Shaping the Microglia in Retinal Degenerative Diseases Using Stem Cell Therapy: Practice and Prospects

Retinal degenerative disease (RDD) refers to a group of diseases with retinal degeneration that cause vision loss and affect people’s daily lives. Various therapies have been proposed, among which stem cell therapy (SCT) holds great promise for the treatment of RDDs. Microglia are immune cells in the retina that have two activation phenotypes, namely, pro-inflammatory M1 and anti-inflammatory M2 phenotypes. These cells play an important role in the pathological progression of RDDs, especially in terms of retinal inflammation. Recent studies have extensively investigated the therapeutic potential of stem cell therapy in treating RDDs, including the immunomodulatory effects targeting microglia. In this review, we substantially summarized the characteristics of RDDs and microglia, discussed the microglial changes and phenotypic transformation of M1 microglia to M2 microglia after SCT, and proposed future directions for SCT in treating RDDs.

Neuregulin-1 Regulates the Conversion of M1/M2 Microglia Phenotype via ErbB4-dependent Inhibition of the NF-κB Pathway

BackgroundThe inflammatory response caused by microglia in the central nervous system plays an important role in Alzheimer's disease. Neuregulin-1 (NRG1) is a member of the neuregulin family and has been demonstrated to have anti-inflammatory properties. The relationship between NRG1, microglia phenotype and neuroinflammation remains unclear.Materials and MethodsBV2 cells were used to examine the mechanism of NRG1 in regulating microglia polarization. Neuronal apoptosis, inflammatory factors TNF-α and iNOS, microglia polarization, ErbB4 and NF-κB p65 expression were assessed.ResultsWe found that exogenous NRG1 treatment or overexpression improved microglial activity and reduced the secretion of the inflammatory factors TNF-α and iNOS in vitro. The expression of Bax in SH-SY5Y neuron cells incubated with medium collected from the NRG1 treatment group decreased. Additionally, our study showed that NRG1 treatment reduced the levels of the M1 microglia markers CD120 and iNOS and increased the levels of the M2 microglia markers CD206 and Arg-1. Furthermore, we observed that NRG1 treatment attenuated Aβ-induced NF-κB activation and promoted the expression of p-ErbB4 and that knockdown of ErbB4 abrogated the effects of NRG1 on NF-κB, Bax levels and M2 microglial polarization. ConclusionNRG1 inhibits the release of inflammatory factors in microglia and regulates the switching of the M1/M2 microglia phenotype, most likely via ErbB4-dependent inhibition of the NF-κB pathway.

Microglia Polarization in Alzheimer’s Disease: Mechanisms and a Potential Therapeutic Target

Neuroinflammation regulated by microglia is one of the important factors involved in the pathogenesis of Alzheimer’s disease (AD). Activated microglia exhibited phenotypes termed as M1 and M2 phenotypes separately. M1 microglia contribute to the development of inflammation via upregulating pro-inflammatory cytokines, while M2 microglia exert anti-inflammation effects through enhancing the expression of anti-inflammation factors. Moreover, M1 and M2 microglia could be mutually transformed under various conditions. Both M1 and M2 microglia are implicated in AD. Amyloid-β (Aβ) and hyperphosphorylated tau are two major components of AD pathological hallmarks, neuritic plaques, and neurofibrillary tangles. Both Aβ and hyperphosphorylated tau were involved in microglial activation and subsequent inflammation, which further contribute to neuronal and synaptic loss in AD. In this review, we summarized the roles of M1 and M2 microglia in AD and underlying mechanisms, which will provide an insight into the role of microglia in the pathogenesis of AD and highlight the therapeutic potential of modulating microglia.

Recruitment of Regulatory T Cells with rCCL17 Promotes M2 Microglia/Macrophage Polarization Through TGFβ/TGFβR/Smad2/3 Pathway in a Mouse Model of Intracerebral Hemorrhage

