Angiopoietin-1/Tie2 signaling pathway inhibits lipopolysaccharide-induced activation of RAW264.7 macrophage cells

Angiopoietin-1 (Ang1) is a ligand for the endothelial-specific tyrosine kinase receptor Tie2 and has been shown to play an essential role in embryonic vasculature development. There have been many studies about the anti-inflammatory effects of Ang1, most of which focus on endothelium cells. In the present study, we explore the role of Ang1-Tie2 signaling in the activation of macrophages upon lipopolysaccharide (LPS) stimulation. We found that Tie2 receptor is expressed on macrophages and Ang1 could inhibit LPS-induced activation of macrophages, as evidenced by cell migration and TNF-α production, specifically through Tie2 receptor. We further investigated the mechanism and found that Ang1-Tie2 could block LPS-induced activation of NF-κB which has been shown to be necessary for macrophage activation with LPS treatment. Thus, we described, for the first time, the role of Ang1-Tie2 signaling in macrophage activation and the possible mechanisms in response to immune stimulation.     

Tick saliva regulates migration, phagocytosis, and gene expression in the macrophage-like cell line, IC-21

We studied the effects of tick saliva on cell migration, cell signaling, phagocytosis, and gene expression in the murine macrophage cell line, IC-21. Saliva increased both basal- and platelet-derived growth factor (PDGF)-stimulated migration in IC-21 cells. However, saliva did not affect PDGF-stimulated extracellular signal-regulated kinase (ERK) activity. Zymosan-mediated interleukin-1 receptor associated kinase (IRAK) activity increased when cells were pretreated with saliva. Saliva suppressed phagocytosis of zymosan particles by IC-21 cells. An RT² Profiler™ PCR Array revealed that saliva regulates gene expression in a manner consistent with an immune response skewed toward a Th2 reaction, which is characterized by production of anti-inflammatory cytokines IL-4 and IL-10. Our results using IC-21 cells suggest that Dermacentor variabilis has evolved a mechanism for regulating macrophage function, which may contribute to the tick’s ability to modulate immune function.     

Protective role of Cytoglobin and Neuroglobin against the Lipopolysaccharide (LPS)-induced inflammation in Leydig cells ex vivo

Leydig cells are responsible for testosterone production in male testis upon stimulation by luteinizing hormone. Inflammation and oxidative stress related Leydig cell dysfunction is one of the major causes of male infertility. Cytoglobin (CYGB) and Neuroglobin (NGB) are two globin family member proteins which protect cells against oxidative stress.In the current study, we established a Lipopolysaccharide (LPS)-induced inflammation model in TM3 Leydig cell culture to study the function of CYGB and NGB proteins under inflammatory conditions. CYGB and NGB were downregulated using siRNA and shRNA based experimental strategies. Overexpression was conducted using lentiviral pLenti-III-CYGB-2A-GFP, and pLenti-III-NGB-2A-GFP vector systems. As testicular macrophages regulate immune function upon inflammation and steroidogenesis of Leydig cells, we generated direct/indirect co-culture systems of TM3 and mouse macrophage (RAW264.7) cells ex vivo.Downregulation of CYGB and NGB induced nitride oxide (NO) release, blocked cell cycle progression, reduced testosterone production and increased inflammatory and apoptotic pathway gene expression in the presence and absence of LPS. On the other hand, CYGB and NGB overexpression reduced TNFα and COX-2 protein expressions and increased the expression of testosterone biogenesis pathway genes upon LPS stimulation. In addition, CYGB and NGB overexpression upregulated testosterone production. The present study successfully established an inflammatory interaction model of TM3 and RAW264.7 cells. Suppression of CYGB and NGB in TM3 cells changed macrophage morphology, enhanced macrophage cell number and NO release in co-culture experiments upon LPS exposure.In summary, these results demonstrate that globin family members might control LPS induced inflammation by regulating apoptotic mechanisms and macrophage response.     

