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Thabo Lapp, Daniel Boehringer, Antonia Hildebrand, Paola Kammrath Betancor, Jiaqi Fan, Thomas Reinhard, Gunther R Schlunck; Immuno-modulative effects of corneal endothelium on innate immune-cells as determined by transcriptome analysis.. Invest. Ophthalmol. Vis. Sci. 2019;60(9):890.
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© ARVO (1962-2015); The Authors (2016-present)
Immune reactions following corneal transplantation are less frequent compared to other forms of organ transplantation. The underlying mechanisms of corneal graft rejection and the effect of corneal endothelial cells (CEC) in this context are not fully understood. Using a human in vitro model, we aimed to assess whether donor CEC modulate the immune response of host innate immune cells.
Human monocytes were isolated from peripheral blood mononuclear cells (PBMC) and differentiated into monocyte-derived macrophages (MDM). A standardized protocol was used for processing of human corneas into fragments of defined sizes. MDMs were stimulated using processed corneal material with or without CEC. Processed human skin samples, lipopolysaccharide (LPS) or interferon-gamma (IFNγ) served as controls. RNA extraction was done using a commercially available RNA purification kit; next-generation sequencing for massive analysis of cDNA ends (MACE) was performed by GenXPro (Frankfurt, Germany). Data analysis and bioinformatic calculations were done using the R platform.
Unsupervised clustering allowed for identification of individual MDM donors as well as discrimination of different stimuli. Control stimuli (LPS and IFNγ) induced differential up- and down-regulation of a large number of known LPS- and IFNγ-regulated gene products. Clustering analysis revealed distinct transcriptome changes induced by phagocytosis of corneal material. Stimulation of MDM with CEC led to up- (n=237) and down-regulation (n=71) of various genes. Furthermore, MDM challenged with corneal allogen showed a distinct upregulation of metallothioneins. Metascape analysis of the 500 most altered genes (log2-fold change ≥2 or ≤-2 and uncorrected -log10 p-value ≥2) indicated enrichment of GO-clusters including PRR-signalling (e.g. TLR), NK-cell-regulation, cytokine-receptor-interactions, and activation of adaptive immune cells.
Human macrophages show a distinct upregulation of various metallothioneins when challenged with human corneal allogen. Furthermore, the presence of corneal endothelium during stimulation induces the upregulation of various genes involved in intracellular signaling, antigen processing, and chemokine- and immune-signaling. These results strongly suggest immuno-modulative effects of corneal endothelial cells.
This abstract was presented at the 2019 ARVO Annual Meeting, held in Vancouver, Canada, April 28 - May 2, 2019.
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