Notch signaling increases PPARγ protein stability and enhances lipid uptake through AKT in IL-4-stimulated THP-1 and primary human macrophages.

Notch signaling and nuclear receptor PPARγ are involved in macrophage polarization, but cross talk between them has not been reported in macrophages. In this study, the effect of Notch signaling on PPARγ in IL-4-stimulated human macrophages (M(IL-4)) was investigated using THP-1-derived macrophages and human monocyte-derived macrophages as models. Human M(IL-4) increased the expression of JAGGED1 and activated Notch signaling. Overexpression of Notch1 intracellular domain (NIC1) increased PPARγ expression, while inhibiting Notch signaling decreased PPARγ levels in M(IL-4). NIC1 overexpression in THP-1-derived macrophages increased PPARγ protein stability by delaying its proteasome-mediated degradation, but did not affect its mRNA. Phosphorylation of AKT was enhanced in NIC1-overexpressing cells, and a specific AKT inhibitor reduced the level of PPARγ. NIC1-overexpressing THP-1 cells exhibited increased CD36 levels via activation of PPARγ, resulting in enhanced intracellular lipid accumulation. In summary, this study provides evidence linking Notch signaling and PPARγ via AKT in M(IL-4).

Regulation of capillary tubules and lipid formation in vascular endothelial cells and macrophages via extracellular vesicle-mediated microRNA-4306 transfer.

OBJECTIVE: This study aimed to examine regulation of capillary tubules and lipid formation in vascular endothelial cells and macrophages via extracellular vesicle-mediated microRNA (miRNA)-4306 transfer. METHODS: Whole blood samples (12 mL) were collected from 53 patients, and miR-4306 levels in extracellular vesicles (EVs) were analyzed by reverse transcription-polymerase chain reaction. Human coronary artery vascular endothelial cells (HCAECs) and human monocyte-derived macrophages (HMDMs) were transfected with a scrambled oligonucleotide, an miR-4306 mimic, or an anti-miR-4306 inhibitor. The direct effect of miR-4306 on the target gene was analyzed by a dual-luciferase reporter assay. RESULTS: EV-contained miR-4306 released from HMDMs was significantly upregulated in coronary artery disease. Oxidized low-density lipoprotein (ox-LDL)-stimulated HMDM-derived EVs inhibited proliferation, migration, and angiogenesis abilities of HCAECs in vitro. However, ox-LDL-stimulated HCAEC-derived EVs enhanced lipid formation of HMDMs. The possible mechanism of these findings was partly due to EV-mediated miR-4306 upregulation of the Akt/nuclear factor kappa B signaling pathway. CONCLUSIONS: Paracrine cellular crosstalk between HCAECs and HMDMs probably supports the pro-atherosclerotic effects of EVs under ox-LDL stress.

Plant-made virus-like particles bearing influenza hemagglutinin (HA) recapitulate early interactions of native influenza virions with human monocytes/macrophages.

INTRODUCTION: Plant-made virus-like particles (VLP) bearing influenza virus hemagglutinins (HA) are novel vaccine candidates that induce cross-reactive humoral and poly-functional T cell responses. To better understand the mechanisms that underlie this broad immunogenicity we studied early interactions of VLPs bearing either H1 (A/California/07/2009 (H1N1)) or H5 (A/Indonesia/05/2005 (H5N1)) with a human monocytoid cell line (U-937 cells) and human monocyte-derived macrophages (MDMs) as model antigen-presenting cells (APC). METHODS AND RESULTS: Using Vibrio cholerae sialidase and lectins that target α2,6- (Sambucus nigra lectin) or α2,3-linked sialic acids (Maackia amurensis lectin I), we demonstrated that VLPs bind to these APCs in a sialic acid-dependent manner. Using lysosomal markers and DiD-labelled VLPs, we found that attachment to the cell surface leads to internalization, trafficking to acidic cell compartments and fusion of the VLP lipid envelope with endosomal membranes. Incubation of MDMs with H1- but not H5-VLPs induced proliferation of autologous peripheral blood mononuclear cells suggesting antigen processing and stimulation of a memory T cell response. CONCLUSIONS: Plant-made VLPs bearing influenza HA not only mimic the structure of influenza virions to some degree but also recapitulate key features of the initial virus-APC interaction. These observations may help to explain the balanced humoral and cellular responses to plant-made VLP vaccines.

