Superoxide Production In Response Research Articles

Effects of neuronal cell adhesion molecule L1 and nanoparticle surface modification on microglia

Microelectrode arrays for neural recording suffer from low yield and stability partly due to the inflammatory host responses. A neuronal cell adhesion molecule L1 coating has been shown to promote electrode-neuron integration, reduce microglia activation and improve recording. Coupling L1 to surface via a nanoparticle (NP) base layer further increased the protein surface density and stability. However, the exact L1-microglia interaction in these coatings has not been studied. Here we cultured primary microglia on L1 modified surfaces (with and without NP) and characterized microglia activation upon phorbol myristate acetate (PMA) and lipopolysaccharide (LPS) stimulation. Results showed L1 coatings reduced microglia's superoxide production in response to PMA and presented intrinsic antioxidant properties. Meanwhile, L1 decreased iNOS, NO, and pro-inflammatory cytokines (TNF alpha, IL-6, IL-1 beta), while increased anti-inflammatory cytokines (TGF beta 1, IL-10) in LPS stimulated microglia. Furthermore, L1 increased Arg-1 expression and phagocytosis upon LPS stimulation. Rougher NP surface showed lower number of microglia attached per area than their smooth counterpart, lower IL-6 release and superoxide production, and higher intrinsic reducing potential. Finally, we examined the effect of L1 and nanoparticle modifications on microglia response in vivo over 8 weeks with 2-photon imaging. Microglial coverage on the implant surface was found to be lower on the L1 modified substrates relative to unmodified, consistent with the in vitro observation. Our results indicate L1 significantly reduces superoxide production and inflammatory response of microglia and promotes wound healing, while L1 immobilization via a nanoparticle base layer brings added benefit without adverse effects. Statement of significanceSurface modification of microelectrode arrays with L1 has been shown to reduce microglia coverage on neural probe surface in vivo and improves neural recording, but the specific mechanism of action is not fully understood. The results in this study show that surface bound L1 reduces superoxide production from cultured microglia via direct reduction reaction and signaling pathways, increases anti-inflammatory cytokine release and phagocytosis in response to PMA or LPS stimulation. Additionally, roughening the surface with nanoparticles prior to L1 immobilization further increased the benefit of L1 in reducing microglia activation and oxidative stress. Together, our findings shed light on the mechanisms of action of nanotextured and neuroadhesive neural implant coatings and guide future development of seamless tissue interface.

RIP2-deficiency induces inflammation in response to SV40 Large T induced genotoxic stress through altered ROS homeostasis

Deciphering signaling pathways that regulate the complex interplay between inflammation and cell death is a key challenge in understanding innate immune responses. Over recent years, receptor interacting protein (RIP) kinases have been described to regulate the interplay between inflammation and cell death. Whereas RIP1 and 3, the most well described members of the RIP kinase family, play important roles in necroptosis, RIP2's involvement in regulating inflammation, cell death processes and cancer is less well described and controversially discussed.Here, we demonstrate that RIP2 exerts immune regulatory functions by regulating mitochondrial damage and mitochondrial superoxide production in response to SV40 LT-induced genotoxic stress by the induction of ULK1-phosphorylation, therefore regulating the expression of interferon stimulated genes (ISGs) and NLRP3-inflammasome dependent IL-1β release. Because RIP2 is upregulated and/or activated in autoimmune/inflammatory disease and cancer, observations from this study promise implications of RIP kinases in human disease.

MIF as a biomarker and therapeutic target for overcoming resistance to proteasome inhibitors in human myeloma

