Itaconate protects ferroptotic neurons by alkylating GPx4 post stroke.

Neuronal ferroptosis plays a key role in neurologic deficits post intracerebral hemorrhage (ICH). However, the endogenous regulation of rescuing ferroptotic neurons is largely unexplored. Here, we analyzed the integrated alteration of metabolomic landscape after ICH using LC-MS and MALDI-TOF/TOF MS, and demonstrated that aconitate decarboxylase 1 (Irg1) and its product itaconate, a derivative of the tricarboxylic acid cycle, were protectively upregulated. Deficiency of Irg1 or depletion of neuronal Irg1 in striatal neurons was shown to exaggerate neuronal loss and behavioral dysfunction in an ICH mouse model using transgenic mice. Administration of 4-Octyl itaconate (4-OI), a cell-permeable itaconate derivative, and neuronal Irg1 overexpression protected neurons in vivo. In addition, itaconate inhibited ferroptosis in cortical neurons derived from mouse and human induced pluripotent stem cells in vitro. Mechanistically, we demonstrated that itaconate alkylated glutathione peroxidase 4 (GPx4) on its cysteine 66 and the modification allosterically enhanced GPx4's enzymatic activity by using a bioorthogonal probe, itaconate-alkyne (ITalk), and a GPx4 activity assay using phosphatidylcholine hydroperoxide. Altogether, our research suggested that Irg1/itaconate-GPx4 axis may be a future therapeutic strategy for protecting neurons from ferroptosis post ICH.

Targeted macrophage phagocytosis by Irg1/itaconate axis improves the prognosis of intracerebral hemorrhagic stroke and peritonitis.

BACKGROUND: Macrophages are innate immune cells whose phagocytosis function is critical to the prognosis of stroke and peritonitis. cis-aconitic decarboxylase immune-responsive gene 1 (Irg1) and its metabolic product itaconate inhibit bacterial infection, intracellular viral replication, and inflammation in macrophages. Here we explore whether itaconate regulates phagocytosis. METHODS: Phagocytosis of macrophages was investigated by time-lapse video recording, flow cytometry, and immunofluorescence staining in macrophage/microglia cultures isolated from mouse tissue. Unbiased RNA-sequencing and ChIP-sequencing assays were used to explore the underlying mechanisms. The effects of Irg1/itaconate axis on the prognosis of intracerebral hemorrhagic stroke (ICH) and peritonitis was observed in transgenic (Irg1flox/flox; Cx3cr1creERT/+, cKO) mice or control mice in vivo. FINDINGS: In a mouse model of ICH, depletion of Irg1 in macrophage/microglia decreased its phagocytosis of erythrocytes, thereby exacerbating outcomes (n = 10 animals/group, p < 0.05). Administration of sodium itaconate/4-octyl itaconate (4-OI) promoted macrophage phagocytosis (n = 7 animals/group, p < 0.05). In addition, in a mouse model of peritonitis, Irg1 deficiency in macrophages also inhibited phagocytosis of Staphylococcus aureus (n = 5 animals/group, p < 0.05) and aggravated outcomes (n = 9 animals/group, p < 0.05). Mechanistically, 4-OI alkylated cysteine 155 on the Kelch-like ECH-associated protein 1 (Keap1), consequent in nuclear translocation of nuclear factor erythroid 2-related factor 2 (Nrf2) and transcriptional activation of Cd36 gene. Blocking the function of CD36 completely abolished the phagocytosis-promoting effects of Irg1/itaconate axis in vitro and in vivo. INTERPRETATION: Our findings provide a potential therapeutic target for phagocytosis-deficiency disorders, supporting further development towards clinical application for the benefit of stroke and peritonitis patients. FUNDING: The National Natural Science Foundation of China (32070735, 82371321 to Q. Li, 82271240 to F. Yang) and the Beijing Natural Science Foundation Program and Scientific Research Key Program of Beijing Municipal Commission of Education (KZ202010025033 to Q. Li).

IL-10 protects against OPC ferroptosis by regulating lipid reactive oxygen species levels post stroke.

