Reactive Oxygen Species In Macrophages Research Articles

Characterization of xanthine dehydrogenase and aldehyde oxidase of Marsupenaeus japonicus and their response to microbial pathogen.

Reactive oxygen species (ROS) play key roles in many physiological processes. In particular, the sterilization mechanism of bacteria using ROS in macrophages is a very important function for biological defense. Xanthine dehydrogenase (XDH) and aldehyde oxidase (AOX), members of the molybdo-flavoenzyme subfamily, are known to generate ROS. Although these enzymes occur in many vertebrates, some insects, and plants, little research has been conducted on XDHs and AOXs in crustaceans. Here, we cloned the entire cDNA sequences of XDH (MjXDH: 4328bp) and AOX (MjAOX: 4425bp) from Marsupenaeus japonicus (kuruma shrimp) using reverse transcriptase-polymerase chain reaction (RT-PCR) and random amplification of cDNA ends (RACE). Quantitative real-time RT-PCR transcriptional analysis revealed that MjXDH mRNA is highly expressed in heart and stomach tissues, whereas MjAOX mRNA is highly expressed in the lymphoid organ and intestinal tissues. Furthermore, expression of MjAOX was determined to be up-regulated in the lymphoid organ in response to Vibrio penaeicida at 48 and 72h after injection; in contrast, hydrogen peroxide (H2O2) concentrations increased significantly at 6, 12, 48, and 72h after injection with white spot syndrome virus (WSSV) and at 72h after injection with V. penaeicida. To the best of our knowledge, this study is the first to have identified and cloned XDH and AOX from a crustacean species.

Tu1283 - Nadph Oxidase 2 is a Source of Reactive Oxygen Species in Macrophages During Helicobacter Pylori Infection

Tu1283 - Nadph Oxidase 2 is a Source of Reactive Oxygen Species in Macrophages During Helicobacter Pylori Infection

Group A Streptococcus (GAS) Inhibits the Oxidative Burst Response in a Human Macrophage‐like Cell Line By Preventing the Formation of NADPH Oxidase Complex

Group A Streptococcus (GAS) is the cause of millions of infections worldwide each year, many of which progress to severe invasive infections such as sepsis, necrotizing fasciitis, and streptococcal toxic shock syndrome. Cells of the human innate immune system typically initiate the oxidative burst response to form reactive oxygen species (ROS) as a killing mechanism in response to pathogens. Other bacterial species including Lactococcus lactis and Staphylococcus aureus engage defense mechanisms such as catalase or pigments to detoxify these ROS. However, GAS lacks such detoxifying enzymes, and its survival in macrophages suggests its use of an alternative mechanism to avoid ROS‐mediated killing. When we infected THP‐1 macrophages with GAS, infected macrophages displayed significantly decreased peroxide production when compared to infection with S. aureus, L. lactis, and Group B Streptococcus. As the NADPH Oxidase (NOX2) complex is responsible for initiating the oxidative burst response and the generation of ROS such as peroxide, these data suggest that GAS may be impairing the production of ROS by either preventing the assembly of the NOX2 complex or its activation. The NOX2 complex forms on the surface of the phagosomal membrane and consists of two membrane proteins, gp91 and p22, which assemble with three cytosolic proteins, p47, p40, and p67. To assess whether GAS prevents the assembly of the NOX2 complex, we performed cell fractionation to isolate phagosomes of GAS and L. lactis‐infected macrophages. Western blot and mass spectrometry data indicate that NOX2 complex formation is indeed impaired in GAS‐infected cells. We are also currently performing immunoprecipitation experiments using infected THP‐1 cell lysates and antibodies for the p22 membrane subunit to look for NOX2 complex formation. Thus, these data will determine if GAS impairs the production of harmful ROS in macrophages by preventing the initial assembly of the NOX2 complex, ultimately allowing their persistence in an innate immune cell designed to kill them.This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

Dual Roles of Graphene Oxide To Attenuate Inflammation and Elicit Timely Polarization of Macrophage Phenotypes for Cardiac Repair.

