Salmonella-induced changes in the level of key immunoregulatory bacteria affect the transcriptional activity of the <i>Foxp3</i> and <i>RORgt</i> genes in the gut-associated lymphoid tissue of rats

Abstract

Intestinal microbiome is involved in multiple host physiological events, contributes to formation and maintenance of immune homeostasis due to regulated immune response aimed at protecting against pathogen invasion. A special role in differentiation of various T cell subsets is played by segmented filamentous bacteria (SFB) capable of inducing proinflammatory Th17 cell differentiation in gut-associated lymphoid tissue (GALT) as well as members of the genus Clostridium (cluster IV and XIVa) and Bacteroides fragilis (polysaccharide A (PSA)), stimulate emergence of regulatory T cells (Treg) and production of suppressive cytokine IL-10. Important B. fragilis-derived metabolites are short-chain fatty acids (SCFA) able to activate GALT cells via the FFAR2 receptor. Lowering SCFA level results in decreased gut Treg count and alters Th17/Treg ratio being immediately coupled in the GALT to downregulated FFAR2 mRNA, Foxp3 expression and increased RORγt. Hence, we wanted to determine level of the key immunoregulatory bacteria within the rat wall intestinal microflora and their effects on GALT transcriptional activity of Foxp3 and RORyt genes upon Salmonella-induced inflammation and as well as after administering vancomycin and B. fragilis. Bacterial genus and species origin and related percentage in rat microflora were assessed by using polymerase chain reaction (PCR-RV) to identify specific 16S rDNA genes. Transcriptional activity of genes was measured by using real-time RT-PCR. It was found that administration of vancomycin and Salmonella spp. increased level of SFB and a decreased A.muciniphila, F.prausnitzii. In addition, infection of vancomycin-pretreated rats with S.enteritidis and S. typhimurium increased SFB amount paralleled with marked decline in Bacteroides-Prevotela group, A.muciniphila, Clostridium spp. clusters XIV, IV, and F.prausnitzii that resulted in downregulated Foxp3 and upregulated Rorγt mRNA expression, respectively. However, inoculating B. fragilis to vancomycin-pretreated animals receiving S. enteritidis or S. typhimurium resulted in decreased level of SFB and Rorγt mRNA, and, conversely, increased amount of Bacteroides-Prevotela group, A.muciniphila, Clostridium spp. clusters XIV, IV, F.prausnitzii and expression of Foxp3 gene evidencing about recovered homeostasis in intestinal microbiome. The data obtained showed that B. fragilis can be successfully used in treatment of inflammatory bowel diseases or diseases with impaired intestinal barrier function.