Cytokine Expression In Human Macrophages Research Articles

Cannflavin A inhibits TLR4-induced chemokine and cytokine expression in human macrophages

Cannflavin A (CFL-A), a flavonoid present in the hemp plant Cannabis sativa L. (C. sativa), has anti-inflammatory and neuroprotective capacity. Research continues to elucidate the anti-inflammatory effects of components of C. sativa, with evidence that plant-derived cannabinoids and terpenes can mediate anti-inflammatory activity by targeting toll-like receptor (TLR) signalling, the sensors of pathogen-associated molecules. This study set out to determine if TLR-mediated inflammatory signalling is a CFL-A target using the endotoxin lipopolysaccharide (LPS) to induce TLR4 signalling in human THP-1-derived macrophages. TLR4 activation promoted the production of the chemokine CXCL10 and cytokines IL-1β and TNFα. Treatment with CFL-A dose-dependently attenuated TLR4-induced CXCL10 and IL-1β secretion, with our findings also indicating that the inhibitory effects of CFL-A on chemokine/cytokine secretion are in line with an NF-κB inhibitor. This study highlights TLR4 signalling as a cannflavin target in macrophages.

Hydroxycarboxylic Acid Receptor Ligands Modulate Proinflammatory Cytokine Expression in Human Macrophages and Adipocytes without Affecting Adipose Differentiation.

Members of the hydroxycarboxylic acid receptor (HCA1-3) family are mainly expressed in adipocytes and immune cells. HCA2 ligand, niacin, has been used for decades as lipid-modifying drug. Recent studies suggest that HCA ligands can be involved in the modulation of inflammatory processes. In this study, we evaluated the effects of HCA1-3 ligands on adipose differentiation and cytokine expression in human adipocytes and macrophages. Simpson-Golabi-Behmel syndrome (SGBS) preadipocytes were induced to differentiate into adipocytes for 8 d in the presence or absence of HCA ligands and evaluated for lipid accumulation and adipogenic gene expression. The inhibitory effects of the ligands on the expression and production of cytokines were measured in lipopolysaccharide (LPS)-stimulated adipocytes and THP-1 macrophage cells. Preadipocytes treated with HCA ligands showed no changes in the capacity to differentiate into adipocytes and no significant alteration in peroxisome proliferator activated receptor γ (PPARγ) or its target gene expression. HCA2-3 ligands significantly downregulated LPS-induced expression of interleukin (IL)-6 (53-64%), tumor necrosis factor-α (TNF-α) (55-69%) and IL-8 (51-59%) in adipocytes and macrophages. IL-1β inhibition (58-68%) by HCA2-3 ligands was observed only in adipocytes. Furthermore, LPS increased the expression of HCA2-3 in adipocytes and macrophages and this expression was decreased by treatment with their ligands. These results suggest that HCA ligands modulated LPS-mediated pro-inflammatory gene expression in both macrophages and adipocytes without affecting adipogenesis. Therefore, targeting HCA2 and HCA3 would be beneficial in treating inflammation conditions associated with atherosclerosis and obesity.

Leptospira interrogans infection leads to IL-1β and IL-18 secretion from a human macrophage cell line through reactive oxygen species and cathepsin B mediated-NLRP3 inflammasome activation

Leptospirosis is a worldwide zoonosis caused by spirochetes from the genus Leptospira. Although there is a large diversity of clinical signs and symptoms, a severe inflammatory response is common to all leptospirosis patients. The mechanism of IL-1β secretion during Leptospira infection has been previously studied in mouse macrophages. However, the outcome of Leptospira infection is very different in human and murine macrophages, and the mechanisms responsible for IL-1β secretion in human macrophages had not been investigated. This study therefore examines the effects of Leptospira interrogans infection on inflammasome activation and proinflammatory cytokine expression in human macrophages. Increased mRNA and protein expression of NLRP3 was observed by real time RT-PCR and flow cytometry at 1 h after co-cultivation. Enzyme-linked immunosorbent assay (ELISA) determination showed that IL-1β and IL-18 are released in the culture supernatants at 1 h after cultivation. The inhibition assay showed that glybenclamide (a K+ efflux inhibitor that blocks NLRP3 inflammasome activation) and N-benzyloxycarbony-Val-Ala-Asp (O-methyl)-fluoromethylketone (Z-VAD-FMK; a caspase-1 inhibitor) and NLRP3 depletion with siRNAs reduced the levels of IL-1β and IL-18 release. Moreover, the levels of IL-1β and IL-18 production decreased in CA-074 (a cathepsin B inhibitor) and NAC (an anti-oxidant) pretreated human macrophages, compared to untreated controls. This study suggests that L. interrogans infection leads to reactive oxygen species (ROS)- and cathepsin B-dependent NLRP3 inflammasome activation, which subsequently mediates caspase-1 activation and IL-1β and IL-18 release.

