Macrophages produce IL-33 by activating MAPK signaling pathway during RSV infection

It has been reported that RSV infection can enhance IL-33 production in lung macrophages. However, little is known about specific signaling pathways for activation of macrophages during RSV infection. In the present study, by using real-time RT-PCR as well as western blot assay, it became clear that RSV infection can enhance not only the expression of mRNAs for MAPK molecules (including p38, JNK1/2, and ERK1/2), but also the levels of MAPK proteins in lung macrophages as well as RAW264.7 cells. Furthermore, infection with RSV resulted in an increased level of phosphorylated MAPK proteins in RAW264.7 cells, suggesting that MAPK signaling pathway may participate in the process of RSV-induced IL-33 secretion by macrophages. In fact, the elevated production of IL-33 in RAW264.7 was attenuated significantly by pretreatment of the cells with special MAPK inhibitor before RSV infection, further confirming the function of MAPKs pathway in RSV-induced IL-33 production in macrophages. In contrast, the expression of NF-κB mRNA as well as the production of NF-κB protein in lung macrophages and RAW264.7 cells was not enhanced markedly after RSV infection. Moreover, RSV infection failed to induce the phosphorylation of NF-κB in RAW264.7 cells, suggesting that NF-κB signaling pathway may be not involved in RSV-induced IL-33 production in macrophages. Conclusion, these results indicate that RSV-induced production of IL-33 in macrophages is dependent on the activation of MAPK signaling pathway.     

Lipoteichoic acid induces nuclear factor-kappaB activation and nitric oxide synthase expression via phosphatidylinositol 3-kinase, Akt, and p38 MAPK in RAW 264.7 macrophages

We previously demonstrated that lipoteichoic acid (LTA) might activate phosphatidylcholine-phospholipase C (PC-PLC) and phosphatidylinositol-phospholipase C (PI-PLC) to induce protein kinase C activation, which in turn initiates nuclear factor-kappaB (NF-kappaB) activation and finally induces inducible nitric oxide synthase (iNOS) expression and nitric oxide (NO) release in RAW 264.7 macrophages. In this study, we further investigated the roles of tyrosine kinase, phosphatidylinositiol 3-kinase (PI3K)/Akt, and p38 mitogen-activated protein kinase (MAPK) in LTA-induced iNOS expression and NO release in RAW 264.7 macrophages. Tyrosine kinase inhibitors (genistein and tyrphostin AG126), PI3K inhibitors (wortmannin and LY 294002), and a p38 MAPK inhibitor (SB 203580) attenuated LTA-induced iNOS expression and NO release in concentration-dependent manners. Treatment of RAW 264.7 macrophages with LTA caused time-dependent activations of Akt and p38 MAPK. The LTA-induced Akt activation was inhibited by wortmannin, LY 294002, genistein, and tyrphostin AG126. The LTA-induced p38 MAPK activation was inhibited by genistein, tyrphostin AG126, wortmannin, LY 294002, and SB 203580. The LTA-induced formation of an NF-kappaB-specific DNA-protein complex in the nucleus was inhibited by wortmannin, LY 294002, genistein, tyrphostin AG126, and SB 203580. Treatment of macrophages with LTA caused an increase in kappaB-luciferase activity, and this effect was inhibited by tyrphostin AG126, wortmannin, LY 294002, the Akt dominant negative mutant (AktDN), and SB 203580. Based on those findings, we suggest that LTA might activate the PI3K/Akt pathway through tyrosine kinase to induce p38 MAPK activation, which in turn initiates NF-kappaB activation, and ultimately induces iNOS expression and NO release in RAW 264.7 macrophages.     

