Induction of IRG1 following ischemic stroke promotes HO-1 and BDNF expression to alleviate neuroinammation and restrain ischemic brain injury

Inammatory stimuli induce immunoresponsive gene 1 (IRG1) expression that in turn catalyzes the production of itaconate through diverting cis-aconitate away from the tricarboxylic acid cycle. The immunoregulatory effect of IRG1/itaconate axis has recently documented in LPS-activated mouse and human macrophages. In addition, dimethyl itaconate, an itaconate derivative, was reported to ameliorate disease severity in the animal models of psoriasis and multiple sclerosis. Currently, whether IRG1/itaconate axis exerts an immunomodulatory effect in ischemic stroke is unknown. Thus, we investigated whether IRG1 plays a role in modulating brain injury in ischemic stroke. In addition, the molecular mechanisms underlying the protective effects of IRG1 in ischemic stroke were elucidated. To further whether the induction of IRG1 following ischemic stroke plays a role in modulating ischemic brain injury, we to compare the level of ischemic brain injury between IRG1 −/− mice and its corresponding WT controls. To do that, we rst determined whether IRG1 deciency may result in alteration of the cerebrovasculature in IRG1 −/− mice, as it would affect the outcome of ischemic brain injury following ischemic insults. Thus, the level of CBF in naïve WT and IRG1 −/− mice was measured and compared. Our results showed no signicant differences in the reading of CBF between WT and IRG1 −/− mice (Fig. S1). In addition, the blood-brain barrier (BBB) integrity of IRG1 −/− mice was assessed and compared with that of WT controls. We observed WT and IRG1 −/− mice exhibited a similar level of BBB integrity, as there was no increased Evans blue leakage detected in the brain of IRG1 −/− mice compared to that of WT controls (Fig. S2). Collectively, our results demonstrate that the CBF and BBB integrity are comparable between WT and IRG1 −/− mice, suggesting that IRG1 deciency does not alter the cerebrovasculature in IRG1 −/− mice. With that, we then assessed and compared the severity of ischemic brain injury between WT and IRG1 −/− MCAO mice. Although the physiological parameters of male WT and IRG1 −/− mice that IRG1 deciency-induced HO-1 reduction may be responsible for the repressed BDNF expression in the ischemic brain of IRG1 −/− stroke animals. To test our hypothesis, we thought to enhance BDNF expression in IRG1 −/− stroke animals by administration of D3T, an Nrf2/HO-1 pathway inducer. Indeed, D3T treatment enhanced BDNF expression in the ischemic brain of IRG1 −/− stroke animals. Importantly, D3T treatment also lessened ischemic brain injury in IRG1 −/− stroke animals. Thus, our results demonstrate that IRG1 deciency-induced BDNF repression and ischemic brain injury exacerbation can be rescued by D3T, and that may be mediated through the effect of D3T on the induction of HO-1 expression in the ischemic brain.


Introduction
Stroke is a leading cause of disability in the U.S. and worldwide. More than 80% of stroke cases belong to ischemic stroke, in which the occlusion of cerebral blood vessels initiates the acute phase of cerebral injury followed by excitotoxicity and oxidative damage (1,2). Tissue plasminogen activator is an FDAapproved therapy for ischemic stroke, and it functions to dissolve the clots that allows reestablishment of cerebral blood ow. However, reperfusion induces reactive oxygen species (ROS) generation and recruits peripheral in ammatory immune cells into the ischemic brain, leading to enhanced neuroin ammation and ischemic brain injury exacerbation (3,4).
In ammatory stimuli induce immunoresponsive gene 1 (IRG1) expression (5)(6)(7). IRG1 has been shown to catalyze the production of itaconate through diverting cis-aconitate away from the tricarboxylic acid cycle (TCA) cycle (8). The immunoregulatory effect of itaconate was recently documented in macrophages (MΦ). For instance, the study showed that itaconate promoted Nrf2 activation, a key player in the antioxidant defense, in mouse and human MΦ stimulated with LPS (6,7). In addition, IRG1 −/− MΦ activated with LPS exhibited augmented in ammatory responses compared to wild type MΦ stimulated with LPS (5). Furthermore, dimethyl itaconate (DMI), an itaconate derivative, was reported to inhibit IL- 17-induced IκBς activation in keratinocytes and ameliorate disease severity in the imiquimod-induced psoriasis animal model (9). Moreover, DMI was shown to offer a protection against myocardial and cerebral ischemic/reperfusion injury in animal models (10,11). Finally, DMI was recently reported by our group to suppress neuroin ammation and ameliorate disease severity in experimental autoimmune encephalomyelitis (EAE), an animal model of multiple sclerosis (12).
