Voluntary exercise attenuates obesity-associated inflammation through ghrelin expressed in macrophages

Chronic low-level inflammation is associated with obesity and a sedentary lifestyle, causing metabolic disturbances such as insulin resistance. Exercise training has been shown to decrease chronic low-level systemic inflammation in high-fat diet (HFD)-induced obesity. However, the molecular mechanisms mediating its beneficial effects are not fully understood. Ghrelin is a peptide hormone predominantly produced in the stomach that stimulates appetite and induces growth hormone release. In addition to these well-known functions, recent studies suggest that ghrelin localizes to immune cells and exerts an anti-inflammatory effect. The purpose of the current study was to investigate the role of ghrelin expressed in macrophages in the anti-inflammatory effects of voluntary exercise training. Expression of tumor necrosis factor-α (TNF-α), monocyte chemotactic protein (MCP)-1 and F4/80 was increased in adipose tissue from mice fed a HFD (HFD mice) compared with mice fed a standard diet (SD mice), whereas the expression of these inflammatory cytokines was markedly decreased in mice performing voluntary wheel running during the feeding of a HFD (HFEx mice). The expression of TNF-α was also increased in peritoneal macrophages by a HFD and exercise training inhibited the increase of TNF-α expression. Interestingly, expression of ghrelin in peritoneal macrophages was decreased by a HFD and recovered by exercise training. Suppression of ghrelin expression by siRNA increased TNF-α expression and LPS-stimulated NF-κB activation in RAW264 cells, which is a macrophage cell line. TNF-α expression by stimulation with LPS was significantly suppressed in RAW264 cells cultured in the presence of ghrelin. These results suggest that ghrelin exerts potent anti-inflammatory effects in macrophages and functions as a mediator of the beneficial effects of exercise training.     

Trans-10, cis-12-conjugated linoleic acid attenuates tumor necrosis factor-α production by lipopolysaccharide-stimulated porcine peripheral blood mononuclear cells through induction of interleukin-10

Conjugated linoleic acid (CLA) can stimulate or inhibit immune cell function, and among CLA isomers, trans-10, cis-12 (t10c12)-CLA was shown to participate in the modulation of pro- or anti-inflammatory cytokine secretion. The objective of this study was to examine the effect of t10c12-CLA on tumor necrosis factor (TNF)-α production by lipopolysaccharide (LPS)-stimulated porcine peripheral blood mononuclear cells (PBMCs). In addition, we determined whether these effects were associated with the induction of interleukin (IL)-10. Treatment of LPS-unstimulated porcine PBMCs with t10c12-CLA increased both TNF-α expression and IL-10 production. However, treatment of LPS-stimulated porcine PBMCs with t10c12-CLA suppressed TNF-α production and increased the levels of IL-10. Furthermore, treatment of LPS-stimulated porcine PBMCs with IL-10 suppressed the production of TNF-α. The effects of t10c12-CLA on TNF-α expression by both LPS-naïve and LPS-stimulated PBMCs were inhibited by IL-10 treatment. The suppressive effects of t10c12-CLA on TNF-α production by LPS-stimulated porcine PBMCs were inhibited by an anti-IL-10 polyclonal antibody. These findings suggest that t10c12-CLA has an immunostimulatory effect on porcine PBMCs mediated via the up-regulation of TNF-α production, and an anti-inflammatory effect in LPS-stimulated PBMCs mediated via the down-regulation of TNF-α production, and that both is likely to be associated with the induction of IL-10.     

Anti-inflammatory activity of myricetin from Diospyros lotus through suppression of NF-κB and STAT1 activation and Nrf2-mediated HO-1 induction in lipopolysaccharide-stimulated RAW264.7 macrophages

