BASIC INVESTIGATION
Effects of Topical Janus Kinase Inhibition on Ocular Surface Inflammation and Immunity William Stevenson, MD,* Zahra Sadrai, MD,* Jing Hua, MD,* Shilpa Kodati, MD,* Jing-Feng Huang, PhD,† Sunil K. Chauhan, PhD, DVM,* and Reza Dana, MD, MPH, MSc*
Purpose: To determine the effects of topical Janus kinase inhibition on ocular surface inflammation and immunity.
Key Words: ocular surface inflammation, corneal thermocautery, dry eye disease, Janus kinase, tofacitinib (CP-690,550) (Cornea 2014;33:177–183)
Methods: Ophthalmic 0.003% tofacitinib (CP-690,550) was administered topically to inhibit Janus kinase activation at the ocular surface. Male BALB/c mice 6 to 8 weeks of age were subjected to corneal thermocautery and randomized to receive tofacitinib, vehicle, or no treatment. Corneas were subsequently excised for fluorescenceactivated cell sorting and quantitative real-time reverse transcription polymerase chain reaction. Female C57BL/6 mice 6 to 8 weeks of age were exposed to desiccating stress to induce experimental dry eye disease and randomized to receive tofacitinib, tofacitinib and vehicle, vehicle, or no treatment. Corneal fluorescein staining was performed to evaluate clinical disease severity. The corneas and conjunctivae were harvested for immunohistochemical staining and quantitative real-time reverse transcription polymerase chain reaction.
Results: After corneal thermocautery, it was found that tofacitinib treatment decreased the corneal infiltration of CD45+, Gr-1+, and CD11b+ cells on days 1 and 3. Transcripts encoding interleukin (IL)-1b and IL-6 were significantly decreased by tofacitinib treatment at post-thermocautery day 3. In experimental dry eye disease, tofacitinib treatment twice per day significantly decreased corneal fluorescein staining on days 12 and 15. The corneal infiltration of CD11b+ cells was significantly decreased by tofacitinib treatment twice per day. Tofacitinib treatment twice per day significantly increased the corneal expression of IL-1RA, and significantly decreased the corneal expression of tumor necrosis factor and IL-23. Further, tofacitinib treatment twice per day significantly decreased the conjunctival expression of IL-17A and significantly increased the conjunctival expression of FoxP3. Conclusions: Topical ophthalmic tofacitinib, a Janus kinase inhibitor, suppressed ocular surface inflammation and immunity in experimental corneal thermocautery and dry eye disease. Received for publication April 17, 2013; revision received September 9, 2013; accepted September 26, 2013. Published online ahead of print December 16, 2013. From the *Schepens Eye Research Institute, Massachusetts Eye and Ear Infirmary, Department of Ophthalmology, Harvard Medical School, Boston, MA; and †Pfizer, Inc, Medicine Development Group, San Diego, CA. This research was supported by NIH Grant EY-20889 and Pfizer, Inc. No authors received compensation for the development of this manuscript. J.-F. Huang was an employee of Pfizer, Inc at the time of this study. The authors have no other funding or conflicts of interest to disclose. W. Stevenson and Z. Sadrai contributed equally to this manuscript and should be viewed as co–first authors. Reprints: Reza Dana, Schepens Eye Research Institute, 20 Staniford St, Boston, MA 02114 912-0117 (e-mail: [email protected]). Copyright © 2013 by Lippincott Williams & Wilkins
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T
he ocular surface provides physical, chemical, and immunological barriers that insulate the eye from the external environment. Intermittent exposure to potential irritants (eg, pathogens or allergens) entails that low-level inflammation is a relatively common occurrence at the ocular surface. Inflammation is an important physiologic response to potentially injurious stimuli; however, conditions such as ocular trauma, infection, allergy, and autoimmune disease have the potential to cause irreversible inflammatory damage in the form of sight-threatening corneal opacification, scarring, or neovascularization.1 A variety of experimental models are available to investigate the innate and adaptive immune responses that mediate ocular surface inflammation. Corneal thermocautery provides an experimental model of acute, antigenindependent ocular surface inflammation characterized by proinflammatory cytokine expression and leukocyte infiltration.2 Experimental dry eye disease (DED) involves innate and adaptive immune responses as evidenced by proinflammatory cytokine expression, antigen-presenting cell (APC) maturation and migration to regional lymphatic tissues, and pathogenic T-cell priming and trafficking to the ocular surface.3 The antiinflammatory medications currently available for ophthalmic use (eg, corticosteroids and nonsteroidal antiinflammatory agents) are associated with a variety of deleterious side effects including cataract, ocular hypertension, and corneal thinning.4 Novel therapeutic