Restraint of proinflammatory cytokine biosynthesis by mitogen-activated protein kinase phosphatase-1 in lipopolysaccharide-stimulated macrophages

Exposure of macrophages to LPS elicits the production of proinflammatory cytokines, such as TNF-alpha, through complex signaling mechanisms. Mitogen-activated protein (MAP) kinases play a critical role in this process. In the present study, we have addressed the role of MAP kinase phosphatase-1 (MKP-1) in regulating proinflammatory cytokine production using RAW264.7 macrophages. Analysis of MAP kinase activity revealed a transient activation of c-Jun N-terminal kinase (JNK) and p38 after LPS stimulation. Interestingly, MKP-1 was induced concurrently with the inactivation of JNK and p38, whereas blocking MKP-1 induction by triptolide prevented this inactivation. Ectopic expression of MKP-1 accelerated JNK and p38 inactivation and substantially inhibited the production of TNF-alpha and IL-6. Induction of MKP-1 by LPS was found to be extracellular signal-regulated kinase dependent and involved enhanced gene expression and increased protein stability. Finally, MKP-1 expression was also induced by glucocorticoids as well as cholera toxin B subunit, an agent capable of preventing autoimmune diseases in animal models. These findings highlight MKP-1 as a critical negative regulator of the macrophage inflammatory response, underscoring its premise as a potential target for developing novel anti-inflammatory drugs.     

Macrophage-colony-stimulating factor-induced proliferation and lipopolysaccharide-dependent activation of macrophages requires Raf-1 phosphorylation to induce mitogen kinase phosphatase-1 expression

Macrophages are key regulators of immune responses. In the absence of an activating signal, murine bone marrow-derived macrophages undergo proliferation in response to their specific growth factor, namely M-CSF. The addition of bacterial LPS results in macrophage growth arrest and their engagement in a proinflammatory response. Although participation of ERKs is required for both macrophage proliferation and activation, ERK phosphorylation follows a more delayed pattern in response to activating agents. In primary macrophages, mitogen kinase phosphatase-1 (MKP-1) is a key regulator of the time course of MAPK activity. Here we showed that MKP-1 expression is dependent on Raf-1 activation. The time course of Raf-1 activation correlated with that of ERK-1/2. However, whereas ERK phosphorylation in response to M-CSF is Raf-1 dependent, in response to LPS, an alternative pathway directs the activation of these kinases. Inhibition of Raf-1 activity increased the expression of cyclin-dependent kinase inhibitors and growth arrest. In contrast, no effect was observed in the expression of proinflammatory cytokines and inducible NO synthase following LPS stimulation. The data reported here reveal new insights into how signaling determines opposing macrophage functions.     

Mycobacterial cord factor enhances migration of neutrophil‐like HL‐60 cells by prolonging AKT phosphorylation

Trehalose 6,6′‐dimycolate (TDM), or cord factor, is a crucial stimulus of immune responses during Mycobacterium tuberculosis infection. Although TDM has immuno‐stimulatory properties, including adjuvant activity and the ability to induce granuloma formation, the mechanisms underlying these remain unknown. We hypothesized that TDM stimulates transendothelial migration of neutrophils, which are the first immune cells to infiltrate the tissue upon infection. In this study, it was shown that TDM enhances N‐formylmethionyl‐leucyl‐phenylalanine (fMLP)‐induced chemotaxis and transendothelial movement by prolonging AKT phosphorylation in human neutrophils. TDM induced expression of macrophage‐inducible C‐type lectin, a receptor for TDM, and induced secretion of pro‐inflammatory cytokines and chemokines in differentiated HL‐60 cells. In 2‐ and 3‐D neutrophil migration assays, TDM‐stimulated neutrophils showed increased fMLP‐induced chemotaxis and transendothelial migration. Interestingly, following fMLP stimulation of TDM‐activated neutrophils, AKT, a crucial kinase for neutrophil polarization and chemotaxis, showed prolonged phosphorylation at serine 473. Taken together, these data suggest that TDM modulates transendothelial migration of neutrophils upon mycobacterial infection through prolonged AKT phosphorylation. AKT may therefore be a promising therapeutic target for enhancing immune responses to mycobacterial infection.

