ α as initial inflammatory response to C. difficile TcdA.…”