targets are needed to address the dearth of safe and effective treatments for pathologic ocular surface inflammation.5 Janus kinase– signal transducer and activator of transcription (Jak-STAT) signaling facilitates the intracellular transmission of extracellular polypeptide signals. Jak1 and Jak3 mediate cellular responses to a variety of immunologically relevant cytokines.6 Tofacitinib (CP-690,550), a small molecule inhibitor of Jak1 and Jak3, has been shown to modulate immune responses in experimental and clinical trials involving conditions such as rheumatoid arthritis and kidney allotransplantation.7 Here, ophthalmic tofacitinib was used to evaluate the effects of topical Jak inhibition on ocular surface inflammation and immunity in experimental corneal thermocautery and DED. www.corneajrnl.com |
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MATERIALS AND METHODS Animals Male BALB/c mice (Charles River Laboratories; Wilmington, MA) 6 to 8 weeks of age were subjected to corneal thermocautery. Female C57BL/6 mice (Charles River Laboratories) 6 to 8 weeks of age underwent DED induction. Mice were housed in a secure, pathogen-free environment at the Schepens Eye Research Institute Animal Care Facility. All the procedures and protocols were approved by the Schepens Eye Research Institute Animal Care and Use Committee. All the animals were treated in accordance with the Association for Research in Vision and Ophthalmology Statement for the Use of Animals in Ophthalmic and Visual Research.
Medication Formulation Ophthalmic 0.003% tofacitinib (CP-690,550), a small molecule inhibitor of Jak1 and Jak3, was supplied in a masked fashion along with its corresponding vehicle by Pfizer, Inc (San Diego, CA).
Murine Corneal Thermocautery Each mouse was anesthetized with an intraperitoneal injection of ketamine (120 mg/kg) and xylazine (20 mg/kg) before corneal thermocautery. The tip of a hand-held thermocautery unit (Aaron Medical Industries, Saint Petersburg, FL) was used to create 6 light burns in the central 50% of the cornea as previously described.2,8 Vetropolycin ophthalmic ointment containing bacitracin–neomycin–polymyxin (Dechra Veterinary Products; Overland Park, KS) was subsequently applied to the affected area. After corneal thermocautery, mice were randomly divided into 3 groups: (1) topical 0.003% tofacitinib treated, (2) topical vehicle treated, and (3) untreated. Experimental groups received 3 mL of their respective treatments via topical ocular instillation twice per day beginning immediately after corneal thermocautery and continuing throughout the study period. Mice were killed using carbon dioxide inhalation on postthermocautery days 1 and 3, and the affected corneas were excised for cellular and molecular analysis.
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tofacitinib once per day and topical vehicle treated once per day, (3) those treated with a topical vehicle twice per day, and (4) those that were untreated (control). Experimental groups received 3 mL of their respective treatments bilaterally by topical ocular instillation from days 3 to 15. Carbon dioxide inhalation was used to euthanize the mice on day 15 before tissue harvesting for cellular and molecular analysis.
Corneal Fluorescein Staining
Corneal fluorescein staining was used to evaluate corneal epitheliopathy in experimental DED (n = 4–5 mice per group). The National Eye Institute’s standardized grading system was used to score punctuate staining.12 Corneal fluorescein staining was performed in a masked fashion on day 0 (before beginning dry eye induction), day 3 (baseline measure before treatment initiation), day 12, and day 15 (final measurement). Corneal fluorescein staining was performed by administering 0.6 mL of 2.5% fluorescein (Sigma–Aldrich Corporation) to the inferior lateral conjunctival sac as was previously described.9–11 Corneal staining was examined 3 minutes later using slit-lamp biomicroscopy under cobalt blue light. The average corneal fluorescein score was calculated for each individual mouse.
Fluorescence-Activated Cell Sorting Corneas were harvested and pooled (n = 7–10 corneas per group) on post-thermocautery days 1 and 3. The corneas were digested with 2 mg/mL of collagenase D (Sigma–Aldrich Corporation) for 50 minutes at 37°C. The suspension was filtered through a 70-mm cell strainer (Becton-Dickinson and Company; Franklin Lakes, NJ), and nonspecific staining was blocked with anti-FcR CD16/CD32 antibody (Becton-Dickinson and Company). The cells were subsequently labeled with CY5-conjugated anti–CD-45 (eBioscience; San Diego, CA), AlexaFluor 488 anti-CD-11b (Becton-Dickinson and Company), and fluorescein isothiocyanate–conjugated anti–Gr-1 (eBioscience) for 45 minutes at 4°C. The cells were then analyzed using a flow cytometer (Beckman Coulter; Fullerton, CA). The percentage of stained cells out of the total corneal cells was calculated with respect to isotype control staining.
