Human Monocyte Adhesion Research Articles

Antiviral and Immunomodulatory Effects of Norantea brasiliensis Choisy on Dengue Virus-2

Background/Aims: Severe dengue fever is a result of exacerbated immune responses and no specific treatments are available. We evaluated the antiviral and immunomodulatory effects of Norantea brasiliensis Choisy. Methods: Human adherent monocytes infected in vitro with dengue virus (DENV)-2 were incubated with the crude ethanol extract from leaves (NB1) or 3 derived fractions: dichloromethane (NB3), ethyl acetate (NB5), and butanolic (NB6) partitions. The antiviral and immunomodulatory activities were determined by intracellular detection of DENV antigen within monocytes and by secreted NS1 viral protein and cytokines. Results: The crude extract alone exhibited both antiviral activities (intracellular and secreted antigens) and all fractions derived from this extract modulated NS1 production. Regarding the immunomodulatory effect, among the secreted factors, TNF-α was inhibited by NB3 and NB6; IL-6 was inhibited by NB1, NB3, and NB6; IL-10 by NB1 and NB3; and IFN-α by NB6. The crude extract (NB1) presented the best antiviral effect, whereas the dichloromethane fraction (NB3) presented an immunomodulatory effect in the inflammatory and anti-inflammatory cytokines. Conclusion: During in vitro DENV infection, N. brasiliensis Choisy exerts both antiviral and immunomodulatory effects that are likely associated, considering that less viral load may lead to less immunostimulation.

Abstract 14296: Site-targeting Nitroglycerin Nanotherapeutic for Local Immunosuppression Without Induction of Tolerance

Nitroglycerin (NTG) markedly enhances nitric oxide (NO) bioavailability. However, its ability to mimic the anti-inflammatory properties of NO remains unknown. Here, we examined whether NTG can suppress endothelial cell (EC) activation during inflammation and developed NTG nanoformulation to simultaneously amplify its anti-inflammatory effects and ameliorate adverse effects associated with high-dose NTG administration. Our findings reveal that NTG significantly inhibits human monocyte adhesion to NO-deficient human microvascular ECs in vitro (EC50 = 0.64 uM) through an increase in endothelial NO and decrease in endothelial ICAM-1 clustering, as determined by NO analyzer, microfluorimetry, and immunofluorescence staining. Nanoliposomal NTG (NTG-NL) was formulated by encapsulating NTG within unilamelar lipid nanoparticles that were ~150 nm in diameter and readily uptaken by ECs, as determined by dynamic light scattering and quantitative fluorescence microscopy, respectively. More importantly, NTG-NL produced an approximately two orders of magnitude greater anti-inflammatory effect than free NTG while preventing excessive mitochondrial superoxide production and loss of arterial vasorelaxation associated with high NTG doses. Finally, to facilitate targeting of NTG-NL to inflamed ICAM-1-expressing vessels, we have tethered a non-immunogenic fragment of ICAM-1 antibody to the surface of NTG-NL. Our preliminary in vitro studies show that NTG-NL modified with anti-ICAM-1 fragment exhibits 6-fold greater binding to inflamed (ICAM-1-expressing) ECs than to normal ECs. Thus, by identifying the superior therapeutic effects of NTG nanoformulation and conferring potent site-targeting capability to it, this study provides the rationale for detailed investigation of NTG nanotherapeutic as a potentially superior anti-inflammatory therapy.

α1-Antitrypsin Combines with Plasma Fatty Acids and Induces Angiopoietin-like Protein 4 Expression.

