MLIF Modulates Microglia Polarization in Ischemic Stroke by Targeting eEF1A1.

Yulan Liu,Yongsheng Yu,Junqin Mao,Zhibing Song,Tiejun Li,Shanshan Deng,Yuefan Zhang,Yuliang Wang,Fayed A K Megahed,Qian Zhang,Tamer A Addissouky,Yuchen Guo

doi:10.3389/fphar.2021.725268

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.

Highlights

Stroke is one of the three leading causes of death and the most common reason for adults’ disability worldwide (Campbell and Khatri, 2020)
Monocyte locomotion inhibitory factor (MLIF) was dissolved in PBS to a final concentration of 4 mg/ml and stored at −80°C. eukaryotic elongation factor 1A1 (eEF1A1) antibody was purchased from Abcam (Cambridge, MA, United States), and Jun N-terminal kinase (JNK), p-JNK, extracellular signal-regulated kinase (ERK), p-ERK, p38, p-p38, iκB, p-iκB, p65, and p-p65 antibodies were purchased from Cell Signaling Technology (Danvers, MA, United States)
To evaluate the effect of MLIF on microglia polarization in ischemic stroke, co-immunostaining was performed with the representative M1 marker CD16/32 or M2 marker CD206 and the microglia/macrophage marker Iba1

Summary

Introduction

Stroke is one of the three leading causes of death and the most common reason for adults’ disability worldwide (Campbell and Khatri, 2020). Numerous studies have suggested that activated microglia with distinct phenotypes are double-edged swords in ischemic stroke Induced by IFN-γ and LPS stimulation, M1 phenotype microglia display enhanced expression of CD16/32, CD86, and iNOS and production of multiple pro-inflammatory cytokines (IL-1β, IL-6, and TNF-α) (Jha et al, 2016; Subhramanyam et al, 2019). The M2 phenotype could be induced by IL-4 or IL-13 and is characterized by enhanced expression of arginase-1, Fizz, Ym1, CD206, insulin-like growth factor (IGF-1), and anti-inflammatory cytokines (IL-10, IL-4, and IL-13) (Prinz et al, 2019; Kwon and Koh, 2020). The M1 phenotype aggravates inflammation and exacerbates neuronal death, while the M2 phenotype contributes to improving neuronal survival and tissue repair (Han et al, 2021; Zhang et al, 2021). Switching microglia from the M1 phenotype toward the M2 phenotype is an effective therapeutic strategy for ischemic stroke (Jiang et al, 2020)

Methods

Results

Conclusion