Stefanie Ebert

Microglia in the healthy and degenerating retina: insights from novel mouse models.

In contrast to the tremendous amount of research data from the central nervous system, relatively little is known about microglial homeostasis in the retina. This may be explained by a strong research bias towards important brain pathologies including Alzheimers disease, Parkinsons disease, and Multiple Sclerosis. In addition, there are specific technical limitations which hampered the analysis of retinal microglia, including their relatively small number in ocular tissue. The lack of experimental tools also prevented direct visualization and molecular analysis of this specialized neuronal macrophage population. Over the last few years, this situation has changed considerably as more and more retinal disorders have come into focus. Many rare monogenic forms as well as more prevalent complex disorders, in particular the age-related macular degeneration involves innate immune mechanisms. As a consequence, new genetic and experimental mouse models have been developed that mimic various forms of human retinal degeneration. In conjunction with these disease models, novel macrophage/microglia-specific reporter mice were established that allow the monitoring of retinal microglia in situ and in vivo. This review summarizes recent findings from these mouse models and thereby provides an overview of microglial homeostasis in the healthy and degenerating retina. Based on this knowledge, microglia-targeted therapies are envisioned which could delay or attenuate degenerative retinal disease.

Luteolin triggers global changes in the microglial transcriptome leading to a unique anti-inflammatory and neuroprotective phenotype

BackgroundLuteolin, a plant derived flavonoid, exerts a variety of pharmacological activities and anti-oxidant properties associated with its capacity to scavenge oxygen and nitrogen species. Luteolin also shows potent anti-inflammatory activities by inhibiting nuclear factor kappa B (NFkB) signaling in immune cells. To better understand the immuno-modulatory effects of this important flavonoid, we performed a genome-wide expression analysis in pro-inflammatory challenged microglia treated with luteolin and conducted a phenotypic and functional characterization.MethodsResting and LPS-activated BV-2 microglia were treated with luteolin in various concentrations and mRNA levels of pro-inflammatory markers were determined. DNA microarray experiments and bioinformatic data mining were performed to capture global transcriptomic changes following luteolin stimulation of microglia. Extensive qRT-PCR analyses were carried out for an independent confirmation of newly identified luteolin-regulated transcripts. The activation state of luteolin-treated microglia was assessed by morphological characterization. Microglia-mediated neurotoxicity was assessed by quantifying secreted nitric oxide levels and apoptosis of 661W photoreceptors cultured in microglia-conditioned medium.ResultsLuteolin dose-dependently suppressed pro-inflammatory marker expression in LPS-activated microglia and triggered global changes in the microglial transcriptome with more than 50 differentially expressed transcripts. Pro-inflammatory and pro-apoptotic gene expression was effectively blocked by luteolin. In contrast, mRNA levels of genes related to anti-oxidant metabolism, phagocytic uptake, ramification, and chemotaxis were significantly induced. Luteolin treatment had a major effect on microglial morphology leading to ramification of formerly amoeboid cells associated with the formation of long filopodia. When co-incubated with luteolin, LPS-activated microglia showed strongly reduced NO secretion and significantly decreased neurotoxicity on 661W photoreceptor cultures.ConclusionsOur findings confirm the inhibitory effects of luteolin on pro-inflammatory cytokine expression in microglia. Moreover, our transcriptomic data suggest that this flavonoid is a potent modulator of microglial activation and affects several signaling pathways leading to a unique phenotype with anti-inflammatory, anti-oxidative, and neuroprotective characteristics. With the identification of several novel luteolin-regulated genes, our findings provide a molecular basis to understand the versatile effects of luteolin on microglial homeostasis. The data also suggest that luteolin could be a promising candidate to develop immuno-modulatory and neuroprotective therapies for the treatment of neurodegenerative disorders.

