Microglial Polarization: Novel Therapeutic Strategy against Ischemic Stroke

Ischemic stroke, which is the second highest cause of death and the leading cause of disability, represents ~71% of all strokes globally. Some studies have found that the key elements of the pathobiology of stroke is immunity and inflammation. Microglia are the first line of defense in the nervous system. After stroke, the activated microglia become a double-edged sword, with distinct phenotypic changes to the deleterious M1 types and neuroprotective M2 types. Therefore, ways to promote microglial polarization toward M2 phenotype after stroke have become the focus of attention in recent years. In this review, we discuss the process of microglial polarization, summarize the alternation of signaling pathways and epigenetic regulation that control microglial polarization in ischemic stroke, aiming to find the potential mechanisms by which microglia can be transformed into the M2 polarized phenotype.


Introduction
Stroke is the second leading cause of death and the highest disabling disease in the world, with an increasing incidence in developing countries [1][2][3]. China suffers the greatest burden of stroke globally, with about 2.4 million new cases and 1.1 million stroke-related deaths annually [4]. Ischemic stroke induced by arterial occlusion is the major cause of strokes and account for ~71% of all strokes in the world. The standard treatment for acute ischemic stroke is intravenous thrombolysis with tissue-type plasminogen activator (t-PA) and endovascular treatment called microglial activation [10]. Activated microglia is one of the most important cellular components of poststroke neuroinflammation, which occurs within an hour to more than a month, developing four morphological states: ramified, intermediate, amoeboid and round [11][12][13]. Age is a critical co-factor for CNS diseases. Interestingly, the function of microglial cells changed with aging and the morphology of the microglia is more de-ramified [14]. Compared with young microglial cells, aged microglia activation is amplified and prolonged [15]. The existence of an aging-related microglial phenotype in the aged human brain is verified and it is involved in pathological processes of CNS diseases [16].
Microglia could present different phenotypes in accordance with the stimulus, the environment, and the period, which is called microglial polarization [17,18]. Similar to macrophages, microglial polarization is divided into classically activated (M1, pro-inflammatory) phenotype and alternatively activated (M2, antiinflammatory) phenotype. Many differences of the polarization of these two cell types have been noted [19].
Polarized microglia differ from polarized macrophages in protein expression, phagocytosis, and injury response. In response to inflammatory factor, M2 microglia are more protective and tend to maintain the M2 phenotype status [20]. The phenotype of microglial cells also changed with aging. Aged microglia demonstrated a propensity for the development of a pro-inflammatory phenotype with increased pro-inflammatory cytokines and inflammatory receptors, which is referred to as primed, reactive or sensitized [15]. Microglia play an important role in various neurological diseases, involving in multiple aspects of neuroinflammation, such as cytotoxicity, repair, immunosuppression and regeneration at the basis of different polarization states [21]. Thus, we make a review to discuss the process of microglial polarization and summarize the alternation of signaling pathways and epigenetic modifications that control microglial polarization in ischemic stroke, aiming to find the potential mechanisms and drugs by which microglia can be shifted from M1 into the M2 polarized type after ischemic stroke.
In response to an immune challenge, the process of microglial polarization shift towards priming with aging [38]. Under the stimulation of LPS, aged microglia showed hyperactive response with higher induction of inflammatory IL-1 and anti-inflammatory IL-10. And aged microglia prolonged the downregulation of the fractalkine receptor and failed to up-regulation of IL-4 receptor [39]. Taken together, the ability of microglia to lower inflammation in the brain is impaired. The understanding of the aged microglia phenotype and function in humans is limited, particularly in the process of microglial polarization. Mounting evidence is needed to confirm the role of aged microglia polarization in ischemia stroke.

