Tumor Necrosis Factor-alpha Secretion Research Articles

Structural characterisation and immunomodulatory activity of a neutral polysaccharide from Sambucus adnata Wall

A neutral polysaccharide, SPW-2, was purified from the leaves of Sambucus adnata Wall. using water extraction and alcohol precipitation, Sevage deproteination, ion exchange chromatography and gel filtration chromatography. This molecule had an average molecular weight of 7040 Da and was composed of arabinose, xylose, mannose, glucose and galactose at a molar ratio of 1.5:0.5:1.2:5.0:1.8. The repetitive structural units of SPW-2 were deduced using methylation analysis and nuclear magnetic resonance (one- and two-dimensional) spectroscopy. In vitro immunological activity test showed that SPW-2 could induce the secretion of nitric oxide, interleukin-1β (IL-1β), IL-6 and tumour necrosis factor-alpha (TNF-α), and increase the mRNA expression level of inducible nitric oxide synthase (iNOS), IL-1β, IL-6 and TNF-α in macrophages. The data supported the notion that SPW-2 exerts an immunomodulatory effect by activating macrophages and enhancing the host immune system function, which enabled it to be used as a novel immunomodulator for application in the treatment of immunological diseases.

Comparative impact of platelet rich plasma and transforming growth factor-β on chondrogenic differentiation of human adipose derived stem cells

Introduction: Transforming growth factor-beta (TGF-β) is known as standard chondrogenic differentiation agent, even though it comes with undesirable side effects such as early hypertrophic maturation, mineralization, and secretion of inflammatory/angiogenic factors. On the other hand, platelet-rich plasma (PRP) is found to have a chondrogenic impact on mesenchymal stem cell proliferation and differentiation, with no considerable side effects. Therefore, we compared chondrogenic impact of TGF-β and PRP on adipose-derived stem cells (ADSCs), to see if PRP could be introduced as an alternative to TGF-β. Methods: Differentiation of ADSCs was monitored using a couple of methods including glycosaminoglycan production, miRNAs expression, vascular endothelial growth factor (VEGF)/tumor necrosis factor alpha (TNFα) secretion, alkaline phosphatase (ALP) and calcium content assays. Results: Accordingly, the treatment of differentiating cells with 5% (v/v) PRP resulted in higher glycosaminoglycan production, enhanced SOX9 transcription, and lowered TNFα and VEGF secretion compared to the control and TGF-β groups. Besides, the application of PRP to the media up-regulated miR-146a and miR-199a in early and late stages of chondrogenesis, respectively. Conclusion: PRP induces in vitro chondrogenesis, as well as TGF-β with lesser inflammatory and hypertrophic side effects.

Induction of tissue factor expression by anti-β2-glycoprotein I is mediated by tumor necrosis factor α.

Antiphospholipid antibodies (aPL) are heterogeneous and there is evidence that binding specificity determines which cellular effects they can trigger. We have therefore hypothesised that the induction of tissue factor (TF) in monocytes and endothelial cells by aPL depends on their binding specificity. To further investigate this, we have analyzed the ability of three human monoclonal aPL with distinctly different binding specificities to induce transcription and cell surface expression of TF in monocytes and endothelial cells. Results with human monoclonal aPL were validated with IgG-fractions obtained from patients with APS. We confirmed previous results that a lipid reactive human monoclonal aPL rapidly induced TF transcription and cell surface expression in monocytes and endothelial cells. A monoclonal aPL reactive against β2 glycoprotein I (β2GPI) induced TF with a delayed time course. This was fully dependent on the induction of tumor necrosis factor alpha (TNFα) secretion as capture of TNFα by adalimumab prevented TF induction. This pattern was confirmed with patient IgG fractions. Both lipid reactive and anti-β2GPI induced TF transcription. Unexpectedly, this activity of anti-β2GPI was mediated fully by TNFα which was secreted in response to incubation with anti-β2GPI. The role of TNFα in mediating TF induction by anti-β2GPI may have wider implications for APS pathogenesis.

P110δ PI3-Kinase Inhibition Perturbs APP and TNFα Trafficking, Reduces Plaque Burden, Dampens Neuroinflammation, and Prevents Cognitive Decline in an Alzheimer's Disease Mouse Model.