RhoA-Mediated Signaling in Mechanotransduction of Osteoblasts

Osteoblasts play a pivotal role in load-driven bone formation by activating Wnt signaling through a signal from osteocytes as a mechanosensor. Osteoblasts are also sensitive to mechanical stimulation, but the role of RhoA, a small GTPase involved in the regulation of cytoskeleton adhesion complexes, in mechanotransduction of osteoblasts is not completely understood. Using MC3T3-E1 osteoblast-like cells under 1 hr flow treatment at 10 dyn/cm², we examined a hypothesis that RhoA signaling mediates the cellular responses to flow-induced shear stress. To test the hypothesis, we conducted genome-wide pathway analysis and evaluated the role of RhoA in molecular signaling. Activity of RhoA was determined with a RhoA biosensor, which determined the activation state of RhoA based on a fluorescence resonance energy transfer between CFP and YFP fluorophores. A pathway analysis indicated that flow treatment activated phosphoinositide 3-kinase (PI3K) and mitogen-activated protein kinase (MAPK) signaling as well as a circadian regulatory pathway. Western blot analysis revealed that in response to flow treatment phosphorylation of Akt in PI3K signaling and phosphorylation of p38 and ERK1/2 in MAPK signaling were induced. FRET measurement showed that RhoA was activated by flow treatment, and an inhibitor to a Rho kinase significantly reduced flow-induced phosphorylation of p38, ERK1/2, and Akt as well as flow-driven elevation of the mRNA levels of osteopontin and cyclooxygenase-2. Collectively, the result demonstrates that in response to 1 hr flow treatment to MC3T3-E1 cells at 10 dyn/cm², RhoA plays a critical role in activating PI3K and MAPK signaling as well as modulating the circadian regulatory pathway.     

RhoA-Mediated Signaling in Mechanotransduction of Osteoblasts

Osteoblasts play a pivotal role in load-driven bone formation by activating Wnt signaling through a signal from osteocytes as a mechanosensor. Osteoblasts are also sensitive to mechanical stimulation, but the role of RhoA, a small GTPase involved in the regulation of cytoskeleton adhesion complexes, in mechanotransduction of osteoblasts is not completely understood. Using MC3T3-E1 osteoblast-like cells under 1 hr flow treatment at 10 dyn/cm(2), we examined a hypothesis that RhoA signaling mediates the cellular responses to flow-induced shear stress. To test the hypothesis, we conducted genome-wide pathway analysis and evaluated the role of RhoA in molecular signaling. Activity of RhoA was determined with a RhoA biosensor, which determined the activation state of RhoA based on a fluorescence resonance energy transfer between CFP and YFP fluorophores. A pathway analysis indicated that flow treatment activated phosphoinositide 3-kinase (PI3K) and mitogen-activated protein kinase (MAPK) signaling as well as a circadian regulatory pathway. Western blot analysis revealed that in response to flow treatment phosphorylation of Akt in PI3K signaling and phosphorylation of p38 and ERK1/2 in MAPK signaling were induced. FRET measurement showed that RhoA was activated by flow treatment, and an inhibitor to a Rho kinase significantly reduced flow-induced phosphorylation of p38, ERK1/2, and Akt as well as flow-driven elevation of the mRNA levels of osteopontin and cyclooxygenase-2. Collectively, the result demonstrates that in response to 1 hr flow treatment to MC3T3-E1 cells at 10 dyn/cm(2), RhoA plays a critical role in activating PI3K and MAPK signaling as well as modulating the circadian regulatory pathway.     

PTEN/PI3k/AKT Regulates Macrophage Polarization in Emphysematous mice

Macrophages play an important role in the pathogenesis of COPD. Macrophage polarization towards the M2 phenotype has been observed in the lung tissues of COPD patients and cigarette smokers. The molecular basis of this process remains unclear, and it has not been completely illuminated in animal models of emphysema. In our study, we combined cigarette smoke (CS) exposure with intraperitoneal injection of cigarette smoke extract (CSE) to build an emphysema model. We found by immunohistochemical staining and flow cytometry that the expression level of CD206 and the ratio of M2 to M1 macrophages was increased in emphysematous mice. We also demonstrated that decreased protein level for phosphatase and tensin homology deleted on chromosome ten (PTEN) and increased total protein levels for phosphorylation ‐protein kinase B (p‐AKT) in the lung tissue of emphysematous mice and in CSE‐treated RAW264.7 cells. In both bone marrow‐derived macrophages (BMDMs) from emphysematous mice and CSE‐treated RAW264.7 cells, we observed by RT‐PCR that the mRNA levels of M2 macrophage‐related markers and cytokines were increased. Furthermore, M1 macrophage‐related markers and cytokines were decreased. Meanwhile we treated BMDMs from emphysematous mice and CSE‐treated RAW264.7 cells with the phosphoinositide 3‐kinase (PI3K)/Akt inhibitor (LY294002), we observed a reduction in RNA levels of M2 macrophage‐related markers and cytokines. In conclusion, we confirmed that macrophage M2 polarization was induced in emphysematous mice generated by CS exposure combined with intraperitoneal injection of CSE. We also showed that M2 polarization was mediated through PTEN/PI3k/AKT pathway activation.     

