Robust Reactive Oxygen Species Production Research Articles

Cigarette smoke impairs the hematopoietic supportive property of mesenchymal stem cells via the production of reactive oxygen species and NLRP3 activation

BackgroundMesenchymal stem cells (MSCs) play important roles in tissue homeostasis by providing a supportive microenvironmental niche for the hematopoietic system. Cigarette smoking induces systemic abnormalities, including an impeded recovery process after hematopoietic stem cell transplantation. However, the role of cigarette smoking-mediated alterations in MSC niche function have not been investigated.MethodsIn the present study, we investigated whether exposure to cigarette smoking extract (CSE) disrupts the hematopoietic niche function of MSCs, and pathways impacted. To investigate the effects on bone marrow (BM)-derived MSCs and support of hematopoietic stem and progenitor cells (HSPCs), mice were repeatedly infused with the CSE named 3R4F, and hematopoietic stem and progenitor cells (HSPCs) supporting function was determined. The impact of 3R4F on MSCs at cellular level were screened by bulk-RNA sequencing and subsequently validated through qRT-PCR. Specific inhibitors were treated to verify the ROS or NLRP3-specific effects, and the cells were then transplanted into the animal model or subjected to coculture with HSPCs.ResultsBoth direct ex vivo and systemic in vivo MSC exposure to 3R4F resulted in impaired engraftment in a humanized mouse model. Furthermore, transcriptomic profile analysis showed significantly upregulated signaling pathways related to reactive oxygen species (ROS), inflammation, and aging in 3R4F-treated MSCs. Notably, ingenuity pathway analysis revealed the activation of NLRP3 inflammasome signaling pathway in 3R4F-treated MSCs, and pretreatment with the NLRP3 inhibitor MCC950 rescued the HSPC-supporting ability of 3R4F-treated MSCs.ConclusionIn conclusion, these findings indicate that exposure to CSE reduces HSPCs supportive function of MSCs by inducing robust ROS production and subsequent NLRP3 activation.

Integration of O2-economised tumour-targeted photosensitive magnetic nanomaterials in the diagnosis and therapy of gastric cancer.

Hypoxia in the tumour microenvironment is a major limiting factor in photodynamic therapy. The present study employed a novel O2-economised photosensitizer, ACSN, to effectively curtail oxygen consumption by impeding the aerobic respiration of tumour cells, thereby increasing the reactive oxygen species (ROS) production in photodynamic therapy. To enhance the efficacy of photodynamic therapy, the active targeting peptide iRGD was employed to facilitate drug accumulation in the tumour tissue. Therefore, we constructed a targeted drug platform, ACSN/Fe3O4@MSNs-iRGD, that integrates diagnosis and treatment. The drug exhibited excellent active targeting ability towards gastric cancer MGC-803 cells and can efficiently penetrate the mitochondria upon cellular internalisation. The photosensitizer ACSN, released from the drug, effectively suppressed mitochondrial aerobic respiration to conserve oxygen and exhibited robust ROS production upon laser excitation. The core-shell structure comprises Fe3O4, which offers excellent T2 dark contrast for real-time tumour monitoring through MRI imaging. By incorporating excellent photodynamic therapy and MRI imaging capabilities, this drug can serve as an effective platform for the integration of tumour diagnosis and treatment, thus addressing the limitations associated with conventional tumour therapies. It is anticipated that this approach will soon be clinically translated.