Development of localized inflammatory environments by M1 macrophages in the cardiac infarction region exacerbates heart failure after myocardial infarction (MI). Therefore, the regulation of inflammation by M1 macrophages and their timely polarization toward regenerative M2 macrophages suggest an immunotherapy. Particularly, controlling cellular generation of reactive oxygen species (ROS), which cause M1 differentiation, and developing M2 macrophage phenotypes in macrophages propose a therapeutic approach. Previously, stem or dendritic cells were used in MI for their anti-inflammatory and cardioprotective potentials and showed inflammation modulation and M2 macrophage progression for cardiac repair. However, cell-based therapeutics are limited due to invasive cell isolation, time-consuming cell expansion, labor-intensive and costly ex vivo cell manipulation, and low grafting efficiency. Here, we report that graphene oxide (GO) can serve as an antioxidant and attenuate inflammation and inflammatory polarization of macrophages via reduction in intracellular ROS. In addition, GO functions as a carrier for interleukin-4 plasmid DNA (IL-4 pDNA) that propagates M2 macrophages. We synthesized a macrophage-targeting/polarizing GO complex (MGC) and demonstrated that MGC decreased ROS in immune-stimulated macrophages. Furthermore, DNA-functionalized MGC (MGC/IL-4 pDNA) polarized M1 to M2 macrophages and enhanced the secretion of cardiac repair-favorable cytokines. Accordingly, injection of MGC/IL-4 pDNA into mouse MI models attenuated inflammation, elicited early polarization toward M2 macrophages, mitigated fibrosis, and improved heart function. Taken together, the present study highlights a biological application of GO in timely modulation of the immune environment in MI for cardiac repair. Current therapy using off-the-shelf material GO may overcome the shortcomings of cell therapies for MI.

Exploring hesperidin-copper complex as an enzyme mimic for monitoring macrophage activity

The present study evaluates the potential of hesperidin-copper complex for sensing superoxide anions, which is an important marker for the diagnosis of oxidative stress-based disorders. The metal ion center present in the complex scavenges the superoxide, which can be detected through electrochemical method. The hesperidin-copper complex was successfully synthesized employing a simple room temperature method and characterized. The cyclic voltammograms recorded using a working electrode modified with hesperidin-copper complex show a reduction peak at − 0.175 V, which can be attributed to the electron transfer involving the copper center present in the complex. The sensing study using amperometric techniques in the presence of different concentrations of superoxide anions reveals that hesperidin-copper complex-coated working electrode exhibits good sensitivity and linearity. The limit of detection (LOD) for this enzyme-less sensor is 0.547 μM and limit of quantification (LOQ) is 1.65 μM. The response time was less than 2 s. This sensor was also not affected by common interferents. The sensor performance was assessed in vitro for the quantification of reactive oxygen species in macrophages under stimulated and unstimulated conditions. The results demonstrate that this sensor can be employed for clinical applications involving diagnosis of inflammatory conditions.

Heterocyclic homoprostanoid derivative attenuates monoarthritis in rats: An in vitro and in vivo preclinical paradigm

From our lab, among the nineteen heterocyclic homoprostanoids (HHPs), three derivatives (compounds 3, 3b and 3c) exerted antioxidant and anti-inflammatory activity. Present study is an extension of the earlier work, and, is designed to establish their therapeutic potential in monoarthritis in rats. In addition, their possible mechanism of action would be investigated. A battery of in vitro tests such as lipopolysaccharide (LPS)-induced nitrite (NO)/reactive oxygen species (ROS) and NO/interleukin (IL)-6 generation in murine macrophages and whole blood (WhB), respectively were conducted. Later, in vitro cyclooxygenase (COX) enzyme inhibitory activity was also evaluated. All the tested compounds showed comparable efficacy against ROS and NO in LPS-stimulated murine macrophages. However, compound 3 did not exert inhibitory effect on LPS-induced NO/IL-6 generation in WhB assay. Compounds (3b and 3c) inhibited the NO generation in LPS-stimulated WhB. However, only compound 3b reversed the raised IL-6 levels in this assay. None of the test compounds inhibited COX iso-enzymes in the in vitro assay. All three HHPs showed comparable efficacy against carrageenan-induced paw inflammation. However, none of them exhibited any dose-dependent effect in this model. Based upon previous reports, compound 3c was explored against adjuvant-induced monoarthritis (AIA) in male Sprague-Dawley rats, where it exerted promising therapeutic effect. In addition to radiological and histological examinations of tibio-tarsal joint, various parameters such as chronic inflammation/pain, clinical score, interleukin (IL)-6 levels and complete blood cell profile were evaluated in AIA rats. Chronic treatment with 3c halted the disease progression in rats, improved the overall health of animals, as demonstrated by haematological, clinical scoring and joint examinations (radiological and histopathological). Inhibitory effect on elevated IL-6 in AIA rats suggested the possible mechanism of 3c on cytokine signalling. Overall, the study supports the anti-arthritic potential of compound 3c.

