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 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 ( 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. 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.

In-Depth Molecular Characterization of Neovascular Membranes Suggests a Role for Hyalocyte-to-Myofibroblast Transdifferentiation in Proliferative Diabetic Retinopathy

“…However, the scaffold provided by the cortical vitreous does not seem to be the only factor facilitating RNV formation. Vitreous hyalocytes, which represent a unique resident myeloid cell population ( 9 ), have been suggested as essential participants in the course of cicatrical contraction in proliferative vitreoretinal disease in the past ( 10 ). Nevertheless, the exact role of vitreous hyalocytes in the pathophysiology of RNV in PDR is largely unknown.…”

The Human Eye Transcriptome Atlas: A Searchable Comparative Transcriptome Database for Healthy and Diseased Human Eye Tissue

“…In addition, the same sequencing protocol was used for all FFPE samples 8 , which minimizes technical variability between different datasets. Retinal microglia and hyalocytes, in contrast, were sequenced using a different protocol, which is optimized for unfixed samples 9 . This may have added some technical variability.…”

“…Resected tissue was processed as previously described [8][9][10] . In brief, samples were either processed unfixed 9 or formalin-fixed and embedded in paraffin 8 immediately after surgery according to routine protocols.…”

“…Histological diagnoses were confirmed by two experienced ophthalmic pathologists. For cell analysis, retinal microglia or vitreous hyalocytes were FACS sorted and isolated according to a standardized protocol 9 . A detailed overview of the analyzed samples, the associated publications and the respective tissue processing manner can be found in Table 1.…”

“…PCR bias was removed using unique molecular identifiers. For unfixed samples, RNA sequencing was performed as previously described 8,9 .…”