Julian Wolf, MD

All Publications

• Transcriptional Profiling Identifies Prognostic Gene Signatures for Conjunctival Extranodal Marginal Zone Lymphoma. Biomolecules Wolf, J., Reinhard, T., Hajdu, R. I., Schlunck, G., Auw-Haedrich, C., Lange, C. 2023; 13 (1)

  Abstract

  This study characterizes the transcriptional profile and the cellular tumor microenvironment of conjunctival extranodal marginal zone lymphoma (EMZL) and identifies prognostically relevant biomarkers. Ten formalin-fixed and paraffin-embedded conjunctival EMZL and eight healthy conjunctival specimens were analyzed by Massive Analysis of cDNA Ends (MACE) RNA sequencing. The 3417 upregulated genes in conjunctival EMZL were involved in processes such as B cell proliferation and Rac protein signaling, whereas the 1188 downregulated genes contributed most significantly to oxidative phosphorylation and UV protection. The tumor microenvironment, as determined by deconvolution analysis, was mainly composed of multiple B cell subtypes which reflects the tumor's B cell lineage. However, several T cell types, including T helper 2 cells and regulatory T cells, as well as innate immune cell types, such as anti-inflammatory macrophages and plasmacytoid dendritic cells, were also strongly enriched in conjunctival EMZL. A 13-biomarker prognostic panel, including S100A8 and S100A9, classified ocular and extraocular tumor recurrence, exceeded prognostic accuracy of Ann Arbor and American Joint Committee on Cancer (AJCC) staging, and demonstrated prognostic value for patient survival in 21 different cancer types in a database of 12,332 tumor patients. These findings may lead to new options of targeted therapy and may improve prognostic prediction for conjunctival EMZL.

  View details for DOI 10.3390/biom13010115

  View details for PubMedID 36671500

• STAT3 signaling induced by IL-6 family cytokines modulates angiogenesis. Journal of cell science Rapp, J., Jung, M., Klar, R. F., Wolf, J., Arnold, J., Gorka, O., Groß, O., Lange, C., Agostini, H., Schlunck, G., Bucher, F. 2022

  Abstract

  Aberrant angiogenesis is a hallmark of cardiovascular and retinal neovascular disease. The STAT3 pathway represents a potential pharmacological target for those diseases due to its impact on angiogenesis. Surprisingly some STAT3 activators including the IL6 cytokine member oncostatin M (OSM) enhance angiogenesis whereas others like ciliary neurotropic factor (CNTF) reduce it. This study aims to clarify those conflicting effects. In contrast to the antiangiogenic cytokine CNTF, the proangiogenic OSM was able to activate intracellular signaling pathways beyond STAT3 including ERK and AKT. These differences translated into transcriptomic and metabolic shifts. siRNA mediated STAT3 knockdown experiments showed a decrease in VEGF-induced endothelial migration and sprouting while enhancing OSMs proangiogenic drive and switching CNTF's antiangiogenic to a proangiogenic response. These effects correlated with a transcriptomic shift representing enhanced STAT1 and ERK activity following STAT3 knockdown including a compensatory prolonged pSTAT1 activity. In conclusion, the angiogenic effect of STAT3 seems to be determined by cytokine-induced STAT3 specificity and simultaneous activity of other intracellular signaling pathways while the STAT3 pathway, predominantly recognized for its proangiogenic phenotypes, reveals novel antiangiogenic potential.

  View details for DOI 10.1242/jcs.260182

  View details for PubMedID 36458801

• Conjunctival Swabs Reveal Higher Detection Rate Compared to Schirmer Strips for SARS-CoV-2 RNA Detection in Tears of Hospitalized COVID-19 Patients. Journal of clinical medicine Sabage, L. E., Sun, Y. J., Wolf, J., Sabage, J., Mazzo, A., Santos, C. F., Mahajan, V. B., Manzoni Lourençone, L. F. 2022; 11 (23)

  Abstract

  To determine the prevalence of SARS-CoV-2 in tear samples and to investigate whether it correlates with ocular findings and patients' prognosis in Brazil.Tears were collected using Schirmer strips (SS) and conjunctival swabs (CS) from patients hospitalized with laboratory-confirmed SARS-CoV-2 infection. Samples were analyzed using qRT-PCR. Demographic and clinical data, ocular symptoms, and Schirmer tests (ST) were collected from patients. Charlson Comorbidity Index (CCI) was used to rate comorbidities, and patients were monitored until hospital discharge or death.There were 61 hospitalized patients, 33 of which were diagnosed with COVID-19. Within the confirmed COVID-19 patients, SARS-CoV-2 was detected in 18.2% (n = 6) of CS and 12.1% (n = 4) of SS samples. Subjective and objective parameters for dry eye syndrome (e.g., ST COVID-19: 8.3 ± 6.4mm, non-COVID-19: 8.9 ± 6.6mm, p > 0.05) were comparable between COVID-19 (n = 33) and non-COVID-19 patients (n = 28). Among the 16 COVID-19 patients exhibiting ocular symptoms, only tearing was reported significantly more frequently when tear samples were positive for SARS-CoV-2 (p < 0.05). Strikingly, patients whose tears tested positive for SARS-CoV-2 had significantly inferior CCI (pos.: 34.0 ± 31.8%, neg.: 67.6 ± 36.4%, p < 0.05) and higher mortality rates (pos.: 50.0%, neg.: 7.4%, p < 0.01).SARS-CoV-2 was detected with a prevalence of 18.2% on the ocular surface. Decreased CCI and increased mortality rate in the positive tear group suggests that viral detection may relate to prognosis and highlight the need of personal protective measures for healthcare professionals. Most of the patients, regardless of COVID-19 diagnosis, had low tear production and eye discomfort, possibly pointing to the need for artificial tear use during hospitalization.

