Corneal tissue induces transcription of metallothioneins in monocyte-derived human macrophages.

Autor: Wolf J; Eye Center, Medical Center, Faculty of Medicine, University of Freiburg, Germany. Electronic address: julian.wolf@uniklinik-freiburg.de., Zhuang X; Eye Center, Medical Center, Faculty of Medicine, University of Freiburg, Germany., Hildebrand A; Eye Center, Medical Center, Faculty of Medicine, University of Freiburg, Germany., Boneva S; Eye Center, Medical Center, Faculty of Medicine, University of Freiburg, Germany., Schwämmle M; Eye Center, Medical Center, Faculty of Medicine, University of Freiburg, Germany., Kammrath Betancor P; Eye Center, Medical Center, Faculty of Medicine, University of Freiburg, Germany., Fan J; Department of Radiation Oncology and Cyberknife Center, Faculty of Medicine and University Hospital Cologne, University of Cologne, Cologne, Germany., Böhringer D; Eye Center, Medical Center, Faculty of Medicine, University of Freiburg, Germany., Maier P; Eye Center, Medical Center, Faculty of Medicine, University of Freiburg, Germany., Lange C; Eye Center, Medical Center, Faculty of Medicine, University of Freiburg, Germany., Reinhard T; Eye Center, Medical Center, Faculty of Medicine, University of Freiburg, Germany., Schlunck G; Eye Center, Medical Center, Faculty of Medicine, University of Freiburg, Germany., Lapp T; Eye Center, Medical Center, Faculty of Medicine, University of Freiburg, Germany. Electronic address: thabo.lapp@uniklinik-freiburg.de.
Jazyk: angličtina
Zdroj: Molecular immunology [Mol Immunol] 2020 Dec; Vol. 128, pp. 188-194. Date of Electronic Publication: 2020 Nov 01.
DOI: 10.1016/j.molimm.2020.10.016
Abstrakt: Purpose: 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.
Methods: 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.
Results: 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.
Conclusions: 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.
(Copyright © 2020 Elsevier Ltd. All rights reserved.)
Databáze: MEDLINE
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