Currently, whether IRG1 exerts a protective effect against ischemic stroke remains unknown. Thus, we accessed whether IRG1 is induced in the ischemic brain following middle cerebral artery occlusion (MCAO), and investigated whether IRG1 exerts an immunomodulatory effect in ischemic brain injury. We then deciphered the molecular mechanisms underlying the protective effects of IRG1 in ischemic stroke.
In this study, we report for the rst time that the induction of IRG1 following ischemic stroke serves as an endogenous protective mechanism to alleviate neuroin ammation and restrain ischemic brain injury, and that may be mediated through the protective effect of IRG1 on the induction of HO-1 and BDNF expression in the ischemic brain. Thus, our ndings suggest that targeting IRG1 may represent a novel approach for the treatment of cerebral ischemia.

Material And Methods
Mice IRG1 −/− and its corresponding wild type (WT) control C57BL/6 mice were purchased from the Jackson Laboratory (Bar Harbor, ME). All animal experimental procedures were approved by the Purdue Animal Care and Use Committee (PACUC) and performed in strict compliance with National Institutes of Health Guidelines for the Care and Use of Laboratory Animals. All mice were housed and bred in the animal facility with controlled humidity, temperature, and 12h: 12 h light: dark cycle with free access to food and water.
Primary microglia (MG) and macrophage (Mϕ) culture Primary MG were generated from P1-P2 neonatal mice previously described (14). Brie y, cerebral cortical cells from P1-P2 neonatal mice were dissociated and plated in 75-cm 2 culture asks in DMEM/F12 supplemented with 10% FBS. The medium was removed and replenished with complete medium containing 10 ng/ml GM-CSF on day 4 and 8 after plating. MG were harvested by shaking the asks at 250 rpm at 37 °C for 30 minutes on day 12. MΦ were generated from bone marrow cells as previous described (15). Brie y, bone marrow cells were cultured in RPMI 1640 medium containing 10 ng/ml M-CSF. Cells were replenished with fresh media containing 10 ng/ml M-CSF at day 3 and collected for experiments at day 7.

Middle cerebral artery occlusion (MCAO) model
Adult male (3-4 months old) C57BL/6 and IRG1 −/− mice were used for cerebral ischemia experiments as previously described (16). The transient ischemic stroke was induced by an intraluminal suture occlusion model. Brie y, mouse was anesthetized by iso urane with a mixture of 70% compressed air and 30% oxygen. During the surgical procedure, the body temperature of the animal was maintained at ~ 37 °C. Cerebral blood ow (CBF) was measured by laser Doppler owmetry (Moor Instrument VMS-LDF2) to con rm the reduction of CBF following suture insertion. After 40 minutes occlusion, the intraluminal suture was removed to reestablish cerebral blow ow. Mice with a total reduction of CBF more than 80% were included in this study.

Infarct volume measurements
The infarct volume of the ischemic brain was determined by TTC staining. Brie y, the ischemic brain harvested from MCAO mice was subjected to 2-mm coronal slicing with a rodent brain matrix followed by 1% TTC staining. After staining, the brain sections were scanned and the infarct volume was calculated by ImageJ as previously described (16).

Isolation of mononuclear cells from mouse brain
Mice subjected to MCAO were anesthetized and perfused with PBS transcardially. After removing the meninges, olfactory bulb and cerebellum, the forebrains were homogenized with 1X HBSS buffer followed by ltration through a 70-µm nylon cell strainer. After centrifugation, cells were resuspended in 30% Percoll underlayering with 70% Percoll. Following centrifugation, the mononuclear cells were then isolated from the interface between 30% and 70% Percoll. The isolated cells were then stained with antibodies and analyzed by ow cytometer (BD FACSVerse).

