Microglial activation plays a central role in poststroke inflammation and causes secondary neuronal damage; however, it also contributes in debris clearance and chronic recovery. Microglial pro- and antiinflammatory responses (or so-called M1-M2 phenotypes) coexist and antagonize each other throughout the disease progress. As a result of this balance, poststroke immune responses alter stroke outcomes. Our previous study found microglial expression of interferon regulatory factor 5 (IRF5) and IRF4 was related to pro- and antiinflammatory responses, respectively. In the present study, we genetically modified the IRF5 and IRF4 signaling to explore their roles in stroke. Both in vitro and in vivo assays were utilized; IRF5 or IRF4 small interfering RNA (siRNA), lentivirus, and conditional knockout (CKO) techniques were employed to modulate IRF5 or IRF4 expression in microglia. We used a transient middle cerebral artery occlusion model to induce stroke and examined both acute and chronic stroke outcomes. Poststroke inflammation was evaluated with flow cytometry, RT-PCR, MultiPlex, and immunofluorescence staining. An oscillating pattern of the IRF5-IRF4 regulatory axis function was revealed. Down-regulation of IRF5 signaling by siRNA or CKO resulted in increased IRF4 expression, enhanced M2 activation, quenched proinflammatory responses, and improved stroke outcomes, whereas down-regulation of IRF4 led to increased IRF5 expression, enhanced M1 activation, exacerbated proinflammatory responses, and worse functional recovery. Up-regulation of IRF4 or IRF5 by lentivirus induced similar results. We conclude that the IRF5-IRF4 regulatory axis is a key determinant in microglial activation. The IRF5-IRF4 regulatory axis is a potential therapeutic target for neuroinflammation and ischemic stroke.

Microglial activation is a key element in initiating and perpetuating inflammatory responses to stroke. Interferon regulatory factor 5 (IRF5) and IRF4 signaling have been found critical in mediating macrophage pro-inflammatory (M1) and anti-inflammatory (M2) phenotypes respectively in peripheral inflammation. We hypothesize that the IRF5/4 regulatory axis also mediates microglial activation after stroke. C57BL6 mice of 8–12 weeks were subject to a 90-minute middle cerebral artery occlusion and the brains evaluated at 24h, 3d, 10d and 30d after reperfusion. Flow cytometry was utilized to examine microglial activation and cytokine expression. RT-PCR was performed for mRNA levels of IRF5/4 in sorted microglia. Microglial expression of IRF5/4 was examined by immunohistochemistry, and brain cytokine levels were determined by ELISA. Our results revealed that the IRF5 mRNA level in sorted microglia increased at 3d of stroke; whereas IRF4 mRNA level exhibited bi-phasic increases, with a transient rise at 24h and a peak at 10d. The same pattern was seen in IRF5/4 protein colocalization with Iba-1+ cells by IHC. Intracellular levels of TNFα and IL-1β in microglia peaked at 3d of stroke, and IL-4+IL-10+ double positive microglia significantly increased at day 10. Brain levels of these cytokines were consistent with microglial cytokine changes. Worse behavior test results were seen at 3d vs. 10d of stroke. We conclude that microglia phenotypes are dynamic to ischemic stroke and IRF5/4 signaling may regulate microglial M1/M2 activation and impact on stroke outcomes.

Neonatal hypoxic–ischemic encephalopathy (HIE) is increasingly recognized as a sexually dimorphic disease. Male infants are not only more vulnerable to ischemic insult; they also suffer more long-term cognitive deficits compared with females with comparable brain damage. The innate immune response plays a fundamental role in mediating acute neonatal HIE injury. However, the mechanism underlying the sex difference in chronic HIE is still elusive. The present study investigated the sex difference in HIE outcomes and inflammatory response in the chronic stage (30 days after HIE). Postnatal day 10 (P10) male and female C57BL/6 pups were subjected to 60-min Rice–Vanucci model (RVM) to induce HIE. Brain atrophy and behavioral deficits were analyzed to measure stroke outcomes at 30 days of HIE. Flow cytometry (FC) was performed to examine central (microglial activation) and peripheral immune responses. Serum levels of cytokines and sex hormones were determined by enzyme-linked immunosorbent assay (ELISA). Neurogenesis was quantified by 5-Bromo-2′-deoxyuridine (BrdU) incorporation with neurons. Results showed males had worse HIE outcomes than females at the endpoint. Female microglia exhibited a more robust anti-inflammatory response that was corresponding to an enhanced expression of CX3C chemokine receptor 1 (CX3CR1) than males. More infiltration of peripheral lymphocytes was seen in male vs. female HIE brains. Cytokine levels of tumor necrosis factor (TNF)-α and interleukin (IL)-10 were more upregulated in males and females respectively than their counterparts. Neurogenesis was more highly induced in females vs. males. No significant difference in circulating hormonal level was found between males and females after HIE. We conclude that a sex dichotomy in pro- and anti-inflammatory response underlies the sex-specific chronic HIE outcomes, and an enhanced neurogenesis in females also contribute to the sex difference.