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Isolation of Cortical Microglia with Preserved Immunophenotype and Functionality From Murine Neonates
Published on: January 30, 2014
Microglia functions in peripheral inflammation-exacerbated cerebral inflammation under hypoxia in a CCL5-dependent
Liang Guo1, Xiangpei Yue1, Xiaochang Zhang1
1Academy of Military Medical Sciences, Beijing 100850, China.
Introduction:
The role of peripheral inflammation (PI) in exacerbating hypoxic neuroinflammation is being recognized, with the underlying molecular mechanism largely unknown.
Objectives:
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).
Methods:
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.
Results:
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.
Conclusion:
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.
Insights
Peripheral inflammation exacerbates hypoxic neuroinflammation via CCL5. This study identifies a key microglial subtype and cytokine, offering potential therapeutic targets for severe hypoxic brain conditions.
Area of Science:
- Neuroscience
- Immunology
- Genomics
Background:
- Peripheral inflammation (PI) is increasingly recognized for worsening hypoxic neuroinflammation.
- The precise molecular mechanisms driving this interaction remain largely unknown.
Purpose of the Study:
- To create the first single-cell atlas of the cerebral immune microenvironment under homeostasis, hypoxia, PI, and combined conditions (Hyp/LPS).
- To elucidate the role of specific immune cells, particularly microglia, and cytokines in PI-exacerbated hypoxic neuroinflammation.
Main Methods:
- Single-cell transcriptomic analysis (10X Genomics) of mouse models simulating homeostasis, hypoxia, LPS-induced PI, and Hyp/LPS.
- Flow cytometry to validate immune cell populations and microglial subtypes.
- Utilized Ccl5-knockout mice and primary microglia to confirm CCL5's role.
Main Results:
- Defined nine distinct microglial subtypes, categorized into homeostatic and disease-associated microglia (DAM) groups.
- Identified Ccl4+ microglia as highly responsive to stimuli within the hippocampus and cortex.
- CCL5 was the most elevated cytokine in microglia during Hyp/LPS, and its absence or neutralization significantly reduced neuroinflammation, confirming its mediating role.
Conclusions:
- Uncovered a critical microglial subtype and the cytokine CCL5 in the interplay between peripheral and cerebral inflammation.
- Provides potential biomarkers and therapeutic targets for severe hypoxic neuroinflammation.