  View details for DOI 10.3390/jcm11236929

  View details for PubMedID 36498504

• Next-Generation-Sequencing in der Augenheilkunde. Die Ophthalmologie Wolf, J., Lange, C., Reinhard, T., Schlunck, G. 2022

  Abstract

  New methods for basic research are finding their way into ophthalmology and expand the options for research, diagnostics and treatment. This article centers on the study of gene activity in cells and tissues by next-generation sequencing (NGS). Following abrief introduction to the basic principles of NGS, this article focuses on transcriptome analysis by RNA sequencing using examples from ophthalmology. The RNA sequencing provides acomprehensive and unbiased overview of gene activity in cells and tissues and thus forms an important foundation for generating new testable scientific hypotheses. It thus contributes to the in-depth characterization of pathological changes and supports the development of new diagnostic and therapeutic approaches.

  View details for DOI 10.1007/s00347-022-01765-6

  View details for PubMedID 36418561

• Transcriptional Profiling Provides New Insights into Organ Culture-Induced Changes in Human Donor Corneas. International journal of molecular sciences Wolf, J., Kammrath Betancor, P., Maier, P., Heinzelmann, S. U., Jiang, J., Lange, C., Reinhard, T., Schlunck, G., Lapp, T. 2022; 23 (23)

  Abstract

  Corneal transplantation is one of the most common forms of tissue transplantation worldwide. Donor corneal tissue used in transplantation is provided by eye banks, which store the tissue in culture medium after procurement. To date, the effects of cell culture on human corneal tissue have not been fully elucidated. Using the 3' RNA sequencing method for massive analysis of cDNA ends (MACE), we show that cultivation of corneal tissue leads to significant changes in a variety of molecular processes in human corneal tissue that go well beyond aspects of previously known culture effects. Functionally grouped network analysis revealed nine major groups of biological processes that were affected by corneal organ culture, among them keratinization, hypoxia, and angiogenesis, with genes from each group being affected by culture time. A cell type deconvolution analysis revealed significant modulations of the corneal immune cell profile in a time dependent manner. The results suggest that current culture conditions should be further refined and that prolonged cultivation may be detrimental. Recently, we showed that MACE enables transcriptional profiling of formalin-fixed and paraffin-embedded (FFPE) conjunctival tissue with high accuracy even after more than 10 years of storage. Here we demonstrate that MACE provides comparable results for native and FFPE corneal tissue, confirming that the technology is suitable for transcriptome analysis of a wide range of archived diseased corneal samples stored in histological archives. Finally, our data underscore the feasibility of bioinformatics cell-type enrichment analysis in bulk RNA-seq data to profile immune cell composition in fixed and archived corneal tissue samples, for which RNA-seq analysis of individual cells is often not possible.

  View details for DOI 10.3390/ijms232314507

  View details for PubMedID 36498835

• Protocol to quantify enzymatic effects on vitreous liquefaction in porcine eyes using a transwell-plate system. STAR protocols Wolf, J., Sabage, L. E., Sun, Y. J., Mahajan, V. B. 2022; 3 (4): 101754

  Abstract

  This protocol describes an exvivo model to quantify enzymatic effects on vitreous liquefaction using porcine eyes in a transwell-plate system via induced syneresis. It provides a standardized dissection process and performs critical steps for gel-liquid separation with high precision, minimal tissue loss, and scalability. The protocol can be applied to other studies investigating vitreous liquefaction or gelatinous tissue analysis and can also serve to study vitreous liquefaction invivo as it may occur during aging or disease progression.

  View details for DOI 10.1016/j.xpro.2022.101754

  View details for PubMedID 36208453

• Web-based gene expression analysis-paving the way to decode healthy and diseased ocular tissue. Die Ophthalmologie Wolf, J., Lapp, T., Reinhard, T., Agostini, H., Schlunck, G., Lange, C. 2022

  Abstract

  Gene expression analysis using RNA sequencing has helped to improve the understanding of many diseases. Databases, such as the Gene Expression Omnibus database of the National Center for Biotechnology Information provide RNA sequencing raw data from various diseased tissue types but their analysis requires advanced bioinformatics skills. Therefore, specific ocular databases provide the transcriptional profiles of different ocular tissues and in addition enable intuitive web-based data analysis.The aim of this narrative review is to provide an overview of ocular transcriptome databases and to compare them with the Human Eye Transcriptome Atlas newly established in Freiburg.PubMed literature search.A total of nine ocular transcriptome databases focusing on different aspects were identified. The iSyTE and Express platforms specialize in gene expression during lens and retinal development in mice, whereas retina.tigem.it, Eye in a Disk, and Spectacle focus on selected ocular tissues such as the retina. Spectacle, UCSC Cell Browser and Single Cell Portal allow intuitive exploration of single cell RNA sequencing data derived from retinal, choroid, cornea, iris, trabecular meshwork and sclera specimens. The microarray profiles of a variety of healthy ocular tissues are included in the Ocular Tissue Database. The Human Eye Transcriptome Atlas provides the largest collection of different ocular tissue types, contains the highest number of ocular diseases and is characterized by a high level of quality achieved by methodological consistency.Ocular transcriptome databases provide comprehensive and intuitive insights into the transcriptional profiles of a variety of healthy and diseased ocular tissues. Thus, they improve our understanding of the underlying molecular mediators, support hypothesis generation and help in the search for new diagnostic and therapeutic targets for various ocular diseases.

