Microarray analyses

Following initial confirmation of the expression of FFAR3 in human brain endothelium (Fig. 1a) and on hCMEC/D3 cells (Fig. 1b), we investigated the effect of exposure of hCMEC/D3 monolayers to 1 μM propionate for 24 h. Such treatment had a significant (P FDR < 0.1) effect on the expression of 1136 genes: 553 upregulated and 583 downregulated (Fig. 1c). Initially, we used SPIA with all the significantly differentially expressed genes to identify KEGG signalling pathways inhibited and activated in the presence of propionate. Protein processing in the endoplasmic reticulum and RNA transport were activated upon exposure of cells to propionate, which was unsurprising given gene expression had been induced. A number of pathways associated with non-specific microbial infections (Gram-negative bacteria, viral) were inhibited by propionate (Fig. 1d), as were the cytosolic DNA-sensing pathway (upregulated by pathogen DNA during microbial infections, triggering innate immune signalling [46]), the NFκB signalling pathway and the Toll-like receptor signalling pathway. Of the 19,309 genes we examined on the array, 203 of the 224 genes known to be associated with the BBB were detected (Additional file 1: Table S1). Eleven of these were significantly differentially expressed, with the majority being associated with the inflammatory response.

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 Full size image

Enrichr [47, 48] was used to examine KEGG pathways significantly associated with the list of significantly differentially expressed genes. All 1136 significantly differentially expressed genes mapped to Enrichr. As with SPIA, the genes were associated with KEGG pathways implicated in non-specific microbial infections and RNA- and endoplasmic reticulum-associated processes (Fig. 1e).

WikiPathways analysis (Enrichr) of all the significantly differentially expressed genes highlighted responses to oxidative stress being associated with propionate treatment (not shown). Closer examination of the data demonstrated this was linked to NRF2 (NFE2L2) signalling, with the significantly upregulated genes closely associated with oxidative stress responses (Fig. 1f).

Pathway validation

Transcriptomic analysis identified two particular clusters of pathways as being regulated by propionate treatment: those involved in the non-specific inflammatory response to microbial products (Fig. 1d, e) and those involved in the response to oxidative stress (Fig. 1f). We, therefore, sought to validate these responses in an in vitro model of the BBB.

TLR-specific pathway

Inhibition of the TLR-specific pathway by propionate suggests this metabolite may have a protective role against exposure of the BBB to bacterial LPS, derived from the cell walls of Gram-negative bacteria. In accord with this hypothesis, exposure of hCMEC/D3 monolayers for 12 h to propionate at physiological concentrations (1 μM) was able to significantly attenuate the permeabilising effects of exposure to Escherichia coli O111:B4 LPS (subsequent 12 h stimulation, 50 ng/ml), measured both through paracellular permeability to a 70-kDa FITC-conjugated dextran tracer (Fig. 2a) and trans-endothelial electrical resistance (Fig. 2b). To determine the specificity of these effects for propionate, we investigated the actions of the closely related SCFAs acetate and butyrate. While physiologically relevant circulating concentrations of butyrate (1 μM) replicated the effects of propionate on both trans-endothelial electrical resistance and paracellular tracer permeability, this was not the case for acetate (65 μM) (Fig. 2a, b).

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 Full size image

Circulating concentrations of propionate are approximately 1 μM at rest, but these may be expected to increase following consumption of, for example, a meal containing high levels of fermentable fibre [1]; consequently, we examined the effects of 10 μM and 100 μM propionate upon the response of hCMEC/D3 monolayers to LPS stimulation. Both LPS-induced deficits in trans-endothelial electrical resistance (Additional file 2: Figure S1a) and paracellular tracer permeability (Additional file 2: Figure S1b) were fully attenuated by higher doses of propionate, without any obvious further effects beyond those seen with 1 μM of the SCFA.

