Retinal endothelial cell phenotypic modifications during experimental autoimmune uveitis: a transcriptomic approach

BMC Ophthalmol. 2020 Mar 17;20(1):106. doi: 10.1186/s12886-020-1333-5.

Abstract

Background: Blood-retinal barrier cells are known to exhibit a massive phenotypic change during experimental autoimmune uveitis (EAU) development. In an attempt to investigate the mechanisms of blood-retinal barrier (BRB) breakdown at a global level, we studied the gene regulation of total retinal cells and retinal endothelial cells during non-infectious uveitis.

Methods: Retinal endothelial cells were isolated by flow cytometry either in Tie2-GFP mice (CD31+ CD45- GFP+ cells), or in wild type C57BL/6 mice (CD31+ CD45- endoglin+ cells). EAU was induced in C57BL/6 mice by adoptive transfer of IRBP1-20-specific T cells. Total retinal cells and retinal endothelial cells from naïve and EAU mice were sorted and their gene expression compared by RNA-Seq. Protein expression of selected genes was validated by immunofluorescence on retinal wholemounts and cryosections and by flow cytometry.

Results: Retinal endothelial cell sorting in wild type C57BL/6 mice was validated by comparative transcriptome analysis with retinal endothelial cells sorted from Tie2-GFP mice, which express GFP under the control of the endothelial-specific receptor tyrosine kinase promoter Tie2. RNA-Seq analysis of total retinal cells mainly brought to light upregulation of genes involved in antigen presentation and T cell activation during EAU. Specific transcriptome analysis of retinal endothelial cells allowed us to identify 82 genes modulated in retinal endothelial cells during EAU development. Protein expression of 5 of those genes (serpina3n, lcn2, ackr1, lrg1 and lamc3) was validated at the level of inner BRB cells.

Conclusion: Those data not only confirm the involvement of known pathogenic molecules but further provide a list of new candidate genes and pathways possibly implicated in inner BRB breakdown during non-infectious posterior uveitis.

Keywords: Blood-retinal barrier; Endothelial cells; Inflammation; Lipocalin 2; RNA-Seq; Transcriptome; Uveitis; ackr1; lrg1; serpina3n.

MeSH terms

  • Animals
  • Autoimmune Diseases / diagnosis*
  • Autoimmune Diseases / immunology
  • Autoimmune Diseases / metabolism
  • Blood-Retinal Barrier
  • Cell Count
  • Disease Models, Animal
  • Endothelial Cells / pathology*
  • Female
  • Flow Cytometry
  • Immunity, Cellular*
  • Male
  • Mice
  • Mice, Inbred C57BL
  • Retina / pathology*
  • T-Lymphocytes / immunology*
  • Uveitis / diagnosis*
  • Uveitis / immunology
  • Uveitis / metabolism