     

Selective regulation of IL-10 signaling and function by zymosan

Balanced activity of pro- and anti-inflammatory cytokines during innate immune responses is required to allow effective host defense while avoiding tissue damage and autoimmunity. Induction of cytokine production after recognition of pathogen-associated molecular patterns (PAMPs) by innate immune cells has been well demonstrated, but modulation of cytokine function by PAMPs is not well understood. In this study we show that stimulation of macrophages with zymosan, which contains PAMPs derived from yeast, rapidly extinguished macrophage responses to IL-10, a suppressive cytokine that limits inflammatory tissue damage but also compromises host defense. The mechanism of inhibition involved protein kinase Cbeta and internalization of IL-10R, and was independent of TLR2 and phagocytosis. Inhibition of IL-10 signaling and function required direct contact with zymosan, and cells in an inflammatory environment that had not contacted zymosan remained responsive to the paracrine activity of zymosan-induced IL-10. These results reveal a mechanism that regulates IL-10 function such that antimicrobial functions of infected macrophages are not suppressed, but the activation of surrounding noninfected cells and subsequent tissue damage are limited. The fate of individual cells in an inflammatory microenvironment is thus specified by dynamic interactions among host cells, microbes, and cytokines that determine the balance between protection and pathology.     

SIRT1 regulates inflammation response of macrophages in sepsis mediated by long noncoding RNA

Molecular mechanisms for macrophage immune responses modulated by SIRT1 during sepsis remain unclear. Here, we show that SIRT1 expression is down-regulated in macrophages from mouse sepsis model or LPS stimulation. SIRT1 expression in macrophages correlates with low levels of a long noncoding RNA (lncRNA)-NONMMUT003701 [named as lncRNA-CCL2]. SIRT1 inhibits lncRNA-CCL2 expression via sustaining a repressive chromatin state in the lncRNA-CCL2 locus. The inflammation cytokines expression is downregulated by knockdown of lncRNA-CCL2. Such inhibition can be reversed partly by decreased SIRT1 activity. Thus, this work uncovers previously unidentified mechanisms in which SIRT1 associates with lncRNA and lncRNA regulates macrophage inflammatory response.     

Immunological Properties of TtT/M-87 Cell Line Established from Murine Pituitary Tumor-Associated Macrophages

TtT/M-87 cell is a macrophage cell line established from thyrotropic pituitary tumor tissues in mouse. In this paper, we report the immunological properties of M-87 cells as a model of tumor-associated macrophage. Contrasting with resident peritoneal macrophages, M-87 cells constitutively secreted small but significant amounts of TNF-α and IL-1α, which were detectable in both biological assays (cytotoxic activity for L929 and co-mitogenic activity for Con A-induced T cell proliferation, respectively) and ELISA, and produced larger amounts of these cytokines upon stimulation with LPS. They expressed MHC class II molecules on their cell surface without stimulation by IFN-γ. The accessory or antigen-presenting cell activity in antibody-producing response of spleen lymphocytes to sheep red blood cells was shown to be much higher in M-87 cells than normal peritoneal macrophages. In addition, when normal spleen lymphocytes were cultured with allogeneic tumor cells, such as EL-4 and S-180, in the presence of M-87 cells, lymphocytes reactive to stimulator cells were activated to manifest inhibitory effect on the tumor cell growth and also to manifest specific cytotoxic effect on the allogeneic tumor cells. These results show that M-87 cells derived from tumor-associated tissue are activated macrophages and that they are inhibitory to tumor cell growth and augmentative in the induction of T-cell-mediated immune responses.     

Red blood cell lysate modulates the expression of extracellular matrix proteins in dermal fibroblasts

Trichinella spiralis is a zoonotic nematode and food borne parasite and infection with T. spiralis leads to suppression of the host immune response and other immunopathologies. The excretory/secretory (ES) products of T. spiralis play important roles in the process of immunomodulation. However, the mechanisms and related molecules are unknown. Macrophages, a target for immunomodulation by the helminth parasite, play a critical role in initiating and modulating the host immune response to parasite infection. In this study, we examined the effect of ES products from different stages of T. spiralis on modulating J774A.1 macrophage activities. ES products from different stages of T. spiralis reduced the capacity of macrophages to express pro-inflammatory cytokines (tumor necrosis factor α, interleukin-1β , interleukin-6 , and interleukin-12) in response to lipopolysaccharide (LPS) challenge. However, only ES products from 3-day-old adult worms and 5-day-old adult worms/new-born larvae significantly inhibited inducible nitric oxide synthase gene expression in LPS-induced macrophages. In addition, ES products alone boosted the expression of anti-inflammatory cytokines interleukin-10 and transforming growth factor-β and effector molecule arginase 1 in J774A.1 macrophages. Signal transduction studies showed that ES products significantly inhibited nuclear factor-κB translocation into the nucleus and the phosphorylation of both extracellular signal-regulated protein kinase 1/2 and p38 mitogen-activated protein kinase in LPS-stimulated J774A.1 macrophages. These results suggest that ES products regulate host immune response at the macrophage level through inhibition of pro-inflammatory cytokines production and induction of macrophage toward the alternative phenotype, which maybe important for worm survival and host health.     

