Toxic Lipid Peroxidation Research Articles

Abstract 4132769: Carnosine Supplementation Improves Walking Ability of Peripheral Arterial Disease Patients

Introduction: Peripheral artery disease (PAD) is a significant cause of cardiovascular morbidity and mortality, characterized by atherosclerosis in the skeletal muscle. Currently no mechanism-based therapeutics are available for this diseased population. In the muscle, histidyl dipeptides, such as carnosine (ranging between 5-10 mM) possess the abilities to bind with toxic lipid peroxidation products, such as acrolein. Preclincial studies show carnosine is depleted in the ischemic leg, increasing intramuscular carnosine improves angiogenesis, and blood flow in the ischemic leg. In humans, carnosine levels are increased in the muscle with carnosine supplementation. However, little is known whether carnosine levels are affected in PAD patients and its effect on their walking ability. Hypothesis: Carnosine supplementation will improve walking ability of PAD patients. Methods: We recruited normal (males: n=48; females: n=52; age: 50 ±10 years) and PAD subjects (males: n=52; females: n=41; age: 67±8 years; ankle brachial index (ABI): 0.65±0.18 measured urinary histidyl dipeptides, histidyl dipeptide-aldehyde conjugates, and oxidative stress markers: N-Acetyl-S-(3-hydroxypropyl)-L-Cysteine (3-HPMA) and N-acetyl-S-(2-carboxyethyl)-L-cysteine (CEMA), both are acrolein metabolites. PAD subjects (n=7; n=5 females and n=2 males; ABI: 0.68±0.08, age: 63±8 years) were supplemented with carnosine 2 g/day for 3 months, measured distance covered in a six-minute walk test (6MWT), ABI, blood profile at baseline and after completion. Results: In PAD subjects urinary carnosine was decreased (normal: 20±29 vs PAD:11±14 nmoles/mg creatinine, p<0.024), carnosine aldehdye conjugates were increased (carnosine propanal: 1.46±1.27 vs normal 0.56±1.08 nmoles/mg creatinine, p<0.0001; and carnosine propanol: 4.66±2.80 vs normal 1.52±1.21 nmoles/mg creatinine, p<0.0001). Oxidative stress markers were increased in the PAD subjects (CEMA: 341±210 vs normal: 119±86 ng/mg creatinine, p<0.0004 and 3HPMA: 6.57±6.32 ng/mg protien vs normal: 5.49±5.28 ng/mg creatinine, p<0.001). Following carnosine supplementation, blood profile was unchanged. Distance covered in 6MWT increased by 166±204 feet (before: 577±129 vs 743±152 feet after, p<0.048) and there was an increasing trend in ABI (before: 0.68±0.08 vs 0.80±0.19; after, p<0.18). Conclusion: Urinary carnosine could serve as a biomarker to evaluate PAD pathology and carnosine supplementation may improve walking ability of PAD patients.

Abstract 4134449: Increasing Histidyl dipeptides in Skeletal Muscle Preserves Muscle Mass and Function During Heart Failure.

