The Role of Osteopontin in Microglia Biology: Current Concepts and Future Perspectives.

The innate immune landscape of the central nervous system (CNS), including the brain and the retina, consists of different myeloid cell populations with distinct tasks to fulfill. Whereas the CNS borders harbor extraparenchymal CNS-associated macrophages whose main duty is to build up a defense against invading pathogens and other damaging factors from the periphery, the resident immune cells of the CNS parenchyma and the retina, microglia, are highly dynamic cells with a plethora of functions during homeostasis and disease. Therefore, microglia are constantly sensing their environment and closely interacting with surrounding cells, which is in part mediated by soluble factors. One of these factors is Osteopontin (OPN), a multifunctional protein that is produced by different cell types in the CNS, including microglia, and is upregulated in neurodegenerative and neuroinflammatory conditions. In this review, we discuss the current literature about the interaction between microglia and OPN in homeostasis and several disease entities, including multiple sclerosis (MS), Alzheimer's and cerebrovascular diseases (AD, CVD), amyotrophic lateral sclerosis (ALS), age-related macular degeneration (AMD) and diabetic retinopathy (DR), in the context of the molecular pathways involved in OPN signaling shaping the function of microglia. As nearly all CNS diseases are characterized by pathological alterations in microglial cells, accompanied by the disturbance of the homeostatic microglia phenotype, the emergence of disease-associated microglia (DAM) states and their interplay with factors shaping the DAM-signature, such as OPN, is of great interest for therapeutical interventions in the future.

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 Cx3cr1GFP/+ 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.

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.

Comparative transcriptome analysis of human and murine choroidal neovascularization identifies fibroblast growth factor inducible-14 as phylogenetically conserved mediator of neovascular age-related macular degeneration.

BACKGROUND: Visual outcome of patients with neovascular age-related macular degeneration has significantly improved during the last years following the introduction of anti-vascular endothelial growth factor (VEGF) therapy. However, about one third of patients show persistent exudation and decreasing visual acuity despite recurrent anti-VEGF treatment, which implies a role of other, still unknown proangiogenic mediators. METHODS: The present study applied transcriptional profiling of human and mouse (C57BL/6J wildtype) choroidal neovascularization (CNV) membranes each with reference to healthy control tissue to identify yet unrecognized mediators of CNV formation. Key factors were further investigated by immunohistochemistry as well as by intravitreal inhibition experiments and multiplex protein assays in the laser-induced CNV mouse model. FINDINGS: Transcriptional profiles of CNV membranes were characterized by enhanced activation of blood vessel development, cytoskeletal organization, and cytokine production, with angiogenesis and wound healing processes predominating in humans and activation of immune processes in mice. Besides several species-specific factors, 95 phylogenetically conserved CNV-associated genes were detected, among which fibroblast growth factor inducible-14 (FN14), a member of the tumor necrosis factor (TNF) receptor family, was identified as a key player of CNV formation. Blocking the pathway by intravitreal injection of a FN14 decoy receptor modulated the cytokine profile - most notably IL-6 - and led to a significant reduction of CNV size in vivo. INTERPRETATION: This study characterizes the transcriptome of human and mouse CNV membranes in an unprejudiced manner and identifies FN14 as a phylogenetically conserved mediator of CNV formation and a promising new therapeutic target for neovascular AMD. FUNDING: This study was funded by the Helmut Ecker Foundation and the Volker Homann Foundation.

Time- and Stimulus-Dependent Characteristics of Innate Immune Cells in Organ-Cultured Human Corneal Tissue.

