Macrophage-mediated Inflammation Research Articles

The broad-spectrum chemokine inhibitor NR58-3.14.3 modulates macrophage-mediated inflammation in the diseased retina.

BackgroundThe activity of macrophages is implicated in the progression of retinal pathologies such as atrophic age-related macular degeneration (AMD), where they accumulate among the photoreceptor layer and subretinal space. This process is aided by the local expression of chemokines, which furnish these cells with directional cues that augment their migration to areas of retinal injury. While these qualities make chemokines a potential therapeutic target in curtailing damaging retinal inflammation, their wide variety and signalling redundancy pose challenges in broadly modulating their activity. Here, we examine the efficacy of the broad-spectrum chemokine inhibitor NR58-3.14.3—a suppressor of Ccl- and Cxcl- chemokine pathways—in suppressing macrophage activity and photoreceptor death, using a light-induced model of outer retinal atrophy and inflammation.MethodsPhoto-oxidative damage was induced in SD rats via exposure to 1000 lux of light for 24 h, after which animals were euthanized at 0- or 7-day post-exposure time points. Prior to damage, NR58-3.14.3 was injected intravitreally. Retinas were harvested and evaluated for the effect of NR58-3.14.3 on subretinal macrophage accumulation and cytokine expression profile, as well as photoreceptor degeneration.ResultsWe report that intravitreal administration of NR58-3.14.3 reduces the accumulation of macrophages in the outer retina following exposure to light damage, at both 0- and 7-day post-exposure time points. Injection of NR58-3.14.3 also reduced the up-regulation of inflammatory markers including of Il6, Ccl3, and Ccl4 in infiltrating macrophages, which are promoters of their pathogenic activity in the retina. Finally, NR58-3.14.3-injected retinas displayed markedly reduced photoreceptor death following light damage, at both 0 and 7 days post-exposure.ConclusionsOur findings indicate that NR58-3.14.3 is effective in inhibiting subretinal macrophage accumulation in light-induced retinal degeneration and illustrate the potential of broad-spectrum chemokine inhibitors as novel therapeutic agents in thwarting retinal inflammation. Although broad-spectrum chemokine inhibitors may not be appropriate for all retinal inflammatory conditions, our results suggest that they may be beneficial for retinal dystrophies in which chemokine expression and subretinal macrophage accumulation are implicated, such as advanced AMD.

Targeting arginase-II protects mice from high-fat-diet-induced hepatic steatosis through suppression of macrophage inflammation

Nonalcoholic fatty liver disease (NAFLD) associates with obesity and type 2 diabetes. Hypoactive AMP-activated protein kinase (AMPK), hyperactive mammalian target of rapamycin (mTOR) signaling, and macrophage-mediated inflammation are mechanistically linked to NAFLD. Studies investigating roles of arginase particularly the extrahepatic isoform arginase-II (Arg-II) in obesity-associated NAFLD showed contradictory results. Here we demonstrate that Arg-II−/− mice reveal decreased hepatic steatosis, macrophage infiltration, TNF-α and IL-6 as compared to the wild type (WT) littermates fed high fat diet (HFD). A higher AMPK activation (no difference in mTOR signaling), lower levels of lipogenic transcription factor SREBP-1c and activity/expression of lipogenic enzymes were observed in the Arg-II−/− mice liver. Moreover, release of TNF-α and IL-6 from bone marrow-derived macrophages (BMM) of Arg-II−/− mice is decreased as compared to WT-BMM. Conditioned medium from Arg-II−/−-BMM exhibits weaker activity to facilitate triglyceride synthesis paralleled with lower expression of SREBP-1c and SCD-1 and higher AMPK activation in hepatocytes as compared to that from WT-BMM. These effects of BMM conditioned medium can be neutralized by neutralizing antibodies against TNF-α and IL-6. Thus, Arg-II-expressing macrophages facilitate diet-induced NAFLD through TNF-α and IL-6 in obesity.

