Gene Expression In Monocyte-derived Macrophages Research Articles

Extracellular Vesicles Secreted by Adipose Tissue during Obesity and Type 2 Diabetes Mellitus Influence Reverse Cholesterol Transport-Related Gene Expression in Human Macrophages.

Obesity is a risk factor for type 2 diabetes mellitus (T2DM) and cardiovascular disease (CVD). Adipose tissue (AT) extracellular vesicles (EVs) could play a role in obesity and T2DM associated CVD progression via the influence of their specific cargo on gene expression in recipient cells. The aim of this work was to evaluate the effects of AT EVs of patients with obesity with/without T2DM on reverse cholesterol transport (RCT)-related gene expression in human monocyte-derived macrophages (MDMs) from healthy donors. AT EVs were obtained after ex vivo cultivation of visceral and subcutaneous AT (VAT and SAT, respectively). ABCA1, ABCG1, PPARG, LXRβ (NR1H2), and LXRα (NR1H3) mRNA levels in MDMs as well as in origine AT were determined by a real-time PCR. T2DM VAT and SAT EVs induced ABCG1 gene expression whereas LXRα and PPARG mRNA levels were simultaneously downregulated. PPARG mRNA levels also decreased in the presence of VAT EVs of obese patients without T2DM. In contrast ABCA1 and LXRβ mRNA levels tended to increase with the addition of obese AT EVs. Thus, AT EVs can influence RCT gene expression in MDMs during obesity, and the effects are dependent on T2DM status.

TNF-α and IL-10 Control CXCL13 Expression in Human Macrophages

The chemokine CXCL13 controls the normal organization of secondary lymphoid tissues and the neogenesis of ectopic lymphoid structures in nonlymphoid organs, particularly the lungs. The progression and severity of idiopathic pulmonary fibrosis (IPF), a fatal and irreversible interstitial lung disease, is predicted by the circulating blood concentrations of CXCL13. Although CXCL13 is produced by pulmonary tissues, it has not been determined which cells are involved. This study examines CXCL13 production by lung tissue macrophages from patients with IPF and the signaling pathways controlling CXCL13 gene expression in human alveolar macrophages (AM) and monocyte-derived macrophages (MoDM). CXCL13 is found in CD68- and CD206-positive AM from patients with IPF, and the CXCL13 gene is induced in these macrophages and MoDM when they are stimulated with LPS. We found that TNF-α and IL-10 control optimal CXCL13 gene expression in MoDM and possibly in AM by activating the NF-κB and JAK/STAT pathways, respectively. We also found that blood TNF-α and CXCL13 concentrations are significantly correlated in patients with IPF, suggesting that TNF-α contributes to CXCL13 production in humans. In conclusion, the results of this study demonstrate that AM from patients with IPF produces CXCL13 and that the NF-κB and JAK/STAT pathways are required to induce the expression of this major chemokine.

Monocyte derived macrophages as an appropriate model for porcine cytomegalovirus immunobiology studies

Porcine cytomegalovirus (PCMV) causes lifelong latent infections in swine. The pathogen is occasionally associated with inclusion body rhinitis and pneumonia in piglets, reproductive disorders in pregnant sows and respiratory disease complex in older pigs. Immunosuppressive potential of PCMV infection is discussed. Macrophages were recognised as one of target cell types where propagation of virus occurs. The aim of present study was to set up model PCMV infection of monocyte derived macrophages (MDMs) in vitro for PCMV immunobiology research. Obtained results showed that PCMV is able to infect and propagate in MDMs. Possible immunosuppressive effect of PCMV on infected macrophages was evaluated by measurement of immune relevant gene expression in MDMs. Infection decreased expression of IL-8 and TNF-α (pro-inflammatory cytokines) and increased expression of IL-10 (anti-inflammatory cytokine) on mRNA transcription level. Obtained data support hypothesis that higher sensitivity of animals to coinfection with other swine pathogens and its more severe clinical manifestations could potentially be the consequence of PCMV infection.

Loperamide Restricts Intracellular Growth of Mycobacterium tuberculosis in Lung Macrophages.