  View details for DOI 10.1007/s00347-022-01721-4

  View details for PubMedID 36098765

  View details for PubMedCentralID PMC9469811

• Hyalocyte functions and immunology EXPERT REVIEW OF OPHTHALMOLOGY Boneva, S. K., Wolf, J., Wieghofer, P., Sebag, J., Lange, C. K. 2022; 17 (4): 249-262

  View details for DOI 10.1080/17469899.2022.2100763

  View details for Web of Science ID 000850876100001

• Single-Cell Protein and Transcriptional Characterization of Epiretinal Membranes From Patients With Proliferative Vitreoretinopathy. Investigative ophthalmology & visual science Laich, Y., Wolf, J., Hajdu, R. I., Schlecht, A., Bucher, F., Pauleikhoff, L., Busch, M., Martin, G., Faatz, H., Killmer, S., Bengsch, B., Stahl, A., Lommatzsch, A., Schlunck, G., Agostini, H., Boneva, S., Lange, C. 2022; 63 (5): 17

  Abstract

  Proliferative vitreoretinopathy (PVR) remains an unresolved clinical challenge and can lead to frequent revision surgery and blindness vision loss. The aim of this study was to characterize the microenvironment of epiretinal PVR tissue, in order to shed more light on the complex pathophysiology and to unravel new treatment options.A total of 44 tissue samples were analyzed in this study, including 19 epiretinal PVRs, 13 epiretinal membranes (ERMs) from patients with macular pucker, as well as 12 internal limiting membranes (ILMs). The cellular and molecular microenvironment was assessed by cell type deconvolution analysis (xCell), RNA sequencing data and single-cell imaging mass cytometry. Candidate drugs for PVR treatment were identified in silico via a transcriptome-based drug-repurposing approach.RNA sequencing of tissue samples demonstrated distinct transcriptional profiles of PVR, ERM, and ILM samples. Differential gene expression analysis revealed 3194 upregulated genes in PVR compared with ILM, including FN1 and SPARC, which contribute to biological processes, such as extracellular matrix (ECM) organization. The xCell and IMC analyses showed that PVR membranes were composed of macrophages, retinal pigment epithelium, and α-SMA-positive myofibroblasts, the latter predominantly characterized by the co-expression of immune cell signature markers. Finally, 13 drugs were identified as potential therapeutics for PVR, including aminocaproic acid and various topoisomerase-2A inhibitors.Epiretinal PVR membranes exhibit a unique and complex transcriptional and cellular profile dominated by immune cells and myofibroblasts, as well as a variety of ECM components. Our findings provide new insights into the pathophysiology of PVR and suggest potential targeted therapeutic options.

  View details for DOI 10.1167/iovs.63.5.17

  View details for PubMedID 35579905

  View details for PubMedCentralID PMC9123517

• Transcriptional and Distributional Profiling of Microglia in Retinal Angiomatous Proliferation. International journal of molecular sciences Schlecht, A., Wolf, J., Boneva, S., Prinz, G., Braunger, B. M., Wieghofer, P., Agostini, H., Schlunck, G., Lange, C. 2022; 23 (7)

  Abstract

  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.

  View details for DOI 10.3390/ijms23073443

  View details for PubMedID 35408803

  View details for PubMedCentralID PMC8998238

• In-Depth Molecular Profiling Specifies Human Retinal Microglia Identity. Frontiers in immunology Wolf, J., Boneva, S., Rosmus, D. D., Agostini, H., Schlunck, G., Wieghofer, P., Schlecht, A., Lange, C. 2022; 13: 863158

  Abstract

  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.

  View details for DOI 10.3389/fimmu.2022.863158

  View details for PubMedID 35371110

  View details for PubMedCentralID PMC8971200

• Deciphering the Molecular Signature of Human Hyalocytes in Relation to Other Innate Immune Cell Populations. Investigative ophthalmology & visual science Wolf, J., Boneva, S., Rosmus, D. D., Agostini, H., Schlunck, G., Wieghofer, P., Schlecht, A., Lange, C. 2022; 63 (3): 9

  Abstract

  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.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.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.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.

  View details for DOI 10.1167/iovs.63.3.9

  View details for PubMedID 35266958

  View details for PubMedCentralID PMC8934546

• The Human Eye Transcriptome Atlas: A searchable comparative transcriptome database for healthy and diseased human eye tissue. Genomics Wolf, J., Boneva, S., Schlecht, A., Lapp, T., Auw-Haedrich, C., Lagrèze, W., Agostini, H., Reinhard, T., Schlunck, G., Lange, C. 2022; 114 (2): 110286

  Abstract

  The applications of deep sequencing technologies in life science research and clinical diagnostics have increased rapidly over the last decade. Although fast algorithms for data processing exist, intuitive, portable solutions for data analysis are still rare. For this purpose, we developed a web-based transcriptome database, which provides a platform-independent, intuitive solution to easily explore and compare ocular gene expression of 100 diseased and healthy human tissue samples from 15 different tissue types collected at the Eye Center of the University of Freiburg. To ensure comparability of expression between different tissues, reads were normalized across all 100 samples. Differentially expressed genes were calculated between each tissue type to determine tissue-specific genes. Unsupervised analysis of all 100 samples revealed an accurate clustering according to different tissue types and a high tissue specificity by analyzing known tissue-specific marker genes. Bioinformatic cell type deconvolution using xCell provided detailed insights into the cellular profiles of each tissue type. Several new tissue-specific marker genes were identified. These genes were involved in tissue- or disease-specific processes, such as myelination for the optic nerve, visual perception for retina, keratinocyte differentiation for conjunctival carcinoma, as well as endothelial cell migration for choroidal neovascularization membranes. The results are accessible at the Human Eye Transcriptome Atlas website at https://www.eye-transcriptome.com. In summary, this searchable transcriptome database enables easy exploration of ocular gene expression in healthy and diseased human ocular tissues without bioinformatics expertise. Thus, it provides rapid access to detailed insights into the molecular mechanisms of various ocular tissues and diseases, as well as the rapid retrieval of potential new diagnostic and therapeutic targets.

