Deniz Hos - CAP 11

Loss of corneal transparency due to severe inflammation and neovascularization is one of the leading causes of blindness worldwide. This project aims to investigate the functional relationship between myeloid cells and corneal neovascularization in various clinically relevant neovascular disease models in mice. The findings of this work will contribute to future immunomodulatory therapeutic interventions promoting corneal repair and preventing corneal disease.

Introduction

The cornea, the transparent front part of the eye, is physiologically avascular but can secondarily be invaded by blood vessels (BV) and lymphatic vessels (LV) as a result of severe trauma, infection, inflammation or degenerative disorders (1). BV can directly impair corneal transparency and vision if growing into the optical zone. Unlike BV, clinically invisible LV do not reduce corneal transparency per se, but contribute to various diseases of the ocular surface including corneal transplant rejection, dry eye disease and ocular allergy. Here, corneal LV facilitate the migration of antigen presenting cells from the ocular surface to the regional lymph nodes, inducing undesired immune responses (2). 

Preclinical evidence indicates that myeloid cells are important mediators of inflammatory corneal neovascularization (3). Yet, the exact mechanisms of blood monocyte recruitment, corneal macrophage activation and the functional consequences of this process for corneal hemangiogenesis (HA) and lymphangiogenesis (LA) are not fully understood. Therefore, the aim of this project is to investigate the specific role of myeloid cells in inflammatory corneal neovascularization in several clinically relevant corneal neovascular disease models.  

Distinct functions of early and late stage corneal wound macrophages in corneal hem- and lymphangiogenesis

The role of macrophages in various stages of the vascular response after corneal damage had not been investigated so far. Using two different mouse models of acute and chronic corneal injury, our group has recently identified distinct functions of early- vs. late-phase macrophages in corneal HA and LA (4). Whereas early-phase macrophages are essential for the initiation and progression of corneal HA and LA, late-phase macrophages control the maintenance of established corneal LV, but not BV (4). Furthermore, we showed that the hem- and lymphangiogenic potential of macrophages is controlled by the type of corneal damage: whereas a perforating corneal incision injury induced primarily macrophages with lymphangiogenic potential, corneal suture placement provoked macrophages with both hem- and lymphangiogenic potential (4). Taken together, our work highlights a previously unknown and injury-context dependent role of early- versus late-phase corneal wound macrophages with potential clinical impact on therapy development for sight-threatening corneal neovascular diseases. 

Corneal lymphangiogenesis driven by macrophages is involved in the resolution of pathological corneal inflammation and edema

We could recently demonstrate an unexpectedly beneficial role for macrophage-driven corneal LA in the resolution of corneal inflammation and edema (5, 6). We showed that IL-10, a multifunctional cytokine that is known for its anti-inflammatory and immune-regulatory effects, induces the expression of the major lymphangiogenic growth factor VEGF-C in corneal macrophages (5). Consistently, corneal injury in IL-10 deficient mice resulted in reduced expression of VEGF-C and decreased corneal LA (5). In addition, IL-10 deficiency resulted in a more severe inflammatory response with increased expression of pro-inflammatory cytokines and increased inflammatory cell numbers in injured corneas when compared to wildtype littermates, which even persisted after the inflammatory stimulus has been removed (5). To further define the role of myeloid-cell derived IL-10 signaling in corneal LA, we generated and analyzed mice with myeloid-specific deletion of Stat3 (Stat3^MKO), which is a central mediator of IL-10 signaling. Corneal injury in Stat3^MKOmice resulted in reduced corneal LA and persistent inflammation, corroborating the critical role of IL-10 positive macrophages in the regulation of corneal LA and inflammation (5).  This indicates that during the corneal inflammatory response, IL-10 leads to an anti-inflammatory but pro-lymphangiogenic VEGF-C secreting macrophage phenotype that induces the growth of LV, which in turn support the egress of inflammatory cells and the termination of the local inflammatory response. To our knowledge, this is the first report demonstrating “beneficial” functions for corneal LV in the promotion of a physiological healing response, as corneal LA had so far been mostly considered as pathological and generally undesirable.

In addition to these findings, we have recently also demonstrated that corneal LA is critically involved in corneal fluid homeostasis (6). For this purpose, we used the mouse model of perforating corneal incision injury, which induces transient corneal edema, opacification, and corneal LA but not HA (4). 

We hypothesized that LA might be involved in the regulation of corneal edema and therefore determined whether inhibition of LA after injury has an impact on corneal edema and transparency. Importantly, inhibition of corneal LA by VEGFR3-blockade resulted in increased central corneal thickness due to delayed drainage of corneal edema and prolonged corneal opacification (6). Corneal LA after this type of injury seems to depend on the presence of macrophages, as early macrophage depletion by local application of clodronate liposomes significantly reduced corneal LA (4; Figure). Together, these findings provide evidence that VEGF-C/VEGFR-3 driven corneal LA plays an unexpectedly beneficial role in the regulation of corneal edema and transparency, which might open new treatment options in blinding diseases associated with corneal edema and transparency loss. Currently, one of our aims is to better define the molecular mechanisms that control myeloid cell-mediated regulation of corneal fluid homeostasis.

Perspectives

Translationally, the findings of this project may provide the rationale and novel therapeutic tools to ameliorate corneal neovascular diseases and promote corneal repair by modulating local immune responses.