SARS‐CoV‐2 is assumed to use angiotensin‐converting enzyme 2 (ACE2) and other auxiliary proteins for cell entry. Recent studies have described conjunctival congestion in 0.8% of patients with laboratory‐confirmed severe acute respiratory syndrome coronavirus‐2 (SARS‐CoV‐2), and there has been speculation that SARS‐CoV‐2 can be transmitted through the conjunctiva. However, it is currently unclear whether conjunctival epithelial cells express ACE2 and its cofactors. In this study, a total of 38 conjunctival samples from 38 patients, including 12 healthy conjunctivas, 12 melanomas, seven squamous cell carcinomas, and seven papilloma samples, were analyzed using high‐throughput RNA sequencing to assess messenger RNA (mRNA) expression of the SARS‐CoV‐2 receptor ACE2 and its cofactors including TMPRSS2, ANPEP, DPP4, and ENPEP. ACE2 protein expression was assessed in eight healthy conjunctival samples using immunohistochemistry. Our results show that the SARS‐CoV‐2 receptor ACE2 is not substantially expressed in conjunctival samples on the mRNA (median: 0.0 transcripts per million [TPM], min: 0.0 TPM, max: 1.7 TPM) and protein levels. Similar results were obtained for the transcription of other auxiliary molecules. In conclusion, this study finds no evidence for a significant expression of ACE2 and its auxiliary mediators for cell entry in conjunctival samples, making conjunctival infection with SARS‐CoV‐2 via these mediators unlikely.

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.

Myeloid cells such as resident retinal microglia (MG) or infiltrating blood‐derived macrophages (Mϕ) accumulate in areas of retinal ischemia and neovascularization (RNV) and modulate neovascular eye disease. Their temporospatial distribution and biological function in this process, however, remain unclarified. Using state‐of‐the‐art methods, including cell‐specific reporter mice and high‐throughput RNA sequencing (RNA Seq), this study determined the extent of MG proliferation and Mϕ infiltration in areas with retinal ischemia and RNV in Cx3cr1^CreERT2:Rosa26‐tdTomato mice and examined the transcriptional profile of MG in the mouse model of oxygen‐induced retinopathy (OIR). For RNA Seq, tdTomato‐positive retinal MG were sorted by flow cytometry followed by Gene ontology (GO) cluster analysis. Furthermore, intraperitoneal injections of the cell proliferation marker 5‐ethynyl‐2′‐deoxyuridine (EdU) were performed from postnatal day (p) 12 to p16. We found that MG is the predominant myeloid cell population while Mϕ rarely appears in areas of RNV. Thirty percent of retinal MG in areas of RNV were EdU‐positive indicating a considerable local MG cell expansion. GO cluster analysis revealed an enrichment of clusters related to cell division, tubulin binding, ATPase activity, protein kinase regulatory activity, and chemokine receptor binding in MG in the OIR model compared to untreated controls. In conclusion, activated retinal MG alter their transcriptional profile, exhibit considerable proliferative ability and are by far the most frequent myeloid cell population in areas of ischemia and RNV in the OIR model thus presenting a potential target for future therapeutic approaches.

Background: Retinal neovascularization (RNV) membranes can lead to a tractional retinal detachment, the primary reason for severe vision loss in end-stage disease proliferative diabetic retinopathy (PDR). The aim of this study was to characterize the molecular, cellular and immunological features of RNV in order to unravel potential novel drug treatments for PDR.Methods: A total of 43 patients undergoing vitrectomy for PDR, macular pucker or macular hole (control patients) were included in this study. The surgically removed RNV and epiretinal membranes were analyzed by RNA sequencing, single-cell based Imaging Mass Cytometry and conventional immunohistochemistry. Immune cells of the vitreous body, also known as hyalocytes, were isolated from patients with PDR by flow cytometry, cultivated and characterized by immunohistochemistry. A bioinformatical drug repurposing approach was applied in order to identify novel potential drug options for end-stage diabetic retinopathy disease.Results: The in-depth transcriptional and single-cell protein analysis of diabetic RNV tissue samples revealed an accumulation of endothelial cells, macrophages and myofibroblasts as well as an abundance of secreted ECM proteins such as SPARC, FN1 and several types of collagen in RNV tissue. The immunohistochemical staining of cultivated vitreal hyalocytes from patients with PDR showed that hyalocytes express α-SMA (alpha-smooth muscle actin), a classic myofibroblast marker. According to our drug repurposing analysis, imatinib emerged as a potential immunomodulatory drug option for future treatment of PDR.Conclusion: This study delivers the first in-depth transcriptional and single-cell proteomic characterization of RNV tissue samples. Our data suggest an important role of hyalocyte-to-myofibroblast transdifferentiation in the pathogenesis of diabetic vitreoretinal disease and their modulation as a novel possible clinical approach.

