The kinase complex mTORC2 promotes the longevity of virus-specific memory CD4+ T cells by preventing ferroptosis.

Antigen-specific memory CD4+ T cells can persist and confer rapid and efficient protection from microbial reinfection. However, the mechanisms underlying the long-term maintenance of the memory CD4+ T cell pool remain largely unknown. Here, using a mouse model of acute infection with lymphocytic choriomeningitis virus (LCMV), we found that the serine/threonine kinase complex mammalian target of rapamycin complex 2 (mTORC2) is critical for the long-term persistence of virus-specific memory CD4+ T cells. The perturbation of mTORC2 signaling at memory phase led to an enormous loss of virus-specific memory CD4+ T cells by a unique form of regulated cell death (RCD), ferroptosis. Mechanistically, mTORC2 inactivation resulted in the impaired phosphorylation of downstream AKT and GSK3ß kinases, which induced aberrant mitochondrial reactive oxygen species (ROS) accumulation and ensuing ferroptosis-causative lipid peroxidation in virus-specific memory CD4+ T cells; furthermore, the disruption of this signaling cascade also inhibited glutathione peroxidase 4 (GPX4), a major scavenger of lipid peroxidation. Thus, the mTORC2-AKT-GSK3ß axis functions as a key signaling hub to promote the longevity of virus-specific memory CD4+ T cells by preventing ferroptosis.

Ironing out the role of ferroptosis in immunity.

Ferroptosis is a type of regulated cell death that drives the pathophysiology of many diseases. Oxidative stress is detectable in many types of regulated cell death, but only ferroptosis involves lipid peroxidation and iron dependency. Ferroptosis originates and propagates from several organelles, including the mitochondria, endoplasmic reticulum, Golgi, and lysosomes. Recent data have revealed that immune cells can both induce and undergo ferroptosis. A mechanistic understanding of how ferroptosis regulates immunity is critical to understanding how ferroptosis controls immune responses and how this is dysregulated in disease. Translationally, more work is needed to produce ferroptosis-modulating immunotherapeutics. This review focuses on the role of ferroptosis in immune-related diseases, including infection, autoimmune diseases, and cancer. We discuss how ferroptosis is regulated in immunity, how this regulation contributes to disease pathogenesis, and how targeting ferroptosis may lead to novel therapies.

Redox homeostasis maintained by GPX4 facilitates STING activation.

Stimulator-of-interferon genes (STING) is vital for sensing cytosolic DNA and initiating innate immune responses against microbial infection and tumors. Redox homeostasis is the balance of oxidative and reducing reactions present in all living systems. Yet, how the intracellular redox state controls STING activation is unclear. Here, we show that cellular redox homeostasis maintained by glutathione peroxidase 4 (GPX4) is required for STING activation. GPX4 deficiency enhanced cellular lipid peroxidation and thus specifically inhibited the cGAS-STING pathway. Concordantly, GPX4 deficiency inhibited herpes simplex virus-1 (HSV-1)-induced innate antiviral immune responses and promoted HSV-1 replication in vivo. Mechanistically, GPX4 inactivation increased production of lipid peroxidation, which led to STING carbonylation at C88 and inhibited its trafficking from the endoplasmic reticulum (ER) to the Golgi complex. Thus, cellular stress-induced lipid peroxidation specifically attenuates the STING DNA-sensing pathway, suggesting that GPX4 facilitates STING activation by maintaining redox homeostasis of lipids.

Renal Purge of Hemolymphatic Lipids Prevents the Accumulation of ROS-Induced Inflammatory Oxidized Lipids and Protects Drosophila from Tissue Damage.

Animals require complex metabolic and physiological adaptations to maintain the function of vital organs in response to environmental stresses and infection. Here, we found that infection or injury in Drosophila induced the excretion of hemolymphatic lipids by Malpighian tubules, the insect kidney. This lipid purge was mediated by a stress-induced lipid-binding protein, Materazzi, which was enriched in Malpighian tubules. Flies lacking materazzi had higher hemolymph concentrations of reactive oxygen species (ROS) and increased lipid peroxidation. These flies also displayed Malpighian tubule dysfunction and were susceptible to infections and environmental stress. Feeding flies with antioxidants rescued the materazzi phenotype, indicating that the main role of Materazzi is to protect the organism from damage caused by stress-induced ROS. Our findings suggest that purging hemolymphatic lipids presents a physiological adaptation to protect host tissues from excessive ROS during immune and stress responses, a process that is likely to apply to other organisms.

