title: Enteric glial cells are susceptible to Clostridium difficile toxin B.
abstract: Clostridium difficile causes nosocomial/antibiotic-associated diarrhoea and pseudomembranous colitis. The major virulence factors are toxin A and toxin B (TcdB), which inactivate GTPases by monoglucosylation, leading to cytopathic (cytoskeleton alteration, cell rounding) and cytotoxic effects (cell-cycle arrest, apoptosis). C. difficile toxins breaching the intestinal epithelial barrier can act on underlying cells, enterocytes, colonocytes, and enteric neurons, as described in vitro and in vivo, but until now no data have been available on enteric glial cell (EGC) susceptibility. EGCs are crucial for regulating the enteric nervous system, gut homeostasis, the immune and inflammatory responses, and digestive and extradigestive diseases. Therefore, we evaluated the effects of C. difficile TcdB in EGCs. Rat-transformed EGCs were treated with TcdB at 0.1-10 ng/ml for 1.5-48 h, and several parameters were analysed. TcdB induces the following in EGCs: (1) early cell rounding with Rac1 glucosylation; (2) early G2/M cell-cycle arrest by cyclin B1/Cdc2 complex inactivation caused by p27 upregulation, the downregulation of cyclin B1 and Cdc2 phosphorylated at Thr161 and Tyr15; and (3) apoptosis by a caspase-dependent but mitochondria-independent pathway. Most importantly, the stimulation of EGCs with TNF-α plus IFN-γ before, concomitantly or after TcdB treatment strongly increased TcdB-induced apoptosis. Furthermore, EGCs that survived the cytotoxic effect of TcdB did not recover completely and showed not only persistent Rac1 glucosylation, cell-cycle arrest and low apoptosis but also increased production of glial cell-derived neurotrophic factor, suggesting self-rescuing mechanisms. In conclusion, the high susceptibility of EGCs to TcdB in vitro, the increased sensitivity to inflammatory cytokines related to apoptosis and the persistence of altered functions in surviving cells suggest an important in vivo role of EGCs in the pathogenesis of C. difficile infection.
authors.affiliation: Department of Experimental Medicine, Histology and Medical Embryology Section, Perugia University, Piazza Lucio Severi 1, Edificio B IV piano, Sant'Andrea delle Fratte, 06132, Perugia, Italy. katia.fettucciari@unipg.it.
authors.forename: Katia
authors.identifier: 0000-0001-8074-8243
authors.initials: K
authors.lastname: Fettucciari
authors.affiliation: Department of Experimental Medicine, Histology and Medical Embryology Section, Perugia University, Piazza Lucio Severi 1, Edificio B IV piano, Sant'Andrea delle Fratte, 06132, Perugia, Italy.
authors.forename: Pamela
authors.initials: P
authors.lastname: Ponsini
authors.affiliation: Department of Experimental Medicine, Histology and Medical Embryology Section, Perugia University, Piazza Lucio Severi 1, Edificio B IV piano, Sant'Andrea delle Fratte, 06132, Perugia, Italy.
authors.forename: Davide
authors.initials: D
authors.lastname: Gioè
authors.affiliation: Department of Experimental Medicine, Physiology and Biochemistry Section, Perugia University, Perugia, Italy.
authors.forename: Lara
authors.initials: L
authors.lastname: Macchioni
authors.affiliation: Department of Clinical Sciences and Translational Medicine, Tor Vergata University, Rome, Italy.
authors.forename: Camilla
authors.initials: C
authors.lastname: Palumbo
authors.affiliation: Gastroenterology Section, Perugia General Hospital, Perugia, Italy.
authors.forename: Elisabetta
authors.initials: E
authors.lastname: Antonelli
authors.affiliation: Department of Medicine, Internal Medicine, Rheumatology and Medical Therapy of Pain Section, Perugia University, District of Terni, Perugia, Italy.
authors.forename: Stefano
authors.initials: S
authors.lastname: Coaccioli
authors.affiliation: Pathology Section, Spedali Civili, Brescia, Italy.
authors.forename: Vincenzo
authors.initials: V
authors.lastname: Villanacci
authors.affiliation: Department of Experimental Medicine, Physiology and Biochemistry Section, Perugia University, Perugia, Italy.
authors.forename: Lanfranco
authors.initials: L
authors.lastname: Corazzi
authors.affiliation: Department of Experimental Medicine, Histology and Medical Embryology Section, Perugia University, Piazza Lucio Severi 1, Edificio B IV piano, Sant'Andrea delle Fratte, 06132, Perugia, Italy.
authors.forename: Pierfrancesco
authors.initials: P
authors.lastname: Marconi
authors.affiliation: Department of Medicine, Gastroenterology, Hepatology and Digestive Endoscopy Section, Perugia University, Perugia, Italy.
authors.forename: Gabrio
authors.initials: G
authors.lastname: Bassotti
mesh_terms: D000818:Animals; D017209:Apoptosis; D001426:Bacterial Proteins / Q000378:metabolism*; D001427:Bacterial Toxins / Q000378:metabolism*; D059447:Cell Cycle Checkpoints; D002460:Cell Line; D016360:Clostridioides difficile / Q000502:physiology*; D004761:Enterocolitis, Pseudomembranous / Q000378:metabolism / Q000382:microbiology* / Q000473:pathology*; D041981:Gastrointestinal Tract / Q000294:innervation* / Q000378:metabolism / Q000382:microbiology / Q000473:pathology; D051100:Glial Cell Line-Derived Neurotrophic Factor / Q000378:metabolism; D009457:Neuroglia / Q000378:metabolism / Q000382:microbiology* / Q000473:pathology*; D051381:Rats
chemical_list: D001426:Bacterial Proteins; D001427:Bacterial Toxins; D051100:Glial Cell Line-Derived Neurotrophic Factor; C031815:toxB protein, Clostridium difficile
keywords: Apoptosis; Cell-cycle arrest; Clostridium difficile toxin B (TcdB); Enteric glial cells (EGCs); Glial cell-derived neurotrophic factor (GDNF); Proinflammatory cytokines; Rac1 glucosylation