Background Intracerebral hemorrhage (ICH) is a devastating neurological disease with high mortality and morbidity. The microglia activation and peripheral inflammatory cells infiltration play an important role in the ICH prognosis. Previous studies have demonstrated that regulatory T cells (Tregs) ameliorated neuroinflammation following experimental ICH. However, the specific molecular mechanism underlying such effects of Tregs remains unclear. In the present study, our aims were to examine the effect of Tregs recruitment induced by recombinant CC chemokine ligand 17(rCCL17) in an intrastriatal autologous blood mouse model of ICH and to determine whether the TGF-β/TGF-βR/Smad2/3 pathway was involved in Tregs promoted M2 microglia/macrophage polarization. Methods A total of 404 adult CD1 mice (male, eight-week-old) were subject to sham surgery or autologous blood injection of ICH. A CD25-specific mouse antibody or isotype control mAb was injected intraperitoneally 48h prior to ICH induction to deplete Tregs. Recombinant CCL17 (rCCL17), a C-C chemokine receptor 4 (CCR4), was delivered intranasally at 1 h post-ICH. SB431542, a specific inhibitor of TGF-β was administered intraperitoneally 1 h before ICH induction. Post-ICH assessments included neuro-behavior evaluation, brain edema, hematoma volume, hemoglobin content, western blotting, double immunofluorescence staining and immunohistochemistry. Results Endogenous brain expressions of CCL17 and Tregs marker Foxp3 as well as the number of Tregs in the perihematomal region were increased following ICH. The Tregs deletion by a CD25 antibody aggravated short-term neurological deficits and brain edema, increased the level of inflammatory cytokines and peripheral inflammatory cells infiltration, exacerbated hematoma expansion and increased M1phenotypes of microglia/macrophage in ICH mice. The rCCL17 treatment increased the number of Tregs in the brain, reduced hematoma expansion and brain edema, promoted microglia/macrophage polarization toward M2 phenotypes. Moreover, the expressions of brain TGF-β/phosphorylated-Smad2/3 were increased. The neuroprotective effects of rCCL17 were abolished by co-administration of the selective TGF-β inhibitor SB431542. Conclusions Our study demonstrated rCCL17 recruited of Tregs to brain in the autologous blood injection model of ICH. Tregs promoted microglia/macrophages polarization toward M2 phenotype and alleviation early brain injury, at least in part, through the TGFβ/TGFβR/Smad2/3 signaling pathway in ICH mice. Thus, rCCL17-mediated Tregs recruitment may provide a promising therapeutic strategy to reduce early brain injury after ICH.

Treadmill Exercise Attenuates Cerebral Ischemia–Reperfusion Injury by Promoting Activation of M2 Microglia via Upregulation of Interleukin-4

Background: Exercise has been proven to be an effective therapy for stroke by reducing the microglia-initiated proinflammatory response. Few studies, however, have focused on the phenotypic changes in microglia cells caused by exercise training. The present study was designed to evaluate the influence of treadmill exercise on microglia polarization and the molecular mechanisms involved.Methods: Male Sprague-Dawley rats were randomly assigned into 3 groups: sham, MCAO and exercise. The middle cerebral artery occlusion (MCAO) and exercise groups received MCAO surgery and the sham group a sham operation. The exercise group also underwent treadmill exercise after the surgery. These groups were studied after 4 and 7 days to evaluate behavioral performance using a modified neurological severity score (mNSS), and infarct conditions using 2,3,5-triphenyl tetrazolium chloride. Quantitative real-time polymerase chain reaction (qRT-PCR) and Luminex was employed to determine the expressions of markers of microglia phenotypes. Western blotting was employed to identify the phosphorylation levels of Janus kinase1 (JAK1) and signal transducer and activator of transcription 6 (STAT6). Immunofluorescence was conducted to identify microglia phenotypes.Results: Treadmill exercise was found to improve neurobehavioral outcomes, mainly motor and balance functions, reduce infarct volumes and significantly increase interleukin-4 (IL-4) expression. In addition, treadmill exercise inhibited M1 microglia and promoted M2 microglia activation as evidenced by decreased M1 and increased M2 markers. Furthermore, an obvious increase in p-JAK1 and p-STAT6 was observed in the exercise group.Conclusions: Treadmill exercise ameliorates cerebral ischemia–reperfusion injury by enhancing IL-4 expression to promote M2 microglia polarization, possibly via the JAK1-STAT6 pathway.

Melatonin Reduces Neuroinflammation and Improves Axonal Hypomyelination by Modulating M1/M2 Microglia Polarization via JAK2-STAT3-Telomerase Pathway in Postnatal Rats Exposed to Lipopolysaccharide