Alzheimer's disease (AD) is associated with the cleavage of the amyloid precursor protein (APP) to produce the toxic amyloid-β (Aβ) peptide. Accumulation of Aβ, together with the concomitant inflammatory response, ultimately leads to neuronal death and cognitive decline. Despite AD progression being underpinned by both neuronal and immunological components, therapeutic strategies based on dual targeting of these systems remains unexplored. Here, we report that inactivation of the p110δ isoform of phosphoinositide 3-kinase (PI3K) reduces anterograde axonal trafficking of APP in hippocampal neurons and dampens secretion of the inflammatory cytokine tumor necrosis factor-alpha by microglial cells in the familial AD APPswe/PS1ΔE9 (APP/PS1) mouse model. Moreover, APP/PS1 mice with kinase-inactive PI3Kδ (δD910A) had reduced Aβ peptides levels and plaques in the brain and an abrogated inflammatory response compared with APP/PS1 littermates. Mechanistic investigations reveal that PI3Kδ inhibition decreases the axonal transport of APP by eliciting the formation of highly elongated tubular-shaped APP-containing carriers, reducing the levels of secreted Aβ peptide. Importantly, APP/PS1/δD910A mice exhibited no spatial learning or memory deficits. Our data highlight inhibition of PI3Kδ as a new approach to protect against AD pathology due to its dual action of dampening microglial-dependent neuroinflammation and reducing plaque burden by inhibition of neuronal APP trafficking and processing.SIGNIFICANCE STATEMENT During Alzheimer's disease (AD), the accumulation of the toxic amyloid-β (Aβ) peptide in plaques is associated with a chronic excessive inflammatory response. Uncovering new drug targets that simultaneously reduce both Aβ plaque load and neuroinflammation holds therapeutic promise. Using a combination of genetic and pharmacological approaches, we found that the p110δ isoform of phosphoinositide 3-kinase (PI3K) is involved in anterograde trafficking of the amyloid precursor protein in neurons and in the secretion of tumor necrosis factor-alpha from microglial cells. Genetic inactivation of PI3Kδ reduces Aβ plaque deposition and abrogates the inflammatory response, resulting in a complete rescue of the life span and spatial memory performance. We conclude that inhibiting PI3Kδ represents a novel therapeutic approach to ameliorate AD pathology by dampening plaque accumulation and microglial-dependent neuroinflammation.

Isolation, Culture and Functional Characterization of Glia and Endothelial Cells From Adult Pig Brain.

Primary cultures of glial and endothelial cells are important tools for basic and translational neuroscience research. Primary cell cultures are usually generated from rodent brain although considerable differences exist between human and rodent glia and endothelial cells. Because many translational research projects aim to identify mechanisms that eventually lead to diagnostic and therapeutic approaches to target human diseases, glia, and endothelial cultures are needed that better reflect the human central nervous system (CNS). Pig brain is easily accessible and, in many aspects, close to the human brain. We established an easy and cost-effective method to isolate and culture different primary glial and endothelial cells from adult pig brain. Oligodendrocyte, microglia, astrocyte, and endothelial primary cell cultures were generated from the same brain tissue and grown for up to 8 weeks. Primary cells showed lineage-specific morphology and expressed specific markers with a purity ranging from 60 to 95%. Cultured oligodendrocytes myelinated neurons and microglia secreted tumor necrosis factor alpha when induced with lipopolysaccharide. Endothelial cells showed typical tube formation when grown on Matrigel. Astrocytes enhanced survival of co-cultured neurons and were killed by Aquaporin-4 antibody positive sera from patients with Neuromyelitis optica. In summary, we established a new method for primary oligodendrocyte, microglia, endothelial and astrocyte cell cultures from pig brain that provide a tool for translational research on human CNS diseases.

Antiviral and immunomodulatory effects of polyphenols on macrophages infected with dengue virus serotypes 2 and 3 enhanced or not with antibodies.