AbstractMultiple myeloma (MM) remains largely incurable despite significant advances in biotherapy and chemotherapy. The development of drug resistance is a major problem in MM management. Macrophage migration inhibitory factor (MIF) expression was significantly higher in purified MM cells from relapsed patients than those with sustained response, and MM patients with high MIF had significantly shorter progression-free survival (PFS) and overall survival (OS). MM cell lines also express high levels of MIF, and knocking out MIF made them more sensitive to proteasome inhibitor (PI)-induced apoptosis not observed with other chemotherapy drugs. Mechanistic studies showed that MIF protects MM cells from PI-induced apoptosis by maintaining mitochondrial function via suppression of superoxide production in response to PIs. Specifically, MIF, in the form of a homotrimer, acts as a chaperone for superoxide dismutase 1 (SOD1) to suppress PI-induced SOD1 misfolding and to maintain SOD1 activity. MIF inhibitor 4-iodo-6-phenylpyrimidine and homotrimer disrupter ebselen, which do not kill MM cells, enhanced PI-induced SOD1 misfolding and loss of function, resulting in significantly more cell death in both cell lines and primary MM cells. More importantly, inhibiting MIF activity in vivo displayed synergistic antitumor activity with PIs and resensitized PI-resistant MM cells to treatment. In support of these findings, gene-profiling data showed a significantly negative correlation between MIF and SOD1 expression and response to PI treatment in patients with MM. This study shows that MIF plays a crucial role in MM sensitivity to PIs and suggests that targeting MIF may be a promising strategy to (re)sensitize MM to the treatment.

Dysregulated NADPH Oxidase Promotes Bone Damage in Murine Model of Autoinflammatory Osteomyelitis.

Autoinflammatory diseases are characterized by dysregulation of the innate immune system, leading to spontaneous inflammation. Pstpip2cmo mouse strain is a well-characterized model of this class of disorders. Because of the mutation leading to the lack of adaptor protein PSTPIP2, these animals suffer from autoinflammatory chronic multifocal osteomyelitis similar to several human syndromes. Current evidence suggests that it is driven by hyperproduction of IL-1β by neutrophil granulocytes. In this study, we show that in addition to IL-1β, PSTPIP2 also negatively regulates pathways governing reactive oxygen species generation by neutrophil NOX2 NADPH oxidase. Pstpip2cmo neutrophils display highly elevated superoxide production in response to a range of stimuli. Inactivation of NOX2 NADPH oxidase in Pstpip2cmo mice did not affect IL-1β levels, and the autoinflammatory process was initiated with similar kinetics. However, the bone destruction was almost completely alleviated, suggesting that dysregulated NADPH oxidase activity is a key factor promoting autoinflammatory bone damage in Pstpip2cmo mice.

Deletion of a Neuronal Drp1 Activator Protects against Cerebral Ischemia.

Mitochondrial fission catalyzed by dynamin-related protein 1 (Drp1) is necessary for mitochondrial biogenesis and maintenance of healthy mitochondria. However, excessive fission has been associated with multiple neurodegenerative disorders, and we recently reported that mice with smaller mitochondria are sensitized to ischemic stroke injury. Although pharmacological Drp1 inhibition has been put forward as neuroprotective, the specificity and mechanism of the inhibitor used is controversial. Here, we provide genetic evidence that Drp1 inhibition is neuroprotective. Drp1 is activated by dephosphorylation of an inhibitory phosphorylation site, Ser637. We identify Bβ2, a mitochondria-localized protein phosphatase 2A (PP2A) regulatory subunit, as a neuron-specific Drp1 activator in vivo Bβ2 KO mice of both sexes display elongated mitochondria in neurons and are protected from cerebral ischemic injury. Functionally, deletion of Bβ2 and maintained Drp1 Ser637 phosphorylation improved mitochondrial respiratory capacity, Ca2+ homeostasis, and attenuated superoxide production in response to ischemia and excitotoxicity in vitro and ex vivo Last, deletion of Bβ2 rescued excessive stroke damage associated with dephosphorylation of Drp1 S637 and mitochondrial fission. These results indicate that the state of mitochondrial connectivity and PP2A/Bβ2-mediated dephosphorylation of Drp1 play a critical role in determining the severity of cerebral ischemic injury. Therefore, Bβ2 may represent a target for prophylactic neuroprotective therapy in populations at high risk of stroke.SIGNIFICANCE STATEMENT With recent advances in clinical practice including mechanical thrombectomy up to 24 h after the ischemic event, there is resurgent interest in neuroprotective stroke therapies. In this study, we demonstrate reduced stroke damage in the brain of mice lacking the Bβ2 regulatory subunit of protein phosphatase 2A, which we have shown previously acts as a positive regulator of the mitochondrial fission enzyme dynamin-related protein 1 (Drp1). Importantly, we provide evidence that deletion of Bβ2 can rescue excessive ischemic damage in mice lacking the mitochondrial PKA scaffold AKAP1, apparently via opposing effects on Drp1 S637 phosphorylation. These results highlight reversible phosphorylation in bidirectional regulation of Drp1 activity and identify Bβ2 as a potential pharmacological target to protect the brain from stroke injury.