Microglia activation and associated inflammation are implicated in the periventricular white matter damage (PWMD) in septic postnatal rats. This study investigated whether melatonin would mitigate inflammation and alleviate the axonal hypomyelination in the corpus callosum in septic postnatal rats. We further explored if this might be related to the modulation of microglial polarization from M1 phenotype to M2 through the JAK2/STAT3/telomerase pathway. We reported here that indeed melatonin not only can it reduce the neurobehavioral disturbances in LPS-injected rats, but it can also dampen microglia-mediated inflammation. Thus, in LPS + melatonin group, the expression of proinflammatory mediators in M1 phenotype microglia was downregulated. As opposed to this, M2 microglia were increased which was accompanied by upregulated expression of anti-inflammatory mediators along with telomerase reverse transcriptase or melatonin receptor 1(MT1). In parallel to this was decreased NG2 expression but increased expression of myelin and neurofilament proteins. Melatonin can improve hypomyelination which was confirmed by electron microscopy. In vitro in primary microglia stimulated by LPS, melatonin decreased the expression of proinflammatory mediators significantly; but it increased the expression of anti-inflammatory mediators. Additionally, the expression levels of p-JAK2 and p-STAT3 were significantly elevated in microglia after melatonin treatment. Remarkably, the effect of melatonin on LPS-treated microglia was blocked by melatonin receptor, JAK2, STAT3 and telomerase reverse transcriptase inhibitors, respectively. Taken together, it is concluded that melatonin can attenuate PWMD through shifting M1 microglia towards M2 via MT1/JAK2/STAT3/telomerase pathway. The results suggest a new therapeutic strategy whereby melatonin may be adopted to convert microglial polarization from M1 to M2 phenotype that would ultimately contribute to the attenuation of PWMD.

The secretome of M1 and M2 microglia differently regulate proliferation, differentiation and survival of adult neural stem/progenitor cell

Microglia has been reported to be able to regulate the proliferation, differentiation and survival of adult Neural stem/progenitor cells (NSPCs) by modulating the microenvironment, which results in different consequences of adult neurogenesis. However, whether the microglial activation is beneficial or harmful to NSPCs is still controversial because of the complexity and variability of microglial activation phenotypes. In this study, we detected the expression levels of M1 marker and M2 marker in IFN-γ- and IL-4-induced microglia at different time, respectively. The phenotypic markers of M1 and M2 microglia were stable for 24 h after removal of IFN-γ and IL-4 intervention, but exhibited different change patterns during the next 24 h. Then, the adult NSPCs were treated by the conditioned medium from IFN-γ- and IL-4-activated microglia. The conditioned medium from IFN-γ-activated microglia promoted apoptosis and astroglial differentiation of NSPCs, while suppressed proliferation and neuronal differentiation of NSPCs. However, the conditioned medium from IL-4-activated microglia exhibited opposite effects on these physiological processes. In addition, the direct treatment of IFN-γ or IL-4 alone did not significantly affect the proliferation, differentiation and survival of NSPCs. These results suggest that the secretome of pro-inflammatory (M1) and anti-inflammatory (M2) microglia differently regulated the proliferation, differentiation and survival of adult NSPCs. These findings will help further study the biological mechanism of microglia regulating neurogenesis, and provide a therapeutic strategy for neurological diseases by regulating microglial phenotypes to affect neurogenesis.

Astrocytes Stimulate Microglial Proliferation and M2 Polarization In Vitro through Crosstalk between Astrocytes and Microglia

Microglia are resident immune cells of the central nervous system that act as brain-specific macrophages and are also known to regulate the innate immune functions of astrocytes through secretory molecules. This communication plays an important role in brain functions and homeostasis as well as in neuropathologic disease. In this study, we aimed to elucidate whether astrocytes and microglia could crosstalk to induce microglial polarization and proliferation, which can be further regulated under a microenvironment mimicking that of brain stroke. Microglia in a mixed glial culture showed increased survival and proliferation and were altered to M2 microglia; CD11b−GFAP+ astrocytes resulted in an approximately tenfold increase in microglial cell proliferation after the reconstitution of astrocytes. Furthermore, GM-CSF stimulated microglial proliferation approximately tenfold and induced them to become CCR7+ M1 microglia, which have a phenotype that could be suppressed by anti-inflammatory cytokines such as IL-4, IL-10, and substance P. In addition, the astrocytes in the microglial co-culture showed an A2 phenotype; they could be activated to A1 astrocytes by TNF-α and IFN-γ under the stroke-mimicking condition. Altogether, astrocytes in the mixed glial culture stimulated the proliferation of the microglia and M2 polarization, possibly through the acquisition of the A2 phenotype; both could be converted to M1 microglia and A1 astrocytes under the inflammatory stroke-mimicking environment. This study demonstrated that microglia and astrocytes could be polarized to M2 microglia and A2 astrocytes, respectively, through crosstalk in vitro and provides a system with which to explore how microglia and astrocytes may behave in the inflammatory disease milieu after in vivo transplantation.