Accumulation of reactive oxygen species (ROS), especially on lipids, induces massive cell death in neurons and oligodendrocyte progenitor cells (OPCs) and causes severe neurologic deficits post stroke. While small compounds, such as deferoxamine, lipostatin-1, and ferrostatin-1, have been shown to be effective in reducing lipid ROS, the mechanisms by which endogenously protective molecules act against lipid ROS accumulation and subsequent cell death are still unclear, especially in OPCs, which are critical for maintaining white matter integrity and improving long-term outcomes after stroke. Here, using mouse primary OPC cultures, we demonstrate that interleukin-10 (IL-10), a cytokine playing roles in reducing neuroinflammation and promoting hematoma clearance, significantly reduced hemorrhage-induced lipid ROS accumulation and subsequent ferroptosis in OPCs. Mechanistically, IL-10 activated the IL-10R/STAT3 signaling pathway and upregulated the DLK1/AMPK/ACC axis. Subsequently, IL-10 reprogrammed lipid metabolism and reduced lipid ROS accumulation. In addition, in an autologous blood injection intracerebral hemorrhagic stroke (ICH) mouse model, deficiency of the endogenous Il-10, specific knocking out Il10r or Dlk1 in OPCs, or administration of ACC inhibitor was associated with increased OPC cell death, demyelination, axonal sprouting, and the cognitive deficits during the chronic phase of ICH and vice versa. These data suggest that IL-10 protects against OPC loss and white matter injury by reducing lipid ROS, supporting further development of potential clinical applications to benefit patients with stroke and related disorders.

H3K9 trimethylation dictates neuronal ferroptosis through repressing Tfr1.

Spontaneous intracerebral hemorrhage (ICH) is a devastating disease with high morbidity and mortality worldwide. We have previously shown that ferroptosis contributes to neuronal loss in ICH mice. The overload of iron and dysfunction of glutathione peroxidase 4 (GPx4) promote neuronal ferroptosis post-ICH. However, how epigenetic regulatory mechanisms affect the ferroptotic neurons in ICH remains unclear. In the current study, hemin was used to induce ferroptosis in N2A and SK-N-SH neuronal cells to mimic ICH. The results showed that hemin-induced ferroptosis was accompanied by an increment of global level of trimethylation in histone 3 lysine 9 (H3K9me3) and its methyltransferase Suv39h1. Transcriptional target analyses indicated that H3K9me3 was enriched at the promoter region and gene body of transferrin receptor gene 1 (Tfr1) and repressed its expression upon hemin stimulation. Inhibition of H3K9me3 with inhibitor or siRNA against Suv39h1 aggravated hemin- and RSL3-induced ferroptosis by upregulating Tfr1 expression. Furthermore, Suv39h1-H3K9me3 mediated repression of Tfr1 contributes to the progression of ICH in mice. These data suggest a protective role of H3K9me3 in ferroptosis post ICH. The knowledge gained from this study will improve the understanding of epigenetic regulation in neuronal ferroptosis and shed light on future clinical research after ICH.

Inhibition of the LRRC8A channel promotes microglia/macrophage phagocytosis and improves outcomes after intracerebral hemorrhagic stroke.

Promoting microglial/macrophage (M/Mφ) phagocytosis accelerates hematoma clearance and improves the prognosis of intracerebral hemorrhagic stroke (ICH). Cation channels such as Piezo1 modulate bacterial clearance by regulating M/Mφ. Whether LRRC8A, an anion channel, affects M/Mφ erythrophagocytosis and functional recovery after ICH was investigated here. We found that LRRC8A is highly expressed on M/Mφ in the perihematomal region of ICH mice. Conditional knockout of Lrrc8a in M/Mφ or treatment with an LRRC8A channel blocker accelerated hematoma clearance, reduced neuronal death, and improved functional recovery after ICH. Mechanistically, the LRRC8A channel inhibition promoted M/Mφ phagocytosis by activating AMP-activated protein kinase (AMPK), thereby inducing nuclear translocation of nuclear factor-erythroid 2 related factor 2 (Nrf2) and increasing Cd36 transcription. Our findings illuminate the regulation of M/Mφ phagocytosis by the LRRC8A channel via the AMPK-Nrf2-CD36 pathway after ICH, suggesting that LRRC8A is a potential target for hematoma clearance in ICH treatment.

IRG1/itaconate increases IL-10 release to alleviate mechanical and thermal hypersensitivity in mice after nerve injury.

