The Effect of 0.1% Topical Ciclosporin A for 12-weeks on the Eye Surface Immune Cells in Dry Eyes

The immune system plays a pivotal role in dry eye disease (DED) pathogenesis. An increased level of several tear cytokines, chemokines, expression of HLA-DR molecules and activation of antigen presenting cells - dendritic cells (DCs), and lymphocytes all plays a central role in DED pathogenesis . The increased expressions of CD4+ and CD8+ T cells in the ocular surface of DED patients suggest a potential role of adaptive immunity in DED chronicity. The presence of antigen-presenting cells in the ocular surface and the upregulation of MHC II, IL-2, IL-17, IL-6 and IFN- γ and other co-stimulatory molecules in the tears of DED patients indicates a possible activation of the local adaptive immune response.

The conjunctiva serves as a primary site for immunological and histochemical analysis in ocular surface diseases . Researchers have employed various techniques to study ocular surface immunology such as impression cytology, conjunctival biopsy and through real-time polymerase chain reaction, immunocytochemistry, and light microscopy. Flow cytometry is a powerful technique that allows for the phenotypic analysis of cells through immunolabeling of extracellular expressions of various immune cell markers. An increased expressions of activation markers like HLA-DR and various immune cell populations including T cells (CD3+, CD4+, and CD8+), B cells, natural killer cells, and monocytes (CD14+ cells) are observed in DED using flow cytometry on conjunctival impression cytology samples .

Historically, studying human ocular surface immune cells required invasive techniques. With the advent of in vivo confocal microscopy (IVCM), has enabled real-time, non-invasive visualization of ocular surface epithelial immune cells (EICs) and corneal nerves. Over the past 20 years, researchers have used IVCM to examine pathologic changes in various ocular surface disorders. It is evident from several IVCM studies that corneal EICs undergoes changes such as increase in density and changes in its morphology in all forms of DED. Conventional static IVCM captures section images at a single time point of the corneal EIC's, and their analysis is limited to its density and morphology. While the in vivo nature of IVCM prevents direct immune cell phenotyping, the observed cells are predominantly believed to be DCs based on their morphological characteristics. Therefore, the authors have used the term EICs to define immune cells density (EIC density) quantified using static-IVCM. Unlike this method, which limits identification of the immune cell type, a recently pioneered, novel technique called functional IVCM (Fun-IVCM) enables identification of the immune cell types in the cornea by observing their morphodynamics. This method involves capturing consecutive sequence and volume scan images over a 30-40-minute period, with intervals of 4-5 minutes. The images are then time-lapsed to identify dynamic behaviour of each immune cell. Based on their dynamic behaviour and morphology, cells can be differentiated into either epithelial T cells (highly motile, smaller body size) or dendritic cells (DC's, less motile, larger body size with dendrites). Anterior corneal stromal macrophages can also be identified. Time-lapsed images allow for the examination of additional cell motility parameters, such as instantaneous speed and displacement speed.

The first-line treatment for DED is artificial tears. But, due to the chronic nature of the disease and recurrent acute exacerbations anti-inflammatory therapies has been largely implemented. Consequently, many of the current therapeutic approaches target various inflammatory pathways, aiming to break the vicious cycle of DED. Both topical corticosteroids and immunosuppressants effectively reduce inflammation in DED. Topical corticosteroids are used in DED to control acute exacerbations of inflammation of the ocular surface, but their longer-term use is limited by their iatrogenic ocular side-effects. To contrast this, immunosuppressive agents such as ciclosporin A (CsA) have potential anti-inflammatory effect, with minimal local side-effects. The use of CsA in chronic DED can significantly reduce ocular surface staining, redness, inflammatory markers and improves dry eye symptoms. In brief, CsA dampens T cell proliferation by inhibiting the phosphatase calcineurin. CsA functions at different steps of the immune response, during antigen presentation and DC-T cell interaction. Innate immune cells are now seen as potentially important targets of CsA and may reflect simultaneous targeting of dendritic cells, macrophages, and neutrophils. In the ocular surface preliminary evidence suggest that CsA can significantly reduce activation markers such as HLA-DR, CD11c, certain apoptosis markers, and conjunctival T lymphocytes accompanied the clinical improvement in CsA-treated DED patients.

While a handful of clinical trials have evidenced the efficacy of CsA in reducing inflammation and improving symptoms of DED, the direct effect of CsA on ocular surface immune cells has not been studied in detail using static and using Fun-IVCM. Given the effect of CsA on both innate and adaptive immune cells, the authors hypothesise that the use of 0.1% CsA will have an inhibitory effect on the ocular surface immune response along with improvement of signs and symptoms of DED compared with its vehicle. This paper highlights the randomized clinical trial protocol to comprehensively study the effect of CsA on the ocular surface innate and adaptive immune system in DED. The study will investigate CsA's impact on DCs (CD11c+ DCs, HLA-DR), T helper cell populations (CD4+ T cells) through analysis of lymphocyte markers (CD45, CD3, CD4) and their associated cytokines (Th1 pathway cytokines), particularly IL-2, IFN- γ and IL-17 which is a primary target of CsA's immunomodulatory action.

1.2. Study aims and hypothesis Aim 1: To investigate and compare the effect of 12-weeks topical 0.1% CsA therapy on ocular surface innate and adaptive immune cells using imaging - Static-IVCM and Fun-IVCM (immune cell density, morphology and morphodynamic behaviours), immune cell phenotyping using conjunctival impression cytology and flow cytometry techniques and tear cytokine analysis (Multiplex bead assay assessing tear concentrations of IL-2, IL-4, IL-6, IL-12, IL-17, IL-23, IFN- γ) Hypothesis: CsA will have an inhibitory effect on the ocular surface innate and adaptive immune cell density, morphology and tear cytokines in DED.

Aim 2: To assess the association between DED signs, symptoms and immune cell characteristics and tear cytokines in DED Hypothesis: A direct relationship between immune cell characteristics, teat cytokines and DED signs, symptoms exist.

Aim 3: To investigate the temporal relationship between immune cell changes and clinical improvement Hypothesis: Changes in immune cell characteristics (density and dynamics) and inflammatory markers precede clinical improvement in signs and symptoms of DED following CsA treatment.