Efficacy of Topical Blockade of Interleukin-1 in Experimental Dry Eye Disease

PURPOSE— To evaluate the therapeutic efficacy of topical IL-1Ra in the treatment of Dry Eye Disease (DED). METHODS— DED was induced in C57BL/6 female mice through exposure to a desiccating environment within a controlled environment chamber. Topical formulations containing 5% IL-1Ra, 1% methylprednisolone, 0.05% cyclosporin A (CsA), and a vehicle control containing carboxymethylcellulose sodium (CMC) were applied following the induction of dry eye. Corneal fluorescein staining (CFS) was performed by a masked observer in the different treatment groups. Immunohistochemical studies were undertaken to enumerate corneal CD11b+ cells, as well as to evaluate corneal lymphangiogenesis. Real-time polymerase reaction was used to quantify the expression of IL-1 β in the cornea. RESULTS— A significant decrease in CFS was observed following topical treatment with 5% IL-1Ra (P<0.01), 1% methylprednisolone (P<0.01), and 0.05% CsA (P<0.03). Additionally, a significant decrease in the numbers of central corneal CD11b+ cells (P<0.05), corneal lymphatic growth (P<0.05), and corneal IL-1 β expression (P<0.003), compared to vehicle treated, were only demonstrated following treatment with 5% IL-1Ra and 1% methylprednisolone, and were absent following CsA treatment. CONCLUSIONS— Topical treatment with IL-1Ra is efficacious in ameliorating the clinical signs of the DED, as well as in reducing underlying inflammation.These effects comparab le to treatment with topical methylprednisolone. Topical IL-1Ra may hold promise as a novel therapeutic strategy in the treatment of dry eye.


INTRODUCTION
Dry eye disease (DED) is an extremely common ocular disorder, and large epidemiological studies, using a variety of definitions, have estimated its prevalence to approximate 10-20% of the adult population. 1 DED is defined as a multifactorial condition of the tears and ocular surface, characterized by symptoms of discomfort, visual disturbance and tear film instability with potential damage to the ocular surface. Moreover, it is accompanied by increased osmolarity of the tear film and inflammation of the ocular surface. 1 DED can affect visual function, and thus common tasks of daily living such as reading, speed and driving are adversely affected by this condition. 2 Inflammation has been recognized as an important component of DED and antiinflammatory therapies have therefore proven useful in decreasing ocular surface inflammation, stimulating tear production, and improving the overall signs and symptoms of dry eye. 3 Moreover, consistent with the concept that inflammation is a key feature in the pathophysiology of DED is the finding that the clinical signs and symptoms of dry eye improve with methylprednisolone and cyclosporin (CsA). 4,5 Unfortunately the long-term use of topical corticosteroids is limited by potential sight-threatening side effects, such as glaucoma, cataract and infection. 6 Furthermore, CsA, the only drug approved by the FDA in the United States for treatment of DED, is associated in some patients with burning and stinging sensation, and requires several months to produce a clinical therapeutic effect. 7 Interleukin-1 receptor antagonist (IL-1Ra) is an endogenous IL-1 receptor antagonist, which is primarily produced by activated monocytes and tissue macrophages, and inhibits the activities of the proinflammatory forms of IL-1 (IL-1α and IL-1β) by competitively binding to the IL-1 receptor-I (IL-1RI). 8 IL-1Ra is typically upregulated in inflammatory conditions, such as in rheumatoid arthritis to down-modulate the inflammatory functions of the IL-1RI ligation by the agonistic ligands, IL-1α and IL-1β. 9 Although, the binding affinity of natural IL-1Ra to its receptor is similar to that of IL-1α/IL-1β, higher concentrations of IL-1Ra relative to agonistic IL-1 ligands have been shown to be required to completely inhibit the cellular effects of IL-1. 10,11 This is probably due to the fact that only 5% receptor availability for IL-1 can initiate inflammatory responses and thus virtually all cellular IL-1RI receptors must be blocked for sufficient suppression of the IL-1 system. 11,12 Normal tear fluid has been found to contain high concentrations of IL-1Ra in concentrations 25,000 and 40,000 times greater than both proinflammatory forms of IL-1, 13 and it is thought that concentration of IL-1Ra in the tear fluid may therefore be a natural homeostatic mechanism for regulation of IL-1-mediated functions on the ocular surface. 14 Given the critical role of inflammation in the pathogenesis of DED 15 , we examined the therapeutic efficacy of topical IL-1Ra in DED using a validated murine model of dry eye 16 . Our data shows a significant amelioration of clinical signs of DED in IL-1Ra treated mice. In addition, we investigated the effect of topical IL-1Ra in modulating infiltration of inflammatory cells, expression of inflammatory cytokine and lymphangiogenesis in dry eye cornea -mechanisms that are of pathophysiologic relevance in DED induction and progression 17, 18 -and demonstrate significant suppression of DED-associated inflammation with topical use of IL-1Ra.