Docosahexaenoic acid attenuates microglial activation and delays early retinal degeneration

Microgliosis is a common phenomenon in neurodegenerative disorders including retinal dystrophies. We performed a detailed characterization of activated microglia in the retinoschisin (Rs1h)‐deficient (Rs1h−/Y) mouse model of inherited retinal degeneration. To visualize and isolate microglia, we crossed Rs1h−/Y animals with transgenic MacGreen mice, which express green fluorescent protein under the control of the macrophage‐specific csf1r promoter. Activated microglia were detected in retinal sections and whole‐mounts of early postnatal MacGreen/Rs1h−/Y mice before the onset of overt neuronal cell death. These activated microglia contained prominent lipid droplets and analysis of the retinal lipid composition showed decreased docosahexaenoic acid (DHA) levels in Rs1h−/Y retinas. To establish a link between microglia activation, reduced DHA levels, and neurodegeneration, a dietary intervention study was performed. Female Rs1h−/− mice and their Rs1h−/Y litter were either subjected to a diet enriched with DHA, or a control chow lacking DHA. Supplementation with DHA enhanced photoreceptor survival and converted activated microglia to a quiescent phenotype. Furthermore, DHA, but not docosapentaenoic acid or adrenic acid reduced pro‐inflammatory gene expression, migration, and lipid accumulation of cultured BV‐2 microglia. We conclude that retinal DHA levels control the activity of microglia and thereby may affect the progression and extent of retinal degeneration.

The Novel Activated Microglia/Macrophage WAP Domain Protein, AMWAP, Acts as a Counter-Regulator of Proinflammatory Response

Microgliosis is a common phenomenon in neurodegenerative disorders, including retinal dystrophies. To identify candidate genes involved in microglial activation, we used DNA-microarray analysis of retinal microglia from wild-type and retinoschisin-deficient (Rs1h−/Y) mice, a prototypic model for inherited retinal degeneration. Thereby, we cloned a novel 76 aa protein encoding a microglia/macrophage-restricted whey acidic protein (WAP) termed activated microglia/macrophage WAP domain protein (AMWAP). The gene consists of three exons and is located on mouse chromosome 11 in proximity to a chemokine gene cluster. mRNA expression of AMWAP was detected in microglia from Rs1h−/Y retinas, brain microglia, and other tissue macrophages. AMWAP transcription was rapidly induced in BV-2 microglia upon stimulation with multiple TLR ligands and IFN-γ. The TLR-dependent expression of AMWAP was dependent on NF-κB, whereas its microglia/macrophage-specific transcription was regulated by PU.1. Functional characterization showed that AMWAP overexpression reduced the proinflammatory cytokines IL-6 and IL-1β and concomitantly increased expression of the alternative activation markers arginase 1 and Cd206. Conversely, small interfering RNA knockdown of AMWAP lead to higher IL-6, IL-1β, and Ccl2 transcript levels, whereas diminishing arginase 1 and Cd206 expression. Moreover, AMWAP expressing cells had less migratory capacity and showed increased adhesion in a trypsin-protection assay indicating antiserine protease activity. In agreement with findings from other WAP proteins, micromolar concentrations of recombinant AMWAP exhibited significant growth inhibitory activity against Escherichia coli, Pseudomonas aeruginosa, and Bacillus subtilis. Taken together, we propose that AMWAP is a counter-regulator of proinflammatory microglia/macrophage activation and a potential modulator of innate immunity in neurodegeneration.