Western Blot Analysis
Protein samples were prepared in RIPA buffer [50 mM Tris-HCl (pH 8.0), 150 mM NaCl, 1% NP-40, 0.5% sodium deoxycholate, 1 mM PMSF and 1X protease inhibitor cocktail] with 0.3% SDS (brain tissue) or 0.1% SDS (cultured cell). The protein concentrations were detected by PierceTM BCA Protein Assay Kit (Thermo Fisher Scienti c, Waltham, MA). Protein electrophoresis was conducted on 10% or 12% SDS-PAGE. After transferred to polyvinylidene di uoride membranes (Millipore Temecula, CA), the blots were reacted with speci c antibodies and detected by using Immobilon Western Chemiluminescent HRP Substrate (Millipore, Temecula, CA). Immunohistochemistry Brain samples were dissected and xed with 4% PFA in PBS at 4 °C overnight. After 6% and 30% sucrose dehydration, brain samples were embedded in OCT and cut into 40 µm cryosections. Cryosections were air-dried at room temperature (RT) and permeabilized in PBS containing 0.5% Triton X-100 for 30 minutes. Following blocking with goat serum (5% goat serum, 0.25% Triton X-100 in PBS) at RT for 1 hour, sections were incubated with primary HO-1 antibody at 4 °C overnight. After wash, sections were then stained with secondary antibody for 2 hours followed by Alexa Fluor 488 anti-Iba1 antibody staining for 2 hours at RT.
After wash, samples were coverslipped with ProLong Gold antifade mountant containing DAPI. The Zstack images of Iba1 and HO-1 co-localization were visualized by confocal microscopy (Fluoview FV10i, Olympus). The number of Iba1 + cells per mm 2 was counted and quanti ed by ImageJ.

Statistical analysis
All results are given as mean ± SEM. The normal distribution of the data in each group was con rmed by Shapiro-Wilk normality test. Comparisons between two groups were done by unpaired t test, whereas comparisons between multiple groups were done by one-way ANOVA followed by Bonferroni post hoc test. Statistical signi cance was determined as p < 0.05.

Results
Ischemic stroke induces IRG1 expression in the ischemic brain, however IRG1 de ciency exacerbates brain injury following ischemic insults To evaluate whether ischemic stroke induces IRG1 expression, C57BL/6 mice were subjected to MCAO, and the ischemic brains were harvested and subjected to Q-PCR analysis to assess IRG1 expression. Our results showed that ischemic stroke induced IRG1 expression in the ischemic brain in which IRG1 was highly upregulated in the ipsilateral but not contralateral hemisphere (Fig. 1A).
To further evaluate whether the induction of IRG1 following ischemic stroke plays a role in modulating ischemic brain injury, we thought to compare the level of ischemic brain injury between IRG1 −/− mice and its corresponding WT controls. To do that, we rst determined whether IRG1 de ciency may result in alteration of the cerebrovasculature in IRG1 −/− mice, as it would affect the outcome of ischemic brain injury following ischemic insults. Thus, the level of CBF in naïve WT and IRG1 −/− mice was measured and compared. Our results showed no signi cant differences in the reading of CBF between WT and IRG1 −/− mice (Fig. S1). In addition, the blood-brain barrier (BBB) integrity of IRG1 −/− mice was assessed and compared with that of WT controls. We observed WT and IRG1 −/− mice exhibited a similar level of BBB integrity, as there was no increased Evans blue leakage detected in the brain of IRG1 −/− mice compared to that of WT controls (Fig. S2). Collectively, our results demonstrate that the CBF and BBB integrity are comparable between WT and IRG1 −/− mice, suggesting that IRG1 de ciency does not alter the cerebrovasculature in IRG1 −/− mice. With that, we then assessed and compared the severity of ischemic brain injury between WT and IRG1 −/− MCAO mice. Although the physiological parameters of male WT and IRG1 −/− mice during MCAO were comparable (Table S1), WT and IRG1 −/− MCAO mice displayed a different pattern of brain injury following ischemic stroke. In WT MCAO mice, the infarct was mainly observed in the striatum of ischemic brain, however in IRG1 −/− MCAO mice, the infarct was further extended to the cortex of ischemic brain (Fig. 1B top). Further analysis of the ischemic brains revealed that IRG1 −/− MCAO mice exhibited a larger infarct volume than WT MCAO controls (IRG1 −/− 55.0 ± 6.5 vs. WT 26.9 ± 4.0) (Fig. 1B bottom). The similar results were observed in female MCAO mice in which female IRG1 −/− MCAO mice also developed a larger infarct than female WT MCAO controls (IRG1 −/− 61.5 ± 7.4 vs. WT 26.4 ± 4.1) (Fig. S3). In addition, the long-term survival rate of WT and IRG1 −/− MCAO mice was assessed. Our results showed that IRG1 −/− MCAO mice displayed a decreased survival rate compared to WT MCAO controls in which IRG1 −/− MCAO mice had a survival rate of 50%, whereas WT MCAO controls had a survival rate of 70% at day 7 post-injury (Fig. 1C). Collectively, our results demonstrate that ischemic stroke induces IRG1 expression in the ischemic brain, however IRG1 de ciency exacerbates brain injury following ischemic insults.