Mer receptor tyrosine kinase negatively regulates lipoteichoic acid-induced inflammatory response via PI3K/Akt and SOCS3

Activation of toll-like receptor (TLR) signaling that initiates an innate immune response to pathogens must be strictly regulated to prevent excessive inflammatory damage in the host. Here, we demonstrate that Mer receptor tyrosine kinase (MerTK) is a negative regulatory molecule in the lipoteichoic acid (LTA)-induced inflammatory response. LTA that activated TLR2 signaling concomitantly induced activation of MerTK signaling in RAW264.7 macrophages, including phosphoinositide 3-kinase (PI3K)/Akt and suppressor of cytokine signaling 3 (SOCS3). Moreover, LTA induced MerTK activation in a time-dependent manner, and LTA-induced MerTK activation was dependent on the ligand Gas6. Additionally, pretreatment with a specific Mer-blocking antibody significantly inhibited LTA-induced phosphorylation of MerTK, while further enhancing LTA-induced phosphorylation of IκB-α and NF-κBp65 as well as production of TNF-α and IL-6. Meanwhile, the antibody blockade of MerTK markedly prevented LTA-induced Akt phosphorylation and SOCS3 expression, both of which were crucial for the inhibition of TLR2-mediated immune response. Collectively, these results suggest, for the first time, that MerTK is an intracellular negative feedback regulator that inhibits the inflammatory response of LTA-stimulated macrophages through the PI3K/Akt pathway and SOCS3 protein.     

CD137 ligand‐mediated reverse signals increase cell viability and cytokine expression in murine myeloid cells: Involvement of mTOR/p70S6 kinase and Akt

Cross‐linking of CD137 ligand (CD137L), a member of the TNF family, with recombinant CD137‐Fc (rCD137‐Fc) protein enhanced adherence of bone marrow‐derived macrophages, and increased the expression of ICAM‐1, IL‐1β, IL‐6, M‐CSF and phosphotyrosine proteins. In RAW264.7 cells, a murine myeloid cell line, rCD137‐Fc not only increased adherence but also cell multiplication, in a manner comparable to LPS or M‐CSF. In addition, it up‐regulated expression of IL‐1β, IL‐1 receptor antagonist, IL‐6, COX2, tenascin C, neuropeptide Y and M‐CSF mRNA. Neutralization of M‐CSF by incubating the RAW264.7 cells with anti‐M‐CSF mAb did not prevent the CD137L signal‐induced viability. Viability was blocked by PP2, an Src tyrosine kinase inhibitor, rapamycin, an mTOR inhibitor and LY294002, a PI3K inhibitor, but not by Wortmannin, another PI3K inhibitor. Cross‐linking of CD137L increased phosphorylation of Akt and p70S6 kinase. The latter was blocked by PP2, rapamycin or LY294002, but not by Wortmannin, whereas phosphorylation of Akt was blocked by LY294002 or Wortmannin. These findings demonstrate that reverse signals evoked by CD137L regulate immune functions in macrophages.     