Induction of haem oxygenase-1 increases infection of dog macrophages by L. infantum.

We aimed to induce and inhibit HO-1, ascertaining its effect on infection rate, parasite load and the levels of superoxide, reactive oxygen species (ROS), nitric oxide (NO), TNF-alpha and IL-10 in cultured macrophages from healthy dogs infected by Leishmania infantum. Macrophages obtained from 15 healthy dogs were cultured alone or infected with L. infantum, with or without association of HO-1 inducer and inhibitor. The infection rate and the parasite load were determined by the number of infected macrophages and number of promastigotes per macrophage, respectively. HO-1 levels and gene expression, as well as IL-10 and TNF-alpha levels were also measured in these cultures. Superoxide, ROS and NO levels in macrophages were measured through flow cytometry. Induction of HO-1 increased the infection rate and parasite load, while its inhibition decreased the infection rate and IL-10 production. There was a positive correlation between HO-1 and infection rate or parasite load. Increased infection rate was associated with decreased superoxide, ROS and NO levels. Induction of HO-1 metabolism in dogs infected by L. infantum is possibly one of the mechanisms responsible for increasing the infection of macrophages, mainly through reduction in the oxidative and nitrosative metabolisms of these cells.

Anti-encephalitogenic effects of cucumber leaf extract

Cucumber (Cucumis sativus) fruit has been used in cuisine worldwide, while its leaves are rich in immunomodulatory compounds. Cucumber leaf extract (CLE) was characterized by the predominance of triterpenoids cucurbitacins and significant levels of phenolics. Effects of CLE on CD4+ T helper (Th) cells and macrophages, as the major encephalitogenic cells in the autoimmunity of the central nervous system were investigated in our study. CLE potently inhibited production of major pathogenic Th cytokines: interferon-gamma and interleukin-17, as well as of nitric oxide and reactive oxygen species in macrophages. Antigen-presenting activity of macrophages and dendritic cells was also affected by CLE. The effects of CLE were co-incident with modulation of NFκB and p38 MAPK signaling. Concentrations of CLE used in vitro did not show toxic effects on zebrafish embryos. Moreover, CLE inhibited generation of encephalitogenic cells in vivo. These results demonstrate that CLE deserve further investigation on its anti-encephalitogenic therapeutic properties.

Leishmania donovani inhibits inflammasome-dependent macrophage activation by exploiting the negative regulatory proteins A20 and UCP2.

In visceral leishmaniasis, we found that the antileishmanial drug Amp B produces a higher level of IL-1β over the infected control. Moreover, administering anti-IL-1β antibody to infected Amp B-treated mice showed significantly less parasite clearance. Investigation revealed that Leishmania inhibits stimuli-induced expression of a multiprotein signaling platform, NLRP3 inflammasome, which in turn inhibits caspase-1 activation mediated maturation of IL-1β from its pro form. Attenuation of NLRP3 and pro-IL-1β in infection was found to result from decreased NF-κB activity. Transfecting infected cells with constitutively active NF-κB plasmid increased NLRP3 and pro-IL-1β expression but did not increase mature IL-1β, suggesting that IL-1β maturation requires a second signal, which was found to be reactive oxygen species (ROS). Decreased NF-κB was attributed to increased expression of A20, a negative regulator of NF-κB signaling. Silencing A20 in infected cells restored NLRP3 and pro-IL-1β expression, but also increased matured IL-1β, implying an NF-κB-independent A20-modulated IL-1β maturation. Macrophage ROS is primarily regulated by mitochondrial uncoupling protein 2 (UCP2), and UCP2-silenced infected cells showed an increased IL-1β level. Short hairpin RNA-mediated knockdown of A20 and UCP2 in infected mice independently documented decreased liver and spleen parasite burden and increased IL-1β production. These results suggest that Leishmania exploits A20 and UCP2 to impair inflammasome activation for disease propagation.-Gupta, A. K., Ghosh, K., Palit, S., Barua, J., Das, P. K., Ukil, A. Leishmania donovani inhibits inflammasome-dependent macrophage activation by exploiting the negative regulatory proteins A20 and UCP2.