Tyrosine kinase Syk associates with toll-like receptor 4 and regulates signaling in human monocytic cells

Toll-like receptor 4 (TLR4) induces an innate immune response in mammals by recognizing lipopolysaccharide (LPS), a component of the cell wall of Gram-negative bacteria. In this study, we show that tyrosine kinase Syk constitutively associates with TLR4 in THP-1 cells. As previously reported in peripheral blood mononuclear cells, TLR4 gets inducibly tyrosine phosphorylated upon LPS engagement in THP-1 cells. Piceatannol, a pharmacological inhibitor of the tyrosine kinase Syk, abrogates TLR4 tyrosine phosphorylation at low doses. The kinetics of TLR4 tyrosine phosphorylation in THP-1 cells coincides with an early wave of Syk tyrosine phosphorylation. Additionally, serine threonine kinase interleukin-1 (IL1) receptor-associated kinase 1 (IRAK-1) is transiently recruited to the complex containing adaptor molecule MyD88, TLR4 and Syk within 1 min of LPS engagement and dissociates by 30 min. Finally, the inhibition of Syk with piceatannol has no effect on LPS-mediated release of cytokines IL6, IL1beta, tumor necrosis factor-alpha, neither on chemokines macrophage inhibitory protein (MIP)1alpha, MIP1beta, monocyte chemoattractant protein -1, IL8, Groalpha and RANTES. However, IL10 and IL12p40 releases are significantly inhibited. Our findings implicate Syk as a novel modulator of LPS-mediated TLR4 responses in human monocytic cells and shed insight into the kinetics of early complex formation upon LPS engagement.     

Liver Kinase B1 Suppresses Lipopolysaccharide-induced Nuclear Factor κB (NF-κB) Activation in Macrophages

Liver kinase B1 (LKB1), a serine/threonine kinase, is a tumor suppressor and metabolic regulator. Recent data suggest that LKB1 is essential in regulating homeostasis of hematopoietic cells and immune responses. However, its role in macrophages and innate immune system remains unclear. Here we report that macrophage LKB1 inhibits pro-inflammatory signaling in response to LPS. LPS-induced pro-inflammatory cytokines and pro-inflammatory enzymes were monitored in bone marrow-derived macrophages isolated from myeloid cell-specific LKB1 knock out mice and their wild type littermate control mice. LPS induced higher levels of pro-inflammatory cytokines and pro-inflammatory enzymes in bone marrow-derived macrophages from LKB1 KO than those from wild type mice. Consistently, LPS induced higher levels of NF-κB activation in LKB1-deficient macrophages than those in wild type. Further, LPS stimulation significantly increased LKB1 phosphorylation at serine 428, which promoted its binding to IκB kinaseβ (IKKβ), resulting in the inhibition of NF-κB. Finally, LPS injection caused higher levels of cytokine release and more severe tissue injury in the lung tissues of LKB1 KO mice than in those of control mice. We conclude that LKB1 inhibits LPS-induced NF-κB activation in macrophages.     

草鱼树突状细胞的分离鉴定及益生芽孢杆菌对其免疫功能的影响

为揭示草鱼(Ctenopharyngodon idellus)树突状细胞(Dendritic cells)的生物学特性及益生芽孢杆菌对其免疫功能的影响, 研究通过草鱼体外细胞培养技术分离获得草鱼DCs, 对其形态学特征、生物学功能及膜表面标记分子的表达进行了分析鉴定; RT-PCR检测了益生芽孢杆菌对草鱼DCs免疫相关细胞因子表达的影响。结果表明草鱼DCs具有典型的树突状形态, 可有效地激活T淋巴细胞的增殖并具有迁移能力。LPS刺激可促进其成熟过程, 显著提升膜表面标记分子CD80/86、CD83的表达。这表明草鱼DCs和哺乳动物DCs在形态和功能上具有高度相似性。RT-PCR结果显示, 在体外条件下使用紫外照射灭活的益生枯草芽孢杆菌对草鱼DCs进行刺激后, 抗炎性因子IL-4, IL-10的表达水平显著提升(P<0.05), 并在12h时达到峰值。这表明枯草芽孢杆菌可通过促进树突状细胞分泌抗炎性因子来影响其免疫功能。以上结果为进一步研究鱼类树突状细胞的生物学特性及益生芽孢杆菌对其免疫功能的影响提供了重要的依据。     