Effect of 7,8-dihydroneopterin mediated CD36 down regulation and oxidant scavenging on oxidised low-density lipoprotein induced cell death in human macrophages.

The role of CD36 in oxidised low-density lipoprotein (oxLDL) mediated cell death was examined by down regulating the receptor level with the macrophage generated antioxidant 7,8-dihydroneopterin. Down regulation of CD36 protein levels in human monocyte derived macrophages by 7,8-dihydroneopterin corresponded to a decrease in CD36-mRNA. The oxidation products of 7,8-dihydroneopterin, dihydroxanthopterin and neopterin did not significantly down regulate CD36. The CD36 down regulation resulted in a decrease in oxLDL uptake measured as 7-ketocholesterol accumulation. Though less oxLDL was taken up by the macrophages as a result of the 7,8-dihydroneopterin induced down regulation in CD36 levels, the cytotoxicity of the oxLDL was not decreased. Addition of 7,8-dihydroneopterin to oxLDL treated macrophages decreased the concentration of intracellular oxidants. In the presence of oxLDL, 7,8-dihydroneopterin was oxidised to neopterin showing that the 7,8-dihydroneopterin was scavenging intracellular oxidants generated in response to the oxLDL. The results show CD36 down regulation does not protect human macrophages form oxLDL cytotoxicity but 7,8-dihydroneopterin intracellular oxidant scavenging is protective.

Atheroprotective Effects of Tumor Necrosis Factor-Stimulated Gene-6.

Tumor necrosis factor-stimulated gene-6 (TSG-6), an anti-inflammatory protein, was shown to be localized in the neointima of injury-induced rat arteries. However, the modulatory effect of TSG-6 on atherogenesis has not yet been reported. We aimed to evaluate the atheroprotective effects of TSG-6 on human endothelial cells (HECs), human monocyte-derived macrophages (HMDMs), human aortic smooth muscle cells (HASMCs) in vitro, and aortic lesions in apolipoprotein E-deficient mice, along with expression levels of TSG-6 in coronary lesions and plasma from patients with coronary artery disease (CAD). TSG-6 was abundantly expressed in HECs, HMDMs, and HASMCs in vitro. TSG-6 significantly suppressed cell proliferation and lipopolysaccharide-induced up-regulation of monocyte chemotactic protein-1, intercellular adhesion molecule-1, and vascular adhesion molecule-1 in HECs. TSG-6 significantly suppressed inflammatory M1 phenotype and suppressed oxidized low-density lipoprotein-induced foam cell formation associated with down-regulation of CD36 and acyl-CoA:cholesterol acyltransferase-1 in HMDMs. In HASMCs, TSG-6 significantly suppressed migration and proliferation, but increased collagen-1 and -3 expressions. Four-week infusion of TSG-6 into apolipoprotein E-deficient mice significantly retarded the development of aortic atherosclerotic lesions with decreased vascular inflammation, monocyte/macrophage, and SMC contents and increased collagen fibers. In addition, it decreased peritoneal M1 macrophages with down-regulation of inflammatory molecules and lowered plasma total cholesterol levels. In patients with CAD, plasma TSG-6 levels were significantly increased, and TSG-6 was highly expressed in the fibrous cap within coronary atherosclerotic plaques. These results suggest that TSG-6 contributes to the prevention and stability of atherosclerotic plaques. Thus, TSG-6 may serve as a novel therapeutic target for CAD.

Regulation of macrophage cholesterol efflux and liver X receptor α activation by nicotine.