Genome-wide siRNA screen of genes regulating the LPS-induced TNF-\u03b1 response in human macrophages

The mammalian innate immune system senses many bacterial stimuli through the toll-like receptor (TLR) family. Activation of the TLR4 receptor by bacterial lipopolysaccharide (LPS) is the most widely studied TLR pathway due to its central role in host responses to gram-negative bacterial infection and its contribution to endotoxemia and sepsis. Here we describe a genome-wide siRNA screen to identify genes regulating the human macrophage TNF-α response to LPS. We include a secondary validation screen conducted with six independent siRNAs per gene to facilitate removal of off-target screen hits. We also provide microarray data from the same LPS-treated macrophage cells to facilitate downstream data analysis. Tertiary screening with multiple TLR ligands and a microbial extract demonstrate that novel screen hits have broad effects on the innate inflammatory response to microbial stimuli. These data provide a resource for analyzing gene function in the predominant pathway driving inflammatory cytokine expression in human macrophages.

Dynamics of IL-12 cytokine expression in human macrophages after dioxin exposure

Interleukin IL-12 is a key proinflammatory cytokine synthesized by macrophages; however, information on the effect of dioxin on its expression is still fragmentary. The presence of previously identified potential dioxin-responsive elements (DREs) in the regulatory regions of IL12A and IL12B genes, encoding IL-12 subunits IL-12p35 and IL-12p40, respectively, suggests direct activation of these genes by binding of the dioxin/AhR/ARNT complex to DREs. In this work the binding capacity of these DREs was demonstrated by gel shift assay. The study of the dynamics of IL12A and IL12B gene expression in the human U937 macrophages revealed no influence of dioxin on IL12A expression. In contrast, activation of IL12B gene expression with subsequent inhibition was noted. The observed dynamics can be explained by direct activation of the expression by the dioxin-containing complex and subsequent inhibition of the expression as a result of oxidative stress caused by dioxin. Thus, the well-known effect of dioxin on the immune system can be associated with its differential influence on the expression dynamics of the genes encoding IL-12 subunits.

Modulation of cytokine expression in human macrophages by endocrine-disrupting chemical Bisphenol-A

Exposure to environmental endocrine-disrupting chemical Bisphenol-A (BPA) is often associated with dysregulated immune homeostasis, but the mechanisms remain unclear. In the present study, the effects of BPA on the cytokines responses of human macrophages were investigated. Treatment with BPA increased pro-inflammation cytokines tumor necrosis factor-α (TNF-α) and interleukin-6 (IL-6) production, but decreased anti-inflammation cytokines interleukin-10 (IL-10) and transforming growth factor-β (TGF-β) production in THP1 macrophages, as well as in primary human macrophages. BPA effected cytokines expression through estrogen receptor α/β (ERα/β)-dependent mechanism with the evidence of ERα/β antagonist reversed the expression of cytokines. We also identified that activation of extracellular regulated protein kinases (ERK)/nuclear factor κB (NF-κB) signal cascade marked the effects of BPA on cytokines expression. Our results indicated that BPA effected inflammatory responses of macrophages via modulating of cytokines expression, and provided a new insight into the link between exposure to BPA and human health.

Effect of the N-butanoyl glutathione (GSH) derivative and acyclovir on HSV-1 replication and Th1 cytokine expression in human macrophages

Macrophages are an important defense against in vivo herpes simplex virus (HSV) infection by early cytokine secretion; however, they can be infected by HSV-1 and they may be compromised in their ability to produce cytokines. In this paper, we studied the expression of two Th1 cytokines, interleukin (IL)-12 and IL-27, upon HSV-1 infection of human macrophages, and how it is regulated by treatment with two antiviral drugs exerting their anti-HSV-1 activity through different mechanisms of action. We found that infection does not alter intra-macrophage thiol content, while it induces mRNA expression of IL-12 p35 and IL-12 p40 as well as of IL-27 p28 and IL-27 EBI3, as revealed by RT-PCR. The increased expression of mRNA is accompanied by increased production of IL-12 p40 and IL-27 p28 protein, as detected in the culture supernatants by ELISA. The two antiviral drugs tested were acyclovir (ACV), commonly used to treat herpes virus infections, and an N-butanoyl glutathione (GSH) derivative, GSH-C4. While ACV inhibits viral DNA polymerase, GSH-C4 inhibits virus replication by interfering with protein folding and maturation of viral particles. Indeed, GSH-C4, altering the intracellular redox state, may modulate the Th1/Th2 balance favoring Th1-type response. Our data show that both drugs inhibit HSV-1 replication in macrophages, without significantly affecting cytokine mRNA levels. Nonetheless, lower levels of IL-12 p40 and IL-27 p28 proteins were found in the supernatants of macrophages treated with either GSH-C4 or ACV, likely as an indirect consequence of inhibited HSV-1 replication.