Anti-parasitic effect of the diuretic and Na+-ATPAse inhibitor furosemide in cutaneous leishmaniasis.

Leishmania amazonensis promastigotes are known to express furosemide (Lasix®)-sensitive P-type membrane Na+-ATPase. In the present study, furosemide activity was studied in intracellular amastigotes and infected BALB/c mice to investigate its efficacy in cutaneous leishmaniasis (CL). Intracellular parasites, but not macrophages, were found to be sensitive to killing by furosemide (IC50 = 87 µ m vs CC50 ≫ 1000 µ m, respectively). Although furosemide did not induce nitric oxide production or intracellular pH changes in infected macrophages, it led to a significant reactive oxygen species (ROS) burst. Freshly isolated tissue parasites expressed a high degree of Na+-ATPase activity that decreased with culture, indicative of a higher enzyme expression in amastigotes than in promastigotes. Both intraperitoneal and oral treatment of L. amazonensis-infected mice with furosemide dosages equivalent to that prescribed as a diuretic significantly reduced the parasite's growth compared with the situation in untreated mice. Combination with oral furosemide increased the efficacy and safety of intraperitoneal treatment with sodium stibogluconate (SSG). To summarize, furosemide control of intracellular leishmanial growth by means of parasite Na+-ATPase inhibition, and macrophage ROS activation may help explain its sole and SSG-combined therapeutic effect against murine CL.

Athero-inflammatory nanotherapeutics: Ferulic acid-based poly(anhydride-ester) nanoparticles attenuate foam cell formation by regulating macrophage lipogenesis and reactive oxygen species generation.

Future development of anti-oxidant formulations for atherosclerosis applications is essential to deliver an efficacious dose while limiting localized concentrations of pro-oxidants. In this study, we illustrate the potential of degradable ferulic acid-based polymer nanoparticles to control macrophage foam cell formation by significantly reducing oxLDL uptake through downregulation of scavenger receptors, CD-36, MSR-1, and LOX-1. Another critical finding is the ability of the degradable ferulate-based polymer nanoparticles to lower macrophage reactive oxygen species (ROS) levels, a precursor to apoptosis and plaque escalation. The degradable ferulic acid-based polymer nanoparticles hold significant promise as a means to alter the treatment and progression of atherosclerosis.

Non-Lethal Sonodynamic Therapy Inhibits Atherosclerotic Plaque Progression in ApoE-/- Mice and Attenuates ox-LDL-mediated Macrophage Impairment by Inducing Heme Oxygenase-1