OBJECTIVE: This study aims to investigate the characteristics of liver X receptor α (LXRα) and its target gene expression, as well as cholesterol efflux in human macrophages treated by nicotine. METHODS: Human monocyte-derived macrophages were collected. Before apoA-I-mediated human monocyte-derived macrophage cholesterol efflux, and mRNA expression of LXRα, and some of its target genes being detected, the macrophages were induced with or without nicotine. RESULTS: Pre-incubation of Human monocyte-derived macrophages with nicotine, cholesterol efflux was suppressed to apolipoprotein AI. Nicotine also inhibited LXRα and some of its target genes mRNA expression involved cholesterol metabolism, and facilitated some inflammatory genes expression. CONCLUSION: The changed function of cholesterol efflux and some genes expression may be the pathogenetic cause, and LXR activity of macrophage may offer potential therapeutic benefit in the treatment of atherosclerosis. Thus nicotine can regulate foam cell formation by inhibiting LXR pathway.

Apoptotic-like Leishmania exploit the host's autophagy machinery to reduce T-cell-mediated parasite elimination.

Apoptosis is a well-defined cellular process in which a cell dies, characterized by cell shrinkage and DNA fragmentation. In parasites like Leishmania, the process of apoptosis-like cell death has been described. Moreover upon infection, the apoptotic-like population is essential for disease development, in part by silencing host phagocytes. Nevertheless, the exact mechanism of how apoptosis in unicellular organisms may support infectivity remains unclear. Therefore we investigated the fate of apoptotic-like Leishmania parasites in human host macrophages. Our data showed--in contrast to viable parasites--that apoptotic-like parasites enter an LC3(+), autophagy-like compartment. The compartment was found to consist of a single lipid bilayer, typical for LC3-associated phagocytosis (LAP). As LAP can provoke anti-inflammatory responses and autophagy modulates antigen presentation, we analyzed how the presence of apoptotic-like parasites affected the adaptive immune response. Macrophages infected with viable Leishmania induced proliferation of CD4(+) T-cells, leading to a reduced intracellular parasite survival. Remarkably, the presence of apoptotic-like parasites in the inoculum significantly reduced T-cell proliferation. Chemical induction of autophagy in human monocyte-derived macrophage (hMDM), infected with viable parasites only, had an even stronger proliferation-reducing effect, indicating that host cell autophagy and not parasite viability limits the T-cell response and enhances parasite survival. Concluding, our data suggest that apoptotic-like Leishmania hijack the host cells' autophagy machinery to reduce T-cell proliferation. Furthermore, the overall population survival is guaranteed, explaining the benefit of apoptosis-like cell death in a single-celled parasite and defining the host autophagy pathway as a potential therapeutic target in treating Leishmaniasis.

Inhibition of Toll-like receptor 2 expression by siRNA in human monocyte-derived macrophages / 中国免疫学杂志

To screen the siRNAs (small interference RNA sequences ) which specifically inhibit the gene expression of TLR2 in human monocyte-derived macrophage , and discuss their prospects on the treatment of HIV at the level of molecular immunology.Methods:We obtained the mRNA sequences of human TLR 2 gene from NCBI gene bank ,then designed three siRNAs by siDESIGNTM software.The siRNA targeting human housekeeping gene GAPDH was used as positive control.The fluorescent labeling missense siRNA sequences (NC-FAM ) was used as negative control.We collected fresh peripheral blood from healthy volunteers and isolated mononuclear cells from the blood samples.The human mononuclear macrophages were then purified from mononuclear cells by utilizing adherence method.Cationic liposome reagent Lipofectamine 2000 was used to mediate siRNAs into the human mononuclear macrophages.The levels of TLR2 mRNA expression of siRNA-transfected monocyte-derived macrophage were determined by q-PCR.Expression of TLR2 protein was determined by Western blot.Results: At 72 h after transfection ,we found that the expression of GAPDH mRNA and protein in positive control group decreased significantly.Also found there existed significant differences between each siRNA group (F=41.957,P<0.001).Compared with negative control ,the relative expression of TLR2 mRNA in all siRNAs groups decreased significantly (P<0.05 ) , and the inhibition rates were 46%, 43%, 43% by three miRNAs respectively.Western blot showed that the expression of TLR 2 protein in siRNAs groups decreased significantly compared with that of control (P<0.05 ).Conclusion: The designed siRNAs in this study could inhibit the expression of TLR 2 gene in human monocyte-derived macrophage ,indicating that mediation of TLR-2 expression by siRNA might be a novel strategy for HIV treatment from the per-spective of molecular immunology.