Background: There is a lack of specific antiviral therapy against dengue virus (DENV) in current use. Therefore, a great proportion of dengue cases progress to severe clinical forms due to a complex interplay between virus and host immune response. It has been hypothesized that heterotypic non-neutralizing antibodies enhance DENV infection in phagocytic cells, and this induces an inflammatory response that is involved in the pathogenesis of severe dengue.Purpose: To identify the antiviral and immunomodulatory effects of polyphenols on dengue virus infection.Methods: Human U937-DC-SIGN macrophages were infected with DENV serotypes 2 or 3 in the presence or not of enhancing antibody 4G2. Viral titers and the secretion of tumor necrosis factor-alpha, IL-6, IL-10 and interferon-alpha were analyzed timely.Results: DENV infection alone induced high production of IL-6 and TNF-α, but in the presence of 4G2 antibody, viral titers and TNF-α secretion were potentiated. Based on anti-inflammatory antecedents, the polyphenols curcumin, fisetin, resveratrol, apigenin, quercetin and rutin were tested for antiviral and immunomodulatory properties. Only quercetin and fisetin inhibited DENV-2 and DENV-3 infection in the absence or presence of enhancing antibody (>90%, p<0.001); they also inhibited TNF-α and IL-6 secretion (p<0.001).Conclusion: Quercetin and fisetin down-regulate the production of proinflammatory cytokines induced by DENV infection enhanced by antibodies a mechanism involved in severe dengue.

S888 TNF‐ALPHA PROMOTES CLONAL EXPANSION OF KIT D816V+ CELLS IN SYSTEMIC MASTOCYTOSIS

Background:Systemic mastocytosis (SM) is characterized by clonal mast cell (MC) infiltration in the bone marrow (BM). Most patients harbor the activating KIT mutation D816V. Cytokines are substantially involved in the interactions between neoplastic cells and the BM microenvironment in SM and in classical myeloproliferative neoplasms (MPN) and have been recognized as a major trigger of disease evolution. The pro‐apoptotic/anti‐proliferative and pro‐inflammatory cytokine tumor necrosis factor alpha (TNFa) has been identified as an important driver for the expansion of JAK2 V617F+ clones and the establishment of MPN.Aims:Investigation of the potential role of TNFa in disease evolution of SM patients.Methods:In vitro and in vivo experiments with MC lines and primary material of SM patients.Results:In a first step, lentivirally‐driven expression of the KIT D816V mutant gene in the human Mo7e cells suggested an oncogene‐dependent regulation of the production of TNFa. Correspondingly, the human neoplastic KIT D816V+ MC lines HMC‐1.2 and ROSAKIT D816V expressed and secreted substantial amounts of TNFa and inhibition of KIT D816V either by the recently approved multi‐kinase inhibitor midostaurin or by RNAi‐mediated knockdown of KIT significantly reduced expression of this cytokine. Mechanistically, we studied auto‐/paracrine effects of TNFa on the clonal selection of neoplastic MC using KIT D816V negative (ROSAKIT WT) and positive (ROSAKIT D816V) subclones of ROSA cells. TNFa reduced proliferation of ROSAKIT WT cells (84.9% ± 4.6% of control, p&lt;0.01) whereas ROSAKIT D816V cells were resistant to TNFa (102.7% ± 3.7% of control, n.s.). Preliminary data also indicate a relative resistance of primary neoplastic cells obtained from KIT D816V+ SM patients against TNFa in colony‐forming unit (CFU) assay. To determine the mechanism of TNFa resistance in KIT D816V+ cells, we screened for key regulators of apoptosis and identified the anti‐apoptotic protein survivin to be highly upregulated in KIT D816V+ ROSA cells upon TNFa treatment but not in KIT D816V‐ ROSA cells. We hypothesize that KIT D816V+ MC produce substantial amounts of TNFa that induces apoptosis and suppresses proliferation of normal BM cells while MC themselves are relatively resistant via upregulation of survivin. In a next step, we studied the effect of TNFa on growth of neoplastic MC in vivo in a xenotransplant model using ROSAKIT D816V cells with and without knockout of TNFa in NOD‐SCID IL‐2Rγ‐null (NSG) mice. CRISPR/Cas9‐mediated knockout completely abolished expression and secretion of TNFa in ROSAKIT D816V cells and resulted in a significantly longer survival of mice compared to mice injected with TNFa expressing controls (median 49 vs. 58 days, p &lt; 0.05). Finally, the clinical relevance of TNFa expression was studied in a SM patient's cohort (n = 45). TNFa levels in sera of patients were significantly higher (median 1.95, range 0.72–16.64 pg/ml, p = 0.002) compared to age‐ and sex‐matched controls (median 1.49, range 0.51–5.63 pg/ml). Importantly, when we stratified SM patients into those with clearly elevated TNFa levels and those with TNFa levels within the reference range of controls (≤3.63 pg/ml) a significantly lower overall survival was observed for SM patients with high TNFa serum levels (median 6.3 years vs. not reached, p &lt; 0.005).Summary/Conclusion:In summary, we have identified TNFa as a critical, KIT D816V‐dependent, cytokine‐mediator that promotes clonal expansion of KIT D816V+ MC and may thereby be involved in clonal evolution and disease progression in SM.