Mitochondrial Metabolic Reprogramming by CD36 Signaling Drives Macrophage Inflammatory Responses.

A hallmark of chronic inflammatory disorders is persistence of proinflammatory macrophages in diseased tissues. In atherosclerosis, this is associated with dyslipidemia and oxidative stress, but mechanisms linking these phenomena to macrophage activation remain incompletely understood. To investigate mechanisms linking dyslipidemia, oxidative stress, and macrophage activation through modulation of immunometabolism and to explore therapeutic potential targeting specific metabolic pathways. Using a combination of biochemical, immunologic, and ex vivo cell metabolic studies, we report that CD36 mediates a mitochondrial metabolic switch from oxidative phosphorylation to superoxide production in response to its ligand, oxidized LDL (low-density lipoprotein). Mitochondrial-specific inhibition of superoxide inhibited oxidized LDL-induced NF-κB (nuclear factor-κB) activation and inflammatory cytokine generation. RNA sequencing, flow cytometry, 3H-labeled palmitic acid uptake, lipidomic analysis, confocal and electron microscopy imaging, and functional energetics revealed that oxidized LDL upregulated effectors of long-chain fatty acid uptake and mitochondrial import, while downregulating fatty acid oxidation and inhibiting ATP5A (ATP synthase F1 subunit alpha)-an electron transport chain component. The combined effect is long-chain fatty acid accumulation, alteration of mitochondrial structure and function, repurposing of the electron transport chain to superoxide production, and NF-κB activation. Apoe null mice challenged with high-fat diet showed similar metabolic changes in circulating Ly6C+ monocytes and peritoneal macrophages, along with increased CD36 expression. Moreover, mitochondrial reactive oxygen species were positively correlated with CD36 expression in aortic lesional macrophages. These findings reveal that oxidized LDL/CD36 signaling in macrophages links dysregulated fatty acid metabolism to oxidative stress from the mitochondria, which drives chronic inflammation. Thus, targeting to CD36 and its downstream effectors may serve as potential new strategies against chronic inflammatory diseases such as atherosclerosis.

Estradiol inhibits fMLP-induced neutrophil migration and superoxide production by upregulating MKP-2 and dephosphorylating ERK

Estrogen has been reported to inhibit neutrophil infiltration related inflammation and suppress neutrophils migration in vitro, but the underlying mechanism is not fully understood. By using HL-60 differentiated neutrophil-like cells (dHL-60) and human neutrophils, we examined the effect of 17-β estradiol (E2) on cell migration and superoxide production in response to chemotactic peptide N-formyl-methionyl-leucyl-phenylalanine (fMLP) and explored the mechanisms involved. We found that fMLP significantly induced dHL-60 cell and neutrophil migration and superoxide production, which was inhibited by ERK inhibitor PD98059. E2 significantly inhibited fMLP-induced dHL-60 cell and neutrophil migration and superoxide production at both physiological and pharmacological concentrations. Mechanistic studies showed that pretreatment of these cells with E2 rapidly elevated the protein level of mitogen-activated protein kinase phosphatase 2 (MKP-2) and inhibited fMLP-induced ERK phosphorylation. Pretreatment of these cells with estrogen receptor (ER) antagonist ICI 182780 reversed the inhibition of fMP-induced cell migration and superoxide production, and the induction of MKP-2 expression and the suppression of fMP-induced ERK phosphorylation by E2. However, pretreatment of cells with G-protein coupled ER antagonist G15 had no such effect. Collectively, these results demonstrate that fMLP stimulates neutrophil chemotaxis and superoxide production through activating ERK, and indicate that ER-mediated upregulation of MKP-2 may dephosphorylate ERK and contribute to the inhibitory effect of E2 on neutrophil activation by fMLP. Our study reveals new mechanisms involved in the anti-inflammatory activity of estrogen.