IRG1 de ciency enhances neuroin ammation in ischemic stroke
To determine whether IRG1 de ciency-exacerbated ischemic brain injury is due to enhanced neuroin ammation, the ischemic brains were harvested from WT and IRG1 −/− MCAO mice to assess MG activation. Mononuclear cells were isolated from the ischemic brains of WT and IRG1 −/− MCAO mice and then subjected to FACS analysis to assess the expression of surface maturation markers, CD80 and CD86, on MG. MG were determined based on their intermediate expression of CD45 (CD45 int ) and positive expression of CD11b (CD11b + ). Although the level of CD80-expressing CD45 int CD11b + cells in the ischemic brain was not altered between WT and IRG1 −/− MCAO mice (data not shown), the number of CD86-expressing CD45 int CD11b + cells in the ischemic brain was signi cantly increased in IRG1 −/− MCAO mice compared to WT MCAO controls ( Fig. 2A). In addition, ischemic brain tissues were harvested and subjected to IHC to assess the level of Iba1 + cells to con rm the observation of enhanced MG activation in IRG1 −/− MCAO mice. Notably, the number of Iba1 + cells in the ipsilateral cortex and striatum of IRG1 −/− MCAO mice was signi cantly higher than that in WT MCAO controls (Fig. 2B), con rming that IRG1 de ciency promotes MG activation in ischemic stroke.
As the peripheral immune cell in ltration of the ischemic brain has been reported to cause the secondary brain injury that in turn aggravates neuroin ammation, we therefore compared the level of peripheral immune cell in ltration in the ischemic brain of WT and IRG1 −/− MCAO mice. Mononuclear cells were isolated from the ischemic brains of WT and IRG1 −/− MCAO mice, and the CNS in ltrating immune cells were determined based on their high expression of CD45 (CD45 hi ) and positive expression of CD11b (CD11b + ). We did not observe increased CD45 hi CD11b + cells in the ipsilateral hemisphere compared to the contralateral hemisphere in WT MCAO mice at 24 h post-injury. However, a signi cant increase of CD45 hi CD11b + cells was observed in the ipsilateral hemisphere of IRG1 −/− MCAO mice when compared to the contralateral hemisphere of IRG1 −/− MCAO mice and the ipsilateral hemisphere of WT MCAO mice at 24 h post-injury (Fig. 2C), suggesting IRG1 de ciency promotes the immune cell in ltration of the CNS in ischemic stroke. Collectively, our results demonstrate that IRG1 de ciency promotes MG activation and immune cell in ltration that lead to enhanced neuroin ammation in ischemic stroke.
