Monocytes promote acute neuroinflammation and become pathological microglia in neonatal hypoxic-ischemic brain injury

Rationale: Monocytes belong to the mononuclear phagocyte system and are immune responders to tissue injury and infection. There were also reports of monocytes transforming to microglia-like cells. Here we explore the roles of monocytes in microglia ontogeny and the pathogenesis of neonatal cerebral hypoxic-ischemic (HI) brain injury in mice. Methods: We used three genetic methods to track the development of monocytes, including CX3CR1(GFP)(/+); CCR2(RFP/)(+) reporter mice, adoptive transfer of GFP +/- monocytes, and fate-mapping with CCR2-CreER mice, in neonatal mouse brains with or without lipopolysaccharide (LPS, 0.3 mg/kg)-sensitized Vannucci HI. We also used genetic (CCR2(RFP/)(RFP), CCR2 knockout) and pharmacological methods (RS102895, a CCR2 antagonist) to test the roles of monocytic influx in LPS/HI brain injury. Results: CCR2(+) monocytes entered the late-embryonic brains via choroid plexus, but rapidly became CX3CR1(+ )amoeboid microglial cells (AMCs). The influx of CCR2(+) monocytes declined after birth, but recurred after HI or LPS-sensitized HI (LPS/HI) brain injury, particularly in the hippocampus. The CCR2-CreER-based fate-mapping showed that CCR2(+) monocytes became CD68(+) TNF alpha(+) macrophages within 4 d after LPS/HI, and maintained as TNF alpha(+) MHCII+ macrophages or persisted as Tmem119(+) Sall1(+) P2RY12(+) ramified microglia for at least five months after injury. Genetic deletion of the chemokine receptor CCR2 markedly diminished monocytic influx, the expression of pro- and anti-inflammatory cytokines, and brain damage. Post-LPS/HI application of RS102895 also reduced inflammatory responses and brain damage, leading to better cognitive functions. Conclusion: These results suggest that monocytes promote acute inflammatory responses and may become pathological microglia long after the neonatal LPS/HI insult. Further, blocking the influx of monocytes may be a potential therapy for neonatal brain injury.