Abstract
Classical or M1 activity of microglia/macrophages has been described in several neurodegenerative and brain inflammatory conditions and has also been linked to expansion of ischemic injury in post-stroke brain. While different pathways of M1 polarization have been suggested to occur in the post-stroke brain, the precise underlying mechanisms remain undefined. Using a transient middle cerebral artery occlusion (MCAO) rat model, we showed a progressive M2 to M1 polarization in the perilesional brain region with M1 cells becoming one of the dominant subsets by day 4 post-stroke. Comparing key receptors involved in M1 polarization (CD8, IFNγR, Clec4, FcγR, TLR3 and TLR4) and their signal transducers (Syk, Stat1, Irf3, and Traf6) at the day 4 time point, we showed a strong upregulation of CD8 along with SYK transducer in dissected perilesional brain tissue. We further showed that CD8 expression in the post-stroke brain was associated with activated (CD68+) macrophages and that progressive accumulation of CD8+CD68+ cells in the post-stroke brain coincided with increased iNOS (M1 marker) and reduced Arg1 (M2 marker) expression on these cells. In vitro ligand-based stimulation of the CD8 receptor caused increased iNOS expression and an enhanced capacity to phagocytose E. coli particles; and interestingly, CD8 stimulation was also able to repolarize IL4-treated M2 cells to an M1 phenotype. Our data suggest that increased CD8 signaling in the post-stroke brain is primarily associated with microglia/macrophages and can independently drive M1 polarization, and that modulation of CD8 signaling could be a potential target to limit secondary post-stroke brain damage.
Highlights
To study microglia/macrophage activation and polarization after ischemic stroke, we utilized the well-established middle cerebral artery occlusion (MCAO) rat model [13, 26], which showed large ischemic lesions in the ipsilateral cortex and striatum (Fig 1A) and corroborated with the strong neurobehavioral decline observed in these animals (S2A and S2B Fig)
We further showed a gradual increase in activated CD68+ cells between 6 hours and 4 days following stroke (Fig 1C) that corroborated with increased transcripts of a general macrophage marker, Mac-1 (P < 0.001; S3B Fig)
We show that specific stimulation of CD8 alone resulted in upregulation of its signal transducers Syk (P < 0.05) and Raptor (P < 0.01; Fig 5E), the latter being part of the mammalian target of rapamycin complex 1 (mTORC1) complex shown to be involved in strokerelated inflammation [21]
Summary
Cerebral ischemia induces neuronal and glial cell death, resulting in extensive local inflammation of the brain parenchyma and microvasculature characterized by production of proinflammatory mediators, rapid activation of resident microglia and infiltration of peripheral. Other activation pathways involved in macrophage polarization in the brain after stroke are described that include toll-like receptor (TLR)-4/TNF receptor-associated factor (TRAF)-6 pathway, TLR3/interferon regulatory factor (IRF)-3 pathway, or spleen tyrosine kinase (SYK) signaling through either CD8, FcγR or Clec receptors [18,19,20]. These signaling pathways converge in activation of NF-κB, resulting in increased expression of proinflammatory cytokines. We show here that CD8 signaling is an important pathway during M1 polarization in post-stroke rat brain
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