Microglial Polarization in Neurological Disorders
Although it is oversimplified to divide microglia into the M1 and M2 phenotypes, the classification has important implications for comprehending the role of microglia in CNS diseases [40]. The role of microglial polarization in a variety of neurological disorders has been illuminated. Targeting M2 phenotype polarization has been proved to be a potential therapeutic strategy. In Alzheimer's disease (AD), studies have shown that the dysfunction of M2 microglia and the excessive activation of M1 microglia promote inflammatory pathological injury. Through polarization moderation, microglia could induce tissue repair and phagocytosis to reduce Aβ levels, alleviating AD pathological damage [22]. In AD mouse models, DSP-8658 and Bexarotene have proved to enhance microglial Aβ phagocytosis [41,42]. In Parkinson's disease (PD), the dopaminergic degeneration is involved in microglial polarization, Rosiglitazone boosts the M2 phenotype over the pro-inflammatory phenotype modulating microglia polarization [43]. Although the pathology of amyotrophic lateral sclerosis (ALS) has still not been completely understood [44], hirsutella sinensis prolongs the lifespan of ALS mice by promoting transition of microglial polarization from M1 to M2 phenotype [45]. In Huntington's disease, microglial polarization affects striatal neuronal dysfunction [46]. In multiple sclerosis (MS), M1 microglia have a greater ability to present antigens, leading to demyelination and neurodegeneration, while M2 microglia protect oligodendrocytes and neurons from damage and ameliorate disease severity [26]. A recent clinical trial showed that anti-pathogenic human endogenous retrovirus type W (pHERVW) envelope protein (ENV)mediated microglial polarization exerts neuroprotective effects in MS [47] (see Table 2).

Polarized Microglia-based Therapy in Ischemic Stroke
While ischemic stroke occurs, the microenvironment of microglia has changed and classic (M1) or alternative (M2) microglia are polarized responding to peripheral inflammation. At the early stage of ischemic stroke, microglia tend to assume the M2 phenotype responding to acute injury, and then microglia transform into the M1 phenotype that induces an inflammatory response [48]. The mechanism of microglial polarization during ischemic stroke involves multiple pathways that have not been entirely clear. Present studies showed that the type of microglial polarization was decided by signaling pathways. Understanding the accurate mechanism of microglial polarization, we can find a breakthrough in the treatment. In the following, we discuss the transcription factors and epigenetic regulation associated with ischemia-induced microglial polarization to find out the mechanism of microglial M1 to M2 transition (see Table  3).
Other transcription factors may regulate microglial polarization by influencing the activity of NF-κB. Notch signaling promote production of IFN-γ through recruitment of p50 and c-Rel, in response to LPS. With NF-κB activation, inflammation and neurotoxicity exacerbate ischemic brain damage [59]. The crosstalk between Notch and NF-κB inhibits the expression of PPARγ which is necessary for the induction of the M2 phenotype [60,61]. STAT1 and STAT3 are able to increase the expression of NF-κB p65. Inhibiting the activation of STAT1 and STAT3 prevents the inflammatory reaction caused by brain ischemia, thereby reducing the occurrence of infarction and edema. In contrast, CREB cooperated with C/EBPβ promote tissue repair by amplification of M2-specific gene [62]. Confoundingly, the expression of M1-specific genes associated with inflammation is also affected by C/EBPβ [63]. The role of C/EBPβ in regulating microglial phenotypes depends on the competitiveness of CREB and NF-κB [64]. CREB-binding protein (CBP) is another competition site. The increase of CREB activity has a negative effect on the combination of CBP and NF-κB [65,66]. With the activation of TLRs, interferon regulatory factor-3 (IRF-3) is phosphorylated and interacts with CBP promoting the M2 polarization. The RelA/CBP/p300 complex is formed at the same time [67][68][69]. In summary, the balance of NF-κB and CREB plays a crucial role in the microglial polarization in cerebral ischemia [49].
In addition, nuclear factor erythroid 2-related factor 2 (Nrf2) is activated and involved in the anti-inflammatory effect of the M2 phenotype microglia, which is a key factor of brain endogenous defense system, in response to oxidative stress [70,71]. After the activation of Nrf2, neuro-inflammation induced by LPS was inhibited both in vivo and in vitro [72,73]. A study concluded that achyranthes bidentata polypeptidek's could inhibit neuroinflammation in BV2 microglia through Nrf2 dependent mechanism [74]. Through the activation of the Nrf2 pathway and the inhibition of the NF-κB pathway, Biochanin A may contribute to the neuro-protection against ischemic injury in rats by anti-oxidative and antiinflammatory actions [75]. Other studies conclude that the disruption of mTORC1 pathway could shift microglial phenotype to decrease brain inflammation [76].