Animals
Eight to ten week old female C57BL/6 mice (Charles River Laboratory, Wilmington, MA) were used in this study.

Induction of Dry Eye
Dry eye was induced in the mice by placing them in a Controlled Environment Chamber (CEC), which allows continuous regulation of relative humidity below 30%, a constant temperature of 21-23ºC and airflow of 15 L/minute, 24 hours a day. 16 To achieve maximum ocular surface dryness, the conditions in CEC were supplemented with topical application of 1% atropine sulfate (Falcon Pharma, Fort Worth, Texas) twice daily for the first 48 hours. Additionally, the mice also received on their dorsal surface subcutaneous injections 0.1ml (5mg/ml) of scopolamine hydrobromide (Sigma-Aldrich, St. Louis, MO) to exacerbate dry eye three times a day (9 AM, 1 PM, 5 PM) for the entire duration of the experiment.

Topical Formulations and Treatment Regimen
The formulations tested included 5% IL-1Ra, 1% methylprednisolone, 0.05% Cyclosporin A (Restasis, Allergan, Irvine, CA). The topical IL-1Ra and methylprednisolone were formulated aseptically from the commercially available intravenous Anakinra (Kineret, Biovitrum, Stockholm, Sweden) and methylprednisolone sodium succinate respectively, and transferred into a sterile, light-protected dropper container. Anakinra (50 mg/ml) was diluted in carboxymethylcellulose sodium (Refresh Liquigel, Allergan, Irvine, CA) and methylprednisolone (10 mg/ml) was diluted in nonpreserved sterile normal saline solution. Forty-eight hours after the induction of dry eye, the mice in the CEC were randomly divided into five different groups (n=8-10 in each group): (i) an untreated group, (ii) a group receiving topical vehicle control (carboxymethylcellulose sodium), (iii) a group receiving 5% IL-1Ra, (iv) a group receiving 0.05% CsA and (v) a group receiving 1% methylprednisolone. Three microliters of the eye drop were applied topically to both eyes of unanesthetized mice three times a day from 48 hours to day 9 (total 21 doses). The untreated group received no eye drops. Ocular signs of dry eye were evaluated using fluorescein staining at day 2, day 6 and day 9. Mice were then euthanized on day 10 for cellular and molecular studies.

Corneal Fluorescein Staining
Fluorescein staining of the corneal epithelium was used as a diagnostic tool to study the effect of desiccating stress on the ocular surface of the mice. Corneal fluorescein staining was performed at baseline (day 0, before placing the mice in the CEC), day 2 (before instillation of topical therapy), day 6 and finally at day 9. 0.7 μL of 2.5% fluorescein (Sigma-Aldrich, St. Louis, MO) was applied to the eye using a micropipette into the inferior conjunctival sac (n= 8 eyes per group) as previously described. 16 After three minutes, punctate staining on the corneal surface was evaluated in a maskedfashion using a slit lamp biomicroscope under cobalt blue light. The National Eye Institute grading system was used to assess the corneal surface staining. 19,20