Background: In heart failure patients muscle wasting is a serious comorbidity. Oxidative stress and inflammatory cytokines, which cause muscle wasting, are increased in the skeletal muscle of heart failure patients. Oxidative stress leads to oxidation of lipids, generating toxic lipid peroxidation products, such as acrolein, which activates TNFa. Whether lipid peroxidation products accumulate in muscle and their removal from muscle influences muscle atrophy during heart failure has not been studied. In the muscle there are histidyl dipeptides, such as carnosine, ranging between 5-10 mM, synthesized via enzyme carnosine synthase (CARNS) that bind lipid peroxidation products. We examined whether intramuscular histidyl dipeptides influences heart failure-induced muscle wasting. Hypothesis: Histidyl dipeptides, alleviates intramuscular inflammation, preserves muscle mass and function during heart failure. Methods: Wild type (WT) and muscle-specific CARNS transgenic (Tg) mice were subjected to sham and transverse aortic constriction (TAC) surgeries. Cardiac function, muscle strength, atrophic and inflammatory markers, aldehyde modified protein, CARNS, and histidyl dipeptides were measured after 12 weeks. Results: In heart failure mice, weight of gastrocnemius (sham: 18.64 ± 1.73 vs TAC: 14.10 ± 0.90 mg, p=0.005), soleus (sham: 6.06 ± 0.82 vs TAC: 4.14 ± 0.70 mg, p=0.001), and tibialis anterior (sham:10.76 ± 1.62 vs TAC: 6.22 ± 1.83 mg, p=0.003), were decreased. Muscle strength was diminished (sham: 2.81 ± 0.36 vs TAC: 1.52 ± 0.27 N, p=0.004). Atrophic marker, atrogin1, aldehyde protein adducts and TNF-a were increased in the gastrocnemius muscle of TAC mice (~2-3-fold vs sham, p<0.05). CARNS expression and carnosine was decreased in different muscles of TAC mice (gastrocnemius; sham: 43.38 ± 20.46 vs TAC: 24.48 ± 6.49 nmoles/mg protein, p=0.03; soleus; sham: 22.04 ± 11.36 vs TAC: 11.02 ± 4.69 nmoles/mg protein, p=0.03; and tibialis; sham: 27.9 ± 11.70 vs TAC: 15.50 ± 3.48 nmoles/mg protein, p=0.01). Gastrocnemius muscle weight (WT TAC: 14.38 ± 1.08 vs Tg TAC: 17.30 ± 1.32 mg, p=0.004) and muscle strength (WT TAC: 3.06 ± 0.32 vs Tg TAC: 3.98 ± 0.41 N) were preserved, atrogin and TNFa expressions were decreased in gastrocnemius muscle of Tg TAC mice (~2-3-fold, p=0.04). Conclusion: Increasing intramuscular histidyl dipeptides alleviates muscle inflammation, preserves muscle mass, and function during heart failure.

Metabolic pathways for removing reactive aldehydes are diminished in the skeletal muscle during heart failure.

Muscle wasting is a serious complication in heart failure patients. Oxidative stress and inflammation are implicated in the pathogenesis of muscle wasting. Oxidative stress leads to the formation of toxic lipid peroxidation products, such as 4-hydroxy-2-nonenal (HNE), which covalently bind with proteins and DNA and activate atrophic pathways. Whether the formation of lipid peroxidation products and metabolic pathways that remove these toxic products are affected during heart failure-associated skeletal muscle wasting has never been studied. Male C57BL/6J mice were subjected to sham and transverse aortic constriction (TAC) surgeries for 4, 8 or 14weeks. Different skeletal muscle beds were weighed, and the total cross-sectional area of the gastrocnemius muscle was measured via immunohistochemistry. Muscle function and muscle stiffness were measured by a grip strength meter and atomic force microscope, respectively. Atrophic and inflammatory marker levels were measured via qRT‒PCR. The levels of acrolein and HNE-protein adducts, aldehyde-removing enzymes, the histidyl dipeptide-synthesizing enzyme carnosine synthase (CARNS), and amino acid transporters in the gastrocnemius muscle were measured via Western blotting and qRT‒PCR. Histidyl dipeptides and histidyl dipeptide aldehyde conjugates in theGastrocnemius and soleus muscles were analyzed by LC/MS-MS. Body weight, gastrocnemius muscle and soleus muscle weights and the total cross-sectional area of the gastrocnemius musclewere decreased after 14weeks of TAC. Heart weight, cardiac function, grip strength and muscle stiffness were decreased in the TAC-operated mice. Expression of the atrophic and inflammatory markers Atrogin1 and TNF-α, respectively, was increased ~ 1.5-2fold in the gastrocnemius muscle after 14weeks of TAC (p < 0.05 and p = 0.004 vs sham). The formation of HNE and acrolein protein adducts was increased, and the expression of the aldehyde-removing enzyme aldehyde dehydrogenase (ALDH2) was decreased in the gastrocnemius muscle of TAC mice. Carnosine (sham: 5.76 ± 1.3 vs TAC: 4.72 ± 0.7nmol/mg tissue, p = 0.04) and total histidyl dipeptide levels (carnosine and anserine; sham: 11.97 ± 1.5 vs TAC: 10.13 ± 1.4nmol/mg tissue, p < 0.05) were decreased in the gastrocnemius muscle of TAC mice. Depletion of histidyl dipeptides diminished the aldehyde removal capacity of the atrophic gastrocnemius muscle. Furthermore, CARNS and TAUT protein expression were decreased in the atrophic gastrocnemius muscle. Our data reveals that reduced expression of ALDH2 and depletion of histidyl dipeptides in the gastrocnemius muscle during heart failure leads to the accumulation of toxic aldehydes and might contribute to muscle wasting.

