Efeito do pré-tratamento com propionato na infecção por Clostridioides difficile e na resposta da glia entérica às toxinas A e B

Abstract

Gastrointestinal (GI) dysfunction following Clostridioides difficile infection (CDI) has been reported in mouse and human. Enteric glia, part of the enteric nervous system, contribute to GI motility and are vulnerable to C. difficile toxins, which cause cell death and trigger an inflammatory response. Little is known about the effect of propionate, a short-chain fatty acid (SCFA). Therefore, this study aimed to evaluate whether pre-treatment with propionate

prevents GI dysfunction after CDI and modulates enteric glia responses (cell death and pro- inflammatory responses) to C. difficile toxins. Regarding the in vivo study, 8-week-old mice

were pre-treated with propionate (150 mM in drinking water) or not, and then infected with C.

difficile. After infection, mice were monitored daily to assess disease severity. On day 14 post- infection, GI function was assessed using the Evans blue method. In vitro, rat enteric glia were

incubated with TcdA (50 ng/mL) or TcdB (1 ng/mL), alone or with propionate (10 mM), one hour before toxin. After 2, 12, and/or 18 hours of incubation with the toxins, cells were collected for analysis of SCFA transporter/receptor expression (MCT1, MCT4, FFAR2, and FFAR3 via qPCR), pro-inflammatory markers (S100B and IL-6 via qPCR and immunofluorescence, respectively), as well as nuclear translocation of pSTAT3 (by immunofluorescence) and cell death. In vivo, infected mice lost weight and developed severe diarrhea, which was completely resolved by day 13 post-infection, accompanied by delayed total gastrointestinal transit (TTGIT) on day 14. On the other hand, pre-treatment with propionate improved survival, reduced weight loss, prevented diarrhea, and decreased delay in TTGIT. In vitro, enteric glial cells express SCFA transporters (MCT1 and MCT4) and receptors (FFAR2 and FFAR3), which were altered by exposure to C. difficile toxins. TcdA or TcdB promoted cell death, increased

caspase 3/7 activity, reduced Bcl2 expression (protein and transcripts), increased levels of pro- inflammatory mediators S100B (transcripts) and IL-6 (protein) in enteric glia, and promoted

nuclear translocation of pSTAT3. Whereas propionate supplementation elevated Bcl2 levels, it did not prevent cell death, but decreased IL-6 protein expression, S100B transcripts, and

reduced nuclear translocation of pSTAT3 challenged with TcdA or TcdB. Overall, pre- treatment with propionate is effective in preventing gastrointestinal dysfunction after CDI,

improving survival, and reducing disease severity in the acute phase. Additionally, propionate supplementation modulates the pro-inflammatory response (reducing IL-6/STAT3 expression and the positive regulation of S100B) induced by TcdA or TcdB in enteric glia. These findings offer new insights into how microbiota-derived products can influence gastrointestinal system function after infection and the enteric glial response to C. difficile toxins.