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. 2022 Aug 12;14(15):6047-6065.
doi: 10.18632/aging.204216. Epub 2022 Aug 12.

Regulation of microglial activation in stroke in aged mice: a translational study

Conelius Ngwa  1 , Abdullah Al Mamun  1 , Shaohua Qi  1 , Romana Sharmeen  1 , Yan Xu  1 , Fudong Liu  1
Affiliations

Regulation of microglial activation in stroke in aged mice: a translational study

Conelius Ngwa et al. Aging (Albany NY). .

Abstract

Numerous neurochemical changes occur with aging and stroke mainly affects the elderly. Our previous study has found interferon regulatory factor 5 (IRF5) and 4 (IRF4) regulate neuroinflammation in young stroke mice. However, whether the IRF5-IRF4 regulatory axis has the same effect in aged brains is not known. In this study, aged (18-20-month-old), microglial IRF5 or IRF4 conditional knockout (CKO) mice were subjected to a 60-min middle cerebral artery occlusion (MCAO). Stroke outcomes were quantified at 3d after MCAO. Flow cytometry and ELISA were performed to evaluate microglial activation and immune responses. We found aged microglia express higher levels of IRF5 and lower levels of IRF4 than young microglia after stroke. IRF5 CKO aged mice had improved stroke outcomes; whereas worse outcomes were seen in IRF4 CKO vs. their flox controls. IRF5 CKO aged microglia had significantly lower levels of IL-1β and CD68 than controls; whereas significantly higher levels of IL-1β and TNF-α were seen in IRF4 CKO vs. control microglia. Plasma levels of TNF-α and MIP-1α were decreased in IRF5 CKO vs. flox aged mice, and IL-1β/IL-6 levels were increased in IRF4 CKO vs. controls. The anti-inflammatory cytokines (IL-4/IL-10) levels were higher in IRF5 CKO, and lower in IRF4 CKO aged mice vs. their flox controls. IRF5 and IRF4 signaling drives microglial pro- and anti-inflammatory response respectively; microglial IRF5 is detrimental and IRF4 beneficial for aged mice in stroke. IRF5-IRF4 axis is a promising target for developing new, effective therapeutic strategies for the cerebral ischemia.

Keywords: IRF; aging; inflammation; microglia; stroke.

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Conflict of interest statement

CONFLICTS OF INTEREST: The authors declare that they have no conflicts of interest.

Figures

Figure 1
Figure 1
IRF5 and IRF4 expression levels in microglia in young and aged C57BL/6 mice. (A, B) Representative flow plots of microglial IRF5 (A) and the mean fluorescence intensity (MFI) (B). (C, D) Representative flow plots of microglial IRF4 (C) and the MFI) (D). n = 4-5 /group sham and 6-7/group stroke; *p<0.0500.
Figure 2
Figure 2
Cell-membrane inflammatory marker levels in IRF5 or IRF4 CKO vs. flox microglia by flow cytometry performed on stroke and sham brains. (A, C) Representative flow plots of IRF5 CKO, and (E, G) IRF4 CKO microglia gated by CD68 and CD206. (B, D, F, H) Quantification data of MFI for CD68 (B for IRF5, and F for IRF4) and CD206 (D for IRF5, and H for IRF4). n = 4 to 5 per sham and 6 to 7 per stroke group; *P < 0.0500.
Figure 3
Figure 3
Intracellular cytokine levels in IRF5 or IRF4 CKO vs. flox microglia by flow cytometry performed on stroke and sham brains. Quantification data are presented as mean MFI. (A–D) Data of IRF5 CKO and (E–H) data of IRF4 CKO microglia, respectively. (A, C) are representative intracellular staining plots for IL-1β/TNF-α in both IRF5 CKO and flox microglia; (E, G) are plots for IL-1β/TNF-α in both IRF4 CKO and flox microglia. MFI of these cytokines were quantified in (B, D, F, H). n = 4 to 5 per sham and 6 to 7 per stroke group; *P < 0.0500.
Figure 4
Figure 4
Microglial phagocytosis by flow cytometry performed on IRF4 CKO stroke and sham mice. Quantification data are presented as mean percentage of bead+ microglia. (A, B) Fluorescence intensity plots for IRF4 KO and flox microglia exposed to FITC fluorescent bioparticles. (C) Percentage fluorescence of phagocytosis in IRF4 CKO vs. flox sham and stroke microglia; and (D) comparative quantified data for the ratio of IRF4 CKO stk/shm vs. IRF4 flox stk/shm in (C). n = 4 to 5 per sham and 6 to 7 per stroke group; *P < 0.0500, **P < 0.0010, ***P < 0.0001.
Figure 5
Figure 5
IRF5 blood plasma levels of inflammatory mediators at 3d after MCAO. Proinflammatory mediators (TNF-α, IL-1β, IL-6, IL-12p40, and MIP-α; A–E) and anti-inflammatory mediators (IL-4 and IL-10; F, G) in IRF5 CKO mice. Each sample was probed in duplicates. n=6 per stroke and 4 per sham group; *P < 0.0500.
Figure 6
Figure 6
IRF4 blood plasma levels of inflammatory mediators at 3d after MCAO. Proinflammatory mediators (TNF-α, IL-1β, IL-6, IL-12p40, and MIP-α; A–E) and anti-inflammatory mediators (IL-4 and IL-10; F, G) in IRF4 CKO mice. Each sample was probed in duplicates. n=6 per stroke and 4 per sham group; *P < 0.0500.
Figure 7
Figure 7
Stroke outcomes in aged IRF5/4 CKO and flox mice at 3d after MCAO. (A, C) Representative IRF5 and IRF4 CKO vs. flox brain slices stained with cresyl violet. (B, D) Quantification of infarct volumes. (E, F) Corner test scores calculated by R/(R+L) x100, where R and L are right and left turn number respectively. (G, H) Neurological deficits scores. n =6/group; *P < 0.0500.
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