Lipid mediators are fatty acid-derived small signaling molecules that are critical in vascular responses and leukocyte trafficking during inflammation. Eicosanoids prostaglandin E2 (PGE2) induces pain and fever responses while leukotriene B4 (LTB4) recruits neutrophils to initiate inflammation, the recruited neutrophils subsequently produce lipoxin A4 (LXA4) to limit the magnitude and duration of acute inflammation. Eicosanoid regulation of innate immune cells is well established, but eicosanoid regulation of adaptive immunity is less understood. Moreover, the bioactions of eicosanoids are often context-dependent. Therefore to fully understand how lipid mediators modulate immune responses and homeostasis, it is vital to assess their immunological mechanisms in cell type- and disease-specific settings.

My dissertation aims to address whether and how LXA4 mediates adaptive immune responses in posterior autoimmune uveitis. In the first Chapter I will introduce arachidonic acid-derived eicosanoids PGE2, LTB4, LXA4 and lipoxin B4 (LXB4) and discuss their multifaceted bioactions in various inflammatory settings, with the focus of their regulation of lymphocytes. In Chapter Two I will provide an overview on the therapeutic effects of special proresolving mediators (SPMs) and LXA4 in ocular diseases and briefly highlight two collaborations that revealed the roles of PGE2 and LTB4 in enhancing inflammatory neutrophil function in peritonitis, and the mechanism in which PGE2 contributes to pruritic responses in atopic dermatitis.

Chapter Three will present my thesis aims in identifying whether LXA4 plays a role during autoimmune uveitis pathogenesis and dissecting the mechanism in which LXA4 modulates disease. Data from my thesis project showed in vivo generation of LXA4 was significantly downregulated in the draining lymph nodes at peak uveitis. Mice lacking 5-lipoxygenase (5-LOX), the rate-limiting enzyme for LXA4 production, exhibited aggravated disease. Ccr7 expression was downregulated in CD4+ T cells of LXA4 deficient mice while glycolytic responses of these T cells were enhanced, resulting in elevated IFN- production and migration of effector CD4+ T cells to the eye. Conversely, LXA4 treatment of immunized mice ameliorated disease. The findings underscore the importance of the LXA4 circuit in guiding adaptive T cell function in autoimmunity, and uncover a novel mechanism of LXA4 regulation in T cell effector function and trafficking from peripheral lymph nodes to the site of inflammation.

Chapter Four demonstrates a novel neuroprotective role of astrocyte-secreted lipoxins A4 and B4 in retinal neurodegeneration. Lipoxins’ anti-inflammatory functions are well established, but they have not been shown to impact neuronal survival. In this project, we determined that LXA4 and LXB4 are synthesized in the inner retina in health, but their endogenous formation becomes reduced following injury. Lipoxin treatment provided neuroprotection following acute injury, while inhibition of the LXA4 pathway exacerbated injury-induced neuronal death. These results identified lipoxins as important neuroprotective factors that maintain neuronal homeostasis and inflammation in the central nervous system (CNS).

In the last chapter, I will conclude with the main findings from my projects and suggestions for future studies on lipid mediators. In this dissertation, we demonstrate the indispensable roles of lipoxins in maintaining homeostasis and mediating inflammatory responses, and their abilities in fine-tuning cellular responses to control the duration and amplitude of inflammation.

The cornea is a transparent, avascular tissue at the ocular surface that refracts light and protects the eye from microbial infection. Inflammation, while a necessary and beneficial part of wound repair, can be detrimental if left unchecked especially in the cornea where transparency is necessary for vision. The cornea has thus evolved tightly regulated circuits to promote wound healing, maintain host defense, and control inflammation. One such pathway that plays a key role in the cornea is the 15-lipoxygenase(LOX)/lipoxin(LX) A₄ lipid circuit that is present endogenously in the uninjured cornea. LXA₄ is a lipid mediator derived from dietary fatty acids that displays potent anti-inflammatory and pro-resolving bioactions. Previous results have demonstrated a key role for LXA₄ in a murine model of corneal epithelial abrasion where 15-LOX (and subsequent LXA₄) deficiency impairs rate of wound repair while topical administration of LXA₄ restores corneal wound healing.

Sex-specific differences have long been observed in an inflammatory and/or autoimmune setting. Females have a higher incidence of many inflammatory diseases including rheumatoid arthritis, systemic lupus erythematosus, and Sjogren's syndrome. Furthermore, there is a sex-specific difference in rate of dermal wound repair that changes post-menopause, showing that inflammation and wound healing are intimately linked. In the eye, Dry Eye Syndrome is a highly widespread inflammatory-based disease that affects predominantly post-menopausal women. Androgen deficiency has been implicated in Dry Eye disease progression. On the other hand, estrogen's role in the eye has been largely ignored despite the presence of estrogen and its two traditional receptors in leukocytes and most major ocular tissues. An epidemiological study of forty thousand female health professionals revealed a correlation between estrogen replacement therapy and an increase in reported dry eye symptoms, implicating estrogen in inflammatory regulation.

The overarching aim of this dissertation was to determine if sex-specific differences are driven by estrogen regulation of the corneal inflammatory reparative response; more specifically, to establish a link, if any, between estrogen signaling and the 15-LOX/LXA₄ lipid circuit. Given the potent protective bioactions of LXA₄ in corneal wound healing, an understanding of sex-steroid regulation of the 15-LOX/LXA₄ circuit may have important implications in ocular therapy and inflammatory disease progression. The following hypothesis was tested: is there a sex-specific difference in the corneal inflammatory reparative response and, if so, what role does estrogen play in the establishment of such differences?

An estrogen-driven sex-specific difference in corneal wound healing was demonstrated for the first time. Estrogen receptor (ER) β signaling downregulated corneal epithelial 15-LOX expression and subsequent LXA₄ formation, the first study to link sex-specific differences and the action of estradiol to the protective anti-inflammatory 15-LOX/LXA₄ circuit. Furthermore, a sex-specific difference in corneal inflammatory tone and leukocyte population upon re-injury was established. In vitro assays of phagocytosis demonstrated for the first time that phagocytosis is a potent stimulus for LXA₄ formation, establishing a positive feedforward loop for LXA₄ action in leukocytes whereby LXA₄ stimulates phagocytosis and subsequent phagocytosis stimulates LXA₄ production. Estrogen signaling via ERβ was also shown to upregulate neutrophil retention and delay clearance by inhibiting the LXA₄-induced stimulation of phagocytosis. Taken together, these results provide evidence for an ERβ-driven upregulation of the inflammatory response and downregulation of the protective 15-LOX/LXA₄ lipid pathway in the corneal wound healing response. Elucidation of these sex-specific and sex steroid-driven differences provides insights into the high female-dominated incidence of inflammatory diseases. Estrogen primes females for an amplified inflammatory response upon a disruption to the ocular surface. This has important ramifications in the cornea where chronic inflammation can lead to blindness.