Oncolytic HSV therapy increases trametinib access to brain tumors and sensitizes them in vivo.

Hyperactivation of the RAS-RAF-MEK-ERK signaling pathway is exploited by glioma cells to promote their growth and evade apoptosis. MEK activation in tumor cells can increase replication of ICP34.5-deleted herpes simplex virus type 1 (HSV-1), but paradoxically its activation in tumor-associated macrophages promotes a pro-inflammatory signaling that can inhibit virus replication and propagation. Here we investigated the effect of blocking MEK signaling in conjunction with oncolytic HSV-1 (oHSV) for brain tumors. Infected glioma cells co-cultured with microglia or macrophages treated with or without trametinib were used to test trametinib effect on macrophages/microglia. Enzyme-linked immunosorbent assay, western blotting, and flow cytometry were utilized to evaluate the effect of the combination therapy. Pharmacokinetic (PK) analysis of mouse plasma and brain tissue was used to evaluate trametinib delivery to the CNS. Intracranial human and mouse glioma-bearing immune deficient and immune competent mice were used to evaluate the antitumor efficacy. Oncolytic HSV treatment rescued trametinib-mediated feedback reactivation of the mitogen-activated protein kinase signaling pathway in glioma. In vivo, PK analysis revealed enhanced blood-brain barrier penetration of trametinib after oHSV treatment. Treatment by trametinib, a MEK kinase inhibitor, led to a significant reduction in microglia- and macrophage-derived tumor necrosis factor alpha (TNFα) secretion in response to oHSV treatment and increased survival of glioma-bearing mice. Despite the reduced TNFα production observed in vivo, the combination treatment activated CD8+ T-cell mediated immunity and increased survival in a glioma-bearing immune-competent mouse model. This study provides a rationale for combining oHSV with trametinib for the treatment of brain tumors.

Cytotoxic and proinflammatory responses induced by ZnO nanoparticles in in vitro intestinal barrier.

ZnO nanoparticles (NPs) are widely used nowadays, thus the gastrointestinal exposure to ZnO NPs is likely to be relevant and the effects on the intestinal barrier should be investigated. Polarized Caco-2 cells were exposed from the apical (Ap) and basolateral (Bl) compartments to increasing concentrations (0, 10, 50 and 100μg/mL) of sonochemical (sono) and commercial ZnO NPs. The transepithelial electrical resistance (TEER), cell viability, proinflammatory cytokine release and presence of protein oxidative damage were evaluated after exposure. TEER was not significantly affected by Ap exposure to either sono or commercial ZnO NPs at any tested concentrations. After Bl exposure to sono ZnO NPs (all the concentrations) and to 100μg/mL of commercial ZnO NPs TEER was decreased (P<0.05). Ap and Bl exposure to 100μg/mL sono ZnO NPs and Ap exposure to 50μg/mL commercial ZnO NPs induced a significant (P<0.05) release of interleukin-6. A significant (P<0.05) release of interleukin-8 was observed after Ap exposure to ZnO NPs at 100μg/mL and after Bl exposure to sono ZnO NPs at 100μg/mL. Ap or Bl exposure to sono or commercial ZnO NPs did not affect tumour necrosis factor-alpha secretion or protein sulphydryl oxidation. In conclusion, the ZnO NP exposure from the Ap compartment appeared almost safe, while the exposure through the basal compartment appeared to be more hazardous and the different NP size and crystallinity seem to affect the mode of action, but further studies are necessary to elucidate better these toxicity mechanisms.

Proinflammatory effects of dust storm and thermal inversion particulate matter (PM10) on human peripheral blood mononuclear cells (PBMCs) in vitro: a comparative approach and analysis.

