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Transcriptional Profiling Uncovers Human Hyalocytes as a Unique Innate Immune Cell Population

ABSTRACT: 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 with disorders of the vitreoretinal interface undergoing vitrectomy at the University Eye Hospital Freiburg between 2018 and 2019. Vitreal hyalocytes were characterized 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 human hyalocyte transcriptome was compared to the transcriptional profile of human macrophages, brain microglia and blood-derived monocytes 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 SPP1, FTL and CD74 as the most abundantly expressed genes in hyalocytes, which was confirmed by immunofluorescence. 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, such as brain microglia or blood-derived monocytes, showed an overall strong similarity (R2 > 0.637, p < 0.001), hyalocytes revealed 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 a clearly underestimated mediator in vitreoretinal disease and for the immune privilege of the eye.

ORGANISM(S): Homo sapiens

PROVIDER: GSE147657 | GEO | 2020/10/05

REPOSITORIES: GEO

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Project description:Purpose: The present study compares the transcriptional profiles of human hyalo-cytes, retinal microglia as well as classical, intermediate and non-classical mono-cytes in a pairwise analysis. Methods: Using fluorescence-activated cell sorting, hyalocytes and retinal microglia were isolated from the vitreous or retinal tissue of enucleated eyes due to melano-ma of the iris, ciliary body or choroid. In addition, classical, intermediate and non-classical monocytes were isolated from whole blood. RNA sequencing was per-formed and the transcriptional profiles of all five immune cell populations were ana-lyzed using bioinformatic analysis. Results: Pairwise analysis indicated distinct differences between hyalocytes and all three subsets of monocytes, whereas a high degree of similarity was apparent when compared to retinal microglia, with comparable expression levels of established MG markers such asTREM2, P2RY12 and TMEM119. Cluster analysis based on signa-ture genes of yolk sac-derived cells as well as hematopoietic stem and progenitor cells indicated that in contrast to monocytes, human hyalocytes and retinal microglia share a common origin in the yolk sac. Among the top expressed genes in hyalo-cytes, SPP1, CD74 and C3 were significantly upregulated when compared to mon-ocytes. Despite the high level of similarity, genes such as TNF, CCL3 and CXCL8 were significantly upregulated in hyalocytes when compared to retinal microglia. In addition, ten hyalocyte-specific genes were identified, among them FOS, DUSP1 and EGR2. Conclusions: This study provides detailed insights into the molecular profile of iso-lated human hyalocytes and, for the first time, human retinal microglia. The expres-sion profile of hyalocytes is similar to that of retinal microglia and indicates that hy-alocytes are, contrary to the prevailing notion, yolk sac-derived cells. The hyalocyte-specific markers identified in this study will contribute in the future to deciphering the different roles of this fascinating cell population in health and diseases of the vitre-oretinal interface.

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Transcriptional Profiling Uncovers Human Hyalocytes as a Unique Innate Immune Cell Population

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