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Updated: Apr 5, 2026
Isolation and Flow Cytometric Assessment of Neuroimmune Interactions in a Mini-Stroke Murine Model
Published on: June 20, 2025
Role for microglia in sex differences after ischemic stroke: importance of M2
Sheetal Bodhankar1,2, Andrew Lapato1,2, Yingxin Chen3
1Neuroimmunology Research, R&D-31, VA Portland Health Care System, 3710 SW U.S. Veterans Hospital Rd., Portland, OR, 97239, USA.
Abstract:
Inflammation plays a critical role in the pathogenesis of ischemic stroke. This process depends, in part, upon proinflammatory factors released by activated resident central nervous system (CNS) microglia (MG). Previous studies demonstrated that transfer of IL-10(+) B-cells reduced infarct volumes in male C57BL/6 J recipient mice when given 24 h prior to or therapeutically at 4 or 24 h after experimental stroke induced by 60 min middle cerebral artery occlusion (MCAO). The present study assesses possible sex differences in immunoregulation by IL-10(+) B-cells on primary male vs. female MG cultured from naïve and ischemic stroke-induced mice. Thus, MG cultures were treated with recombinant (r)IL-10, rIL-4 or IL-10(+) B-cells after lipopolysaccharide (LPS) activation and evaluated by flow cytometry for production of proinflammatory and anti-inflammatory factors. We found that IL-10(+) B-cells significantly reduced MG production of TNF-α, IL-1β and CCL3 post-MCAO and increased their expression of the anti-inflammatory M2 marker, CD206, by cell-cell interactions. Moreover, MG from female vs. male mice had higher expression of IL-4 and IL-10 receptors and increased production of IL-4, especially after treatment with IL-10(+) B-cells. These findings indicate that IL-10-producing B-cells play a crucial role in regulating MG activation, proinflammatory cytokine release and M2 phenotype induction, post-MCAO, with heightened sensitivity of female MG to IL-4 and IL-10. This study, coupled with our previous demonstration of increased numbers of transferred IL-10(+) B-cells in the ischemic hemisphere, provide a mechanistic basis for local regulation by secreted IL-10 and IL-4 as well as direct B-cell/MG interactions that promote M2-MG.
Insights
Interleukin-10-producing B-cells reduce inflammation after ischemic stroke by modulating microglia activation. Female mice show heightened sensitivity to these regulatory effects, suggesting sex-specific therapeutic potential.
Area of Science:
- Neuroimmunology
- Stroke Pathophysiology
- Cellular Immunology
Background:
- Inflammation is key in ischemic stroke, driven by activated microglia releasing proinflammatory factors.
- Previous work showed Interleukin-10 (IL-10)-producing B-cells reduce stroke damage in male mice.
- Sex differences in immune responses to stroke are increasingly recognized.
Purpose of the Study:
- To investigate sex-based differences in the immunoregulatory effects of IL-10-producing B-cells on microglia.
- To determine how IL-10-producing B-cells influence microglia activation and cytokine production in male versus female mice post-stroke.
Main Methods:
- Primary microglia cultures from male and female mice (naïve and stroke-induced) were treated with IL-10, IL-4, or IL-10(+) B-cells post-lipopolysaccharide activation.
- Flow cytometry was used to assess the production of pro- and anti-inflammatory factors and M2 marker expression (CD206).
- Receptor expression for IL-4 and IL-10 on microglia was also evaluated.
Main Results:
- IL-10(+) B-cells significantly decreased microglia production of TNF-α, IL-1β, and CCL3 after middle cerebral artery occlusion (MCAO).
- IL-10(+) B-cells enhanced the expression of the anti-inflammatory M2 marker CD206 on microglia via cell-cell interactions.
- Female microglia exhibited higher expression of IL-4 and IL-10 receptors and increased IL-4 production, particularly after IL-10(+) B-cell treatment.
Conclusions:
- IL-10-producing B-cells are crucial in regulating microglia activation, reducing proinflammatory cytokine release, and inducing an M2 phenotype post-stroke.
- Female microglia demonstrate heightened sensitivity to IL-4 and IL-10, indicating sex-specific mechanisms in stroke immunomodulation.
- These findings provide a mechanistic basis for B-cell-mediated neuroprotection in stroke, highlighting sex differences.
