Abstract
Immune cells display a high degree of phenotypic plasticity, which may facilitate their participation in both the progression and resolution of injury-induced inflammation. The purpose of this study was to investigate the temporal expression of genes associated with classical and alternative polarization phenotypes described for macrophages and to identify related cell populations in the brain following neonatal hypoxia-ischemia (HI). HI was induced in 9-day old mice and brain tissue was collected up to 7 days post-insult to investigate expression of genes associated with macrophage activation. Using cell-markers, CD86 (classic activation) and CD206 (alternative activation), we assessed temporal changes of CD11b+ cell populations in the brain and studied the protein expression of the immunomodulatory factor galectin-3 in these cells. HI induced a rapid regulation (6 h) of genes associated with both classical and alternative polarization phenotypes in the injured hemisphere. FACS analysis showed a marked increase in the number of CD11b+CD86+ cells at 24 h after HI (+3667%), which was coupled with a relative suppression of CD11b+CD206+ cells and cells that did not express neither CD86 nor CD206. The CD11b+CD206+ population was mixed with some cells also expressing CD86. Confocal microscopy confirmed that a subset of cells expressed both CD86 and CD206, particularly in injured gray and white matter. Protein concentration of galectin-3 was markedly increased mainly in the cell population lacking CD86 or CD206 in the injured hemisphere. These cells were predominantly resident microglia as very few galectin-3 positive cells co-localized with infiltrating myeloid cells in Lys-EGFP-ki mice after HI. In summary, HI was characterized by an early mixed gene response, but with a large expansion of mainly the CD86 positive population during the first day. However, the injured hemisphere also contained a subset of cells expressing both CD86 and CD206 and a large population that expressed neither activation marker CD86 nor CD206. Interestingly, these cells expressed the highest levels of galectin-3 and were found to be predominantly resident microglia. Galectin-3 is a protein involved in chemotaxis and macrophage polarization suggesting a novel role in cell infiltration and immunomodulation for this cell population after neonatal injury.
Highlights
Hypoxic-ischemic (HI) brain injury is an important contributor to neonatal mortality as well as permanent neurological impairments in surviving infants
Using total cortical homogenates we performed a qRT-PCR assessment of temporal expression profiles of genes associated with classical activation (CD86, IL-6, IL-1β, Cox2, inducible nitric oxide synthase (iNOS)) and alternative activation (CD206, IL-10, Fizz1, Arg1) in macrophages, as well as the immunomodulatory factor galectin3 (Gal3) following HI (Figure 1)
Post-hoc analysis revealed acute regulation in the ipsilateral hemisphere compared with the contralateral hemisphere of genes associated with classical activation: CD86 (4.0-fold; p < 0.001), IL-6 (8.6-fold; p < 0.001), IL-1β (46.3-fold; p < 0.001); as well as genes associated with alternative activation: IL-10 (6.0-fold; p < 0.001), Fizz1 (2.1-fold; p < 0.001), Arg1 (4.3-fold; p < 0.001) at 6 h after HI, with no significant regulation at later time points
Summary
Hypoxic-ischemic (HI) brain injury is an important contributor to neonatal mortality as well as permanent neurological impairments in surviving infants. HI triggers an imbalance of CNS homeostasis and initiates peripheral and central inflammatory responses, which can be detected within 2–3 h of insult in rodent models (Hedtjärn et al, 2004; Bonestroo et al, 2013). Microglia are the primary immune competent and phagocytic cells of the brain (Kreutzberg, 1996). Experimental evidence from adult models show that brain injury rapidly activates microglia and lead to increased phagocytic activity and altered production of cytokines and reactive oxygen metabolites (Hanisch, 2002), features that are well documented in neonatal HI (Hedtjärn et al, 2004). In the adult brain there is a considerable contribution of infiltrating peripheral immune cells to the brain after strokelike injury (Iadecola and Anrather, 2011). Little infiltration of peripheral cells is seen acutely after neonatal stroke (Denker et al, 2007), it remains unclear to what extent peripheral immune cells contribute to the inflammatory response after neonatal hypoxia-ischemia (Mallard and Vexler, 2015)
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