AMPKα1 in B Cells Dampens Primary Antibody Responses yet Promotes Mitochondrial Homeostasis and Persistence of B Cell Memory.

Emerging evidence indicates that metabolic programs regulate B cell activation and Ab responses. However, the metabolic mediators that support the durability of the memory B cell and long-lived plasma cell populations are not fully elucidated. Adenosine monophosphate-activated protein kinase (AMPK) is an evolutionary conserved serine/threonine kinase that integrates cellular energy status and nutrient availability to intracellular signaling and metabolic pathways. In this study, we use genetic mouse models to show that loss of ΑMPKα1 in B cells led to a weakened recall Ab response associated with a decline in the population of memory-phenotype B cells. AMPKα1-deficient memory B lymphocytes exhibited aberrant mitochondrial activity, decreased mitophagy, and increased lipid peroxidation. Moreover, loss of AMPKα1 in B lymphoblasts was associated with decreased mitochondrial spare respiratory capacity. Of note, AMPKα1 in B cells was dispensable for stability of the bone marrow-resident, long-lived plasma cell population, yet absence of this kinase led to increased rates of Ig production and elevated serum Ab concentrations elicited by primary immunization. Collectively, our findings fit a model in which AMPKα1 in B cells supports recall function of the memory B cell compartment by promoting mitochondrial homeostasis and longevity but restrains rates of Ig production.

Comparative effect of bovine buttermilk, whey, and lactoferrin on the innate immunity receptors and oxidative status of intestinal epithelial cells.

Milk contains bioactive molecules with important functions as defensive proteins; among them are the whey protein lactoferrin and proteins of the milk fat globule membrane (MFGM) present in buttermilk. The aim of this study has been to investigate the effects of lactoferrin, whey, and buttermilk as modulators of intestinal innate immunity and oxidative stress on intestinal epithelial cells, to evaluate its potential use for the development of functional foods. The mRNA expression levels of innate immune system Toll-like receptors (TLR2, TLR4, and TLR9), lipid peroxidation (malondialdehyde + 4-hydroxyalkenals) and protein expression levels of carbonyl were analyzed in enterocyte-like Caco-2/TC7 cells treated for 24 h with different concentrations of lactoferrin, whey, or buttermilk. None of the substances analyzed caused oxidative damage; however, whey significantly decreased the levels of lipid peroxidation. Furthermore, both lactoferrin and whey reduced the oxidative stress induced by lipopolysaccharide. With respect to TLR receptors, lactoferrin, whey, and buttermilk specifically altered the expression of TLR2, TLR4, and TLR9 receptors, with a strong decrease in the expression levels of TLR4. These results suggest that lactoferrin, whey, and buttermilk are potentially interesting ingredients for functional foods because they seem to modulate oxidative stress and the inflammatory response induced by the activation of TLRs.

PPARG-mediated ferroptosis in dendritic cells limits antitumor immunity.

Dendritic cells (DCs) are antigen-presenting cells of the immune system, which play a key role in antitumor immunity by activating cytotoxic T cells. Here, we report that elevated ferroptosis, a lipid peroxidation-mediated cell death, impairs the maturation of DCs and their function in tumor suppression. Ferroptosis is selectively induced in DCs by the GXP4 inhibitor RSL3, but not the SLC7A11 inhibitor erastin. Ferroptotic DCs lose their ability to secrete pro-inflammatory cytokines (TNF and IL6) and express MHC class I in response to the maturation signal of lipopolysaccharide. Moreover, ferroptotic DCs fail to induce CD8+ T cells to produce IFNG/IFNγ. Mechanistically, PPARG/PPARγ, a nuclear receptor involved in the regulation of lipid metabolism, is responsible for RSL3-induced ferroptosis in DCs. Consequently, the genetic depletion of PPARG restores the maturation and function of DCs. Using immunogenic cell death-based DC vaccine models, we further demonstrate that PPARG-mediated ferroptosis of DCs limits antitumor immunity in mice. Together, these findings demonstrate a novel role of ferroptotic DCs in driving an immunosuppressive tumor microenvironment.