  View details for DOI 10.1016/j.ygeno.2022.110286

  View details for PubMedID 35124170

• [Web-based gene expression analysis-paving the way to decode healthy and diseased ocular tissue]. Der Ophthalmologe : Zeitschrift der Deutschen Ophthalmologischen Gesellschaft Wolf, J., Lapp, T., Reinhard, T., Agostini, H., Schlunck, G., Lange, C. 2022

  Abstract

  Gene expression analysis using RNA sequencing has helped to improve the understanding of many diseases. Databases, such as the Gene Expression Omnibus database of the National Center for Biotechnology Information provide RNA sequencing raw data from various diseased tissue types but their analysis requires advanced bioinformatics skills. Therefore, specific ocular databases provide the transcriptional profiles of different ocular tissues and in addition enable intuitive web-based data analysis.The aim of this narrative review is to provide an overview of ocular transcriptome databases and to compare them with the Human Eye Transcriptome Atlas newly established in Freiburg.PubMed literature search.A total of nine ocular transcriptome databases focusing on different aspects were identified. The iSyTE and Express platforms specialize in gene expression during lens and retinal development in mice, whereas retina.tigem.it, Eye in a Disk, and Spectacle focus on selected ocular tissues such as the retina. Spectacle, UCSC Cell Browser and Single Cell Portal allow intuitive exploration of single cell RNA sequencing data derived from retinal, choroid, cornea, iris, trabecular meshwork and sclera specimens. The microarray profiles of a variety of healthy ocular tissues are included in the Ocular Tissue Database. The Human Eye Transcriptome Atlas provides the largest collection of different ocular tissue types, contains the highest number of ocular diseases and is characterized by a high level of quality achieved by methodological consistency.Ocular transcriptome databases provide comprehensive and intuitive insights into the transcriptional profiles of a variety of healthy and diseased ocular tissues. Thus, they improve our understanding of the underlying molecular mediators, support hypothesis generation and help in the search for new diagnostic and therapeutic targets for various ocular diseases.

  View details for DOI 10.1007/s00347-022-01592-9

  View details for PubMedID 35194679

  View details for PubMedCentralID PMC8863098

• 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. Biochimica et biophysica acta. Molecular basis of disease Wolf, J., Schlecht, A., Rosmus, D. D., Boneva, S., Agostini, H., Schlunck, G., Wieghofer, P., Lange, C. 2022; 1868 (4): 166340

  Abstract

  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.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.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.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.This study was funded by the Helmut Ecker Foundation and the Volker Homann Foundation.

  View details for DOI 10.1016/j.bbadis.2022.166340

  View details for PubMedID 35032596

• Immunosenescence in Choroidal Neovascularization (CNV)-Transcriptional Profiling of Naïve and CNV-Associated Retinal Myeloid Cells during Aging. International journal of molecular sciences Schlecht, A., Thien, A., Wolf, J., Prinz, G., Agostini, H., Schlunck, G., Wieghofer, P., Boneva, S., Lange, C. 2021; 22 (24)

  Abstract

  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) Cx3cr1GFP/+ 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.

  View details for DOI 10.3390/ijms222413318

  View details for PubMedID 34948115

  View details for PubMedCentralID PMC8707893

• In-Depth Molecular Characterization of Neovascular Membranes Suggests a Role for Hyalocyte-to-Myofibroblast Transdifferentiation in Proliferative Diabetic Retinopathy. Frontiers in immunology Boneva, S. K., Wolf, J., Hajdú, R. I., Prinz, G., Salié, H., Schlecht, A., Killmer, S., Laich, Y., Faatz, H., Lommatzsch, A., Busch, M., Bucher, F., Stahl, A., Böhringer, D., Bengsch, B., Schlunck, G., Agostini, H., Lange, C. A. 2021; 12: 757607

  Abstract

  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.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.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.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.

  View details for DOI 10.3389/fimmu.2021.757607

  View details for PubMedID 34795670

  View details for PubMedCentralID PMC8593213

• The role of interferon regulatory factor 8 for retinal tissue homeostasis and development of choroidal neovascularisation. Journal of neuroinflammation Zhang, P., Schlecht, A., Wolf, J., Boneva, S., Laich, Y., Koch, J., Ludwig, F., Boeck, M., Thien, A., Härdtner, C., Kierdorf, K., Agostini, H., Schlunck, G., Prinz, M., Hilgendorf, I., Wieghofer, P., Lange, C. 2021; 18 (1): 215

  Abstract

  Microglia cells represent the resident innate immune cells of the retina and are important for retinal development and tissue homeostasis. However, dysfunctional microglia can have a negative impact on the structural and functional integrity of the retina under native and pathological conditions.In this study, we examined interferon-regulatory factor 8 (Irf8)-deficient mice to determine the transcriptional profile, morphology, and temporospatial distribution of microglia lacking Irf8 and to explore the effects on retinal development, tissue homeostasis, and formation of choroidal neovascularisation (CNV).Our study shows that Irf8-deficient MG exhibit a considerable loss of microglial signature genes accompanied by a severely altered MG morphology. An in-depth characterisation by fundus photography, fluorescein angiography, optical coherence tomography and electroretinography revealed no major retinal abnormalities during steady state. However, in the laser-induced CNV model, Irf8-deficient microglia showed an increased activity of biological processes critical for inflammation and cell adhesion and a reduced MG cell density near the lesions, which was associated with significantly increased CNV lesion size.Our results suggest that loss of Irf8 in microglia has negligible effects on retinal homeostasis in the steady state. However, under pathological conditions, Irf8 is crucial for the transformation of resident microglia into a reactive phenotype and thus for the suppression of retinal inflammation and CNV formation.

