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Three-dimensional Confocal Analysis of Microglia/macrophage Markers of Polarization in Experimental Brain Injury
Published on: September 4, 2013
Neuronal chemokine-like-factor 1 (CKLF1) up-regulation promotes M1 polarization of microglia in rat brain after
Xin Zhou1, Ya-Ni Zhang2, Fang-Fang Li1
1State Key Laboratory of Bioactive Substances and Functions of Natural Medicines, Institute of Materia Medica and Neuroscience Center, Chinese Academy of Medical Sciences and Peking Union Medical College, Beijing, 100050, China.
Abstract:
The phenotypic transformation of microglia in the ischemic penumbra determines the outcomes of ischemic stroke. Our previous study has shown that chemokine-like-factor 1 (CKLF1) promotes M1-type polarization of microglia. In this study, we investigated the cellular source and transcriptional regulation of CKLF1, as well as the biological function of CKLF1 in ischemic penumbra of rat brain. We showed that CKLF1 was significantly up-regulated in cultured rat cortical neurons subjected to oxygen-glucose deprivation/reoxygenation (ODG/R) injury, but not in cultured rat microglia, astrocytes and oligodendrocytes. In a rat model of middle cerebral artery occlusion, we found that CKLF1 was up-regulated and co-localized with neurons in ischemic penumbra. Furthermore, the up-regulated CKLF1 was accompanied by the enhanced nuclear accumulation of NF-κB. The transcriptional activity of CKLF1 was improved by overexpression of NF-κB in HEK293T cells, whereas application of NF-κB inhibitor Bay 11-7082 (1 μM) abolished it, caused by OGD/R. By using chromatin-immunoprecipitation (ChIP) assay we demonstrated that NF-κB directly bound to the promoter of CKLF1 (at a binding site located at -249 bp to -239 bp of CKLF1 promoter region), and regulated the transcription of human CKLF1. Moreover, neuronal conditional medium collected after OGD/R injury or CKLF1-C27 (a peptide obtained from secreted CKLF1) induced the M1-type polarization of microglia, whereas the CKLF1-neutralizing antibody (αCKLF1) or NF-κB inhibitor Bay 11-7082 abolished the M1-type polarization of microglia. Specific knockout of neuronal CKLF1 in ischemic penumbra attenuated neuronal impairments and M1-type polarization of microglia caused by ischemic/reperfusion injury, evidenced by inhibited levels of M1 marker CD16/32 and increased expression of M2 marker CD206. Application of CKLF1-C27 (200 nM) promoted the phosphorylation of p38 and JNK in microglia, whereas specific depletion of neuronal CKLF1 in ischemic penumbra abolished ischemic/reperfusion-induced p38 and JNK phosphorylation. In summary, CKLF1 up-regulation in neurons regulated by NF-κB is one of the crucial mechanisms to promote M1-type polarization of microglia in ischemic penumbra.
Insights
Chemokine-like-factor 1 (CKLF1), upregulated in neurons by NF-κB during ischemic stroke, drives M1-type microglia polarization. This neuronal CKLF1 promotes microglial activation, exacerbating stroke injury.
Area of Science:
- Neuroscience
- Immunology
- Molecular Biology
Background:
- Microglial phenotype in the ischemic penumbra is critical for stroke outcomes.
- Previous research indicated chemokine-like-factor 1 (CKLF1) promotes M1-type microglia polarization.
Purpose of the Study:
- To investigate the cellular source and transcriptional regulation of CKLF1.
- To elucidate the biological function of CKLF1 in the ischemic penumbra of rat brains.
Main Methods:
- Oxygen-glucose deprivation/reoxygenation (ODG/R) in cultured rat neurons.
- Middle cerebral artery occlusion (MCAO) rat model.
- Chromatin-immunoprecipitation (ChIP) assay.
- NF-κB manipulation and inhibition (Bay 11-7082).
- Neuronal CKLF1 knockout and CKLF1 peptide/antibody application.
Main Results:
- CKLF1 was significantly upregulated in neurons, not other glial cells, following ODG/R.
- NF-κB directly binds to the CKLF1 promoter, enhancing its transcription.
- Neuronal CKLF1 induces M1-type microglia polarization, neuronal impairment, and p38/JNK phosphorylation, which is attenuated by CKLF1 knockout.
Conclusions:
- Neuronal CKLF1, transcriptionally regulated by NF-κB, is a key factor promoting M1-type microglia polarization in the ischemic penumbra.
- Targeting neuronal CKLF1 may offer a therapeutic strategy for ischemic stroke.