α1-Antitrypsin (A1AT) purified from human plasma upregulates expression and release of angiopoietin-like protein 4 (Angptl4) in adherent human blood monocytes and in human lung microvascular endothelial cells, providing a mechanism for the broad immune-regulatory properties of A1AT independent of its antiprotease activity. In this study, we demonstrate that A1AT (Prolastin), a potent inducer of Angptl4, contains significant quantities of the fatty acids (FA) linoleic acid (C18:2) and oleic acid (C18:1). However, only trace amounts of FAs were present in preparations that failed to increase Angplt4 expression, for example, A1AT (Zemaira) or M-type A1AT purified by affinity chromatography. FA pull-down assays with Western blot analysis revealed a FA-binding ability of A1AT. In human blood-adherent monocytes, A1AT-FA conjugates upregulated expression of Angptl4 (54.9-fold, p < 0.001), FA-binding protein 4 (FABP4) (11.4-fold, p < 0.001), and, to a lesser degree, FA translocase (CD36) (3.1-fold, p < 0.001) relative to A1AT devoid of FA (A1AT-0). These latter effects of A1AT-FA were blocked by inhibitors of peroxisome proliferator-activated receptor (PPAR) β/δ (ST247) and PPARγ (GW9662). When compared with controls, cell pretreatment with ST247 diminished the effect of A1AT-LA on Angptl4 mRNA (11.6- versus 4.1-fold, p < 0.001) and FABP4 mRNA (5.4- versus 2.8-fold, p < 0.001). Similarly, preincubation of cells with GW9662 inhibited inducing effect of A1AT-LA on Angptl4 mRNA (by 2-fold, p < 0.001) and FABP4 mRNA (by 3-fold, p < 0.001). Thus, A1AT binds to FA, and it is this form of A1AT that induces Angptl4 and FABP4 expression via a PPAR-dependent pathway. These findings provide a mechanism for the unexplored area of A1AT biology independent of its antiprotease properties.

Arsenic Exposure Increases Monocyte Adhesion to the Vascular Endothelium, a Pro-Atherogenic Mechanism.

Epidemiological studies have shown that arsenic exposure increases atherosclerosis, but the mechanisms underlying this relationship are unknown. Monocytes, macrophages and platelets play an important role in the initiation of atherosclerosis. Circulating monocytes and macrophages bind to the activated vascular endothelium and migrate into the sub-endothelium, where they become lipid-laden foam cells. This process can be facilitated by platelets, which favour monocyte recruitment to the lesion. Thus, we assessed the effects of low-to-moderate arsenic exposure on monocyte adhesion to endothelial cells, platelet activation and platelet-monocyte interactions. We observed that arsenic induces human monocyte adhesion to endothelial cells in vitro. These findings were confirmed ex vivo using a murine organ culture system at concentrations as low as 10 ppb. We found that both cell types need to be exposed to arsenic to maximize monocyte adhesion to the endothelium. This adhesion process is specific to monocyte/endothelium interactions. Hence, no effect of arsenic on platelet activation or platelet/leukocyte interaction was observed. We found that arsenic increases adhesion of mononuclear cells via increased CD29 binding to VCAM-1, an adhesion molecule found on activated endothelial cells. Similar results were observed in vivo, where arsenic-exposed mice exhibit increased VCAM-1 expression on endothelial cells and increased CD29 on circulating monocytes. Interestingly, expression of adhesion molecules and increased binding can be inhibited by antioxidants in vitro and in vivo. Together, these data suggest that arsenic might enhance atherosclerosis by increasing monocyte adhesion to endothelial cells, a process that is inhibited by antioxidants.

Abstract 211: IL-35 Suppresses Endothelial Cell Activation by Inhibiting Histone H3K14 Acetylation and AP-1

Background: IL-35 is a newly identified anti-inflammatory cytokine, which inhibits inflammation and immune responses by inducing regulatory T cells and regulatory B cells and suppressing effector T cells and macrophages. We previously reported that IL-35 is a responsive cytokine that is not constitutively expressed in human tissues, but can be induced by proinflammatory stimuli in vascular endothelial cells (EC), smooth muscle cells and monocytes. However, the functions of IL-35 on non-lymphocytes such as vascular EC remain unknown. EC activation induced by proatherogenic stimuli including lysophosphatidylcholine (LPC) is considered as the initiation step of monocyte recruitment and atherosclerosis. In this study, we examined the expression of IL-35 during early atherosclerosis and the role of IL-35 in LPC-induced EC activation. Methods and Results: Using microarray method, we found that two IL-35 subunits and their receptor subunits are all significantly induced during early atherosclerosis in the aortas of atherosclerotic mouse model, apolipoprotein E (ApoE) knockout mice fed with 3 weeks of high fat diet. In addition, we found that IL-35 can suppress LPC-induced monocyte adhesion to human aortic ECs (HAEC). Mechanistic, our analysis using EC biology gene array shows that IL-35 selectively inhibits LPC-induced expressions of EC adhesion molecules ICAM-1, VCAM-1, and proinflammatory cytokine IL-1β in human aortic EC (HAEC). To further dissect the molecular mechanisms underlying the IL-35 inhibitory effects on LPC-induced EC activation gene upregulation, we performed mass spectrum analysis and electrophoretic mobility shift assay. We found that IL-35 blocked LPC-induced histone H3 lysine 14 (H3K14) acetylation as well as the binding of pro-inflammatory transcription factor AP-1 to the promoters of EC adhesion molecules in HAEC. Conclusions: IL-35 is induced in the aortas during early atherogenesis and suppresses human EC activation and monocyte adhesion by inhibiting histone acetylation and AP-1 nuclear translocation.

