Enteric glial cells counteract Clostridium difficile Toxin B through a NADPH oxidase/ROS/JNK/caspase-3 axis, without involving mitochondrial pathways

Abstract

Enteric glial cells (EGCs) are components of the intestinal epithelial barrier essential for regulating the enteric nervous system. Clostridium difficile is the most common cause of antibiotic-associated colitis, toxin B (TcdB) being the major virulence factor, due to its ability to breach the intestinal epithelial barrier and to act on other cell types. Here we investigated TcdB effects on EGCs and the activated molecular mechanisms. Already at 2 hours, TcdB triggered ROS formation originating from NADPH-oxidase, as demonstrated by their reduction in the presence of the NADPH-oxidase inhibitor ML171. Although EGCs mitochondria support almost completely the cellular ATP need, TcdB exerted weak effects on EGCs in terms of ATP and mitochondrial functionality, mitochondrial ROS production occurring as a late event. ROS activated the JNK signalling and overexpression of the proapoptotic Bim not followed by cytochrome c or AIF release to activate the downstream apoptotic cascade. EGCs underwent DNA fragmentation through activation of the ROS/JNK/caspase-3 axis, evidenced by the ability of ML171, N-acetylcysteine, and the JNK inhibitor SP600125 to inhibit caspase-3 or to contrast apoptosis. Therefore, TcdB aggressiveness towards EGCs is mainly restricted to the cytosolic compartment, which represents a peculiar feature, since TcdB primarily influences mitochondria in other cellular types.

Dysbiosis-Associated Enteric Glial Cell Immune-Activation and Redox Imbalance Modulate Tight Junction Protein Expression in Gulf War Illness Pathology

“…Furthermore, we observed that a reactive EGC phenotype can also have detrimental effects on the EGCs as shown by increased DNA fragmentation and cell death through the increased inflammation and ROS generated. This ultimately may result in programmed cell death in glia by pyroptosis or apoptosis as shown elsewhere (Macchioni et al, 2017). The general loss in enteric glia could lead to suboptimal functioning of enteric neurons and even enteric neuropathy (Bassotti et al, 2018).…”

“…Studies have showed that NADPH oxidases are activated in response to pathogenic stimuli in human EGC (Macchioni et al, 2017). We found that treatment of EGC with LPS or HMGB1 significantly increased their expression of NOX-2 (Figures 11A–D) ( P < 0.05).…”

“…Increased pathogenic stimuli were found to initiate apoptosis in EGC (Macchioni et al, 2017). We also investigated the fate of these reactive EGCs when continually exposed to PAMPs or DAMPs though a tunel assay to detect fragmented DNA.…”

Protein Kinase A/CREB Signaling Prevents Adriamycin-Induced Podocyte Apoptosis via Upregulation of Mitochondrial Respiratory Chain Complexes

“…The results of our previous study indicated that mitochondrial function was associated with ADR-induced apoptosis (12). It is well known that mitochondria are essential for oxidative phosphorylation and generation of reactive oxygen species during ATP production (35). Zhu et al reported that the strong apoptotic response in ADR-treated cells is associated with increased mitochondrial damage and ROS production (36).…”

The Enteric Glial Network Acts in the Maintenance of Intestinal Homeostasis and in Intestinal Disorders

“…It was demonstrated that TcdB stimulates morphological alteration and apoptosis in EGCs in vitro [98,103]. TcdB-induced apoptosis of EGCs involves the NADPH oxidase/ ROS/JNK/caspase-3 signaling pathway independently of the mitochondrial pathway [103]. In addition, TcdB induces senescence in EGCs.…”