Dry eye disease (DED), an inflammatory autoimmune disorder affecting the ocular surface, degrades visual performance and the quality of life of >10 million people in the United States alone. The primary limitation in the effective treatment of DED is an incomplete understanding of its specific cellular and molecular pathogenic elements. Using a validated mouse model of DED, herein we functionally characterize the different T cell subsets, including regulatory T cells (Tregs) and pathogenic effector T cells, and determine their contribution to the pathogenesis of DED. Our data demonstrate the presence of dysfunctional Tregs and the resistance of pathogenic T cells, particularly Th17 cells, to Treg suppression in DED. In addition, we clearly show that in vivo blockade of IL-17 significantly reduces the severity and progression of disease, which is paralleled by a reduction in the expansion of Th17 cells and restoration of Treg function. Our findings elucidate involvement of a previously unknown pathogenic T cell subset (Th17) in DED that is associated specifically with Treg dysfunction and disease pathogenesis and suggest a new target for dry eye therapy.

Purpose Dry eye disease (DED) is associated with ocular surface inflammation that is thought to be primarily mediated by CD4 T cells. The purpose of this study was to investigate whether this T cell mediated immune response is generated in the lymphoid compartment, and to characterize the functional phenotype of the T cells activated in DED. Methods DED was induced in female C57BL/6 mice by exposure to a desiccating environment in the controlled environment chamber (CEC) and to systemic scopolamine. T cells from regional draining lymph nodes (LN) of DED mice and normal mice were analyzed for surface activation markers (CD69 and CD154), chemokine and cytokine receptors, and for their proliferation potential. Results Draining LN of DE mice showed increased frequencies of CD69 and CD154 expressing T cells with higher proliferative capacity. In addition, these LN T cells primarily showed a Th-1 phenotype, expressing significantly higher levels of IFN-γ and IL-12Rβ2 but not IL-4R. Similarly, the LN of DE mice showed significantly increased frequencies of T cells expressing CXCR3 and CCR5, but not CCR4, suggesting a bias toward a Th-1 phenotype. Conclusions Our data demonstrate that a Th-1 type immune response is induced in the regional LN of DE mice. The identification of specific cytokine/chemokine receptors overexpressed by these T cells may signify potential novel targets/strategies for the treatment of DED.

Background The lymphatic system plays a key role in tissue fluid homeostasis and lymphatic dysfunction due to genetic defects or lymphatic vessel obstruction can cause lymphedema, disfiguring tissue swellings often associated with fibrosis and recurrent infections without available cures to date. In this study, retinoic acids (RAs) were determined to be a potent therapeutic agent that is immediately applicable to reduce secondary lymphedema. Methods and Results We report that RAs promote proliferation, migration and tube formation of cultured lymphatic endothelial cells (LECs) by activating FGF-receptor signaling. Moreover, RAs control the expression of cell-cycle checkpoint regulators such as p27Kip1, p57Kip2 and the aurora kinases through both an Akt-mediated non-genomic action and a transcription-dependent genomic action that is mediated by Prox1, a master regulator of lymphatic development. Moreover, 9-cisRA was found to activate in vivo lymphangiogenesis in animals based on mouse trachea, matrigel plug and cornea pocket assays. Finally, we demonstrate that 9-cisRA can provide a strong therapeutic efficacy in ameliorating the experimental mouse tail lymphedema by enhancing lymphatic vessel regeneration. Conclusions These in vitro and animal studies demonstrate that 9-cisRA potently activates lymphangiogenesis and promotes lymphatic regeneration in an experimental lymphedema model, presenting it as a promising novel therapeutic agent to treat human lymphedema patients.

Key Points• Afferent lymphatic vessels express interleukin-7.• Interleukin-7 supports lymphatic drainage.The cytokine interleukin (IL)-7 exerts essential roles in lymph node (LN) organogenesis and lymphocyte development and homeostasis. Recent studies have identified lymphatic endothelial cells (LECs) as a major source of IL-7 in LNs. Here, we report that LECs not only produce IL-7, but also express the IL-7 receptor chains IL-7Ra and CD132. Stimulation with recombinant IL-7 enhanced LEC in vitro activity and induced lymphangiogenesis in the cornea of wild-type (WT) mice. Whereas in IL-7Ra 2/2 mice, dermal lymphatic vessels (LVs)were abnormally organized and lymphatic drainage was compromised, transgenic overexpression of IL-7 in mice resulted in an expanded dermal LV network with increased drainage function. Moreover, systemic treatment with recombinant IL-7 enhanced lymphatic drainage in the skin of WT mice and of mice devoid of lymphocytes. Experiments in IL-7Rabone marrow chimeras demonstrated that the drainage-enhancing activity of IL-7 was exclusively dependent on IL-7Ra expression in stromal but not in hematopoietic cells. Finally, near-infrared in vivo imaging performed in IL-7Ra 2/2 mice revealed that the pumping activity of collecting vessels was normal but fluid uptake into lymphatic capillaries was defective. Overall, our data point toward an unexpected new role for IL-7 as a potential autocrine mediator of lymphatic drainage. (Blood. 2013;122(13):2271-2281

Blood and lymphatic vessels are not evenly distributed in normal limbal areas. Furthermore, corneal LG and HG respond differently to inflammatory stimuli. These new findings will shed some light on corneal physiology and pathogenesis and on the development of experimental models and therapeutic strategies for corneal diseases.

These novel findings together indicate that VLA-1 is critically involved in the processes of lymphangiogenesis. Further investigation on this factor may provide novel therapies for corneal inflammation, transplant rejection, and other lymphatic-related disorders in the body.

Angiopoietin-2 is critically involved in lymphatic processes in vivo and in vitro. Further investigation of the Ang-2 pathway may provide novel insights and therapeutic strategies for lymphatic-related disorders, which occur both inside and outside the eye.

To study the effect of topical application of very late antigen 4 (VLA-4) small-molecule antagonist (anti-VLA-4 sm) in a mouse model of dry eye disease. Methods: Anti-VLA-4 sm (or control vehicle) was applied topically to mice placed in a controlledenvironment chamber. Corneal fluorescein staining and conjunctival T-cell enumeration were performed in the different treatment groups. Real-time polymerase chain reaction was used to quantify expression of inflammatory cytokines in the cornea and conjunctiva. Results: Dry eye syndrome induced increased corneal fluorescein staining, corneal and conjunctival tumor necrosis factor ␣ messenger RNA expression, and T-cell infiltration into the conjunctiva. Very late antigen 4 blockade significantly decreased corneal fluorescein staining compared with the untreated dry eye disease and control vehicle-treated groups (PϽ.001 and P=.02, respectively). In addition, VLA-4 blockade was associated with a significant decrease in conjunctival T-cell numbers (PϽ .001 vs control vehicle-treated group) and tumor necrosis factor-␣ transcript levels in the cornea (P=.04 vs control vehicle-treated group) and conjunctiva (P=.048 vs control vehicle-treated group). Conclusion: Application of topical anti-VLA-4 sm led to a significant decrease in dry eye signs and suppression of inflammatory changes at the cellular and molecular levels. Clinical Relevance: Topical blockade of VLA-4 may be a novel therapeutic approach to treat the clinical signs and inflammatory changes accompanying dry eye disease.