Murine iPSC-derived microglia and macrophage cell culture models recapitulate distinct phenotypical and functional properties of classical and alternative neuro-immune polarisation.

The establishment and validation of reliable induced pluripotent stem cell (iPSC)-derived in vitro models to study microglia and monocyte/macrophage immune function holds great potential for fundamental and translational neuro-immunology research. In this study, we first demonstrate that ramified CX3CR1+ iPSC-microglia (cultured within a neural environment) and round-shaped CX3CR1- iPSC-macrophages can easily be differentiated from newly established murine CX3CR1eGFP/+CCR2RFP/+ iPSC lines. Furthermore, we show that obtained murine iPSC-microglia and iPSC-macrophages are distinct cell populations, even though iPSC-macrophages may upregulate CX3CR1 expression when cultured within a neural environment. Next, we characterized the phenotypical and functional properties of murine iPSC-microglia and iPSC-macrophages following classical and alternative immune polarisation. While iPSC-macrophages could easily be triggered to adopt a classically-activated or alternatively-activated phenotype following, respectively, lipopolysaccharide + interferon γ or interleukin 13 (IL13) stimulation, iPSC-microglia and iPSC-macrophages cultured within a neural environment displayed a more moderate activation profile as characterised by the absence of MHCII expression upon classical immune polarisation and the absence of Ym1 expression upon alternative immune polarisation. Finally, extending our preceding in vivo studies, this striking phenotypical divergence was also observed for resident microglia and infiltrating monocytes within highly inflammatory cortical lesions in CX3CR1eGFP/+CCR2RFP/+ mice subjected to middle cerebral arterial occlusion (MCAO) stroke and following IL13-mediated therapeutic intervention thereon. In conclusion, our study demonstrates that the applied murine iPSC-microglia and iPSC-macrophage culture models are able to recapitulate in vivo microglia and monocyte/macrophage ontogeny and corresponding phenotypical/functional properties upon classical and alternative immune polarisation, and therefore represent a valuable in vitro platform to further study and modulate microglia and (infiltrating) monocyte immune responses under neuro-inflammatory conditions within a neural environment.

Targeted intracerebral delivery of the anti-inflammatory cytokine IL13 promotes alternative activation of both microglia and macrophages after stroke.

BACKGROUND: Subtle adjustment of the activation status of CNS resident microglia and peripheral macrophages, to promote their neuroprotective and neuroregenerative functions, may facilitate research towards curing neurodegenerative disorders. In the present study, we investigated whether targeted intracerebral delivery of the anti-inflammatory cytokine interleukin (IL)13, by means of transplanting IL13-expressing mesenchymal stem cells (IL13-MSCs), can promote a phenotypic switch in both microglia and macrophages during the pro-inflammatory phase in a mouse model of ischemic stroke. METHODS: We used the CX3CR1eGFP/+ CCR2RFP/+ transgenic mouse model to separately recognize brain-resident microglia from infiltrated macrophages. Quantitative immunohistochemical analyses were applied to characterize polarization phenotypes of both cell types. RESULTS: Distinct behaviors of both cell populations were noted dependent on the anatomical site of the lesion. Immunohistochemistry revealed that mice grafted with IL13-MSCs, in contrast to non-grafted and MSC-grafted control mice, were able to drive recruited microglia and macrophages into an alternative activation state, as visualized by a significant increase of Arg-1 and a noticeable decrease of MHC-II expression at day 14 after ischemic stroke. Interestingly, both Arg-1 and MHC-II were expressed more abundantly in macrophages than in microglia, further confirming the distinct behavior of both cell populations. CONCLUSIONS: The current data highlight the importance of controlled and localized delivery of the anti-inflammatory cytokine IL13 for modulation of both microglia and macrophage responses after ischemic stroke, thereby providing pre-clinical rationale for the application of L13-MSCs in future investigations of neurodegenerative disorders.

