Abstract
Clostridium difficile infection (CDI) is the leading cause of nosocomial infection in hospitalized patients receiving long-term antibiotic treatment. An excessive host inflammatory response is believed to be the major mechanism underlying the pathogenesis of C. difficile infection, and various proinflammatory cytokines such as IL-1β are detected in patients with C. difficile infection. IL-1β is known to be processed by caspase-1, a cysteine protease that is regulated by a protein complex called the inflammasome, which leads to a specialized form of cell death called pyroptosis. The function of inflammasome activation-induced pyroptosis is to clear or limit the spread of invading pathogens via infiltrated neutrophils. Here, we focused on inflammasome activation induced by intact C. difficile to re-evaluate the nature of inflammasome activation in CDI pathogenesis, which could provide information that leads to an alternative therapeutic strategy for the treatment of this condition in humans. First, we found that caspase-1-dependent IL-1β production was induced by C. difficile pathogens in macrophages and increased in a time-dependent manner. Moreover, intracellular toxigenic C. difficile was essential for ATP-P2X7 pathway of inflammasome activation and subsequent caspase-1-dependent pyroptotic cell death, leading to the loss of membrane integrity and release of intracellular contents such as LDH. Notably, we also observed that bacterial components such as surface layer proteins (SLPs) were released from pyroptotic cells. In addition, pro-IL-1β production was completely MyD88 and partially TLR2 dependent. Finally, to investigate the role of the caspase-1-dependent inflammasome in host defense, we found that colonic inflammasome activation was also induced by CDI and that caspase-1 inhibition by Ac-YVAD-CMK led to increased disease progression and C. difficile load. Taken together, the present results suggest that MyD88 and TLR2 are critical component in pro-IL-1β production and intracellular C. difficile following the ATP-P2X7 pathway of inflammasome activation and pyroptosis, which play important roles in host defense through the utilization of inflammation-mediated bacterial clearance mechanisms during C. difficile infection.
Highlights
Clostridium difficile infection (CDI) is the most well-known condition among the etiologies of nosocomial infectious diarrhea in hospitalized patients receiving long-term antibiotic treatment (Rupnik et al, 2009)
The results showed that mature IL-1β and caspase-1 were obviously observed at an multiplicity of infection (MOI) of 0.5 for 6 h and increased in a time-dependent manner in the supernatants of the infected macrophages (Figures 1A,B)
To demonstrate the role of caspase-1 in C. difficile-induced IL-1β release, we further infected the macrophages in the presence of Ac-YVAD-cmk, an irreversible specific caspase-1 inhibitor, and found that this procedure remarkably abolished the release of IL-1β and caspase-1 activation (Figure 1D)
Summary
Clostridium difficile infection (CDI) is the most well-known condition among the etiologies of nosocomial infectious diarrhea in hospitalized patients receiving long-term antibiotic treatment (Rupnik et al, 2009). C. difficile is the major cause of nosocomial antibiotic-associated diarrhea through the production of toxin A (TcdA) and toxin B (TcdB). The pathogenesis of C. difficile has been largely attributed to the production of TcdA and TcdB by the bacteria, vaccination with these toxins has not provided complete protection against the disease in animals (Torres et al, 1995), which suggests that other factors contribute to the disease severity. Patients with the TLR4 rs1927914 polymorphism (GG genotype) had a higher risk of C. difficile colonization (Hung et al, 2013), suggesting that CDI is associated with the innate immune status of the host. Various proinflammatory cytokines, such as IL-1β, are detected in patients with CDI (Steiner et al, 1997; Steele et al, 2012)
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