PURPOSE: The pattern of immune cells infiltrating the corneal stroma has been extensively studied in mice, but data on human tissue have been far less elaborate. To further characterize the number and differentiation state of resident immune cells in organ-cultured human corneal tissue, we employed a comprehensive bioinformatic deconvolution (xCell) of bulk RNA-sequencing (RNA-seq) data, immunohistochemistry (IHC), and flow cytometry (FC). METHODS: A transcriptome-based analysis of immune cell types in human corneal samples was performed. The results were validated by IHC, focusing on the identification of pro-inflammatory (M1) and regulatory (M2) macrophages. A protocol was established to identify these 2 different macrophage populations in human corneal tissue by means of FC. Subsequently, corneal samples in organ culture were differentially stimulated by IL-10, IL-4 & IL-13, or LPS and macrophage populations were evaluated regarding their response to these stimuli. Furthermore, cell survival was analyzed in correlation with time in organ culture. RESULTS: xCell-based mathematical deconvolution of bulk RNA-seq data revealed the presence of CD8 T cells, Th17 cells, dendritic cells, and macrophages as the predominant immune cell types in organ-cultured human corneal tissue. Furthermore, RNA-seq allowed the detection of different macrophage marker genes in corneal samples, including PTPRC (CD45), ITGAM (CD11b), CD14, and CD74. Our RNA-seq data showed no evidence of a relevant presence of monocytes in human corneal tissue. The presence of different macrophage subtypes was confirmed by IHC. The disintegration and subsequent FC analysis of human corneal samples showed the presence of both M1 (HLA-DR+, CD282+, CD86+, and CD284+) and M2 (CD163+ and CD206+) macrophage subtypes. Furthermore, we found that the total number of macrophages in corneal samples decreased more than the total cell count with increasing tissue culture time. Treatment with IL-10 led to higher total cell counts per cornea and to an increased expression of the M2 marker CD163 (p < 0.05) while expression levels of various M1 macrophage markers were not significantly reduced by interleukin treatment. CONCLUSIONS: Regarding different macrophage populations, untreated human corneas showed more M1 than M2 macrophages. With increasing organ culture time, these macrophages decreased. In terms of cell dynamics, adding interleukins to the organ culture medium influenced the phenotype of macrophages within the cornea as detected by FC. Modifying the immunomodulatory properties of human grafts appears a promising approach to further reduce the risk of graft rejection in patients. In this context, treatment with interleukins was more effective in upregulating M2 macrophages than in suppressing M1 macrophages in corneal tissue.

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 < 0.001) are dominating in vitreal hyalocytes. While the comparison of the gene expression profiles of hyalocytes and other myeloid cell populations showed an overall strong similarity (R2 > 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.

Secreted Phosphoprotein 1 Expression in Retinal Mononuclear Phagocytes Links Murine to Human Choroidal Neovascularization.

Age-related macular degeneration (AMD) represents the most common cause of blindness in the elderly in the Western world. An impairment of the outer blood-retina barrier and a localized inflammatory microenvironment cause sprouting of choroidal neovascular membranes (CNV) in neovascular AMD that are in intimate contact with surrounding myeloid cells, such as retinal microglia, and ultimately lead to visual impairment. The discovery of novel target molecules to interfere with angiogenesis and inflammation is vital for future treatment approaches in AMD patients. To explore the transcriptional profile and the function of retinal microglia at sites of CNV, we performed a comprehensive RNA-seq analysis of retinal microglia in the mouse model of laser-induced choroidal neovascularization (mCNV). Here, we identified the angiogenic factor Osteopontin (Opn), also known as "secreted phosphoprotein 1" (Spp1), as one of the most highly expressed genes in retinal microglia in the course of CNV formation. We confirmed the presence of SPP1 at the lesion site in recruited retinal microglia in Cx3cr1 CreER:Rosa26-tdTomato reporter mice by confocal microscopy and in whole retinal tissue lysates by ELISA highlighting a massive local production of SPP1. Inhibition of SPP1 by intravitreal injection of an anti-SPP1 antibody significantly increased the lesion size compared to IgG-treated control eyes. In line with our results in rodents, we found an increased SPP1 mRNA expression in surgically extracted human choroidal neovascular (hCNV) membranes by the quantitative RNA-seq approach of massive analysis of cDNA ends (MACE). Numerous IBA1+SPP1+ myeloid cells were detected in human CNV membranes. Taken together, these results highlight the importance of SPP1 in the formation of CNV and potentially offer new opportunities for therapeutic intervention by modulating the SPP1 pathway.