Hydroperoxylated vs Dihydroxylated Lipids: Differentiation of Isomeric Cardiolipin Oxidation Products by Multidimensional Separation Techniques.

In recent years, cardiolipin (CL) oxidation products were recognized as potential markers for mitochondrial dysfunction in conjunction with age related diseases. The analysis of oxidized CL requires powerful analysis techniques due to high structural diversity. In addition, low concentrations of partly labile compounds pose a special challenge, supplemented by the occurrence of isomeric compounds, e.g., hydroperoxylated vs dihydroxylated products. Therefore, we present a hyphenated method based on liquid chromatography coupled to trapped ion mobility spectrometry (TIMS) for separation and tandem mass spectrometry (MS/MS) for structural characterization. This enables comprehensive analysis of an artificially oxidized CL extract of bovine heart. Isomeric oxidation products could be differentiated by mobility-resolved MS/MS fragmentation experiments. Our developed method could help to better understand the physiological role of oxidized CL.

IFN-γ ameliorates autoimmune encephalomyelitis by limiting myelin lipid peroxidation.

Evidence has suggested both a pathogenic and a protective role for the proinflammatory cytokine IFN-γ in experimental autoimmune encephalomyelitis (EAE). However, the mechanisms underlying the protective role of IFN-γ in EAE have not been fully resolved, particularly in the context of CNS antigen-presenting cells (APCs). In this study we examined the role of IFN-γ in myelin antigen uptake by CNS APCs during EAE. We found that myelin antigen colocalization with APCs was decreased substantially and that EAE was significantly more severe and showed a chronic-progressive course in IFN-γ knockout (IFN-γ-/-) or IFN-γ receptor knockout (IFN-γR-/-) mice as compared with WT animals. IFN-γ was a critical regulator of phagocytic/activating receptors on CNS APCs. Importantly, "free" myelin debris and lipid peroxidation activity at CNS lesions was increased in mice lacking IFN-γ signaling. Treatment with N-acetyl-l-cysteine, a potent antioxidant, abolished lipid peroxidation activity and ameliorated EAE in IFN-γ-signaling-deficient mice. Taken together the data suggest a protective role for IFN-γ in EAE by regulating the removal of myelin debris by CNS APCs and thereby limiting the substrate available for the generation of neurotoxic lipid peroxidation products.

The double-edged role of 12/15-lipoxygenase during inflammation and immunity.

12/15-Lipoxygenase (12/15-LOX) mediates the enzymatic oxidation of polyunsaturated fatty acids, thereby contributing to the generation of various bioactive lipid mediators. Although 12/15-LOX has been implicated in the pathogenesis of multiple chronic inflammatory diseases, its physiologic functions seem to include potent immune modulatory properties that physiologically contribute to the resolution of inflammation and the clearance of inflammation-associated tissue damage. This review aims to give a comprehensive overview about our current knowledge on the role of this enzyme during the regulation of inflammation and immunity. This article is part of a Special Issue entitled: Lipid modification and lipid peroxidation products in innate immunity and inflammation edited by Christoph J. Binder.

[Human selenium-containing single-chain variable fragment with glutathione peroxidase activity protects NIH3T3 fibroblast against oxidative damage].

Ultraviolet B (UVB medium wave, 280-315 nm) induces cellular oxidative damage and apoptosis by producing reactive oxygen species (ROS). Glutathione peroxidase functions as an antioxidant by catalyzing the reduction of hydrogen peroxide, the more important member of reactive oxygen species. A human selenium-containing single-chain variable fragment (se-scFv-B3) with glutathione peroxidase activity of 1288 U/µmol was generated and investigated for its antioxidant effects in UVB-induced oxidative damage model. In particular, cell viability, lipid peroxidation extent, cell apoptosis, the change of mitochondrial membrane potential, caspase-3 activity and the levels of intracellular reactive oxygen species were assayed. Human se-scFv-B3 protects NIH3T3 cells against ultraviolet B-induced oxidative damage and subsequent apoptosis by prevention of lipid peroxidation, inhibition of the collapse of mitochondrial membrane potential as well as the suppression of the caspase-3 activity and the level of intracellular ROS. It seems that antioxidant effects of human se-scFv-B3 are mainly associated with its capability to scavenge reactive oxygen species, which is similar to that of the natural glutathione peroxidase.

