Lactate modulates microglial inflammatory responses through HIF-1α-mediated CCL7 signaling after cerebral ischemia in mice

Abstract

Lactate is a potent regulator of neuroinflammation. We recently demonstrated that lactate alleviated neuronal injury via HIF-1α-regulated microglial inflammation after oxygen-glucose deprivation (OGD). However, the underlying mechanisms and the effect of lactate on microglial responses after ischemic stroke remained unknown. Mouse acute cerebral ischemia-reperfusion injury was induced by middle cerebral artery occlusion (MCAO). L-lactate (100 mM, 2 μl) was intracerebroventricularly administrated 30 min after the reperfusion. Microglia responses were evidenced by the expression of multiple markers such as CD86, iNOS, arginase-1, CD206 and Ym1 in the peri-infarction 24 h after MCAO using western blot analysis and quantitative real-time PCR. Inflammatory factors IL-6, TNF-α, TGF-β and IL-10, as well as NF-κB signaling were also detected. Infarct size and neuronal apoptosis in the peri-infarction at 24 h, mice survival within 7 days and long-term neurobehavioral function were evaluated. The involvement of HIF-1α in lactate-mediated microglial inflammation after MCAO was assessed using a HIF-1α inhibitor. Additionally, transcriptome analysis was used to identify the potential lactate targets in BV2 cells after OGD. The recombinant product of the identified CCL7 gene was used to verify its effect on cerebral ischemia-reperfusion injury in vivo. Lactate supplementation reduced infarction volume, neuronal apoptosis and neurological deficits. Lactate reduced the expression of CD86, iNOS, IL-6, TNF-α and elevated the expression of arginase-1, CD206, Ym1, TGF-β and IL-10 in the peri-infarction at 24 h after reperfusion. Consistently, lactate inhibited the NF-κB signaling. Additionally, lactate upregulated HIF-1α in microglia 24 h after reperfusion, while inhibition of HIF-1α reversed the effects of lactate on brain damage and neuroinflammation after cerebral ischemia. Furthermore, CCL7 was identified as the top down-regulated inflammatory gene induced by lactate in OGD-treated BV2 cells. It was also found high expression of CCL7 in the peri-infarction at 24 h after reperfusion and lactate treatment inhibited CCL7 expression. However, HIF-1α inhibitor reversed the effect of lactate treatment on CCL7 expression. Finally, supplementation of recombinant CCL7 reversed the mitigated neuroinflammation and neuroprotective effect rendered by lactate treatment after MCAO. We concluded that treatment with lactate modulated the microglia inflammatory responses and alleviated cerebral ischemia injury. The inhibition of CCL7/NF-κB signaling by HIF-1α might be involved in the beneficial effect of lactate treatment.

Keywords: Cerebral ischemia; Hypoxia-inducible factor 1-alpha; Inflammatory response; Lactate; Microglia.