cAMP modulates macrophage development by suppressing M-CSF-induced MAPKs activation

M-CSF is a key cytokine in macrophage development by inducing MAPKs activation, and cAMP can inhibit MAPKs activation induced by inflammatory stimuli. To explore the effects of cAMP on M-CSF-induced MAPKs activation and on macrophage development, the model of bone marrow-derived murine macrophages （BMMs） was used. The effects of cAMP on M-CSF-induced MAPKs activation were analyzed by Western blotting assay, and the effects of cAMP on CD14 and F4/80 expression during macrophage development were examined by FACS analysis. Macrophage morphology showed the successful establishment of the model of macrophage development. Western blotting assay revealed that M-CSF activated ERK, JNK and p38 in both mature and immature macrophages, and cAMP inhibited M-CSF-induced ERK, JNK and p38 activation in a time-dependent manner. FACS analysis revealed that macrophage development was impaired with cAMP pretreatment. In conclusion, cAMP modulates macrophage development by suppressing M-CSF-induced MAPKs activation. Cellular ＆ Molecular Immunology.     

PYNOD对LPS活化的BV2小胶质细胞炎症因子释放的影响

 目的:观察PYNOD对LPS活化的BV2小胶质细胞炎症因子释放的影响。方法: 将表达PYNOD的重组质粒pEGFP-C2-PYNOD瞬时转染BV2细胞后,加入LPS作用24 h,Griess 法检测一氧化氮（nitric oxide, NO）的释放,实时荧光定量PCR（real-time PCR）检测诱导型一氧化氮合酶（inducible NO synthase, iNOS）和白细胞介素-1β（interleukin-1β,IL-1β）mRNA的表达,此外Western blotting和ELISA法检测iNOS和IL-1β的蛋白表达。结果: 转染PYNOD重组质粒能显著抑制LPS诱导的BV2小胶质细胞炎症因子NO的释放（P＜0.05）。Real-time PCR证实PYNOD可抑制iNOS和 IL-1β 的mRNA表达,差异有统计学意义（P＜0.05）。ELISA和Western blotting证实PYNOD可下调iNOS和 IL-1β 蛋白的表达（P＜0.05）。结论: PYNOD蛋白可以在转录水平和翻译水平显著抑制LPS刺激的BV2小胶质细胞活化产生的炎症反应。     

P物质通过MAPKs信号通路缓解早产大鼠高氧肺损伤

目的探索神经肽P物质(SP)对高氧暴露早产鼠肺组织的作用及与丝裂原活化蛋白激酶家族(MAPKs)信号传导机制的关系。方法将早产鼠随机分为常氧组、常氧+SP干预组、高氧组和高氧+SP干预组;实验第3、7及14天时,观察肺组织病理改变;测肺湿/干重;放免法测肺组织中SP的含量;分别采用硫代巴比妥酸法、亚硝酸盐法和二硝基苯甲酸法检测丙二醛(MDA)、过氧化物歧化酶(SOD)和谷胱甘肽-过氧化物酶(GSH-Px)水平;TUNEL法检测肺组织凋亡细胞;Western blot法检测MAPKs蛋白含量。结果高氧暴露后肺组织损伤,湿/干重增加,SP含量降低,MDA含量增加SOD和GSH-Px含量降低(P0.05),凋亡细胞增多,并随着高氧暴露时间延长改变越明显,SP干预后肺损伤有所改善,湿/干重回降,MDA含量降低,SOD、GSH-Px含量增加(P0.05),TUNEL阳性细胞减少;高氧组细胞外信号调节蛋白激酶(ERK)、c-Jun氨基末端激酶(JNK)及P38激酶(P38)蛋白表达明显高于空气组(P0.05),且随时间的延长变化增大,而SP干预后ERK蛋白表达越加增强(P0.05),JNK、P38蛋白表达明显减弱(P0.05)。结论高氧可引起早产鼠肺组织氧化损伤;SP可通过干预MAPKs的表达从而保护氧化应激状态下肺组织。     

Cinobufagin exerts anti-proliferative and pro-apoptotic effects through the modulation ROS-mediated MAPKs signaling pathway