De ciency in IRG1 results in decreased cerebral HO-1 expression following ischemic stroke IRG1 induces the production of itaconate, and two itaconate derivatives, DMI and 4-octyl-itaconate, have been shown to induce Nrf2 activation and subsequent HO-1 upregulation (5,8,9). In addition, previous studies have demonstrated that the induction of HO-1 exerts a protective effect against ischemic brain injury (16,18,19). We therefore evaluated whether ischemic stroke induces HO-1 expression in the ischemic brain. Our results showed that HO-1 was highly upregulated in the ipsilateral but not contralateral hemisphere at both mRNA and protein levels in WT MCAO mice (Fig. 3A and B). To further investigate whether IRG1 de ciency affects cerebral HO-1 expression following ischemic stroke, we compared the level of HO-1 expression in the ischemic brain between WT and IRG1 −/− MCAO mice. We found that HO-1 expression was largely decreased in the ischemic brain of IRG1 −/− MCAO mice compared to that in WT MCAO controls (Fig. 3C). As MG were reported to be the main producers of HO-1 (20), we then assessed whether MG produce HO-1 in the ischemic brain. We observed the co-localization of HO-1 and Iba1 immunoreactivity in the cortex and striatum of WT and IRG1 −/− MCAO mice (Fig. 3D). In addition, we found that the level of HO-1 immunoreactivity was higher in the ischemic brain of WT MCAO mice than that of IRG1 −/− MCAO mice, and that is consistent with our results of western blot analysis ( Fig. 3C and D). Altogether, our results demonstrate that ischemic stroke induces HO-1 expression, and de ciency in IRG1 results in decreased HO-1 expression in the ischemic brain.
MG and MΦ de cient in IRG1 exhibit reduced HO-1 expression following LPS challenge To con rm our in vivo ndings that IRG1 de ciency decreases cerebral HO-1 expression following ischemic stroke, MG and MΦ were generated from WT and IRG1 −/− mice and then stimulated with LPS to assess IRG1 and HO-1 expression. At the mRNA level, IRG1 was highly upregulated at 5 h and declined at 8 h after LPS stimulation in both WT MG and MΦ (Fig. 4A). At the protein level, IRG1 was slightly induced at 8 h and strongly upregulated at 16 h after LPS stimulation in both MG and MΦ (Fig. 4B left panel). LPS treatment, the level of HO-1 expression was signi cantly lower in IRG1 −/− MΦ compared to that in WT MΦ (Fig. 4D). Collectively, our results demonstrate that IRG1 induces HO-1 expression, and de ciency in IRG1 results in reduced HO-1 expression in MG and MΦ following in ammatory stimuli. IRG1 de ciency represses BDNF expression in the ischemic brain, however IRG1 de ciency-induced BDNF repression and ischemic brain injury exacerbation can be rescued by D3T HO-1 induction has been shown to attenuate ischemic stroke induced-hippocampal neuronal injury via the activation of BDNF-TrkB-PI3K/Akt signaling pathway (21). Our observation of decreased HO-1 expression in the ischemic brain of IRG1 −/− MCAO mice prompted us to investigate whether IRG1 de ciency may lead to repressed BDNF expression in the ischemic brain. Indeed, we found that BDNF expression at both mRNA and protein levels were repressed in the ischemic brain of IRG1 −/− MCAO mice compared to that in WT MCAO controls ( Fig. 5A and C). To further evaluate whether IRG1 de ciencyinduced BDNF repression can be rescued by the induction of HO-1 expression, IRG1 −/− MCAO mice were administered with either vehicle or D3T, an Nrf2/HO-1 pathway inducer (16,(22)(23)(24), and the ischemic brains were then harvested to assess BDNF expression. D3T strongly upregulated BDNF expression in the ischemic brain of IRG1 −/− MCAO mice at both mRNA and protein levels ( Fig. 5B and C). Strikingly, IRG1 de ciency-exacerbated ischemic brain injury was lessened by D3T treatment in IRG1 −/− MCAO mice.