Hepatocytes release ceramide-enriched pro-inflammatory extracellular vesicles in an IRE1α-dependent manner

Nonalcoholic steatohepatitis (NASH) is a lipotoxic disease wherein activation of endoplasmic reticulum (ER) stress response and macrophage-mediated hepatic inflammation are key pathogenic features. However, the lipid mediators linking these two observations remain elusive. We postulated that ER stress-regulated release of pro-inflammatory extracellular vesicles (EVs) from lipotoxic hepatocytes may be this link. EVs were isolated from cell culture supernatants of hepatocytes treated with palmitate (PA) to induce lipotoxic ER stress, characterized by immunofluorescence, Western blotting, electron microscopy, and nanoparticle tracking analysis. Sphingolipids were measured by tandem mass spectrometry. EVs were employed in macrophage chemotaxis assays. PA induced significant EV release. Because PA activates ER stress, we used KO hepatocytes to demonstrate that PA-induced EV release was mediated by inositol requiring enzyme 1α (IRE1α)/X-box binding protein-1. PA-induced EVs were enriched in C16:0 ceramide in an IRE1α-dependent manner, and activated macrophage chemotaxis via formation of sphingosine-1-phosphate (S1P) from C16:0 ceramide. This chemotaxis was blocked by sphingosine kinase inhibitors and S1P receptor inhibitors. Lastly, elevated circulating EVs in experimental and human NASH demonstrated increased C16:0 ceramide. PA induces C16:0 ceramide-enriched EV release in an IRE1α-dependent manner. The ceramide metabolite, S1P, activates macrophage chemotaxis, a potential mechanism for the recruitment of macrophages to the liver under lipotoxic conditions.

Hemopexin therapy reverts heme-induced proinflammatory phenotypic switching of macrophages in a mouse model of sickle cell disease

AbstractHemolytic diseases, such as sickle cell anemia and thalassemia, are characterized by enhanced release of hemoglobin and heme into the circulation, heme-iron loading of reticulo-endothelial system macrophages, and chronic inflammation. Here we show that in addition to activating the vascular endothelium, hemoglobin and heme excess alters the macrophage phenotype in sickle cell disease. We demonstrate that exposure of cultured macrophages to hemolytic aged red blood cells, heme, or iron causes their functional phenotypic change toward a proinflammatory state. In addition, hemolysis and macrophage heme/iron accumulation in a mouse model of sickle disease trigger similar proinflammatory phenotypic alterations in hepatic macrophages. On the mechanistic level, this critically depends on reactive oxygen species production and activation of the Toll-like receptor 4 signaling pathway. We further demonstrate that the heme scavenger hemopexin protects reticulo-endothelial macrophages from heme overload in heme-loaded Hx-null mice and reduces production of cytokines and reactive oxygen species. Importantly, in sickle mice, the administration of human exogenous hemopexin attenuates the inflammatory phenotype of macrophages. Taken together, our data suggest that therapeutic administration of hemopexin is beneficial to counteract heme-driven macrophage-mediated inflammation and its pathophysiologic consequences in sickle cell disease.

Macrophage-mediated inflammation and glial response in the skeletal muscle of a rat model of familial amyotrophic lateral sclerosis (ALS)

Amyotrophic lateral sclerosis (ALS) is a fatal neurodegenerative disease characterized by progressive motor dysfunction and loss of large motor neurons in the spinal cord and brain stem. While much research has focused on mechanisms of motor neuron cell death in the spinal cord, degenerative processes in skeletal muscle and neuromuscular junctions (NMJs) are also observed early in disease development. Although recent studies support the potential therapeutic benefits of targeting the skeletal muscle in ALS, relatively little is known about inflammation and glial responses in skeletal muscle and near NMJs, or how these responses contribute to motor neuron survival, neuromuscular innervation, or motor dysfunction in ALS. We recently showed that human mesenchymal stem cells modified to release glial cell line-derived neurotrophic factor (hMSC-GDNF) extend survival and protect NMJs and motor neurons in SOD1G93A rats when delivered to limb muscles. In this study, we evaluate inflammatory and glial responses near NMJs in the limb muscle collected from a rat model of familial ALS (SOD1G93A transgenic rats) during disease progression and following hMSC-GDNF transplantation. Muscle samples were collected from pre-symptomatic, symptomatic, and end-stage animals. A significant increase in the expression of microglial inflammatory markers (CD11b and CD68) occurred in the skeletal muscle of symptomatic and end-stage SOD1G93A rats. Inflammation was confirmed by ELISA for inflammatory cytokines interleukin-1 β (IL-1β) and tumor necrosis factor-α (TNF-α) in muscle homogenates of SOD1G93A rats. Next, we observed active glial responses in the muscle of SOD1G93A rats, specifically near intramuscular axons and NMJs. Interestingly, strong expression of activated glial markers, glial fibrillary acidic protein (GFAP) and nestin, was observed in the areas adjacent to NMJs. Finally, we determined whether ex vivo trophic factor delivery influences inflammation and terminal Schwann cell (TSC) response during ALS. We found that intramuscular transplantation of hMSC-GDNF tended to exhibit less inflammation and significantly maintained TSC association with NMJs. Understanding cellular responses near NMJs is important to identify suitable cellular and molecular targets for novel treatment of ALS and other neuromuscular diseases.