Abstract

Cinobufagin (CBG) is a cardiotoxic bufanolide steroid secreted by the skin and parotid venom glands of the Asiatic toad Bufo bufo gargarizans (called Chan-Su). Although CBG is known to exhibit anti-cancer activities, very little is known about its potential mechanism(s) of action. In this study, we investigated whether CBG mediates its effect through the modulation of the mitogen-activated protein kinases (MAPKs) signaling pathway in human multiple myeloma (MM) U266 cells. We found that CBG caused the significant activation of ERK, JNK and p38 MAPK in U266 cells. CBG showed much higher cytotoxicity against U266 cells as compared to peripheral blood mononuclear cells (PBMC). Induction of CBG increased reactive oxygen species (ROS) generation from mitochondria, which is associated with the induction of apoptosis as characterized by increased sub-G1 DNA contents of cell cycle, positive Annexin V binding, activation of caspase-3 and cleavage of PARP. Inhibition of ROS generation by N-acetyl-l-cysteine (NAC) significantly prevented CBG-induced ERK, JNK and p38 MAPK activation and apoptosis. CBG also down-regulated the expression of various downstream gene products that mediate cell proliferation, survival, angiogenesis and metastasis. Interestingly, ERK, JNK and p38MAPK pharmacological inhibitors blocked CBG-induced MAPKs activation and ERK inhibitor (PD98059) also prevented the CBG-induced caspase-3 activation and PARP cleavage in U266 cells. Taken together, these findings suggest that CBG can act as a potent anticancer agent against MM and possibly exerts its effects through the ROS-mediated activation of ERK, JNK and p38 MAPK leading to the activation of caspase-3 in U266 cells.     

经CR2激活MAPKs与抑制HIV-gp160处理的CD4+细胞增殖

目的建立CR2稳定表达的HOS-CR2、HOS-CD4CR2细胞系,研究细胞中MAPKs的活化和细胞增殖变化,阐明经CR2的信号传导、CR2的表达对HIV-gp160等所致的CD4+细胞的影响.方法用哺乳动物细胞稳定转染法建立稳定表达CR2的HOS-CR2、HOS-CD4CR2细胞系,细胞经磁珠阳性纯化后,用FACS和Western blot对CR2的表达进行鉴定.用PMA、10% NHS、HIV-gp160等处理细胞后,经Western blot检测细胞的MAPKs的活化水平;用Cell Titer 96 Aqueous One Solution Reagent检测和分析处理后细胞的增殖差异.结果 FACS检测和统计学方法分析结果表明CR2表达的阳性率高达96%以上;Western blot结果显示所建HOS-CR2、HOS-CD4CR2细胞系的CR2的表达水平与Raji细胞相近;细胞经PMA、预先激活补体的10% NHS处理细胞,发现在处理10 min时ERK、JNK、P38的活化达高峰;PD98059、Wortmanin和抗-CR2能阻断ERK、JNK、P38的活化.HIV-gp160、预先激活补体的10% NHS能相应激活HOS-CR2, HOS-CD4 和HOS-CD4CR2细胞ERK、JNK、P38,这些激活均可被相应的抗体所阻断;细胞增殖实验结果显示HIV-gp160能抑制细胞增殖(P＜0.01),CR2的表达能加重HIV-gp160所致的细胞增殖抑制(P＜0.01).结论磁珠阳性纯化法可浓集稳定表达靶基因的细胞,提高阳性率和表达水平.刺激因素可经由CR2激活MAPKs,CR2的表达促进HIV-gp160所诱导的细胞死亡,与HIV感染所致的CD4+细胞缺损密切相关,CD4+细胞缺失机制的阐明将有助于AIDS致病机制和防治策略的研究.     