  View details for DOI 10.1186/s12974-021-02230-y

  View details for PubMedID 34544421

  View details for PubMedCentralID PMC8454118

• Imaging mass cytometry for high-dimensional tissue profiling in the eye. BMC ophthalmology Schlecht, A., Boneva, S., Salie, H., Killmer, S., Wolf, J., Hajdu, R. I., Auw-Haedrich, C., Agostini, H., Reinhard, T., Schlunck, G., Bengsch, B., Lange, C. A. 2021; 21 (1): 338

  Abstract

  Imaging mass cytometry (IMC) combines the principles of flow cytometry and mass spectrometry (MS) with laser scanning spatial resolution and offers unique advantages for the analysis of tissue samples in unprecedented detail. In contrast to conventional immunohistochemistry, which is limited in its application by the number of possible fluorochrome combinations, IMC uses isoptope-coupled antibodies that allow multiplex analysis of up to 40 markers in the same tissue section simultaneously.In this report we use IMC to analyze formalin-fixed, paraffin-embedded conjunctival tissue. We performed a 18-biomarkers IMC analysis of conjunctival tissue to determine and summarize the possibilities, relevance and limitations of IMC for deciphering the biology and pathology of ocular diseases.Without modifying the manufacturer's protocol, we observed positive and plausible staining for 12 of 18 biomarkers. Subsequent bioinformatical single-cell analysis and phenograph clustering identified 24 different cellular clusters with distinct expression profiles with respect to the markers used.IMC enables highly multiplexed imaging of ocular samples at subcellular resolution. IMC is an innovative and feasible method, providing new insights into ocular disease pathogenesis that will be valuable for basic research, drug discovery and clinical diagnostics.

  View details for DOI 10.1186/s12886-021-02099-8

  View details for PubMedID 34544377

  View details for PubMedCentralID PMC8454101

• Subretinal fibrosis in neovascular age-related macular degeneration: current concepts, therapeutic avenues, and future perspectives. Cell and tissue research Tenbrock, L., Wolf, J., Boneva, S., Schlecht, A., Agostini, H., Wieghofer, P., Schlunck, G., Lange, C. 2021

  Abstract

  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.

  View details for DOI 10.1007/s00441-021-03514-8

  View details for PubMedID 34477966

• Time- and Stimulus-Dependent Characteristics of Innate Immune Cells in Organ-Cultured Human Corneal Tissue. Journal of innate immunity Zhuang, X., Schlunck, G., Wolf, J., Rosmus, D. D., Bleul, T., Luo, R., Böhringer, D., Wieghofer, P., Lange, C., Reinhard, T., Lapp, T. 2021: 1-14

  Abstract

  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).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.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.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.

  View details for DOI 10.1159/000516669

  View details for PubMedID 34182556

• Characterization of the Cellular Microenvironment and Novel Specific Biomarkers in Pterygia Using RNA Sequencing. Frontiers in medicine Wolf, J., Hajdu, R. I., Boneva, S., Schlecht, A., Lapp, T., Wacker, K., Agostini, H., Reinhard, T., Auw-Hädrich, C., Schlunck, G., Lange, C. 2021; 8: 714458

  Abstract

  With a worldwide prevalence of ~12%, pterygium is a common degenerative and environmentally triggered ocular surface disorder characterized by wing-shaped growth of conjunctival tissue onto the cornea that can lead to blindness if left untreated. This study characterizes the transcriptional profile and the cellular microenvironment of conjunctival pterygia and identifies novel pterygia-specific biomarkers. Formalin-fixed and paraffin-embedded pterygia as well as healthy conjunctival specimens were analyzed using MACE RNA sequencing (n = 8 each) and immunohistochemistry (pterygia n = 7, control n = 3). According to the bioinformatic cell type enrichment analysis using xCell, the cellular microenvironment of pterygia was characterized by an enrichment of myofibroblasts, T-lymphocytes and various antigen-presenting cells, including dendritic cells and macrophages. Differentially expressed genes that were increased in pterygia compared to control tissue were mainly involved in autophagy (including DCN, TMBIM6), cellular response to stress (including TPT1, DDX5) as well as fibroblast proliferation and epithelial to mesenchymal transition (including CTNNB1, TGFBR1, and FN1). Immunohistochemical analysis confirmed a significantly increased FN1 stromal immunoreactivity in pterygia when compared to control tissue. In addition, a variety of factors involved in apoptosis were significantly downregulated in pterygia, including LCN2, CTSD, and NISCH. Furthermore, 450 pterygia-specific biomarkers were identified by including transcriptional data of different ocular surface pathologies serving as controls (training group), which were then validated using transcriptional data of cultured human pterygium cells. Among the most pterygia-specific factors were transcripts such as AHNAK, RTN4, TPT1, FSTL1, and SPARC. Immunohistochemical validation of SPARC revealed a significantly increased stromal immunoreactivity in pterygia when compared to controls, most notably in vessels and intravascular vessel wall-adherent mononuclear cells. Taken together, the present study provides new insights into the cellular microenvironment and the transcriptional profile of pterygia, identifies new and specific biomarkers and in addition to fibrosis-related genes, uncovers autophagy, stress response and apoptosis modulation as pterygium-associated processes. These findings expand our understanding of the pathophysiology of pterygia, provide new diagnostic tools, and may enable new targeted therapeutic options for this common and sight-threatening ocular surface disease.

  View details for DOI 10.3389/fmed.2021.714458

  View details for PubMedID 35174178

  View details for PubMedCentralID PMC8841401

• Viral S protein histochemistry reveals few potential SARS-CoV-2 entry sites in human ocular tissues. Scientific reports Martin, G., Wolf, J., Lapp, T., Agostini, H. T., Schlunck, G., Auw-Hädrich, C., Lange, C. A. 2021; 11 (1): 19140

