Title:Modulation of Proinflammatory Bacteria- and Lipid-Coupled Intracellular Signaling Pathways in a Transwell Triple Co-Culture Model by Commensal Bifidobacterium Animalis R101-8
Volume: 20
Issue: 2
Author(s): Darab Ghadimi*, Annegret Nielsen, Mohamed Farghaly Yoness Hassan, Regina Fölster-Holst, Michael Ebsen, Sven Olaf Frahm, Christoph Röcken, Michael de Vrese and Knut J. Heller
Affiliation:
- Department of Microbiology and Biotechnology, Max Rubner-Institute, Hermann-Weigmann-Str 1, D-24103 Kiel,Germany
Keywords:
Metaflammation, bifidobacteria, IL-6, adipokine, metabolism, cells.
Abstract:
Background and Aims: 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.
Material and Methods: 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.
Results: 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.
Conclusion: 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.