T-cell immunoglobulin- and mucin-domain-containing molecule-4 maintains adipose tissue homeostasis by orchestrating M2 macrophage polarization via nuclear factor kappa B pathway

Obesity is generally associated with low-grade inflammation. Adipose tissue macrophages (ATMs) orchestrate metabolic inflammation. The classical (M1-like) or alternative (M2-like) activation of ATMs is functionally coupled with the metabolic status of fat tissues. It has been found that T-cell immunoglobulin- and mucin-domain-containing molecule-4 (Tim-4) inhibits inflammation by regulating macrophages. However, the exact role of Tim-4 in macrophage polarization and obesity remains unknown. Here, we identified Tim-4 as a critical switch governing macrophage M1/M2 polarization and energy homeostasis. Tim-4 deletion led to spontaneous obesity in elder mice and promoted obesity severity of db/db mice. Obesity microenvironment enhanced the expression of Tim-4 in white adipose tissue and ATMs. In vitro, we detected an increase in M1-like cells and decrease in M2-like cells in both peritoneal macrophages and bone marrow-derived macrophages from Tim-4 knockout mice. Mechanistically, we demonstrated that Tim-4 promoted M2-like macrophages polarization via suppressing nuclear factor kappa B (NF-κB) signaling pathway. In addition, we found that Tim-4 promoted TLR4 internalization, which might contribute to regulation of NF-κB signaling. Collectively, these results indicated that Tim-4 maintained adipose tissue homeostasis by regulating macrophage polarization via NF-κB pathway, which would provide a new target for obesity intervention.     

Adenosine receptor activation promotes macrophage class switching from LPS-induced acute inflammatory M1 to anti-inflammatory M2 phenotype

Lipopolysaccharide induced monocytes/macrophages exhibit a pro-inflammatory M1 phenotype. Elevated levels of the purine nucleoside adenosine play a major role in this response. The role of adenosine receptor modulation in directing the macrophage phenotype switch from proinflammatory classically activated M1 phenotype to an anti-inflammatory alternatively activated M2 phenotype is investigated in this study. The mouse macrophage cell line RAW 264.7 was used as the experimental model and stimulated with Lipopolysaccharide (LPS) at a dose of 1 μg/ml. Adenosine receptors were activated by treating cells with the receptor agonist NECA (1 μM). Adenosine receptor stimulation in macrophages is found to suppress LPS-induced production of proinflammatory mediators (pro-inflammatory cytokines, Reactive Oxygen Species and nitrite levels). M1 marker CD38 (Cluster of Differentiation 38) and CD83 (Cluster of Differentiation 83) were significantly decreased while M2 markers Th2 cytokines, Arginase, TIMP (Tissue Inhibitor of Metalloproteinases) and CD206 (Cluster of Differentiation 206) exhibited an increase. Hence from our study we observed that activation of adenosine receptors can program the macrophages from a pro-inflammatory classically activated M1 phenotype to an anti-inflammatory alternatively activated M2 phenotype. We report the significance and a time course profile of phenotype switching by receptor activation. Adenosine receptor targeting may be explored as a therapeutic intervention strategy in addressing acute inflammation.     

Anti-inflammatory effects of isoacteoside from Abeliophyllum distichum

Abstract

Isoacteoside, a dihydroxypheynylethyl glycoside, is a major bioactive component of Abeliophyllum distichum (White Forsythia) which is a deciduous shrub native to the south and central areas of Korea. The present study is designed to evaluate the anti-inflammatory activities and underlying mechanisms of isoacteoside in human mast cell line, HMC-1 cells. We isolated isoacteoside from A. distichum. The anti-inflammatory effect of isoacteoside was investigated in HMC-1 cells by studying the following markers: phorbol 12-myristate 13-acetate and calcium ionophore A23187 (PMACI)-induced interleukin (IL)-1β, IL-6, IL-8, and tumor necrosis factor alpha (TNF-α) secretion and mRNA expression by ELISA and RT-PCR, respectively. In addition, mechanism related to anti-inflammatory was investigated by Western blotting. Isoacteoside significantly suppressed the production and mRNA expression of proinflammatory cytokines including IL-1β, IL-6, IL-8 and TNF-α in PMACI-stimulated HMC-1 cells without cytotoxicity. It was found that anti-inflammatory effects of isoacteoside are mediated by action on caspase-1, mitogen-activated protein kinases (c-Jun N-terminal kinase, p38, extracellular signal-regulated protein kinase) and nuclear factor-kappa B pathways. Taken together, the present findings provide new insights that isoacteoside may be a promising anti-inflammatory agent for inflammatory disorders.     