Particulate matter (PM) as the carcinogenic air pollutants can lead to aggravated health outcomes. Epidemiological studies demonstrated that PM can be engaged in different diseases such as cardiovascular, respiratory and cancer. The in vitro secretion of proinflammatory cytokines by human peripheral blood mononuclear cells (PBMCs) has been used to assess the effects of PM with an aerodynamic diameter < 10μm (PM10). This study compared the proinflammatory cytokines tumor necrosis factor alpha (TNF-α), interleukin 6 (IL-6), and interleukin 1-beta (IL1-β) secretions of PBMCs exposed to PM10 of dust storm and inversion. We collected PM10 samples during the spring and autumn seasons in two locations. Isolated PBMCs were exposed separately to 50, 150, and 300μg/ml of different type of PM10 for 4 and 24h. The mean concentrations of TNF-α for the PM of dust storm and inversion were 6305.61 ± 2421 and 6651.74 ± 2820, respectively. Also the mean concentrations of IL1-β for the PM of dust storm and inversion were 556.86 ± 162 and 656.35 ± 196, respectively. Furthermore, these values for the production of IL-6 were 12,655 ± 5661 and 16,685 ± 8069, respectively. Although no significant difference was observed between the PM of dust storm and that of inversion with regard to PBMCs, the results showed a significant increase in the proinflammatory cytokine secretion of both PMs compared with the controls. Moreover, TNF-α, IL1-β, and IL-6 secreted in cells exposed to PM10 of dust storm were about 10 times more than the controls, these values for cells exposed to PM10 of inversion were around 10, 12, and 14 times more than the controls, respectively. It can be concluded that the PM10 of both dust storm and inversion can play a significant role in proinflammatory cytokine secretion due to its harmful effect on human health. Graphical abstractThis picture shows the Proinflammatory cytokine producing potential of PM10 with two sources (dust storm and urban air pollution) in exposure with human PBMCs in vitro.

MCC950 blocks enhanced interleukin-1β production in patients with NLRP3 low penetrance variants

ObjectiveTo determine the role of the NLRP3 inflammasome by using the selective NLRP3 inhibitor MCC950 in patients with NLRP3 low penetrance variants and clinical symptoms suggestive for an autoinflammatory syndrome including central nervous system (CNS) involvement. MethodsNineteen symptomatic patients with low penetrance NLRP3 variants (Q703K n = 17, V198M n = 2) recruited between 2011 and 2017 were included in this monocentric study. A functional inflammasome activation assay was performed in patients in comparison to healthy controls (HC), including the determination of interleukin-1beta (IL-1β), interleukin-6 (IL-6) and tumor-necrosis factor alpha (TNF-α) secretion in the presence of the NLRP3 selective small-molecule inhibitor MCC950. Detailed clinical features were assessed and anti-IL-1 treatment response was determined. ResultsPeripheral blood mononuclear cells (PBMC) from patients with low penetrance NLRP3 variants displayed enhanced IL-1β levels following inflammasome activation compared to HC. Furthermore, IL-1β release was NLRP3-dependent as it was blocked by MCC950. The production of IL-6 and TNF-α was also increased in patients with low penetrance NLRP3 variants. Clinically, they presented with a heterogenous spectrum of neurological manifestations, while cranial nerve inflammation was the most common feature. Overall inflammasome activation did not correlate with disease severity. Eight of ten treated patients responded to anti IL-1 treatment, however a complete response was only documented in four patients. ConclusionPBMC of several patients with NLRP3 low penetrance variants and CNS manifestation showed increased NLRP3-specific IL-1β release upon stimulation and elevated NLRP3-independent IL-6 and TNF-α levels as those were not suppressed by MCC950. Our data suggest that beside the possible causal involvement of the NLRP3 inflammasome additional, yet unidentified genetic or environmental factors may contribute to the multi-organ inflammation in our patients and explain the partial response to IL-1 targeting therapies.

Assessing the translocation of silver nanoparticles using an in vitro co-culture model of human airway barrier

The lung has been recognized as one of the main target organs for nanoparticles (NPs) exposure. Cellular uptake of nanoparticles into pulmonary components has been routinely evaluated in the conventional monoculture format, which lacks relevant cell to cell communications and interactions that are vital in the physiological environment. A more physiologically relevant co-culture model has thus been developed and described here to study the translocation of NPs across human airway barrier. The model consists of human bronchial epithelial cells (Calu-3), endothelial cells (EA.hy926) and macrophage-like cells (differentiated Thp-1) in a two-chamber system. Silver nanoparticles (AgNPs) coated with tannic acid were used as an example nanoparticle. These AgNPs were applied to the co-culture system where their movement and resultant toxicity were monitored. Cellular uptake and translocation of AgNPs through the modeled barrier were confirmed using analytical methods. Mild cytotoxicity at the given dosage levels was also observed, accompanied by reduced secretion of interleukin-6 (IL-6), interleukin-8 (IL-8), and tumor necrosis factor-alpha (TNF-α). This human airway model provides researchers with an alternative method for the quantitative evaluation of uptake, translocation and toxicity of aerosol contaminants or nano-sized drug delivery systems in a more relevant in vitro format.