Experimental Dry Eye Disease
Real-Time Polymerase Chain Reaction
Mice were housed in a controlled environment chamber that allowed for the continuous regulation of airflow (15 L/min), relative humidity (15%–20%), and temperature (21–23°C) as previously described with minor modifications.9–11 Atropine sulfate, 1.0% (Bausch & Lomb; Rochester, NY) was administered via topical ocular instillation twice per day for the first 3 days of DED induction.10 Subcutaneous injections of 0.1 mL of 5.0 mg/mL of scopolamine hydrobromide (Sigma– Aldrich Corporation; St Louis, MO) were administered 3 times per day for the duration of the experiment.10,11 After 3 days of dry eye induction, topical atropine treatment was discontinued, and the mice were randomly divided into the following experimental groups: (1) those treated with topical 0.003% tofacitinib twice per day, (2) those treated with topical 0.003%
Total RNA was isolated from corneas (n = 2–4 corneas per group) or conjunctivae (n = 3–4 conjunctivae per group) using a commercially available kit (RNeasy; Qiagen; Valencia, CA). The first strand of complementary DNA was synthesized by reverse transcriptase using random hexamers (SuperScript III; Life Technologies; Carlsbad, CA). Real-time polymerase chain reaction (PCR) was performed in duplicate or triplicate using Taqman Universal PCR Mastermix and dye-labeled, predesigned primers for interleukin (IL)-1b (Mm00434228_m1), IL-6 (Mm00446190_m1), IL-1Ra (Mm00446186_m1), TNF (Mm99999068_m1), IL-23 (Mm00518984_m1), interferon (IFN)-g (Mm01168134_m1), IL-17A (Mm00439619_m1), FoxP3 (Mm00475156_m1), and GAPDH (Mm99999915_g1) (Applied Biosystems; Foster
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City, CA). The glyceraldehyde-3-phosphate dehydrogenase gene was used as the endogenous reference for each reaction, and the results were normalized to the untreated groups.
Immunohistochemistry Whole-mount corneas were harvested on day 15 of the DED experiment and stained for CD11b+ cells, as previously described.10 Immunostained corneas were mounted using Vector Shield mounting media with 4’, 6-diamidino-2-phenylindole (Vector Laboratories; Burlingame, CA). Using a confocal microscope (Leica TCS–SP5; Leica Microsystems; Buffalo Grove, IL) at 40· magnification and Z-stack images were taken through the entire thickness of the cornea. CD11b+ cells were enumerated in a masked fashion in 3 areas in the periphery (0.5 mm from the limbus) and 2 areas in the center (central 2 mm) of each cornea.10 To account for differences in stromal thickness, cell counts were converted to CD11b+ cells per cubic millimeters of cornea, after which they were averaged.
Statistical Analysis
The 2-tailed t test was used for data analysis, and P # 0.05 was considered statistically significant. Because of the exploratory nature of the analyses, P values were not adjusted for multiple endpoints/comparisons. Representative results from 2 to 3 trials are presented as the mean 6 SEM.
Jak Inhibition in Ocular Surface Inflammation
RESULTS Topical Jak Inhibition Decreased Leukocyte Infiltration After Corneal Thermocautery Corneal thermocautery is a well-described murine model of acute corneal inflammation and immunity.2,8 Fluorescenceactivated cell sorting (FACS) was performed on postthermocautery days 1 and 3 to evaluate the corneal infiltration of CD45+, GR-1+, and CD11b+ cells (n = 7–10 corneas per group). On postthermocautery day 1, there was a marked reduction in the corneal infiltration of CD45+, GR-1+, and CD11b+ cells in the tofacitinib-treated group as compared with that in the vehicle-treated and untreated groups (Fig. 1A). The differences were less pronounced on postthermocautery day 3 when comparing the tofacitinib-treated with the vehicletreated groups (Fig. 1B).