Robust reactive oxygen species production in interfacial reaction between organic acids and biochar: The combined effect of electron acceptance and electron conduction

Interfacial electron transport and reactive oxygen species (ROS) generation in the redox action between biochar (BC) and low-molecular-weight organic acids (LMWOAs) have been overlooked during the utilization of BC in soil amelioration/remediation. Herein, BC and N-doped BC (NBC) with various physicochemical properties were prepared at pyrolysis temperatures of 350, 550 and 750 °C (namely BC/NBC350, 550 and 750) and their interactions with LMWOAs were systematically investigated. Results of ROS quenching and electrochemical cell experiments revealed that BC/NBC could strongly interact with LMWOAs, especially ascorbic acid (AA), and acted as an electron shuttle to mediate one electron transfer from AA to oxygen to generate O2•−, followed by the robust generation of H2O2 and •OH. Interestingly, the generation rates of •OH and H2O2 in the NBC-AA system were significantly higher than that in the BC-AA system, of which the NBC550-AA system exhibited the best performance, enabling its superior ability in bisphenol A degradation. Different from conventional wisdom, the electron shuttle capacity depended on a combined aromatization degree and electron-accepting capacity of BC/NBC. These findings complement the interfacial electron transfer mechanism during the natural BC-LMWOAs interaction and provide new inspiration for the development of green and efficient organic pollutant removal technologies.

Design of One-for-All Near-Infrared Aggregation-Induced Emission Nanoaggregates for Boosting Theranostic Efficacy.

Fluorescence-guided phototherapy, including photodynamic and photothermal therapy, is considered an emerging noninvasive strategy for cancer treatments. Organic molecules are promising theranostic agents because of their facile construction, simple modification, and good biocompatibility. Organic systems that integrated multifunctionalities in a single component and achieved high efficiency in both imaging and therapies are rarely reported as the inherently competitive energy relaxation pathways are hard to modulate, and fluorescence quenching occurs upon molecular aggregation. Herein, a versatile theranostic platform with near-infrared emission, high fluorescence quantum yield, robust reactive oxygen species production, and excellent photothermal conversion efficiency was developed based on an aggregation-induced emission luminogen, namely, TPA-TBT. In vivo studies revealed that the TPA-TBT nanoaggregates exhibit outstanding photodynamic and photothermal therapy efficacy to ablate tumors inoculated in a mouse model. This work offers a design strategy to develop one-for-all cancer theranostic agents by modulating and utilizing the relaxation energy of excitons in full.

Ferritin reduction is essential for cerebral ischemia-induced hippocampal neuronal death through p53/SLC7A11-mediated ferroptosis

Cerebral ischemia is the most common cause of hippocampal neuronal death and the most prevalent cause of stroke with high mortality rate. Ferroptosis has been suggested to affect the role of hippocampal neurons. This study explores the influence of lentivirus infection-induced ferritin overexpression in hippocampal neuronal injury and death through simulations in August Copenhagen Irish rat models. Twenty-four-hour cerebral ischemia–reperfusion injury was induced in the rats after 90-min middle cerebral artery occlusion (MCAO). Ferritin overexpression was induced through lentivirus infection. The Morris Water Maze (MWM) test and tau hyperphosphorylation test were performed on hippocampal neurons to establish a MCAO model. The effect of ferritin overexpression on hippocampal neuronal death was evaluated using hematoxylin–eosin staining and annexin V/propidium iodide flow cytometry. The MWM test revealed that MCAO modeling decreased the cognitive and locomotor capacity of the rats, whereas ferritin overexpression partially reversed the effect of MCAO. In addition, the hyperphosphorylation of tau caused by MCAO was reduced by ferritin. Pathogenic changes, impaired viability, increased apoptosis, and elevated caspase-9 cleavage in hippocampal neurons were clearly recovered by ferritin. Moreover, robust reactive oxygen species production and glutathione consumption, which was induced by MCAO modeling, were ameliorated by ferritin. Furthermore, two key modulators of ferroptosis, p53 and SLC7A11, were demonstrated to be upregulated by MCAO modeling and downregulated by ferritin. Ferritin reduction is essential for cerebral ischemia-induced hippocampal neuronal ferroptosis mediated via p53 and SLC7A11.

