The Anti-inflammatory Effects of Short Chain Fatty Acids on Lipopolysaccharide- or Tumor Necrosis Factor ?-Stimulated Endothelial Cells via Activation of GPR41/43 and Inhibition of HDACs.
ABSTRACT: Background and Aim: Previously, we found that short chain fatty acids (SCFA) inhibit LPS or TNF?-induced endothelial inflammatory responses and excessive vascular cell adhesion molecule-1 (VCAM-1) expression, two important steps in the development of atherosclerosis. However, the mechanisms involved are still unclear. We hypothesized that the effects of SCFA are associated with activation of G-protein coupled receptor 41/43 (GPR41/43) and/or inhibition of histone deacetylases (HDACs). Methods: The expression and location of GPR41/43 and HDAC3 in human umbilical vein endothelial cells (HUVEC) were confirmed. HUVEC were pre-incubated with acetate, butyrate or propionate alone or in combination with GLPG0974 (GLPG, antagonist of GPR43) or ?-hydroxybutyrate (SHB, antagonist of GPR41) and then exposed to LPS or TNF?. Interleukin (IL)-6 and IL-8 levels and VCAM-1 expression were measured. HDAC activity was measured after treatment with butyrate, propionate and trichostatin A (TSA, HDAC inhibitor). The peripheral blood mononuclear cell (PBMC) adhesive level was also determined after TSA treatment. Results: GPR41/43 were expressed on the membrane of HUVEC and HDAC3 was located in cytoplasm and nucleus. The GLPG and/or SHB treatments restored the inhibitory effects of acetate on IL-6 and IL-8 production and the inhibitory effects of butyrate or propionate on IL-6 production, but not on IL-8. In contrast, GLPG and/or SHB treatments did not affect the inhibitory effects of butyrate or propionate on TNF?-induced VCAM-1 expression. TSA showed similar effects on IL-8 production and VCAM-1 expression as butyrate and propionate. In addition, TSA significantly inhibited the adhesion of PBMC to an endothelial monolayer. Conclusion: Activation of GPR41/43 mediates the effects of acetate on IL-6 and IL-8 production and the effects of butyrate and propionate on IL-6 production. Furthermore, inhibition of HDACs mediates the effects of butyrate and propionate on IL-8 production, VCAM-1 expression, and PBMC adhesion to an endothelial monolayer. These data indicate the beneficial roles of SCFA in preventing vascular inflammation and relevant diseases by activation of GPR41/43 and inhibition of HDACs.
Project description:Background and Aim: Endothelial activation is characterized by excessive production of cytokines and chemokines as well as adhesion molecules expression which is involved in the development of atherosclerosis. The aim of our study is to investigate the effects of short chain fatty acids (SCFA) on lipopolysaccharide (LPS) or tumor necrosis factor alpha (TNF?)-induced endothelial activation. Methods and Results: Human umbilical vein endothelial cells (HUVEC) were pre-treated with acetate (10 mM), butyrate (0.1 mM) or propionate (0.3 mM) for 1, 16, or 24 h and then stimulated with LPS (1 or 10 ?g/ml) or TNF? (100 pg/ml or 1 ng/ml) for 6, 12, or 24 h. Cytokines in the supernatant were measured by ELISA. HUVEC were pre-treated with acetate (10 mM), butyrate (5 mM) or propionate (10 mM) for 24 h and then stimulated with LPS (1 ?g/ml) or TNF? (1 ng/ml) for 8 h. The expression of the adhesion molecules intracellular adhesion molecule-1 (ICAM-1) and vascular cell adhesion molecule-1 (VCAM-1) was detected by flow cytometry. The human blood mononuclear cell adhesive level to HUVEC monolayer was measured. LPS and TNF? induced a significant increase in the release of interleukin-6 (IL-6) and IL-8. Acetate, butyrate and propionate reduced IL-6 and IL-8 levels and the magnitude was dependent on the incubation times. LPS or TNF? increased ICAM-1 and VCAM-1 expression. Pre-incubation with acetate had no effect. In contrast, butyrate and propionate decreased VCAM-1 expression in TNF? stimulated cells but showed no effects on ICAM-1 expression. Butyrate significantly inhibited the adhesion of mononuclear cells to an endothelial monolayer and propionate was less effective. Conclusion: SCFA, including acetate, butyrate and propionate, influenced LPS- or TNF?-induced endothelial activation by inhibiting the production of IL-6 and IL-8, and reducing the expression of VCAM-1 and subsequent cell adhesion. Results were dependent on the concentrations and pre-incubation time of each SCFA and stimulation time of LPS or TNF?.
