Modulation of Proinflammatory Bacteria- and Lipid-Coupled Intracellular Signaling Pathways in a Transwell Triple Co-Culture Model by Commensal Bifidobacterium Animalis R101-8.

Abstract

Following a fat-rich diet, alterations in gut microbiota contribute to enhanced gut permeability, metabolic endotoxemia, and low grade inflammation-associated metabolic disorders. To better understand whether commensal bifidobacteria influence the expression of key metaflammation-related biomarkers (chemerin, MCP-1, PEDF) and modulate the pro-inflammatory bacteria- and lipid-coupled intracellular signaling pathways, we aimed at i) investigating the influence of the establishment of microbial signaling molecules-based cell-cell contacts on the involved intercellular communication between enterocytes, immune cells, and adipocytes, and ii) assessing their inflammatory mediators' expression profiles within an inflamed adipose tissue model. Bifidobacterium animalis R101-8 and Escherichia coli TG1, respectively, were added to the apical side of a triple co-culture model consisting of intestinal epithelial HT-29/B6 cell line, human monocyte-derived macrophage cells, and adipose-derived stem cell line in the absence or presence of LPS or palmitic acid. mRNA expression levels of key lipid metabolism genes HILPDA, MCP-1/CCL2, RARRES2, SCD, SFRP2 and TLR4 were determined using TaqMan qRT-PCR. Protein expression levels of cytokines (IL-1β, IL-6, and TNF-α), key metaflammation-related biomarkers including adipokines (chemerin and PEDF), chemokine (MCP- 1) as well as cellular triglycerides were assessed by cell-based ELISA, while those of p-ERK, p-JNK, p-p38, NF-κB, p-IκBα, pc-Fos, pc-Jun, and TLR4 were assessed by Western blotting. B. animalis R101-8 inhibited LPS- and palmitic acid-induced protein expression of inflammatory cytokines IL-1β, IL-6, TNF-α concomitant with decreases in chemerin, MCP-1, PEDF, and cellular triglycerides, and blocked NF-kB and AP-1 activation pathway through inhibition of p- IκBα, pc-Jun, and pc-Fos phosphorylation. B. animalis R101-8 downregulated mRNA and protein levels of HILPDA, MCP-1/CCL2, RARRES2, SCD and SFRP2 and TLR4 following exposure to LPS and palmitic acid. B. animalis R101-8 improves biomarkers of metaflammation through at least two molecular/signaling mechanisms triggered by pro-inflammatory bacteria/lipids. First, B. animalis R101-8 modulates the coupled intracellular signaling pathways via metabolizing saturated fatty acids and reducing available bioactive palmitic acid. Second, it inhibits NF-kB's and AP-1's transcriptional activities, resulting in the reduction of pro-inflammatory markers. Thus, the molecular basis may be formed by which commensal bifidobacteria improve intrinsic cellular tolerance against excess pro-inflammatory lipids and participate in homeostatic regulation of metabolic processes in vivo.