Autoimmune uveitis is a major cause of blindness and experimental autoimmune uveitis (EAU) is mediated by interphotoreceptor retinoid-binding protein specific effector CD4+ T cells that infiltrate the retina. At least two MHC Class II (MHC II) antigen-presenting cell (APC) events are required for uveitis to develop. The first occurs in the secondary lymphoid organs when dendritic cells (DCs) activate and expand effector CD4+ T cells that enter the circulation and migrate systemically. The second APC event occurs when DC-primed effector CD4+ T cells infiltrate the retina and are restimulated by the relevant autoantigen. Importantly, if this second restimulation does not occur, then uveitis does not develop. However, it is still unclear which cell type(s) function as APCs within the retina. There are two candidate MHC II+ cell types-resident microglia and infiltrating DCs. We used the inducible Cre-lox approach to develop mouse strains in which MHC II was knocked out specifically on microglia using either the P2ry12 or Tmem119 gene to drive recombination. We also used Itgax (CD11c encoding gene) to drive recombination in DCs. Using this approach, we uncovered that the second APC event was mediated by MHC II+ microglia and not infiltrating MHC II+ DCs. Therefore, microglia are an important therapeutic target that can prevent and/or diminish uveitis even in the presence of circulating retinal autoantigen-specific effector CD4+ T cells.

Glaucoma is a leading cause of irreversible blindness worldwide, characterized by progressive retinal ganglion cell (RGC) loss and optic nerve degeneration. Although elevated intraocular pressure (IOP) is a major risk factor, disease progression can occur despite normal IOP, highlighting the need for neuroprotective strategies beyond IOP reduction. Neuroinflammation has been implicated in glaucomatous neurodegeneration through complement system activation via the classical and lectin pathways. However, the role of the alternative pathway (AP), which functions as an amplification loop for central complement component 3 (C3) activation, in glaucoma is unclear. In this study, we investigated the role of the AP in glaucoma using a microbead-induced mouse model of glaucoma in mice deficient in either complement factor B (Cfb-/-), to selectively block the AP, or in C3 (C3-/-) to block all three complement pathways. Our results indicate the AP is critical for glaucoma development, and blocking this pathway resulted in significant neuroprotection, preventing loss of RGCs, axons, and visual acuity, which coincided with reduced glial activation and inflammation. Blocking the AP provided comparable neuroprotection to blocking all three complement pathways, indicating that the AP amplification loop is an essential component of destructive neuroinflammation in glaucoma. Furthermore, blocking the AP also conferred neuroprotection in the optic nerve crush model, suggesting a broader role for the AP in optic neuropathies. These findings establish the AP as a key driver of complement-mediated neurodegeneration in glaucoma and highlight the therapeutic potential of targeting the AP in glaucoma and other neurodegenerative diseases.

Autoimmune uveitis is a major cause of blindness in the working-age population of developed countries. Experimental autoimmune uveitis (EAU) depends on activation of interphotoreceptor retinoid-binding protein (IRBP) specific CD4 + effector T cells that migrate systemically and infiltrate into the retina. Following systemic induction of retinal antigen-specific T cells, the development of EAU can be broken down into three phases: early phase when inflammatory cells begin to infiltrate the retina, amplification phase, and peak phase. Although studied extensively, the function of local antigen-presenting cells (APCs) within the retina remains unclear. Two potential types of APCs are present during uveitis, resident microglia and infiltrating CD11c + dendritic cells (DCs). MHC class II (MHC II) is expressed within the retina on both CD11c + DCs and microglia during the amplification phase of EAU. Therefore, we used microglia specific (P2RY12 and TMEM119) and CD11c + DC specific MHC II knockout mice to study the function of APCs within the retina using the conventional and adoptive transfer methods of inducing EAU. Microglia were essential during all phases of EAU development: the early phase when microglia were MHC Il negative, and amplification and peak phases when microglia were MHC II positive. Unexpectedly, retinal infiltrating MHC Il + CD11c + DCs were present within the retina but their antigen-presenting function was not required for all phases of uveitis. Our data indicate microglia are the critical APCs within the retina and an important therapeutic target that can prevent and/or diminish uveitis even in the presence of circulating IRBP-specific CD4 + effector T cells.