Maackiapterocarpan B from Sophora tonkinensis Suppresses Inflammatory Mediators via Nuclear Factor-κB and Mitogen-Activated Protein Kinase Pathways.

Maackiapterocarpan B, one of the pterocarpan analogs found in Sophora tonkinensis, is known to display pharmacological activities. However, the anti-inflammatory effects of maackiapterocarpan B and its molecular mechanism have yet to be clearly elucidated. In the present study, the effects of maackiapterocarpan B on macrophage-mediated inflammation in vitro were assessed. Maackiapterocarpan B inhibited the production of nitric oxide, the expression of tumor necrosis factor α, colony stimulating factor 2, interleukin-1β and interleukin-6, and the activation of nuclear factor-κB and mitogen-activated protein kinases in lipopolysaccharide-stimulated macrophages. These observations suggest the potential of maackiapterocarpan B in the treatment of inflammatory diseases.

Caspase-1 inhibition attenuates activation of BV2 microglia induced by LPS-treated RAW264.7 macrophages.

Neuroinflammation has been recognized as a factor in the pathogenesis of neurodegenerative diseases. Emerging evidence suggests that peripheral inflammation, besides neuroinflammation, functions as a modulator of disease progression and neuropathology in several neurodegenerative diseases. However, detailed correlations among peripheral inflammation, neuroinflammation and neurodegeneration remain unknown. In the present study, we prepared a peripheral inflammation model with lipopolysaccharides (LPS)-stimulated RAW264.7 macrophages to explore its activation on BV2 microglia. We found that LPS induced the production of IL-1β, IL-6 and TNF-α in the culture medium of RAW264.7 macrophages. We further showed that LPS plus ATP activated inflammasome, evidenced by the upregulation of caspase-1 and IL-1β, which was suppressed by ZYVAD, a caspase-1 inhibitor. Furthermore, the conditioned medium obtained from LPS-treated RAW264.7 macrophages activated BV2 microglia, stimulating the release of IL-1β, IL-6 and TNF-α from BV2 cells. ZYVAD pretreatment markedly suppressed BV2 microglia activation induced by RAW264.7 cells conditioned medium. Taken together, our study indicates that macrophage-mediated peripheral inflammation subsequently evokes neuroinflammation and may aggravate neural damage. Inflammasome and caspase-1 may be potential targets for modulating systemic inflammatory responses in neurodegenerative diseases.

Hypoxia Potentiates Palmitate-induced Pro-inflammatory Activation of Primary Human Macrophages

Pro-inflammatory cytokines secreted by adipose tissue macrophages (ATMs) contribute to chronic low-grade inflammation and obesity-induced insulin resistance. Recent studies have shown that adipose tissue hypoxia promotes an inflammatory phenotype in ATMs. However, our understanding of how hypoxia modulates the response of ATMs to free fatty acids within obese adipose tissue is limited. We examined the effects of hypoxia (1% O2) on the pro-inflammatory responses of human monocyte-derived macrophages to the saturated fatty acid palmitate. Compared with normoxia, hypoxia significantly increased palmitate-induced mRNA expression and protein secretion of IL-6 and IL-1β. Although palmitate-induced endoplasmic reticulum stress and nuclear factor κB pathway activation were not enhanced by hypoxia, hypoxia increased the activation of JNK and p38 mitogen-activated protein kinase signaling in palmitate-treated cells. Inhibition of JNK blocked the hypoxic induction of pro-inflammatory cytokine expression, whereas knockdown of hypoxia-induced transcription factors HIF-1α and HIF-2α alone or in combination failed to reduce IL-6 and only modestly reduced IL-1β gene expression in palmitate-treated hypoxic macrophages. Enhanced pro-inflammatory cytokine production and JNK activity under hypoxia were prevented by inhibiting reactive oxygen species generation. In addition, silencing of dual-specificity phosphatase 16 increased normoxic levels of IL-6 and IL-1β and reduced the hypoxic potentiation in palmitate-treated macrophages. The secretome of hypoxic palmitate-treated macrophages promoted IL-6 and macrophage chemoattractant protein 1 expression in primary human adipocytes, which was sensitive to macrophage JNK inhibition. Our results reveal that the coexistence of hypoxia along with free fatty acids exacerbates macrophage-mediated inflammation.