Fluticasone propionate-induced regulation of the balance within macrophage subpopulations

In asthma, treatment with inhaled corticosteroids reduces chronic peribronchial inflammation and restores the balance within macrophage subpopulations. This study investigates whether corticosteroids can regulate monocyte differentiation in vitro and thereby influence the balance of functionally distinct macrophages. Graded doses of fluticasone propionate (FP) were added to cultures of normal peripheral blood monocytes in the presence or absence of IL-4. Cells were harvested after 7 days' culture. Double immunofluorescence studies were performed on cytospins of differentiated macrophages using the MoAbs RFD1 and RFD7 to distinguish inductive and suppressive macrophages by their respective phenotypes. Macrophage function was determined by quantifying allostimulation in a mixed leucocyte reaction and by measuring tumour necrosis factor-alpha (TNF-alpha) production. FP reduced the number of mature cells with a D1+ antigen-presenting phenotype and up-regulated the development of cells with the D1/D7+ and D7+ phenotypes. Functionally, this was associated with reduced stimulation of T cell proliferation in a mixed leucocyte reaction (MLR). Fluticasone also reversed the increase in both D1+ expression and TNF-alpha production induced by IL-4. The effect of FP persisted for 24 h after removal of FP from the culture medium. These results suggest that FP treatment of asthmatics may have a direct beneficial effect by normalizing the macrophage subset imbalance that contributes to the chronic peribronchial inflammation present in this condition.     

Phosphatase regulation of macrophage activation

Macrophages are innate immune cells that play critical roles in tissue homeostasis and the immune response to invading pathogens or tumor cells. A hallmark of macrophages is their “plasticity,” that is, their ability to respond to cues in their local microenvironment and adapt their activation state or phenotype to mount an appropriate response. During the inflammatory response, macrophages may be required to mount a profound anti-bacterial or anti-tumor response, an anti-inflammatory response, an anti-parasitic response, or a wound healing response. To do so, macrophages express cell surface receptors for growth factors, chemokines and cytokines, as well pathogen and danger associated molecular patterns. Downstream of these cell surface receptors, cell signalling cascades are activated and deactivated by reversible and competing activities of lipid and protein kinases and phosphatases. While kinases drive the activation of cell signalling pathways critical for macrophage activation, the strength and duration of the signalling is regulated by phosphatases. Hence, gene knockout mouse models have revealed critical roles for lipid and protein phosphatases in macrophage activation. Herein, we describe our current understanding and the key roles of specific cellular phosphatases in the regulation of the quality of macrophage polarization as well as the quantity of cytokines produced by activated macrophages.     

二甲双胍激活AMPK通路减轻饱和脂肪酸诱导的RAW264.7巨噬细胞炎性反应

目的 观察二甲双胍对饱和脂肪酸(SFA)诱导的RAW264.7巨噬细胞炎性反应中炎性因子的影响及探讨其可能机制.方法 SFA干预RAW264.7巨噬细胞建立体外炎性反应模型;实验分为对照组、SFA干预组、二甲双胍+ SFA干预组、AMPK抑制剂Compound C+二甲双胍+SFA干预组;实时定量PCR检测TNF-α和IL-6 mRNA的表达,ELISA法检测TNF-α和IL-6蛋白的分泌,Western blot分析腺苷酸活化蛋白激酶(AMPK)的磷酸化水平.结果 与对照组比较,SFA干预组RAW264.7巨噬细胞TNF-α、IL-6 mRNA表达及蛋白分泌水平均显著升高(P＜0.05);与SFA组比较,二甲双胍+SFA干预组TNF-α、IL-6 mRNA表达水平及蛋白分泌水平均降低(P＜0.05),而细胞AMPK的磷酸化水平增强(P＜0.05);与二甲双胍+SFA干预组比,Compound C+二甲双胍+SFA干预组TNF-α、IL-6 mRNA表达及蛋白分泌水平均升高,AMPK磷酸化水平降低(P＜0.05).结论 二甲双胍激活AMPK降低饱和脂肪酸诱导的RAW264.7巨噬细胞炎性因子TNF-α、IL-6的分泌.     