Elevated Superoxide Production in Response to Angiotensin II by Proximal Tubules of Rats Consuming a Moderately Enriched Fructose Diet

Dietary fructose consumption has increased dramatically over the past four decades in part due to increased consumption of sugary beverages. Rats ingesting a diet that includes 20 % fructose in the drinking water develop salt‐sensitive hypertension. This is in part due to an increased sensitivity of the proximal nephron to angiotensin II (Ang II), which stimulates Na reabsorption in this segment at physiological concentrations via increases in reactive oxygen species such as superoxide (O2−). Thus we hypothesized that dietary fructose enhances the ability of Ang II to stimulate O2− production by proximal tubules. To test this hypothesis, we first measured the effect of Ang II on O2− production by proximal tubule suspensions from rats fed either a control diet or one in which 20% fructose was included in the drinking water. Ang II (10 nM) increased O2− production by 351 ± 85 relative light units (RLU)/mg protein/s in tubules from fructose‐fed rats but only by 131 ± 55 RLU/mg/s in tubules from control animals (Δ 220 ± 101 RLU/mg/s; p < 0.01, n=8). To test whether the increase was due to activation of NADPH oxidase (NOX), we tested the ability of apocynin to block the effects of Ang II. In the presence of apocynin, Ang II did not significantly change O2− production by tubules from either fructose‐fed (Δ 27 ± 19 RLU/mg/s) or control (Δ 29 ± 39 RLU/mg/s) rats. We next tested whether the fructose‐dependent increase was due to enhanced NOX4 activity by examining the effects of the NOX1 and 4 inhibitor GKT136901 on O2− production and NOX4 expression by Western blot. In the presence of GKT136901, Ang II did not significantly change O2− production by tubules from either fructose‐fed (Δ 36 ± 20 RLU/mg/s) or control (Δ 82 ± 78 RLU/mg/s) animals. Fructose feeding increased NOX4 expression in proximal tubules by 55 ± 13% compared to controls (p < 0.01, n=6). Together these data indicate that a diet including 20% fructose in the drinking water enhances the ability to increase O2− production by proximal tubules in response to Ang II due to activation of NOX4. Given that O2− is a potent oxidant, we studied whether the primary antioxidant in proximal tubule cells, reduced glutathione was decreased under basal conditions or in response to Ang II. We found no significant differences in either reduced or total glutathione in proximal tubules from fructose‐fed and control rats. However, Ang II decreased the ratio of reduced to total glutathione by 10 ± 3 % (p<0.03; n=19) in tubules from fructose‐fed rats and by only 1 ± 3 % in in tubules from controls. We conclude that elevated dietary fructose enhances O2− production by proximal tubules in response to Ang II. This increase is likely mediated by enhanced activity and expression of NOX4. The elevated oxidative stress caused by augmented O2− production results in diminished antioxidant defenses. In total these effects increase proximal nephron Na reabsorption and contribute to salt‐sensitive hypertension.Support or Funding InformationThis work was supported in part by a grant from the Heart, Lung and Blood Institute of the National Institutes of Health to J.L.G. (HL128053).This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

Anti-inflammatory treatment improves high-density lipoprotein function in rheumatoid arthritis

ObjectivePatients with rheumatoid arthritis (RA) are at increased cardiovascular risk. Recent studies suggest that high-density lipoprotein (HDL) may lose its protective vascular phenotype in inflammatory conditions. However, the effects of...