New approaches for improving tuberculosis (TB) control using adjunct host-directed cellular and repurposed drug therapies are needed. Autophagy plays a crucial role in the response to TB, and a variety of autophagy-inducing drugs that are currently available for various medical conditions may serve as an adjunct treatment in pulmonary TB. Here, we evaluated the potential of loperamide, carbamazepine, valproic acid, verapamil, and rapamycin to enhance the antimicrobial immune response to Mycobacterium tuberculosis (Mtb). Human monocyte-derived macrophages (MDMs) and murine alveolar cells (MACs) were infected with Mtb and treated with loperamide, carbamazepine, valproic acid, verapamil, and rapamycin in vitro. Balb/c mice were intraperitoneally administered loperamide, valproic acid, and verapamil, and MACs were infected in vitro with Mtb. The induction of autophagy, the containment of Mtb within autophagosomes and the intracellular Mtb burden were determined. Autophagy was induced by all of the drugs in human and mouse macrophages, and loperamide significantly increased the colocalization of microtubule-associated protein 1 light chain 3 with Mtb in MDMs. Carbamazepine, loperamide, and valproic acid induced microtubule-associated protein 1 light chain 3 and autophagy related 16- like protein 1 gene expression in MDMs and in MACs. Loperamide also induced a reduction in TNF-α production. Loperamide and verapamil induced autophagy, which was associated with a significant reduction in the intracellular growth of Mtb in MACs and alveolar macrophages. The intraperitoneal administration of loperamide and valproic acid induced autophagy in freshly isolated MACs. The antimycobacterial activity in MACs was higher after loperamide treatment and was associated with the degradation of p62. In conclusion, loperamide shows potential as an adjunctive therapy for the treatment of TB.

Reduced expression of TLR3, TLR10 and TREM1 by human macrophages in Chronic cavitary pulmonary aspergillosis, and novel associations of VEGFA, DENND1B and PLAT

Chronic cavitary pulmonary aspergillosis (CCPA) is an uncommon but serious pulmonary disease of humans, with an annual mortality rate of 10–30%. It is caused by the fungus Aspergillus fumigatus. Patients are overtly immunocompetent; however, some immunogenetic defect is likely. To investigate this, we performed a genetic association study analysing biologically plausible candidate genes in 112 CCPA patients and 279 healthy controls, and investigated gene expression in monocyte-derived macrophages from patients and controls at baseline and during stimulation with A. fumigatus. Single-nucleotide polymorphisms (SNPs) associated with CCPA were found in TLR1, CLEC7A (dectin-1), PLAT (n = 2), VEGFA, and DENND1B. Macrophages from CCPA patients showed low TLR3 and TLR10 expression and high TREM1 expression at baseline, as compared with macrophages from healthy subjects, with major expression differences being seen in most Toll-like receptors (TLRs) during 9 h of co-culture with A. fumigatus. The differences in baseline expression between the healthy and CCPA groups suggest roles for TLR3 and TLR10 in susceptibility to CCPA, and the association of SNPs in PLAT (n = 2), VEGFA and DENND1B supports novel roles for plasminogen activation and angiogenesis and of these genes specifically in susceptibility to CCPA.

A prominent role for the IL1 pathway and IL15 in susceptibility to chronic cavitary pulmonary aspergillosis

Chronic cavitary pulmonary aspergillosis (CCPA) is a progressive lung condition with a 10–30% annual mortality. Although overtly immunocompetent, some immunogenetic defect in patients is likely. To investigate a possible immunogenetic defect in CCPA, we analysed biologically plausible candidate genes in 112 CCPA patients and 279 healthy controls in a genetic association study of genes involved in the post-recognition immune response to Aspergillus fumigatus. We also compared gene expression in monocyte-derived macrophages from subjects with and without disease, both at baseline and during stimulation with A. fumigatus. Compared with macrophages from healthy subjects, CCPA macrophages showed unrestrained rises in IL1A, IL1B, IL6, IRAK2 and TRAF6 throughout the experiment, and a lack of expression of TGFB1 at 9 h. Single nucleotide polymorphisms (SNPs) associated with CCPA were found in IL1B (n = 2), IL1RN and IL15 (n = 3). Uncontrolled expression of IL1 and IL6 and continuing high levels of these cytokines may result in continuing cellular influx and pro-inflammatory responses, inhibiting disease resolution and contributing to disease progression in CCPA. The association of SNPs in IL1B, IL1RN and IL15 with CCPA supports a role for the IL1 pathway, as well as implicating the IL15 gene, in susceptibility to CCPA.