The prion protein inhibits monocytic cell migration by stimulating β1 integrin adhesion and uropod formation

The broad tissue distribution and evolutionary conservation of the glycosylphosphatidylinositol (GPI)-anchored prion protein (PrP, also known as PRNP) suggests that it plays a role in cellular homeostasis. Given that integrin adhesion determines cell behavior, the proposed role of PrP in cell adhesion might underlie the various in vitro and in vivo effects associated with PrP loss-of-function, including the immune phenotypes described in PrP(-/-) mice. Here, we investigated the role of PrP in the adhesion and (transendothelial) migration of human (pro)monocytes. We found that PrP regulates β1-integrin-mediated adhesion of monocytes. Additionally, PrP controls the cell morphology and migratory behavior of monocytes: PrP-silenced cells show deficient uropod formation on immobilized VCAM and display bleb-like protrusions on the endothelium. Our data further show that PrP regulates ligand-induced integrin activation. Finally, we found that PrP controls the activation of several proteins involved in cell adhesion and migration, including RhoA and its effector cofilin, as well as proteins of the ERM family. We propose that PrP modulates β1 integrin adhesion and migration of monocytes through RhoA-induced actin remodeling mediated by cofilin, and through the regulation of ERM-mediated membrane-cytoskeleton linkage.

Milk-derived bioactive peptides inhibit human endothelial-monocyte interactions via PPAR-γ dependent regulation of NF-κB.

BackgroundMilk-derived bioactive peptides retain many biological properties and have therapeutic effects in cardiovascular disorders such as atherosclerosis. Under inflammatory conditions the expression of endothelial cells adhesion molecules is induced, increasing monocyte adhesion to human vessel wall, a critical step in the pathogenesis of atherosclerosis. In the present work we explored the effects of milk-derived bioactive peptides on the expression of the inflammatory phenotype of human endothelial cells and their effects on monocyte adherence to endothelial cells.ResultsTreatment of endothelial cells with milk-derived hydrolysate inhibited their production of inflammatory proteins MCP-1 and IL-8 and expression of VCAM-1, ICAM-1 and E-selectin. Milk derived hydrolysate also attenuated the adhesion of human monocytes to activated endothelial cells. The effect was similar to that obtained in endothelial cells treated with troglitazone, a ligand of peroxisome proliferators-activator receptor-gamma (PPAR-γ). PPAR-γ is a transcription factor which when activated antagonises the pro-inflammatory capability of nuclear factor κB (NF-κB). We further examined whether the effects of milk-derived hydrolysates on endothelial cells may be mediated through NF-κB activation via a PPAR-γ dependent mechanism. The specific PPAR-γ inhibitor, GW9662 blocked the effects of the hydrolysate on the NF-κB-mediated chemokines and adhesion molecules expression in endothelial cells.ConclusionsThese results suggest that milk-derived bioactive peptides work as anti-atherogenic agents through the inhibition of endothelial-dependent adhesive interactions with monocytes by inhibiting the NF-κB pathway through a PPAR-γ dependent mechanism.Electronic supplementary materialThe online version of this article (doi:10.1186/s12950-014-0044-1) contains supplementary material, which is available to authorized users.

Suppressive effects of induced pluripotent stem cell-conditioned medium on in vitro hypertrophic scarring fibroblast activation.

Hypertrophic scarring (HS) is a type of fibrosis that occurs in the skin, and is characterized by fibroblast activation and excessive collagen production. However, at present, therapeutic strategies for this condition are ineffective. Previous studies have identified that the mutual regulation of chronic inflammation, mechanical force and fibroblast activation leads to the formation of HS. Induced pluripotent stem cells (iPSCs) are novel bioengineered embryonic-like stem cells, initially created from mouse adult fibroblasts. The current study demonstrated that iPSC-conditioned medium (iPSC-CM) may significantly suppress hypertrophic scar fibroblast activation. It was observed that in the presence of iPSC-CM, the level of collagen I was markedly reduced and α-smooth muscle actin, a marker for myofibroblasts (activated fibroblasts that mediate mechanical force-induced HS formation), exhibited a significantly lower level of expression in human dermal fibroblasts (HDFs) activated with transforming growth factor-β1. Additionally, iPSC-CM attenuated the local inflammatory cell response by blocking the adhesion of human acute monocytic leukemia cell monocytes and fibroblasts in vitro. In addition, the contractile ability of HDFs may be reduced by iPSC-CM. These observations suggest that iPSC-CM may protect against processes leading to hypertrophic scarring by attenuating fibroblast activation, blocking inflammatory cell recruitment and adhesion and reducing the contractile ability of fibroblasts.

