cells Article Phosphorylation of Microglial IRF5 and IRF4 by IRAK4 Regulates Inflammatory Responses to Ischemia Conelius Ngwa, Abdullah Al Mamun, Yan Xu, Romana Sharmeen and Fudong Liu * Department of Neurology, McGovern Medical School, The University of Texas Health Science Center at Houston, Houston, TX 77030, USA; [email protected] (C.N.); [email protected] (A.A.M.); [email protected] (Y.X.); [email protected] (R.S.) * Correspondence: [email protected]; Tel.: +1-713-500-7038; Fax: +1-713-500-0660 Abstract: Background: Interferon Regulatory Factor (IRF) 5 and 4 play a determinant role in regu- lating microglial pro- and anti-inflammatory responses to cerebral ischemia. How microglial IRF5 and IRF4 signaling are activated has been elusive. We hypothesized that interleukin-1 receptor associated kinase 4 (IRAK4) phosphorylates and activates IRF5 and IRF4 in ischemic microglia. We aimed to explore the upstream signals of the two IRFs, and to determine how the IRAK4-IRF signaling regulates the expression of inflammatory mediators, and impacts neuropathology. Meth- ods: Spontaneously Immortalized Murine (SIM)-A9 microglial cell line, primary microglia and neurons from C57BL/6 WT mice were cultured and exposed to oxygen-glucose deprivation (OGD), followed by stimulation with LPS or IL-4. An IRAK4 inhibitor (ND2158) was used to examine IRAK40s effects on the phosphorylation of IRF5/IRF4 and the impacts on neuronal morphology by co-immunoprecipitation (Co-IP)/Western blot, ELISA, and immunofluorescence assays. Results: We confirmed that IRAK4 formed a Myddosome with MyD88/IRF5/IRF4, and phosphorylated both IRFs, which subsequently translocated into the nucleus. Inhibition of IRAK4 phosphorylation quenched microglial pro-inflammatory response primarily, and increased neuronal viability and Citation: Ngwa, C.; Mamun, A.A.; neurite lengths after ischemia. Conclusions: IRAK4 signaling is critical for microglial inflammatory Xu, Y.; Sharmeen, R.; Liu, F. responses and a potential therapeutic target for neuroinflammatory diseases including cerebral Phosphorylation of Microglial IRF5 ischemia. and IRF4 by IRAK4 Regulates Inflammatory Responses to Ischemia. Keywords: inflammation; IRAK4; IRF5; IRF4; ischemia; microglia Cells 2021, 10, 276. https://doi.org/ 10.3390/cells10020276 Academic Editor: Yasu-Taka Azuma 1. Introduction Received: 11 December 2020 Immune responses are a fundamental pathophysiological procedure in cerebral is- Accepted: 27 January 2021 Published: 30 January 2021 chemia [1], and microglial activation plays a central role in initiating and perpetuating the inflammatory response [2]. After cerebral ischemia, microglia are activated producing Publisher’s Note: MDPI stays neutral and releasing a plethora of cytokines, chemokines, cytotoxic mediators and trophic fac- with regard to jurisdictional claims tors, which consequently mediate pro- and anti-inflammatory responses [3–7]. Microglia in published maps and institutional with the pro-inflammatory phenotype are associated with detrimental outcomes [8,9]. In affiliations. contrast, microglia of the anti-inflammatory phenotype promote tissue repair and confer neuroprotective effects [10–13]. Over-activation of microglia exacerbates cerebral ischemic injury [11,14,15]; therefore, it has high translational value to modulate microglial activation via inhibiting or skewing the pro-inflammatory to the anti-inflammatory phenotype [11,12]. Our previous in vivo studies using genetic manipulations in animals have found inter- Copyright: © 2021 by the authors. Licensee MDPI, Basel, Switzerland. feron regulatory factor 5 (IRF5) and 4 (IRF4) play critical roles in mediating microglial pro- This article is an open access article and anti-inflammatory response respectively after stroke. However, through which path- distributed under the terms and way IRF5/IRF4 mediate the microglial response remains elusive. Systemic inflammation conditions of the Creative Commons studies on macrophage activation have suggested interleukin-1 receptor-associated kinase Attribution (CC BY) license (https:// 4 (IRAK-4) and adaptor protein MyD88 are crucial in phosphorylation of IRFs [14–20]. We creativecommons.org/licenses/by/ hypothesize that the IRAK4-IRF signaling phosphorylates the two IRFs in microglia, and 4.0/). facilitates IRFs’ translocation from the cytoplasm to the nucleus to regulate gene expression Cells 2021, 10, 276. https://doi.org/10.3390/cells10020276 https://www.mdpi.com/journal/cells Cells 2021, 10, x FOR PEER REVIEW 2 of 16 tor-associated kinase 4 (IRAK-4) and adaptor protein MyD88 are crucial in phosphoryla- Cells 2021, 10, 276 tion of IRFs [14–20]. We hypothesize that the IRAK4-IRF signaling phosphorylates2 ofthe 16 two IRFs in microglia, and facilitates IRFs’ translocation from the cytoplasm to the nu- cleus to regulate gene expression of inflammatory mediators. In this study, we specifi- callyof inflammatory performed a mediators. series of in Invitro this assays study, in we microglia specifically culture performed exposed ato series oxygen of glucosein vitro deprivatioassays in microglian (OGD), culturean in vitro exposed ischemia to oxygen model, glucoseto elucidate deprivation the molecular (OGD), mechanis an in vitrom. Theischemia interaction model, toof elucidateIRF5/IRF4 the with molecular MyD88 mechanism. and IRAK4 The was interaction examined. of IRF5/IRF4 The effects with of IRAK4MyD88 inhibition and IRAK4 on was the examined. activation Theof the effects two ofIRFs IRAK4 and inhibitionthe impacts on on the ischemic activation neurons of the weretwo IRFs determined and the. impacts on ischemic neurons were determined. 2. Materials and Methods 2.1. Spontaneously Immortalized Murine Micro Microgliaglia Culture Spontaneously immortalized murine microglia, SIM-A9_CRL-3265SIM-A9_CRL-3265 (American(American Type Culture Collection, ATCC ATCC,, Manassas, VA VA,, USA USA)) cells were cultured as previously de- scribed [21] [21].. Briefly Briefly cells were cultured in DMEM/F DMEM/F-12-12 medium_DFL15 medium_DFL15 (Caisson (Caisson Labor- Labo- atories,ratories, Smithfield, Smithfield, UT UT,, USA USA)) containing containing 10% 10% FBS, 5% HS, andand 1%1% P/S.P/S. Next, cells werewere gently shaken and detached from the culture vessel with phosph phosphateate buffered saline (PBS) containing 1 mM EDTA, 1 mM EGTA and 1 mg/mL D-glucose. The detached cells were containing 1 mM EDTA, 1 mM EGTA and 1 mg/mL D-glucose. The detached cells were counted with a hemocytometer, and rested for at least 24 h in a Forma Steri-Cycle CO2 counted with a hemocytometer, and rested for at least 24◦ h in a Forma Steri-Cycle CO2 incubator (Thermo Scientific, Waltham, MA, USA) at 37 C, 95% humidity and 5% CO2, incubator (Thermo Scientific, Waltham, MA, USA) at 37 °C, 95% humidity and 5% CO2, before treatments. The cell line can yield more than 5 million cells after culture and was before treatments. The cell line can yield more than 5 million cells after culture and was used for western blotting experiments (Figures1–3). used for western blotting experiments (Figures 1–3). Figure 1. IRF5 and IRF4 bindFigure to 1. MyD88 IRF5 and or IRAK4.IRF4 bind (A to, MyD88B) SIM-A9 or IRAK4. cell homogenates (A, B) SIM-A9 were cell subjectedhomogenates to Co-IP were withsubjected to Co-IP with anti-IRAK4 antibody followed by immunoblotting for p-IRF5/p-IRF4, t-IRF5/t-IRF4, anti-IRAK4 antibody followed by immunoblotting for p-IRF5/p-IRF4, t-IRF5/t-IRF4, and MyD88. (C, D) WB optical and MyD88. (C, D) WB optical density quantification of the ratio of p-IRF5 over t-IRF5 (C) and density quantification of the ratio of p-IRF5 over t-IRF5 (C) and p-IRF4 over t-IRF4 (D). (E, F) SIM-A9 cell homogenates p-IRF4 over t-IRF4 (D). (E, F) SIM-A9 cell homogenates were subjected to Co-IP with anti-MyD88 were subjected to Co-IP withantibody anti-MyD88 to detect antibody p-IRF5/p to detect-IRF4p-IRF5/p-IRF4 and t-IRF5/t-IRF4. and ( t-IRF5/t-IRF4.G, H) WB optical (G ,densityH) WB quantification optical density of the quantification of the ratio of p-IRF5ratio of over p-IRF5 t-IRF5 over (G t)- andIRF5 p-IRF4 (G) and over p-IRF4 t-IRF4 over (H). t- IgGIRF4 controls H). IgG were controls from were the same from homogenates the same ho- of each treatment. n = 3 independentmogenates of experiments/per each treatment. n condition. = 3 independent IB, immunoblot; experiments/per p, phosphorylated; condition. IB, immunoblot; t, total; IgG, p, immunoglobulin negative control.phosphorylated; One-way ANOVA.t, total; IgG, immunoglobulin negative control. One-way ANOVA. Cells 2021, 10, 276 3 of 16 CellsCells 22021021,, 1010,, xx FORFOR PEERPEER REVIEWREVIEW 33 ofof 1616 Figure 2. IRAK4 phosphorylates IRF5/IRF4. (A) SIM-A9 cells were treated with 8 μM ND2158, Figure 2. IRAK4Figure phosphorylates 2. IRAK4 phosphorylates IRF5/IRF4. (A IRF5/IRF4.) SIM-A9 cells(A) SIM were-A9 treated cells were with treated 8 µM ND2158, with 8 μM chosen ND2158, by MTS gradient chosen by MTS gradient viability assay. (B) After treatments, cell homogenates were immunob- viability assay.chosen (B) After by MTS treatments, gradient cellviability homogenates assay. (B) wereAfter immunoblotted.treatments, cell homogenates ImageJ quantified were imm ratiosunob- of p-IRAK4/t- lotted.lotted. ImageJImageJ quantifiedquantified ratiosratios ofof pp--IRAK4/tIRAK4/t--IRAK4IRAK4 ((CC),), pp--IRF5/tIRF5/t--IRF5IRF5 ((DD)),, andand pp--IRF4/tIRF4/t--IRF4IRF4 ((EE)) IRAK4 (C), p-IRF5/t-IRF5 (D), and p-IRF4/t-IRF4 (E) in (B). Data were obtained from 3 independent experiments.