Therapeutic benefits of apocynin in mice with lipopolysaccharide/D-galactosamine-induced acute liver injury via suppression of the late stage pro-apoptotic AMPK/JNK pathway

The excessive generation of reactive oxygen species (ROS) plays crucial roles in the development of acute liver injury. Nicotinamide adenine dinucleotide phosphate oxidase (NOX) is responsible for the robust production of ROS under inflammatory circumstance, but the pathological roles of NOX and the pharmacological significance of NOX inhibitor in acute liver injury remains unclear. In the present study, the potential roles of NOX in acute liver injury were investigated in a mouse model with lipopolysaccharide (LPS)/D-galactosamine (D-Gal)-induced acute liver injury. The results indicated that LPS/D-Gal exposure time-dependently increased the level of ROS in liver tissue. Pretreatment with the NOX inhibitor apocynin suppressed LPS/D-Gal induced upregulation of ROS, 8-hydroxy-2-deoxyguanosine (8-OH-dG), protein carbonyl content and thiobarbituric acid reactive substances (TBARS). Pretreatment with apocynin also suppressed LPS/D-Gal-induced elevation of aminotransferase, alleviated histological abnormalities, inhibited the production of pro-inflammatory cytokine tumor necrosis factor α (TNF-α), blocked the activation of caspase cascade, reduced the count of TUNEL-positive cells and prevented LPS/D-Gal-induced mortality. Interestingly, post insult treatment with apocynin also suppressed LPS/D-Gal-induced oxidative stress, hepatocyte apoptosis, liver damage but improved the survival rate. Mechanistically, posttreatment with apocynin prohibited LPS/D-Gal-induced activation of the late stage pro-apoptotic AMP-activated protein kinase (AMPK)/c-Jun N-terminal kinase (JNK) pathway. Post-insult treatment with the antioxidant N-acetylcysteine also resulted in suppressed activation of AMPK/JNK, mitigated apoptosis and alleviated liver injury. These data suggest that NOX-derived ROS might be a crucial late stage detrimental factor in LPS/D-Gal-induced acute liver injury via promoting the activation of the pro-apoptotic AMPK/JNK pathway, and the NOX inhibitor might have important value in the pharmacological intervention of inflammation-base liver damage.

RANKL treatment of vascular endothelial cells leading to paracrine pro-calcific signaling involves ROS production.

Numerous studies have highlighted the causal link between over-production of reactive oxygen species (ROS) and cardiovascular complications such as vascular calcification (VC). Receptor-activator of nuclear factor-κB ligand (RANKL) has previously been shown to act on endothelial cells, eliciting the production/release of paracrine pro-calcific signals that act, in-turn, upon underlying vascular smooth muscle cells (VSMCs) to induce osteoblastic differentiation and VC. A role for endothelial ROS signaling in this process has not been established however. In the current paper, we investigate the possibility that RANKL leads to ROS signaling within the endothelial layer as part of the RANKL-driven VC signaling cascade. Human aortic endothelial cells (HAECs) were treated with RANKL (25ng/ml, 72h) and monitored for ROS production, in parallel with various pro-calcific signaling indices. Antioxidant co-treatments included TRAIL (5ng/ml), apocynin (10mM) and N-acetylcysteine (5mM). Treatment of HAECs with RANKL-induced robust ROS production. This surge could be partially attenuated by TRAIL and strongly attenuated by apocynin and N-acetylcysteine. RANKL also elicited a range of signaling events in HAECs that we have previously demonstrated are coupled to osteoblastic differentiation in underlying VSMCs. These include non-canonical NF-κB/p52 activation, elevated BMP-2 release and increased alkaline phosphatase (ALP) enzyme activity (cellular and extracellular). Importantly, these RANKL-induced signaling events could be completely prevented by co-treatment of HAECs with antioxidants. In summary, RANKL elicits ROS generation in HAECs with direct consequences for generation of paracrine pro-calcific signals known to effect calcification in underlying VSMCs.

Morphine induces dysfunction of PINK1/Parkin-mediated mitophagy in spinal cord neurons implying involvement in antinociceptive tolerance.

