title: 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.
authors.affiliation: Department of Experimental Medicine, University of Perugia, Perugia, Italy.
authors.forename: Lara
authors.initials: L
authors.lastname: Macchioni
authors.affiliation: Scientific and educational center of Terni, University of Perugia, Perugia, Italy.
authors.forename: Magdalena
authors.initials: M
authors.lastname: Davidescu
authors.affiliation: Department of Experimental Medicine, University of Perugia, Perugia, Italy.
authors.forename: Katia
authors.initials: K
authors.lastname: Fettucciari
authors.affiliation: Department of Experimental Medicine, University of Perugia, Perugia, Italy.
authors.forename: Maya
authors.initials: M
authors.lastname: Petricciuolo
authors.affiliation: Scientific and educational center of Terni, University of Perugia, Perugia, Italy.
authors.forename: Leonardo
authors.initials: L
authors.lastname: Gatticchi
authors.affiliation: Department of Experimental Medicine, University of Perugia, Perugia, Italy.
authors.forename: Davide
authors.initials: D
authors.lastname: Gioè
authors.affiliation: Pathology Section, Spedali Civili di Brescia, Brescia, Italy.
authors.forename: Vincenzo
authors.initials: V
authors.lastname: Villanacci
authors.affiliation: Department of Gastroenterology, University of Pisa, Pisa, Italy.
authors.forename: Massimo
authors.initials: M
authors.lastname: Bellini
authors.affiliation: Department of Experimental Medicine, University of Perugia, Perugia, Italy.
authors.forename: Pierfrancesco
authors.initials: P
authors.lastname: Marconi
authors.affiliation: Department of Experimental Medicine, University of Perugia, Perugia, Italy.
authors.forename: Rita
authors.initials: R
authors.lastname: Roberti
authors.affiliation: Department of Medicine, University of Perugia, Perugia, Italy.
authors.forename: Gabrio
authors.initials: G
authors.lastname: Bassotti
authors.affiliation: Department of Experimental Medicine, University of Perugia, Perugia, Italy.
authors.forename: Lanfranco
authors.initials: L
authors.lastname: Corazzi
mesh_terms: D000818:Animals; D001426:Bacterial Proteins / Q000633:toxicity*; D001427:Bacterial Toxins / Q000633:toxicity*; D053148:Caspase 3 / Q000378:metabolism*; D002460:Cell Line; D048670:MAP Kinase Kinase 4 / Q000378:metabolism*; D019255:NADPH Oxidases / Q000378:metabolism*; D009457:Neuroglia / Q000187:drug effects* / Q000201:enzymology / Q000378:metabolism; D051381:Rats; D017382:Reactive Oxygen Species / Q000378:metabolism*; D015398:Signal Transduction
chemical_list: D001426:Bacterial Proteins; D001427:Bacterial Toxins; D017382:Reactive Oxygen Species; C031815:toxB protein, Clostridium difficile; D019255:NADPH Oxidases; D048670:MAP Kinase Kinase 4; D053148:Caspase 3