The Heme Scavenger Hemopexin Reverts Heme-Driven Pro-Inflammatory Phenotypic Switching of Macrophages in Sickle Cell Disease

Hemolytic diseases, such as sickle cell anemia and thalassemia, are characterized by enhanced release of hemoglobin and heme into the circulation, heme-iron loading of reticulo-endothelial system macrophages as well as chronic inflammation. Here we demonstrate that in addition to activating the vascular endothelium, hemoglobin and heme alter the macrophage phenotype in sickle cell disease.We show that exposure of cultured macrophages to hemolytic aged red blood cells, heme or iron causes their functional phenotypic change towards a pro-inflammatory phenotype, with increased expression levels of inflammatory markers such as IL-6 (P<0.001), TNFα (P<0.0001), IL-1β (P<0.001) MHCII (P<0.001), CD86 (P<0.05) and CD14 (P<0.0001) and decreased expression levels of anti-inflammatory markers such as IL-10 (P<0.0001), CD206 (P<0.0001), Arginase-1 and Ym1. Interestingly, hemolysis and macrophage heme/iron accumulation in a mouse model of sickle cell disease triggers similar pro-inflammatory phenotypic alterations in hepatic macrophages, increasing the expression levels of IL-6 (P<0.0001), MHCII (P<0.05), CD86 (P<0.0001) and iNOS (P<0.01). On the mechanistic level, heme-induced pro-inflammatory phenotype switching of macrophages critically depends on ROS production and activation of the TLR4 signaling pathway.We further demonstrate that the heme scavenger hemopexin protects reticulo-endothelial macrophages from heme overload in heme-loaded Hx-null mice (P<0.05) and reduces production of cytokines (IL-6, TNFα: P<0.01) and reactive oxygen species (P<0.001). Importantly, in sickle mice the administration of human exogenous hemopexin attenuates the inflammatory phenotype of macrophages, by decreasing the expression of IL-6 (P<0.05), MHCII (P<0.01), CD86 (P<0.001) and iNOS (P<0.05).Taken together, our data suggest that therapeutical administration of hemopexin is beneficial to counteract heme-driven macrophage-mediated inflammation in sickle cell disease. DisclosuresBrinkman:CSL Behring: Employment. Zuercher:CSL Behring: Employment.

Resveratrol Protects Against Pathological Preterm Birth by Suppression of Macrophage-Mediated Inflammation.

Inflammatory cytokines play a major role in spontaneous preterm birth. Resveratrol has strong anti-inflammatory effects, but its effect on preterm birth in vivo is unknown. We investigated whether resveratrol protects against preterm birth in the lipopolysaccharide (LPS)-induced preterm mouse model. Twelve-day-old pregnant mice were fed 20 to 40 mg/kg resveratrol daily. On day 15, 10 μg of LPS was injected into uterine cervices. Resveratrol administration significantly decreased the rate of preterm birth. Resveratrol administration abolished LPS-induced elevation of tumor necrosis factor α (TNF-α) and interleukin (IL) 1β but not IL-6 levels. The TNF-α messenger RNA levels were decreased in the cervices of resveratrol-administered mice compared with controls. Resveratrol treatment suppressed the elevation in TNF-α and IL-1β levels in LPS-exposed peritoneal macrophages. Further resveratrol treatment eradicated the proinflammatory cytokine-mediated elevation in cyclooxygenase 2 (COX-2) in peritoneal macrophages. Resveratrol may protect against pathological preterm birth by suppression of elevated proinflammatory cytokines and consequent elevation of COX-2 in macrophages.