Regulation of Pattern Recognition Receptors by the Apolipoprotein A-I Mimetic Peptide 4F

The apolipoprotein A-I (apoA-I) mimetic peptide 4F favors the differentiation of human monocytes to an anti-inflammatory phenotype and attenuates lipopolysaccharide (LPS)-induced inflammatory responses. We investigated the effects of LPS on the Toll-like receptor (TLR) signaling pathway in 4F-differentiated monocyte-derived macrophages. Monocyte-derived macrophages were pretreated with 4F or vehicle for 7 days. 4F downregulated cell-surface TLRs (4, 5, and 6) as determined by flow cytometry. 4F attenuated the LPS-dependent upregulation of genes encoding TLR1, 2, and 6 and genes of the MyD88-dependent (CD14, MyD88, TRAF6, interleukin-1 receptor-associated kinase 4, and inhibitor of kappa light polypeptide gene enhancer in B-cells, kinase beta) and MyD88-independent (interferon regulatory factor 3, TANK-binding kinase 1, and Toll-interleukin 1 receptor domain-containing adaptor-inducing interferon-β) pathways as determined by microarray analysis and quantitative reverse transcriptase polymerase chain reaction. Functional analyses of monocyte-derived macrophages showed that 4F reduced LPS-dependent TLR4 recycling, phosphorylation of nuclear factor of kappa light polypeptide gene enhancer in B-cells inhibitor, alpha, activation and translocation of nuclear factor-κB and inhibited the secretion of tumor necrosis factor-α and interleukin-6 induced by LPS or lipoteichoic acid. These changes were associated with depletion of cellular cholesterol and caveolin, components of membrane lipid rafts. These data suggest that disruption of rafts by 4F alters the assembly of TLR-ligand complexes in cell membranes and inhibits proinflammatory gene expression in monocyte-derived macrophages, thus attenuating the responsiveness of macrophages to LPS.

A large-scale study of differential gene expression in monocyte-derived macrophages infected with several strains of Mycobacterium avium subspecies paratuberculosis

Mycobacterium avium subspecies paratuberculosis (MAP) is a significant concern to the American and European dairy industries and possibly to human health. MAP possesses the rare ability to survive and replicate in infected macrophages, cells that are typically able to destroy pathogens. Little is known about what changes occur in MAP-infected macrophages that prevent phagosome maturation and lead to intracellular survival of the bacteria. In this study, a bovine immunologically specific cDNA microarray was used to study genes whose expression was altered in monocyte-derived macrophages (MDM) when these cells were infected with 10 different strains of MAP bacteria. Although we used MAP strains isolated from four different host species, cluster analysis of each strains influence in infected MDMs showed no species of origin specific MAP alterations in the host transcriptome. However, MAP strain K10 was observed as a clear outlier in the cluster analysis. Additionally, we observed two SuperShedder MAP strains clustering very closely together compared to the other strains in this study. Overall, microarray analysis yielded 78 annotated genes whose expression was altered by MAP infection, regardless of strain. Few of these genes have been previously studied in the context of Johne's disease or other mycobacterium-caused diseases. Large groups of apoptosis genes, transcription factors and cytokines were found to be differentially expressed in infected monocyte-derived macrophages as well as several genes not previously linked to MAP-host interactions. Identifying novel host genes affected by MAP infection of macrophages may lead to a more complete picture of this complex host-pathogen interaction.

Increased expression of the macrophage markers and of 11β-HSD-1 in subcutaneous adipose tissue, but not in cultured monocyte-derived macrophages, is associated with liver fat in human obesity

To determine whether increased expression of macrophage markers and of inflammatory markers in subcutaneous adipose tissue is associated with liver fat in human obesity. We also determined whether expression of TNF (gene encoding TNF-alpha), HSD11B1 (gene encoding 11beta-HSD-1) and RETN (gene encoding resistin) in cultured monocyte-derived macrophages differs between obese/overweight and non-obese subjects. Cross-sectional comparison of obese/overweight and non-obese subjects with respect to adipose tissue gene expression, gene expression in monocyte-derived macrophages, liver fat content and in vivo insulin sensitivity. Adipose tissue gene expression, gene expression in monocyte-derived macrophages, liver fat content and in vivo insulin sensitivity: 10 healthy non-obese (24.2+/-1.0 kg/m(2)) and 10 healthy obese/overweight (33.1+/-1.7 kg/m(2)) women. Gene expression in monocyte-derived macrophages: seven healthy non-obese (22.1+/-0.7 kg/m(2)) and seven healthy obese/overweight (36.9+/-2.2 kg/m(2)) women. Adipose tissue biopsies and blood samples for isolation of peripheral mononuclear cells were taken after an overnight fast. Liver fat content was measured using magnetic resonance proton spectroscopy. Whole body insulin sensitivity was measured using the hyperinsulinemic euglycemic clamp technique. Expression levels of TNF, HSD11B1, RETN and the macrophage markers CD68 and ITGAM were determined by real-time PCR. In adipose tissue, expression of HSD11B1, ITGAM and CD68 was significantly increased in the obese/overweight as compared to the non-obese group. Expression of all these genes was closely positively correlated with liver fat content and inversely correlated with whole body insulin sensitivity. The associations between expression of CD68, ITGAM and HSD11B1 and liver fat were independent of obesity. There were no differences in TNF, HSD11B1, RETN or CD68 gene expression basally or after stimulation with lipopolysaccharide in monocyte-derived macrophages between obese/overweight and non-obese subjects. Accumulation of fat in the liver is associated with increased adipose tissue inflammation independent of obesity.