Project description:The gut microbiota produces metabolites such as short-chain fatty acids (SCFAs) that regulate the energy homeostasis and impact on immune cell function of the host. Recently, innovative approaches based on the oral administration of SCFAs have been discussed for therapeutic modification of inflammatory immune responses in autoimmune diseases. So far, most studies have investigated the SCFA-mediated effects on CD4<sup>+</sup> T cells and antigen presenting cells. Here we show that butyrate and, to a lesser degree, propionate directly modulate the gene expression of CD8<sup>+</sup> cytotoxic T lymphocytes (CTLs) and Tc17 cells. Increased IFN-? and granzyme B expression by CTLs as well as the molecular switch of Tc17 cells towards the CTL phenotype was mediated by butyrate independently of its interaction with specific SCFA-receptors GPR41 and GPR43. Our results indicate that butyrate strongly inhibited histone-deacetylases (HDACs) in CD8<sup>+</sup> T cells thereby affecting the gene expression of effector molecules. Accordingly, the pan-HDAC inhibitors trichostatin A (TSA) and sodium valproate exerted similar influence on CD8<sup>+</sup> T cells. Furthermore, higher acetate concentrations were also able to increase IFN-? production in CD8<sup>+</sup> T lymphocytes by modulating cellular metabolism and mTOR activity. These findings might have significant implications in adoptive immunotherapy of cancers and in anti-viral immunity.
Project description:Short-chain fatty acids (SCFAs) are among the main classes of bacterial metabolic products and are mainly synthesized in the colon through bacterial fermentation. Short-chain fatty acids, such as acetate, butyrate, and propionate, reduce endothelial activation induced by proinflammatory mediators, at least in part, by activation of G protein-coupled receptors (GPRs): GPR41 and GPR43. The objective of the study was to analyze the possible protective effects of SCFAs on endothelial dysfunction induced by angiotensin II (AngII). Rat aortic endothelial cells (RAECs) and rat aortas were incubated with AngII (1 μM) for 6 h in the presence or absence of SCFAs (5-10 mM). In RAECs, we found that AngII reduces the production of nitric oxide (NO) stimulated by calcium ionophore A23187; increases the production of reactive oxygen species (ROS), both from the nicotinamide adenine dinucleotide phosphate oxidase system and the mitochondria; diminishes vasodilator-stimulated phosphoprotein (VASP) phosphorylation at Ser239; reduces GPR41 and GPR43 mRNA level; and reduces the endothelium-dependent relaxant response to acetylcholine in aorta. Coincubation with butyrate and acetate, but not with propionate, increases both NO production and pSer239-VASP, reduces the concentration of intracellular ROS, and improves relaxation to acetylcholine. The beneficial effects of butyrate were inhibited by the GPR41 receptor antagonist, β-hydroxybutyrate, and by the GPR43 receptor antagonist, GLPG0794. Butyrate inhibited the down-regulation of GPR41 and GPR43 induced by AngII, being without effect acetate and propionate. Neither β-hydroxybutyrate nor GLPG0794 affects the protective effect of acetate in endothelial dysfunction. In conclusion, acetate and butyrate improve endothelial dysfunction induced by AngII by increasing the bioavailability of NO. The effect of butyrate seems to be related to GPR41/43 activation, whereas acetate effects were independent of GPR41/43.
Project description:Elucidating the mechanisms by which short chain fatty acids (SCFA) reduce body weight may assist in the development of an effective weight control strategy. Dietary supplementation of acetate, propionate, butyrate or their admixture was shown to significantly inhibit the body weight gain induced by high-fat diet feeding. Supplementation of SCFAs caused significant changes in the expressions of G-protein coupled receptor 43 (GPR43) and GPR41 characterized by increases in the adipose tissue and reductions in the colon. Additionally, they influenced the bacterial community structure in feces, with a reduction in the proportion of Firmicutes and an increase in the proportion of Bacteroidetes. The effects of dietary SCFAs on the GPR expression and gut microbiota composition may further result in body weight reduction by enhancing triglyceride hydrolysis and FFA oxidation in the adipose tissue, promoting beige adipogenesis and mitochondrial biogenesis, and inhibiting chronic inflammation.
