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MLIF Modulates Microglia Polarization in Ischemic Stroke by Targeting eEF1A1
Yulan Liu1,2, Shanshan Deng1, Zhibing Song3
1School of Medicine, Shanghai University, Shanghai, China.
Abstract:
Monocyte locomotion inhibitory factor (MLIF) is a heat-stable pentapeptide from Entamoeba histolytica. Our previous study found that MLIF protects against ischemic stroke in rats and mice and exerts a neuroprotection effect in human neuroblastoma SH-SY5Y cells. Microglia/macrophage polarization has been proven to be vital in the pathology of ischemic stroke. Nevertheless, whether MLIF is able to modulate microglia/macrophage polarization remains unclear. We performed middle cerebral artery occlusion (MCAO) on C57BL/6J male mice and induced cultured BV2 microglia by oxygen-glucose deprivation (OGD), respectively. Immunfluorescence was utilized to detect the M1/2 markers, such as CD206 and CD16/32. qPCR and ELISA were used to detect the signature gene change of M1/2. The MAPK and NF-κB pathway associated proteins were measured by Western blot. To identify the protein target of MLIF, a pull-down assay was performed. We found that MLIF promoted microglia transferring from a "sick" M1 phenotype to a "healthy" M2 phenotype in vivo or in vitro. Furthermore, we proved that eukaryotic elongation factor 1A1 (eEF1A1) was involved in the modulation of microglia/macrophage polarization. Knocking down eEF1A1 by siRNA exhibited the M1 promotion effect and M2 inhibition effect. Taken together, our results demonstrated MLIF modulated microglia/macrophage polarization by targeting eEF1A1 in ischemic stroke.
Insights
Monocyte locomotion inhibitory factor (MLIF) shifts microglia from a pro-inflammatory M1 to a protective M2 state. This neuroprotective effect in ischemic stroke involves targeting eukaryotic elongation factor 1A1 (eEF1A1).
Area of Science:
- Neuroscience
- Immunology
- Molecular Biology
Background:
- Microglia/macrophage polarization is critical in ischemic stroke pathology.
- Monocyte locomotion inhibitory factor (MLIF) shows neuroprotective effects against ischemic stroke.
- The role of MLIF in modulating microglia/macrophage polarization is not well understood.
Purpose of the Study:
- To investigate MLIF's effect on microglia/macrophage polarization in ischemic stroke.
- To identify the molecular mechanisms and protein targets underlying MLIF's action.
Main Methods:
- Middle cerebral artery occlusion (MCAO) model in mice and oxygen-glucose deprivation (OGD) in BV2 microglia.
- Immunofluorescence for M1/M2 markers (CD206, CD16/32).
- Quantitative PCR (qPCR), ELISA, Western blot, and pull-down assays to analyze gene/protein expression and interactions.
Main Results:
- MLIF promoted the M1 to M2 phenotype shift in microglia both *in vivo* and *in vitro*.
- Eukaryotic elongation factor 1A1 (eEF1A1) was identified as a key mediator in MLIF's polarization modulation.
- siRNA-mediated knockdown of eEF1A1 reversed the beneficial M2 shift, promoting M1 and inhibiting M2 phenotypes.
Conclusions:
- MLIF effectively modulates microglia/macrophage polarization towards a protective M2 phenotype in ischemic stroke.
- The mechanism involves MLIF targeting eEF1A1, influencing the M1/M2 balance.
- This finding highlights MLIF as a potential therapeutic agent for ischemic stroke via immune modulation.