  Abstract

  Despite the reported low expression of the primary SARS-CoV-2 receptor ACE2 in distinct ocular tissues, some clinical evidence suggests that SARS-CoV-2 can infect the eye. In this study, we explored potential entry sites for SARS-CoV-2 by viral S protein histochemistry on various ocular tissues and compared the staining patterns with RNA and protein expression of TMPRSS2 and ACE2. Potential viral entry sites were investigated by histochemistry using tagged recombinant viral S protein on 52 ocular tissue samples including specimens of the cornea, conjunctiva, lid margin, lacrimal gland tissue, retina, choroid, and RPE. In addition, ACE2 and TMPRSS2 immunohistochemistry were performed on the same ocular tissue, each with distinct antibodies binding to different epitopes. Lung tissue samples were used as positive controls. Finally, bulk RNA sequencing (RNA-Seq) was used to determine the expression of ACE2 and its auxiliary factors in the tissues mentioned above. S protein histochemistry revealed a positive staining in lung tissue but absent staining in the cornea, the conjunctiva, eye lid samples, the lacrimal glands, the retina and the optic nerve which was supported by hardly any immunoreactivity for ACE2 and TMPRSS2 and scarce ACE2 and TMPRSS2 RNA expression. Negligible staining with antibodies targeting ACE2 or TMPRSS2 was seen in the main and accessory lacrimal glands. In contrast, ocular staining (S protein, ACE2, TMPRSS2) was distinctly present in pigmented cells of the RPE and choroid, as well as in the ciliary body and the iris stroma. S protein histochemistry revealed hardly any SARS-CoV-2 entry sites in all ocular tissues examined. Similarly, no significant ACE2 or TMPRSS2 expression was found in extra- and intraocular tissue. While this study suggest a rather low risk of ocular infection with SARS-CoV-2, it should be noted, that potential viral entry sites may increase in response to inflammation or in certain disease states.

  View details for DOI 10.1038/s41598-021-98709-y

  View details for PubMedID 34580409

  View details for PubMedCentralID PMC8476534

• What is the significance of the conjunctiva as a potential transmission route for SARS-CoV-2 infections? Der Ophthalmologe : Zeitschrift der Deutschen Ophthalmologischen Gesellschaft Lange, C., Wolf, J., Auw-Haedrich, C., Schlecht, A., Boneva, S., Lapp, T., Agostini, H., Martin, G., Reinhard, T., Schlunck, G. 2021; 118 (Suppl 1): 85-88

  Abstract

  Recent studies have described conjunctivitis in approximately 1% of COVID-19 patients and speculated that SARS-CoV‑2 can be transmitted via the conjunctiva. In this article we recapitulate the molecular mechanisms of host cell entry of SARS-CoV‑2 and discuss the current evidence for a potential conjunctival transmission of SARS-CoV‑2. The current body of evidence indicates that SARS-CoV‑2 requires the membrane-bound angiotensin-converting enzyme 2 (ACE2) and the membrane-bound serine protease TMPRSS2 to enter cells. Recent studies suggest that COVID-19 patients rarely exhibit viral RNA in tear film and conjunctival smears and that, ACE2 and TMPRSS2 are only expressed in small amounts in the conjunctiva, making conjunctival infection with SARS-CoV‑2 via these mediators unlikely. Nevertheless, we consider the current evidence to be still too limited to provide a conclusive statement and recommend appropriate protective measures for healthcare personnel who are in close contact with suspected and confirmed COVID-19 patients.

  View details for DOI 10.1007/s00347-020-01255-7

  View details for PubMedID 33141332

  View details for PubMedCentralID PMC7607541

• MACE RNA sequencing analysis of conjunctival squamous cell carcinoma and papilloma using formalin-fixed paraffin-embedded tumor tissue. Scientific reports Boneva, S., Schlecht, A., Zhang, P., Boehringer, D., Lapp, T., Mittelviefhaus, H., Reinhard, T., Auw-Haedrich, C., Schlunck, G., Wolf, J., Lange, C. 2020; 10 (1): 21292

  Abstract

  Recent advances in the field of biomedical research allow for elucidation of the transcriptional signature of rare tumors such as conjunctival squamous cell carcinoma (SCC). In this study we compare its expression profile to conjunctival papilloma (Pap) and healthy conjunctival tissue (Ctrl) and develop a classification tool to differentiate these entities. Seven conjunctival SCC, seven Pap and ten Ctrl were formalin-fixed and paraffin-embedded (FFPE) and analyzed using Massive Analysis of cDNA Ends (MACE) RNA sequencing. Differentially expressed genes (DEG) and gene ontology (GO) clusters were explored and the abundance of involved cell types was quantified by xCell. Finally, a classification model was developed to distinguish SCC from Pap and Ctrl. Among the most prominent DEG in SCC a plethora of keratins were upregulated when compared to Pap and Ctrl. xCell analysis revealed an enrichment of immune cells, including activated dendritic cells and T-helper type 1 cells (Th1), in SCC when compared to Ctrl. The generated classification model could reliably discriminate between the three entities according to the expression pattern of 30 factors. This study provides a transcriptome-wide gene expression profile of rare conjunctival SCC. The analysis identifies distinct keratins, as well as dendritic and Th1 cells as important mediators in SCC. Finally, the provided gene expression classifier may become an aid to the conventional histological classification of conjunctival tumors in uncertain cases.

  View details for DOI 10.1038/s41598-020-78339-6

  View details for PubMedID 33277602

  View details for PubMedCentralID PMC7718249

• Corneal tissue induces transcription of metallothioneins in monocyte-derived human macrophages. Molecular immunology Wolf, J., Zhuang, X., Hildebrand, A., Boneva, S., Schwämmle, M., Kammrath Betancor, P., Fan, J., Böhringer, D., Maier, P., Lange, C., Reinhard, T., Schlunck, G., Lapp, T. 2020; 128: 188-194

