Deciphering the molecular signature of human hyalocytes in relation to other innate immune cell populations

PURPOSE: Hyalocytes are the tissue-resident innate immune cell population of the vitreous body with important functions in health and vitreoretinal disease. The purpose of this study is to gain new insights into the biology and function of human hyalocytes in comparison to other innate immune cells. METHODS: The present study applies fluorescence-activated cell sorting and RNA sequencing to compare the transcriptional profiles of human hyalocytes, retinal microglia (rMG) and classical, intermediate, and non-classical monocytes isolated from the same patients. Immunohistochemistry was applied for morphological characterization of human hyalocytes. RESULTS: Pairwise analysis indicates distinct differences between hyalocytes and monocytes, whereas a high degree of similarity to rMG is apparent, with comparable expression levels of established microglia markers, such as TREM2, P2RY12, and TMEM119. Among the top expressed genes in hyalocytes, SPP1, CD74, and C3, were significantly upregulated when compared with monocytes. Despite the high level of similarity of hyalocytes and rMG, ten highly expressed genes in hyalocytes compared to microglia were identified, among them FOS, DUSP1, and EGR2. CONCLUSIONS: This study reveals a high degree of similarity between hyalocytes and retinal microglia. Nevertheless, hyalocytes exhibit some expression differences that may adapt them to the specific needs of the vitreous and provide the basis for deciphering the multiple roles of this fascinating cell population in health and vitreoretinal diseases

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

Immunosenescence in choroidal neovascularization (CNV) — transcriptional profiling of naïve and CNV-associated retinal myeloid cells during aging

Immunosenescence is considered a possible factor in the development of age-related macular degeneration and choroidal neovascularization (CNV). However, age-related changes of myeloid cells (MCs), such as microglia and macrophages, in the healthy retina or during CNV formation are ill-defined. In this study, Cx3cr1-positive MCs were isolated by fluorescence-activated cell sorting from six-week (young) and two-year-old (old) Cx3cr1(GFP)(/+) mice, both during physiological aging and laser-induced CNV development. High-throughput RNA-sequencing was performed to define the age-dependent transcriptional differences in MCs during physiological aging and CNV development, complemented by immunohistochemical characterization and the quantification of MCs, as well as CNV size measurements. These analyses revealed that myeloid cells change their transcriptional profile during both aging and CNV development. In the steady state, senescent MCs demonstrated an upregulation of factors contributing to cell proliferation and chemotaxis, such as Cxcl13 and Cxcl14, as well as the downregulation of microglial signature genes. During CNV formation, aged myeloid cells revealed a significant upregulation of angiogenic factors such as Arg1 and Lrg1 concomitant with significantly enlarged CNV and an increased accumulation of MCs in aged mice in comparison to young mice. Future studies need to clarify whether this observation is an epiphenomenon or a causal relationship to determine the role of immunosenescence in CNV formation

Transcriptional and distributional profiling of microglia in retinal angiomatous proliferation

Macular neovascularization type 3, formerly known as retinal angiomatous proliferation (RAP), is a hallmark of age-related macular degeneration and is associated with an accumulation of myeloid cells, such as microglia (MG) and infiltrating blood-derived macrophages (MAC). However, the contribution of MG and MAC to the myeloid cell pool at RAP sites and their exact functions remain unknown. In this study, we combined a microglia-specific reporter mouse line with a mouse model for RAP to identify the contribution of MG and MAC to myeloid cell accumulation at RAP and determined the transcriptional profile of MG using RNA sequencing. We found that MG are the most abundant myeloid cell population around RAP, whereas MAC are rarely, if ever, associated with late stages of RAP. RNA sequencing of RAP-associated MG showed that differentially expressed genes mainly contribute to immune-associated processes, including chemotaxis and migration in early RAP and proliferative capacity in late RAP, which was confirmed by immunohistochemistry. Interestingly, MG upregulated only a few angiomodulatory factors, suggesting a rather low angiogenic potential. In summary, we showed that MG are the dominant myeloid cell population at RAP sites. Moreover, MG significantly altered their transcriptional profile during RAP formation, activating immune-associated processes and exhibiting enhanced proliferation, however, without showing substantial upregulation of angiomodulatory factors