The development of opioid-induced analgesic tolerance is a clinical challenge in long-term use for managing chronic pain. The mechanisms of morphine tolerance are poorly understood. Mitochondria-derived reactive oxygen species (ROS) is a crucial signal inducing analgesic tolerance and pain. Chronic administration of morphine leads to robust ROS production and accumulation of damaged mitochondria, which are immediately removed by mitophagy. Here, we show that morphine inhibits mitochondria damage-induced accumulation of PTEN-induced putative kinase 1 (PINK1) in neurons. It interrupts the recruitment of Parkin to the impaired mitochondria and inhibits the ubiquitination of mitochondrial proteins catalyzed by Parkin. Consequently, morphine suppresses the recognition of autophagosomes to the damaged mitochondria mediated by LC3 and sequestosome-1 (SQSTM1/p62). Thus, morphine inhibits autophagy flux and leads to the accumulation of SQSTM1/p62. Finally, the impaired mitochondria cannot be delivered to lysosomes for degradation and ultimately induces robust ROS production and morphine tolerance. Our findings suggest that the dysfunction of mitophagy is involved in morphine tolerance. The deficiency of PINK1/Parkin-mediated clearance of damaged mitochondria is crucial for the generation of excessive ROS and important to the development of analgesic tolerance. These findings suggest that the compounds capable of stabilizing PINK1 or restoring mitophagy may be utilized to prevent or reduce opioid tolerance during chronic pain management.

Carbon nanotubes and crystalline silica stimulate robust ROS production, inflammasome activation, and IL-1β secretion in macrophages to induce myofibroblast transformation.

Pulmonary exposure to inhaled particulates elicits complex inflammatory and fibrotic responses that may progress to chronic fibrosis. The fibrogenic potentials of respirable particulates are influenced by their physicochemical properties and their interactions with major pathways to drive fibrotic development in the lung. Macrophages were exposed to six carbon nanotubes (CNTs) of varying dimensions, crystalline silica, or carbon black (CB), with lipopolysaccharide (LPS) and transforming growth factor (TGF)-β1 as positive controls. Macrophage-conditioned media was collected and applied to cultures of human pulmonary fibroblast line WI38-VA13 to induce myofibroblast transformation. Multi-walled and single-walled CNTs (MWCNTs and SWCNTs, respectively) and silica, but not CB, stimulated robust myofibroblast transformation through macrophage-conditioned media. On an equal weight basis, MWCNTs induced higher induction than SWCNTs. High induction was observed for MWCNTs with a long and slender or a short and rigid shape, and silica, atlevelscomparable to those by LPS and TGF-β1. Fibrogenic particulates induced high levels of IL-1β mRNA expression and protein secretion that are required for macrophage-guided myofibroblast transformation. Induction of IL-1β is dependent on the activation of the NLRP3 (NOD-like receptor family, pyrin domain containing 3) inflammasome and ROS (reactive oxygen species) production in macrophages, as inhibition of NLRP3 by MCC950 and reduction of ROS production by N-acetylcysteine blocked NLRP3 activation,IL-1β induction, and fibroblast activation anddifferentiation. Therefore, fibrogenic CNTs and silica, but not CB, elicit robust macrophage-guided myofibroblast transformation, which depends on the induction of IL-1β through the NLRP3 inflammasome pathway andthe increased production of ROS in macrophages.

Oxidative stress-induced mitochondrial dysfunction in a normal colon epithelial cell line.