Abstract 12495: Enhanced Thrombin Activity in Biglycan-deficient Mice Promotes Vascular Inflammation in Murine Atherosclerosis

Introduction: Thrombin signalling initiates inflammatory events directly and through activation of platelets. Endogenous and pharmacologic inhibitors of thrombin are therefore of relevance during atheroprogression and for therapeutic intervention. The small leucine-rich proteoglycan biglycan (BGN) is thought to inhibit thrombin activity by activation of heparin cofactor II. Hypothesis: Genetic deletion of BGN affects thrombin activity, inflammation and atherosclerosis. Methods:Male mice deficient in apolipoprotein E and BGN (ApoE-/-/Bgn-/0) were compared with their ApoE-/- littermates. Results: In ApoE-/- mice BGN was detected in the plasma and subendothelial matrix of capillaries, arterioles and in atherosclerotic plaques. In line with a role of BGN in balancing thrombin activity, ApoE-/-/Bgn-/0 mice exhibited higher activity of circulating thrombin and greater platelet activation than did ApoE-/- mice. Furthermore, higher concentrations of circulating cytokines in ApoE-/-/Bgn-/0 mice suggested a proinflammatory phenotype. Accordingly, isolated peritoneal macrophages released elevated concentrations of proinflammatory cytokines in response to Toll-like receptor stimulation. Immunohistochemistry and FACS analysis of the aorta demonstrated increased macrophage content in atherosclerotic lesions. Furthermore, ApoE-/-/Bgn-/0 mice exhibited higher aortic plaque burden and larger atherosclerotic lesions at the aortic root. Treatment with the thrombin inhibitor argatroban reversed platelet activation and aortic macrophage accumulation in ApoE-/-/Bgn-/0 mice. Finally, ApoE-/-/Bgn-/0 mice exhibited progressive dilation of the aortic arch, a pathological finding corroborated by markedly reduced collagen fibril density. Conclusions: The present results indicate that BGN plays a previously unappreciated protective role during progression of atherosclerosis by inhibiting thrombin activity and platelet activation, and ultimately macrophage-mediated plaque inflammation.

Abstract P023: Toll-like Receptor 4 Deficiency Reduces Macrophage Mediated Renal Injury in Ang-II Induced Hypertension

Infiltration of macrophages has been amply demonstrated in the kidney of humans and experimental models of hypertension however, its role in tissue injury and repair remains unclear. Furthermore, recent studies implicate upregulation of toll-like receptor 4 (TLR4) and inflammation in hypertension and vascular remodeling. Previously, we observed that C3H/HeJ mice lacking functional TLR4 were hyporesponsive to Ang-II induced hypertension and renal damage. In this study, we hypothesized that TLR4 deficiency inhibits macrophage mediated inflammation and TGF-β-induced fibroblast accumulation to reduce renovascular injury and fibrosis. Male C3H/HeOuJ (functional TLR4) and C3H/HeJ mice (dysfunctional TLR4) aged 8-10 weeks underwent subcutaneous osmotic pump insertion for infusion of Angiotensin-II (Ang-II) at 1000 ng/kg/min or vehicle (0.9% saline) for 4 weeks. Blood pressure (BP) was measured twice weekly by non-invasive tail-cuff plethysmography. Ang-II treatment increased mean BP in both groups but to a greater degree in C3H/HeOuJ mice compared to C3H/HeJ (138.6± 6.3 vs. 103.4±7.2 mm/Hg). Plasma creatinine in saline treated C3H/HeOuJ and C3H/HeJ mice was normal (0.37±0.026 and 0.32±0.023 mg/dL respectively) in contrast to ang-II treated mice (1.34±0.075 vs. 0.78±0.052 mg/dL). Flow cytometry revealed increased F4/80+ and CD40+ cells indicating pro-inflammatory M1 macrophage in Ang-II treated C3H/HeOuJ compared to C3H/HeJ mice. Similarly, Ang-II increased the mRNA and protein expression of CD40, MCP-1, TNF-α and TGF-β in C3H/HeOuJ compared to C3H/HeJ mice. Immunostaining revealed increased colocalization of CD45 and procollagen-1 in renal sections of C3H/HeOuJ mice suggesting increased accumulation of myeloid fibroblasts than in C3H/HeJ mice. The expression of α-smooth muscle actin an indicator of myofibroblast and extracellular matrix proteins, collagen type I and fibronectin (Western blot and immunostaining) was increased in C3H/HeOuJ compared to C3H/HeJ mice. In conclusion, our data suggests that in C3H/HeJ mice, deficiency of functional TLR4 reduces macrophage activation and inflammation which in turn decreases TGF-β-induced renal fibrotic response in Ang-II induced hypertension.

Suppression of Adaptive Immune Cell Activation Does Not Alter Innate Immune Adipose Inflammation or Insulin Resistance in Obesity.