Project description:The gut microbiota is essential for human health and plays an important role in the pathogenesis of several diseases. Short-chain fatty acids (SCFA), such as acetate, butyrate and propionate, are end-products of microbial fermentation of macronutrients that distribute systemically via the blood. The aim of this study was to investigate the transcriptional response of immature and LPS-matured human monocyte-derived DC to SCFA. Our data revealed distinct effects exerted by each individual SCFA on gene expression in human monocyte-derived DC, especially in the mature ones. Acetate only exerted negligible effects, while both butyrate and propionate strongly modulated gene expression in both immature and mature human monocyte-derived DC. An Ingenuity pathway analysis based on the differentially expressed genes suggested that propionate and butyrate modulate leukocyte trafficking, as SCFA strongly reduced the release of several pro-inflammatory chemokines including CCL3, CCL4, CCL5, CXCL9, CXCL10, and CXCL11. Additionally, butyrate and propionate inhibited the expression of lipopolysaccharide (LPS)-induced cytokines such as IL-6 and IL-12p40 showing a strong anti-inflammatory effect. This work illustrates that bacterial metabolites far from the site of their production can differentially modulate the inflammatory response and generally provides new insights into host-microbiome interactions.
Project description:Short-chain fatty acids (SCFAs) are major products of gut microbial fermentation and profoundly affect host health and disease. SCFAs generate IL-10(+) regulatory T cells, which may promote immune tolerance. However, SCFAs can also induce Th1 and Th17 cells upon immunological challenges and, therefore, also have the potential to induce inflammatory responses. Because of the seemingly paradoxical SCFA activities in regulating T cells, we investigated, in depth, the impact of elevated SCFA levels on T cells and tissue inflammation in mice. Orally administered SCFAs induced effector (Th1 and Th17) and regulatory T cells in ureter and kidney tissues, and they induced T cell-mediated ureteritis, leading to kidney hydronephrosis (hereafter called acetate-induced renal disease, or C2RD). Kidney hydronephrosis in C2RD was caused by ureteral obstruction, which was, in turn, induced by SCFA-induced inflammation in the ureteropelvic junction and proximal ureter. Oral administration of all major SCFAs, such as acetate, propionate, and butyrate, induced the disease. We found that C2RD development is dependent on mammalian target of rapamycin activation, T cell-derived inflammatory cytokines such as IFN-? and IL-17, and gut microbiota. Young or male animals were more susceptible than old or female animals, respectively. However, SCFA receptor (GPR41 or GPR43) deficiency did not affect C2RD development. Thus, SCFAs, when systemically administered at levels higher than physiological levels, cause dysregulated T cell responses and tissue inflammation in the renal system. The results provide insights into the immunological and pathological effects of chronically elevated SCFAs.
Project description:<h4>Background</h4>Eosinophilic esophagitis (EoE) is a food allergen driven disease that is accompanied by interleukin (IL) 13 overexpression and esophageal barrier dysfunction allowing transepithelial food allergen permeation. Nutraceuticals, such as short-chain fatty acids (SCFAs) that restore barrier function and increase immune fitness may be a promising tool in the management of EoE. Here, we investigated the effects of the SCFAs acetate, propionate, and butyrate on an IL-13-compromised human esophageal epithelial barrier, including the mechanisms involved.<h4>Methods</h4>An air-liquid interface culture model of differentiated human EPC2-hTERT (EPC2) was used to study whether SCFAs could restore barrier function after IL-13-induced impairment. Esophageal epithelial barrier function was monitored by transepithelial electrical resistance (TEER) and FITC-dextran paracellular flux, and was further examined by qPCR and immunohistochemical analysis. G protein-coupled receptor (GPR) GPR41, GPR43, GPR109a, or histone deacetylase (HDAC) (ant)agonists were used to assess mechanisms of action of SCFAs.<h4>Results</h4>IL-13 stimulation decreased TEER and increased FITC flux, which was counteracted by butyrate and propionate, but not acetate treatment. Barrier proteins FLG and DSG1 mRNA expression was upregulated following butyrate and propionate treatment, whereas expression of eosinophil chemoattractant CCL26 and protease CAPN14 was downregulated. Similarly, butyrate and propionate restored FLG and DSG1 protein expression. Similar effects were observed with an HDAC antagonist but not with GPR agonists.<h4>Conclusion</h4>Nutraceuticals butyrate and propionate restore the barrier function of esophageal epithelial cells after an inflammatory insult and may be of therapeutic benefit in the management of EoE.