Amelogenins modulate cytokine expression in LPS-challenged cultured human macrophages

Amelogenins are enamel matrix proteins with a proven ability to restore tissues in patients with advanced periodontitis and chronic skin wounds. To explore the mechanisms of action of amelogenins in wound inflammation, the in vitro effect on the expression of selected cell mediators involved in inflammation and tissue repair from human monocyte-derived macrophages was studied. Macrophages were treated with amelogenins in serum-enriched medium with simultaneous lipopolysaccharide (LPS) stimulation, for 6, 24 and 72 h, and the conditioned culture medium was analysed for 28 different cytokines. Amelogenin treatment directed the LPS-induced release of both pro- and anti-inflammatory cytokines towards an alternatively activated macrophage phenotype. This change in activation was also demonstrated by the amelogenin-induced secretion of alternative macrophage activation-associated CC chemokine-1 (AMAC-1, also known as CCL18; p<0.001), a well-documented marker of alternative activation. Amelogenins were also shown significantly to increase the macrophage expression of vascular endothelial growth factor and, to a lesser but significant extent, insulin-like growth factor-1 after 24h of culture. The results of the present in vitro study show that monocyte-derived macrophages stimulated by inflammatory agonist LPS respond to the treatment with amelogenins by reducing the pro-inflammatory activity and increasing the expression of tissue repair mediators.     

Anti-Inflammatory Effect of Buckwheat Sprouts in Lipopolysaccharide-Activated Human Colon Cancer Cells and Mice

In conducting an in vitro screening of ethanol extracts from various natural foods using a human colon cancer cell line (CoLoTC cells), an extract of buckwheat sprouts (ExtBS) was found to express significant anti-inflammatory activity. The anti-inflammatory activity of ExtBS was confirmed by oral administration of lipopolysaccharide (LPS) to mice. Inflammatory cytokines (interleukin 6 and tumor necrosis factor alpha) were markedly up-regulated in the spleen and liver from LPS-administrated mice, and combinatory treatment with LPS and ExtBS decreased up-regulation of them in both cytokines. Both serum cytokine levels corresponded to their gene expressions in tissues, but no anti-inflammatry effect in mice was observed when ExtBS was treated intraperitoneally. ExtBS oral administration also showed protective activity as to hepatic injury induced by galactosamine/LPS treatment. Based on these data, we suggest that ExtBS contains anti-inflammatory compounds.     

Critical roles of the WASP N-terminal domain and Btk in LPS-induced inflammatory response in macrophages

While Wiskott-Aldrich syndrome protein (WASP) plays critical roles in TCR signaling as an adaptor molecule, how it transduces innate immune signals remains to be elucidated. To investigate the roles of WASP in innate immune cells, we established bone marrow-derived macrophage (BMDM) cell lines from WASP15 transgenic (Tg) mice overexpressing the WASP N-terminal region (exons 1-5). Upon LPS stimulation, WASP15 Tg BMDM cell lines produce lower levels of inflammatory cytokines, such as TNF-α, IL-6, and IL-12p40 than the wild-type BMDM cell line. In addition, the production of nitric oxide by WASP15 Tg BMDM cells in response to LPS and IFN-γ was significantly impaired. Furthermore, we uncovered that the WASP N-terminal domain associates with the Src homology (SH) 3 domain of Bruton's tyrosine kinase (Btk). Overexpression of the WASP N-terminal domain diminishes the extent of tyrosine phosphorylation of endogenous WASP in WASP15 Tg BMDM cells, possibly by interfering with the specific binding between endogenous WASP and Btk during LPS signaling. These observations strongly suggest that the interaction between WASP N-terminal domain and Btk plays important roles in the LPS signaling cascade in innate immunity.