Obesity-induced inflammation in visceral adipose tissue (VAT) is a major contributor to insulin resistance and type 2 diabetes. Whereas innate immune cells, notably macrophages, contribute to visceral adipose tissue (VAT) inflammation and insulin resistance, the role of adaptive immunity is less well defined. To address this critical gap, we used a model in which endogenous activation of T cells was suppressed in obese mice by blocking MyD88-mediated maturation of CD11c+ antigen-presenting cells. VAT CD11c+ cells from Cd11cCre + Myd88 fl/fl vs. control Myd88 fl/fl mice were defective in activating T cells in vitro, and VAT T and B cell activation was markedly reduced in Cd11cCre + Myd88 fl/fl obese mice. However, neither macrophage-mediated VAT inflammation nor systemic inflammation were altered in Cd11cCre + Myd88 fl/fl mice, thereby enabling a focused analysis on adaptive immunity. Unexpectedly, fasting blood glucose, plasma insulin, and the glucose response to glucose and insulin were completely unaltered in Cd11cCre + Myd88 fl/fl vs. control obese mice. Thus, CD11c+ cells activate VAT T and B cells in obese mice, but suppression of this process does not have a discernible effect on macrophage-mediated VAT inflammation or systemic glucose homeostasis.

An injectable hydrogel derived from small intestine submucosa as a stem cell carrier.

Small intestine submucosa (SIS) is used as an attractive biomaterial in the field of regenerative medicine because it possesses numerous bioactive factors such as cell adhesion proteins, growth factors, and glycosaminoglycans. In most cases, SIS has been used as sheet and sponge-like forms. The goal of present study was to prepare an injectable SIS hydrogel and to examine the feasibility as an in vivo stem cell carrier of injectable SIS hydrogel form. The rat muscle-derived stem cells (rMDSCs) were simply incorporated into SIS hydrogel by mixing. The SIS hydrogel showed a porous microstructure. In vitro cytotoxicity test, attachment, and proliferation of rMDSCs on the SIS hydrogel were higher than those on tissue culture dishes. Then rMDSCs-loaded SIS hydrogel was subcutaneously injected into rats. The in vivo formed rMDSCs-loaded SIS hydrogel implant induced the ingrowth of new vessels and cell proliferation. In addition, SIS and rMDSCs suppressed macrophage-mediated inflammation. In conclusion, we confirmed that injectable SIS hydrogel could serve as a minimally invasive stem cell carrier. © 2015 Wiley Periodicals, Inc. J Biomed Mater Res Part B: Appl Biomater, 104B: 1544-1550, 2016.

Aldosterone induced macrophage movement through IL-6/Rho-ROCK pathway

Background/aim: Primary aldosteronism (PA) patients are correlated to higher cardiovascular events, endothelial function, and altered cardiac structure. Macrophage-mediated low grade inflammation plays important role of aldosterone-induced myocardial and vascular fibrosis. However, theeffect of aldosterone on macrophage movementis still unclear. Methods: We investigated the effect of aldosterone on macrophage movement and its mechanism. We used THP-1 as cell model of macrophage, and transwell assay to evaluate the migration and infiltration of cell. Two periods (16 hour and 48 hour) wereused to evaluate the migration and infiltration of macrophage. Results: Aldosterone increased THP-1 migration and infiltrationsince 10−8 M at 48 hours but not 16 hours. Aldosterone induced both galectin-3 and IL-6 production in THP-1 cell. In contrast, aldosterone does not affect the production of matrix metalloproteinase-2 and tissue inhibitor of metalloproteinase-1in THP-1. The increased migration and infiltrationby aldosterone was inhibited partially by IL-6 siRNA but not galectin-3 siRNA. Aldosterone also induced Rho-ROCK up-regulation but also partially inhibited by IL-6 siRNA. The aldosterone induced cell migration and infiltration was blocked by Y27632 (Rho-ROCK inhibitor). Conclusions: Aldosterone increased microphage migration and infiltration. The process was partially involved IL-6/Rho-ROCK pathway.

Vasoactive intestinal peptide suppresses macrophage-mediated inflammation by downregulating interleukin-17A expression via PKA- and PKC-dependent pathways.

