Experimental & Molecular Medicine · 10 authors, 7 centres
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This study identifies NFAT5 as a negative regulator of corneal edema resolution after perforating injury in mice. NFAT5 expression shifts from corneal fibroblasts to macrophages after injury, and its deletion in myeloid cells accelerates edema resorption by enhancing macrophage pinocytosis. Targeting NFAT5 may offer a novel nonsurgical therapy for edema-induced corneal blindness.
**Background:** Corneal edema, a leading cause of blindness, results from fluid imbalance and loss of transparency. Current treatment relies on corneal transplantation, but donor shortages limit access. The osmosensitive transcription factor NFAT5 (TonEBP) regulates salt/water balance in skin via macrophage-driven VEGF-C secretion and lymphangiogenesis. Its role in corneal edema was unknown. This study investigated NFAT5 expression and function in naive and injured corneas using a mouse model of perforating corneal injury (PCI).
**Methods:** NFAT5 expression was analyzed by confocal microscopy in corneal whole mounts from naive and PCI-injured mice, with colocalization to vimentin (fibroblasts) and F4/80 (macrophages). Ubiquitous (UbcCre/NFAT5^fl/fl^) and myeloid cell-specific (LysMCre/NFAT5^fl/fl^) NFAT5 knockout mice were generated. Corneal thickness (mean corneal thickness, MCT) was measured by in vivo optical coherence tomography (OCT) at baseline and 1 week after PCI. Vegfc, Aqp1, and Aqp5 mRNA expression was quantified by qPCR. Lymphatic vessel ingrowth was assessed by LYVE-1 staining. Pinocytosis capacity was evaluated in bone marrow-derived macrophages (BMDMs) using pHrodo Green Dextran uptake.
**Key Results:** In naive corneas, NFAT5 colocalized strongly with vimentin (correlation 0.45 ± 0.11) but weakly with F4/80 (0.11 ± 0.03). After PCI, colocalization shifted: NFAT5 with vimentin decreased to 0.15 ± 0.02 (p < 0.0001), while with F4/80 increased to 0.45 ± 0.02 (p < 0.0001). In vitro, LPS/IFN-γ stimulation upregulated Nfat5 mRNA in BMDMs (2.83 ± 1.15-fold; p < 0.01). Ubiquitous NFAT5 knockout did not alter baseline MCT (UbcCre^+^/NFAT5^fl/fl^: 89.52 ± 5.59 µm vs. controls 92.59 ± 3.76 µm; p > 0.05) but increased corneal macrophage numbers (F4/80^+^ area: 24.76 ± 3.54% vs. 16.14 ± 0.36%; p < 0.01). After PCI, MCT was significantly lower in UbcCre^+^/NFAT5^fl/fl^ mice (97.10 ± 10.0 µm) compared to UbcCre^-^/NFAT5^fl/fl^ mice (137.1 ± 28.8 µm; p < 0.01). Similarly, in myeloid-specific knockout mice, MCT after PCI was 126.0 ± 16.6 µm in LysMCre^+^/NFAT5^fl/fl^ vs. 161.3 ± 17.8 µm in LysMCre^-^/NFAT5^fl/fl^ (p < 0.001). Vegfc mRNA was increased in injured wild-type corneas (5.61 ± 0.55-fold; p < 0.0001) but not further upregulated in NFAT5-deficient mice (3.32 ± 0.52-fold; p > 0.05 vs. uninjured knockout). Lymphatic vessel density and morphology were unchanged. Aqp1 and Aqp5 expression did not differ between groups. Pinocytosis capacity was significantly higher in LysMCre^+^/NFAT5^fl/fl^ BMDMs (1.45 ± 0.28% saturated cells) vs. controls (0.61 ± 0.18%; p < 0.0001).
**Clinical Implications:** This study reveals NFAT5 as a novel negative regulator of corneal edema resolution, acting through myeloid cells. NFAT5 deficiency enhances macrophage pinocytosis, accelerating fluid clearance without affecting lymphangiogenesis or aquaporin expression. Targeting NFAT5 could provide a nonsurgical therapy for acute corneal edema (e.g., after injury) and chronic conditions like Fuchs endothelial dystrophy or keratoconus hydrops, potentially reducing the need for corneal transplantation. Further research is needed to translate these findings to human corneas and develop safe NFAT5 inhibitors.