Modulation of proinflammatory bacteria- and lipid – coupled intracellular signaling pathways by commensal Bifidobacterium animalis R101-8

Background and aims Following a high-fat diet, changes in gut microbiota contribute to increased gut permeability, metabolic endotoxemia, and low grade inflammation-induced 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 profile 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 cells 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 of 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 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 downregulated mRNA and protein levels of HILPDA, MCP-1/CCL2, RARRES2, SCD and SFRP2 and TLR4 following exposure to LPS and palmitic acid.

Abbreviations

ASCs,	Human adipose-derived stem cells
B.animalis,	Bifidobacterium animalis
BSA,	Bovine serum albumin
CFU,	Colony forming units
ddH2O,	Double-distilled water
DMEM,	Dulbecco’s Modified Eagle’s Medium
DMEM/F-12,	Dulbecco’s Modified Eagle Medium/Nutrient Mixture F-12
DMSO,	Dimethyl sulfoxi
ELISA,	Enzyme-linked immunosorbent assay
FBS,	Fetal bovine serum
FFAs,	Free fatty acids
GAPDH,	Glycerol aldehydephosphate dehydrgenase
HFD,	High-fat diet
HILPDA,	Hypoxia inducible lipid droplet-associated
HMDM,	Human monocyte-derived macrophage
IκBα,	Nuclear factor of kappa light polypeptide gene enhancer in B-cells inhibitor, alpha
LAB,	Lactic acid bacteria
LC-SFAs,	Long-chain saturated fatty acids
IL-1β,	Interleukin-1β
IL-6,	Interleukin-6
IL-6R,	Interleukin-6 receptor
JNK,	c-Jun N-terminal kinase
LPS,	Lipopolysaccharide
LTA,	Lipoteichoic acid
MAMPs,	Microbe-associated molecular patterns
MCP-1,	Monocyte chemoattractant protein-1
MOI,	Multiplicity of infection
MTT,	3-(4,5–dimethyl-thiazol-2-yl) -2,5-diphenyltetrazoliumbromide
NAFLD,	Non-alcoholic fatty liver disease
NASH,	Non-alcoholic steatohepatitis
NF-kB,	Nuclear factor ‘kappa-light-chain-enhancer’ of activated B-cells
PEDF,	Pigment epithelium derived factor
p38 MAPK,	p38 mitogen activated proteinkinases
PBS,	Phosphate buffer saline
PGN,	Peptidoglycan
PVDF,	Polyvinylidene difluoride
qRT-PCR,	Quantitative reverse transcription polymerase chain reaction
RARRES2,	Retinoic acid receptor responder protein 2
SCD,	Stearoyl-CoA desaturase
SEM,	Standard error of the mean
SFRP2,	Frizzled-related protein 2
TEER,	Transepithelial/transendothelial electrical resistance
TG,	Triglycerides
TLRs,	Pattern-recognition receptors including toll-like receptors
EU/mL,	Endotoxin units per milliliter
WB,	Western blotting

Conclusion B. animalis ameliorates biomarkers of metaflammation through at least two molecular/signaling mechanisms that are triggered by pro-inflammatory bacteria/lipids. First, B. animalis modulates the coupled intracellular signaling pathways via metabolizing saturated fatty acids and reducing available bioactive palmitic acid. Second, it inhibits activation of nuclear transcription factors NF-kB and AP-1, resulting in reduction of pro-inflammatory cytokines, adipokines and chemokiens. This may be the molecular basis by which commensal bifidobacteria enhance intrinsic cellular tolerance against excess pro-inflammatory lipids, and participate in homeostatic regulation of metabolic processes in vivo.

Publication History

Article published online:
10 June 2021