Background: Previous studies from our group showed that low-intensity sonodynamic therapy (SDT) has protective effects on atherosclerosis (AS). However, because the intensity of ultrasound passing through tissue is attenuated, the consequences of very low-intensity SDT, referred to as non-lethal SDT (NL-SDT), on atherosclerotic plaques are unclear. The aim of this study was to determine whether NL-SDT affects atherosclerotic plaques and to elucidate the possible underlying mechanisms. Methods: An AS model was established using ApoE^-/- mice fed a western diet. En face Oil Red O staining was used to measure atherosclerotic plaque size. Hematoxylin and eosin staining and immunohistochemical staining were used to observe plaque morphology and assess the location of macrophages and heme oxygenase 1 (HO-1). HO-1 mRNA and protein levels in AS plaques were evaluated by real-time PCR and western blotting. Human THP-1 cells and mouse peritoneal macrophages were used in this study. Western blotting was used to investigate the expression of cellular proteins after NL-SDT. Macrophage apoptosis was evaluated by TUNEL assays and flow cytometry with Annexin V/PI double staining. Intracellular reactive oxygen species (ROS) and mitochondrial membrane potential (MMP) were measured with 2′-7′-dichlorofluorescein diacetate (DCFH-DA) and 5,5′,6,6′-tetrachloro-1,1′,3,3′-tetraethyl benzimidazolyl carbocyanine iodide (JC-1) staining, respectively. Results: NL-SDT significantly inhibited AS progression and reduced the necrotic core area. NL-SDT induced HO-1 expression in lesional macrophages and in cultured macrophages. NL-SDT activated the protein kinase B (AKT) and extracellular signal-related protein kinase (ERK) pathways and the transcription factor NF-E2-related factor 2 (Nrf2).NL-SDT significantly reduced oxidized LDL (ox-LDL)-induced macrophage MMP collapse, ROS production and cell apoptosis. Zinc protoporphyrin (ZnPP), a HO-1-specific inhibitor, reversed the protective effects of NL-SDT. Conclusion: NL-SDT inhibits atherosclerotic plaque progression and increases plaque stability. In vitro, NL-SDT has a protective effect on ox-LDL-induced macrophage impairment via HO-1.

Abstract 12019: Inactivation of AMP-activated Protein Kinase Sensitizes Heart to Pressure Overload-induced Hypertrophy

Introduction: Hypertrophy triggers activation of adenosine monophosphate-activated protein kinase (AMPK), an energy sensor in the heart. We have identified activated protein C (APC) is an endogenous AMPK agonist, and the non-anticoagulant APC-2Cys mutant but not the non-signaling APC-E170A mutant stimulates cardiac AMPK activation as well. Hypothesis: AMPK play a critical role in preventing heart from hypertrophy induced by pressure overload. APC as a AMPK agonist inhibits pressure overload-induced hypertrophy via APC’s cytoprotective signaling activity. Methods: Wild-type (WT) and AMPK-kinase dead (KD) transgenic mice were subjected to transverse aortic constriction (TAC) surgery. Echocardiography was performed to evaluate the heart function, and histology staining revealed the morphological changes. Real-time PCR and immunoblotting were used to detect mRNA and protein levels of signaling changes. Results: There is no phenotype difference between wild type (WT) and AMPK-kinase dead (AMPK KD) mice under normal physiological conditions. However after 4 weeks of TAC surgery, AMPK-KD mice demonstrated significantly bigger heart as compared to WT mice (p<0.05). The cardiac functions measured by echocardiography in AMPK-KD hearts were significantly impaired as compared with WT hearts (p<0.05). The immunohistochemical staining showed that the increased macrophage infiltration and reactive oxygen species (ROS) in AMPK-KD versus WT hearts after 4 weeks of TAC surgery. The immunoblotting results demonstrated that the redox status markers such as p66 shc , 4-hydroxynonenal and extracellular signal-regulated kinase (ERK) were up-regulated in the AMPK-KD hearts after 4 weeks of TAC surgery (all p<0.05 versus WT hearts). APC and APC-2Cys but not APC-E170A administration significantly attenuated hypertrophy and fibrosis caused by pressure overload. The macrophage infiltration and p66 shc activation caused by pressure overload were also inhibited by APC and APC-2Cys but not by APC-E170A. Conclusions: Cardiac AMPK inactivation aggravates pressure overload induced hypertrophy. APC-2Cys without anticoagulant activity could be a therapeutic drug for treatment of hypertension-related hypertrophy patients without bleeding risk.