RNA Isolation and Molecular analysis using Real Time Polymerase Chain Reaction
Total RNA was isolated from the cornea tissues using the RNeasy microkit (Qiagen, Valencia, CA; catalog No. 74004). Two corneas per group were isolated, pooled together and stored at −80°C in Trizol (Invitrogen, Carlsbad, California; catalog No. 15596026) until future use. The first strand of cDNA was synthesized with random hexamers using SuperScriptTM III Reverse Trancriptase (Invitrogen, Carlsbad, CA; catalog No.18080) according to the manufacturer's recommendations, and utilizing 500ng of total RNA. Real-Time PCR was performed using FAM-MGB dye labeled predesigned primers (Applied Biosystem, Foster City, CA) for GAPDH (assay ID.Mm99999915_g1), IL-1β (assay ID.Mm00434228_m1). Two and half μL of cDNA was loaded in each well and assays were performed in duplicates. A non-template control was included in all the experiments to evaluate DNA contamination of the reagent used. The GAPDH gene was used as the endogenous reference for each reaction. The results were normalized by the cycle threshold (CT) of GAPDH and the relative mRNA level in the untreated group was used as the normalized control for the others groups.

Immunohistochemical Staining
The following primary antibodies were used for immunohistochemical staining: rat antimouse CD11b-FITC for monocytes/macrophages (BD Pharmingen, San Diego, California, 1:100), goat anti-mouse CD31 FITC as pan-endothelial marker (Santa Cruz Biotechnology, Santa Cruz, California, 1:100) and purified rabbit anti-mouse LYVE-1 as lymphatic endothelial marker (Abcam, MA, USA, 1:400). Respective isotypes were used as negative controls. The secondary antibody used was Rhodamine conjugated goat anti-rabbit (BD Pharmingen, San Diego, California, 1:100). For whole mount immunofluorescence corneal staining, freshly excised corneas were washed in PBS and acetone fixed for 15 minutes and then stained with CD11b or double stained with CD31 and LYVE-1 as described previously. 21 To analyze infiltration of CD11b + cells, corneas from three mice from each group were taken and cells were counted in 6 areas in the periphery (0.5 μm area from the limbus) and the center (central 2 μm area) of the cornea in a masked fashion, using confocal microscope (Leica TCS 4D; Lasertechnik, Heidelberg, Germany) at 40X magnification ( Figure 2A). The mean number of cells was obtained by averaging the total number of cells in all the areas studied and the result was expressed as the number of positive cells per mm 2 .

Quantification of Corneal Lymphangiogenesis
The lymphatics were analyzed by an automated image analysis program using Matlab (The Mathworks, Inc., Natick, MA). 22 Lymphatics were isolated from 12 digitized immunofluorescent micrographs with this program, and subsequently analyzed the Lymphatic Area (LA). LA represents the total surface area of the lymphatic vessels when projected into the plane of the image.

Statistical Analysis
A two-tailed Student's t-test was performed and P-values less than 0.05 were deemed statistically significant. Results are presented as the mean ± standard error of the mean (SEM) of at least three experiments.

Amelioration of clinical signs of dry eye
The clinical signs of dry eye were measured by corneal fluorescein staining using the National Eye Institute grading scheme. On day 0, mice were placed in the CEC and after 48 hours all mice showed an increase in corneal staining corresponding to dry eye induction (Fig 1A). Topical treatment with the 5% IL-1Ra, 1% methylprednisolone, 0.05% CsA and the vehicle control (CMC) were started on day 2 and corneal fluorescein staining scores were measured at days 6 and 9. Treatment with topical 5% IL1-Ra, 1% methylprednisolone and 0.05% CsA led to a significant decrease in corneal fluorescein staining compared to the untreated and vehicle group at day 6 (P < 0.01) and day 9 (P < 0.003) (Fig 1A). Moreover, 5% IL-1Ra and 1% methylprednisolone showed a statistically significant greater reduction in the CFS score compared to 0.05% CsA at day 9 (55% and 60% vs. 34%; respectively; P < 0.01) (Fig 1B).