Dynamic monitoring of SO2 changes during ferroptosis using a light-controlled lipid droplets-targeting probe

During the process of ferroptosis, the generation of toxic lipid peroxides triggers severe inflammatory responses. Despite sulfur dioxide (SO2) being recognized as an important signal molecule and antioxidant, its specific physiological mechanism in ferroptosis remains unclear. Here, we developed a novel probe, SP-TB, tailored for detecting SO2 within lipid droplets (LDs). SP-TB features a triphenylamine moiety for LD targeting, coupled with a spiropyran serving as both the SO2 reaction site and a light-responsive switch. Under normal conditions, SP-TB exhibits yellow fluorescence at 570 nm with its SO2 reaction site closed. Upon UV light activation, the spiropyran moiety undergoes isomerization to the merocyanine form (MC-TB), resulting in red fluorescence emission at 634 nm. During this photoisomerization, the exposed CC-CN+ fragment enables specific Michael addition reactions with SO2, quenching the red fluorescence of MC-TB and enhancing yellow fluorescence. The probe exhibits stable photoreversibility and excellent LDs targeting, along with remarkable SO2 selectivity with a low detection limit of 3.5 μM. Notably, the controllability of the SO2 reaction site minimizes false positives. This probe was successfully used to monitor the changes of SO2 concentrations in LDs during ferroptosis.

Brucella rough RB51 infection activates P53-Slc7a11-Gpx4/GSH pathway to induce ferroptosis to attenuate the intracellular survival on macrophages

B. abortus is a facultative intracellular bacterium that replicates within macrophages. Intracellular survival is one of the important indexes to evaluate the virulence of Brucella. Ferroptosis is a type of programmed cell death induced by the accumulation of free iron, reactive oxygen species (ROS), and toxic lipid peroxides, play roles on cancers, cardiovascular diseases, and inflammatory diseases. In this study, we found that Brucella rough strain RB51 induced ferroptosis on macrophages with reduced levels of host glutathione and glutathione peroxidase 4 (Gpx4), together with increased ferrous iron, lipid peroxidation, and ROS. The inhibitor ferrostatin-1 significantly reduced the ferroptosis of RB51-infected macrophages, confirming that ferroptosis occurred during infection with Brucella RB51. Furthermore, we found that RB51 infection induced ferroptosis is regulated by P53-Slc7a11-Gpx4/GSH signal pathway. Inhibiting P53 decreased the levels of ROS and lipid peroxidation, while the levels of Slc7a11, Gpx4 and GSH were rescued. More importantly, inhibiting ferroptosis by different ferroptosis inhibitors increased the intracellular survival of Brucella RB51, indicating ferroptosis functions on the attenuation of Brucella intracellular survival. Collectively, our observations demonstrate that Brucella RB51 infection induces ferroptosis on macrophages, which is regulated by P53-Slc7a11-Gpx4/GSH signal pathway and functions on the attenuation of intracellular survival of Brucella.

Sonoinduced Tumor Therapy and Metastasis Inhibition by a Ruthenium Complex with Dual Action: Superoxide Anion Sensitization and Ligand Fracture.

Photoresponsive ruthenium(II) complexes have recently emerged as a promising tool for synergistic photodynamic therapy and chemotherapy in oncology, as well as for antimicrobial applications. However, the limited penetration power of photons prevents the treatment of deep-seated lesions. In this study, we introduce a sonoresponsive ruthenium complex capable of generating superoxide anion (O2•-) via type I process and initiating a ligand fracture process upon ultrasound triggering. Attaching hydroxyflavone (HF) as an "electron reservoir" to the octahedral-polypyridyl-ruthenium complex resulted in decreased highest occupied molecular orbital (HOMO)-lowest unoccupied molecular orbital (LUMO) energy gaps and triplet-state metal to ligand charge transfer (3MLCT) state energy (0.89 eV). This modification enhanced the generation of O2•- under therapeutic ultrasound irradiation at a frequency of 1 MHz. The produced O2•- rapidly induced an intramolecular cascade reaction and HF ligand fracture. As a proof-of-concept, we engineered the Ru complex into a metallopolymer platform (PolyRuHF), which could be activated by low-power ultrasound (1.5 W cm-2, 1.0 MHz, 50% duty cycle) within a centimeter range of tissue. This activation led to O2•- generation and the release of cytotoxic ruthenium complexes. Consequently, PolyRuHF induced cellular apoptosis and ferroptosis by causing mitochondrial dysfunction and excessive toxic lipid peroxidation. Furthermore, PolyRuHF effectively inhibited subcutaneous and orthotopic breast tumors and prevented lung metastasis by downregulating metastasis-related proteins in mice. This study introduces the first sonoresponsive ruthenium complex for sonodynamic therapy/sonoactivated chemotherapy, offering new avenues for deep tumor treatment.