Dynamics of extracellular superoxide production by Trichodesmium colonies from the Sargasso Sea

AbstractReactive oxygen species (ROS) are key players in the health and biogeochemistry of the ocean and its inhabitants. The vital contribution of microorganisms to marine ROS levels, particularly superoxide, has only recently come to light, and thus the specific biological sources and pathways involved in ROS production are largely unknown. To better understand the biogenic controls on ROS levels in tropical oligotrophic systems, we determined rates of superoxide production under various conditions by natural populations of the nitrogen‐fixing diazotroph Trichodesmium obtained from various surface waters in the Sargasso Sea. Trichodesmium colonies collected from eight different stations all produced extracellular superoxide at high rates in both the dark and light. Colony density and light had a variable impact on extracellular superoxide production depending on the morphology of the Trichodesmium colonies. Raft morphotypes showed a rapid increase in superoxide production in response to even low levels of light, which was not observed for puff colonies. In contrast, superoxide production rates per colony decreased with increasing colony density for puff morphotypes but not for rafts. These findings point to Trichodesmium as a likely key source of ROS to the surface oligotrophic ocean. The physiological and/or ecological factors underpinning morphology‐dependent controls on superoxide production need to be unveiled to better understand and predict superoxide production by Trichodesmium and ROS dynamics within marine systems.

Involvement of PI3Kγ in Superoxide Anion Production in Response to IL-8, RANTES, and fMLP in Human Peripheral Blood Neutrophils

Involvement of PI3Kγ in Superoxide Anion Production in Response to IL-8, RANTES, and fMLP in Human Peripheral Blood Neutrophils Neutrophils are essential components of the immune system and have a critical role in combating bacterial and fungal infections. A key weapon in the neutrophil armory is the “respiratory burst,” which is the generation of reactive oxygen species (ROS) by a multicomponent oxidase complex. It is well established that pre-exposure of human neutrophils to proinflammatory cytokines and chemokines markedly augments the production of reactive oxygen species (ROS) to subsequent stimuli. In inflammatory reactions there are complex interactions of protein mediators (cytokines) and mediators derived from lipids. An important event in inflammation is superoxide production, in relation to microbicidal activity as well as tissue damage. A better understanding of phenomena involved in the regulation of NADPH oxidase could help developing novel therapeutic agents for inflammatory diseases involving abnormal neutrophil superoxide. Therefore, stimulating superoxide production by human neutrophils was investigated for this reason and because it sheds a light on intracellular signals that activate this response. Pretreatment of human neutrophils with N-formyl-methionyl-leucyl-phenylalanine (fMLP), interleukin-8 (IL-8), and regulated on activation, normal T cell expressed and secreted (RANTES) markedly augmented the amount of superoxide anion produced, which was inhibited completely (IL-8 and fMLP) or partially (RANTES) by a specific isoform of phosphoinositide 3-kinase (PI3K), the PI3KγII inhibitor.

Aerobic exercise training reduces cardiac function in adult male offspring exposed to prenatal hypoxia.

Intrauterine growth restriction (IUGR) has been associated with increased susceptibility to myocardial ischemia-reperfusion (I/R) injury. Exercise is an effective preventive intervention for cardiovascular diseases; however, it may be detrimental in conditions of compromised health. The aim of this study was to determine whether exercise training can improve cardiac performance after I/R injury in IUGR offspring. We used a hypoxia-induced IUGR model by exposing pregnant Sprague-Dawley rats to 21% oxygen (control) or hypoxic (11% oxygen; IUGR) conditions from gestational day 15 to 21. At 10 wk of age, offspring were randomized to a sedentary group or to a 6-wk exercise protocol. Transthoracic echocardiography assessments were performed after 6 wk. Twenty-four hours after the last bout of exercise, ex vivo cardiac function was determined using a working heart preparation. With exercise training, there was improved baseline cardiac performance in male control offspring but a reduced baseline cardiac performance in male IUGR exercised offspring (P < 0.05). In male offspring, exercise decreased superoxide generation in control offspring, while in IUGR offspring, it had the polar opposite effect (interaction P ≤ 0.05). There was no effect of IUGR or exercise on cardiac function in female offspring. In conclusion, in male IUGR offspring, exercise may be a secondary stressor on cardiac function. A reduction in cardiac performance along with an increase in superoxide production in response to exercise was observed in this susceptible group.