Reduction of CD11b + cells infiltration in dry eye cornea
The normal cornea has a resident population of bone marrow-derived immature CD11b + antigen-presenting cells 23 , and the induction of dry eye is associated with a significant increase in the number of corneal CD11b + cells 24 . Using confocal microscopy of corneal whole mounts, we investigated CD11b + cells infiltrating the cornea of different regimen treated groups (Fig 2A). A significant decrease was seen in the number of CD11b+ cells, both in the periphery and the center, of dry eye corneas which were treated with 5% IL-1Ra (176.2 ± 6.5 and 125.0 ± 3.4) and 1% methylprednisolone (176.1 ± 5.4 and 132.1 ± 3.5) compared to vehicle treated dry eye corneas (260.7 ± 11.0 and 164 ± 7.1) (Fig 2B). However, 0.05% CsA treated group only showed a significant decrease in the number of CD11b + cells in the peripheral corneas compared to vehicle treated corneas (209.5 ± 6.8 vs. 260.7 ± 11.0; P = 0.004) (Fig 2B). No difference in the CD11b+ cell numbers was observed between untreated and vehicle treated dry eye corneas.

Reduction in the expression of IL-1β in dry eye cornea
DED is accompanied by an increase in the proinflammatory forms of IL-1. 13 Therefore, to determine the relative efficacy of the different treatment regimens on the gene expression of IL-1β, real-time PCR was performed to quantify the transcript expression of IL-1β in the cornea of different groups (n = 4 to 6 per group). Analysis of relative expression of IL-1β among different groups showed that treatment with 5% IL-Ra and 1% methylprednisolone significantly decreased the levels of IL-1β in the cornea compared to CsA (P = 0.02) and vehicle control (P = 0.003) treated groups (Fig 3).

Inhibition of lymphatics ingrowth in dry eye cornea
The normal human cornea is avascular and alymphatic, thus suppressing both the afferent (lymphatic) and efferent (vascular) arms of the immune cycle. 25,26 The lymphatic system plays an essential role in generating immune-inflammatory responses by directing the antigen-bearing immunocytes (e.g. dendritic cells) from the periphery to the draining lymph nodes where the T cells are primed and expanded. 27 Our data on corneal lymphatic ingrowth showed a significant increase in the lymphatic area (LA) in the dry eye corneas compared to normal corneas (0.14 ± 0.018 vs. 0.01 ± 0.002, respectively; P = 0.02) (Fig 4A). A significant decreased in the LA was observed only in 5% IL-1Ra (0.05 ± 0.001) and 1% methylprednisolone (0.06 ± 0.006) treated groups compared to the vehicle treated group (0.14 ± 0.01; P < 0.04). ( Figure 4B).

