Access Type

Dissertation/Thesis

Date of Award

January 2019

Degree Type

Dissertation

Degree Name

Ph.D.

Department

Anatomy and Cell Biology

First Advisor

Susmit Suvas

Abstract

Herpes stromal keratitis (HSK) is a chronic immuno-inflammatory ocular disease caused by Herpes simplex virus-1 (HSV-1) infection in the cornea. HSK is characterized by the development of corneal opacity and angiogenesis accompanied by the loss of corneal transparency. Despite extensive studies on inhibiting corneal angiogenesis and strategies to reduce HSK disease development, a key underlying mechanism to maintain an intact and a transparent corneal surface during HSK disease progression, remain obscure. Our study has addressed possible causes for the HSK disease severity by identifying the key factors such as the development of inflammatory hypoxia, reduced tear volume, Inflammation of extra-orbital lacrimal gland and the conjunctiva and the transdifferentiation of the corneal epithelium in the HSK developing eyes. Intriguingly, our study also emphasizes the protective role of IGFBP-3 in inhibiting the severe disease progression of HSK.

During the pre-clinical and the clinical disease phase of HSK, the influx of neutrophils have been studied as the most prominent innate immune cell type. Development of corneal hem- and lymph-angiogenesis, opacity and epithelial defects hinders the clear vision and is known to be the cause of the increased neutrophil influx in HSV-1 infected cornea. Previous studies have indicated that the influx of neutrophils into mucosal tissue can re-model the development of inflammatory hypoxia. Therefore, the aim of this study was to investigate the development of hypoxia and hypoxia-associated gene expression during the progression of HSK disease. Our results showed an increased influx of neutrophils during the clinical disease of HSK that leads to the development of hypoxia, and the upregulation of hypoxia-associated genes. This study provided us novel insights on the prevalence of the glycolytic metabolism in the HSK developing eyes. Additionally, our data demonstrated that the blocking of Hypoxia-Inducible Factor (HIF) with Acriflavine alone might not be an efficacious drug in reducing HSK disease severity. Overall, these results pave way for using novel therapeutic targets that play a dual role to block HIF and control corneal inflammation during HSK.

Maintaining a healthy corneal epithelium, retaining a steady tear volume and its secretion could be the other factors that may aid in alleviating HSK disease severity. Mounting evidence corroborates corneal nerves as a critical factor in the maintenance of corneal integrity and homeostasis of the ocular surface by mediating the reflex actions of blinking and tearing. In support, in a mouse model during HSV-1 ocular infection, corneal nerve damage has been demonstrated. Hence, corneal transparency and clear vision are critically dependent upon the integrity of corneal tissue maintained by the intricate network of corneal nerves, the presence of intact corneal epithelium and tear film that is controlled by a cross-talk between the components of the lacrimal functional unit. Our study showed a reduced measurement of tear volume in the HSK developing eyes correlating to the inflammation in the extra-orbital lacrimal gland (EoLG) and the conjunctiva. Atrophy of tear secreting acinar cells in the EoLG, the significant loss of goblet cells in the conjunctiva and the influx of leukocytes and CD4 T cells were the major events occurring during the HSV-1 infection. These events caused dysfunction to the lacrimal functional unit, thereby inducing severe HSK and ‘dry eye’ conditions. Nevertheless, topical treatment of lacritin (tear glycoprotein) delayed but did not prevent the reduction in tear volume nor reduced the severity of HSK.

Existence of a healthy, smooth and lubricated corneal surface is critical for the maintenance of transparent cornea to aid clear vision. Corneal epithelium comprises of non-keratinized stratified squamous epithelium. Nevertheless, under pathological conditions non-keratinized epithelium transforms into a keratinized epithelium that closely matches the features of skin epithelium (epidermis). The keratin expression profile determines this switch in keratinization in the cornea. Cytokeratin-12 (K12) is one of the keratins specific to the corneal epithelium, which is replaced by epidermis-specific cytokeratin-10 (K10) expression under pathological conditions. Intriguingly, our data showed the loss of K12 expression with the emergence of K10 expression during the clinical stage of HSK disease. Our data suggested the occurrence of corneal epithelial transformation. Another prominent marker, PAX-6 (paired box -6 transcription factor) has a crucial role in the maintenance of corneal epithelial identity by regulation of its differentiation. Our data showed a dramatic loss of PAX-6 expression in the HSK developing eyes. ‘Squamous metaplasia’ is the pathological phenomenon characterized by abnormal differentiation of corneal epithelium into the skin like epithelium. Overall, our results signified the ‘trans-differentiation of corneal epithelial cells to the skin like epithelium, in HSV-1 infected mouse cornea.

Corneal hem-angiogenesis is one of the hallmarks of HSK. Hence, it is essential to understand better for the molecules involved in regulating hem-angiogenesis, immune cell survival, and its effector function. Intriguingly, angiogenesis array data showed an increased expression of IGFBP-3 in the HSV-1 infected corneas of C57BL/6 (B6) mice. Therefore, we were inclined to study the role of IGFBP-3 molecule in HSV-1 infected B6 corneas. IGFBP-3 is one of the six highly conserved IGF-binding proteins. IGFBP-3 binds to 75% of the serum IGF-I and IGF-II in complexes and regulates IGF-1R signaling by sequestering IGF molecule. IGF binding to IGF-1R results in phosphorylation of tyrosine residues in IGF-1Rβ chain. IGF-1R signaling that has been reported to promote angiogenesis (hem- and lymph-angiogenesis). Our data indicated severe HSK disease with robust angiogenesis in IGFBP-3-/- mice associated with a significant increase in IGF-1R phosphorylation in the infiltrated leukocytes. These results suggest the protective role of IGFBP-3 in inhibiting IGF-1R phosphorylation that leads to the development of severe HSK disease progression.

Altogether, this study broadens our knowledge on understanding several factors involved in the alterations of the ocular surface system during the pathogenesis of HSK. In summary, this study highlights on five crucial factors that may play an essential role in reducing HSK disease pathogenesis in the cornea; 1) regulating the development of inflammatory hypoxia 2) maintaining of the stable tear volume 3) reducing the lacrimal and conjunctival inflammation 4) maintaining the corneal epithelial integrity 3) alleviating corneal hem-angiogenesis (Figure 42).

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