Abstract 20: Role of IRF5-IRF4 Regulatory Axis in Microglial Polarization After Neonatal Stroke

Abstract

Background: Perinatal arterial ischemic stroke (PAIS) is an important cause of neonatal death and long-term disability. Once PAIS occurs, microglia are activated and innate immune responses are mounted in the brain. Microglial activation is characterized as M1 (pro-inflammatory) or M2 (anti-inflammatory) phenotype depending on the progression of brain injury. Recent studies with adult models of peripheral inflammation have found that interferon regulatory factor 5 (IRF5) regulates expression of pro-inflammatory cytokines, while IRF4 controls M2 macrophage polarization. The balance of the IRF5-IRF4 regulatory axis is critical in establishing M1/M2 macrophage phenotypes; however, it is unknown if microglial activation is regulated by the same mechanism in neonatal brains after PAIS. We hypothesize that IRF5-IRF4 regulatory axis directs M1/M2 microglial polarization and manipulation of the axis confers neuroprotection against PAIS. Methods: IRF5/IRF4 inducible conditional knockout (ICKO) mice in which IRF5 or IRF4 was deleted specifically in microglia by Tamoxifen induction were subjected to 60-minute Rice-Vanucci Modeling (RVM) at post-natal day 10 (P10) to induce PAIS. Stroke outcomes were evaluated at 7d of RVM. C57BL6 P10 pups will also be used for examination of microglial activation and IRF5/IRF4 expression by flow cytometry (FC), RT-PCR, IHC, and western blotting. Results: Microglia exhibited high levels of M2 marker CD206 at 3d and M1 marker MHCII at 7d of PAIS by FC analysis (n= 6st, 3sh/gp). IRF4 mRNA in FC-sorted microglia was upregulated 24h after stroke and peaked at 3d, but declined to baseline at 7d. Conversely, IRF5 was not upregulated until 3d and peaked at 7d. Correspondingly, IRF4 and IRF5 expression in the nuclear fraction was increased at 3d and 7d respectively. Similar patterns of IRF5/IRF4 expression was seen in Iba-1 + cells in the brain by IHC (n= 8st, 4sh/gp). IRF4 ICKO mice had worsened stroke outcomes while IRF5 ICKO showed protective effects 7d after PAIS. Conclusions: PAIS led to a transient M2 phenotype of microglia that switched to M1 polarization at a later time. IRF5-IRF4 regulatory axis plays an important role in mediating M1/M2 polarization of microglia and is a promising target of interventional therapy for PAIS.