In-depth molecular profiling specifies human retinal microglia identity

Microglia are the tissue-resident macrophages of the retina and brain, being critically involved in organ development, tissue homeostasis, and response to cellular damage. Until now, little is known about the molecular signature of human retinal microglia and how it differs from the one of brain microglia and peripheral monocytes. In addition, it is not yet clear to what extent murine retinal microglia resemble those of humans, which represents an important prerequisite for translational research. The present study applies fluorescence-activated cell sorting to isolate human retinal microglia from enucleated eyes and compares their transcriptional profile with the one of whole retinal tissue, human brain microglia as well as classical, intermediate and non-classical monocytes. Finally, human retinal microglia are compared to murine retinal microglia, isolated from Cx3cr1 GFP/+ mice. Whereas human retinal microglia exhibited a high grade of similarity in comparison to their counterparts in the brain, several enriched genes were identified in retinal microglia when compared to whole retinal tissue, as well as classical, intermediate, and non-classical monocytes. In relation to whole retina sequencing, several risk genes associated with age-related macular degeneration (AMD) and diabetic retinopathy (DR) were preferentially expressed in retinal microglia, indicating their potential pathophysiological involvement. Although a high degree of similarity was observed between human and murine retinal microglia, several species-specific genes were identified, which should be kept in mind when employing mouse models to investigate retinal microglia biology. In summary, this study provides detailed insights into the molecular profile of human retinal microglia, identifies a plethora of tissue-specific and species-specific genes in comparison to human brain microglia and murine retinal microglia, and thus highlights the significance of retinal microglia in human retinal diseases and for translational research approaches

Cre recombinase expression or topical tamoxifen treatment do not affect retinal structure and function, neuronal vulnerability or glial reactivity in the mouse eye

Mice with a constitutive or tamoxifen-induced Cre recombinase (Cre) expression are frequently used research tools to allow the conditional deletion of target genes via the Cre-loxP system. Here we analyzed for the first time in a comprehensive and comparative way, whether retinal Cre expression or topical tamoxifen treatment itself would cause structural or functional changes, including changes in the expression profiles of molecular markers, glial reactivity and photoreceptor vulnerability. To this end, we characterized the transgenic alpha-Cre, Lmop-Cre and the tamoxifen-inducible CAGG-CreER (TM) mouse lines, all having robust Cre expression in the neuronal retina. In addition, we characterized the effects of topical tamoxifen treatment itself in wildtype mice. We performed morphometric analyses, immunohistochemical staining, in vivo ERG and angiography analyses and realtime RT-PCR analyses. Furthermore, the influence of Cre recombinase or topical tamoxifen exposure on neuronal vulnerability was studied by using light damage as a model for photoreceptor degeneration. Taken together, neither the expression of Cre, nor topical tamoxifen treatment caused detectable changes in retinal structure and function, the expression profiles of investigated molecular markers, glial reactivity and photoreceptor vulnerability. We conclude that the Cre-loxP system and its induction through tamoxifen is a safe and reliable method to delete desired target genes in the neural retina. (C) 2016 IBRO. Published by Elsevier Ltd. All rights reserved

Transcriptional Profiling Identifies Upregulation of Neuroprotective Pathways in Retinitis Pigmentosa

Hereditary retinal degenerations like retinitis pigmentosa (RP) are among the leading causes of blindness in younger patients. To enable in vivo investigation of cellular and molecular mechanisms responsible for photoreceptor cell death and to allow testing of therapeutic strategies that could prevent retinal degeneration, animal models have been created. In this study, we deeply characterized the transcriptional profile of mice carrying the transgene rhodopsin V20G/P23H/P27L (VPP), which is a model for autosomal dominant RP. We examined the degree of photoreceptor degeneration and studied the impact of the VPP transgene-induced retinal degeneration on the transcriptome level of the retina using next generation RNA sequencing (RNASeq) analyses followed by weighted correlation network analysis (WGCNA). We furthermore identified cellular subpopulations responsible for some of the observed dysregulations using in situ hybridizations, immunofluorescence staining, and 3D reconstruction. Using RNASeq analysis, we identified 9256 dysregulated genes and six significantly associated gene modules in the subsequently performed WGCNA. Gene ontology enrichment showed, among others, dysregulation of genes involved in TGF-β regulated extracellular matrix organization, the (ocular) immune system/response, and cellular homeostasis. Moreover, heatmaps confirmed clustering of significantly dysregulated genes coding for components of the TGF-β, G-protein activated, and VEGF signaling pathway. 3D reconstructions of immunostained/in situ hybridized sections revealed retinal neurons and Müller cells as the major cellular population expressing representative components of these signaling pathways. The predominant effect of VPP-induced photoreceptor degeneration pointed towards induction of neuroinflammation and the upregulation of neuroprotective pathways like TGF-β, G-protein activated, and VEGF signaling. Thus, modulation of these processes and signaling pathways might represent new therapeutic options to delay the degeneration of photoreceptors in diseases like RP

Transcriptional profiling uncovers human hyalocytes as a unique innate immune cell population