AIMTo determine how a normal human colon cell line reacts to microbial challenge as a way to study oxidative stress-induced responses associated with inflammatory bowel disease.METHODSNormal human colon epithelial cells (ATCC® CRL.1790™) were stimulated with either heat killed E. coli or heat killed murine cecal contents (HKC) and examined for several relevant biomarkers associated with inflammation and oxidative stress including cytokine production, mitochondrial autophagy and oxidant status. TNFα, IL-1β and IL-8 protein concentrations were measured within the supernatants. Fluorescent microscopy was performed to quantify the production of reactive oxygen species (ROS) using an oxidation responsive fluorogenic probe. Mitochondrial morphology and mitochondrial membrane potential was assessed by dual staining using COXIV antibody and a dye concentrating in active mitochondria. Mitochondrial ROS scavenger was used to determine the source of ROS in stimulated cells. Autophagy was detected by staining for the presence of autophagic vesicles. Positive controls for autophagy and ROS/RNS experiments were treated with rapamycin and chloroquine. Mitochondrial morphology, ROS production and autophagy microscopy experiments were analyzed using a custom acquisition and analysis microscopy software (ImageJ).RESULTSExposing CRL.1790 cells to microbial challenge stimulated cells to produce several relevant biomarkers associated with inflammation and oxidative stress. Heat killed cecal contents treatment induced a 10-12 fold increase in IL-8 production by CRL.1790 cells compared to unstimulated controls at 6 and 12 h (P < 0.001). Heat killed E. coli stimulation resulted in a 4-5 fold increase in IL-8 compared to the unstimulated control cells at each time point (P < 0.001). Both heat killed E. coli and HKC stimulated robust ROS production at 6 (P < 0.001), and 12 h (P < 0.01). Mitochondrial morphologic abnormalities were detected at 6 and 12 h based on reduced mitochondrial circularity and decreased mitochondrial membrane potential, P < 0.01. Microbial stimulation also induced significant autophagy at 6 and 12 h, P < 0.01. Lastly, blocking mitochondrial ROS generation using mitochondrial specific ROS scavenger reversed microbial challenge induced mitochondrial morphologic abnormalities and autophagy.CONCLUSIONThe findings from this study suggest that CRL.1790 cells may be a useful alternative to other colon cancer cell lines in studying the mechanisms of oxidative stress events associated with intestinal inflammatory disorders.

Priming of the neutrophil respiratory burst: role in host defense and inflammation.

Neutrophils are the major circulating white blood cells in humans. They play an essential role in host defense against pathogens. In healthy individuals, circulating neutrophils are in a dormant state with very low efficiency of capture and arrest on the quiescent endothelium. Upon infection and subsequent release of pro-inflammatory mediators, the vascular endothelium signals to circulating neutrophils to roll, adhere, and cross the endothelial barrier. Neutrophils migrate toward the infection site along a gradient of chemo-attractants, then recognize and engulf the pathogen. To kill this pathogen entrapped inside the vacuole, neutrophils produce and release high quantities of antibacterial peptides, proteases, and reactive oxygen species (ROS). The robust ROS production is also called 'the respiratory burst', and the NADPH oxidase or NOX2 is the enzyme responsible for the production of superoxide anion, leading to other ROS. In vitro, several soluble and particulate agonists induce neutrophil ROS production. This process can be enhanced by prior neutrophil treatment with 'priming' agents, which alone do not induce a respiratory burst. In this review, we will describe the priming process and discuss the beneficial role of controlled neutrophil priming in host defense and the detrimental effect of excessive neutrophil priming in inflammatory diseases.

Conjugation to the sigma-2 ligand SV119 overcomes uptake blockade and converts dm-Erastin into a potent pancreatic cancer therapeutic.

Cancer-selective drug delivery is an important concept in improving treatment while minimizing off-site toxicities, and sigma-2 receptors, which are overexpressed in solid tumors, represent attractive pharmacologic targets. Select sigma-2 ligands have been shown to be rapidly internalized selectively into cancer cells while retaining the capacity to deliver small molecules as drug cargoes. We utilized the sigma-2-based drug delivery concept to convert Erastin, a clinically underperforming drug, into a potent pancreatic cancer therapeutic. The Erastin derivative des-methyl Erastin (dm-Erastin) was chemically linked to sigma-2 ligand SV119 to create SW V-49. Conjugation increased the killing capacity of dm-Erastin by nearly 35-fold in vitro and reduced the size of established tumors and doubled the median survival in syngeneic and patient-derived xenograft models when compared to non-targeted dm-Erastin. Mechanistic analyses demonstrated that cell death was associated with robust reactive oxygen species production and could be efficiently antagonized with antioxidants. Mass spectrometry was employed to demonstrate selective uptake into pancreatic cancer cells. Thus, targeted delivery of dm-Erastin via conjugation to the sigma-2 ligand SV119 produced efficient tumor control and prolonged animal survival with minimal off-target toxicities, and SW V-49 represents a promising new therapeutic with the potential to advance the fight against pancreatic cancer.