Biodistribution of polyacrylic acid-coated iron oxide nanoparticles is associated with proinflammatory activation and liver toxicity.

Iron oxide nanoparticles (IONs) have physical and chemical properties that render them useful for several new biomedical applications. Still, so far, in vivo safety studies of IONs with coatings of biomedical interest are still scarce. The aim of this study, therefore, was to clarify the acute biological effects of polyacrylic acid (PAA)-coated IONs, by determining their biodistribution and their potential proinflammatory and toxic effects in CD-1 mice. The biodistribution of PAA-coated IONs in several organs (liver, spleen, kidneys, brain, heart, testes and lungs), the plasma cytokines, chemokine and aminotransferases levels, white blood cell count, oxidative stress parameters, adenosine triphosphate and histologic features of liver, spleen and kidneys were evaluated 24 h after a single acute (8, 20 or 50 mg kg(-1) ) intravenous administration of PAA-coated IONs in magnetite form. The obtained results showed that these IONs accumulate mainly in the liver and spleen and, to a lesser extent, in the lungs. Although our data showed that PAA-coated IONs do not cause severe organ damage, an inflammatory process was triggered in vivo, as evidenced by as evidenced by increased neutrophils and large lymphocytes in the differential blood count. Moreover, an accumulation of iron in macrophages of the liver and spleen was observed and hepatic lipid peroxidation was elicited, showing that the IONs are able to induce oxidative stress. The effects of these nanoparticles need to be further investigated regarding the mechanisms involved and the long-term consequences of intravenous administration of PAA-coated IONs. Copyright © 2016 John Wiley & Sons, Ltd.

Cytotoxicity, oxidative stress, and inflammation in human Hep G2 liver epithelial cells following exposure to gold nanorods.

The gold nanorods (GNRs) are great potentials in imaging, therapy, biosensing, and many other commercial applications. However, GNRs interactions with human cells and potential health risks remain not well known. The present investigation aimed to evaluate the in vitro toxicity of 10 and 25 nm GNRs (10-50 µg/mL) following exposure for 48 h in human Hep G2 liver epithelial cells using 3-(4,5-dimethyl thiazol-2-yl)-2,5-diphenyl tetrazolium bromide (MTT), lactate dehydrogenase (LDH) leakage, glutathione (GSH) estimation, lipid peroxidation (TBARS), caspase-3 levels, and interleukin-8 (IL-8) release assays. Exposure of GNRs to cells results in decrease in cell viability and causes cell membrane damage through LDH leakage results in cytotoxicity. The IC50 (concentration required to inhibit 50% of cells) values of 10 nm GNRs, 25 nm GNRs, and quartz (toxic control)-treated cells were found to be 19.9, 26.8, and 36.35 µg/mL, suggesting the higher cytotoxicity of GNRs. The GNRs exposure to liver cells found in depleted GSH levels, increased lipid peroxidation, and increased caspase-3 levels leads to induction of oxidative stress. In addition, enhanced levels of IL-8 were found, a sign of inflammation. The 10 nm GNRs have shown significant toxicity against all biochemical assays when compare to 25 nm GNRs and quartz-treated cells. Finally, the data indicate that the concentration size-dependent in vitro toxicity of GNRs toward liver Hep G2 cells. The toxicity of GNRs may be due to cell membrane damage, induction of oxidative stress, and inflammatory mediator release. Further investigations are necessitated to elucidate the in vivo toxicity of GNRs.

THE RELATIONSHIP OF THE IMMUNE AND METABOLIC DISORDERS DURING VARIOUS METHODS OF MULTICOMPONENT GENERAL ANESTHESIA IN LAPAROSCOPIC CHOLECYSTECTOMY.