PHOSPHATIDYLINOSITOL 3-KINASE REGULATES PROLIFERATION OF RAW 264.7 MACROPHAGES

Phosphatidylinositol 3-kinase (PI3-kinase) is an enzyme that acts as a direct biochemical link between a novel phosphatidylinositol pathway and a number of proteins containing intrinsic or associated kinase activities. Here we demonstrate that wortmannin, PI3-kinase inhibitor, decreases the proliferation of RAW 264.7 macrophages and that another structurally unrelated inhibitor of PI3-kinase, LY294002, also inhibits the proliferation. These results indicate a possible involvement of PI3-kinase in RAW 264.7 macrophages growth regulation. Wortmannin stimulation of RAW 264.7 macrophages is followed by sustained expression of the mRNA of c-fos and a transient expression of the mRNA of c-jun. We also show that the wortmannin and LY294002 induce a cell cycle arrest in asynchronously growing cells leading to an inhibition of cell proliferation after 12 h of treatment. In addition, wortmannin or LY294002 inhibited the phorbol 12-myristate 13-acetate-induced macrophages proliferation potently. These results suggest that PI3-kinase plays an important role in growth regulation of RAW 264.7 macrophages and that protein kinase C is a down stream effector of PI3-kinase.     

鲍曼不动杆菌外膜蛋白A通过Akt/mTOR/p70S6K信号通路引起RAW264.7细胞自噬

目的:分析鲍曼不动杆菌标准株ATCC 19606(Acinetobacter baumannii ATCC 19606)外膜蛋白A(outer membrane protein A,Omp A)对RAW264. 7细胞的自噬诱导作用。方法:建立Omp A刺激RAW264. 7细胞的模型,通过细胞免疫荧光染色、Western blot和透射电子显微镜检测Omp A对RAW264. 7细胞自噬的影响。结果:Omp A可以引起自噬蛋白LC3B-II表达升高,并抑制Akt/mTOR/p70S6K的磷酸化水平;雷帕霉素可以进一步降低mTOR和p70S6K磷酸化,并提高Omp A引起的LC3B-II表达升高。结论:鲍曼不动杆菌Omp A通过Akt/mTOR/p70S6K信号通路引起RAW264. 7细胞自噬。这为将来进一步研究鲍曼不动杆菌引起自噬的分子机制及找到对抗其感染的新方法提供理论依据。     

PHOSPHATIDYLINOSITOL 3-KINASE REGULATES PROLIFERATION OF RAW 264.7 MACROPHAGES

Phosphatidylinositol 3-kinase (PI3-kinase) is an enzyme that acts as a direct biochemical link between a novel phosphatidylinositol pathway and a number of proteins containing intrinsic or associated kinase activities. Here we demonstrate that wortmannin, PI3-kinase inhibitor, decreases the proliferation of RAW 264.7 macrophages and that another structurally unrelated inhibitor of PI3-kinase, LY294002, also inhibits the proliferation. These results indicate a possible involvement of PI3-kinase in RAW 264.7 macrophages growth regulation. Wortmannin stimulation of RAW 264.7 macrophages is followed by sustained expression of the mRNA of c-fos and a transient expression of the mRNA of c-jun. We also show that the wortmannin and LY294002 induce a cell cycle arrest in asynchronously growing cells leading to an inhibition of cell proliferation after 12 h of treatment. In addition, wortmannin or LY294002 inhibited the phorbol 12-myristate 13-acetate-induced macrophages proliferation potently. These results suggest that PI3-kinase plays an important role in growth regulation of RAW 264.7 macrophages and that protein kinase C is a down stream effector of PI3-kinase.     

Cot/tpl2 activity is required for TLR-induced activation of the Akt p70 S6k pathway in macrophages: Implications for NO synthase 2 expression