Differences in Cytokine Production in Human Macrophages and in Virulence in Mice Are Attributable to the Acidic Polymerase Protein of Highly Pathogenic Influenza A Virus Subtype H5N1

The pathogenesis of influenza A virus subtype H5N1 (hereafter, "H5N1") infection in humans is not completely understood, although hypercytokinemia is thought to play a role. We previously reported that most H5N1 viruses induce high cytokine responses in human macrophages, whereas some H5N1 viruses induce only a low level of cytokine production similar to that induced by seasonal viruses. To identify the viral molecular determinants for cytokine induction of H5N1 viruses in human macrophages, we generated a series of reassortant viruses between the high cytokine inducer A/Vietnam/UT3028II/03 clone 2 (VN3028IIcl2) and the low inducer A/Indonesia/UT3006/05 (IDN3006) and evaluated cytokine expression in human macrophages. Viruses possessing the acidic polymerase (PA) gene of VN3028IIcl2 exhibited high levels of hypercytokinemia-related cytokine expression in human macrophages, compared with IDN3006, but showed no substantial differences in viral growth in these cells. Further, the PA gene of VN3028IIcl2 conferred enhanced virulence in mice. These results demonstrate that the PA gene of VN3028IIcl2 affects cytokine production in human macrophages and virulence in mice. These findings provide new insights into the cytokine-mediated pathogenesis of H5N1 infection in humans.

Sex hormone modulation of proinflammatory cytokine and C-reactive protein expression in macrophages from older men and postmenopausal women

Inflammation plays a central role in the development and progression of coronary heart disease (CHD). The sex hormones estrogen and testosterone have been shown to modify the inflammatory response by influencing cytokine expression in human macrophages obtained from younger individuals. The effect of these hormones on the expression of proinflammatory markers in macrophages obtained from a CHD age-relevant population has not been studied. Human monocyte-derived macrophages (HMDMs) were obtained from healthy normolipidemic men and postmenopausal women (age 50-70 years), and cultured in autologous serum along with both physiological and supraphysiological concentrations of estrogen or testosterone. HMDMs were stimulated with oxidized low-density lipoproteins, and the expression of the cytokines tumor necrosis factor alpha (TNF-alpha or TNF), interleukin (IL)6, and IL-1 beta (IL1B) and of the acute-phase protein C-reactive protein (CRP) was measured. Both physiological and supraphysiological concentrations of testosterone reduced the expression and secretion of TNF-alpha and reduced the expression of IL-1 beta, but did not affect the expression of IL6 or CRP. Estrogen did not modify the expression of TNF-alpha, IL6, and IL-1 beta. Estrogen caused a variable response in CRP expression that was positively associated with the plasma small dense LDL-cholesterol concentration of the donors. There were no gender differences in any of the observed effects. Our results indicate that testosterone may exert anti-inflammatory effects by reducing macrophage TNF-alpha expression, while the effects of estrogen on macrophage CRP expression may depend upon the extracellular lipid environment.

Plasmin Triggers Cytokine Induction in Human Monocyte-Derived Macrophages

Fibrinolytic activity is upregulated in atherosclerotic lesions, yet little is known about the role of plasmin in macrophage function. We postulated a direct effect of plasmin on human monocyte-derived macrophages. Plasmin activates macrophages via the annexin A2 heterotetramer composed of annexin A2 and S100A10 with subsequent stimulation of Janus kinase JAK1/TYK2 signaling. JAK1/TYK2 leads to STAT3 activation, Akt-dependent NF-kappaB activation, and phosphorylation of extracellular signal-regulated kinase 1/2 and mitogen-activated kinase p38. These signaling pathways trigger nuclear translocation of STAT3 and p65 transcription factors and the induction of the proinflammatory cytokines tumor necrosis factor-alpha and IL-6. Inhibitors of JAK, p38, and NF-kappaB revealed that these signaling pathways are indispensable for the plasmin-mediated tumor necrosis factor-alpha and IL-6 induction. By contrast, the extracellular signal-regulated kinase 1/2 activation is essential only for the IL-6 expression. The activation clearly depends on the proteolytic activity of plasmin, which cleaves the A2 subunit of the annexin A2 heterotetramer. Downregulation of each of the receptor subunits by antisense oligodeoxynucleotides abolished the plasmin-induced expression of proinflammatory cytokines stressing the crucial role the annexin A2 heterotetramer. Plasmin generated at sites of inflammation such as atherosclerotic lesions will trigger cytokine expression in human macrophages.

The Contact of Human Macrophages with Extracellular Matrix Proteins Selectively Induces Expression of Proinflammatory Cytokines

The interaction of macrophages with proteins of the extracellular matrix (ECM) is important for the regulation of the immune and nonimmune functions displayed by these cells. Little, however, is known about the ability of different ECM proteins to transmit inflammatory signals into macrophages. Here we investigated the effect of the ECM proteins collagen type I, fibrin and fibronectin on the expression of the proinflammatory cytokines interleukin-1β (IL-1β) and IL-8 using RT-PCR, Northern and Western blot analysis and ELISA technique. It was found that collagen strongly induced IL-1β and IL-8 expression in the macrophages. Fibronectin also stimulated cytokine expression, however, the amounts of the specific mRNAs were significantly lower compared to those induced by collagen. On the protein level IL-1β revealed a close correlation to the mRNA expression. In contrast, fibrin did not elicit any IL-1β and IL-8 response. These data show that different ECM proteins vary in their ability to induce proinflammatory cytokine expression in human macrophages suggesting that the protein composition of the ECM might be crucial in the initiation of inflammatory processes.