A genome-wide CRISPR screen reveals that antagonism of glutamine metabolism sensitizes head and neck squamous cell carcinoma to ferroptotic cell death

Glutamine is a conditionally essential amino acid for the growth and survival of rapidly proliferating cancer cells. Many cancers are addicted to glutamine, and as a result, targeting glutamine metabolism has been explored clinically as a therapeutic approach. Glutamine-catalyzing enzymes are highly expressed in primary and metastatic head and neck squamous cell carcinoma (HNSCC). However, the nature of the glutamine-associated pathways in this aggressive cancer type has not been elucidated. Here, we explored the therapeutic potential of a broad glutamine antagonist, DRP-104 (sirpiglenastat), in HNSCC tumors and aimed at shedding light on glutamine-dependent pathways in this disease. We observed a potent antitumoral effect of sirpiglenastat in HPV- and HPV + HNSCC xenografts. We conducted a whole-genome CRISPR screen and metabolomics analyses to identify mechanisms of sensitivity and resistance to glutamine metabolism blockade. These approaches revealed that glutamine metabolism blockade results in the rapid buildup of polyunsaturated fatty acids (PUFAs) via autophagy nutrient-sensing pathways. Finally, our analysis demonstrated that GPX4 mediates the protection of HNSCC cells from accumulating toxic lipid peroxides; hence, glutamine blockade sensitizes HNSCC cells to ferroptosis cell death upon GPX4 inhibition. These findings demonstrate the therapeutic potential of sirpiglenastat in HNSCC and establish a novel link between glutamine metabolism and ferroptosis, which may be uniquely translated into targeted glutamine-ferroptosis combination therapies.

Recent Progress of Glutathione Peroxidase 4 Inhibitors in Cancer Therapy.

Ferroptosis is a novel type of programmed cell death that relies on the build-up of intracellular iron and leads to an increase in toxic lipid peroxides. Glutathione Peroxidase 4 (GPX4) is a crucial regulator of ferroptosis that uses glutathione as a cofactor to detoxify cellular lipid peroxidation. Targeting GPX4 in cancer could be a promising strategy to induce ferroptosis and kill drugresistant cancers effectively. Currently, research on GPX4 inhibitors is of increasing interest in the field of anti-tumor agents. Many reviews have summarized the regulation and ferroptosis induction of GPX4 in human cancer and disease. However, insufficient attention has been paid to GPX4 inhibitors. This article outlines the molecular structures and development prospects of GPX4 inhibitors as novel anticancer agents.

Ferroptosis inhibitor improves outcome after early and delayed treatment in mild spinal cord injury

We show that redox active iron can induce a regulated form of non-apoptotic cell death and tissue damage called ferroptosis that can contribute to secondary damage and functional loss in the acute and chronic periods after spinal cord injury (SCI) in young, adult, female mice. Phagocytosis of red blood cells at sites of hemorrhage is the main source of iron derived from hemoglobin after SCI. Expression of hemeoxygenase-1 that induces release of iron from heme, is increased in spinal cord macrophages 7 days after injury. While iron is stored safely in ferritin in the injured spinal cord, it can, however, be released by NCOA4-mediated shuttling of ferritin to autophagosomes for degradation (ferritinophagy). This leads to the release of redox active iron that can cause free radical damage. Expression of NCOA4 is increased after SCI, mainly in macrophages. Increase in the ratio of redox active ferrous (Fe2+) to ferric iron (Fe3+) is also detected after SCI by capillary electrophoresis inductively coupled mass spectrometry. These changes are accompanied by other hallmarks of ferroptosis, i.e., deficiency in various elements of the antioxidant glutathione (GSH) pathway. We also detect increases in enzymes that repair membrane lipids (ACSL4 and LPCAT3) and thus promote on-going ferroptosis. These changes are associated with increased levels of 4-hydroxynonenal (4-HNE), a toxic lipid peroxidation product. Mice with mild SCI (30 kdyne force) treated with the ferroptosis inhibitor (UAMC-3203-HCL) either early or delayed times after injury showed improvement in locomotor recovery and secondary damage. Cerebrospinal fluid and serum samples from human SCI cases show evidence of increased iron storage (ferritin), and other iron related molecules, and reduction in GSH. Collectively, these data suggest that ferroptosis contributes to secondary damage after SCI and highlights the possible use of ferroptosis inhibitors to treat SCI.

