Basic Study
Copyright ©The Author(s) 2022. Published by Baishideng Publishing Group Inc. All rights reserved.
World J Gastroenterol. Aug 14, 2022; 28(30): 4075-4088
Published online Aug 14, 2022. doi: 10.3748/wjg.v28.i30.4075
P2X7 receptor blockade decreases inflammation, apoptosis, and enteric neuron loss during Clostridioides difficile toxin A-induced ileitis in mice
Ana A Q A Santos, Deiziane V S Costa, Danielle A Foschetti, Antoniella S G Duarte, Conceição S Martins, Pedro M G Soares, Patricia Castelucci, Gerly A C Brito
Ana A Q A Santos, Deiziane V S Costa, Conceição S Martins, Pedro M G Soares, Department of Morphology, School of Medicine, Federal University of Ceara, Fortaleza 60430-170, Ceara, Brazil
Deiziane V S Costa, Department of Physiology and Pharmacology, School of Medicine, Federal University of Ceará, Fortaleza 60430-170, Ceara, Brazil
Danielle A Foschetti, Department of Pathology and Legal Medicine, School of Medicine, Federal University of Ceara, Fortaleza 60430-170, Ceara, Brazil
Antoniella S G Duarte, Department of Morphology (UFC), Federal University of Ceara, Fortaleza 60430-170, Ceara, Brazil
Patricia Castelucci, Department of Anatomy, Institute of Biomedical Sciences, University of São Paulo, Sao Paulo 05508-270, Brazil
Gerly A C Brito, Department of Morphology, Federal University of Ceara, Fortaleza 60140-170, Ceara, Brazil
Author contributions: Santos AAQA and Costa DVS contributions equally; Santos AAQA participated in the design and performed the experiments, analyzed the data and wrote the manuscript; Costa DVS analyzed the data and wrote the manuscript; Foschetti DA, Duarte ASG, Martins CS, and Soares PMG helped in the acquisition of the data and review of the manuscript; Castelucci P participated in the initial experimental design and helped to revise the manuscript; Brito GAC, the main investigator of the laboratory where the experiments were performed, conceptualized the main ideas, supervised the study and reviewed the manuscript.
Supported by PRONEX CNPq/FUNCAP, No. PR2-0101-00060.01.00/15; São Paulo Research Foundation (FAPESP), No. 2014/25927-2 and No. 2018/07862-1.
Institutional animal care and use committee statement: All mouse procedures were conducted according to current regulations regarding animal experiments approved by the local Animal Care and Use Committee, No. 31/2015.
Conflict-of-interest statement: All the authors report no relevant conflicts of interest for this article.
Data sharing statement: No additional data are available.
ARRIVE guidelines statement: The authors have read the ARRIVE guidelines, and the manuscript was prepared and revised according to the ARRIVE guidelines.
Open-Access: This article is an open-access article that was selected by an in-house editor and fully peer-reviewed by external reviewers. It is distributed in accordance with the Creative Commons Attribution NonCommercial (CC BY-NC 4.0) license, which permits others to distribute, remix, adapt, build upon this work non-commercially, and license their derivative works on different terms, provided the original work is properly cited and the use is non-commercial. See:
Corresponding author: Gerly A C Brito, MD, PhD, Professor, Department of Morphology, Federal University of Ceara, Rua Delmiro de Farias, Fortaleza 60140-170, Ceara, Brazil.
Received: November 9, 2021
Peer-review started: November 9, 2021
First decision: April 16, 2022
Revised: May 4, 2022
Accepted: July 11, 2022
Article in press: July 11, 2022
Published online: August 14, 2022

Clostridioides difficile (C. difficile) is the most common pathogen causing health care-associated infections. C. difficile TcdA and TcdB have been shown to activate enteric neurons; however, what population of these cells is more profoundly influenced and the mechanism underlying these effects remain unknown.


To characterize a specific population of TcdA-affected myenteric neurons and investigate the role of the P2X7 receptor in TcdA-induced ileal inflammation, cell death, and the changes in the enteric nervous system in mice.


Swiss mice were used to model TcdA-induced ileitis in ileal loops exposed to TcdA (50 μg/Loop) for 4 h. To investigate the role of the P2X7 receptor, Brilliant Blue G (50 mg/kg, i.p.), which is a nonspecific P2X7 receptor antagonist, or A438079 (0.7 μg/mouse, i.p.), which is a competitive P2X7 receptor antagonist, were injected one hour prior to TcdA challenge. Ileal samples were collected to analyze the expression of the P2X7 receptor (by quantitative real-time polymerase chain reaction and immunohistochemistry), the population of myenteric enteric neurons (immunofluorescence), histological damage, intestinal inflammation, cell death (terminal deoxynucleotidyltransferase-mediated dUTP-biotin nick end labeling), neuronal loss, and S100B synthesis (immunohistochemistry).


TcdA upregulated (P < 0.05) the expression of the P2X7 receptor gene in the ileal tissues, increasing the level of this receptor in myenteric neurons compared to that in control mice. Comparison with the control mice indicated that TcdA promoted (P < 0.05) the loss of myenteric calretinin+ (Calr) and choline acetyltransferase+ neurons and increased the number of nitrergic+ and Calr+ neurons expressing the P2X7 receptor. Blockade of the P2X7 receptor decreased TcdA-induced intestinal damage, cytokine release [interleukin (IL)-1β, IL-6, IL-8, and tumor necrosis factor-α], cell death, enteric neuron loss, and S100B synthesis in the mouse ileum.


Our findings demonstrated that TcdA induced the upregulation of the P2X7 receptor, which promoted enteric neuron loss, S100B synthesis, tissue damage, inflammation, and cell death in the mouse ileum. These findings contribute to the future directions in understanding the mechanism involved in intestinal dysfunction reported in patients after C. difficile infection.

Keywords: Clostridioides difficile, Clostridioides difficile toxin A, P2X7 receptor, Enteric nervous system, Enteric neuron, Enteric glia

Core Tip: There is a knowledge gap regarding the population of enteric neurons affected by TcdA and the role of the P2X7 receptor, which is a low-sensitivity adenosine triphosphate-gated cation channel, in TcdA-induced alterations in enteric neurons and enteric glial cell (EGC)-derived mediators, particularly S100B. The findings of the present study demonstrated the mechanism of P2X7 receptor-driven enteric neuronal loss induced by TcdA in the mouse ileum. TcdA promoted the upregulation of the P2X7 receptor, which promoted cell death in enteric neurons and induced the release of proinflammatory mediators, which in turn promoted S100B synthesis in EGCs. However, the blockade of the P2X7 receptor abrogated ileal damage induced by TcdA.