Inflammation in Salt-Sensitive Hypertension and Renal Damage.

Low-grade inflammation drives elevations in blood pressure (BP) and consequent target organ damage in diverse experimental models of hypertension. Here, we discuss recent advances elucidating immune-mediated mechanisms of BP elevation and associated target organ damage. Inflammatory mediators produced by immune cells or target organs act on the kidney, vasculature, skin, and nervous system to modulate hypertension. For example, cells of the innate immune system, including monocytes, neutrophils, and dendritic cells (DCs), can all promote BP elevation via actions in the vasculature and kidney. Macrophages expressing VEGF-C impact non-osmotic sodium storage in the skin that in turn regulates salt sensitivity. Within the adaptive immune system, activated T cells can secrete tumor necrosis factor-alpha (TNF-α), interleukin-17a (IL-17a), and interferon-gamma (IFN-γ), each of which has augmented BP and renal damage in pre-clinical models. Inversely, deficiency of IL-17a in mice blunts the hypertensive response and attenuates renal sodium retention via a serum- and glucocorticoid-regulated kinase 1 (SGK1)-dependent pathway. Linking innate and adaptive immune responses, dendritic cells activated by augmented extracellular sodium concentrations stimulate T lymphocytes to produce pro-hypertensive cytokines. By contrast, regulatory T cells (Tregs) can protect against hypertension and associated kidney injury. Rodent studies reveal diverse mechanisms via which cells of the innate and adaptive immune systems drive blood pressure elevation by altering the inflammatory milieu in the kidney, vasculature, and brain.

Effect of Grifola frondosa polysaccharide on anti-tumor activity in combination with 5-Fu in Heps-bearing mice

GP11 had been reported to have effectively anti-tumor activity by improving the immune function in our previous study. To avoid drawbacks of the 5-Fu, GP11 in combination with 5-Fu was investigated in this study. The results demonstrated that such synergism displayed enhance the anti-tumor activity of 5-Fu. Additionally, a strength effect was also observed in regulating immune function of GP11 and 5-Fu simultaneous administration, such as enhancing serum interleukin-2 (IL-2) and tumor necrosis factor-alpha (TNF-α) secretion, and increasing immune organs weights. Moreover, GP11 could improve the haematological and biochemical parameters deterioration, superoxide dismutase (SOD) activities reduction and malondialdehyde (MDA) levels enhancement in non-immune organs induced by 5-Fu. All these results illustrated that GP11 exhibited attenuated and synergized effect on 5-Fu by improving the immune function. It could be developed as an auxiliary preparation for chemotherapeutic drugs.

The host cellular immune response to cytomegalovirus targets the endothelium and is associated with increased arterial stiffness in ANCA-associated vasculitis