Poly(ADP-ribose) polymerase-1 inhibition in brain endothelium protects the blood-brain barrier under physiologic and neuroinflammatory conditions.

Blood-brain barrier (BBB) dysfunction seen in neuroinflammation contributes to mortality and morbidity in multiple sclerosis, encephalitis, traumatic brain injury, and stroke. Identification of molecular targets maintaining barrier function is of clinical relevance. We used a novel in vivo model of localized aseptic meningitis where tumor necrosis factor alpha (TNFα) was introduced intracerebrally and surveyed cerebral vascular changes and leukocyte-endothelium interactions by intravital videomicroscopy. Poly(ADP-ribose) polymerase-1 (PARP) inhibition significantly reduced leukocyte adhesion to and migration across brain endothelium in cortical microvessels. PARP inactivation diminished BBB permeability in an in vivo model of systemic inflammation. PARP suppression in primary human brain microvascular endothelial cells (BMVEC), an in vitro model of BBB, enhanced barrier integrity and augmented expression of tight junction proteins. PARP inhibition in BMVEC diminished human monocyte adhesion to TNFα-activated BMVEC (up to 65%) and migration (80-100%) across BBB models. PARP suppression decreased expression of adhesion molecules and decreased activity of GTPases (controlling BBB integrity and monocyte migration across the BBB). PARP inhibitors down-regulated expression of inflammatory genes and dampened secretion of pro-inflammatory factors increased by TNFα in BMVEC. These results point to PARP suppression as a novel approach to BBB protection in the setting of endothelial dysfunction caused by inflammation.

Haemozoin enhances MMP-9 production through MAPK p38-dependent mechanisms in human adherent monocytes

In human adherent monocytes matrix metalloproteinase-9 (MMP-9) expression and secretion is upregulated by the lipid moiety of natural haemozoin (nHz, malarial pigment). A role for 15-(S, R)-hydroxy-6, 8, 11, 13-eicosatetraenoic acid (15-HETE), a nHZ lipoperoxidation product, has been suggested. Here, the underlying mechanisms were investigated, focusing on the involvement of mitogen-activated protein kinases (MAPKs). Either early or late p38 MAPK phosphorylation was induced by nHz, which did not modify basal phosphorylation and expression ratios of extracellular signal-regulated kinase-1/2 and c-jun N-terminal kinase-1/2. 15-HETE mimicked nHZ effects on p38 MAPK. Lipid-free synthetic (s)HZ and delipidized (d)HZ did not. Both nHZ and 15-HETE promoted the phosphorylation of MAPK-activated protein kinase-2, a known substrate of p38 MAPK. Such an effect was abolished by SB203580 (synthetic p38 MAPK inhibitor). SB203580 also abrogated the nHZ-dependent and 15-HETE-dependent enhancement of MMP-9 mRNA and protein levels in cell lysates and supernatants. These data suggest that nHZ and 15-HETE upregulate MMP-9 expression and secretion through the activation of p38 MAPK pathway in human adherent monocytes. This work provides new evidence on the mechanisms underlying MMP-9 deregulation in malaria. These data might help to design new specific drugs for adjuvant therapy in complicated malaria.

Contribution of homeostatic chemokines CCL19 and CCL21 and their receptor CCR7 to coronary artery disease.