Abstract LB299: Dihydrouridine synthase 2 sustains levels of tRNACys and prevents ferroptosis in lung cancer

Abstract Dihydrouridine is a universally conserved tRNA modification installed by enzymes that are important for human health. High expression of dihydrouridine synthase 2 (DUS2) predicts poor patient outcomes in lung adenocarcinoma (LUAD) for reasons that are not yet clear. Here, we show in human cells and mouse xenografts that DUS2 suppresses ferroptosis, a metal-dependent non-apoptotic form of cell death that is emerging as a therapeutic target in lung cancer. Elevated DUS2 correlates with resistance to ferroptosis inducers and loss of DUS2 causes increased sensitivity with concomitant accumulation of toxic lipid peroxides. Mechanistically, DUS2 is required to maintain tRNA CysGCA levels, supporting translation of cysteine-rich proteins including metallothioneins, key regulators of metal and redox homeostasis Notably, DUS2 KO cells cannot sustain normal levels of metallothioneins, a class of very cysteine rich proteins that serve as key regulators of both metal and redox homeostasis. Accordingly, DUS2 knockout cells are more susceptible to zinc intoxication and have lower levels of reduced glutathione, which partially explains their sensitivity to ferroptosis. Our findings demonstrate that DUS2 is required to support tRNACys levels and fend off ferroptosis in lung cancer cells. This highlights that individual tRNA substrates can play an outsized role in the biological functions of tRNA modifying enzymes, and demonstrates the therapeutic potential of targeting DUS2 in cancer. Citation Format: Austin Draycott, Matthew C. Wang, Diana Martínez Saucedo, Luisa Escobar-Hoyos, Wendy Gilbert. Dihydrouridine synthase 2 sustains levels of tRNACys and prevents ferroptosis in lung cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 2 (Late-Breaking, Clinical Trial, and Invited Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(7_Suppl):Abstract nr LB299.

Effective therapy of advanced breast cancer through synergistic anticancer by paclitaxel and P-glycoprotein inhibitor

Multi-drug resistance (MDR) in advanced breast cancer (ABC) is triggered by the high expression of P-glycoprotein (P-gp), which reduces intracellular concentration of anti-tumor drugs, in turn preventing oxidative stress damage to cytoplasmic and mitochondrial membranes. It is therefore of clinical relevance to develop P-gp-specific targeted nanocarriers for the treatment of drug resistant ABC. Herein, a drug carrier targeting CD44 and mitochondria was synthesised for the delivery of encequidar (ER, P-gp inhibitor) and paclitaxel (PTX). HT@ER/PTX nanoparticles (ER:PTX molar ratio 1:1) had excellent P-gp inhibition ability and targeted mitochondria to induce apoptosis in MCF-7/PTX cells in vitro. Furthermore, HT@ER/PTX nanocarriers showed more anti-tumor efficacy than PTX (Taxol®) in a xenograft mouse model of MCF-7/PTX cells; the tumor inhibitory rates of HT@ER/PTX nanoparticles and Taxol® were 72.64% ± 4.41% and 32.36% ± 4.09%, respectively. The survival of tumor-bearing mice administered HT@ER/PTX nanoparticles was prolonged compared to that of the mice treated with Taxol®. In addition, HT@ER/PTX not only inhibited P-gp-mediated removal of toxic lipid peroxidation byproducts resulting from anti-tumor drugs but also upregulated the expression of mitochondrial dynamics-related protein, fostering oxidative stress damage, which induced activation of the Caspase-3 apoptosis pathway. Our findings indicate that mitochondria targeted co-delivery of anti-tumor drugs and P-gp inhibitors could be a practical approach in treating multi-drug resistance in ABC.

Vildagliptin Treatment Ameliorates Renal Interstitial Fibrosis in a Murine Model of Unilateral Ureteral Obstruction.