An mRNA atlas of G protein-coupled receptor expression during primary human monocyte/macrophage differentiation and lipopolysaccharide-mediated activation identifies targetable candidate regulators of inflammation.

G protein-coupled receptors (GPCRs) are among the most important targets in drug discovery. In this study, we used TaqMan Low Density Arrays to profile the full GPCR repertoire of primary human macrophages differentiated from monocytes using either colony stimulating factor-1 (CSF-1/M-CSF) (CSF-1 Mϕ) or granulocyte macrophage colony stimulating factor (GM-CSF) (GM-CSF Mϕ). The overall trend was a downregulation of GPCRs during monocyte to macrophage differentiation, but a core set of 10 genes (e.g. LGR4, MRGPRF and GPR143) encoding seven transmembrane proteins were upregulated, irrespective of the differentiating agent used. Several of these upregulated GPCRs have not previously been studied in the context of macrophage biology and/or inflammation. As expected, CSF-1 Mϕ and GM-CSF Mϕ exhibited differential inflammatory cytokine profiles in response to the Toll-like Receptor (TLR)4 agonist lipopolysaccharide (LPS). Moreover, 15 GPCRs were differentially expressed between these cell populations in the basal state. For example, EDG1 was expressed at elevated levels in CSF-1 Mϕ versus GM-CSF Mϕ, whereas the reverse was true for EDG6. 101 GPCRs showed differential regulation over an LPS time course, with 65 of these profiles being impacted by the basal differentiation state (e.g. GPRC5A, GPRC5B). Only 14 LPS-regulated GPCRs showed asynchronous behavior (divergent LPS regulation) with respect to differentiation status. Thus, the differentiation state primarily affects the magnitude of LPS-regulated expression, rather than causing major reprogramming of GPCR gene expression profiles. Several GPCRs showing differential profiles between CSF-1 Mϕ and GM-CSF Mϕ (e.g. P2RY8, GPR92, EMR3) have not been widely investigated in macrophage biology and inflammation. Strikingly, several closely related GPCRs displayed completely opposing patterns of regulation during differentiation and/or activation (e.g. EDG1 versus EDG6, LGR4 versus LGR7, GPRC5A versus GPRC5B). We propose that selective regulation of GPCR5A and GPCR5B in CSF-1 Mϕ contributes to skewing toward the M2 macrophage phenotype. Our analysis of the GPCR repertoire expressed during primary human monocyte to macrophage differentiation and TLR4-mediated activation provides a valuable new platform for conducting future functional analyses of individual GPCRs in human macrophage inflammatory pathways.

Proinflammatory cytokine and nitric oxide production by human macrophages stimulated with Trichomonas vaginalis.

Trichomonas vaginalis commonly causes vaginitis and perhaps cervicitis in women and urethritis in men and women. Macrophages are important immune cells in response to T. vaginalis infection. In this study, we investigated whether human macrophages could be involved in inflammation induced by T. vaginalis. Human monocyte-derived macrophages (HMDM) were co-cultured with T. vaginalis. Live, opsonized-live trichomonads, and T. vaginalis lysates increased proinflammatory cytokines, such as TNF-alpha, IL-1beta, and IL-6 by HMDM. The involvement of nuclear factor (NF)-kappaB signaling pathway in cytokine production induced by T. vaginalis was confirmed by phosphorylation and nuclear translocation of p65 NF-kappaB. In addition, stimulation with live T. vaginalis induced marked augmentation of nitric oxide (NO) production and expression of inducible NO synthase (iNOS) levels in HMDM. However, trichomonad-induced NF-kappaB activation and TNF-alpha production in macrophages were significantly inhibited by inhibition of iNOS levels with L-NMMA (NO synthase inhibitor). Moreover, pretreatment with NF-kappaB inhibitors (PDTC or Bay11-7082) caused human macrophages to produce less TNF-alpha. These results suggest that T. vaginalis stimulates human macrophages to produce proinflammatory cytokines, such as IL-1, IL-6, and TNF-alpha, and NO. In particular, we showed that T. vaginalis induced TNF-alpha production in macrophages through NO-dependent activation of NF-kappaB, which might be closely involved in inflammation caused by T. vaginalis.