Project description:Emerging data indicate that excessive short chain fatty acids can mediate the downstream mitogen-activated protein kinase pathways by activating G-protein coupled receptor 41/43 (GPR41/43) to initiate the inflammatory response. The current study was conducted to investigate if a high concentrate (HC) supplemented with sodium butyrate can alleviate the inflammation and if an epigenetic mechanism is involved in regulating the expression of the key GPR41/43 genes in the cecum. Twelve lactating goats were randomly divided into two groups: the control group fed the HC diet and the treatment group fed the HC diet supplemented with sodium butyrate (HCB). Our results suggested that the supplementation of sodium butyrate significantly increased the pH value in the rumen and cecum, downregulated the expression of GPR41/43 and related inflammatory cytokines, upregulated the expression of tight junction proteins, and reduced the protein expression levels of GPR 41/43, ERK1/2, and p38. Moreover, the ratios of DNA methylation and chromatin compaction in the promoter region of the GPR41/43 genes were altered due to the addition of sodium butyrate. In brief, dietary addition of sodium butyrate can reduce the inflammatory injury to the cecal mucosa in lactating goats and can affect the expression of GPR41/43 via epigenetic modification.
Project description:Short-chain fatty acids (SCFA) are bacterial metabolites that can be found in periodontal pockets. The expression of adhesion molecules such as intercellular adhesion molecule-1 (ICAM-1) within the epithelium pocket is considered to be a key event for the selective transmigration of leucocytes towards the gingival sulcus. However, the impact of SCFA on ICAM-1 expression by oral epithelial cells remains unclear. We therefore exposed the oral squamous carcinoma cell line HSC-2, primary oral epithelial cells and human gingival fibroblasts to SCFA, namely acetate, propionate and butyrate, and stimulated with known inducers of ICAM-1 such as interleukin-1-beta (IL1?) and tumor necrosis factor-alfa (TNF?). We report here that butyrate but not acetate or propionate significantly suppressed the cytokine-induced ICAM-1 expression in HSC-2 epithelial cells and primary epithelial cells. The G-protein coupled receptor-43 (GPR43/ FFAR2) agonist but not the histone deacetylase inhibitor, trichostatin A, mimicked the butyrate effects. Butyrate also attenuated the nuclear translocation of p65 into the nucleus on HSC-2 cells. The decrease of ICAM-1 was independent of Nrf2/HO-1 signaling and phosphorylation of JNK and p38. Nevertheless, butyrate could not reverse an ongoing cytokine-induced ICAM-1 expression in HSC-2 cells. Overall, these observations suggest that butyrate can attenuate cytokine-induced ICAM-1 expression in cells with epithelial origin.
Project description:Probiotic Bifidobacterium longum subspecies infantis (Bifidobacterium infantis) consumes human milk oligosaccharides (MO) and protects intestinal permeability thereby having anti-inflammatory effects (Underwood et al., 2015; Bode, 2006; Asakuma et al., 2011) [1-3]. Via the gut-liver axis, gut barrier disruption and dysbiosis lead to hepatic inflammation (Sheng et al., 2017; Jena et al., 2017) [4,5,6]. Our published data revealed that butyrate, as well as synbiotics of B. infantis in combination with MO, had protective effects against cancer-prone non-alcoholic steatohepatitis (NASH) mouse models, i.e., Western diet (WD)-fed bile acid receptor FXR (farnesoid x receptor) knockout (KO) mice (Jena et al., 2018) [6,7]. In addition, MO was particularly effective in increasing the blooming of butyrate-generating bacteria (Jena et al., 2018) . In the present study, we further showed that the reduced ileal short chain fatty acid (SCFA) signaling found in WD-fed FXR KO mice could be reversed by B. infantis and/or MO treatment. Moreover, ileal mRNA levels of SCFA receptors i.e. Gpr41 (Ffar3), Gpr109 (Hcar2), and Gpr43 (Ffar2) were increased in B. infantis and/or MO-treated mice suggesting increased SCFA signaling (Fig. 1). Further, nuclear magnetic resonance (NMR) data revealed that MO and B. Infantis plus MO increased intestinal acetate, propionate, butyrate, and valerate levels (Fig. 2). In addition, B. infantis and/or MO reduced the abundance of genus Bilophila and the relative copy number of bacterial genes including dissimilatory sulfite reductase (dsrA) and methyl coenzyme M reductase A (mcrA), which were all increased in cancer-prone FXR KO mice (Fig. 3).