BackgroundCardiovascular disease is a leading cause of death in ANCA-associated vasculitis (AAV). An expansion of CD4+CD28null T cells is seen mainly in cytomegalovirus (CMV)-seropositive individuals and has been linked to increased cardiovascular disease risk in other conditions. The aims of this study were to phenotype CD4+CD28null T cells in AAV with respect to their pro-inflammatory capacity and ability to target and damage the endothelium and to investigate their relationship to arterial stiffness, a marker of cardiovascular mortality.MethodsCD4+CD28null T cells were phenotyped in 53 CMV-seropositive AAV patients in stable remission and 30 age-matched CMV-seropositive healthy volunteers by flow cytometry following stimulation with CMV lysate. The expression of endothelial homing markers and cytotoxic molecules was evaluated in unstimulated CD4+CD28null T cells. Arterial stiffness was measured by carotid-to-femoral pulse wave velocity (PWV) in patients with AAV.ResultsCD4+CD28null T cells were CMV-specific and expressed a T helper 1 (Th1) phenotype with high levels of interferon-gamma (IFN-γ) and tumour necrosis factor-alpha (TNF-α) secretion. They also co-expressed the endothelial homing markers CX3CR1, CD49d and CD11b and cytotoxic molecules perforin and granzyme B. CD4+CD28null T cells were phenotypically similar in patients with AAV and healthy volunteers but their proportion was almost twice as high in patients with AAV (11.3% [3.7–19.7] versus 6.7 [2.4–8.8]; P = 0.022). The size of the CD4+CD28null T-cell subset was independently linked to increased PWV in AAV (0.66 m/s increase per 10% increase in CD4+CD28null cells, 95% confidence interval 0.13–1.19; P = 0.016).ConclusionThe host cellular immune response to CMV leads to the expansion of cytotoxic CD4+CD28null T cells that express endothelial homing markers and are independently linked to increased arterial stiffness, a marker of cardiovascular mortality. Suppression of CMV in AAV may be of therapeutic value in reducing the risk of cardiovascular disease.

Oregano Essential Oil Attenuates RAW264.7 Cells from Lipopolysaccharide-Induced Inflammatory Response through Regulating NADPH Oxidase Activation-Driven Oxidative Stress.

Oregano is an aromatic plant widely distributed throughout the Mediterranean area and in Asia. Recent studies have revealed that the anti-inflammatory effect of essential oil in this plant. However, the mechanisms underlying the therapeutic potential have not been well elucidated. This study determined whether oregano essential oil (OEO) exerts an anti-inflammatory effect on lipopolysaccharide (LPS)-treated murine macrophage cells (RAW264.7 cells) in vitro and elucidated the possible underlying molecular mechanisms. The results showed that OEO (2.5–10 μg/mL) inhibited the expression and secretion of interleukin-1 beta (IL-1β), interleukin-6 (IL-6), and tumor necrosis factor-alpha (TNF-α) in RAW264.7 cells treated with LPS (1 μg/mL). Consistent with the pro-inflammatory gene expression, the OEO treatment efficiently reduced the LPS-induced activation of mitogen-activated protein kinase, protein kinase B, and nuclear factor κB in RAW264.7 cells. Nicotinamide adenine dinucleotide phosphate (NADPH) oxidase inhibition in Nox2 protein-silenced cells attenuated the mRNA expression of IL-1β, IL-6, and TNF-α in the LPS-induced RAW264.7 cells. The OEO inhibited the LPS-induced elevation of NADPH oxidase and oxidative stress. This result suggests that LPS induces RAW264.7 cell inflammation through the NADPH oxidase-mediated production of reactive oxygen species (ROS). In conclusion, OEO protects against the LPS-induced RAW264.7 cell inflammatory response through the NADPH oxidase/ROS pathway.

Abstract 109: Exosomes from Mesenchymal Stem Cells Suppress Aortic Elastin Induced Inflammation in Macrophages

Introduction: Mesenchymal Stem Cells (MSCs) from bone marrow have been shown to attenuate aortic aneurysm formation in a variety of rodent models. These effects appear to rely on modulation of systemic inflammation, including the attenuation of macrophage mediated inflammation. Elastin appears to play a significant role in inducing this inflammation and stimulating macrophages to express an M1 phenotype. Here we evaluate the effects human elastin and elastin break down products on macrophage phenotype. We also evaluate the role of human exosomes derived from MSCs on modulating the effect of elastin on macrophage phenotype. Methods: Human peripheral blood monocytes were collected and cultured for six days to isolate human macrophages. The cells were then treated with digested elastin from human aortic tissue or the human peptide VGVAPG in combination with human MSCs or exosomes derived from human MSCs. The phenotype of the macrophages was then analyzed using enzyme linked immune sandwich assays for tumor necrosis factor alpha (TNF-a) secretion, quantitative real time PCR and flow-cytometry for cellular proteins. Results: Macrophages cultured with digested elastin from aortic tissue showed a significant dose response increase in TNF-a production and cell surface markers consistent with an M1 phenotype. In addition, transcripts for pro-inflammatory cytokines significantly increased after exposure to digested elastin. Similar results were obtained using the elastin peptide sequence VGVAPG. Co-culture with MSCs significantly attenuated elastin induced macrophage TNF-a production, production of pro-inflammatory transcripts and M1 related proteins. When MSC derived exosomes were cultured with macrophages, pro-inflammatory markers and TNF-a production was reduced below baseline levels, even after stimulation with elastin peptide. Conclusions: Macrophages develop an M1 phenotype in response to digested aortic elastin and elastin derived peptides. This response is significantly attenuated by exosomes derived from human MSCs

Association of genetic variations in PTPN2 and CD122 with ocular Behcet’s disease

BackgroundProtein tyrosine phosphatases (PTPs) play critical roles in human autoimmunity. Previous studies found that PTPN2 may be the key regulatory factor in the T-cell-mediated immune response. PTPN2 regulates the Janus...