Our aim was to identify the role of the homeostatic chemokines CCL19 and CCL21 and their common receptor CCR7 in atherogenesis and to study the relationships between CCL19, CCL21, and CCR7 gene variants and coronary artery disease in a Chinese Han population. Immunohistochemical analysis of samples with atherosclerosis of various stages showed increased CCL19, CCL21, and CCR7 expression in atherosclerotic coronary plaques compared with nonatherosclerotic controls. Expression levels increased in positive correlation with coronary lesion stage. Cell adhesion assays confirmed that CCL19 promoted monocyte adhesion, which was induced by CCR7, to human umbilical vein endothelial cells, an effect partially antagonized by atorvastatin. After the human umbilical vein endothelial cells were treated with CCR7-neutralizing antibody, both CCL19- and CCL21-induced monocyte to human umbilical vein endothelial cell migration and CCL19-induced monocyte to human umbilical vein endothelial cell adhesion were abolished. The associations between genetic variants of CCL19, CCL21, CCR7, and coronary artery disease in a Chinese Han population were determined by matrix-assisted laser desorption/ionization time-of-flight mass spectrometry. The following single nucleotide polymorphisms were associated with coronary artery disease: CCL19 rs2227302, CCL21 rs2812377, and CCR7 rs588019. Individuals with the CCL19 rs2227302 T allele or CCL21 rs2812377 G allele had higher plasma CCL19 levels than those with C/C genotype and higher CCL21 levels than those with T/T genotype in both case and control subjects. CCL19/CCL21-CCR7 is a novel homeostatic chemokine system that modulates human monocyte adhesion and migration, promoting atherogenesis. It is associated with coronary artery disease risk in Chinese Han individuals. These data suggest that the CCL19/CCL21-CCR7 axis plays an important role in atherosclerosis progression.

Hypoxia signaling regulates macrophage migration inhibitory factor (MIF) expression in stroke.

The macrophage migration inhibitory factor (MIF) is a multifaceted cytokine involved in many processes, including cellular responses to ischemia/reperfusion injury in the heart and brain. This study was undertaken to determine whether human MIF expression is induced following cerebral ischemia and its role therein. To examine whether the induction of MIF gene expression was mediated by its transcriptional upregulation, the human MIF gene promoter was cloned and a luciferase assay was used to determine the presence of a hypoxia-responsive region in the human MIF promoter. We found that human MIF promoter activity was significantly upregulated by hypoxia. A functional hypoxia-inducible factor 1α-binding site was identified using an electrophoretic mobility shift assay (EMSA). MIF has a protective effect on cortical neurons under oxygen-glucose deprivation (OGD) treatment. MIF significantly reduced OGD-induced cell death. To determine whether the expression of MIF in the human brain is altered following ischemia, brain sections from 10 stroke patients were examined with an antibody against MIF. Blood vessel endothelial cells in the peri-infarct region of ischemic brain displayed strong MIF immunoreactivity with no MIF immunoreactivity in control brains. Furthermore, we found that treatment of human brain endothelial cells with MIF had no effect on human monocyte adhesion to endothelium. Our study demonstrates that MIF gene expression is altered during stroke and dysregulation of the hypoxia signaling-induced MIF expression plays an important role in neuronal death in stroke.

Role of nanostructured gold surfaces on monocyte activation and Staphylococcus epidermidis biofilm formation

The role of material surface properties in the direct interaction with bacteria and the indirect route via host defense cells is not fully understood. Recently, it was suggested that nanostructured implant surfaces possess antimicrobial properties. In the current study, the adhesion and biofilm formation of Staphylococcus epidermidis and human monocyte adhesion and activation were studied separately and in coculture in different in vitro models using smooth gold and well-defined nanostructured gold surfaces. Two polystyrene surfaces were used as controls in the monocyte experiments. Fluorescent viability staining demonstrated a reduction in the viability of S. epidermidis close to the nanostructured gold surface, whereas the smooth gold correlated with more live biofilm. The results were supported by scanning electron microscopy observations, showing higher biofilm tower formations and more mature biofilms on smooth gold compared with nanostructured gold. Unstimulated monocytes on the different substrates demonstrated low activation, reduced gene expression of pro- and anti-inflammatory cytokines, and low cytokine secretion. In contrast, stimulation with opsonized zymosan or opsonized live S. epidermidis for 1 hour significantly increased the production of reactive oxygen species, the gene expression of tumor necrosis factor-α (TNF-α), interleukin-1β (IL-1β), IL-6, and IL-10, as well as the secretion of TNF-α, demonstrating the ability of the cells to elicit a response and actively phagocytose prey. In addition, cells cultured on the smooth gold and the nanostructured gold displayed a different adhesion pattern and a more rapid oxidative burst than those cultured on polystyrene upon stimulation. We conclude that S. epidermidis decreased its viability initially when adhering to nanostructured surfaces compared with smooth gold surfaces, especially in the bacterial cell layers closest to the surface. In contrast, material surface properties neither strongly promoted nor attenuated the activity of monocytes when exposed to zymosan particles or S. epidermidis.