BACKGROUND: In addition to operating injury in the pathogenesis of immunological and metabolic disorders after surgical interventions anesthesia plays an important role. THE AIM: to establish the relationship of the immune and metabolic disorders during various methods ofmulticomponent general anesthesia in conditions of laparoscopic cholecystectomy in patients with cholelithiasis. MATERIALS AND METHODS: Under constant observation there were 68 women admitted to the hospital for surgical treatment of cholelithiasis. Patients were divided into 3 groups depending on multicomponent general anesthesia (halothane, propofol, sevoflurane). We determined the concentration of cytokines (TNFa, IL-la, IL-i/8, IL-4, IL-iRA, IL-2, IFNy), components of the complement system (C,, C3, C4, C, and C, factor H, C,-inhibitor), the activity of neutrophilsperipheral blood, the concentration of the products ofperoxidation, catalase, superoxide dismutase in blood plasma. RESULTS: The level of immune-inflammation and metabolic disorders in patients with cholelithiasis was higher in patients operated with the use of halothane. The use of sevoflurane has had the most positive effect on the studied indices. CONCLUSION: The close correlation between the investigated immune and metabolic parameters on the background of the use of different schemes of multicomponent general anesthesia in patients with cholelithiasis have let to the conclusion that in the conditions of use of sevoflurane has the least place a "tension" immune and oxidative status.

Adaptive immune responses triggered by oxidative stress contribute to hepatic inflammation in NASH.

UNLABELLED: Previous studies have shown that human nonalcoholic steatohepatitis (NASH) is often associated with the presence of circulating antibodies against protein adducted by lipid peroxidation products. Here we used the methionine-choline deficient (MCD) model of NASH to characterize the possible involvement of adaptive immunity in NASH. In mice fed up to 8 weeks with the MCD diet the extension of liver injury and lobular inflammation paralleled the development of immunoglobulin G (IgG) against malonyldialdehyde (MDA) and 4-hydroxynonenal (4-HNE)-derived antigens as well as with the hepatic recruitment of CD4(+) and CD8(+) T-lymphocytes responsive to the same antigens. Moreover, in these animals the individual IgG reactivity against MDA-adducts positively correlated with transaminase release and hepatic tumor necrosis factor alpha (TNF-α) expression. To substantiate the role of immune responses triggered by oxidative stress in the progression of NASH, mice were immunized with MDA-adducted bovine serum albumin (MDA-BSA) before feeding the MCD diet. MDA-BSA immunization did not affect control mice livers, but further stimulated transaminase release, lobular inflammation, and the hepatic expression of proinflammatory cytokine in MCD-fed mice. The increased severity of NASH in immunized MCD-fed mice involved liver recruitment and the T helper (Th)-1 activation of CD4(+) T cells that, in turn, further stimulated macrophage M1 responses. Moreover, hepatic fibrosis was also evident in these animals in relation with an IL-15-mediated increase of natural killer T-cells (NKT) and the up-regulation in liver production of osteopontin by NKT cells and hepatic macrophages. CONCLUSION: These results indicate that oxidative stress can contribute to the progression of NASH by stimulating both humoral and cellular immune responses, pointing to the possible role of adaptive immunity in the pathogenesis of the disease.

Ameliorative effect of N-acetyl cysteine on alpha-cypermethrin-induced pulmonary toxicity in male rats.

Alpha-cypermethrin (α-CYP) is one of the most widely used insecticides. It may become an air pollutant and adversely affect the health. The present study was designed to determine whether treatment with N-acetyl cysteine (NAC), a well-known antioxidant, can be useful for the management of the deleterious effects of α-CYP on lung tissues. For this purpose, thirty two male rats were divided into four different groups (eight rats for each). Group (I) gavaged with corn oil (control group), group (II) gavaged daily with NAC (150 mg kg(-1) body weight), group (III) gavaged with α-CYP (14.5 mg kg(-1) body weight/day, dissolved in corn oil), group (IV) gavaged with NAC then with α-CYP 2 h later for 12 weeks. α-CYP significantly increased serum lactate dehydrogenase (LDH) and pulmonary malondialdehyde (MDA) levels, while decreased the activities of catalase (CAT) and superoxide dismutase (SOD) as well as reduced glutathione (GSH) content in lung. It also provoked higher levels of serum nitric oxide (NO), lung interleukin-1 beta (IL-1ß), tumor necrosis factor-alpha (TNF-α), hydroxyproline (Hyp) as well as heme oxygenase-1 (HO-1), inducible nitric oxide synthase (iNOS) and nuclear factor-kappa B (NF-К B) gene expression in lung tissues. Histopathological alterations in lung with congestion, cellular infiltration, necrotic changes and thickening of inter-alveolar septa were observed following α-CYP administration. NAC reduced the adverse effects of α-CYP on lung tissues and improved the histological architecture of lung since it showed antioxidant, anti-inflammatory and antifibrotic effects on lung tissues. Our results indicate that NAC exerts a potent protective effect against α-CYP-induced oxidative damage and inflammation in lung tissues.