Renal interstitial fibrosis in mice can be modeled using unilateral ureteral obstruction (UUO). Here, we investigated the anti-fibrotic effects of the dipeptidyl peptidase-4 inhibitor vildagliptin in this model. We found that vildagliptin given in the drinking water at 10.6 ± 1.5 mg/kg/d prevented fibrosis. Mechanistically, UUO was associated with extracellular signal-regulated kinase (ERK) phosphorylation and with the accumulation of the toxic lipid peroxidation product expression of 4-hydroxy-2-nonenal (4-HNE). Both were significantly inhibited by vildagliptin. Similarly, UUO caused reductions in heme oxygenase-1 (HO-1) mRNA in the kidney, whereas interleukin-6 (IL-6) and cyclooxygenase-1 (COX-1) mRNA were increased; these effects were also prevented by vildagliptin. Taking these data together, we propose that vildagliptin reduces renal interstitial fibrosis resulting from UUO by means of its effects on ERK phosphorylation and the amounts of 4-HNE, HO-1, IL-6 and COX-1 in the kidney.

Ultrasound-propelled nanomotors for efficient cancer cell ferroptosis.

Ferroptosis is a non-apoptotic form of cell death that is dependent on the accumulation of intracellular iron that causes elevation of toxic lipid peroxides. Therefore, it is crucial to improve the levels of intracellular iron and reactive oxygen species (ROS) in a short time. Here, we first propose ultrasound (US)-propelled Janus nanomotors (Au-FeOx/PEI/ICG, AFPI NMs) to accelerate cellular internalization and induce cancer cell ferroptosis. This nanomotor consists of a gold-iron oxide rod-like Janus nanomotor (Au-FeOx, AF NMs) and a photoactive indocyanine green (ICG) dye on the surface. It not only exhibits accelerating cellular internalization (∼4-fold) caused by its attractive US-driven propulsion but also shows good intracellular motion behavior. In addition, this Janus nanomotor shows excellent intracellular ROS generation performance due to the synergistic effect of the "Fenton or Fenton-like reaction" and the "photochemical reaction". As a result, the killing efficiency of actively moving nanomotors on cancer cells is 88% higher than that of stationary nanomotors. Unlike previous passive strategies, this work is a significant step toward accelerating cellular internalization and inducing cancer-cell ferroptosis in an active way. These novel US-propelled Janus nanomotors with strong propulsion, efficient cellular internalization and excellent ROS generation are suitable as a novel cell biology research tool.

Mechanisms and regulations of ferroptosis.

Regulation of cell mortality for disease treatment has been the focus of research. Ferroptosis is an iron-dependent regulated cell death whose mechanism has been extensively studied since its discovery. A large number of studies have shown that regulation of ferroptosis brings new strategies for the treatment of various benign and malignant diseases. Iron excess and lipid peroxidation are its primary metabolic features. Therefore, genes involved in iron metabolism and lipid metabolism can regulate iron overload and lipid peroxidation through direct or indirect pathways, thereby regulating ferroptosis. In addition, glutathione (GSH) is the body's primary non-enzymatic antioxidants and plays a pivotal role in the struggle against lipid peroxidation. GSH functions as an auxiliary substance for glutathione peroxidase 4 (GPX4) to convert toxic lipid peroxides to their corresponding alcohols. Here, we reviewed the researches on the mechanism of ferroptosis in recent years, and comprehensively analyzed the mechanism and regulatory process of ferroptosis from iron metabolism and lipid metabolism, and then described in detail the metabolism of GPX4 and the main non-enzymatic antioxidant GSH in vivo.

IFP35 aggravates Staphylococcus aureus infection by promoting Nrf2-regulated ferroptosis

IntroductionSerious Staphylococcus aureus (SA) infection is one of the most life-threatening diseases. Interferon-induced protein 35 (IFP35) is a pleiotropic factor that participates in multiple biological functions, however, its biological role in SA infection is not fully understood. Ferroptosis is a new type of regulated cell death driven by the accretion of free iron and toxic lipid peroxides and plays critical roles in tissue damage. Whether ferroptosis is involved in SA-induced immunopathology and its regulatory mechanisms remain unknown. ObjectivesWe aimed to determine the role and underlying mechanisms of IFP35 in SA-induced lung infections. MethodsSA infection models were established using wild-type (WT) and IFP35 knockout (Ifp35−/−) mice or macrophages. Histological analysis was performed to assess lung injury. Quantitative real-time PCR, western blotting, flow cytometry, and confocal microscopy were performed to detect ferroptosis. Co-IP and immunofluorescence were used to elucidate the molecular regulatory mechanisms. ResultsWe found that IFP35 levels increased in the macrophages and lung tissue of SA-infected mice. IFP35 deficiency protected against SA-induced lung damage in mice. Moreover, ferroptosis occurred and contributed to lung injury after SA infection, which was ameliorated by IFP35 deficiency. Mechanically, IFP35 facilitated the ubiquitination and degradation of nuclear factor E2-related factor 2 (Nrf2), aggravating SA-induced ferroptosis and lung injury. ConclusionsOur data demonstrate that IFP35 promotes ferroptosis by facilitating the ubiquitination and degradation of Nrf2 to exacerbate SA infection. Targeting IFP35 may be a promising approach for treating infectious diseases caused by SA.