Anti-inflammatory and anti-oxidant effects of combination between sulforaphane and acetaminophen in LPS-stimulated RAW 264.7 macrophage cells

Objectives: Accumulating evidence indicates that combination of therapeutic agents may increase their pharmacological properties with fewer undesired side effects. Acetaminophen (APAP) has been widely used to treat pain and fever in many countries. However, APAP only possesses a weak anti-inflammatory property at therapeutic dose, and exhibits hepatotoxicity at high dose. On other hand, sulforaphane (SFN) has been well-known as a potential anti-inflammatory and antioxidant agent. In this study, we investigated the anti-inflammatory and antioxidant effects of combination between APAP and SFN in LPS-stimulated RAW 264.7 macrophage cells.

Methods: Nitric oxide (NO) assay was determined using the Griess assay. Reactive oxygen species (ROS) formation was measured using an ROS-sensitive fluorescence indicator, DCFH-DA. The protein expression was determined by western blot analysis.

Results: Our results showed that the combination of SFN and APAP exhibited an inhibitory effect on inflammatory markers such as NO, iNOS, COX-2, and IL-1β, and this effect was more pronounced than the compound was used alone. In addition, the combination of SFN and APAP at low doses decreased intracellular ROS formation and increased the protein levels of CAT, GPx, Nrf2, NQO1, and HO-1, which were much better than APAP alone and were equivalent to SFN at full dose.

Conclusions: Our findings suggest that the combination of APAP and SFN enhanced anti-inflammatory and anti-oxidant activities in stimulated macrophages, which provide an important rationale to utilize drug and food in combination for prevention and/or treatment inflammation-related diseases.     

Anti-inflammatory and antipyretic effects of boldine

Boldine, and antioxidant alkaloid isolated fromPeumus boldus, exhibits a dose-dependent anti-inflammatory activity in the carrageenan-induced guinea pig paw edema test with an oral ED₅₀ of 34 mg/kg. Boldine also reduces bacterial pyrogen-induced hyperthermia in rabbits to an extent which varied between 51% and 98% at a dose of 60 mg/kg p.o.In vitro studies carried out in rat aortal rings revealed that boldine is an effective inhibitor of prostaglandin biosynthesis, promoting 53% inhibition at 75 M. The latterin vitro effect may be mechanistically linked to the anti-inflammatory and antipyretic effects of boldine exertedin vivo.     

DAPT阻断Notch通路对动脉粥样硬化型缺血性脑卒中大鼠的保护作用

目的:探讨DAPT阻断Notch通路对动脉粥样硬化(AS)基础上缺血性脑卒中模型大鼠的保护作用。方法:24只健康雄性SD大鼠随机分为对照(control,n=6)组和模型(n=18)组,后者建立AS模型,然后再随机分为AS假手术(AS-sham)组、AS缺血性脑卒中(AS-ischemia)组以及AS缺血性脑卒中DAPT处理(AS-ischemiaDAPT)组,每组各6只;HE染色观察大鼠颈动脉组织病理变化,全自动生化分析仪检测大鼠血液甘油三酯(TG)、总胆固醇(TC)、高密度脂蛋白胆固醇(HDL-C)和低密度脂蛋白胆固醇(LDL-C)水平;酶联免疫反应检测大鼠血清炎症因子白细胞介素6(IL-6)和肿瘤坏死因子α(TNF-α)的水平;Western blot检测大鼠颈动脉组织中Notch1和Hes1以及脑组织中核因子κB (NF-κB)和Toll样受体4 (TLR4)的蛋白表达水平。结果:Notch信号通路抑制剂DAPT能显著缓解AS大鼠动脉内膜层增厚以及血管狭窄,减少AS斑块形成。与AS-ischemia组大鼠相比,AS-ischemia-DAPT组大鼠血脂和炎症因子水平均显著降低(P 0. 05),且颈动脉组织中Notch1和Hes1以及脑组织中NF-κB和TLR4的蛋白表达也显著降低(P 0. 05)。结论:DAPT阻断Notch通路不仅可以改善AS缺血性脑卒中大鼠的血脂水平,还可以抑制血清炎症因子释放以及NF-κB/TLR4通路的激活,从而改善AS缺血性脑卒中的症状。     