Efficient Isolation and Expansion of Limbal Melanocytes for Tissue Engineering

Limbal melanocytes (LMs) are found in the corneoscleral limbus basal epithelial layer and interact with neighboring limbal epithelial progenitor cells. The difficulty of isolating and cultivating LMs is due to the small fraction of LMs in the overall limbal population and the frequent contamination of primary cultures by other cell types. This has limited the research on freshly isolated LMs and the investigation of their biological significance in the maintenance of the limbal stem cell niche. Here, we describe an optimized protocol for the efficient isolation and expansion of LMs from cadaveric corneal limbal tissue using CD90 and CD117 as selective markers in fluorescence-activated cell sorting to obtain a pure population of LMs (CD90− CD117+) with self-renewal capacity and sustained melanin production. The isolation of pure LMs from a single preparation enables direct transcriptomic and proteomic analyses, as well as functional studies on freshly isolated LMs, which can be considered the proper counterparts of LMs in vivo and have potential applications in tissue engineering

PAX6 Expression Patterns in the Adult Human Limbal Stem Cell Niche

Paired box 6 (PAX6), a nuclear transcription factor, determines the fate of limbal epithelial progenitor cells (LEPC) and maintains epithelial cell identity. However, the expression of PAX6 in limbal niche cells, primarily mesenchymal stromal cells (LMSC), and melanocytes is scarce and not entirely clear. To distinctly assess the PAX6 expression in limbal niche cells, fresh and organ-cultured human corneoscleral tissues were stained immunohistochemically. Furthermore, the expression of PAX6 in cultured limbal cells was investigated. Immunostaining revealed the presence of PAX6-negative cells which were positive for vimentin and the melanocyte markers Melan-A and human melanoma black-45 in the basal layer of the limbal epithelium. PAX6 staining was not observed in the limbal stroma. Moreover, the expression of PAX6 was observed by Western blot in cultured LEPC but not in cultured LMSC or LM. These data indicate a restriction of PAX6 expression to limbal epithelial cells at the limbal stem cell niche. These observations warrant further studies for the presence of other PAX isoforms in the limbal stem cell niche

TGF-beta 2-Induced Invadosomes in Human Trabecular Meshwork Cells

Primary open-angle glaucoma (POAG) is a leading cause of blindness due to chronic degeneration of retinal ganglion cells and their optic nerve axons. It is associated with disturbed regulation of intraocular pressure, elevated intraocular levels of TGF-β2, aberrant extracellular matrix (ECM) deposition and increased outflow resistance in the trabecular meshwork (TM). The mechanisms underlying these changes are not fully understood. Cell-matrix interactions have a decisive role in TM maintenance and it has been suggested that TGF-β-induced inhibition of matrix metalloproteases may drive aberrant ECM deposition in POAG. Invadopodia and podosomes (invadosomes) are distinct sites of cell-matrix interaction and localized matrix-metalloprotease (MMP) activity. Here, we report on the effects of TGF-β2 on invadosomes in human trabecular meshwork cells. Human TM (HTM) cells were derived from donor tissue and pretreated with vehicle or TGF-β2 (2 ng/ml) for 3d. Invadosomes were studied in ECM degradation assays, protein expression and MMP-2 activity were assessed by western blot and zymography and ECM protein transcription was detected by RT-qPCR. HTM cells spontaneously formed podosomes and invadopodia as detected by colocalization of Grb2 or Nck1 to sites of gelatinolysis. Pretreatment with TGF-β2 enhanced invadosomal proteolysis and zymographic MMP-2 activity as well as MMP-2, TIMP-2 and PAI-1 levels in HTM cell culture supernatants. Rho-kinase inhibition by H1152 blocked the effects of TGF-β2. Concomitant transcription of fibronectin and collagens-1, -4 and -6 was increased by TGF-β2 and fibrillar fibronectin deposits were observed in areas of invadosomal ECM remodelling. In contrast to a current hypothesis, our data indicate that TGF-β2 induces an active ECM remodelling process in TM cells, characterized by concurrent increases in localized ECM digestion and ECM expression, rather than a mere buildup of material due to a lack of degradation. Invadosomal cell adhesion and signaling may thus have a role in POAG pathophysiology

Subretinal fibrosis in neovascular age-related macular degeneration: current concepts, therapeutic avenues, and future perspectives

Age-related macular degeneration (AMD) is a progressive, degenerative disease of the human retina which in its most aggressive form is associated with the formation of macular neovascularization (MNV) and subretinal fibrosis leading to irreversible blindness. MNVs contain blood vessels as well as infiltrating immune cells, myofibroblasts, and excessive amounts of extracellular matrix proteins such as collagens, fibronectin, and laminin which disrupts retinal function and triggers neurodegeneration. In the mammalian retina, damaged neurons cannot be replaced by tissue regeneration, and subretinal MNV and fibrosis persist and thus fuel degeneration and visual loss. This review provides an overview of subretinal fibrosis in neovascular AMD, by summarizing its clinical manifestations, exploring the current understanding of the underlying cellular and molecular mechanisms and discussing potential therapeutic approaches to inhibit subretinal fibrosis in the future