Although hCMEC/D3 cells are a widely used in vitro model of the BBB, they are not without limitations, particularly in terms of their higher inherent permeability when compared with other non-human model systems [49]. To ensure the validity of our findings using hCMEC/D3 cells, we repeated these experiments using primary human brain microvascular endothelial cells (HBMECs). As with hCMEC/D3 cells, exposure of HBMEC monolayers for 12 h to propionate (1 μM) significantly attenuated the permeabilising effects of LPS exposure (subsequent 12 h stimulation, 50 ng/ml), in terms of both paracellular permeability to a 70-kDa FITC-conjugated dextran tracer (Additional file 3: Figure S2a) and trans-endothelial electrical resistance (Additional file 3: Figure S2b). Given this confirmation, subsequent experiments focused solely on the hCMEC/D3 cells as an in vitro BBB model.

Paracellular permeability and trans-endothelial electrical resistance are in large part dependent upon the integrity of inter-endothelial tight junctions [50], which are known to be disrupted following exposure to LPS [51]. We, therefore, examined the intracellular distribution of the key tight junction components occludin, claudin-5 and zona occludens-1 (ZO-1) following treatment with propionate and/or LPS. Exposure of hCMEC/D3 monolayers to propionate alone (1 μM, 24 h) had no noticeable effect on the intracellular distribution of any of the studied tight junction components, whereas treatment with LPS (50 ng/ml, 12 h) caused a marked disruption in the localisation of all three major tight junction molecules, characterised by a loss of peri-membrane immunoreactivity (Fig. 2c). Notably, these effects of LPS were substantially protected against by prior treatment for 12 h with 1 μM propionate.

LPS initiates a pro-inflammatory response through binding to Toll-like receptor 4, TLR4, in a complex with the accessory proteins CD14 and LY96 (MD2) [52]; we, therefore, examined expression of TLR4 signalling components as an explanation for the protective effects of propionate upon this pathway. While propionate treatment of hCMEC/D3 cells (1 μM, 24 h) had no significant effect upon expression of mRNA for TLR4 or LY96 (data not shown), such treatment significantly downregulated expression of CD14 mRNA (Fig. 2d), an effect replicated at the level of cell surface CD14 protein expression (Fig. 2e, f).

NFE2L2 (NRF2) signalling and protection from oxidative stress

Enrichr (WikiPathways) analysis indicated that exposure of hCMEC/D3 cells to propionate resulted in the regulation of a number of antioxidant systems. Of known human anti-oxidant genes [53], 58 were detected on the array. We had also identified an additional six genes via [54] (Additional file 4: Table S2). Searches of the genes associated with each of the individual pathways referenced in Fig. 1f strongly indicated these changes occurred downstream of the transcription factor nuclear factor, erythroid 2-like 2–NFE2L2 (Fig. 3a). Supporting this analysis, exposure of hCMEC/D3 cells for 24 h to 1 μM propionate caused a marked translocation of NFE2L2 from the cytoplasm to the nucleus (Fig. 3b). Functional analysis of antioxidant pathway activity was assessed by monitoring reactive oxygen species production in hCMEC/D3 cells following exposure to the mitochondrial complex I inhibitor rotenone (2.5 μM, 2 h). Pre-exposure of cells to 1 μM propionate for 24 h significantly attenuated the rate of fluorescent tracer accumulation, indicative of reduced levels of intracellular reactive oxygen species (Fig. 3c).

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 Full size image

Efflux transporter expression and activity

A key feature of the BBB is the expression of a wide array of efflux transporter proteins, which limit entry of numerous endogenous and xenobiotic agents to, and promote their export from, the brain. Amongst these, the proteins P-glycoprotein, BCRP and LRP-1 are prominent examples. We investigated the ability of propionate to both modify expression of these transporters and, in the case of the ABC transporter proteins P-glycoprotein and BCRP, serve as a direct inhibitor or substrate for the protein. Exposure of hCMEC/D3 monolayers to propionate at physiological levels (1 μM) for 24 h significantly suppressed expression of LRP-1 without modulating expression of either BCRP or P-glycoprotein (Additional file 2: Figure S1a, b). Similarly, propionate had neither a stimulatory nor inhibitory effect upon either BCRP or P-glycoprotein activity, at concentrations between 12 nM and 27 μM (Additional file 2: Figure S1c–f).