Topical Jak Inhibition Suppressed Proinflammatory Cytokine Expression After Corneal Thermocautery Corneal thermocautery has been shown to upregulate the expression of numerous proinflammatory cytokines, including IL-1 and IL-6.2 These cytokines are produced by a variety of cell types (eg, epithelial and inflammatory cells) in response to injurious stimuli.13 Real-time PCR was used to
FIGURE 1. Leukocyte infiltration after corneal thermocautery. FACS (n = 7–10 corneas per group) results on (A) postthermocautery day 1 and (B) postthermocautery day 3 revealed reduced corneal infiltration of CD45+ and GR-1+ cells in the tofacitinib-treated group. Values represent labeled cells as a percentage of the total cell population (BID = twice daily). 2013 Lippincott Williams & Wilkins
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quantify the corneal expression of transcripts encoding IL-1b and IL-6 on days 1 and 3 postthermocautery (n = 2–3 corneas per group). On postthermocautery day 1, intergroup comparisons did not reveal any significant differences in the corneal expression of IL-1b and IL-6 (data not shown). However, on postthermocautery day 3, there were significant reductions in the expression of IL-1b (P = 0.02) and IL-6 (P = 0.02) in the tofacitinib-treated group as compared with that in the vehicletreated group (Fig. 2).
Topical Jak Inhibition Decreased Corneal Fluorescein Staining in Experimental DED
Corneal fluorescein staining was used to evaluate corneal epitheliopathy. The desiccating challenge began on day 0, and mice were randomized to 4 distinct groups on day 3, including (1) topical 0.003% tofacitinib treatment twice per day, (2) topical 0.003% tofacitinib treatment once per day and topical vehicle-treatment once per day, (3) topical vehicle treatment twice per day, and (4) no treatment (control; n = 4–5 mice per group). On day 12, there were significant differences (P , 0.05) when comparing the corneal fluorescein staining scores of both tofacitinib-treated groups with the corneal fluorescein staining scores of the vehicle-treated and untreated groups (Fig. 3). However, by day 15, only the group that received tofacitinib treatment twice per day exhibited a reduction in corneal fluorescein staining from baseline (246.0 6 15.2%), and this was significant (P , 0.05) when compared with all the other groups.
Topical Jak Inhibition Decreased Corneal Infiltration of CD11b+ Cells in Experimental DED The cell surface glycoprotein CD11b is expressed by a variety of leukocytes including monocytes, macrophages, granulocytes, and natural killer cells. Immunohistochemical
FIGURE 2. Proinflammatory cytokine expression after corneal thermocautery. Real-time PCR results (n = 2–3 corneas per group) from postthermocautery day 3 demonstrated reduced IL-1b and IL-6 mRNA expression in the tofacitinib-treated group relative to the vehicle-treated and untreated groups. Results were normalized to the untreated group (*P , 0.05; BID = twice daily).
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FIGURE 3. Corneal fluorescein staining in experimental DED. Corneal fluorescein staining (n = 4–5 mice per group) demonstrated that tofacitinib treatment twice per day (BID) reduced average corneal fluorescein staining scores on days 12 and 15 (*P , 0.05 as compared with that for vehicle-treated and untreated groups; BID = twice daily; SID = once daily).
staining was used to quantify CD11b+ cells in DED corneas on day 15. Treatment with topical tofacitinib twice per day significantly reduced the number of CD11b+ cells in the peripheral and central cornea as compared with the vehicletreated (P = 0.029 and 0.015, respectively) and untreated (P = 0.015 and 0.032, respectively) groups (Fig. 4). Although tofacitinib treatment once per day also decreased the corneal infiltration of CD11b+ cells relative to the vehicle-treated and untreated groups, these differences were not statistically significant (P . 0.05).
Topical Jak Inhibition Decreased the Corneal and Conjunctival Expression of Proinflammatory Cytokines in Experimental DED Real-time PCR was used to evaluate the expression of mRNA transcripts encoding a variety of cytokines in dry eye corneas and conjunctivae (n = 3–4 corneas per group). The corneal expression of IL-1b was decreased in all the treatment groups relative to that in the untreated group, but there were no significant differences in IL-1b expression between the various treatment groups (data not shown). Tofacitinib treatment twice per day significantly increased (P , 0.03) the expression of the specific receptor antagonist IL-1RA relative to all other groups (Fig. 5A). Tofacitinib treatment twice per day also decreased the corneal expression of the proinflammatory cytokines TNF and IL-23 as compared with vehicle treatment (P = 0.019 and 0.009, respectively). There were no significant intergroup differences in the conjunctival expression of IFN-g (data not shown). Both the tofacitinib treatment regimens significantly decreased (P # 0.002) the conjunctival expression of IL-17A, but only tofacitinib treatment twice per day significantly increased (P # 0.005) the conjunctival expression of the regulatory T cell (Treg)-expressed transcription factor FoxP3 as compared with that in the vehicle-treated and untreated groups (Fig. 5B). 2013 Lippincott Williams & Wilkins
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FIGURE 4. Corneal CD11b+ cell enumeration in DED. A, CD11b+ cell enumeration demonstrated that tofacitinib treatment twice per day significantly decreased the corneal infiltration of CD11b+ cells as compared with the vehicle treatment and no treatment in the peripheral and central cornea. B, Representative grayscale images of CD11b+ staining cells immediately subjacent to the corneal epithelium (*P , 0.05; BID = twice daily; SID = once daily).