Histamine upregulates the expression of histamine receptors and increases the neuroprotective effect of astrocytes

BackgroundAstrocytes have attracted increasing attention over recent decades for their role in neuroinflammation. Histamine, a major aminergic brain neurotransmitter, has an important influence on the main activities of astrocytes, such as ion homeostasis, energy metabolism, and neurotransmitter clearance. However, little is known about the impact of histamine on astrocyte immunomodulatory function.MethodsThe expression of all known histamine receptor subtypes was examined in primary astrocytes. Then, primary astrocytes were pretreated with selective histamine receptor antagonists and stimulated with histamine. Cellular activation, proinflammatory cytokine production, and expression of neurotrophic factors were assessed.ResultsAstrocytes could constitutively express three histamine receptors (H1R, H2R, and H3R), and these three histamine receptors could be selectively upregulated to varying degrees upon histamine treatment. Histamine also dose-dependently stimulated astrocyte activation and subsequent production of glial cell-derived neurotrophic factor (GDNF), whereas it suppressed the secretion of the proinflammatory factors tumor necrosis factor-alpha (TNF-α) and interleukin-1β (IL-1β). The effects of histamine were completely abolished by either an H1R or H3R antagonist, while an H2R antagonist attenuated the effects partly.ConclusionsThe present study identified the expression of H1R, H2R, and H3R on astrocytes. We also demonstrated that negative regulation of astrocytic TNF-α and IL-1β production and the enhancement of astrocytic GDNF stimulated by histamine were receptor-mediated processes in which all three of the expressed histamine receptors (H1R, H2R, and H3R) were involved. These findings may further clarify the involvement and mechanism of astrocyte activation in neuroinflammation.

Contrasting Function of Structured N-Terminal and Unstructured C-Terminal Segments of Mycobacterium tuberculosis PPE37 Protein.

ABSTRACTPathogens frequently employ eukaryotic linear motif (ELM)-rich intrinsically disordered proteins (IDPs) to perturb and hijack host cell networks for a productive infection. Mycobacterium tuberculosis has a relatively high percentage of IDPs in its proteome, the significance of which is not known. The Mycobacterium-specific PE-PPE protein family has several members with unusually high levels of structural disorder and disorder-promoting Ala/Gly residues. PPE37 protein, a member of this family, carries an N-terminal PPE domain capable of iron binding, two transmembrane domains, and a disordered C-terminal segment harboring ELMs and a eukaryotic nuclear localization signal (NLS). PPE37, expressed as a function of low iron stress, was cleaved by M. tuberculosis protease into N- and C-terminal segments. A recombinant N-terminal segment (P37N) caused proliferation and differentiation of monocytic THP-1 cells, into CD11c, DC-SIGN (dendritic cell-specific intercellular adhesion molecule-3-grabbing nonintegrin)-positive semimature dendritic cells exhibiting high interleukin-10 (IL-10) but negligible IL-12 and also low tumor necrosis factor alpha (TNF-α) secretion—an environment suitable for maintaining tolerogenic immune cells. The C-terminal segment entered the macrophage nucleus and induced caspase-3-dependent apoptosis of host cells. Mice immunized with recombinant PPE37FL and PPE37N evoked strong anti-inflammatory response, validating the in vitro immunostimulatory effect. Analysis of the IgG response of PPE37FL and PPE37N revealed significant immunoreactivities in different categories of TB patients, viz. pulmonary TB (PTB) and extrapulmonary TB (EPTB), vis-a-vis healthy controls. These results support the role of IDPs in performing contrasting activities to modulate the host processes, possibly through molecular mimicry and cross talk in two spatially distinct host environments which may likely aid M. tuberculosis survival and pathogenesis.