Fibrinogen drives dystrophic muscle fibrosis via a TGFbeta/alternative macrophage activation pathway

In the fatal degenerative Duchenne muscular dystrophy (DMD), skeletal muscle is progressively replaced by fibrotic tissue. Here, we show that fibrinogen accumulates in dystrophic muscles of DMD patients and mdx mice. Genetic loss or pharmacological depletion of fibrinogen in these mice reduced fibrosis and dystrophy progression. Our results demonstrate that fibrinogen-Mac-1 receptor binding, through induction of IL-1beta, drives the synthesis of transforming growth factor-beta (TGFbeta) by mdx macrophages, which in turn induces collagen production in mdx fibroblasts. Fibrinogen-produced TGFbeta further amplifies collagen accumulation through activation of profibrotic alternatively activated macrophages. Fibrinogen, by engaging its alphavbeta3 receptor on fibroblasts, also directly promotes collagen synthesis. These data unveil a profibrotic role of fibrinogen deposition in muscle dystrophy.     

Interferon gamma suppresses glucocorticoid augmentation of macrophage clearance of apoptotic cells

One of beneficial effects of glucocorticoids (GC) in inflammation may be the augmentation of macrophages' capacity for phagocytosis of apoptotic cells, a process that has a central role in resolution of inflammation. Here we define the phenotype of GC-treated monocyte-derived macrophages, comparing to IFN-gamma-treated and IL-4-treated monocyte-derived macrophages and combinatorial treatment. Our data indicate that the cytokine microenvironment at an inflammatory site will critically determine monocyte functional capacity following treatment with GC. In particular, whilst GC exert dominant regulatory effects over IFN-gamma in terms of cell surface receptor repertoire and morphology, the acquisition of a macrophage capacity for clearance of apoptotic cells is prevented by combined treatment. In terms of mechanism, GC augmentation of phagocytosis was reversed even when monocytes were pre-incubated with GC for the first 24 h of culture, a period that is critical for induction of a highly phagocytic macrophage phenotype. These findings have important implications for the effectiveness of GC in promoting acquisition of a pro-phagocytic macrophage phenotype in inflammatory diseases associated with high levels of IFN-gamma     

Anti-inflammatory effects of Hwang-Heuk-San,a traditional Korean herbal formulation,on lipopolysaccharide-stimulated murine macrophages

Background

Hwang-Heuk-San (HHS), a Korean traditional herbal formula comprising four medicinal herbs, has been used to treat patients with inflammation syndromes and digestive tract cancer for hundreds of years; however, its anti-inflammatory potential is poorly understood. The aim of the present study was to investigate the anti-inflammatory effects of HHS using a lipopolysaccharide (LPS)-activated RAW 264.7 macrophage model.

Methods

The inhibitory effects of HHS on LPS-induced nitric oxide (NO), interleukin-1β (IL-1β) and tumor necrosis factor-α (TNF-α) production were examined using Griess reagent and enzyme-linked immunosorbent assay (ELISA) detection kits. The effects of HHS on the expression of inducible NO synthase (iNOS), IL-1β and TNF-α, their upstream signal proteins, including nuclear factor κB (NF-κB), mitogen-activated protein kinases (MAPKs), and activator protein (AP-1), were also investigated.

Results

A noncytotoxic concentration of HHS significantly reduced the production of NO, IL-1β and TNF-α in LPS-stimulated RAW 264.7 cells, which was correlated with reduced expression of iNOS, IL-1β and TNF-α at the mRNA and protein levels. HHS efficiently blocked the phosphorylation of MAPKs, especially that of extracellular signal-regulated kinase (ERK) and c-Jun NH2-terminal kinase (JNK) but not that of the p38 MAPK. The reduced production of inflammatory molecules by HHS was followed by decreased activity of NF-κB and AP-1.

Conclusions

These results suggest that HHS may offer therapeutic potential for treating inflammatory diseases accompanied by macrophage activation.