Epigenetic Modifications
Besides the transcription factors above, the polarization and functional status of microglia require precise regulation of target gene expression, which can be achieved by epigenetic modifications. Epigenetics refers to modifications that do not alter the genetic code but control how information is encoded in DNA in a tissueand context-specific manner developmentally or environmentally [77]. The mechanisms of epigenetic modifications are usually mediated by modifications of histones and other chromatin proteins (such as methylation, acetylation, and phosphorylation), methylation of CpG DNA motifs, hydroxymethylation, and non-coding RNA [78,79]. The epigenetic markers histone modification and miRNA involved in microglial polarization and activation processes are reportedly more than the others [80]. The following summarizes the recent findings on the role of epigenetic modifications regulating microglial polarization.

MiRNA
MicroRNAs (miRNAs) are small non-coding RNA molecules that regulate gene expression posttranscriptionally. MiRNAs repress gene expression by combining with the 3'-untranslated region, coding sequence, or 5'UTR of target genes [81,82]. A total of 30%-90% of human genes are regulated by miRNAs that modulate cell growth, activation, and differentiation [83]. M1-and M2-polarized microglia exhibit distinct miRNA profiles. Recent research has also defined a role for miRNA in microglial polarization [84]. With the development of miRNA research, more and more miRNAs are related to microglial polarization phenotypes.
It is well accepted that miRNA-155 expression promotes M1 polarization by suppressing M2-signature genes and that miRNA-124 enhances the M2 phenotype by targeting M1 genes [85][86][87]. In MACO mice, miR-124 proved to increase the survival of neuron and M2 microglial polarization [81]. In IL-4 stimulated microglia, miR-145 was the most increased miRNA, facilitating the M2 phenotype in microglia [88]. Overexpression of miR-146a contributed to polarization transitions from M1 to M2 phenotype in microglia [89]. Isosteviol sodium can downregulate miRNA-181b to protect mouse brain with ischemia stroke by repressing NF-κB signaling pathways, providing a novel therapy for ischemic stroke [90]. MiRNA-128 could reduce the M1 phenotypic markers and increase the M2 phenotypic markers, promoting the viability of microglia [91]. Overall, targeting proinflammatory or anti-inflammatory miRNAs to regulate the microglial polarization provides new direction in the treatment of ischemic stroke. However, further studies are badly in need to clarify the function of miRNAs in the switch of microglial phenotype. Additionally, how to deliver miRNAs to the central nervous system (CNS) through the blood brain barrier (BBB) and prevent the degradation of miRNAs are also unsolved. With mechanisms of microglial polarization unveiled, targeting specific miRNAs may provide major restorative therapies and microglial polarization-based therapy will be potential future research field of the treatment of ischemia stroke [92].

DNA Methylation
DNA methylation is an epigenetic process catalyzed by DNA methyltransferases (DNMTs). Methyl groups are added to DNA nucleotides, which leads to chromatin condensation and alteration of gene expression [93]. DNMT maintains cytosine methylation through mitotic and meiotic cell divisions and is widely expressed in brain tissue. The whole DNA methylation in brain is upregulated after cerebral ischemia, which may control gene expression profile in cerebral ischemia injury [94,95]. Aberrant DNA methylation patterns have been proved in cerebral ischemia. Reduced DNA methylation play a neuroprotective role in ischemic stroke. Inhibition of DNMT1 expression affects chromatin structure and increases expression and combination of transcription factors (such as hypoxia-inducible factor-1 (HIF-1)) with neuroprotective genes [96,97]. It has been reported that there is an intrinsic link between DNA methylation in microglia and aging-mediated cognitive deficits [98]. However, the role of DNA methylation has remained to be further elucidated in microglial polarization in ischemic stroke. DNA methylation is a modifiable regulation and it is possible that in the future methylated or unmethylated genes could be a drug target for stroke treatment.