This study aims to compare the potential of standard RNA-sequencing (RNA-Seq) and 3′ massive analysis of c-DNA ends (MACE) RNA-sequencing for the analysis of fresh tissue and describes transcriptome profiling of formalin-fixed paraffin-embedded (FFPE) archival human samples by MACE. To compare MACE to standard RNA-Seq on fresh tissue, four healthy conjunctiva from four subjects were collected during vitreoretinal surgery, halved and immediately transferred to RNA lysis buffer without prior fixation and then processed for either standard RNA-Seq or MACE RNA-Seq analysis. To assess the impact of FFPE preparation on MACE, a third part was fixed in formalin and processed for paraffin embedding, and its transcriptional profile was compared with the unfixed specimens analyzed by MACE. To investigate the impact of FFPE storage time on MACE results, 24 FFPE-treated conjunctival samples from 24 patients were analyzed as well. Nineteen thousand six hundred fifty-nine transcribed genes were detected by both MACE and standard RNA-Seq on fresh tissue, while 3251 and 2213 transcripts were identified explicitly by MACE or RNA-Seq, respectively. Standard RNA-Seq tended to yield longer detected transcripts more often than MACE technology despite normalization, indicating that the MACE technology is less susceptible to a length bias. FFPE processing revealed negligible effects on MACE sequencing results. Several quality-control measurements showed that long-term storage in paraffin did not decrease the diversity of MACE libraries. We noted a nonlinear relation between storage time and the number of raw reads with an accelerated decrease within the first 1000 days in paraffin, while the numbers remained relatively stable in older samples. Interestingly, the number of transcribed genes detected was independent on FFPE storage time. RNA of sufficient quality and quantity can be extracted from FFPE samples to obtain comprehensive transcriptome profiling using MACE technology. We thus present MACE as a novel opportunity for utilizing FFPE samples stored in histological archives.

Recent studies have deciphered the transcriptional profile of choroidal neovascularisation (CNV) in body donor eyes with neovascular age-related macular degeneration (nAMD) and were thus limited by the time span from death to preservation and the associated 5'-RNA degradation. Therefore, this study used CNV and control specimens which had been formalin-fixed and paraffin-embedded immediately after surgical extraction and analyzed them using a 3' RNA sequencing approach. Transcriptome profiles were analyzed and used to estimate content of immune and stromal cells and to define disease-associated gene signatures using statistical and bioinformatic methods. We identified 158 differentially-expressed genes (DEG) that were significantly increased in CNV compared to control tissue. Cell type enrichment analysis revealed a diverse cellular landscape with an enrichment of immune and stroma cell types in CNV including endothelial cells, macrophages, T cells and NKT cells. Gene ontology enrichment analysis demonstrated that DEG contributed to Blood Vessel Development, Extracellular Structure Organization, Response to Wounding and several immune-related terms. The S100 calcium-binding protein A8 (S100A8) and S100A9 emerged among the top DEG, as confirmed by immunohistochemistry on CNV tissue and protein analysis of vitreous samples. This study provides a high-resolution RNA-sequencing-based transcriptional signature of human CNV, characterizes its compositional pattern of immune and stromal cells and reveals S100A8/A9 as a novel biomarker and promising target for AMD-directed therapeutics and diagnostics.