Short-chain fatty acids act as antiinflammatory mediators by regulating prostaglandin E(2) and cytokines

Abstract

Aim: To investigate the effect of short-chain fatty acids (SCFAs) on production of prostaglandin E(2) (PGE(2)), cytokines and chemokines in human monocytes.

Methods: Human neutrophils and monocytes were isolated from human whole blood by using 1-Step Polymorph and RosetteSep Human Monocyte Enrichment Cocktail, respectively. Human GPR41 and GPR43 mRNA expression was examined by quantitative real-time polymerase chain reaction. The calcium flux assay was used to examine the biological activities of SCFAs in human neutrophils and monocytes. The effect of SCFAs on human monocytes and peripheral blood mononuclear cells (PBMC) was studied by measuring PGE(2), cytokines and chemokines in the supernatant. The effect of SCFAs in vivo was examined by intraplantar injection into rat paws.

Results: Human GPR43 is highly expressed in human neutrophils and monocytes. SCFAs induce robust calcium flux in human neutrophils, but not in human monocytes. In this study, we show that SCFAs can induce human monocyte release of PGE(2) and that this effect can be enhanced in the presence of lipopolysaccharide (LPS). In addition, we demonstrate that PGE(2) production induced by SCFA was inhibited by pertussis toxin, suggesting the involvement of a receptor-mediated mechanism. Furthermore, SCFAs can specifically inhibit constitutive monocyte chemotactic protein-1 (MCP-1) production and LPS-induced interleukin-10 (IL-10) production in human monocytes without affecting the secretion of other cytokines and chemokines examined. Similar activities were observed in human PBMC for the release of PGE(2), MCP-1 and IL-10 after SCFA treatment. In addition, SCFAs inhibit LPS-induced production of tumor necrosis factor-alpha and interferon-gamma in human PBMC. Finally, we show that SCFAs and LPS can induce PGE(2) production in vivo by intraplantar injection into rat paws (P < 0.01).

Conclusion: SCFAs can have distinct antiinflammatory activities due to their regulation of PGE(2), cytokine and chemokine release from human immune cells.

Figure 1

GPR43 is highly expressed in human neutrophils and monocytes. Human neutrophils and monocytes were isolated from human whole blood as described in Materials and Methods. Isolated human neutrophils or monocytes were stimulated with 100 ng/mL of lipopolysaccharide (LPS) for 3 h. RNAs were isolated and evaluated by Taqman analysis for absolute quantities of GPR43 and GPR41 mRNA molecules.

Figure 2

Short-chain fatty acids (SCFAs) induce robust calcium flux in human neutrophils, but not in human monocytes. Human neutrophils (A) and monocytes (B) were isolated and exposed to the indicated concentrations of formate, acetate and propionate for a calcium flux assay as described in Materials and Methods. Interleukin-8 (IL-8) is included as a positive control for human neutrophils, and monocyte chemotactic protein-1 (MCP-1) and ATP for human monocytes. The dose-response curves and EC₅₀ values were obtained by nonlinear regression using Graph Pad Prism 4 software.

Figure 3

SCFAs strongly induce PGE₂ production in human monocytes and the effect is receptor-mediated. Freshly isolated human monocytes (1 × 10⁹ cells/L) were incubated with the indicated concentrations of SCFAs or 100 nmol/L dexamethasone (Dex), and then cultured for 22 h without (A) or with (B) 100 ng/mL LPS. In a separate experiment (C, D), human monocytes were incubated with the indicated concentrations (in the parentheses) of inhibitors (described below) for 30 min, then with or without 2 mmol/L butyrate for another 30 min, and finally cultured for 22 h without (C) or with (D) 100 ng/mL LPS. The media and DMSO samples were used as controls with no inhibitors. All culture supernatants were assayed for PGE₂ by ELISA. MLnFP (methyl α-linolenyl fluorophosphonate): Phospholipase inhibitor; 1400W: Selective iNOS inhibitor; SC-560: Selective cyclooxygenase-1 (COX-1) inhibitor; CAY10404: Selective COX-2 inhibitor; PTX: Pertussis toxin. (Cayman Chemical, Ann Arbor, MI). The tested concentrations of each inhibitor (except PTX) were chosen to be about 100 times its IC₅₀ as reported in the data sheet provided by the manufacturer.

Figure 4

SCFAs specifically inhibit LPS-induced IL-10 production and constitutive MCP-1 expression in human monocytes. The culture supernatants as in Figures 3A and B were analyzed for 8 proinflammatory cytokines (including IL-1β, IL-2, IL-6, IL-8, IL-10, IL-12p70, IFN-γ, TNF-α) or 9 proinflammatory chemokines (including MCP-1, MCP-4, eotaxin, eotaxin-3, IL-8, IFN-γ inducible protein-10, macrophage inflammatory protein-1β, macrophage-derived chemokine, thymus and activation-regulated chemokine) by Meso Scale Multi-Spot Discovery Technology. Only the data for IL-10 (A, B) and MCP-1 (C, D) are shown here. Similar results were obtained from 10 human donors.

Figure 5

SCFAs inhibit LPS-induced production of TNF-α and IFN-γ in human peripheral blood mononuclear cells (PBMC) in a dose-dependent manner. Human PBMC isolated by Ficoll-Hypaque centrifugation procedure (1 × 10⁶ cells/mL) were incubated with the indicated concentrations of short-chain fatty acids (SCFA) or 100 nmol/L dexamethasone (Dex), and then cultured for 22 h with or without 100 ng/mL LPS. Culture supernatants were analyzed for 8 proinflammatory cytokines including IL-1β, IL-2, IL-6, IL-8, IL-10, IL-12p70, IFN-γ, TNF-α by Meso Scale Multi-Spot Discovery Technology. The data for TNF-α (A) and IFN-γ (B) with LPS stimulation are shown here.

Figure 6

SCFAs induce PGE₂ production by intraplantar injection into rat paws. The paw of male Sprague-Dawley rats was injected intraplantarly with 0.1 mL of 100 mmol/L formate, acetate, propionate or butyrate. Three micrograms of LPS in saline was also injected alone or in combination with 0.1 mL of 200 mmol/L butyrate. There were 4 rats per group. The normal group was not injected. Three hours post injection, rat paws were punched and fluids were collected in phenylmethanesulphonyl fluoride buffer with indomethacin. All punch fluids were assayed for PGE₂ by ELISA. P < 0.01 for butyrate + LPS group vs normal group, Mann-Whitney U test.