PURPOSE: To decipher the transcriptional signature of macrophages of the human vitreous, also known as hyalocytes, and compare it to the profiles of other myeloid cell populations including human blood-derived monocytes, macrophages, and brain microglia. METHODS: This study involves a total of 13 patients of advanced age with disorders of the vitreoretinal interface undergoing vitrectomy at the University Eye Hospital Freiburg between 2018 and 2019. Vitreal hyalocytes were analyzed by fluorescence-activated cell sorting (FACS) and isolated as CD45+CD11b+CX3CR1+Mat-Mac+ cells using a FACS-based sorting protocol. RNA extraction, library preparation and RNA sequencing were performed and the sequencing data was analyzed using the Galaxy web platform. The transcriptome of human hyalocytes was compared to the transcriptional profile of human blood-derived monocytes, macrophages and brain microglia obtained from public databases. Protein validation for selected factors was performed by immunohistochemistry on paraffin sections from three human donor eyes. RESULTS: On average, 383 ± 233 hyalocytes were isolated per patient, resulting in 128 pg/μl ± 76 pg/μl total RNA per sample. RNA sequencing revealed that SPP1, FTL, CD74, and HLA-DRA are among the most abundantly expressed genes in hyalocytes, which was confirmed by immunofluorescence for CD74, FTL, and HLA-DRA. Gene ontology (GO) enrichment analysis showed that biological processes such as “humoral immune response,” “leukocyte migration,” and “antigen processing and presentation of peptide antigen” (adjusted p 0.637, p < 0.001), hyalocytes demonstrated significant differences with respect to common leukocyte-associated factors. In particular, transcripts involved in the immune privilege of the eye, such as POMC, CD46, and CD86, were significantly increased in hyalocytes compared to other myeloid cell subsets. CONCLUSION: Human hyalocytes represent a unique and distinct innate immune cell population specialized and adapted for the tissue-specific needs in the human vitreous. Vitreal hyalocytes are characterized by a strong expression of genes related to antigen processing and presentation as well as immune modulation. Thus, hyalocytes may represent an underestimated mediator in vitreoretinal disease and for the immune privilege of the eye

Deficiency in retinal TGFβ signaling aggravates neurodegeneration by modulating pro-apoptotic and MAP kinase pathways

Transforming growth factor β (TGFβ) signaling has manifold functions such as regulation of cell growth, differentiation, migration, and apoptosis. Moreover, there is increasing evidence that it also acts in a neuroprotective manner. We recently showed that TGFβ receptor type 2 (Tgfbr2) is upregulated in retinal neurons and Müller cells during retinal degeneration. In this study we investigated if this upregulation of TGFβ signaling would have functional consequences in protecting retinal neurons. To this end, we analyzed the impact of TGFβ signaling on photoreceptor viability using mice with cell type-specific deletion of Tgfbr2 in retinal neurons and Müller cells (Tgfbr2$_{ΔOC}$) in combination with a genetic model of photoreceptor degeneration (VPP). We examined retinal morphology and the degree of photoreceptor degeneration, as well as alterations of the retinal transcriptome. In summary, retinal morphology was not altered due to TGFβ signaling deficiency. In contrast, VPP-induced photoreceptor degeneration was drastically exacerbated in double mutant mice (Tgfbr2$_{ΔOC}$; VPP) by induction of pro-apoptotic genes and dysregulation of the MAP kinase pathway. Therefore, TGFβ signaling in retinal neurons and Müller cells exhibits a neuroprotective effect and might pose promising therapeutic options to attenuate photoreceptor degeneration in humans

Deficiency in Retinal TGF&beta; Signaling Aggravates Neurodegeneration by Modulating Pro-Apoptotic and MAP Kinase Pathways

Transforming growth factor &beta; (TGF&beta;) signaling has manifold functions such as regulation of cell growth, differentiation, migration, and apoptosis. Moreover, there is increasing evidence that it also acts in a neuroprotective manner. We recently showed that TGF&beta; receptor type 2 (Tgfbr2) is upregulated in retinal neurons and M&uuml;ller cells during retinal degeneration. In this study we investigated if this upregulation of TGF&beta; signaling would have functional consequences in protecting retinal neurons. To this end, we analyzed the impact of TGF&beta; signaling on photoreceptor viability using mice with cell type-specific deletion of Tgfbr2 in retinal neurons and M&uuml;ller cells (Tgfbr2&Delta;OC) in combination with a genetic model of photoreceptor degeneration (VPP). We examined retinal morphology and the degree of photoreceptor degeneration, as well as alterations of the retinal transcriptome. In summary, retinal morphology was not altered due to TGF&beta; signaling deficiency. In contrast, VPP-induced photoreceptor degeneration was drastically exacerbated in double mutant mice (Tgfbr2&Delta;OC; VPP) by induction of pro-apoptotic genes and dysregulation of the MAP kinase pathway. Therefore, TGF&beta; signaling in retinal neurons and M&uuml;ller cells exhibits a neuroprotective effect and might pose promising therapeutic options to attenuate photoreceptor degeneration in humans