Chondroitin sulfate disaccharide stimulates microglia to adopt a novel regulatory phenotype

A disaccharide degradation product of chondrotin sulfate proteoglycan‐disaccharide (CSPG‐DS) has been implicated previously in the inhibition of neurodegeneration by influencing microglia activation. In this study, genome‐wide microarray analysis was used to identify specific gene expression profiles of CSPG‐DS‐stimulated BV‐2 microglia‐like cells. Gene products involved in phagocytosis, detoxification, migration, immune regulation, and antigen presentation were found to be altered significantly. These findings were replicated and compared with IFN‐γ‐stimulated primary microglia using real‐time quantitative RT‐PCR validation. Importantly, a unique transcriptional phenotype with anti‐inflammatory and IFN‐γ counter‐regulatory properties partially related to alternatively activated macrophages was identified. Using functional cell assays, we found that CSPG‐DS‐stimulated microglia possess increased phagocytic capacity but lack direct cytotoxic effects such as secretion of NO. Furthermore, conditioned media from CSPG‐DS‐treated microglia did not diminish the viability or cause apoptosis of cultured photoreceptor cells and partially rescued these cells from IFN‐γ‐induced apoptosis. Taken together, our data provide a unique transcript dataset and important in vitro findings about the functional properties of CSPG‐DS‐activated microglia. These might be starting points to explore the in vivo role of CSPG‐DS as a bioactive microglia regulator and its potential, therapeutic application in immune‐related, neurodegenerative disorders.

High glucose, unsaturated and saturated fatty acids differentially regulate expression of ATP-binding cassette transporters ABCA1 and ABCG1 in human macrophages.

The ATP-binding cassette transporters ABCA1 and ABCG1 are highly expressed in macrophage-derived foam cells and promote reverse cholesterol efflux via biogenesis of high-density lipoproteins. The aim of this study was to analyze the direct effects of bioactive factors related to the metabolic syndrome on macrophage transcript levels of all 47 human ABC transporters. Using in vitro M-CSF predifferentiated macrophages and TaqMan low density arrays we could show that linoleic acid, palmitic acid, and high glucose levels have a major impact on ABCA1 and ABCG1 expression but do not strongly affect most other human ABC transporters. In Western blot experiments we demonstrate that ABCA1 and ABCG1 protein levels are synchronously suppressed by high glucose levels and the ω6-unsaturated fatty acid linoleic acid. We conclude that metabolites associated with the metabolic syndrome enhance the formation of atherosclerotic lesions by diminishing the reverse cholesterol transport function of ABCA1 and ABCG1.

Dap12 expression in activated microglia from retinoschisin- deficient retina and its PU.1-dependent promoter regulation

Several alterations in the expression of immune‐related transcripts were identified recently in the degenerating retina of the retinoschisin knockout (Rs1h−/Y) mouse, including the strong expression of the adaptor protein Dap12. As Dap12 is found in leukocytes, we hypothesized that its disease‐related expression may be confined to activated retinal microglia cells. To test this hypothesis, we established a procedure for isolation and culture of retinal microglia cells and performed genome‐wide expression profiling from Rs1h−/Y and control microglia. While retaining their activated state in culture, ex vivo microglia expressed high levels of Dap12 and the transcription factor PU.1. The activation‐dependent induction of Dap12 was also confirmed in the microglia cell line BV‐2 following in vitro stimulation. To examine the transcriptional regulation of Dap12 further, macrophage cell lines were transfected with several Dap12 reporter constructs. Promoter deletion assays and site‐directed mutagenesis experiments demonstrated an essential role of evolutionarily conserved PU.1 consensus sites in the proximal −104/+118 Dap12 promoter. In vitro and in vivo binding of PU.1 to this promoter region was demonstrated using EMSA and chromatin immunoprecipitation. Knockdown of PU.1 by RNA interference caused a significant reduction of endogenous Dap12 expression and re‐expression, and activation of PU.1 in PU.1−/− progenitor cells induced Dap12 transcription. Taken together, our results indicate that activated microglia from degenerating retinae express high levels of Dap12 and PU.1, and PU.1 controls the myeloid‐specific regulation of Dap12 directly and may also play a general role in microglia gene expression during retinal degeneration.