Abstract 025: AMP-activated Protein Kinase Mediates Pressure Overload-induced Hypertrophy

Hypertension is a major risk factor of death and disability from heart and vascular diseases. Heart hypertrophy caused by pressure overload is characterized by the activation of adenosine monophosphate-activated protein kinase (AMPK), which is the major energy sensor in the heart. However, the anti-inflammatory effect of AMPK has not been investigated in the hypertrophy model. Activated protein C (APC) is a vitamin-K dependent plasma serine protease which inhibits blood clotting, and APC is an endogenous AMPK agonist. This study was designed to examine the role of AMPK in hypertrophy and the underlying mechanisms by which APC inhibits heart hypotrophy by pressure overload. We hypothesize that AMPK play a role in preventing heart from hypertrophy induced by pressure overload. APC could inhibit high blood pressure-induced hypertrophy via activation of AMPK signaling pathway. Wild-type (WT) and AMPK-kinase dead (KD) transgenic mice were subjected to transverse aortic constriction (TAC) surgery. Echocardiography was performed to evaluate the heart function, and histology staining revealed the morphological changes. Real-time PCR and western blotting were used to detect the signaling changes of both mRNA and protein expression levels. There is no phenotype difference between WT and AMPK-KD mice under normal physiological conditions. However after 4 weeks of TAC surgery, AMPK-KD mice demonstrated significantly bigger heart than WT mice (p<0.05), and the cardiac functions measured by echocardiography in AMPK-KD hearts were significantly impaired as compared with WT hearts (p<0.05). The immunohistochemical staining showed that the increased macrophage infiltration and reactive oxygen species (ROS) including activated p66shc, 4-HNE and ERK were observed in the AMPK-KD hearts after 4 weeks of TAC surgery (all p<0.05 versus WT hearts). APC administration significantly attenuated hypertrophy and fibrosis caused by pressure overload, and macrophage infiltration and p66shc activation were also inhibited by APC treatment. Therefore, Cardiac AMPK deficiency aggravates hypertrophy caused by pressure overload. AMPK activator APC could be a therapeutic drug for treatment of hypertrophy by high blood pressure.

Associations between microvascular function and short-term exposure to traffic-related air pollution and particulate matter oxidative potential.

BackgroundShort-term exposure to ambient air pollution has been associated with acute increases in cardiovascular hospitalization and mortality. However, causative chemical components and underlying pathophysiological mechanisms remain to be clarified. We hypothesized that endothelial dysfunction would be associated with mobile-source (traffic) air pollution and that pollutant components with higher oxidative potential to generate reactive oxygen species (ROS) would have stronger associations.MethodsWe carried out a cohort panel study in 93 elderly non-smoking adults living in the Los Angeles metropolitan area, during July 2012-February 2014. Microvascular function, represented by reactive hyperemia index (RHI), was measured weekly for up to 12 weeks (N = 845). Air pollutant data included daily data from regional air-monitoring stations, five-day average PM chemical components and oxidative potential in three PM size-fractions, and weekly personal nitrogen oxides (NOx). Linear mixed-effect models estimated adjusted changes in microvascular function with exposure.ResultsRHI was inversely associated with traffic-related pollutants such as ambient PM2.5 black carbon (BC), NOx, and carbon monoxide (CO). An interquartile range change increase (1.06 μg/m3) in 5-day average BC was associated with decreased RHI, −0.093 (95 % CI: −0.151, −0.035). RHI was inversely associated with other mobile-source components/tracers (polycyclic aromatic hydrocarbons, elemental carbon, and hopanes), and PM oxidative potential as quantified in two independent assays (dithiothreitol and in vitro macrophage ROS) in accumulation and ultrafine PM, and transition metals.ConclusionsOur findings suggest that short-term exposures to traffic-related air pollutants with high oxidative potential are major components contributing to microvascular dysfunction.Electronic supplementary materialThe online version of this article (doi:10.1186/s12940-016-0157-5) contains supplementary material, which is available to authorized users.

Development of Thiolated-Graphene Quantum Dots for Regulation of ROS in macrophages.