  Abstract

  Immune reactions following corneal transplantation are the most common cause of transplant failure. However, the underlying mechanisms of corneal graft rejection are not yet fully understood but increasing evidence points to a crucial role of the innate immune system in this context. Using a human in vitro model, we aimed to assess the response of human macrophages to stimulation with human corneal tissue and whether corneal endothelial cells (CEC) have immune-modulating properties.Human monocytes were isolated from peripheral blood mononuclear cells and differentiated into monocyte-derived macrophages (MDM). A standardized protocol was used for disaggregation of human corneas into fragments of defined sizes. MDMs were stimulated using processed corneal material with or without CEC. Lipopolysaccharide (LPS) or interferon-gamma (IFNγ) served as controls. RNA sequencing was applied to analyze the impact of differential stimulation of MDMs on their transcriptional profile. RNA sequencing results were validated using digital PCR.The transcriptional profile of MDMs was significantly modulated by the type of stimulus used for MDM activation as well as by the individual MDM donor. LPS- or IFNγ-stimulation resulted in distinct transcriptional alterations compared to unstimulated MDMs including an upregulation of various cytokines such as CCL3, 4, 5, 19 or CXCL9. Corneal tissue induced the differential expression of 45 genes when compared to unstimulated MDMs, with several metallothioneins (MTs) among the upregulated factors (MT1A, MT1E, MT1F, MT1G, MT1H, MT1L, MT1M, MT1X, MT2A). This effect was independent of the presence or absence of CEC. PCR validation confirmed induction of 3 different metallothioneins (MT1G, MT1H and MT2A) in MDMs stimulated by corneal tissue.The MDM in vitro model proved to be a robust tool to study the effects of LPS, IFNγ and corneal tissue homogenates on the transcriptional activity of MDM. Human macrophages showed a distinct upregulation of various MTs when challenged with human corneal allogen with or without corneal endothelium, which might have an immune-modulatory effect. As a general observation, it appears that in MDM-based studies a significant donor-dependent effect on the transcriptional profile of MDMs needs to be considered and adjusted before downstream analysis.

  View details for DOI 10.1016/j.molimm.2020.10.016

  View details for PubMedID 33137607

• Transcriptional characterization of conjunctival melanoma identifies the cellular tumor microenvironment and prognostic gene signatures. Scientific reports Wolf, J., Auw-Haedrich, C., Schlecht, A., Boneva, S., Mittelviefhaus, H., Lapp, T., Agostini, H., Reinhard, T., Schlunck, G., Lange, C. A. 2020; 10 (1): 17022

  Abstract

  This study characterizes the transcriptome and the cellular tumor microenvironment (TME) of conjunctival melanoma (CM) and identifies prognostically relevant biomarkers. 12 formalin-fixed and paraffin-embedded CM were analyzed by MACE RNA sequencing, including six cases each with good or poor clinical outcome, the latter being defined by local recurrence and/or systemic metastases. Eight healthy conjunctival specimens served as controls. The TME of CM, as determined by bioinformatic cell type enrichment analysis, was characterized by the enrichment of melanocytes, pericytes and especially various immune cell types, such as plasmacytoid dendritic cells, natural killer T cells, B cells and mast cells. Differentially expressed genes between CM and control were mainly involved in inhibition of apoptosis, proteolysis and response to growth factors. POU3F3, BIRC5 and 7 were among the top expressed genes associated with inhibition of apoptosis. 20 genes, among them CENPK, INHA, USP33, CASP3, SNORA73B, AAR2, SNRNP48 and GPN1, were identified as prognostically relevant factors reaching high classification accuracy (area under the curve: 1.0). The present study provides new insights into the TME and the transcriptional profile of CM and additionally identifies new prognostic biomarkers. These results add new diagnostic tools and may lead to new options of targeted therapy for CM.

  View details for DOI 10.1038/s41598-020-72864-0

  View details for PubMedID 33046735

  View details for PubMedCentralID PMC7550331

• 3' MACE RNA-sequencing allows for transcriptome profiling in human tissue samples after long-term storage. Laboratory investigation; a journal of technical methods and pathology Boneva, S., Schlecht, A., Böhringer, D., Mittelviefhaus, H., Reinhard, T., Agostini, H., Auw-Haedrich, C., Schlunck, G., Wolf, J., Lange, C. 2020; 100 (10): 1345-1355

  Abstract

  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.

  View details for DOI 10.1038/s41374-020-0446-z

  View details for PubMedID 32467590

  View details for PubMedCentralID PMC7498368

• Expression of the COVID-19 receptor ACE2 in the human conjunctiva. Journal of medical virology Lange, C., Wolf, J., Auw-Haedrich, C., Schlecht, A., Boneva, S., Lapp, T., Horres, R., Agostini, H., Martin, G., Reinhard, T., Schlunck, G. 2020; 92 (10): 2081-2086

  Abstract

  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.

  View details for DOI 10.1002/jmv.25981

  View details for PubMedID 32374427

  View details for PubMedCentralID PMC7267303

• Transcriptional Profiling Uncovers Human Hyalocytes as a Unique Innate Immune Cell Population. Frontiers in immunology Boneva, S. K., Wolf, J., Rosmus, D. D., Schlecht, A., Prinz, G., Laich, Y., Boeck, M., Zhang, P., Hilgendorf, I., Stahl, A., Reinhard, T., Bainbridge, J., Schlunck, G., Agostini, H., Wieghofer, P., Lange, C. A. 2020; 11: 567274

  Abstract

  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.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.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 (R 2 > 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.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.

  View details for DOI 10.3389/fimmu.2020.567274

  View details for PubMedID 33042148

  View details for PubMedCentralID PMC7517040

• Temporospatial distribution and transcriptional profile of retinal microglia in the oxygen-induced retinopathy mouse model. Glia Boeck, M., Thien, A., Wolf, J., Hagemeyer, N., Laich, Y., Yusuf, D., Backofen, R., Zhang, P., Boneva, S., Stahl, A., Hilgendorf, I., Agostini, H., Prinz, M., Wieghofer, P., Schlunck, G., Schlecht, A., Lange, C. 2020; 68 (9): 1859-1873

  Abstract

  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 Cx3cr1CreERT2 :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.

