Introduction and goals: Hypoxia, regulates cell proliferation, differentiation and effector function, which is mainly driven by hypoxia inducible factors (HIFs). In pathological conditions hypoxia facilitates tissue damage and increases disease severity. The aim of our project is to investigae whether C. difficile toxins A (TcdA) and B (TcdB) influence hypoxic factors (HIF-1/2 α) and tissue damage in C. difficile infection.

Materials and methods: The C2BBe1 colonic epithelial cell line was exposed to C. difficile TcdA and TcdB under hypoxic and normoxic conditions. Hypoxia was induced by atmospheric oxygen reduction (1% O2) or chemically with CoCl2 or deferoxamine (DFO). We assessed production of proinflammatory cytokines, cell cytotoxicity, HIF1/2α levels and epithelial barrier permeability.

Results: While C2BBe1 cells challenged with TcdA and TcdB produced IL-8, exposure to 1% O2 - but not CoCl2 - reduced the amount of IL-8 secreted by C2BBe1 cells upon TcdA stimulation. We further observed an increase in cell cytotoxicity and epithelial permeability under 1% O2 with TcdA and TcdB when compared to normoxia and CoCl2 treatment. Furthermore, we saw that toxins did not affect HIF1α and HIF-2α stabilized protein levels. To understand the contribution of HIF factors on toxin-mediated permeability increase, we next assessed activation of either HIF-1α by CoCl2 or HIF-1α and HIF-2α by DFO. Interestingly, highest permeability increase was observed in 1% O2 hypoxia, whereas CoCl2 and DFO exhibited a similar level of damage as under normoxia.

Conclusion: TcdA and TcdB-mediated effects on the cytokine response and epithelial barrier function are altered by hypoxia. The effects observed with chemically-induced hypoxia differ from those obtained at 1% O2. Thus, these effects are most likely not due to direct engagement of HIF factors, but rather to increased susceptibility of epithelial cells to toxins under hypoxic conditions. Future work aims to investigate the underlying molecular mechanisms.

Funding: Innovative Medicines Initiative 2 Joint Undertaking (grant agreement No 101007799 (Inno4Vac)).

Key words C. difficile, gastrointestinal mucosa, hypoxia, HIF