Activation of human monocytes/macrophages by OHR/AVR118 promotes both pro- and Anti-Inflammatory phenotypes

The immunomodulator OHR/AVR118 has been shown to increase IL-8 and MCP-1 secretion from non-activated human monocytes and U937 pro-monocytic cells, but to decrease MCP-1 secretion from LPS-activated monocytes, suggesting its effect depends on immune cell environment and/or activation state. We therefore assessed the effect of OHR/AVR118 on cytokine secretion by human PBMCs and adherent monocytes. OHR/AVR118 increased IL-6, IL-1β, and TNF-α secretion byPHA/IL-2-primed PBMCs, but did not alter IL-12 secretion. In contrast, treatment of LPS-activated monocytes decreased TNF-α and IL-12, increased IL-6, but did not alter IL-1β, secretion. To further show that the effect of OHR/AVR118 depends on cellular environment, we monitored U937 differentiation towards mature macrophages in the presence of drug. OHR/AVR118 promoted a pro-inflammatory response in PMA-activated cells, as demonstrated by increased expression of the maturation markers CD86, CD32, and CD87 and by increased IL-8, MCP-1, and GM-CSF secretion. In undifferentiated U937 cells, OHR/AVR118 did not alter phagocytosis of opsonized S. aureus and IL-10 secretion. Whereas, after activation, OHR/AVR118 induced an anti-inflammatory phenotype, as indicated by reduced phagocytosis and increased IL-10 secretion. Overall, these findings suggest that OHR/AVR118 has a dual action on monocyte/macrophage function depending on cellular activation state, resulting in either further activation or suppression.

Ouabain affects the expression of activation markers, cytokine production, and endocytosis of human monocytes.

Ouabain is a steroid capable of binding to and inhibiting Na+,-K+-ATPase. Studies have demonstrated some actions of ouabain on immune cells, which indicated both pro- and anti-inflammatory properties of this molecule. Nevertheless, its effects on human monocytes are still poorly understood. Thus, the present work investigated effects of ouabain in the activation and function of human adherent monocytes. Our results show that there is an increase in intracellular calcium levels already 5 minutes following monocyte treatment with 10−7 M of ouabain. Furthermore, monocytes expressed increased amounts of surface activation markers such as CD69, HLA-DR, CD86, and CD80 and also presented an augmented endocytic activity of dextran-FITC particles after 24 hours of culture in the presence of ouabain. However, monocytes treated with ouabain did not have an increased stimulatory capacity in allogeneic mixed leukocyte reaction. Ouabain-treated monocytes produced higher levels of IL-1β and TNF-α as reported before. A novel observation was the fact that ouabain induced IL-10 and VEGF as well. Collectively, these results suggest that ouabain impacts monocyte activation and modulates monocyte functions, implying that this steroid could act as an immunomodulator of these cells.

The Kaposi's Sarcoma-Associated Herpesvirus (KSHV)-Induced 5-Lipoxygenase-Leukotriene B4 Cascade Plays Key Roles in KSHV Latency, Monocyte Recruitment, and Lipogenesis

Kaposi's sarcoma-associated herpesvirus (KSHV) is etiologically associated with Kaposi's sarcoma (KS) and primary effusion lymphoma (PEL). KS lesions are characterized by endothelial cells with multiple copies of the latent KSHV episomal genome, lytic replication in a low percentage of infiltrating monocytes, and inflammatory cytokines plus growth factors. We demonstrated that KSHV utilizes inflammatory cyclooxygenase 2/prostaglandin E2 to establish and maintain latency (Sharma-Walia, N., A. G. Paul, V. Bottero, S. Sadagopan, M. V. Veettil, N. Kerur, and B. Chandran, PLoS Pathog 6:e1000777, 2010 [doi:10.1371/journal.ppat.1000777]). Here, we evaluated the role of 5-lipoxygenase (5LO) and its chemotactic metabolite leukotriene B4 (LTB4) in KSHV biology. Abundant staining of 5LO was detected in human KS tissue sections. We observed elevated levels of 5LO and high levels of secretion of LTB4 during primary KSHV infection of endothelial cells and in PEL B cells (BCBL-1 and BC-3 cells). Blocking the 5LO/LTB4 cascade inhibited viral latent ORF73, immunomodulatory K5, viral macrophage inflammatory protein 1 (MIP-1), and viral MIP-2 gene expression, without much effect on lytic switch ORF50, immediate early lytic K8, and viral interferon-regulatory factor 2 gene expression. 5LO inhibition significantly downregulated latent viral Cyclin and latency-associated nuclear antigen 2 levels in PEL cells. 5LO/LTB4 inhibition downregulated TH2-related cytokine secretion, elevated TH1-related cytokine secretion, and reduced human monocyte recruitment, adhesion, and transendothelial migration. 5LO/LTB4 inhibition reduced fatty acid synthase (FASN) promoter activity and its expression. Since FASN, a key enzyme required in lipogenesis, is important in KSHV latency, these findings collectively suggest that 5LO/LTB4 play important roles in KSHV biology and that effective inhibition of the 5LO/LTB4 pathway could potentially be used in treatment to control KS/PEL.

