Cell-Free Culture Supernatant of Bifidobacterium breve CNCM I-4035 Decreases Pro-Inflammatory Cytokines in Human Dendritic Cells Challenged with Salmonella typhi through TLR Activation

Abstract

Dendritic cells (DCs) constitute the first point of contact between gut commensals and our immune system. Despite growing evidence of the immunomodulatory effects of probiotics, the interactions between the cells of the intestinal immune system and bacteria remain largely unknown. Indeed,, the aim of this work was to determine whether the probiotic Bifidobacterium breve CNCM I-4035 and its cell-free culture supernatant (CFS) have immunomodulatory effects in human intestinal-like dendritic cells (DCs) and how they respond to the pathogenic bacterium Salmonella enterica serovar Typhi, and also to elucidate the molecular mechanisms involved in these interactions. Human DCs were directly challenged with B. breve/CFS, S. typhi or a combination of these stimuli for 4 h. The expression pattern of genes involved in Toll-like receptor (TLR) signaling pathway and cytokine secretion was analyzed. CFS decreased pro-inflammatory cytokines and chemokines in human intestinal DCs challenged with S. typhi. In contrast, the B. breve CNCM I-4035 probiotic strain was a potent inducer of the pro-inflammatory cytokines and chemokines tested, i.e., TNF-α, IL-8 and RANTES, as well as anti-inflammatory cytokines including IL-10. CFS restored TGF-β levels in the presence of Salmonella. Live B.breve and its supernatant enhanced innate immune responses by the activation of TLR signaling pathway. These treatments upregulated TLR9 gene transcription. In addition, CFS was a more potent inducer of TLR9 expression than the probiotic bacteria in the presence of S. typhi. Expression levels of CASP8 and IRAK4 were also increased by CFS, and both treatments induced TOLLIP gene expression. Our results indicate that the probiotic strain B. breve CNCM I-4035 affects the intestinal immune response, whereas its supernatant exerts anti-inflammatory effects mediated by DCs. This supernatant may protect immune system from highly infectious agents such as Salmonella typhi and can down-regulate pro-inflammatory pathways.

Effect of Bifidobacterium on the mRNA expression levels of TRAF6, GSK-3β, and microRNA-146a in LPS-stimulated rat intestinal epithelial cells

“…As with dendritic cells, IEC-6 can also express TLR-9. Recent studies have shown that Bifidobacterium can secret the TLR-9 ligand, unmethylated CpG DNA fragments (Bermudez-Brito et al, 2012, 2013. The above information and the results of this study suggest that the supernatant of Bifidobacterium may bind to TLR-9 in IEC-6 through a non-methylated CpG DNA fragment, activating the TLR-9 pathway of the IEC-6 cells and NF-κB, and thereby causing the generation of miRNA-146a.…”

“…Bermudez-Brito et al (2013) found that Bifidobacterium supernatant can upregulate TLR-9 in intestinal dendritic cells, thereby activating the TLR pathways to regulate the body's innate immune response and inflammation (Taganov et al, 2006). As with dendritic cells, IEC-6 can also express TLR-9.…”

“…According to the method of Bermudez-Brito et al (2013), infantis Bifidobacterium was anaerobically cultured in liquid medium for 48-72 h and centrifuged at 12,000 g and 4°C for 10 min. The supernatant was collected and 1 N sodium hydroxide solution was used to adjust the pH of supernatant to approximately 7.0.…”

“…Recent meta-analyses have shown that for high-risk preterm children, prophylactic use of probiotics (Lactobacillus and Bifidobacterium) can reduce the incidence of NEC and mitigate the severity of the disease (Lin et al, 2005(Lin et al, , 2008. Studies have shown that Bifidobacterium and its culture supernatant may have a beneficial effect on intestinal cells, but the mechanisms by which they act are not identical (McElroy et al, 2012;Bermudez-Brito et al, 2013). In this study, we treated rat small intestinal epithelial cells (IEC-6s) that had been co-cultured with lipopolysaccharides (LPS) with inactivated Bifidobacterium or Bifidobacterium culture supernatant, to explore the effect of Bifidobacterium on LPSstimulated IEC-6 and to provide experimental evidence for the application of Bifidobacterium in NEC prevention.…”

Modulation of immunity and inflammatory gene expression in the gut, in inflammatory diseases of the gut and in the liver by probiotics

“…Recently, our research group [55] co-incubated intestinal-like human DCs from cord blood CD34+ progenitor cells [56] with B. breve CNCM I-4035 or its cell-free supernatant (CFS), S. typhi or a combination of these treatments for 4 h. These treatments upregulated TLR-9 gene transcription. In addition, CFS was a more powerful inducer of TLR-9 expression than were the probiotic bacteria in the presence of S. typhi.…”