Interleukin (IL)-17A is a pro-inflammatory cytokine that markedly enhances inflammatory responses in the lungs by recruiting neutrophils and interacting with other pro-inflammatory mediators. Reducing the expression of IL-17A could attenuate inflammation in the lungs. However, whether VIP exerts its anti-inflammatory effects by regulating the expression of IL-17A has remained unclear. Here, we show that there is a remarkable increase of IL-17A in bronchoalveolar lavage fluid (BALF) and lung tissue of mice with acute lung injury (ALI). Moreover, lipopolysaccharides (LPS) stimulated elevated expression of IL-17A, which was evident by the enhanced levels of mRNA and protein observed. Furthermore, we also found that VIP inhibited LPS-mediated IL-17A expression in a time- and dose-dependent manner in an in vitro model of ALI and that this process might be mediated via the phosphokinase A (PKA) and phosphokinase C (PKC) pathways. Taken together, our results demonstrated that VIP might be an effective protector during ALI by suppressing IL-17A expression.

The PPAR-γ antagonist GW9662 elicits differentiation of M2c-like cells and upregulation of the MerTK/Gas6 axis: a key role for PPAR-γ in human macrophage polarization.

BackgroundThe nuclear receptors PPAR-γ and LXRs regulate macrophage lipid metabolism and macrophage mediated inflammation. We examined the influence of these molecules on macrophage alternative activation, with particular focus on differentiation of “M2c” anti-inflammatory cells.MethodsWe cultured human monocytes in M0, M1, M2a or M2c macrophage differentiating conditions, in the presence or absence of PPAR-γ and LXR ligands. Flow cytometry was used to analyze membrane expression of phenotypic markers. Basal and LPS-stimulated production of soluble mediators was measured by ELISA. Efferocytosis assays were performed by coincubating monocytes/macrophages with apoptotic neutrophils.ResultsWe found that PPAR-γ inhibition, using the PPAR-γ antagonist GW9662, elicits differentiation of M2c-like (CD206+ CD163+ CD16+) cells and upregulation of the MerTK/Gas6 axis. Exposure of differentiating macrophages to IFN-γ, GM-CSF or LPS (M1 conditions), however, hampers GW9662 induction of MerTK and Gas6. When macrophages are differentiated with IL-4 (M2a conditions), addition of GW9662 results into an M2a (CD206+ CD209+ CD163− MerTK−) to M2c (CD206high CD209− CD163+ MerTK+) polarization shift. Conversely, in the presence of dexamethasone (M2c conditions), the PPAR-γ agonist rosiglitazone attenuates CD163 and MerTK upregulation. The LXR agonist T0901317 induces MerTK independently of M2c polarization; indeed, CD206, CD163 and CD16 are downregulated. GW9662-differentiated M2c-like cells secrete high levels of Gas6 and low amounts of TNF-α and IL-10, mimicking dexamethasone effects in vitro. However, unlike conventional M2c cells, GW9662-differentiated cells do not show enhanced efferocytic ability.ConclusionsOur results provide new insights into the role of PPAR-γ and LXR receptors in human macrophage activation and reveal the existence of different patterns regulating MerTK expression. Unexpectedly, PPAR-γ appears to negatively control the expansion of a discrete subset of M2c-like anti-inflammatory macrophages.

Abstract 693: TLR3 Modulates Inflammation and Lipolysis in Visceral Adipose Tissue in High Fat Diet-fed Mice

Inflammation in insulin sensitive tissues, the visceral adipose tissue (VAT), is a central abnormality in obesity/insulin resistance (IR), with recruitment of innate immune cells such as monocytes into adipose tissue driving the development of glucose and lipoprotein dysregulation. We evaluated the role of Toll like receptor 3 (TLR3) in high fat diet-induced obesity and IR. Wild-type C57BL/6 and TLR3 -/- male mice were fed a high fat diet for 15 weeks. High fat feeding resulted in increased TLR3 expression in VAT. TLR3 deficiency attenuated the high fat diet-increased body weight, fasting blood glucose, whole body IR and impaired glucose tolerance. Morphologically, high fat diet induced adiposity and enlarged adipocyte area in VAT, which were attenuated in TLR3 -/- mice. Functionally, high fat diet induced dysregulation of adipocytokines such as downregulation of adiponectin and resistin, upregulation of leptin in VAT, with the disturbance of adiponectin and leptin was corrected in TLR3-/- mice. In addition, high fat diet inhibited insulin pathway, accompanied with decreased phosphorylation of AMPK and lowered expression of lipolysis-related enzymes such as HSL and ATGL, both at the mRNA levels and protein levels, all of which was corrected by TLR3 deficiency. Finally, TLR3 deletion suppressed the high fat feeding-mediated macrophage polarization, evidenced by increased type M1 macrophage (F4/80+/CD11c+/CD206-) infiltration and upregulation of M1 genes such as IL-6 and TNFα. TLR3 modulates high fat diet-induced IR and obesity by suppressing M1 macrophage-mediated VAT inflammation, facilitating secretion of adipocyte-derived hormones, thus enhanced AMPK activity and adipose lipolysis. These findings provide new mechanistic links between dietary factors-mediated IR and associated abnormalities in lipid metabolism and adipose inflammation.