A Comparison of Different Methods of RBC Leukoreduction and Their Ability to Minimize Neutrophil Priming Activity during Storage

Background:Pre-storage leukoreduction (LR) of whole blood (WB) or red blood cells (RBC) reduces white blood cell (WBC) and platelet contamination and decreases the amount of biologic response modifiers (BRMs) implicated in the development of transfusion-related acute lung injury (TRALI). There are two primary methods of LR used world-wide, however there has not yet been a side-by-side comparison of the buffy coat method to the in-line filtration, in terms of the ability to minimize the neutrophil (PMN) priming activity in the supernatant. We hypothesize that LR by buffy coat is equal to LR by filtration in minimizing PMN priming activity.Method:Five units of WB were drawn from healthy volunteer donors. Plasma was removed by centrifugation and the RBC units were equally divided by weight; 50% were LR by buffy coat removal and 50% were LR by in-line filtration (Haemonetics RC2D) into bags containing AS-3 additive solution (110 ml) and stored at 2-6oC per AABB criteria. Sterile couplers were used to obtain samples weekly during storage. The supernantant was isolated by centrifugation (5,000g for 7 min, followed by 12,000g for 6 min) and stored at -80oC. PMNs were isolated from heparinized WB from healthy volunteer donors by standard techniques. PMNs were incubated with plasma samples for 5 min at 37oC then activated with 1uM formyl-MLF (fMLF) and the maximal rate of O2- was measured. Priming activity is the augmentation of the maximal rate of superoxide production in response to fMLF. Supernatants were immunoblotted for peroxiredoxin-6, an intracellular RBC enzyme.Results:LR by filtration decreased WBC amount by >3 logs and platelets by >2 logs whereas LR by buffy coat reduced both WBC and platelets by only a single log. The supernatants from both buffy coat and filtration LR showed no difference in PMN priming vs. albumin or buffer-treated controls on day (D) 1 and demonstrated an increase in O2- production on D1 vs. D42 of storage (Table 1). However, the supernatants from buffy coat LR RBCs demonstrated significantly greater PMN priming activity as compared to supernatants from filter LR RBCs (2.4-fold). Samples from D14 and D28 also showed a statistically significant greater increase in O2- production in buffy coat LR samples compared to filtered samples. Additionally, there was increased immunoreactivity for peroxiredoxin-6 in buffy coat supernatants vs. filtered RBC supernatants on identical days of storage (Fig.1).Discussion:Filter LR is more effective in reducing WBC and platelet contamination, and significantly decreases the accumulation of PMN priming activity and peroxiredoxin-6 immunoreactivity in the RBC supernatant. LR via filtration appears to produce RBC units with less cellular contamination, decreased BRMs and less release of intracellular enzymes. Further testing is required to determine if these LR RBC units are clinically safer than RBCs which undergo buffy coat LR. [Display omitted] [Display omitted] DisclosuresNo relevant conflicts of interest to declare.

Symmetric dimethylarginine alters endothelial nitric oxide activity in glomerular endothelial cells

Circulating symmetric dimethylarginine (SDMA) is increased in patients with chronic kidney disease. SDMA is considered an inert metabolite, but because it can transported into cells, we studied the effect of SDMA on glomerular endothelial cells. SDMA suppressed VEGF-induced endothelial nitric oxide synthase (eNOS) phosphorylation and nitric oxide production, but not VEGFR2 activation and signaling leading to eNOS activation. SDMA caused eNOS uncoupling and increased superoxide anion production in response to VEGF. All these effects were blocked by preventing cellular uptake of SDMA with a molar excess of arginine. These data show that SDMA interferes with nitric oxide production by uncoupling eNOS and leads to oxidative stress in glomerular endothelial cells.In conclusion, our data show that SDMA is not an inert metabolite and that it could contribute to oxidative stress in the renal endothelium.