Supplementary Figure from Midkine Promotes Metastasis and Therapeutic Resistance via mTOR/RPS6 in Uveal Melanoma

Glaucoma, a chronic neurodegenerative disease, is a leading cause of age-related blindness worldwide and characterized by the progressive loss of retinal ganglion cells (RGCs) and their axons. Previously, we developed a novel epigenetic rejuvenation therapy, based on the expression of the three transcription factors Oct4, Sox2, and Klf4 (OSK), which safely rejuvenates RGCs without altering cell identity in glaucomatous and old mice after 1 month of treatment. In the current year-long study, mice with continuous or cyclic OSK expression induced after glaucoma-induced vision damage had occurred were tracked for efficacy, duration, and safety. Surprisingly, only 2 months of OSK fully restored impaired vision, with a restoration of vision for 11 months with prolonged expression. In RGCs, transcription from the doxycycline (DOX)-inducible Tet-On AAV system, returned to baseline 4 weeks after DOX withdrawal. Significant vision improvements remained for 1 month post switching off OSK, after which the vision benefit gradually diminished but remained better than baseline. Notably, no adverse effects on retinal structure or body weight were observed in glaucomatous mice with OSK continuously expressed for 21 months providing compelling evidence of efficacy and safety. This work highlights the tremendous therapeutic potential of rejuvenating gene therapies using OSK, not only for glaucoma but also for other ocular and systemic injuries and age-related diseases.

Uveal melanoma (UM) is a highly malignant tumor of the eye. Metastatic spread of UM occurs almost exclusively via blood vessels and is of tremendous interest, as half of the patients with uveal melanoma die of metastasis in the long run. The tumor microenvironment consists of all cellular and non-cellular compounds of a solid tumor, except for the tumor cells. This study aims to provide a more detailed understanding of the tumor microenvironment of UM to build the foundation for new therapeutic targets. Fluorescence immunohistochemistry was performed to examine the localization of various cell types in the tumor microenvironment in UM. Furthermore, the presence of LAG-3 and its ligands Galectine-3 and LSECtin was examined to evaluate the potential efficacy of immune checkpoint inhibitor-based therapies. The main findings are that blood vessels are mainly located in the middle of the tumor, and that immune cells are mostly found in the outer section of the tumor. LAG-3 and Galectine-3 were found to be highly represented, whereas LSECtin barely occurred in UM. Both the predominant location of tumor-associated macrophages in the outer section of the tumor and the high presence of LAG-3 and Galectine-3 in the UM serve as attainable therapeutic targets.

Objective: This review serves as a comprehensive description and summary of currently available preclinical models of three tumors in ophthalmic oncology: conjunctival melanoma (CM), uveal melanoma (UM), and retinoblastoma. Background: Malignant melanomas are the most common tumors of the eye in adults, most often localized in the uvea and conjunctiva. Although the primary tumor can be successfully eliminated in many cases, nearly one in two UMs-and one in three CMs-are fatal to the patient due to metastasis. Effective therapies for metastatic uveal and CMs are unfortunately still not available, so there is an urgent need for new therapeutic strategies to improve prognosis quoad vitam and prolong the survival of melanoma patients. Another widely known tumor of the eye is retinoblastoma, which is the most common pediatric ocular malignancy, occurring in approximately 1 in 15,000-18,000 live births. Overall, it is considered well treatable, with a survival rate of approximately 90% at 3 years, although fatal if untreated. For a long time, enucleation was also considered the treatment of choice, with bilateral cases having one eye irradiated and the eye with the more advanced tumor removed. Since the 1990s, however, systemic chemotherapy has been predominantly used to preserve the quality of life and vision of young patients, although the cellular activity of the retinoblastoma often remains after treatment with chemotherapeutic agents. Prognosis of the disease is immensely depending on the stage and time of diagnosis and is varying between countries due to different developmental status of health care systems. Methods: We review recent advances in the available literature on established preclinical models in CM, UM, and retinoblastoma. In addition, we discuss the advantages and limitations of these models and provide an overview of current alternatives to animal testing in preclinical studies. Conclusions: In the case of all three diseases, further research is needed for improved therapeutic options. Animal models in particular are indispensable for cancer research in order to mimic the extremely complex processes of human carcinogenesis, physiology and progression. Certainly, animal studies do not easily translate to human diseases due to biological differences and limitations. However, they continue to serve as the primary source and link between in vitro testing and clinical studies in patients. In order to minimize animal experiments and possibly even replace them in the future, alternatives such as 3D cell cultures and in silico predictions are useful and insightful additions and require further development. Still, no currently available preclinical model can be fully translated to some of here described diseases. Nevertheless, they all provide essential insights and knowledge that should be of use in the future for better understanding and pursuit of new therapeutic strategies.