The current study was designed to develop thiolated-graphene quantum dots (SH-GQDs) as a theranostic nanocarrier and evaluate its potential for the optimal scavenging of reactive oxygen species (ROS) in macrophages. SH-GQDs were prepared by hydrothermal pyrolysis of carbon source (citric acid) in the presence of reduced-glutathione (GSH). Raw264.7 cells were treated with varying concentrations of oxLDL (0.5, 1 and 2μg/ml) in the presence or absence of SH-GQDs and cells were stained with peroxide-sensitive fluorescent dye (DCFDA). Flow cytometry analysis was performed to investigate the expression of MSR and ATP-binding cassette transporter (ABCA1) after such treatments as the negative control, oxLDL treatment and oxLDL treatment in the presence of either GQDs or SH-GQDs. SH-GQDs had a size ranging from 10 to 30nm with an average size of 21.3 ± 5.2nm. The elemental analysis indicated that SH-GQDs are mainly composed of carbon, nitrogen, oxygen and sulfur. The expression levels of ABCA1 in macrophages treated with either LDL or oxLDL were lower than those treated with the media control (the negative control: 100 ± 7.6%; LDL: 82.7 ± 1.2%; and oxLDL: 79.2 ± 1.7%). The level of ABCA1 expression increased as cells were incubated with SH-GQDs (SH-GQDs: 101.5 ± 3.1%). The level of MSR on the surface of macrophages upon being treated with SH-GQDs was lower than those with oxLDL (oxLDL: 112.1 ± 8.8% and SH-GQDs: 91.5 ± 4.2%). The enhancement of lipid efflux and down-regulation of MSR in macrophages by SH-GQDs supported its promising usage as a theranostic nanocarrier to prevent foam cell formation and plaque development.

Associations of oxidative stress and inflammatory biomarkers with chemically-characterized air pollutant exposures in an elderly cohort

BackgroundExposure to air pollution has been associated with cardiorespiratory morbidity and mortality. However, the chemical constituents and pollution sources underlying these associations remain unclear. MethodWe conducted a cohort panel study involving 97 elderly subjects living in the Los Angeles metropolitan area. Airway and circulating biomarkers of oxidative stress and inflammation were measured weekly over 12 weeks and included, exhaled breath condensate malondialdehyde (EBC MDA), fractional exhaled nitric oxide (FeNO), plasma oxidized low-density lipoprotein (oxLDL), and plasma interleukin-6 (IL-6). Exposures included 7-day personal nitrogen oxides (NOx), daily criteria-pollutant data, five-day average particulate matter (PM) measured in three size-fractions and characterized by chemical components including transition metals, and in vitro PM oxidative potential (dithiothreitol and macrophage reactive oxygen species). Associations between biomarkers and pollutants were assessed using linear mixed effects regression models. ResultsWe found significant positive associations of airway oxidative stress and inflammation with traffic-related air pollutants, ultrafine particles and transition metals. Positive but nonsignificant associations were observed with PM oxidative potential. The strongest associations were observed among PM variables in the ultrafine range (PM <0.18µm). It was estimated that an interquartile increase in 5-day average ultrafine polycyclic aromatic hydrocarbons was associated with a 6.3% (95% CI: 1.1%, 11.6%) increase in EBC MDA and 6.7% (95% CI: 3.4%, 10.2%) increase in FeNO. In addition, positive but nonsignificant associations were observed between oxLDL and traffic-related pollutants, ultrafine particles and transition metals while plasma IL-6 was positively associated with 1-day average traffic-related pollutants. ConclusionOur results suggest that exposure to pollutants with high oxidative potential (traffic-related pollutants, ultrafine particles, and transition metals) may lead to increased airway oxidative stress and inflammation in elderly adults. This observation was less clear with circulating biomarkers.

Impaired macrophage autophagy induces systemic insulin resistance in obesity.

Obesity-induced insulin resistance and diabetes are significantly associated with infiltrates of inflammatory cells in adipose tissue. Previous studies recognized the involvement of autophagy in the regulation of metabolism in multiple tissues, including β-cells, hepatocytes, myocytes, and adipocytes. However, despite the importance of macrophages in obesity-induced insulin resistance, the role of macrophage autophagy in regulating insulin sensitivity is seldom addressed. In the present study, we show that macrophage autophagy is important for the regulation of systemic insulin sensitivity. We found that macrophage autophagy is downregulated by both acute and chronic inflammatory stimuli, and blockade of autophagy significantly increased accumulation of reactive oxygen species (ROS) in macrophages. Macrophage-specific Atg7 knockout mice displayed a shift in the proportion to pro-inflammatory M1 macrophages and impairment of insulin sensitivity and glucose homeostasis under high-fat diet conditions. Furthermore, inhibition of ROS in macrophages with antioxidant recovered adipocyte insulin sensitivity. Our results provide evidence of the underlying mechanism of how macrophage autophagy regulates inflammation and insulin sensitivity. We anticipate our findings will serve as a basis for development of therapeutics for inflammatory diseases, including diabetes.

