Introduction

Clostridioides difficile (C. difficile) is the leading cause of hospital/antibiotic-associated diarrhoea and pseudomembranous colitis, with dramatic incidence/mortality worldwide. C. difficile produces two toxins, Toxin A and B (TcdB) which cause cytopathic/cytotoxic effects and inflammation in different cell types and TcdB is generally more potent (1000-fold) than TcdA. Recently, we have demonstrated that also enteric glial cells (EGCs) are susceptible to TcdB cytotoxicity, and that inflammatory cytokines (TNF-a plus IFN-g) increased apoptosis of EGCs induced by TcdB, which was correlated with an increased caspase-3, -7, -9 and PARP activation without changes in Bax and Bcl-X_L expression. These results were suggestive because: a) TcdB lead to pro-inflammatory activity, b) cells localized in the intestinal wall, including EGCs, are subjected to the action of the TcdB in a environment characterized by pro-inflammatory cytokines such as IL-1b, TNF-a and IFN-g; c) EGCs, which are key regulators of enteric nervous system, gut homeostasis, the immune and inflammatory responses, and digestive and extradigestive diseases, could undergo an enhancement of the cytotoxic activity by the synergistic interaction of TcdB with pro-inflammatory cytokines.

Therefore, we analyzed the role of proteases (caspases, calpains and cathepsins) in the enhanced susceptibility to apoptosis of EGCs induced by TcdB plus TNF-a and IFN-g.

Methods
Rat-transformed EGCs were treated with 0.1ng/ml TcdB for 1.5h then stimulated for 24h with TNF-a (50ng/ml) plus IFN-g (50ng/ml). In some experiments, the cells were pretreated with proteases inhibitors BAF (pan-caspase inhibitor), z-DEVD-fmk (effector caspase inhibitor) z-IETD-fmk (caspase-8 inhibitor), PD150606 (calpain inhibitor) or CA-074Me (cathepsin B inhibitor). After 24h were analyzed: the percentage of apoptosis by flow cytometry, the caspase-3 and PARP activation by western blot.

Results
Apoptosis induced by TcdB plus TNF-a and IFN-g (TcdB+CK) was prevented by BAF, strongly reduced by PD150606, only partially reduced by z-IETD-fmk and CA-074Me indicating that apoptosis is caspase-, calpain- and cathepsin-dependent. The involvement of calpains was confirmed by analysis of fragments of α-spectrin specifically generated by activation of calpains. Indeed, TcdB alone and TcdB+CK induced a strong activation of calpains and also of caspases and the use of PD150606 reduced the generation of both α-spectrin fragments, suggesting that calpains are also involved in caspase activation. We found that caspase-3 activation was strongly increased by TcdB+CK in EGC (2-fold increase) and was significantly inhibited by PD150606 and Z-IETD.fmk, not affect by z-DEVD-fmk and increased by BAF. PARP activation was significantly inhibited by all inhibitors used. Surprisingly, the cathepsin inhibitor CA-074Me increased caspase-3 and PARP activation

Conclusions
These results indicate that the enhanced susceptibility of EGCs to TcdB induced apoptosis by TNF-a and IFN-g is mediated by caspases, calpains, and cathepsins that which mutually regulate their activation and the activation of the final effectors. These data could have important implication for C. difficile infection in vivo and could offer a rational for interventions of regulation/modulation of pro-inflammatory responses during this infection.