Microbiome-host systems interactions: protective effects of propionate upon the blood-brain barrier

Abstract

Background: Gut microbiota composition and function are symbiotically linked with host health and altered in metabolic, inflammatory and neurodegenerative disorders. Three recognised mechanisms exist by which the microbiome influences the gut-brain axis: modification of autonomic/sensorimotor connections, immune activation, and neuroendocrine pathway regulation. We hypothesised interactions between circulating gut-derived microbial metabolites, and the blood-brain barrier (BBB) also contribute to the gut-brain axis. Propionate, produced from dietary substrates by colonic bacteria, stimulates intestinal gluconeogenesis and is associated with reduced stress behaviours, but its potential endocrine role has not been addressed.

Results: After demonstrating expression of the propionate receptor FFAR3 on human brain endothelium, we examined the impact of a physiologically relevant propionate concentration (1 μM) on BBB properties in vitro. Propionate inhibited pathways associated with non-specific microbial infections via a CD14-dependent mechanism, suppressed expression of LRP-1 and protected the BBB from oxidative stress via NRF2 (NFE2L2) signalling.

Conclusions: Together, these results suggest gut-derived microbial metabolites interact with the BBB, representing a fourth facet of the gut-brain axis that warrants further attention.

Fig. 1

Effects on gene expression of exposure of the hCMEC/D3 cell line to propionate (1 μM, 24 h). a Representative images of FFAR3 immunoreactivity within endothelial cells of capillaries (i) and larger post-capillary (ii) blood vessels in control human brains post-mortem; scale bar 20 μm, sections are 5 μm thick; images are representative of five independent cases; areas of particular immunoreactivity are highlighted by black arrowheads. b Surface expression of FFAR3/GPR41 by hCMEC/D3 cells (grey line, unstained cells, black line secondary antibody control, red line FFAR3); data are representative of three independent experiments. c Volcano plot showing significantly (P_FDR < 0.1, red dots) differentially expressed genes. The top 20 up- and downregulated genes are labelled. d SPIA evidence plot for the 1136 significantly differentially expressed genes. Only those human KEGG pathways associated with non-specific microbial infections are labelled. The pathways at the right of the red oblique line are significant (P < 0.2) after Bonferroni correction of the global P values, pG, obtained by combining the pPERT and pNDE using the normal inversion method. The pathways at the right of the blue oblique line are significant (P < 0.2) after a FDR correction of the global P values, pG. 04810. Regulation of actin cytoskeleton (inhibited); 04064, NF-kappa B signalling pathway (inhibited); 04978, mineral absorption (inhibited); 03013, RNA transport (activated); 04141, protein processing in endoplasmic reticulum (activated); 04350, TGF-beta signalling pathway (activated); 04623, cytosolic DNA-sensing pathway (inhibited). e Association of all significantly differentially expressed genes (n = 1136) with KEGG pathways, Enrichr. f Association of all significantly upregulated genes (n = 553) with WikiPathways, Enrichr. e, f The lighter the colour and the longer the bars, the more significant the result is. Significance of data was determined using rank-based ranking; only the top 10 results are shown in each case

Fig. 2

Protective effects of propionate against LPS-induced barrier disruption. a Assessment of the paracellular permeability of hCMEC/D3 monolayers to 70 kDa FITC–dextran following treatment for 24 h with 65 μM acetate, 1 μM butyrate or 1 μM propionate, with or without inclusion of 50 ng/ml LPS for the last 12 h of incubation; data are mean ± SEM, n = 3 independent experiments. b Trans-endothelial electrical resistance of hCMEC/D3 monolayers following treatment for 24 h with 65 μM acetate, 1 μM butyrate or 1 μM propionate, with or without inclusion of 50 ng/ml LPS for the last 12 h of incubation; data are mean ± SEM, n = 3 independent experiments. c Confocal microscopic analysis of expression of the tight junction components claudin-5, occludin and zona occludens-1 (ZO-1) in hCMEC/D3 cells following treatment for 24 h with 1 μM propionate, with or without inclusion of 50 ng/ml LPS for the last 12 h of incubation. Scale bar (10 μm) applies to all images. Images are representative of at least three independent experiments. d Expression of CD14 mRNA in control and propionate-treated (1 μM; 24 h) hCMEC/D3 cells according to microarray data (data are mean ± SEM, n = 3). e Surface expression of CD14 protein on control and propionate-treated hCMEC/D3 cells (grey line, unstained cells, black line secondary antibody control, red line FFAR3); data are representative of three independent experiments. f Median fluorescence intensity of surface expression of CD14 protein on control and propionate-treated hCMEC/D3 cells; dashed line indicates isotype control fluorescence intensity; data are mean ± SEM, n = 3 independent experiments

Fig. 3

Protective effects of propionate against oxidative stress. a Representation of stress response genes significantly upregulated in the current study and directly influenced by NFE2L2, the master regulator of antioxidant responses [54]. b Confocal microscopic analysis of expression of NFE2L2 (Nrf2) in hCMEC/D3 cells following treatment for 24 h with 1 μM propionate; scale bar (10 μm) applies to all images. Images are representative of at least three independent experiments. c Production of reactive oxygen species (ROS) in control and propionate pre-treated (1 μM, 24 h) hCMEC/D3 cells treated for 30 min with the mitochondrial complex I inhibitor rotenone (2.5 μM). Data are mean ± SEM, n = 3 independent experiments

Fig. 4

Production of propionate by the human gut microbiota. Propionate can be produced directly or indirectly by cross-feeding from succinate and lactate producers (e.g. Selenomonas, Megasphaera and Veillonella spp.). Image produced using information taken from [57]. *Akkermansia muciniphila is known to produce propionate; it is thought to do this via the succinate pathway [57]