DISCUSSION
In recent years, inflammation has been shown to be key in the pathogenesis and amplification of dry eye disease severity. 15 Among the various treatment options, 0.05% CsA has become one of the standard treatments for patients with moderate to severe DED in the United States; however CsA use is associated with tolerability issues such as stinging and burning in some patients. 5,7 In addition to CsA, there are several studies that support the efficacy of topical corticosteroids in the treatment of keratoconjunctivitis sicca. 4 However, the long-term use of topical corticosteroids is limited by numerous side effects including ocular hypertension, cataract, and corneal thinning. 6 The current study demonstrates the beneficial effect of IL-1 blockade via the topical application of IL-1Ra in treating the ocular signs and reversing the inflammatory changes associated with dry eye at both the molecular and cellular levels.
IL-1α and IL-1β are both constitutively expressed by the corneal epithelium, and are potent mediators of corneal inflammation following their release in states of epithelial injury. 28 Specifically, of relevance to DED, hyperosmolality stress is known to induce IL-1β expression in corneal epithelial cells. 29 IL-1 has been implicated in inducing inflammatory cytokine and chemokine expression (including IL-6, monocyte chemotactic protein-1, and granulocyte stimulating factor), as well as matrix metalloproteinase synthesis by keratocytes. 30 In accord with these findings, is the demonstration that levels of proinflammatory forms of IL-1 are directly correlated with the intensity of corneal fluorescein staining, 13 and that IL-1β may contribute to the loss of corneal epithelial barrier function associated with ocular surface inflammation. 31 We show herein that IL-1Ra treated eyes had a significant reversal in corneal epithelial damage, indicated by decreased fluorescein staining, as compared with the untreated and vehicle groups.
The therapeutic efficacy of IL-1Ra is further supported by decreased corneal infiltration of CD11b + cells (which are rich sources of IL-1β 32 observed in mice treated with IL-1Ra. While macrophages are known to reside in the corneal stroma, 33 our laboratory has also previously shown infiltration by mature major histocompatibility complex class IIexpressing antigen-presenting cells in DED. 24 IL-1 is indeed known to mobilize corneal APC infiltration 34 , likely via CC chemokine receptor expression by the APC. 35 These observations are consistent with reported findings by Stapleton et al., showing reduced CD11b+ influx into the corneal stroma following the topical application of IL-1Ra in a mouse model of corneal epithelial scrape injury. 36 Thus, the blockade of IL-1, also suppressed corneal inflammation by subverting APC infiltration. These changes are important in disease pathogenesis and severity since the corneal APC mobilization is known to have a significant effect on the degree and rapidity of corneal immune responses, including graft rejection and herpes keratitis. 37 Desiccating stress in DED initiates an immuno-inflammatory response that is sustained by the ongoing interplay between the ocular surface and various pathogenic immune cells, primarily of the CD4+ T compartment. 38 Evidence suggests that the induction of pathogenic, autoreactive T cells primarily occurs in the regional lymph nodes of DED mice. 39 Recently, Goyal et al. demonstrated that there is an ingrowth of lymphatic vessels in dry eye corneas 17,18 which serve as potential conduits for the migration antigen presenting cells from ocular surface to draining lymph nodes where these cells prime naïve T cells to yet unknown ocular surface autoantigen(s). In the present study, we noticed that dry eye corneas treated with IL-1Ra showed a significant decrease in lymphatic growth. Downregulation of IL-1β could reduce the lymphangiogenesis in the mouse cornea through diminished production of potent pro-lymphangiogenic factors, vascular endothelial growth factor (VEGF)-C and VEGF-D. 40 In addition, decreased macrophage infiltration in response to low levels of IL-1 on ocular surface could reduce corneal lymphangiogenesis. Previously, It has been shown that macrophages play a crucial role in corneal inflammatory lymphangiogenesis by potentially serving as precursor cells for de novo formation of lymphatic vessels. 41,42 We and other groups have recently identified IL-17 producing T helper cells (Th17) as dominant effectors capable of disrupting the corneal epithelial barrier in DED. 43,44 Furthermore, these Th17 cells are dysfunctional in that they are more resistant to suppression by T regulatory cells. 45 Since IL-1 is known to potentiate the differentiation and survival of Th17 cells by synergizing with IL-6 and IL-23 46, 47 , we suggest that treatment with IL-1Ra may also act by suppressing Th17 activity at the ocular surface through inhibiting the effect of IL-1 on the survival of Th17 cells.
In conclusion, our study shows the therapeutic effects of topical IL-1Ra in the amelioration of the clinical signs and the underlying inflammatory changes seen in dry eyes, comparable to that seen with topical methylprednisolone. These data suggest that the use of topical IL-1Ra could hold promise as a therapeutic modality for dry eyes disease and possibly in other inflammatory conditions of the cornea and ocular surface. Topical IL-1Ra effectively reduced corneal fluorescein staining scores in dry eye disease. (Top) Topical 5% IL-1Ra, 1% methylprednisolone and 0.05% CsA treatment produced a significant decrease in corneal fluorescein staining compared to the CMC (vehicle) treated group at day 6 (* P < 0.01) and day 9 ( † P < 0.003). (Bottom) Mean reduction of corneal fluorescein staining scores compared to untreated (dry eye) group at day 9 showed that 5% IL-1Ra and 1% methylprednisolone led to a statistically significant greater reduction in fluorescein staining compared to 0.05% CsA. Data are presented as mean ± SEM. (* P < 0.01).