KEAP1-Dependent Synthetic Lethality Induced by AKT and TXNRD1 Inhibitors in Lung Cancer

Intrinsic resistance to agents targeting phosphoinositide 3-kinase (PI3K)/AKT pathway is one of the major challenges in cancer treatment with such agents. The objective of this study is to identify the genes or pathways that can be targeted to overcome the resistance of non-small cell lung carcinoma (NSCLC) to the AKT inhibitor MK2206, which is currently being evaluated in phase I and II clinical trials. Using a genome-wide siRNA library screening and biologic characterization, we identified that inhibition of thioredoxin reductase-1 (TXNRD1), one of the key antioxidant enzymes, with siRNAs or its inhibitor, auranofin, sensitized NSCLC cells to MK2206 treatment in vitro and in vivo. We found that simultaneous inhibition of TXNRD1 and AKT pathways induced robust reactive oxygen species production, which was involved in c-jun-NH2-kinase (JNK; MAPK8) activation and cell apoptosis. Furthermore, we found that the synthetic lethality interaction between the TXNRD1 and AKT pathways occurred through the KEAP1/NRF2 cellular antioxidant pathway. Finally, we found that synthetic lethality induced by TXNRD1 and AKT inhibitors relied on wild-type KEAP1 function. Our study indicates that targeting the interaction between AKT and TXNRD1 antioxidant pathways with MK2206 and auranofin, a U.S. Food and Drug Administration-approved drug, is a rational strategy to treat lung cancer and that KEAP1 mutation status may offer a predicative biomarker for such combination approaches.

Modulation of Experimental Herpes Encephalitis-Associated Neurotoxicity through Sulforaphane Treatment

Reactive oxygen species (ROS) produced by brain-infiltrating macrophages and neutrophils, as well as resident microglia, are pivotal to pathogen clearance during viral brain infection. However, unchecked free radical generation is also responsible for damage to and cytotoxicity of critical host tissue bystander to primary infection. These unwanted effects of excessive ROS are combated by local cellular production of antioxidant enzymes, including heme oxygenase-1 (HO-1) and glutathione peroxidase 1 (Gpx1). In this study, we showed that experimental murine herpes encephalitis triggered robust ROS production, as well as an opposing upregulation of the antioxidants HO-1 and Gpx1. This antioxidant response was insufficient to prevent tissue damage, neurotoxicity, and mortality associated with viral brain infection. Previous studies corroborate our data supporting astrocytes as the major antioxidant producer in brain cell cultures exposed to HSV-1 stimulated microglia. We hypothesized that stimulating opposing antioxidative responses in astrocytes, as well as neurons, would mitigate the effects of ROS-mediated neurotoxicity both in vitro and during viral brain infection in vivo. Here, we demonstrate that the addition of sulforaphane, a potent stimulator of antioxidant responses, enhanced HO-1 and Gpx1 expression in astrocytes through the activation of nuclear factor-E2-related factor 2 (Nrf2). Additionally, sulforaphane treatment was found to be effective in reducing neurotoxicity associated with HSV-stimulated microglial ROS production. Finally, intraperitoneal injections of sulforaphane into mice during active HSV infection reduced neuroinflammation via a decrease in brain-infiltrating leukocytes, macrophage- and neutrophil-produced ROS, and MHCII-positive, activated microglia. These data support a key role for astrocyte-produced antioxidants in modulating oxidative stress and neuronal damage in response to viral infection.

Nonhematopoietic toll-like receptor 2 contributes to neutrophil and cardiac function impairment during polymicrobial sepsis.

