Anti-inflammatory effects of short chain fatty acids in IFN-γ-stimulated RAW 264.7 murine macrophage cells: Involvement of NF-κB and ERK signaling pathways

Abstract

The overactivation of macrophages causes abnormal cell death and chronic inflammatory diseases. Therefore, the modulation of macrophage-mediated cytotoxicity is expected to become a new therapeutic strategy for various inflammatory diseases. In this study, three types of short chain fatty acids (sodium butyrate (NaB), sodium phenylbutyrate (NaPB), sodium phenylacetate (NaPA)) were found to have anti-inflammatory effects in IFN-γ-stimulated RAW 264.7 cells. They inhibited the expression of iNOS, TNF-α, and IL-6 induced by IFN-γ, while they enhanced the expression of the anti-inflammatory cytokine, IL-10. Their potency as anti-inflammatory agents was in the order of NaB > NaPB > NaPA. Further mechanistic studies revealed these three agents to repress the DNA binding and transcriptional activities of NF-κB, which is an important modulator of inflammation. In addition, these agents repressed the IFN-γ-induced ERK1/2 phosphorylation without affecting the Jak/STAT activities. The potency of NF-κB and ERK inhibition was also in the order of NaB > NaPB > NaPA. The results suggest that the NF-κB and ERK signaling pathways are at least in part involved in the anti-inflammatory activities of these SCFAs. Considering that SCFAs are normally present in the body and have few side effects, they might be promising agents for the prevention and/or treatment of various inflammatory diseases.

Introduction

Macrophages play important roles in various inflammatory responses, immunity, host defense, and tissue repair. Once activated, macrophages secrete a large number of pro-inflammatory cytokines, chemokines, nitric oxide and proteases and induce the apoptosis of resident stromal and parenchymal cells [1]. Macrophages remove apoptotic cells by phagocytosis and secrete anti-inflammatory mediators and growth factors, which leads to wound healing and repair [1], [2]. Therefore, macrophage cells have ambiguous functions, such as inducing or alleviating inflammation depending on their activation status. A correct balance between these two populations is needed for healing and resolution. However, an excess number of activated macrophages can induce abnormal cell death and chronic inflammatory diseases such as rheumatoid arthritis, glomerulonephritis, atherosclerosis and various neurodegenerative diseases including Alzheimer's diseases [3], [4], [5], [6]. Therefore, the modulation of the macrophage-mediated inflammatory responses is emerging as a promising new therapeutic approach against these inflammatory diseases.

Short chain fatty acids (SCFA) are the principal by-products of fiber fermentation in the gastrointestinal tract and have attracted considerable attention in maintaining the bowel homeostasis because they can prevent inflammation and have anti-proliferative and apoptotic effects in colon cancer cells [7], [8], [9]. SCFAs are circulated in the blood at concentrations in the 1–2 mM range [7], [10]. The representative SCFAs are acetate, propionate, butyrate and aromatic fatty acids such as phenylbutyrate (PB) and phenylacetate (PA). These SCFAs regulate cell growth, differentiation and proliferation as well as induce cell cycle arrest and apoptosis [9], [11]. Recently, it was reported that PB and butyrate function as histone deacetylase (HDAC) inhibitors, which regulate gene transcription in the context of the chromatin structure [12], [13]. Based on their function as HDAC inhibitors, PB and butyrate have been developed as anti-cancer drugs and currently under a phase I clinical evaluation [14], [15]. The aromatic short chain fatty acids, PA and PB, have shown anti-tumor activity in colorectal carcinoma cells, which was mediated via the activation of peroxisome proliferators-activated receptor-γ (PPAR-γ) [16]. PB, which is metabolized to PA in humans, was also recently shown to benefit cancer patients who have failed conventional therapy [17], [18], [19]. For both drugs, large drug doses are needed to reach the therapeutic plasma concentrations (mM range). However, these doses are well tolerated, with the dose-limiting toxicity being somnolence.

Besides these various activities, several groups have reported the anti-inflammatory effects of butyrate. In particular, butyrate has been reported to have a prominent anti-inflammatory effect in colitis by modulating IL-8, macrophage inhibitory protein 2 (MIP-2) activities and suppressing the NF-κB activity [19], [20]. In addition, it showed anti-inflammatory effects in human monocytes by inhibiting IL-12 and upregulating IL-10 production [7]. In LPS-stimulated RAW 264.7 murine macrophage cells, a high concentration of butyrate significantly reduced the level of NO production and prevented NF-κB activation by stabilizing IκBα and IκBβ [21]. In addition, butyrate enemas or a high-fiber diet that increases the colonic butyrate concentration have been shown to be effective in treating mucosal inflammation in humans as well as in animal models of colitis [22], [23].

There are no reports on the anti-inflammatory effects of SCFAs (NaB, NaPB, NaPA) on IFN-γ-stimulated macrophage cells. Moreover, the molecular mechanisms and signal transduction pathways underlying the anti-inflammatory effects of SCFAs have not been clearly demonstrated. Therefore, this study compared the anti-inflammatory effects of NaB and two aromatic SCFAs (NaPB and NaPA) in IFN-γ-stimulated RAW 264.7 macrophage cells and analyzed the molecular mechanisms involved. Considering that IFN-γ plays a major role in the pathophysiology of many intestinal inflammatory diseases such as ulcerative colitis, pouchitis, and Crohn's diseases [24], [25], the inhibition of the IFN-γ-mediated inflammatory responses by SCFAs may have therapeutic potential for various inflammatory diseases.