LPS stimulation activates IKK and different MAP kinase pathways, as well as the PI3K‐Akt‐mTOR‐p70 S6k pathway, a negative regulator of these MyD88‐dependent intracellular signals. Here, we show that Cot/tpl2, a MAP3K responsible for the activation of the MKK1‐Erk1/2, controls P‐Ser473 Akt and P‐Thr389 p70 S6k phosphorylation in LPS‐stimulated macrophages. Analysis of the intracellular signalling in Cot/tpl2 KO macrophages versus WT macrophages reveals lower IκBα recovery and higher phosphorylation of JNK and p38α after 1 h of LPS stimulation. Moreover, Cot/tpl2 deficiency increases LPS‐induced NO synthase 2 (NOS2) expression in macrophages. Inhibition of the PI3K pathway abolishes the differences in IκBα and NOS2 expression between Cot/tpl2 KO and WT macrophages following LPS administration. Furthermore, in zymosan‐ and polyI:C‐stimulated macrophages, Cot/tpl2 mediates P‐Ser473 Akt phosphorylation, increases IκBα levels and decreases NOS2 expression. In conclusion, these data reveal a novel role for the Cot/tpl2 pathway in mediating TLR activation of the Akt‐mTOR‐p70 S6k pathway, allowing Cot/tpl2 to fine‐control the activation state of other signalling pathways.     

Caveolin-1 confers antiinflammatory effects in murine macrophages via the MKK3/p38 MAPK pathway

Caveolin-1 has been reported to regulate apoptosis, lipid metabolism, and endocytosis in macrophages. In the present study, we demonstrate that caveolin-1 can act as a potent immunomodulatory molecule. We first observed caveolin-1 expression in murine alveolar macrophages by Western blotting and immunofluorescence microscopy. Loss-of-function experiments using small interfering RNA showed that down regulating caveolin-1 expression in murine alveolar and peritoneal macrophages increased LPS-induced proinflammatory cytokine TNF-alpha and IL-6 production but decreased anti-inflammatory cytokine IL-10 production. Gain-of-function experiments demonstrated that overexpression of caveolin-1 in RAW264.7 cells decreased LPS-induced TNF-alpha and IL-6 production and augmented IL-10 production. p38 mitogen-activated protein kinase (MAPK) phosphorylation was increased by overexpressing caveolin-1 in RAW264.7 cells, whereas c-Jun N-terminal kinase, extracellular signal-regulated kinase MAPK, and Akt phosphorylation were inhibited. The antiinflammatory modulation of LPS-induced cytokine production by caveolin-1 was significantly abrogated by the administration of p38 inhibitor SB203580 in RAW264.7 cells. Peritoneal macrophages isolated from MKK3 null mice did not demonstrate any modulation of LPS-induced cytokine production by caveolin-1. LPS-induced activation of NF-kappaB and AP-1 determined by electrophoretic mobility shift assay were significantly reduced by overexpressing caveolin-1 in RAW264.7 cells. The reductions were attenuated by the administration of p38 inhibitor SB203580. Taken together, our data suggest that caveolin-1 acts as a potent immunomodulatory effector molecule in immune cells and that the regulation of LPS-induced cytokine production by caveolin-1 involves the MKK3/p38 MAPK pathway.     

Immune stimulatory effects of Loranthi ramulus on macrophages through the increase of NO and TNF-α

The activation of macrophages by microorganisms plays an important role in host defense and immunopathology. Loranthi ramulus (LR) is commonly used as a traditional drug and health food in Korea. Here, we investigated the regulatory effects of LR on macrophage-mediated immune responses. Treatment of macrophages with LR resulted in the enhanced cell-surface expression of CD80, CD86 and major histocompatibility complex (MHC) class II, as well as the enhanced production of nitric oxide (NO) and tumor necrosis factor (TNF)-α, and also iNOS and TNF-α mRNA expression. These alterations of LR-treated cells were associated with the activation of NF-κB and mitogen-activated protein kinases (MAPKs). LR increased the phosphorylation of MAPKs (JNK, ERK1/2, p38 MAPK) and the activation of NF-κB in Raw 264.7 cells. These results suggest that LR has increased NO and TNF-α production through phosphorylation of all three MAPKs following IκBα degradation and NF-κB activation. In conclusion, our results demonstrate that LR can effectively promote the activation of macrophages, suggesting that LR may possess the potential to regulate immune responses.     

