O papel do proteoglicano de sulfato de condroitina (CSPG4) na infecção por Clostridioides difficile e na mediação dos efeitos das toxinas A e B na Glia entérica

Abstract

Clostridioides difficile infection (CDI) is a growing public health problem, characterized by severe colitis, a high recurrence rate, and significant costs to healthcare systems. Over the past ten years, a significant increase in community-acquired cases and in pediatric patients, a group previously considered less vulnerable, has been observed globally. Although most studies have focused on the acute effects of infection and its impact on intestinal epithelial cells, the effects of CDI on enteric glia, an important component of the enteric nervous system involved in homeostasis and intestinal motility, as well as the long-term consequences associated with exposure to C. difficile toxins, remain poorly explored. This study aimed to investigate the role of chondroitin sulfate proteoglycan receptor 4 (CSPG4) in mediating the effects of C. difficile toxins A (TcdA) and B (TcdB) on enteric glial cells. Initially, an exploratory analysis of the public RNA sequencing database using the Single Cell Portal platform was performed to characterize the gene expression profiles of different C. difficile toxin receptors (Cspg4, Pvrl3, Fzd1, Lrp1, Fzd2, Tfpi, Fzd7, and Ldlr) in enteric glial cells from the colon of mice and humans. The distribution of these receptors in rat glial cells (CRL2690) was identified using real-time PCR (qPCR). The impact of exposure to TcdA (50 ng/mL) or TcdB (1 ng/mL) on the expression of these receptors in enteroglial cells after 2, 12, and 18 hours of incubation with these toxins was also investigated. The distribution of CSPG4 in large intestine samples from humans and mice, infected or not with C. difficile, was analyzed by immunohistochemistry. In vitro, to investigate the involvement of CSPG4 in the inflammatory response, cell death, and senescence caused by TcdA and TcdB, rat glial cells (CRL2690) were incubated with the toxins, with or without pretreatment with anti-CSPG4 antibody (0.001 μg/mL), whose concentration was previously determined by the Annexin V assay. Gene expression of IL-6 and S100B, both considered markers of inflammation, was quantified by qPCR, while extracellular S100B levels were determined by ELISA. Activation of the transcription factors NFκB and STAT3, as well as senescence-associated β-galactosidase (SA-β-Gal) activity, were assessed by immunofluorescence. The results demonstrated that enteric glia from mice, rats, and humans constitutively express all receptors for C. difficile toxins. CSPG4 constitutively upregulated, with a significant increase in its expression after exposure to toxins. Higher receptor expression was also observed in the myenteric plexus of C. difficile-infected mice compared to the uninfected group. CSPG4 blockade reduced the release of IL-6 and S100B, as well as the nuclear translocation of NFκB and STAT3 induced by TcdB. Although TcdB-induced apoptosis was not affected, the cellular senescence phenotype was significantly attenuated by receptor inhibition. Taken together, the data indicate that CSPG4 plays a relevant role in the enteric glial response to C. difficile infection, particularly in modulating inflammatory and senescence pathways, constituting a potential therapeutic target to mitigate the deleterious effects of bacterial toxins on the enteric nervous system.