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
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.
Section snippets
Reagents and cell cultures
Recombinant mouse IFN-γ, NaB, NaPB and NaPA were obtained from Calbiochem (La Jolla, CA, USA). RAW 264.7 cells, murine macrophages, were obtained from the American Type Culture Collection (Rockville, MD). These cells were cultured in Dulbecco's modified Eagle's medium (DMEM) supplemented with 10% heat-inactivated endotoxin free fetal bovine serum (FBS), 2 mM glutamine, 100 μg/ml streptomycin and 100 units/ml penicillin under a humidified 5% CO2 atmosphere at 37 °C.
Nitrite quantification
NO production from activated
Short chain fatty acids suppresses IFN-γ-induced NO production
RAW 264.7 cells were pretreated with three types of SCFAs (NaB, NaPB, NaPA) for 30 min prior to stimulation with IFN-γ (20 U/ml) for 24 h. As shown in Fig. 1A, the SCFAs significantly repressed IFN-γ-mediated NO generation. The potency of this inhibition was in the order of NaB > NaPB > NaPA. At 1.0 mM, NaB almost completely inhibited NO production, whereas only 50% and 30% inhibition was observed in the NaPB and NaPA treated cells, respectively. Western blot analyses consistently showed that NaB
Discussion
Macrophages play an important role in the initiation and amplification of a variety of inflammatory diseases. Therefore, development of the means to reduce the number of activated macrophages, to inhibit the activation signals and/or their specific macrophage receptors, or to selectively counteract the macrophage products that act as disease amplifiers has been suggested as a promising therapeutic approach against various inflammatory diseases [1], [4].
This study demonstrated the
Acknowledgement
This work was supported by a grant from Neurobiology Research Program (M10412000010-05N1200-01000) and in part by a grant (M103KV010005 03K2201 00510) from Brain Research Center of the 21st Century Frontier Research Program funded by the Ministry of Science and Technology, Republic of Korea. JS Park and EJ Lee are recipients of BK21 grant from Korea Ministry of Education.
References (37)
Inflammation and neurodegenerative diseases
Am J Clin Nutr
(2006)- et al.
Effects of dietary oat brain and diabetes on plasma and caecal volatile fatty acids in the rat
Nutr Res
(1983) Inhibition of histone deacetylase activity by butyrate
J Nutr
(2003)- et al.
The effects of short-chain fatty acids on human colon cancer cell phenotype are associated with histone hyperacetylation
J Nutr
(2002) - et al.
Selective modulation of lipopolysaccharide-stimulated cytokine expression and mitogen-activated protein kinase pathways by dibutyryl-cAMP in BV2 microglial cells
Mol Brain Res
(2003) - et al.
An interferon-γ-activated site (GAS) is necessary for full expression of the mouse iNOS gene in response to interferon-γ and lipopolysaccharide
J Biol Chem
(1997) - et al.
Repression of interferon-γ-induced inducible nitric oxide synthase (iNOS) gene expression in microglia by sodium butyrate is mediated through specific inhibition of ERK signaling pathways
J Neuroimmunol
(2005) - et al.
Sodium butyrate upregulates Kupffer cell PGE2 production and modulates immune function
J Surg Res
(1998) Transcriptional regulation of the tumor necrosis factor alpha gene
Immunobiology
(1995)The inflammatory macrophage: a story of Jekyll and Hyde
Clin Sci
(2003)
Alternative versus classical activation of macrophages
Pathobiology
Tumor necrosis factor production by glomerular macrophages in anti-glomerular basement membrane glomerulonephritis in rabbits
Lab Invest
Macrophages in rheumatoid arthritis
Arthritis Res
Consequences and therapeutic implications of macrophage apoptosis in atherosclerosis: the importance of lesion stage and phagocytic efficiency
Arterioscler Thromb Vasc Biol
Anti-inflammatory effects of sodium butyrate on human monocytes: potent inhibition of IL-12 and upregulation of IL-10 production
FASEB J
Butyrate affects differentiation, maturation and function of human monocyte-derived dendritic cells and macrophages
Clin Exp Immunol
Inhibition of interferon γ signaling by the short chain fatty acid butyrate
Mol Cancer Res
Short-chain fatty acid-initiated cell cycle arrest and apoptosis of colonic epithelial cells is linked to mitochondrial function
Cell Growth Differ
Cited by (225)
Gut microbiota as a key regulator of intestinal mucosal immunity
2024, Life SciencesThe role and mechanism of action of microbiota-derived short-chain fatty acids in neutrophils: From the activation to becoming potential biomarkers
2023, Biomedicine and PharmacotherapyThe impact of microbiota-derived short-chain fatty acids on macrophage activities in disease: Mechanisms and therapeutic potentials
2023, Biomedicine and PharmacotherapyKonjac-mulberry leaf compound powder alleviates OVA-induced allergic rhinitis in BALB/c mice
2023, Food Science and Human WellnessThe importance of gut-brain axis and use of probiotics as a treatment strategy for multiple sclerosis
2023, Multiple Sclerosis and Related DisordersEnvironmental pesticide exposure and the risk of irritable bowel syndrome: A case-control study
2023, Environmental Toxicology and Pharmacology