Nox4 contribution to H2O2 but not in superoxide production in response to increased tubular flow in the mTAL of Dahl salt‐sensitive rats (1134.3)

Studies were carried out to determine the relative contribution of Nox4 in the production of H2O2 vs. superoxide (O2‐) in the renal medullary thick ascending limb of Henle (mTAL) of Dahl salt‐sensitive (SS) rats. We have previously presented that Nox4 is the major source of H2O2 production in mTAL in response to increased luminal flow. Specifically, knock out of Nox4 in SS rats (SSNox4‐/‐) resulted in significant reduction of intracellular H2O2 production compared to SS. In the present study, using fluorescence microscopy (Eth/DHE ratio), we examined the contribution of Nox4 to O2‐ production in isolated microperfused mTAL in response to an increase of luminal flow (from 5 to 20 nl/min) in SS and SSNox4‐/‐ rats fed a 4% NaCl diet for 3 days. NADPH oxidase activity in outer medullary tissue was determined with DHE fluorescence assay. Results: 1) O2‐ production increased significantly and to the same extent in mTAL of both SS and SSNox4‐/‐ rats. 2) No differences were observed in NADPH activity between the two strains. We conclude that Nox4 contributes only to the production of intracellular H2O2 and not O2‐ in response to increased mTAL luminal flow in SS rats fed a high salt diet.

P.3.006 Genetic risk factors for cytokine-induced depression

Ageing is accompanied by reduced functioning of the immune system, termed immunesenescence which is associated with increased risk of infection and mortality. However the immune system does not operate in isolation and can be modified by many environmental factors, including stress. In this study we determined whether physical stress (hip fracture) and psychological distress (depressive symptoms) had additive effects upon the aged immune system, specifically on monocyte numbers and function. We assessed immune function in 101 hip fracture patients (81 female) 6 weeks and 6 months after injury and 43 healthy age matched controls (28 females). Thirty-eight of the hip fracture group were found to be depressed at the 6 week sampling. No differences in peripheral monocyte count, distribution of monocyte subsets or TNFα secretion were observed between hip fracture patients and healthy controls. However we observed significantly reduced superoxide production in response to Escherichia coli in the monocytes of hip fracture patients who developed depressive symptoms compared with non-depressed hip fracture patients (p = 0.002) or healthy controls (p = 0.008) 6 weeks after the fracture which remained decreased 6 months following injury. In previous studies we have shown an effect of depression on neutrophil superoxide generation in hip fracture patients, suggesting a particular susceptibility of this aspect of immune cell function to psychological stress.

Depressive symptoms in hip fracture patients are associated with reduced monocyte superoxide production

Ageing is accompanied by reduced functioning of the immune system, termed immunesenescence which is associated with increased risk of infection and mortality. However the immune system does not operate in isolation and can be modified by many environmental factors, including stress. In this study we determined whether physical stress (hip fracture) and psychological distress (depressive symptoms) had additive effects upon the aged immune system, specifically on monocyte numbers and function. We assessed immune function in 101 hip fracture patients (81 female) 6weeks and 6months after injury and 43 healthy age matched controls (28 females). Thirty-eight of the hip fracture group were found to be depressed at the 6week sampling. No differences in peripheral monocyte count, distribution of monocyte subsets or TNFα secretion were observed between hip fracture patients and healthy controls. However we observed significantly reduced superoxide production in response to Escherichia coli in the monocytes of hip fracture patients who developed depressive symptoms compared with non-depressed hip fracture patients (p=0.002) or healthy controls (p=0.008) 6weeks after the fracture which remained decreased 6months following injury. In previous studies we have shown an effect of depression on neutrophil superoxide generation in hip fracture patients, suggesting a particular susceptibility of this aspect of immune cell function to psychological stress.

Nutrient deprivation increases vulnerability of endothelial cells to proinflammatory insults

