Role of Pannexin-1-P2X7R signaling on cell death and pro-inflammatory mediator expression induced by Clostridioides difficile toxins in enteric glia

Abstract

Clostridioides difficile (C. difficile) produces toxins A (TcdA) and B (TcdB), both associated with intestinal damage and diarrhea. Pannexin-1 (Panx1) channels allows the passage of messenger molecules, such as adenosine triphosphate (ATP), which in turn activate the P2X7 receptors (P2X7R) that regulate inflammation and cell death in inflammatory bowel diseases. The aim of this study was to verify the effect of C. difficile infection (CDI) in the expression of Panx1 and P2X7R in intestinal tissues of mice, as well as their role in cell death and IL-6 expression induced by TcdA and TcdB in enteric glial cells (EGCs). Male C57BL/6 mice (8 weeks of age) were infected with C. difficile VPI10463, and the control group received only vehicle per gavage. After three days post-infection (p.i.), cecum and colon samples were collected to evaluate the expression of Panx1 by immunohistochemistry. In vitro, EGCs (PK060399egfr) were challenged with TcdA or TcdB, in the presence or absence of the Panx1 inhibitor (10Panx trifluoroacetate) or P2X7R antagonist (A438079), and Panx1 and P2X7R expression, caspase-3/7 activity and phosphatidylserine binding to annexin-V, as well as IL-6 expression were assessed. CDI increased the levels of Panx1 in cecum and colon of mice compared to the control group. Panx1 inhibitor decreased caspase-3/7 activity and phosphatidylserine-annexin-V binding, but not IL-6 gene expression in TcdA and TcdB-challenged EGCs. P2X7 receptor antagonist accentually reduced caspase-3/7 activity, phosphatidylserine-annexin-V binding, and IL-6 gene expression in TcdA and TcdB-challenged EGCs. In conclusion, Panx1 is increased during CDI and plays an important role in the effects of C. difficile toxins in EGCs, participating in cell death induced by both toxins by promoting caspase-3/7 activation via P2X7R, which is also involved in IL-6 expression induced by both toxins.

Contribution of large-pore channels to inflammation induced by microorganisms

“…Similarly, endotoxemia induces de novo expression of Cxs and upregulates Panx1 in skeletal muscles, where they are likely to form hemichannels ( Balboa et al, 2018 ). Clostridioides difficile ( C. difficile ) is a Gram-positive, anaerobic toxin-producing bacillus that causes nosocomial diarrhea associated with antibiotic use ( Loureiro et al, 2022 ). Infecting C57BL/6 mice with C. difficile (VPI10463 strain) was shown to increase levels of Panx1 in the cecum and colon ( Loureiro et al, 2022 ).…”

“…Clostridioides difficile ( C. difficile ) is a Gram-positive, anaerobic toxin-producing bacillus that causes nosocomial diarrhea associated with antibiotic use ( Loureiro et al, 2022 ). Infecting C57BL/6 mice with C. difficile (VPI10463 strain) was shown to increase levels of Panx1 in the cecum and colon ( Loureiro et al, 2022 ). Blocking Panx1 with mimetic peptide 10 Panx1 was observed to decrease caspase-3/7 activity and phosphatidylserine-annexin-V binding in toxin A- and toxin B-challenged enteric neurons and enteric glial cells ( Loureiro et al, 2022 ).…”

“…Infecting C57BL/6 mice with C. difficile (VPI10463 strain) was shown to increase levels of Panx1 in the cecum and colon ( Loureiro et al, 2022 ). Blocking Panx1 with mimetic peptide 10 Panx1 was observed to decrease caspase-3/7 activity and phosphatidylserine-annexin-V binding in toxin A- and toxin B-challenged enteric neurons and enteric glial cells ( Loureiro et al, 2022 ).…”

“…In bacterial infections, PAMPs activate hemichannels formed by Cx43, Cx26, or Panx1, causing an outflow of ATP. The latter, through the activation of P2X 7 receptors, induces the activation of caspase 3/7 ( Loureiro et al, 2022 ) and the production of IL-6 ( Robertson et al, 2010 ; Loureiro et al, 2022 ). By activating P2X 1 receptors, it can increase the concentration of cytosolic Ca 2+ ions ( Wang et al, 2017 ).…”

Role of the Alteration in Calcium Homeostasis in Cell Death Induced by Clostridioides difficile Toxin A and Toxin B

“…Necrosis Apoptosis Pyroptosis Refs. [8][9][10][11][12]46,47,[61][62][63]73,82,84,86,91,94,[102][103][104][105]107,108,110,111,[126][127][128][129][130][131][132][133]140,142,144,145] Destruction of the intercellular junctions…”