Corneal edema resorption after injury is regulated by the transcription factor NFAT5
Authors: Hadrian Karina , Musial Gwen , Schönberg Alfrun , Georgiev Tihomir , Küper Christoph , Bock Felix , Cursiefen Claus , Eming Sabine A. , Hos Deniz 
Published: December 2022

In cornea, injury or inflammation leads to an ingrowth of blood‐ and lymphatic vessels associated with corneal edema. That can lead to a loss of transparency which is the second most common cause for blindness worldwide. So far, the only treatment is corneal transplantation, resulting in more than a million people suffering from corneal blindness due to shortage of donor corneas. Recently we showed a positive role of lymphatic vessels in the resolution of corneal edema. The nuclear factor of activated T‐cells 5 (NFAT5/TonEBP) is an important transcription factor regulating lymphangiogenesis (LA) by modulating the expression of pro‐lymphangiogenic vascular endothelial growth factor C (VEGF‐C) in skin macrophages. It has not been investigated whether NFAT5 is expressed in the cornea and which role it plays in the inflammatory response after corneal injury.

In this study, two NFAT5‐knockout mouse models were used: tamoxifen‐inducible systemic (UbcCre/NFAT5fl/fl) and a myeloid cell specific (LysMCre/NFAT5fl/fl). Both underwent perforating corneal injury (PCI) in the central cornea. Seven days after surgery, corneal edema and its resolution were measured using in vivo optical coherence tomography (OCT). The mean corneal thickness (MCT) was evaluated using MatLab®. Corneal inflammation was assayed via immunofluorescence. Bone marrow derived macrophages (BMDMs) from LysMCre/NFAT5fl/fl mice were stimulated with LPS/IFN‐γ for 24 h and used for pinocytosis assays using FITC‐conjugated dextran. Pinocytosis capacity was measured by calculating the ratio of FITC‐saturated cells to the total number of cells using MatLab®.

In naive corneas, NFAT5 was mainly expressed in fibroblasts. After PCI, NFAT5 expression in fibroblasts was downregulated and mainly expressed by macrophages (Correlation coefficient of TonEBP+/Vimentin+: 0.446 ± 0.111 in naive corneas vs 0.153 ± 0.024 after PCI [p < 0.0001]; TonEBP+/F480+: 0.113 ± 0.032 in naive cornea vs. 0.446 ± 0.018 after PCI [p < 0.0001]). In uninjured corneas, NFAT5 knockout did not lead to changes in corneal thickness, however, the number of macrophages was significantly increased (24.76 ± 3.54% in knockout vs. 16.14 ± 0.36%; p = 0.0094 of corneal area covered by F480+ cells). After PCI, the resolution of corneal edema was enhanced in both, systemic NFAT5 knockout mice (Mean corneal thickness: 97.1 ± 10.0 μm in knockout vs. 137.1 ± 28.8 μm [p = 0.002]) and myeloid cell NFAT5 knockout mice (Mean corneal thickness: 97.1 ± 10.0 μm in knockout vs. 126.0 ± 16.6 μm; [p < 0.001]). After incubation of BMDMs from LysMCre/NFAT5fl/fl mice with fluorescent dextran, the percentage of cells saturated which incorporated the fluorescent dextran in inflammatory stimulated macrophages was significantly higher in LysMCre+/NFAT5fl/fl macrophages compared to LysMCre−/NFAT5fl/fl macrophages (1.45 ± 0.28% vs. 0.61 ± 0.18%; [p < 0.001]).

Our results demonstrate that NFAT5 is highly expressed in the cornea and its expression shows considerable changes after PCI. Mechanistically, we found that myeloid cell‐derived NFAT5 is crucial for controlling corneal edema, as edema resorption after PCI was significantly enhanced in mice with conditional loss of NFAT5 in the myeloid cell lineage, presumably due to increased pinocytosis of corneal macrophages. Collectively, we uncover a suppressive role for NFAT5 in corneal edema resorption, thereby identifying a novel therapeutic target to combat edema‐induced corneal blindness.