Diospyros lotus is traditionally used for the treatment of diabetes, diarrhea, tumor, and hypertension. The purpose of this study was to investigate the anti-inflammatory effect and underlying molecular mechanisms of myricetin in lipopolysaccharide (LPS)-stimulated RAW264.7 macrophages. Myricetin dose-dependently suppressed the production of pro-inflammatory mediators (NO, iNOS, PGE₂, and COX-2) in LPS-stimulated RAW264.7 macrophages. Myricetin administration decreased the production of NO, iNOS, TNF-α, IL-6, and IL-12 in mice. Myricetin decreased NF-κB activation by suppressing the degradation of IκBα, nuclear translocation of p65 subunit of NF-κB, and NF-κB DNA binding activity in LPS-stimulated RAW264.7 macrophages. Moreover, myricetin attenuated the phosphorylation of STAT1 and the production of IFN-β in LPS-stimulated RAW264.7 macrophages. Furthermore, myricetin induced the expression of HO-1 through Nrf2 translocation. In conclusion, these results suggest that myricetin inhibits the production of pro-inflammatory mediators through the suppression of NF-κB and STAT1 activation and induction of Nrf2-mediated HO-1 expression in LPS-stimulated RAW264.7 macrophages.     

MK2 posttranscriptionally regulates TNF-α-induced expression of ICAM-1 and IL-8 via tristetraprolin in human pulmonary microvascular endothelial cells

Tristetraprolin (TTP), a substrate of p38 mitogen-activated protein kinase (MAPK)-activated protein kinase 2 (MK2), is an RNA-binding protein that binds to AU-rich elements (AREs) in the 3'-untranslated region (3'-UTR) of its target mRNAs and accelerates mRNA degradation. A previous study by our group showed that MK2 regulates tumor necrosis factor-α (TNF-α)-induced expression of intercellular adhesion molecule-1 (ICAM-1) and interleukin-8 (IL-8) in human lung microvascular endothelial cells; however, the downstream protein of MK2 remains unknown. Interestingly, both ICAM-1 and IL-8 have AREs in the 3'-UTR of their mRNAs. In the present study, we performed experiments to determine whether MK2 regulates TNF-α-induced expression of ICAM-1 and IL-8 via TTP in human pulmonary microvascular endothelial cells (HPMECs). The study revealed that MK2 silencing significantly reduced the half-lives of ICAM-1 and IL-8 mRNAs in TNF-α-stimulated HPMECs. TTP phosphorylation levels were decreased in MK2-silenced cells. TTP silencing led to mRNA stabilization of ICAM-1 and IL-8 and upregulation of protein production following TNF-α stimulation. These results, together with our previous study and others, suggest that MK2, in HPMECs, regulates TNF-α-induced expression of ICAM-1 and IL-8 via TTP at the mRNA decay level.     

A New IRAK-M-Mediated Mechanism Implicated in the Anti-Inflammatory Effect of Nicotine via α7 Nicotinic Receptors in Human Macrophages

Nicotine stimulation of α7 nicotinic acetylcholine receptor (α7 nAChR) powerfully inhibits pro-inflammatory cytokine production in lipopolysaccharide (LPS)-stimulated macrophages and in experimental models of endotoxemia. A signaling pathway downstream from the α7 nAChRs, which involves the collaboration of JAK2/STAT3 and NF-κB to interfere with signaling by Toll-like receptors (TLRs), has been implicated in this anti-inflammatory effect of nicotine. Here, we identifiy an alternative mechanism involving interleukin-1 receptor-associated kinase M (IRAK-M), a negative regulator of innate TLR-mediated immune responses. Our data show that nicotine up-regulates IRAK-M expression at the mRNA and protein level in human macrophages, and that this effect is secondary to α7 nAChR activation. By using selective inhibitors of different signaling molecules downstream from the receptor, we provide evidence that activation of STAT3, via either JAK2 and/or PI3K, through a single (JAK2/PI3K/STAT3) or two convergent cascades (JAK2/STAT3 and PI3K/STAT3), is necessary for nicotine-induced IRAK-M expression. Moreover, down-regulation of this expression by small interfering RNAs specific to the IRAK-M gene significantly reverses the anti-inflammatory effect of nicotine on LPS-induced TNF-α production. Interestingly, macrophages pre-exposed to nicotine exhibit higher IRAK-M levels and reduced TNF-α response to an additional LPS challenge, a behavior reminiscent of the ‘endotoxin tolerant’ phenotype identified in monocytes either pre-exposed to LPS or from immunocompromised septic patients. Since nicotine is a major component of tobacco smoke and increased IRAK-M expression has been considered one of the molecular determinants for the induction of the tolerant phenotype, our findings showing IRAK-M overexpression could partially explain the known influence of smoking on the onset and progression of inflammatory and infectious diseases.     