Effective targeting of breast cancer by the inhibition of P-glycoprotein mediated removal of toxic lipid peroxidation byproducts from drug tolerant persister cells

Therapy resistance has long been considered to occur through the selection of pre-existing clones equipped to survive and quickly regrow, or through the acquisition of mutations during chemotherapy. Here we show that following in vitro treatment by chemotherapy, epithelial breast cancer cells adopt a transient drug tolerant phenotype characterized by cell cycle arrest, epithelial-to-mesenchymal transition (EMT) and the reversible upregulation of the multidrug resistance (MDR) efflux transporter P-glycoprotein (P-gp). The drug tolerant persister (DTP) state is reversible, as cells eventually resume proliferation, giving rise to a cell population resembling the initial, drug-naïve cell lines. However, recovery after doxorubicin treatment is almost completely eliminated when DTP cells are cultured in the presence of the P-gp inhibitor Tariquidar. Mechanistically, P-gp contributes to the survival of DTP cells by removing reactive oxygen species-induced lipid peroxidation products resulting from doxorubicin exposure. In vivo, prolonged administration of Tariquidar during doxorubicin treatment holidays resulted in a significant increase of the overall survival of Brca1−/−;p53−/− mammary tumor bearing mice. These results indicate that prolonged administration of a P-gp inhibitor during drug holidays would likely benefit patients without the risk of aggravated side effects related to the concomitantly administered toxic chemotherapy. Effective targeting of DTPs through the inhibition of P-glycoprotein may result in a paradigm shift, changing the focus from countering drug resistance mechanisms to preventing or delaying therapy resistance.

Ferroptosis in immunostimulation and immunosuppression.

Ferroptosis is a form of iron-dependent regulated cell death characterized by the accumulation of toxic lipid peroxides, particularly in the plasma membrane, leading to lytic cell death. While it plays a crucial role in maintaining the overall health and proper functioning of multicellular organisms, it can also contribute to tissue damage and pathological conditions. Although ferroptotic damage is generally recognized as an immunostimulatory process associated with the release of damage-associated molecular patterns (DAMPs), the occurrence of ferroptosis in immune cells or the release of immunosuppressive molecules can result in immune tolerance. Consequently, there is ongoing exploration of targeting the upstream signals or the machinery of ferroptosis to therapeutically enhance or inhibit the immune response. In addition to introducing the core molecular mechanisms of ferroptosis, we will focus on the immune characteristics of ferroptosis in pathological conditions, particularly in the context of infection, sterile inflammation, and tumor immunity.

1506-P: Effect of Beta-Agonist Treatment on Selenium Metabolism in Differentiated Brown Adipocytes

Selenium (Se) is an essential trace element important to several body processes such as metabolism, thyroid function, and curbing of oxidative stress. Se is utilized as the amino acid known selenocysteine (Sec), decomposed by the enzyme selenocysteine lyase (Scly). Selenoproteins have a broad range of functions, including protecting against lipid peroxidation and apoptosis, metabolic dysregulation such as glucose intolerance, and other immune responses. In brown adipocytes, the selenoprotein glutathione peroxidase 4 (GPX4) plays a key role in lipid peroxidation which in turn allows for proper thermogenic responses triggered by adrenergic signals. GPX4 removes toxic lipid peroxides by converting them into non-toxic alcohol and water. When Se levels are insufficient in brown adipocytes, the selenoprotein GPX4 may be downregulated, leading cells to accumulate a lethal amount of lipid peroxidases and triggering ferroptosis, a type of cell death. Ferroptosis activation, in turn, negatively impact pathways dependent on adrenergic responses in brown adipocytes, such as thermogenesis activation. To determine how Se levels regulate the expression of Scly and GPX4 upon treatment with adrenergic agonist in differentiated PAZ6 brown adipocytes. Human PAZ6 pre-adipocytes were differentiated for 21 days, then treated with insufficient (20 nM), adequate (80 nM), and high sodium selenite (160 nM) levels for 48h. Differentiated brown adipocytes were also treated with 1 μM CL-316243, a β3-adrenergic receptor agonist, for 24h after selenite treatment. Cells were then harvested for qPCR and Western blot analysis. Differentiated PAZ6 cells had an increase in UCP1 expression, a marker of brown adipocytes. Upon Se treatment, both Scly and GPX4 expression were unchanged in PAZ6 cells. Treatment with β3-adrenergic receptor agonist also did not affect the expression levels of both proteins. In differentiated brown adipocytes, expression of Scly and GPX4 is not dependent on Se nor β-agonist signals. Disclosure S.Swanson: None. Funding National Institutes of Health (R01DK128390)

