Immune responses reflect a complex interplay of cellular and extracellular components which define the microenvironment of a tissue. Therefore, factors that locally influence the microenvironment and re‐establish tolerance might be beneficial to mitigate immune‐mediated reactions, including the rejection of a transplant. In this study, we demonstrate that pre‐incubation of donor tissue with the immune modulator soluble CD83 (sCD83) significantly improves graft survival using a high‐risk corneal transplantation model. The induction of tolerogenic mechanisms in graft recipients was achieved by a significant upregulation of Tgfb, Foxp3, Il27, and Il10 in the transplant and an increase of regulatory dendritic cells (DCs), macrophages (Mφ), and T cells (Tregs) in eye‐draining lymph nodes. The presence of sCD83 during in vitro DC and Mφ generation directed these cells toward a tolerogenic phenotype leading to reduced proliferation‐stimulating activity in MLRs. Mechanistically, sCD83 induced a tolerogenic Mφ and DC phenotype, which favors Treg induction and significantly increased transplant survival after adoptive cell transfer. Conclusively, pre‐incubation of corneal grafts with sCD83 significantly prolongs graft survival by modulating recipient Mφ and DCs toward tolerance and thereby establishing a tolerogenic microenvironment. This functional strategy of donor graft pre‐treatment paves the way for new therapeutic options in the field of transplantation.

Macrophages are critical mediators of tissue vascularization both in health and disease. In multiple tissues, macrophages have been identified as important regulators of both blood and lymphatic vessel growth, specifically following tissue injury and in pathological inflammatory responses. In development, macrophages have also been implicated in limiting vascular growth. Hence, macrophages provide an important therapeutic target to modulate tissue vascularization in the clinic. However, the molecular mechanisms how macrophages mediate tissue vascularization are still not entirely resolved. Furthermore, mechanisms might also vary among different tissues. Here we review the role of macrophages in tissue vascularization with a focus on their role in blood and lymphatic vessel formation in the barrier tissues cornea and skin. Comparing mechanisms of macrophage-mediated hem- and lymphangiogenesis in the angiogenically privileged cornea and the physiologically vascularized skin provides an opportunity to highlight similarities but also tissue-specific differences, and to understand how macrophage-mediated hem- and lymphangiogenesis can be exploited for the treatment of disease, including corneal wound healing after injury, graft rejection after corneal transplantation or pathological vascularization of the skin.

The osmosensitive transcription factor nuclear factor of activated T cells 5 (NFAT5; or tonicity-responsive enhancer binding protein; TonEBP) plays a key role in macrophage-driven regulation of cutaneous salt and water balance. In the immune-privileged and transparent cornea, disturbances in fluid balance and pathological edema result in corneal transparency loss, which is one of the main causes of blindness worldwide. The role of NFAT5 in the cornea has not yet been investigated. We analyzed the expression and function of NFAT5 in naive corneas and in an established mouse model of perforating corneal injury (PCI), which causes acute corneal edema and transparency loss. In uninjured corneas, NFAT5 was mainly expressed in corneal fibroblasts. In contrast, after PCI, NFAT5 expression was highly upregulated in recruited corneal macrophages. NFAT5 deficiency did not alter corneal thickness in steady state; however, loss of NFAT5 led to accelerated resorption of corneal edema 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 uncovered a suppressive role for NFAT5 in corneal edema resorption, thereby identifying a novel therapeutic target to combat edema-induced corneal blindness.

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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...

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The limbus, the vascularized junction between the cornea and conjunctiva, is thought to function as a barrier against corneal neovascularization. However, the exact mechanisms regulating this remain unknown. In this study, the limbal epithelial stem cell (LESC) marker ABCB5 was used to investigate the role of LESCs in corneal neovascularization. In an ABCB5KO model, a mild but significant increase of limbal lymphatic and blood vascular network complexity was observed in developing mice (4 weeks) but not in adult mice. Conversely, when using a cornea suture model, the WT animals exhibited a mild but significant increase in the number of lymphatic vessel sprouts compared to the ABCB5KO, suggesting a contextual anti-lymphangiogenic effect of ABCB5 on the limbal vasculature during development, but a pro-lymphangiogenic effect under inflammatory challenge in adulthood. In addition, conditioned media from ABCB5-positive cultured human limbal epithelial cells (ABCB5+) stimulated human blood and lymphatic endothelial cell proliferation and migration. Finally, a proteomic analysis demonstrated ABCB5+ cells have a pro(lymph)angiogenic as well as an anti-inflammatory profile. These data suggest a novel dual, context-dependent role of ABCB5+ LESCs, inhibiting development but promoting inflammatory (lymph)angiogenesis in adulthood and exerting pro(lymph)angiogenic but anti-inflammatory effects. These findings are of high clinical relevance in relation to LESC therapy against blindness.