In-situ and label-free optical monitoring of the adhesion and spreading of primary monocytes isolated from human blood: Dependence on serum concentration levels

Adhesion and spreading of primary monocytes isolated from human blood were monitored utilizing optical waveguide lightmode spectroscopy (OWLS); a highly sensitive label-free biosensor technique using evanescent optical waves generated at a biocompatible surface. Appropriate development on a custom built setup enabled the OWLS cuvette to be operated as a 1.5ml mini-incubator, controlling both temperature and CO2 levels. The incubator-equipped OWLS is readily applicable for delicate and long-term studies on sensitive primary cells, demonstrated here through monitoring the serum dependence of the adhesion and spreading of human monocytes. Moreover, the custom-built setup enables the simultaneous monitoring of the position and overall width of the OWLS resonant peaks. This unique feature makes it possible to distinguish the refractive index variations induced by the adsorption of secreted material from refractive index changes provoked by cellular spreading. A definite attachment and spreading activity was observed on the substratum (glassy silica–titania), when the serum level of the culturing medium was 0.0–0.01%. Increasing serum concentration resulted in a steep fall in monocyte surface adhesion and spreading. 1.0% serum level practically abolished all spreading activity measured by OWLS, and the number of attached cells was significantly decreased, too. Serum addition to fully spread cells provoked a reduction in the cell–substratum contact area, clearly detectable by the biosensor. Cell spreading was inhibited by pre-coating the sensor surface with considerable amounts of serum proteins. These findings suggest that monocyte spreading is inhibited by the adsorption of serum biomolecules to the substratum, rather than by soluble factors present in the serum. All of these results were obtained completely noninvasively with real time monitoring; demonstrating the capabilities of OWLS to sensitively monitor the adhesion properties of immune cells isolated from human blood. The current study is, therefore, a significant step towards the application of label-free optical biosensors in medical diagnostics.

Natural Haemozoin Induces Expression and Release of Human Monocyte Tissue Inhibitor of Metalloproteinase-1

Recently matrix metalloproteinase-9 (MMP-9) and its endogenous inhibitor (tissue inhibitor of metalloproteinase-1, TIMP-1) have been implicated in complicated malaria. In vivo, mice with cerebral malaria (CM) display high levels of both MMP-9 and TIMP-1, and in human patients TIMP-1 serum levels directly correlate with disease severity. In vitro, natural haemozoin (nHZ, malarial pigment) enhances monocyte MMP-9 expression and release. The present study analyses the effects of nHZ on TIMP-1 regulation in human adherent monocytes. nHZ induced TIMP-1 mRNA expression and protein release, and promoted TNF-α, IL-1β, and MIP-1α/CCL3 production. Blocking antibodies or recombinant cytokines abrogated or mimicked nHZ effects on TIMP-1, respectively. p38 MAPK and NF-κB inhibitors blocked all nHZ effects on TIMP-1 and pro-inflammatory molecules. Still, total gelatinolytic activity was enhanced by nHZ despite TIMP-1 induction. Collectively, these data indicate that nHZ induces inflammation-mediated expression and release of human monocyte TIMP-1 through p38 MAPK- and NF-κB-dependent mechanisms. However, TIMP-1 induction is not sufficient to counterbalance nHZ-dependent MMP-9 enhancement. Future investigation on proteinase-independent functions of TIMP-1 (i.e. cell survival promotion and growth/differentiation inhibition) is needed to clarify the role of TIMP-1 in malaria pathogenesis.