Histone Modifications
The electrostatic interaction of positive charges on histones and negative charges on DNA inhibits tightly packed chromatin structures [96]. The acetylation of histones on lysine residues can neutralize the positive charge, thereby disrupting the stability of the histone-DNA interaction, and subsequently changing the concentrated chromatin into an open, loosely packed chromatin structure, allowing gene recruitment activators or inhibitors of transcription and it can be reversed by histone deacetylases (HDACs) activity [99,100]. It has been reported that HDAC inhibitors (HDACi) have antiinflammatory effects in neuroprotection [101]. The protection of HDACi on microglia polarization is involved in its anti-inflammatory effect in the early phase of cerebral ischemia, reducing the activation of microglia and promote activated microglia to protective phenotype, providing a promising therapeutic intervention [102,103]. It has been reported that the inhibition of HDAC1 and HDAC2 activity after transient cerebral ischemia promotes microglia polarization towards M2 Phenotype [104]. Valproic acid treatment attenuated the inflammatory response by modulating microglia polarization through STAT1-mediated acetylation of the NF-κB pathway, dependent of HDAC3 activity [105]. Enhancer of zeste homolog-2 (EZH2), a histone methyltransferase, has been recognized to promote M1 microglial polarization but repress M2 microglial polarization probably via activating STAT3 [106]. On the contrary, histone 3 lysine 27 (H3K27) demethylase Jumonji d3 (Jmjd3) promotes M2 microglial polarization but represses M1 microglia polarization [107,108]. Dehydroepiandrosterone (DHEA) is the most abundant circulating steroid hormone in humans, TrkA signaling activated by DHEA is an effective regulator of inflammation through Jmjd3-dependent pathway, providing potential treatments for neuroinflammatory diseases (Fig. 1) [109].
Besides above, there are other epigenetic regulations contributing to the polarization of microglia in the ischemic stroke. Long noncoding RNA H19 promotes neuroinflammation by driving HDAC1-dependent M1 microglial polarization, suggesting a novel H19-based diagnosis and therapy for ischemic stroke [110]. MiR-30d-5p-enhanced adipose-derived stem cells (ADSC) derived exosomes prevent cerebral injury by inhibiting microglial polarization to M1 [111]. Investigation of epigenetic regulation of microglia polarization and function is at an early stage and there are many unknown areas for future research. Finally, recent breakthroughs have opened a new door to epigenetic therapy of ischemic stroke.
More and more evidence has revealed that modulators of microglial phenotypes may be a promising therapeutic approach for the treatment of ischemic stroke. However, fundamental differences of the cellular environment and damage-response between macrophages and microglia exist, the M1/M2 oversimple classification may not be applicable to microglia. Unbiased methods such as genome-wide transcriptomics, epigenomics and proteomics are urgent needed to aid research progress [112].
Comprehensive single-cell RNA analysis of CNS immune cells identified disease-associated microglia (DAM), which is a kind of microglia with specifically transcription and function. The emergence of DAM may provide a new explanation for the contradictory views on the detrimental or beneficial effects of microglia in recent years [113].

Conclusion
In cerebral ischemia, the neuroprotective effects of M2polarized microglia cells include clearing debris as well as promoting tissue repair. Increasing evidence indicates that shifting microglial phenotype from the proinflammatory M1 state toward the anti-inflammatory M2 phenotype may be an effective therapeutic strategy for ischemic stroke. Importantly, several signalling pathways-such as NF-κB, and Wnt/β-catenin-may be critically involved in microglial polarization in ischemic stroke. The underlying mechanisms of microglial polarization in ischemic stroke are still not well understood and need to be further elucidated. Figure 1. Microglia polarization after ischemic stroke. M1 microglia produce pro-inflammatory cytokines to exacerbate neural death, astrocyte apoptosis, and blood brain barrier (BBB) disruption. Conversely, M2 microglia produce anti-inflammatory cytokines to maintain BBB integrity, promote the proliferation and differentiation of neural cells and tissue repair.