Toll-like receptor 2 (TLR2) has been implicated in neutrophil and cardiac dysfunction during sepsis. Here we tested the hypothesis that nonhematopoietic (parenchymal) and hematopoietic TLR2 play distinct roles in sepsis pathogenesis. To achieve this, we generated two groups of chimeric mice with TLR2 deletions either in nonhematopoietic cells (knockout [KO] mice with wild-type [WT] bone marrow [BM]) or in BM cells (WT mice with KO-BM). Polymicrobial sepsis was created by cecal ligation and puncture (CLP). Neutrophil functions, cytokine production, and bacterial clearance were investigated following CLP or sham procedures. Cardiac contractile function was measured in a Langendorff apparatus. Intracellular reactive oxygen species (ROS) were measured using redox-sensitive dye and flow cytometry. Cecal ligation and puncture mice had markedly increased peritoneal neutrophil recruitment compared with the sham-operated mice. Toll-like receptor 2 KO mice, regardless their TLR2 phenotypes (WT vs. KO) in their BM-derived hematopoietic cells, had markedly increased neutrophil migration as well as phagocytosis and reduced cytokine productions compared with TLR2 WT mice following polymicrobial peritonitis. These changes in the chimeric TLR2 KO mice were associated with enhanced blood bacterial clearance and markedly improved cardiac contractile function. Moreover, CLP induced a robust ROS production in the peritoneal leukocytes isolated from WT mice but not from TLR2 KO mice. Taken together, these data indicate that TLR2, particularly that of nonhematopoietic cells, plays a major role in sepsis pathogenesis by impairing neutrophil migratory and phagocytic function, promoting cytokine production, and mediating cardiac contractile dysfunction during polymicrobial sepsis. Toll-like receptor 2 also mediates critical ROS production during polymicrobial sepsis.

Microglia enhance manganese chloride-induced dopaminergic neurodegeneration: Role of free radical generation

Exposure to elevated levels of manganese has been shown to cause neuronal damage in the midbrain and the development of Parkinsonian symptoms. Activation of microglia and release of neurotoxic factors in particular free radicals are known to contribute to neurodegeneration. We have recently reported that manganese chloride (MnCl2) stimulates microglia to produce reactive oxygen species (ROS). The aim of this study is to determine the role of microglia in the MnCl2-induced degeneration of dopaminergic (DA) neurons that are particularly vulnerable to oxidative insult. MnCl2 (10–300 μM; 7 days) was markedly more effective in damaging DA neurons in the rat mesencephalic neuron–glia cultures than the neuron-enriched (microglia-depleted) cultures. In addition, the microglia-enhanced MnCl2 toxicity was found to be preferential to DA neurons. The microglial enhancement of DA neurotoxicity was further supported by the observation that replenishment of microglia to the neuron-enriched cultures significantly increased the susceptibility of DA neurons to the MnCl2-induced damage. Analysis of the temporal relationship between microglial activation and DA neurodegeneration revealed that MnCl2-stimulated microglial activation preceded DA neurodegeneration. Mechanistically, MnCl2 (10–300 μM) stimulated a concentration- and time-dependent robust production of ROS and moderate production of nitric oxide but no detectable release of tumor necrosis factor-alpha and interleukin-1beta. Application of free radical scavengers including superoxide dismutase/catalase, glutathione, N-acetyl cysteine and an inhibitor of nitric oxide biosynthesis significantly protected DA neurons against the MnCl2-induced degeneration. These results demonstrate that microglial activation and the production of reactive nitrogen and oxygen free radicals promote the MnCl2-induced DA neurodegeneration.