Individual in vivo Profiles of Microglia Polarization After Stroke, Represented by the Genes iNOS and Ym1.

Microglia are the brain-innate immune cells which actively surveil their environment and mediate multiple aspects of neuroinflammation, due to their ability to acquire diverse activation states and phenotypes. Simplified, M1-like microglia are defined as pro-inflammatory cells, while the alternative M2-like cells promote neuroprotection. The modulation of microglia polarization is an appealing neurotherapeutic strategy for stroke and other brain lesions, as well as neurodegenerative diseases. However, the activation profile and change of phenotype during experimental stroke is not well understood. With a combined magnetic resonance imaging (MRI) and optical imaging approach and genetic targeting of two key genes of the M1- and M2-like phenotypes, iNOS and Ym1, we were able to monitor in vivo the dynamic adaption of the microglia phenotype in response to experimental stroke.

MiRNA-124 induces neuroprotection and functional improvement after focal cerebral ischemia.

microRNA-124 (miR-124), the most abundant miRNA of the CNS, was recently shown to modulate the polarization of activated microglia and infiltrating macrophages towards the anti-inflammatory M2 phenotype and protect neurons in various ways after brain disease. In ischemic stroke, microglia and macrophages of a detrimental and persistent pro-inflammatory M1 phenotype have been shown to aggravate the secondary injury. Thus, shifting the polarization of microglia/macrophages into the beneficial, anti-inflammatory M2-like phenotype is considered neuroprotective after stroke onset. Here, we have induced 30 min transient occlusion of the right middle cerebral artery (MCAO) in 34 male, C57BL/6 mice. Lesion development was monitored with T2-weighted MRI. Liposomated miR-124 was injected in 11 animals at 48 h and in 5 animals at 10 days after MCAO. Arg-1, a marker for M2 phenotype, was co-stained with Iba-1, NeuN or GFAP. The distribution of astrocytes, neurons and microglia/macrophages and their expression of Arg-1 were quantified. Early miR-124 injection resulted in a significantly increased neuronal survival and a significantly increased number of M2-like polarized microglia/macrophages. Moreover, the lesion core, delineated by reactive astrocytes, was significantly reduced over time upon early miR-124 injection. These neuroprotective and anti-inflammatory effects of the early miR-124 treatment were pronounced during the first week with Arg-1. Number of Arg-1+ microglia/macrophages correlated with neuronal protection and with functional improvement during the first week. Thus, our present results demonstrate that miR-124 may serve as a novel therapeutic strategy for neuroprotection and functional recovery upon stroke onset.

Dynamic Modulation of Microglia/Macrophage Polarization by miR-124 after Focal Cerebral Ischemia.

Mononuclear phagocytes respond to ischemic stroke dynamically, undergoing an early anti-inflammatory and protective phenotype followed by the pro-inflammatory and detrimental type. These dual roles of microglia/macrophages suggest the need of subtle adjustment of their polarization state instead of broad suppression. The most abundant brain-specific miRNA, miR-124, promotes neuronal differentiation but can also modulate microglia activation and keeps them in a quiescent state. We addressed whether the intracerebral injection of miR-124 in a mouse model of ischemic stroke before or after the peak phase of the pro-inflammatory polarization modifies the pro-/anti- inflammatory balance. In the sub-acute phase, 48 h after stroke, liposomated miR-124 shifted the predominantly pro-inflammatory polarized microglia/macrophages toward the anti-inflammatory phenotype. The altered immune response improved neurological deficit at day 6 after stroke. When miR-124 was injected 10 days after stroke, the pro-/anti- inflammatory ratio was still significantly reduced although to a lower degree and had no effect on recovery at day 14. This study indicates that miR-124 administration before the peak of the pro-inflammatory process of stroke is most effective in support of increasing the rehabilitation opportunity in the sub-acute phases of stroke. Our findings highlight the important role of immune cells after stroke and the therapeutic relevance of their polarization balance.