  View details for DOI 10.1002/glia.23810

  View details for PubMedID 32150307

• Transcriptomic Characterization of Human Choroidal Neovascular Membranes Identifies Calprotectin as a Novel Biomarker for Patients with Age-Related Macular Degeneration. The American journal of pathology Schlecht, A., Boneva, S., Gruber, M., Zhang, P., Horres, R., Bucher, F., Auw-Haedrich, C., Hansen, L., Stahl, A., Hilgendorf, I., Agostini, H., Wieghofer, P., Schlunck, G., Wolf, J., Lange, C. A. 2020; 190 (8): 1632-1642

  Abstract

  Recent studies deciphering the transcriptional profile of choroidal neovascularization (CNV) in body donor eyes with neovascular age-related macular degeneration are limited by the time span from death to preservation and the associated 5'-RNA degradation. This study therefore used CNV and control specimens that were formalin-fixed and paraffin-embedded immediately after surgical extraction and analyzed them by a 3'-RNA sequencing approach. Transcriptome profiles were analyzed to estimate content of immune and stromal cells and to define disease-associated gene signatures by using statistical and bioinformatics methods. This study identified 158 differentially expressed genes (DEGs) that were significantly increased in CNV compared with control tissue. Cell type enrichment analysis revealed a diverse cellular landscape with an enrichment of endothelial cells, macrophages, T cells, and natural killer T cells in the CNV. Gene ontology enrichment analysis found that DEGs contributed to blood vessel development, extracellular structure organization, response to wounding, and several immune-related terms. The S100 calcium-binding proteins A8 (S100A8) and A9 (S100A9) emerged among the top DEGs, 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 to be a novel biomarker and promising target for therapeutics and diagnostics directed at age-related macular degeneration.

  View details for DOI 10.1016/j.ajpath.2020.04.004

  View details for PubMedID 32339498

• RNA Sequencing of Formalin-Fixed and Paraffin-Embedded Tissue as a Complementary Method in Ophthalmopathology. Klinische Monatsblatter fur Augenheilkunde Schlunck, G., Boneva, S., Wolf, J., Schlecht, A., Reinhard, T., Auw-Hädrich, C., Lange, C. 2020; 237 (7): 860-866

  Abstract

  The high-throughput method of "Next Generation Sequencing" (NGS) allows cost-effective decoding of the nucleotide sequences of millions of RNA molecules in a sample. This makes it possible to determine the number of distinct RNA molecules in tissues or cells and to use these data to draw conclusions. The entirety of RNAs, in particular mRNAs (messenger RNAs) as potential precursors of proteins, provides a comprehensive insight into the functional state of the cells and tissues under investigation. In addition to cell cultures or unfixed tissue, formalin-fixed and paraffin-embedded (FFPE) tissue can also be analysed for this purpose using specific methods. In this overview, the methodological strategy and its application to the field of ophthalmic histopathology are presented.

  View details for DOI 10.1055/a-1187-1590

  View details for PubMedID 32659839

• [What is the importance of the conjunctiva as a potential transmission pathway for SARS-CoV-2 infections?] Der Ophthalmologe : Zeitschrift der Deutschen Ophthalmologischen Gesellschaft Lange, C., Wolf, J., Auw-Haedrich, C., Schlecht, A., Boneva, S., Lapp, T., Agostini, H., Martin, G., Reinhard, T., Schlunck, G. 2020; 117 (7): 626-630

  Abstract

  Recent studies have described conjunctivitis in approximately 1% of COVID-19 patients and speculated that SARS-CoV‑2 can be transmitted via the conjunctiva. In this article we recapitulate the molecular mechanisms of host cell entry of SARS-CoV‑2 and discuss the current evidence for a potential conjunctival transmission of SARS-CoV‑2. The current body of evidence indicates that SARS-CoV‑2 requires the membrane-bound angiotensin-converting enzyme 2 (ACE2) and the membrane-bound serine protease TMPRSS2 to enter cells. Recent studies suggest that COVID-19 patients rarely exhibit viral RNA in tear film and conjunctival smears and that, ACE2 and TMPRSS2 are only expressed in very small amounts in the conjunctiva, making conjunctival infection with SARS-CoV‑2 via these mediators unlikely. Nevertheless, we consider the current evidence to be still too limited to provide a conclusive statement and recommend appropriate protective measures for healthcare personnel who are in close contact with suspected and confirmed COVID-19 patients.

  View details for DOI 10.1007/s00347-020-01150-1

  View details for PubMedID 32572552

  View details for PubMedCentralID PMC7306648

• Novel insights into retinal neovascularization secondary to central serous chorioretinopathy using 3D optical coherence tomography angiography. American journal of ophthalmology case reports Gruber, M., Wolf, J., Stahl, A., Ness, T., Scholl, H., Agostini, H., Maloca, P., Lange, C. 2020; 18: 100609

  Abstract

  To describe the clinical presentation and novel anatomical features of a patient with chronic central serous chorioretinopathy (CSCR) complicated by retinal neovascularization (RNV).A 48 year-old patient with a long-standing history of bilateral CSCR presented to our clinic complaining about a sudden onset of tiny floaters. Multimodal imaging including fundus autofluorescence (FAF), fundus fluorescein (FA) and ICG angiography (ICG) and spectral domain optical coherence tomography (SD-OCT) confirmed the diagnosis of CSCR and revealed a pre-retinal neovascularization and concurring vitreous hemorrhage. Swept source OCT angiography (OCTA) and 3D reconstruction virtual reality determined the retinal origin of the neovascularization. Follow-up examination revealed clearing of the vitreous hemorrhage and spontaneous obliteration of the RNV without any treatment three months following the initial presentation.To the best of our knowledge, this is the first report of a RNV associated with CSCR which was determined by three-dimensional (3D) OCTA reconstruction.

  View details for DOI 10.1016/j.ajoc.2020.100609

  View details for PubMedID 32123772

  View details for PubMedCentralID PMC7036447

• Secreted Phosphoprotein 1 Expression in Retinal Mononuclear Phagocytes Links Murine to Human Choroidal Neovascularization. Frontiers in cell and developmental biology Schlecht, A., Zhang, P., Wolf, J., Thien, A., Rosmus, D. D., Boneva, S., Schlunck, G., Lange, C., Wieghofer, P. 2020; 8: 618598

  Abstract

  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.

  View details for DOI 10.3389/fcell.2020.618598

  View details for PubMedID 33585455

  View details for PubMedCentralID PMC7876283