Although the physiological parameters of vehicle-and D3T-treated IRG1 −/− stroke animlas during MCAO were comparable (Table S2), the infarct volume of D3T-treated IRG1 −/− MCAO mice was signi cantly lower than that of vehicle-treated IRG1 −/− MCAO mice (D3T 24.9 ± 2.7 vs. Vehicle 55.6 ± 7.1) (Fig. 5D). Altogether, our results demonstrate that IRG1 de ciency represses BDNF expression in the ischemic brain following ischemic stroke, and D3T, an Nrf2/HO-1 pathway inducer, is able to enhance BDNF expression and lessen ischemic brain injury in IRG1 −/− stroke animals. Discussion IRG1, a mitochondrial enzyme, mediates the production of itaconate during in ammatory responses in myeloid lineage cells (25). Recent studies revealed the unique function of IRG1/itaconate axis in modulating in ammation and further demonstrated the anti-in ammatory effects of itaconate on the suppression of succinate dehydrogenase (SDH) activities, activation of anti-oxidant and antiin ammatory Nrf2/HO-1 pathways, and amelioration of disease severity in the psoriasis animal model (8,9,19). Presently, the immunomodulatory effects of IRG1/itaconate axis were mainly evaluated in the peripheral in ammatory immune responses. Although we have recently reported that DMI, an itaconate derivative, suppressed neuroin ammation and ameliorated disease severity in the chronic CNS disease, EAE (12), whether IRG1/itaconate axis modulates the acute CNS disease, ischemic stroke, remains unexplored. Hence, in this study we investigated the potential immunomodulatory effects of IRG1 on the alleviation of ischemic brain injury and elucidated the molecular mechanisms underlying the protective effect of IRG1 in ischemic stroke. We observed that IRG1 was highly induced in the injured brain following ischemic insults. Importantly, we found that IRG1 −/− stroke animals developed a much severe brain injury compared to WT stroke controls. Thus, we identify a novel function of IRG1 in the acute CNS disease and demonstrate that the induction of IRG1 following ischemic stroke may serve as an endogenous protective mechanism to restrain ischemic brain injury.
Neuroin ammation plays a pivotal role in brain injury following ischemic stroke. MG activation and peripheral immune cell in ltration have been shown to contribute to the induction and aggravation of neuroin ammation in ischemic stroke (14,16). Since we observed exacerbated ischemic brain injury in IRG1 −/− stroke animals, we speculate that IRG1 de ciency may promote neuroin ammation in ischemic stroke. Indeed, we observed increased CD86-expressing MG as well as elevated Iba1 + cells in the ischemic brain of IRG −/− stroke animals, indicating IRG1 de ciency enhances MG activation. In addition, we found that increased peripheral immune cells in ltrate into the ischemic brain of IRG1 −/− stroke animals when compared to that of WT stroke controls. Collectively, our ndings suggest that one of the protective mechanisms of IRG1 in ischemic stroke may be mediated through its's immunomodulatory effect on the suppression of MG activation and inhibition of peripheral immune cell in ltration.
Previous studies demonstrate that the induction of HO-1 exerts a protective effect against ischemic stroke (16,(26)(27)(28)(29). In addition, a study shows that HO-1 plays the essential role in ischemic preconditioning (IPC)-induced protection against brain ischemia, as IPC fails to protect HO-1 de cient mice against permanent ischemic brain injury (30). In this study, we found that both IRG1 and HO-1 were highly induced in the ipsilateral but not the contralateral hemisphere of WT stroke animals. Most importantly, we observed HO-1 expression was largely decreased in the ischemic brain of IRG1 −/− stroke animals compared to that of WT stroke controls. These observations correlate with our results showing IRG1 −/− MCAO mice developed a much severe brain injury than WT MCAO controls, and further con rm the protective effect of HO-1 in ischemic stroke. In addition, to verify the essential role of IRG1 in the induction of endogenous HO-1 expression, primary MG and MΦ were generated from WT and IRG1 −/− mice to determine HO-1 expression. We found IRG1 −/− MG stimulated with LPS expressed a very lower level of HO-1 compared to WT MG stimulate with LPS, and the similar results were also observed in MΦ.
Thus, our results demonstrate that IRG1 is required for endogenous HO-1 expression following in ammatory stimuli in vitro and in vivo, and suggest that the induction of IRG1 following ischemic stroke promotes HO-1 expression to restrain ischemic brain injury.
BDNF participates in neuronal plasticity and promotes neuronal survival and growth (31). Interestingly, we found that following ischemic stroke the cerebral BDNF expression at both mRNA and protein levels were signi cantly lower in IRG1 −/− stroke animals compared to WT stroke controls. As HO-1 has been shown to promote BDNF expression (21), we speculate that IRG1 de ciency-induced HO-1 reduction may be responsible for the repressed BDNF expression in the ischemic brain of IRG1 −/− stroke animals. To test our hypothesis, we thought to enhance BDNF expression in IRG1 −/− stroke animals by administration of D3T, an Nrf2/HO-1 pathway inducer. Indeed, D3T treatment enhanced BDNF expression in the ischemic brain of IRG1 −/− stroke animals. Importantly, D3T treatment also lessened ischemic brain injury in IRG1 −/− stroke animals. Thus, our results demonstrate that IRG1 de ciency-induced BDNF repression and ischemic brain injury exacerbation can be rescued by D3T, and that may be mediated through the effect of D3T on the induction of HO-1 expression in the ischemic brain.
