IRG1, a mitochondrial enzyme, mediates the production of itaconate during inflammatory responses in myeloid lineage cells (25). Recent studies revealed the unique function of IRG1/itaconate axis in modulating inflammation and further demonstrated the anti-inflammatory effects of itaconate on the suppression of succinate dehydrogenase (SDH) activities, activation of anti-oxidant and anti-inflammatory 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 inflammatory immune responses. Although we have recently reported that DMI, an itaconate derivative, suppressed neuroinflammation 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.
Neuroinflammation plays a pivotal role in brain injury following ischemic stroke. MG activation and peripheral immune cell infiltration have been shown to contribute to the induction and aggravation of neuroinflammation in ischemic stroke (14, 16). Since we observed exacerbated ischemic brain injury in IRG1−/− stroke animals, we speculate that IRG1 deficiency may promote neuroinflammation 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 deficiency enhances MG activation. In addition, we found that increased peripheral immune cells infiltrate into the ischemic brain of IRG1−/− stroke animals when compared to that of WT stroke controls. Collectively, our findings 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 infiltration.
Previous studies demonstrate that the induction of HO-1 exerts a protective effect against ischemic stroke (16, 26–29). 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 deficient 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 confirm 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 inflammatory 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 significantly 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 deficiency-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 deficiency-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 deficiency-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-inflammatory effects (6, 35, 36), it would be important to further investigate whether IRG1-inudced A20 expression plays a role in modulating ischemic brain injury.