Inflammation plays an important role in the occurrence and development of neuropathic pain. Immune-responsive gene 1 (IRG1) decarboxylates cis-aconitate to produce itaconate in the mitochondria. Itaconate serves as an immunomodulator of macrophages and represses inflammation in infectious diseases. Recently, a study showed that an itaconate derivative inhibits neuroinflammation and reduces chronic pain in mice. However, the function and molecular mechanisms of endogenous itaconate in neuropathic pain have not been fullyelucidated. In this study, the content of itaconate in the ipsilateral spinal cord after nerve-injured mice was detected with mass spectrometry. The Irg1-/- mouse was constructed to determine the role of endogenous itaconate in the chronic constriction nerve injury (CCI) model. The analgesic effect of exogenous itaconate was assessed with intraperitoneal and intrathecal administration in both male and female CCI mice. The spinal application of 4-OI also reduced the evoked responses of wide dynamic range neurons in CCI mice. The potential analgesic mechanism of itaconate was explored through molecular biology experiments and verified in Interleukin (IL)-10-/- mice. We found the levels of itaconate and IRG1 in the spinal cord significantly increased after CCI. Irg1 deficiency aggravated the mechanical and heat hypersensitivity, while the exogenous administration of the itaconate derivative 4-OI alleviated the neuropathic pain in male and female CCI mice. Mechanistically, the treatment of 4-OI increased the level of IL-10 and activates STAT3/ß-endorphin pathway in the spinal cord, and the analgesia effect of itaconate was impaired in IL-10-/- mice. Finally, we showed that the upregulation of IL-10 induced by 4-OI was mainly from spinal neurons through Nrf2 pathway. This study demonstrated the analgesic effect of endogenous and exogenous itaconate in the neuropathic pain model, suggesting that the spinal IL-10/STAT3/ß-endorphin pathway might mediate the analgesia effect of itaconate.

Ferroptosis in oligodendrocyte progenitor cells mediates white matter injury after hemorrhagic stroke.

Oligodendrocyte progenitor cells (OPCs) differentiate to myelin-producing mature oligodendrocytes and enwrap growing or demyelinated axons during development and post central nervous diseases. Failure of remyelination owing to cell death or undifferentiation of OPCs contributes to severe neurologic deficits and motor dysfunction. However, how to prevent the cell death of OPCs is still poorly understood, especially in hemorrhagic diseases. In the current study, we injected autologous blood into the mouse lateral ventricular to study the hemorrhage-induced OPC cell death in vivo. The integrity of the myelin sheath of the corpus callosum was disrupted post intraventricular hemorrhage (IVH) assessed by using magnetic resonance imaging, immunostaining, and transmission electron microscopy. Consistent with the severe demethylation, we observed massive cell death of oligodendrocyte lineages in the periventricular area. In addition, we found that ferroptosis is the major cell death form in Hemin-induced OPC death by using RNA-seq analysis, and the mechanism was glutathione peroxidase 4 activity reduction-resulted lipid peroxide accumulation. Furthermore, inhibition of ferroptosis rescued OPC cell death in vitro, and in vivo attenuated IVH-induced white matter injury and promoted recovery of neurological function. These data demonstrate that ferroptosis is an essential form of OPC cell death in hemorrhagic stroke, and rescuing ferroptotic OPCs could serve as a therapeutic target for stroke and related diseases.

Reduction of lactoferrin aggravates neuronal ferroptosis after intracerebral hemorrhagic stroke in hyperglycemic mice.

Diabetic hyperglycemia aggravates the prognosis of intracerebral hemorrhagic stroke (ICH) in the clinic. In addition to hematoma expansion and increased inflammation, how diabetic hyperglycemia affects the outcomes of ICH is still unclear. We found that streptozotocin-induced diabetic hyperglycemia not only increased neutrophil infiltration, but also changed the gene expression profile of neutrophils, including lactoferrin (Ltf) encoding gene Ltf. Peroxisome proliferator-activated receptor γ (PPARγ) transcribed Ltf and the lack of neutrophilic Ltf transcription and secretion exacerbated neuronal ferroptosis by accumulating intraneuronal iron. Furthermore, the administration of recombinant Ltf protected against neuronal ferroptosis and improved neurobehavior in hyperglycemic ICH mice, and vice versa. These results indicate that supplementing Ltf or inhibiting neuronal ferroptosis are promising potential strategies to improve the acute outcomes of diabetic ICH in the clinic.