Sphingosine-1-phosphate and toll-like receptor 4-cooperate to induce inflammatory and adhesive responses in human aortic valve endothelial cells

Purpose: Given the association of Toll-like receptor (TLR)-4 to calcific aortic stenosis and the relevant function of the lipid mediator sphingosine-1-phosphate (S1P) in the cardiovascular system, the putative role of TLR4 ligands and S1P in the induction of pro-inflammatory and pro-adhesive responses in endothelial cells from human aortic (AVEC) and pulmonary valves (PVEC) was investigated. Methods: AVEC and PVEC obtained from n =5 heart transplant patients with no valve disease, and AVEC derived from stenotic aortic valves collected from n = 5 patients with degenerative aortic stenosis and indication for valve replacement were studied. Reverse transcriptase PCR assays were used to evaluate the mRNA expression of TLR and S1P receptors. Cytokine secretion and adhesion molecule expression were evaluated by antibody arrays and Western blot, and adhesion of human monocytes to AVEC monolayers was assayed in vitro. Comparison between stenotic and control aortic valve and between aortic and pulmonar valve were performed. Results: Reverse transcriptase PCR assays revealed the expression of TLRs at the mRNA level in AVEC, being TLR4 the most abundant, as well as the expression of S1P receptors, the S1P1 being the most abundant subtype. As regards pro-inflammatory molecules, the exposure of control AVECs to the bacterial lipopolysaccharide (LPS), a TLR4 ligand, induced the secretion of several cytokines such as interleukin (IL)-6, granulocyte macrophage colony-stimulating factor (GM-CSF), and monocyte chemotactic protein-1. Moreover, LPS cooperated with S1P to further up-regulate IL-6 and GM-CSF secretion. In addition, AVEC exposure to LPS induced the expression of the adhesion molecule ICAM-1, which was further up-regulated in the presence of S1P. Interestingly, the effect was more prominent in endothelial cells from the aortic valve than from the pulmonary valve, which rarely undergoes stenosis, and more potent in cells from stenotic valves. The cooperative effect on ICAM-1 up-regulation was inhibited by S1P1 receptor antagonists. In adhesion assays, LPS and S1P cooperated to increase the number of monocytes that adhered to the activated AVEC monolayers, and the effect was more marked in stenotic than in control AVECs. Conclusions: TLR4 and S1P1 receptor cooperate to up-regulate pro-inflammatory/pro-adhesive molecules in AVECs, what might be relevant for the pathogenesis of aortic stenosis given the stronger effect on stenotic aortic valves and the lower effect on pulmonary valves, and could open the way for new therapeutic approaches for the disease.

Enhancement of CCL15 Expression and Monocyte Adhesion to Endothelial Cells (ECs) after Hypoxia/Reoxygenation and Induction of ICAM-1 Expression by CCL15 via the JAK2/STAT3 Pathway in ECs

CCL15, a member of the CC chemokine family, is a potent chemoattractant for leukocytes and endothelial cells (ECs). Given that chemokines play key roles in vascular inflammation, we investigated the effects of hypoxia/reoxygenation (H/R) on expression of human CCL15 and a role of CCL15 in upregulating ICAM-1 in ECs. We found that exposure of ECs to H/R increased expression of CCL15 and ICAM-1, which resulted in an increase in monocyte adhesivity to the ECs. Further studies revealed that knockdown of CCL15 or CCR1 attenuated expression of ICAM-1 in ECs after H/R, suggesting that expression of ICAM-1 is upregulated by CCL15. Stimulation of ECs with CCL15 significantly increased expression of ICAM-1 predominantly via the CCR1 receptor. We observed that phosphorylation of JAK2 and STAT3 was stimulated by CCL15 treatment of ECs. Results from reporter and chromatin immunoprecipitation assays revealed that CCL15 activates transcription from the IFN-γ activation site promoter and stimulates binding of STAT3 to the ICAM-1 promoter. Our data also showed that CCL15 increased cell adhesion of human monocytes to ECs under static and shear-stress conditions. Pretreatment of these cells with inhibitors for JAK, PI3K, and AKT prevented the CCL15-induced expression of ICAM-1 and monocyte adhesion to ECs, suggesting the involvement of those signaling molecules in ICAM-1 gene activation by CCL15. The results suggest that CCR1 and its ligands may be a potential target for treating inflammatory diseases involving upregulation of cell adhesion molecules.