During respiration with normal oxygen consumption, SDH functions to break down succinate in the TCA cycle. During ischemia, SDH works reversely that leads to succinate accumulation. Indeed, a high level of succinate can be detected in the ischemic tissues of heart, live, kidney and brain (32). The accumulated succinate can then be rapidly oxidized following reperfusion that results in increased mitochondrial ROS production. Notably, the inhibition of SDH activity was reported to offer a protection against ischemic injury (32,33). As IRG1/itaconate axis has been shown to suppress SDH activity (8,34), further studies would be required to investigate whether IRG1 de ciency-exacerbated ischemic brain injury may be partly due to elevated succinate accumulation and increased ROS production in the ischemic brain. In addition, as IRG1 has been shown to induce A20 expression to elicit anti-in ammatory effects (6,35,36), it would be important to further investigate whether IRG1-inudced A20 expression plays a role in modulating ischemic brain injury.

Conclusions
In this study, we report for the rst time that the induction of IRG1 in the ischemic brain following ischemic stroke represents an endogenous protective mechanism to restrain ischemic brain injury. We identify that IRG1 de ciency exacerbates ischemic brain injury exhibited with elevated MG activation and increased peripheral immune cell in ltration in the ischemic brain. Most importantly, we demonstrate that IRG1 is required for optimal cerebral HO-1 and BDNF expression following ischemic stroke, as the expression of cerebral HO-1 and BDNF was repressed in IRG1 −/− stroke animals compared to that in WT stroke controls. We further demonstrate that the repressed expression of cerebral HO-1 and BDNF may be responsible for exacerbated ischemic brain injury in IRG1 −/− stroke animals, as the administration of D3T, an Nrf2/HO-1 pathway inducer, induces cerebral BDNF expression and lessens ischemic brain injury in IRG1 −/− stroke animals (Fig. 6). In summary, our study reveals a novel function of IRG1 in modulating ischemic brain injury through the induction of cerebral HO-1 and BDNF expression following ischemic insults, ant that suggests targeting IRG1 and/or IRG1-mediated singling pathways may represent a novel strategy for the treatment of ischemic stroke.  Ischemic stroke induces IRG1 expression in the ischemic brain, however IRG1 de ciency exacerbates brain injury following ischemic insults. (A) C57BL/6 mice were subjected to 40 minutes MCAO (n=8), and the ischemic brains were harvested at 6 hours post-reperfusion. The contralateral (cont.) and ipsilateral (ipsi.) hemisphere of the ischemic brains were separated and subjected to Q-PCR analysis for Irg1 expression. **p<0.01 by unpaired t test. (B) WT and IRG1-/-mice were subjected to MCAO (n=10/group). 24 hours after injury, mice were sacri ced, and ischemic brains were harvested and sliced (2 mm) followed by TTC staining. Three representative TTC-stained samples of WT and IRG1-/-MCAO mice were shown. The infarct volumes were also measured. **p<0.01 by unpaired t test. (C) The Kaplan-Meier survival curve of WT and IRG1-/-MCAO mice (n=10/group) was evaluated up to day 7 post-injury.   MG and MΦ de cient in IRG1 exhibit reduced HO-1 expression following LPS challenge. (A) Primary MG and MΦ were stimulated with LPS 100 ng/ml for 5 hours and 8 hours followed by Q-PCR analysis for IRG1 expression. ***p<0.001 by one way ANOVA. (B) Primary MG and MΦ generated from WT or IRG1-/mice were stimulated with LPS 100 ng/ml for a time course followed by western blot analysis for IRG1 expression. (C) Primary MG and (D) MΦ generated from WT or IRG1-/-mice were treated with LPS 100 ng/ml for a time course and then subjected to Q-PCR analysis for HO-1 expression. The representative results of 3-4 independent experiments were shown. ***p<0.001 by one way ANOVA. D3T-treated IRG1-/-MCAO mice was also measured (n=10/group). **p<0.01 by unpaired t test.