Proinflammatory Cytokine and Nitric Oxide Production by Human Macrophages Stimulated with Trichomonas vaginalis

Trichomonas vaginalis commonly causes vaginitis and perhaps cervicitis in women and urethritis in men and women. Macrophages are important immune cells in response to T. vaginalis infection. In this study, we investigated whether human macrophages could be involved in inflammation induced by T. vaginalis. Human monocyte-derived macrophages (HMDM) were co-cultured with T. vaginalis. Live, opsonized-live trichomonads, and T. vaginalis lysates increased proinflammatory cytokines, such as TNF-alpha, IL-1beta, and IL-6 by HMDM. The involvement of nuclear factor (NF)-kappaB signaling pathway in cytokine production induced by T. vaginalis was confirmed by phosphorylation and nuclear translocation of p65 NF-kappaB. In addition, stimulation with live T. vaginalis induced marked augmentation of nitric oxide (NO) production and expression of inducible NO synthase (iNOS) levels in HMDM. However, trichomonad-induced NF-kappaB activation and TNF-alpha production in macrophages were significantly inhibited by inhibition of iNOS levels with L-NMMA (NO synthase inhibitor). Moreover, pretreatment with NF-kappaB inhibitors (PDTC or Bay11-7082) caused human macrophages to produce less TNF-alpha. These results suggest that T. vaginalis stimulates human macrophages to produce proinflammatory cytokines, such as IL-1, IL-6, and TNF-alpha, and NO. In particular, we showed that T. vaginalis induced TNF-alpha production in macrophages through NO-dependent activation of NF-kappaB, which might be closely involved in inflammation caused by T. vaginalis.

Proinflammatory Cytokine and Nitric Oxide Production by Human Macrophages Stimulated with Trichomonas vaginalis

Trichomonas vaginalis commonly causes vaginitis and perhaps cervicitis in women and urethritis in men and women. Macrophages are important immune cells in response to T. vaginalis infection. In this study, we investigated whether human macrophages could be involved in inflammation induced by T. vaginalis. Human monocyte-derived macrophages (HMDM) were co-cultured with T. vaginalis. Live, opsonized-live trichomonads, and T. vaginalis lysates increased proinflammatory cytokines, such as TNF-alpha, IL-1beta, and IL-6 by HMDM. The involvement of nuclear factor (NF)-kappaB signaling pathway in cytokine production induced by T. vaginalis was confirmed by phosphorylation and nuclear translocation of p65 NF-kappaB. In addition, stimulation with live T. vaginalis induced marked augmentation of nitric oxide (NO) production and expression of inducible NO synthase (iNOS) levels in HMDM. However, trichomonad-induced NF-kappaB activation and TNF-alpha production in macrophages were significantly inhibited by inhibition of iNOS levels with L-NMMA (NO synthase inhibitor). Moreover, pretreatment with NF-kappaB inhibitors (PDTC or Bay11-7082) caused human macrophages to produce less TNF-alpha. These results suggest that T. vaginalis stimulates human macrophages to produce proinflammatory cytokines, such as IL-1, IL-6, and TNF-alpha, and NO. In particular, we showed that T. vaginalis induced TNF-alpha production in macrophages through NO-dependent activation of NF-kappaB, which might be closely involved in inflammation caused by T. vaginalis.