Date of Award

5-2005

Degree Type

Dissertation

Degree Name

Doctor of Philosophy

Major

Comparative and Experimental Medicine

Major Professor

Barry T. Rouse

Committee Members

Robert N. Moore, Albert T. Ichiki, Mark Y. Sangster

Abstract

Herpes simplex viruses (HSV) infection is a common cause of ocular disease and can result in a chronic inflammatory reaction that impairs vision. This latter manifestation is called herpetic stromal keratitis (HSK) and usually occurs as a consequence of virus reactivation from latency in the trigeminal ganglion. The pathogenesis process involves complex interactions of cellular and molecular events. HSK in humans, and certainly its murine experimental model, appears to be an immunopathologic disease process. The crucial cell type orchestrating the inflammation is a CD4+ Tcell that has a Thl cytokineproducing profile. However, prior to this immunoinflammatory phase, multiple events occur that set the stage for subsequent pathology. These include production of cytokines and chemokines, infiltration of innate immune cells and neovascularization of the avascular cornea.

Current understanding about human and murine HSK pathogenesis is reviewed in Part I. Part II progresses this knowledge using knockout and transgenic mice. Results in Part II clarify the essential role of IL-l, a proinflammtory cytokine, produced as a consequence of virus replication, in HSK pathogenesis. Part III, in addition to Part II, elucidate the mechanisms involved in IL-l and IL-6 mediated polymorphonuclear leukocyte (PMN) influx and neovascularization of cornea following virus infection. These cytokines were shown to be produced quite early after infection and capable of inducing angiogenic factors, resulting in angiogenesis of the cornea. In addition, the role of IL-l in inducing proinflammatory arachidonic acid metabolites has been discussed in PART IV. Cycloxygenase 2 enzyme produced from resident corneal cells in presence of IL-l was shown to be crucial for creating an inflammatory milieu and facilitate early PMN influx in murine cornea. These results provide novel insights into the link between viral infections, pro-inflammatory mediators, PMN migration, angiogenesis and HSK development. Our understanding about the role of IL-l in HSK pathogenesis was evaluated clinically in Part V using an IL-l receptor antagonist (IL-l ra) protein. Mice receiving IL-l ra h~d diminished disease severity. The administration of IL-l ra was shown to reduce the influx into the cornea of cells of both the innate and adaptive immune response. In addition, the treatment also diminished corneal VEGF levels resulting reduced angiogenic response. Our results show the iluportance of targeting early proinflammatory molecules such as IL-l to counteract HSK and advocate IL-l ra as an effective agent to achieve this. In Part VI, a novel flow cytometry based assay has been used to quantify corneal angiogenesis following ocular HSV infection. Our results indicate that, this assay was sensitive enough to able to pick up the difference in angiogenic response between mice. Thus, estimation of angiogenesis on the basis of number of endothelial cells proved to be a useful approach to quantify corneal neovascularization.

This dissertation presents research aimed at elucidating both molecular and cellular events in HSK pathogenesis. These results will serve as guidelines for future development of more efficient prophylactic and therapeutic strategies.

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