The effect of different C. difficile MLST strains on viability and activity of macrophages

Objectives: Clostridioides difficile is the most common infectious agent of nosocomial diarrhea. C. difficile infection (CDI) pathogenesis and disease severity depend on its toxins (toxins A, B and binary) and on the host's immune response, especially the innate immune system. The current study examined the efficacy of macrophage activity, macrophages viability and cytokine secretion levelsin response to different sequence type (ST) strains of C. difficile. Methods: RAW 264.7 macrophages were exposed to six different strains of C. difficile as well as to both toxins A and B and macrophage viability was measured. The levels of four secreted cytokines were determined by RT-PCR and ELISA. Morphological changes to the macrophages were investigated by fluorescent microscopy. Results: Strains ST37 and ST42 affected macrophages’ vitality the most. Toxins A and B led to a significant reduction in macrophages' vitality at most time points. In addition, starting at 30-min post-exposure to 5 ng/μl of both toxins led to significant differences in macrophage viability versus at lower concentrations. Furthermore, cytokine secretion levels, including IL-12, IL-6 and TNF-α, increased dramatically when macrophages were exposed to strains ST42 or ST104. Finally, gene expression surveys point to increases in IL-12 gene expression in response to both ST42 and ST104. Conclusions: C. difficile strains with higher toxins levels induced an increased activation of the innate immune system and may activate macrophages more profoundly resulting in secretion of higher levels of pro-inflammatory cytokines. However, higher toxin levels may also damage macrophages’ normal skeletal structure, reducing macrophage viability.