Microglia functions in peripheral inflammation-exacerbated cerebral inflammation under hypoxia in a CCL5-dependent...

The role of peripheral inflammation (PI) in exacerbating hypoxic neuroinflammation is being recognized, with the underlying molecular mechanism largely unknown.

This study is the first to map the single-cell atlas of cerebral immune microenvironment in mice under homeostasis, acute hypoxia (Hyp), LPS-induced PI, and PI-exacerbated hypoxic brain inflammation (Hyp/LPS).

Six- to eight-week-old male C57BL/6J mice were subjected to hypoxic conditions in a decompression chamber (simulating 6000 m altitude) and/or intraperitoneal injection of LPS. Then 10X Genomics chromium single-cell transcriptomic analysis was employed to map the cerebral immune microenvironment of different mouse models, followed by flow cytometry analysis to validate the immune cells and microglial subpopulations identified in the brain and peripheral blood. Ccl5-knockout mice and primary microglia were also used to confirm the critical role of CCL5 in Hyp/LPS.

Nine transcriptionally distinct microglial subtypes are defined and classified into three functional groups based on the expression pattern of disease-associated microglia (DAM) and homeostatic genes. Ccl4⁺ microglia of DAM group, residing within hippocampus and cortex, represents the most responsive subtype to different stimuli. CCL5, with an increased serum level, is the top elevated cytokine in microglia upon Hyp/LPS. Hyp/LPS-induced cerebral inflammation is markedly reduced in Ccl5-knockout mice, and CCL5 neutralization restrains Hyp/LPS-increased expression of pro-inflammatory cytokines in primary microglia, indicating that Hyp/LPS-exacerbated neuroinflammation is mediated by CCL5.

Our study uncovers the key microglial subtype and cytokine in the peripheral and cerebral inflammation interaction in response to different stimuli, providing potential biomarker and therapeutic target for severe hypoxic neuroinflammation.