Abstract Uveal melanoma is a rare form of melanoma that originates in the eye, exerts widespread therapeutic resistance, and displays an inherent propensity for hepatic metastases. Because metastatic disease is characterized by poor survival, there is an unmet clinical need to identify new therapeutic targets in uveal melanoma. Here, we show that the pleiotropic cytokine midkine is expressed in uveal melanoma. Midkine expression in primary uveal melanoma significantly correlates with poor survival and is elevated in patients that develop metastatic disease. Monosomy 3 and histopathologic staging parameters are associated with midkine expression. In addition, we demonstrate that midkine promotes survival, migration across a barrier of hepatic sinusoid endothelial cells and resistance to AKT/mTOR inhibition. Furthermore, midkine is secreted and mediates mTOR activation by maintaining phosphorylation of the mTOR target RPS6 in uveal melanoma cells. Therefore, midkine is identified as a uveal melanoma cell survival factor that drives metastasis and therapeutic resistance, and could be exploited as a biomarker as well as a new therapeutic target. Implications: Midkine is identified as a survival factor that drives liver metastasis and therapeutic resistance in melanoma of the eye.

Autoimmune arthritis is characterized by impaired regulatory T (Treg) cell migration into inflamed joint tissue and by dysregulation of the balance between Treg cells and Th17 cells. Interleukin-6 (IL-6) is known to contribute to this dysregulation, but the molecular mechanisms behind impaired Treg cell migration remain largely unknown. In this study, we assessed dynamic changes in membrane-bound IL-6 receptor (IL6R) expression levels on Th17 cells by flow cytometry during the development of collagen-induced arthritis (CIA). In a next step, bioinformatics analysis based on proteomics was performed to evaluate potential pathways affected by altered IL-6R signaling in autoimmune arthritis. Our analysis shows that membrane-bound IL-6R is upregulated on Th17 cells and is inversely correlated with IL-6 serum levels in experimental autoimmune arthritis. Moreover, IL-6R expression is significantly increased on Th17 cells from untreated patients with rheumatoid arthritis (RA). Interestingly, CD4+ T cells from CIA mice and RA patients show reduced phosphorylation of vasodilator-stimulated phosphoprotein (VASP). Bioinformatics analysis based on proteomics of CD4+ T cells with low or high phosphorylation levels of VASP revealed that integrin signaling and related pathways are significantly enriched in cells with low phosphorylation of VASP. Specific inhibition of p-VASP reduces the migratory function of Treg cells but has no influence on effector CD4+ T cells. Importantly, IL-6R blockade restores the phosphorylation level of VASP, thereby improving the migratory function of Treg cells from RA patients. Thus, our results establish a link between IL6R signaling and phosphorylation of VASP, which controls Treg cell migration in autoimmune arthritis.

PURPOSEWe analyzed the effects of short-term ultraviolet A (UVA) irradiation on the putative limbal stem cell phenotype, limbal fibroblasts, corneal inflammation, and corneal (lymph)angiogenic privilege.METHODSPrimary human limbal epithelial cells and fibroblasts were irradiated with 5.2 J/cm2 of UVA. The limbal epithelial cell phenotype was assessed using P63a, cytokeratin 15, integrin b1 (marking stem and transient amplifying cells), and cytokeratin 3 (a differentiation marker) as well as by a colony-forming efficiency (CFE) assay. An epithelial-fibroblast coculture model was used to compare the ability of irradiated and nonirradiated fibroblasts to support the putative limbal stem cell phenotype. The effects of the conditioned media of irradiated and nonirradiated cells on proliferation and tube formation of human lymphatic and blood endothelial cells also were tested. The levels of factors related to angiogenesis and inflammation were assessed in a protein array and using ELISA.RESULTSUltraviolet A induced phenotypical changes of limbal epithelial cells, as their CFE and putative stem cell/transient amplifying marker expression decreased. Limbal epithelial cells cocultured with UVA-irradiated limbal fibroblasts also exhibited differentiation and CFE decrease. Conditioned media from irradiated limbal epithelial cells and fibroblasts inhibited lymphatic endothelial cell proliferation and tube network complexity. Levels of monocyte chemoattractant protein 1 (MCP1) were reduced following UVA irradiation of both cell populations, while levels of IFN-γ increased in irradiated limbal epithelial cells.CONCLUSIONSThese data imply a key role of cellular components of the limbal niche following short-term UVA irradiation. Overall, UVA irradiation leads to dysfunction of these cells and a anti(lymph)angiogenic and anti-inflammatory micromilieu.