Nutrient deprivation is a stimulus for oxidative stress and is an established method for induction of cell autophagy and apoptosis. The aims of this study were to identify conditions that evoke superoxide production in cultured human umbilical vein endothelial cells (HUVECs), determine the mechanism of action for this response, and examine whether the stimulus might facilitate the adhesion of human isolated neutrophils to the HUVECs. HUVECs were incubated in M199 medium under conditions of serum starvation (serum-free M199 medium), low serum (medium containing 2% fetal calf serum), and high serum (medium containing 20% fetal calf serum). HUVECs were also incubated under proinflammatory conditions, in medium supplemented with 50ng/ml tumor necrosis factor-α (TNF-α) or neutrophils preactivated with 10nM phorbol 12-myristate 13-acetate (PMA). Superoxide production was increased fourfold in serum-starved HUVECs compared to cells incubated in 20% medium, and this was reduced by inhibitors of the mitochondrial electron transport chain and mitochondrial Ca2+ uniporter. Superoxide production was 23.6% higher in HUVECs incubated with TNF-α in 2% medium compared to 2% medium alone, but unchanged with TNF-α in 20% medium. PMA-activated neutrophils adhered to morphologically aberrant HUVECs, which were mainly evident under the low-serum condition. The findings show a role of mitochondrial enzymes in superoxide production in response to nutrient deprivation and suggest that proinflammatory responses in HUVECs become manifest when HUVECs are in an already-compromised state.

Add Another Killer for Interfered Vascular Function

The predominant physiological role of the renin–angiotensin system is to regulate blood pressure and water/sodium homeostasis. Excessive angiotensin II (Ang II), the major bioactive angiotensin peptide of this system, leads to vascular dysfunction that contributes to many cardiovascular diseases. Increasing evidence supports a concept that overactivation of the renin–angiotensin system disrupts normal immune/inflammatory responses. Although this overview of adverse responses to Ang II is widely accepted, how alterations in inflammation contribute to vascular dysfunction is not completely understood. See accompanying article on page 1313 Alterations in immune responses to vascular function were reported >4 decades ago but less advancement in understanding how immune-mediated or inflammation-induced responses have been identified.1 Recently, Guzik et al2 reported that RAG-1−/− mice, lacking both functional T and B lymphocytes, were protected from Ang II–induced loss of endothelium-dependent vasodilatation. Conversely, adoptive transfer of T lymphocytes led to vascular dysfunction that were attributed to impaired endothelium-dependent vasodilatation and an increase in vascular superoxide production in response to Ang II. The authors interpreted these findings as one mechanism of Ang II–induced high blood pressure. After this initial report, other groups have confirmed contribution of immune/inflammation to Ang II–induced vascular dysfunction beyond …

Erratum: Phosphoinositide 3-kinase couples NMDA receptors to superoxide release in excitotoxic neuronal death

Sustained activation of neuronal N-methly D-aspartate (NMDA)-type glutamate receptors leads to excitotoxic cell death in stroke, trauma, and neurodegenerative disorders. Excitotoxic neuronal death results in part from superoxide produced by neuronal NADPH oxidase (NOX2), but how NMDA receptors are coupled to neuronal NOX2 activation is not well understood. Here, we identify a signaling pathway coupling NMDA receptor activation to NOX2 activation in primary neuron cultures. Calcium influx through the NR2B subunit of NMDA receptors leads to the activation of phosphoinositide 3-kinase (PI3K). Formation of phosphatidylinositol (3,4,5)-triphosphate (PI(3,4,5)P3) by PI3K activates the atypical protein kinase C, PKC zeta (PKCζ), which in turn phosphorylates the p47(phox) organizing subunit of neuronal NOX2. Calcium influx through NR2B-containing NMDA receptors triggered mitochondrial depolarization, NOX2 activation, superoxide formation, and cell death. However, equivalent magnitude calcium elevations induced by ionomycin did not induce NOX2 activation or neuronal death, despite causing mitochondrial depolarization. The PI3K inhibitor wortmannin prevented NMDA-induced NOX2 activation and cell death, without preventing cell swelling, calcium elevation, or mitochondrial depolarization. The effects of wortmannin were circumvented by exogenous supply of the PI3K product, PI(3,4,5)P3, and by transfection with protein kinase M, a constitutively active form of PKCζ. These findings demonstrate that superoxide formation and excitotoxic neuronal death can be dissociated from mitochondrial depolarization, and identify a novel role for PI3K in this cell death pathway. Perturbations in this pathway may either increase or decrease superoxide production in response to NMDA receptor activation, and may thereby impact neurological disorders, in which excitotoxicity is a contributing factor.