P3-286: Aging appears to enhance response of primate microglia to the amyloid-β peptide

We have shown that aging renders the aged primate brain selectively vulnerable to the toxicity of fibrillar amyloid–β peptide (fAβ). Injections of small quantities (200 pg) of fAβ in the cerebral cortex caused significant activation of microglia and neuronal loss in the aged rhesus, but were without effect in the young rhesus or aged rat. Given that activated microglia are known to exert both beneficial and harmful effects, we investigated the contribution of recruitment and activation of microglia to fAβ toxicity in the aged rhesus. Various concentrations of macrophage / microglia inhibitory factor (MIF) were either co–injected with fAβ or delivered via an Alzet pump in the frontal cortex of aged rhesus monkeys and the effects on fAβ toxicity and recruitment of microglia examined. To determine the possible mechanism of microglia contribution to fAβ toxicity, generation of reactive oxygen species (ROS) in response to fAβ was investigated in microglia isolated from aged rhesus cortex. fAβ resulted in significantly larger stereologically determined volume of damage in aged rhesus cortex when compared with injections of vehicle. Co–injection of MIF with fAβ or delivery of MIF via Alzet pump resulted in a dose–dependent reduction in the number of microglia and the volume of damage caused by fAβ determined using unbiased stereological methods. Microglia isolated from the aged rhesus cortex displayed robust production of ROS following stimulation with fAβ. Significantly, MIF resulted in a dose dependent reduction in the production of ROS by aged rhesus microglia following fAβ stimulation. These observations indicate that fAβ , which is deposited in plaques in Alzheimer's disease, is likely to exert toxic effects on aging neurons in the primate. Microglia appear to make a major contribution to the age–related vulnerability of neurons to fAβ toxicity in the primate brain, most likely through enhanced production of reactive oxygen species. The absence of microglia activation following low dose fAβ injections in the young rhesus and inhibition of toxicity of such injections in the aged rhesus following MIF treatment suggest that aged primate microglia may display an enhanced response to fAβ stimulation.

Exopolysaccharides from Burkholderia cenocepacia Inhibit Neutrophil Chemotaxis and Scavenge Reactive Oxygen Species

Bacteria belonging to the Burkholderia cepacia complex are important opportunistic pathogens in compromised hosts, particularly patients with cystic fibrosis or chronic granulomatous disease. Isolates of B. cepacia complex may produce large amounts of exopolysaccharides (EPS) that endow the bacteria with a mucoid phenotype and appear to facilitate bacterial persistence during infection. We showed that EPS from a clinical B. cenocepacia isolate interfered with the function of human neutrophils in vitro; it inhibited chemotaxis and production of reactive oxygen species (ROS), both essential components of innate neutrophil-mediated host defenses. These inhibitory effects were not due to cytotoxicity or interference with intracellular calcium signaling. EPS also inhibited enzymatic generation of ROS in cell-free systems, indicating that it scavenges these bactericidal products. B. cenocepacia EPS is structurally distinct from Pseudomonas aeruginosa alginate, yet they share the capacity to scavenge ROS and inhibit chemotaxis. These properties could explain why the two bacterial species resist clearance from the infected cystic fibrosis lung.

Burkholderia cenocepaciaInduces Neutrophil Necrosis in Chronic Granulomatous Disease

Burkholderia cepacia complex is a life-threatening group of pathogens for patients with chronic granulomatous disease (CGD), whose phagocytes are unable to produce reactive oxygen species (ROS). Unlike other CGD pathogens, B. cepacia complex is particularly virulent, characteristically causing septicemia, and is the bacterial species responsible for most fatalities in these patients. We found that a nonmucoid Burkholderia cenocepacia (a predominant species in the B. cepacia complex) isolate was readily ingested by normal human neutrophils under nonopsonic conditions and promoted apoptosis in these cells. The proapoptotic effect was not due to secreted bacterial products, but was dependent on bacterial viability. Phagocytosis was associated with a robust production of ROS, and the apoptotic neutrophils could be effectively cleared by monocyte-derived macrophages. The proapoptotic effect of B. cenocepacia was independent of ROS production because neutrophils from CGD patients were rendered apoptotic to a similar degree as control cells after challenge. More importantly, neutrophils from CGD patients, but not from normal individuals, were rendered necrotic after phagocytosis of B. cenocepacia. The extreme virulence of B. cepacia complex bacteria in CGD, but not in immunocompetent hosts, could be due to its necrotic potential in the absence of ROS.