Molecular mechanism for adiponectin-dependent M2 macrophage polarization: link between the metabolic and innate immune activity of full-length adiponectin

The anti-inflammatory effects of globular adiponectin (gAcrp) are mediated by IL-10/heme oxygenase 1 (HO-1)-dependent pathways. Although full-length (flAcrp) adiponectin also suppresses LPS-induced pro-inflammatory signaling, its signaling mechanisms are not yet understood. The aim of this study was to examine the differential mechanisms by which gAcrp and flAcrp suppress pro-inflammatory signaling in macrophages. Chronic ethanol feeding increased LPS-stimulated TNF-α expression by Kupffer cells, associated with a shift to an M1 macrophage polarization. Both gAcrp and flAcrp suppressed TNF-α expression in Kupffer cells; however, only the effect of gAcrp was dependent on IL-10. Similarly, inhibition of HO-1 activity or siRNA knockdown of HO-1 in RAW264.7 macrophages only partially attenuated the suppressive effects of flAcrp on MyD88-dependent and -independent cytokine signatures. Instead, flAcrp, acting via the adiponectin R2 receptor, potently shifted the polarization of Kupffer cells and RAW264.7 macrophages to an M2 phenotype. gAcrp, acting via the adiponectin R1 receptor, was much less effective at eliciting an M2 pattern of gene expression. M2 polarization was also partially dependent on AMP-activated kinase. flAcrp polarized RAW264.7 macrophages to an M2 phenotype in an IL-4/STAT6-dependent mechanism. flAcrp also increased the expression of genes involved in oxidative phosphorylation in RAW264.7 macrophages, similar to the effect of flAcrp on hepatocytes. In summary, these data demonstrate that gAcrp and flAcrp utilize differential signaling strategies to decrease the sensitivity of macrophages to activation by TLR4 ligands, with flAcrp utilizing an IL-4/STAT6-dependent mechanism to shift macrophage polarization to the M2/anti-inflammatory phenotype.     

Suppression of thymus- and activation-regulated chemokine (TARC/CCL17) production by 1,2,3,4,6-penta-O-galloyl-β-d-glucose via blockade of NF-κB and STAT1 activation in the HaCaT cells

Keratinocytes, one of major cell types in the skin, can be induced by TNF-α and IFN-γ to express thymus- and activation-regulated chemokine (TARC/CCL17), which is considered to be a pivotal mediator in the inflammatory responses during the development of inflammatory skin diseases, such as atopic dermatitis (AD). In this study, we examined the effect of 1,2,3,4,6-penta-O-galloyl-β-d-glucose (PGG), isolated from the barks of Juglans mandshurica, on TNF-α/IFN-γ induced CCL17 expression in the human keratinocyte cell line HaCaT. Pretreatment of HaCaT cells with PGG suppressed TNF-α/IFN-γ-induced protein and mRNA expression of CCL17. PGG significantly inhibited TNF-α/IFN-γ-induced NF-κB activation as well as STAT1 activation. Furthermore, pretreatment with PGG resulted in significant reduction in expression of CXCL9, 10, and 11 in the HaCaT cells treated with IFN-γ. These results suggest that PGG may exert anti-inflammatory responses by suppressing TNF-α and/or IFN-γ-induced activation of NF-κB and STAT1 in the keratinocytes and might be a useful tool in therapy of skin inflammatory diseases.     

The Suppressive Effect of β-Glucan on the Production of Tumor Necrosis Factor-Alpha in BV2 Microglial Cells

β-Glucan was recently shown to have the ability to enhance and stimulate the immune system in humans, but little is known about its the anti-inflammatory effects. We investigated the effect of β-glucan on the production of tumor necrosis factor-alpha (TNF-α), a major pro-inflammatory mediator, in lipopolysaccharide (LPS)-stimulated BV2 microglial cells. β-Glucan decreased the production and expression of TNF-α. In addition, it blocked LPS-stimulated activation of nuclear factor kappa B (NF-κB). Hence β-glucan might suppress LPS-stimulated TNF-α production by inhibiting NF-κB in BV2 microglial cells.