Peroxisome Proliferator-activated Receptor γ Agonists Inhibit HIV-1 Replication in Macrophages by Transcriptional and Post-transcriptional Effects

Previous studies have demonstrated that cyclopentenone prostaglandins (cyPG) inhibit human immunodeficiency virus type 1 (HIV-1) replication in various cell types. We investigated the role of PG in the replication of HIV-1 in primary macrophages. The cyPG, PGA(1) and PGA(2), inhibited HIV-1 replication in acutely infected human monocyte-derived macrophages (MDM). Because PGA(1) and PGA(2) have previously been shown to be peroxisome proliferator-activated receptor gamma (PPARgamma) agonists, we examined the effect of synthetic PPARgamma agonists on HIV replication. The PPARgamma agonist ciglitazone inhibited HIV-1 replication in a dose-dependent manner in acutely infected human MDM. In addition, cyPG and ciglitazone reduced HIV replication in latently infected and viral entry-independent U1 cells, suggesting an effect at the level of HIV gene expression. Ciglitazone also suppressed HIV-1 mRNA levels as measured by reverse transcriptase PCR, in parallel with the decrease in reverse transcriptase activity. Co-transfection of PPARgamma wild type vectors and treatment with PPARgamma agonists inhibited HIV-1 promoter activity in U937 cells. Activation of PPARgamma also decreased HIV-1 mRNA stability following actinomycin D treatment. In summary, our experimental findings implicate PPARgamma as an important factor in the suppression of HIV-1 gene expression in MDM by cyPG. Thus natural and synthetic PPARgamma agonists may play a role in controlling HIV-1 infection in macrophages.

N-acetyl-L-cysteine-induced up-regulation of HIV-1 gene expression in monocyte-derived macrophages correlates with increased NF-kappaB DNA binding activity.

Nuclear factor kappaB (NF-kappaB) is an important cellular regulator of human immunodeficiency virus (HIV) gene expression. In T cells, N-acetyl-L-cysteine (NAC) inhibits the induction of NF-kappaB and transcription of HIV-1. However, NAC up-regulates HIV-1 replication in monocyte-derived macrophages (MDM). In this study we demonstrate that NAC treatment of MDM transfected with a chloramphenicol acetyltransferase (CAT) construct under transcriptional control of the HIV-1 long terminal repeat resulted in an up-regulation of CAT activity. Furthermore, MDM transfected with a HIV-1-NF-kappaB-CAT construct also produced increased CAT activity after NAC treatment. In addition, electrophoretic mobility shift assays revealed that nuclei of NAC-treated MDM contained increased binding activity to wild-type, but not mutant, kappaB oligonucleotides. Components of the binding activity were identified with antibodies as the NF-kappaB subunits p50 and p65. These data indicate that NAC-induced enhancement of HIV-1 replication in MDM is regulated at the level of viral gene expression and mediated by NF-kappaB.

Variations in lentiviral gene expression in monocyte-derived macrophages from naturally infected sheep.

Seventy-nine 1-year-old lambs from three individual farms and a feedlot were examined for natural lentivirus infection. We used three different methods to detect infection and to identify the stage of the ovine lentivirus life cycle in blood-derived macrophages. Cytopathic infectious virus was obtained from 14/14 Border Leicester animals obtained from a naturally infected flock. Neither virus particles, virus proteins, virus specific antibodies nor viral DNA were detected in samples from 34 lambs from two South Kansas City farms. However, among 31 feedlot lambs, we identified 11 infected animals. Specific viral proteins were immunoprecipitated from macrophages of one animal, but no infectious cytopathic virus was isolated from these cells. Cells from ten of the other feedlot animals harboured viral DNA but neither viral particles nor proteins could be detected by our techniques. Thus, in these naturally infected animals, the virus life cycle either proceeded to completion, subject to differentiation of infected precursor cells in blood, or remained arrested at the DNA stage despite maturation of monocytes to macrophages. Sequence analysis of the env gene of viral genomes from two of the ten feedlot sheep showed sequences distinct from those of known ovine and caprine lentiviruses. Surprisingly, these sequences have a higher identity (of nucleotide and derived amino acid sequences) to caprine arthritis-encephalitis virus than to the ovine prototype, maedi-visna virus. These data suggest that the ovine and caprine lentiviruses found in North American sheep may have a common ancestral genotype that is closely related to the caprine virus.