Induction of Early Growth Response-1 Mediates Microglia Activation In Vitro But is Dispensable In Vivo

We have previously identified activation of microglia and induction of the early growth response gene 1 (Egr1) in the retina of retinoschisin-deficient (Rs1h−/Y) mice. We hypothesized that microglial expression of Egr1 might support retinal microgliosis. To test this, Egr1 transcript levels were determined in RNAs isolated from early postnatal retinas and primary microglia from Rs1h−/Y mice and wild-type controls. Egr1 mRNA expression was strongly induced in retinoschisin-deficient retinas as well as in ex vivo isolated microglia. Increased microglial Egr1 protein expression was concordantly detected in retinal sections of Rs1h−/Y mice using immunohistochemistry. Prominent activation-dependent Egr1 mRNA and protein expression was also confirmed in murine BV-2 microglia. Using binding site prediction and chromatin immunoprecipitation, we identified that the Egr1 promoter itself and the microglial marker genes Clec7a and Caspase11 are direct transcriptional targets of Egr1. Over-expression of Egr1 in BV-2 cells by adenoviral infection promoted Clec7a and Caspase11 mRNA synthesis, whereas expression of the Egr1 repressor NAB2 blocked the transcription of these genes. To analyze whether Egr1 was absolutely required for microglial marker expression in vivo, transcript levels were quantified in Rs1h−/Y/Egr1−/− retinas. No significant differences in activation marker expression could be measured in retinal tissue from Rs1h−/Y/Egr1−/− mice compared to Rs1h−/Y mice, suggesting that lack of Egr1 does not impair transcription of microglia genes in vivo. Taken together, our findings suggest that increased Egr1 expression is present in activated retinal microglia and contributes to their activation. However, up-regulation of Egr1 is not absolutely required for retinal microglia activation in vivo.

Induction of STAP-1 promotes neurotoxic activation of microglia.

Activated microglia contribute to neurodegenerative processes in the brain and the retina. Via DNA-microarray analysis, we have previously identified up-regulation of several immune-related genes in the dystrophic retina of retinoschisin-deficient (Rs1h(-/Y)) mice. Here we report a strong overexpression of transcripts for the signal-transducing adaptor protein-1 (STAP-1) in isolated Rs1h(-/Y) microglia. Furthermore, STAP-1 expression was induced in activated bone marrow-derived macrophages as well as LPS-, interferon-gamma-, and CpG-stimulated myeloid cell lines. Ectopic expression of STAP-1 in BV-2 microglia changed the morphology and cytoskeletal organization of the cells and transformed ramified cells to an activated state. STAP-1 overexpression also leads to an interaction with the M-CSF receptor/c-Fms diminishing its ligand-dependent phosphorylation. Finally, STAP-1 expressing cells showed strongly reduced migration with increased cytotoxicity against 661W photoreceptor like cells. Taken together, our study implicates a previously unknown role of STAP-1 in pro-inflammatory microglia activation potentially contributing to neuronal apoptosis and degeneration.

Microglial Activation and Transcriptomic Changes in the Blue Light-Exposed Mouse Retina

Microglia are important components of the ocular immune system and may contribute to age-related macular degeneration. Exposure to blue light induces oxidative protein modifications similar to those observed in drusen and elicits retinal immune responses. To study the underlying cellular events, we analyzed microglial activation and monitored transcriptomic changes in blue light-induced retinal lesions. MacGreen mice with EGFP-tagged retinal microglia were exposed to blue light. At different time intervals, eyes were prepared for immunofluorescence microscopy, microarray analysis, and qRT-PCR. Retinal whole mounts and cross sections showed that EGFP-labeled microglia rapidly migrated toward the retinal lesion. Prominent transcriptomic changes occurred after 12 h, peaked at 24 h, and declined at 72 h. We identified more than 100 differentially expressed genes, including transcripts related to microglial activation, apoptosis, and regenerative signaling. A comparison of our results with published datasets from white light damage indicates overlapping but also distinct molecular mechanisms. This study extends our knowledge of transcriptomic changes in light-induced models of retinal degeneration.