Vimentin is required for tumor progression and metastasis in a mouse model of non–small cell lung cancer

Vimentin is highly expressed in metastatic cancers, and its expression correlates with poor patient prognoses. However, no causal in vivo studies linking vimentin and non–small cell lung cancer (NSCLC) progression existed until now. We use three complementary in vivo models to show that vimentin is required for the progression of NSCLC. First, we crossed LSL-KrasG12D; Tp53fl/fl mice (KPV+/+) with vimentin knockout mice (KPV−/−) to demonstrate that KPV−/− mice have attenuated tumor growth and improved survival compared with KPV+/+ mice. Next, we therapeutically treated KPV+/+ mice with withaferin A (WFA), an agent that disrupts vimentin intermediate filaments (IFs). We show that WFA suppresses tumor growth and reduces tumor burden in the lung. Finally, luciferase-expressing KPV+/+, KPV−/−, or KPVY117L cells were implanted into the flanks of athymic mice to track cancer metastasis to the lung. In KPVY117L cells, vimentin forms oligomers called unit-length filaments but cannot assemble into mature vimentin IFs. KPV–/– and KPVY117L cells fail to metastasize, suggesting that cell-autonomous metastasis requires mature vimentin IFs. Integrative metabolomic and transcriptomic analysis reveals that KPV–/– cells upregulate genes associated with ferroptosis, an iron-dependent form of regulated cell death. KPV–/– cells have reduced glutathione peroxidase 4 (GPX4) levels, resulting in the accumulation of toxic lipid peroxides and increased ferroptosis. Together, our results demonstrate that vimentin is required for rapid tumor growth, metastasis, and protection from ferroptosis in NSCLC.

Regulation of myeloid cells and inflammation marks increased susceptibility in a genetic model of acute viral infection-induced tissue damage.

Abstract M.H2 k/bmice are identical to MA/My parent strain aside from a C57L-derived region within the MHC, which causes these mice to develop significant splenic tissue damage during acute murine cytomegalovirus (MCMV) infection. To understand how MCMV infection leads to the induction of tissue damage, we probed immunological differences between the M.H2 k/band MA/My mice over time. Using tissue imaging and flow cytometry, we observed increased infiltration of neutrophils to the marginal zones of the M.H2 k/bmice aligning with the appearance of necrosis at 2 days post infection (dpi). While TUNEL staining identified increased cell death within these clusters, apoptosis was reduced, suggesting an inflammatory death pathway in the M.H2 k/bneutrophils. In line with this hypothesis, multiplex ELISA identified increased IL-6 and TGF-β in the spleens of M.H2 k/bmice at this time. Intriguingly, depletion of neutrophils prior to infection did not alleviate M.H2 k/bhistopathology or inflammation. Seeing the differential regulation of neutrophils, we assessed the state of other myeloid populations in the spleen during acute MCMV infection. We observed significant loss of marginal zone and red pulp macrophages (MZMs, RPMs) beginning at 2dpi and worsening over time in the M.H2 k/bmice. Further exploration into how the loss of these populations may contribute to tissue damage identified buildup of toxic lipid peroxidation byproduct 4HNE early in the MZMs and progressing through the RPMs alongside the development of histopathology. In conclusion, we have identified increased inflammation, oxidative stress, and macrophage loss in M.H2 k/bspleens during acute MCMV infection that tracks with localization and temporal appearance of histopathology. NIH NIAID R01AI50072, Department of Medicine/Nephrology, Center for Immunity Inflammation and Regenerative Medicine, Beirne B Carter Center for Immunology Research.