Phosphatidylinositol-3-kinase and Akt are required for RIG-I-mediated anti-viral signalling through cross-talk with IPS-1

Retinoic acid-inducible gene I (RIG-I) is a cytosolic pattern-recognition receptor that recognizes viruses and triggers anti-viral immune responses. Activation of intracellular RIG-I signalling is mediated through interferon-β (IFN-β) promoter stimulator-1 (IPS-1), an adaptor of RIG-I, which induces IFN regulatory factor (IRF) 3 activation and type I IFN expression. The phosphatidylinositol-3-kinase (PI3K) and Akt pathway is activated in host immune cells upon viral infection. However, the mechanism as to how they work in RIG-I signalling has not been fully elucidated. Therefore, we investigated the role of PI3K and Akt in the regulation of RIG-I-mediated IRF3 activation and type I IFN expression in macrophages. Our results show that Sendai virus infection, which is recognized by RIG-I, led to IRF3 activation and IFN-β expression and these responses were attenuated by the PI3K inhibitor (LY294002) and an Akt dominant-negative mutant in the macrophage cell line(RAW264.7). IRF3 phosphorylation and dimerization as well as IFN-β expression induced by a synthetic RIG-I agonist, short poly(I:C), were suppressed by LY294002 or siRNA-Akt in bone marrow-derived macrophages. Suppression of PI3K and Akt using a dominant-negative mutant and siRNA knockdown resulted in attenuation of IRF3 activation and IFN-β expression induced by RIG-I itself or its adaptor, IPS-1. Association of Akt with IPS-1 increased with short poly(I:C) stimulation and required the pleckstrin homology domain of Akt and caspase-recruitment domain in IPS-1. Collectively, our results show that PI3K and Akt are required downstream of IPS-1 for RIG-I-mediated anti-viral immune responses. The results describe a novel, interactive relationship between RIG-I downstream signalling molecules resulting in efficient anti-viral immunity.     

Notch signaling regulates the phosphorylation of Akt and survival of lipopolysaccharide-activated macrophages via regulator of G protein signaling 19 (RGS19)

Macrophages play critical roles in innate immune defense by sensing microbes using pattern-recognition receptors. Lipopolysaccharide (LPS) stimulates macrophages via TLR, which leads to activation of downstream signaling cascades. In this study, we investigated the roles of a conserved signaling pathway, Notch signaling, in regulating the downstream signaling cascades of the LPS/TLR4 pathways in macrophages. Using a phospho-proteomic approach and a gamma-secretase inhibitor (GSI) to suppress the processing and activation of Notch signaling, we identified regulator of G protein signaling 19 (RGS19) as a target protein whose phosphorylation was affected by GSI treatment. RGS19 is a guanosine triphosphatase (GTPase)-activating protein that functions to negatively regulate G protein-coupled receptors via G_αi/G_αq-linked signaling. Stimulation of RAW264.7 cells with LPS increased the level of the phosphorylated form of RGS19, while LPS stimulation in the presence of GSI decreased its level. GSI treatment did not alter the mRNA level of rgs19. Treatment with GSI or silencing of rgs19 in macrophages impaired the phosphorylation of Akt Thr³⁰⁸ upon LPS stimulation. Furthermore, targeted deletion of a DNA-binding protein and binding partner of the Notch receptor, RBP-Jκ/CSL, in macrophages resulted in delayed and decreased Akt phosphorylation. Because the PI3K/Akt pathway regulates cell survival in various cell types, the cell cycle and cell death were assayed upon GSI treatment, phosphatidylinositol 3 kinase (PI3K) inhibitor treatment or silencing of rgs19. GSI treatment resulted in decreased cell populations in the G1 and S phases, while it increased the cell population of cell death. Similarly, silencing of rgs19 resulted in a decreased cell population in the G1 phase and an increased cell population in the subG1 phase. Inhibition of Akt phosphorylation by PI3K inhibitor in LPS-stimulated macrophages increased cell population in G1 phase, suggesting a possible cell cycle arrest. Taken together, these results indicate that Notch signaling positively regulates phosphorylation of Akt, possibly via phosphorylation of RGS19, and inhibition of both molecules affects the cell survival and cell cycle of macrophages upon LPS stimulation.