&lt;i&gt;Aralia elata&lt;/i&gt; (Miquel) Seemann Suppresses Inflammatory Responses in Macrophage Cell by Regulation of NF-kappa B Signalling

Purpose: To investigate the antioxidant and anti-inflammatory effects of Aralia elata extract (AEE) in lipopolysaccharide (LPS)-stimulated Raw264.7 cells. Methods : Antioxidant activity was measured using 1, 1-diphenyl-2-picryl-hydrazyl (DPPH) radical scavenging assay. Cell viability was evaluated using 3-(4, 5-dimethylthiazol-2-yl)-2, 5- diphenyltetrazolium bromide (MTT) assay. RAW264.7 cells were stimulated with LPS to study protein expression and production of inflammatory mediators, determined by Western blot analysis. Results: AEE significantly inhibited DPPH-generated free radicals showing maximum inhibition at 40 μg/mL (p < 0.001). AEE alone did not exhibit any signs of cytotoxicity to RAW264.7 cells up to 200 μg/mL concentration. The LPS-induced increase in the production of nitric oxide was concentrationdependently suppressed with half-maximal concentration (IC 50 ) of 91.5 ug/ mL of AEE (p < 0.05 at 10 μg/mL, p < 0.01 at 20 μg/mL and p < 0.001 at 40 μg/mL, respectively). AEE also inhibited dosedependently the LPS-induced increase in inducible nitric oxide synthase (iNOS) and cyclooxygenase-2 (COX-2) expressions with IC 50 of 72.5 ug/ mL. Furthermore, the production of pro-inflammatory cytokines, viz, tumor necrosis factor-α by LPS-stimulation in RAW264.7 cells was inhibited dosedependently with IC 50 of 34.9 ug/ mL by AEE pretreatment. Mechanistic studies revealed that AEE acts by regulation of nuclear factor kappa-B signaling pathway in LPS-stimulated RAW264.7 cells. Conclusion: This study shows, for the first time, that AEE possesses antioxidant and anti-inflammatory effects and can be developed as a potential therapeutic agent for ameliorating macrophage-mediated inflammation. Keywords : Aralia elata , Anti-inflammatory activity, Macrophage cells, Inducible nitric oxide synthase, nuclear factor kappa-B signaling

M2 Macrophage Polarization Mediates Anti-inflammatory Effects of Endothelial Nitric Oxide Signaling.

Endothelial nitric oxide (NO) signaling plays a physiological role in limiting obesity-associated insulin resistance and inflammation. This study was undertaken to investigate whether this NO effect involves polarization of macrophages toward an anti-inflammatory M2 phenotype. Mice with transgenic endothelial NO synthase overexpression were protected against high-fat diet (HFD)-induced hepatic inflammation and insulin resistance, and this effect was associated with reduced proinflammatory M1 and increased anti-inflammatory M2 activation of Kupffer cells. In cell culture studies, exposure of macrophages to endothelial NO similarly reduced inflammatory (M1) and increased anti-inflammatory (M2) gene expression. Similar effects were induced by macrophage overexpression of vasodilator-stimulated phosphoprotein (VASP), a key downstream mediator of intracellular NO signaling. Conversely, VASP deficiency induced proinflammatory M1 macrophage activation, and the transplantation of bone marrow from VASP-deficient donor mice into normal recipients caused hepatic inflammation and insulin resistance resembling that induced in normal mice by consumption of an HFD. These data suggest that proinflammatory macrophage M1 activation and macrophage-mediated inflammation are tonically inhibited by NO → VASP signal transduction, and that reduced NO → VASP signaling is involved in the effect of HFD feeding to induce M1 activation of Kupffer cells and associated hepatic inflammation. Our data implicate endothelial NO → VASP signaling as a physiological determinant of macrophage polarization and show that signaling via this pathway is required to prevent hepatic inflammation and insulin resistance.