Abstract 319: Cardiac Deficiency of AMPK Exacerbates Heart Hypertrophy by Pressure Overload

Introduction: Heart hypertrophy caused by pressure overload is characterized by the activation of adenosine monophosphate-activated protein kinase (AMPK), which is a major energy sensor in the heart. However, the anti-inflammatory effect of AMPK has not been determined in the hypertrophy model. Activated protein C (APC) is a vitamin-K dependent plasma serine protease which inhibits blood clotting, and APC is an endogenous AMPK agonist. This study was designed to examine the role of AMPK in hypertrophy and the underlying mechanisms by which APC inhibits heart hypotrophy by pressure overload. Hypothesis: AMPK play a role in preventing heart from hypertrophy induced by pressure overload. APC could inhibit high blood pressure-induced hypertrophy via activation of AMPK signaling pathway. Methods: Wild-type (WT) and AMPK-kinase dead (KD) transgenic mice were subjected to transverse aortic constriction (TAC) surgery. Echocardiography was performed to evaluate the heart function, and histology staining revealed the morphological changes. Real-time PCR and western blotting were used to determine the signaling changes of mRNA and protein expression levels. Results: There is no phenotype difference between WT and AMPK-KD mice under normal physiological conditions. However after 4 weeks of TAC surgery, AMPK-KD mice demonstrated significantly bigger heart than WT mice (p&lt;0.05), and the cardiac functions measured by echocardiography in AMPK-KD hearts were significantly impaired as compared with WT hearts (p&lt;0.05). The immunohistochemical staining showed that the increased macrophage infiltration and reactive oxygen species (ROS) including activated p66shc, 4-Hydroxynonenal accumulation and phosphorylation of extracellular signal-regulated kinase (ERK) were observed in the AMPK-KD hearts after 4 weeks of TAC surgery (all p&lt;0.05 versus WT hearts). APC administration significantly attenuated hypertrophy and fibrosis caused by pressure overload, and macrophage infiltration and p66shc activation were also inhibited by APC treatment. Conclusions: Cardiac AMPK deficiency aggravates hypertrophy caused by pressure overload. AMPK activator APC could be a therapeutic drug for treatment of hypertrophy by high blood pressure.

Antioxidant Defenses of Francisella tularensis Modulate Macrophage Function and Production of Proinflammatory Cytokines

Francisella tularensis, the causative agent of a fatal human disease known as tularemia, has been used in the bioweapon programs of several countries in the past, and now it is considered a potential bioterror agent. Extreme infectivity and virulence of F. tularensis is due to its ability to evade immune detection and to suppress the host's innate immune responses. However, Francisella-encoded factors and mechanisms responsible for causing immune suppression are not completely understood. Macrophages and neutrophils generate reactive oxygen species (ROS)/reactive nitrogen species as a defense mechanism for the clearance of phagocytosed microorganisms. ROS serve a dual role; at high concentrations they act as microbicidal effector molecules that destroy intracellular pathogens, and at low concentrations they serve as secondary signaling messengers that regulate the expression of various inflammatory mediators. We hypothesized that the antioxidant defenses of F. tularensis maintain redox homeostasis in infected macrophages to prevent activation of redox-sensitive signaling components that ultimately result in suppression of pro-inflammatory cytokine production and macrophage microbicidal activity. We demonstrate that antioxidant enzymes of F. tularensis prevent the activation of redox-sensitive MAPK signaling components, NF-κB signaling, and the production of pro-inflammatory cytokines by inhibiting the accumulation of ROS in infected macrophages. We also report that F. tularensis inhibits ROS-dependent autophagy to promote its intramacrophage survival. Collectively, this study reveals novel pathogenic mechanisms adopted by F. tularensis to modulate macrophage innate immune functions to create an environment permissive for its intracellular survival and growth.