Protective effect of aquacultured flounder fish-derived peptide against oxidative stress in zebrafish.

This study investigates the protective effect of aquacultured flounder fish-derived peptide (AFFP) against 2,2-azobis-(2-amidinopropane) hydrochloride (AAPH)-induced oxidative damage in a zebrafish model. Zebrafish embryos were evaluated for the protective effect by heartbeat rate, survival rate, ROS generation, lipid peroxidation, and cell death. In the results, the AAPH group showed a low survival rate, whereas the AFFP and AAPH co-treated group increased a survival rate. Also, AFFP dose-dependently reduced AAPH-induced intracellular ROS and lipid peroxidation, and decreased cell death in AAPH-induced zebrafish. These results revealed that AFFP could be used as a natural antioxidant, and that the zebrafish provides an alternative in vivo model to efficiently evaluate the antioxidative effects of peptides on fishes.

Integral indicator of oxidative stress in human blood.

Here we performed individual evaluation of the oxidative stress index that serves as an integral criterion for the balance of LPO-antioxidant defense system in women with endocrine pathology (type 1 diabetes mellitus and infertility with hyperprolactinemia). The state of the LPO-antioxidant defense system was estimated from blood levels of LPO substrates with conjugated double bonds, conjugated dienes, ketodienes, conjugated trienes, thiobarbituric acid-reactive substances, retinol, α-tocopherol, reduced and oxidized glutathione, and SOD activity. The use of this oxidative stress index allowed us to diagnose oxidative stress in female patients with endocrine pathology.

[On the usefulness of including general biolong baths in the combined therapeutic and rehabiliatative treatment of the patients presenting with obstructive diseases of the respiratory organs].

Bearing in mind the important role of oxidative stress and intensification of lipid peroxidation processes in the development and progression of obstructive pathology of the respiratory organs, we deemed it appropriate to evaluate the therapeutic effect of general baths containing biolong, an agent showing the antihypoxic and antioxidative properties. The clinical and functional studies that involved 109 patients (52 with chronic obstructive pulmonary disease (COPD) and 57 with bronchial asthma (BA)) have demonstrated the advantages of application of the hydrotherapeutic modality in the patients with bronchial asthma. The therapeutic effect was due to degradation of the allergic inflammation processes, the well apparent decrease in the activity of lipid peroxidation processes, the improvement of humoral immunity, generalized reduction of bronchial obstruction, enhanced physical working capacity, and psychoemotional adaptation.

Immunogenicity of ferroptosis in cancer: a matter of context?

Ferroptosis is a variant of regulated cell death (RCD) elicited by an imbalance of cellular redox homeostasis that culminates with extensive lipid peroxidation and rapid plasma membrane breakdown. Since other necrotic forms of RCD, such as necroptosis, are highly immunogenic, ferroptosis inducers have attracted considerable attention as potential tools to selectively kill malignant cells while eliciting therapeutically relevant tumor-targeting immune responses. However, rather than being consistently immunogenic, ferroptosis mediates context-dependent effects on anticancer immunity. The inability of ferroptotic cancer cells to elicit adaptive immune responses may arise from contextual deficiencies in intrinsic aspects of the process, such as adjuvanticity and antigenicity, or from microenvironmental defects imposed by ferroptotic cancer cells themselves or elicited by the induction of ferroptosis in immune cells.