DISCUSSION Jak-STAT signaling facilitates the transmission of extracellular polypeptide signals (eg, growth factors and cytokines) from receptors on the cell surface to gene promoters in the nucleus.14 In mammals, there are 4 members of the Jak family (Jak1, Jak2, Jak3, and Tyk2) and 7 members of the STAT family (STAT1—5a, 5b, and 6).6 The widely expressed tyrosine kinase Jak1 associates with the IL-2, IL-4, gp130 (eg, IL-6), type I (IFN-a/b) IFN, and type II (IFN-g) IFN receptor families.6 Jak1 knockout mice are deficient in T- and B-cell production and experience perinatal death.15 Leukocyte-expressed Jak3 associates with receptors that employ the common g-chain, that is, the IL-2, IL-4, IL-7, IL-9, IL-15, and IL-21 receptors.6 The most common form of inherited immunodeficiency in humans, X-linked severe combined immunodeficiency, is the result of a common g-chain mutation.16 Jak3-deficient patients suffer from severe combined immunodeficiency manifesting as a profound lack of T and natural killer cells, and dysfunctional B cells.17,18 The Jak inhibitor tofacitinib (CP-690,550) has demonstrated functional specificity 2013 Lippincott Williams & Wilkins
Jak Inhibition in Ocular Surface Inflammation
FIGURE 5. Corneal and conjunctival cytokine expression in DED. A, Reduced corneal mRNA expression (n = 3–4 corneas per group) of proinflammatory TNF, and IL-23, and increased corneal mRNA expression of antiinflammatory IL-1RA after tofacitinib treatment twice per day. B, Reduced conjunctival mRNA expression (n = 3–4 conjunctivae per group) of Th17associated IL-17A and increased conjunctival mRNA expression of Treg-associated FoxP3 after tofacitinib treatment twice per day. The results are normalized to the untreated group (*P , 0.05; BID = twice daily; SID = once daily).
for Jak1 and Jak3 in cellular assays.19 Tofacitinib shows promise in the treatment of immune-mediated conditions such as rheumatoid arthritis, kidney transplantation, and psoriasis.20–22 This study evaluated the effects of topical ophthalmic 0.003% tofacitinib on experimental corneal thermocautery and DED. Corneal thermocautery was used to assess the effects of topical Jak inhibition on acute ocular surface inflammation. The normal cornea contains a heterogeneous population of bone marrow–derived cells that includes epithelial Langerhans cells, anterior stromal dendritic cells, and posterior stromal macrophages.23 Corneal thermocautery provides an antigen-independent inflammatory stimulus that promotes the infiltration and maturation of leukocytes.2,8,24–26 FACS demonstrated that tofacitinib treatment markedly decreased the corneal infiltration of CD45+ leukocytes, and GR-1+ neutrophils and CD11b macrophages after corneal thermocautery. Numerous proinflammatory cytokines are upregulated in response to corneal thermocautery, including IL-1b and IL-6.2 On postthermocautery day 1, there were no significant www.corneajrnl.com |
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differences in the corneal expression of IL-1b and IL-6, potentially because of variable expression at this early time point. However, tofacitinib treatment significantly decreased the corneal expression of IL-1b and IL-6 mRNA as compared with the untreated and vehicle-treated corneas on postthermocautery day 3. Corneal leukocyte infiltration and proinflammatory cytokine expression are closely related; inflammatory cells are rich sources of proinflammatory cytokines, and these cytokines in turn act as chemoattractants for a variety of inflammatory cells.24–26 Moreover, other cells such as corneal epithelial cells express IL-1b and IL-6 in response to injury and inflammation.13 The decreased expression of IL-1b and IL-6 on postthermocautery day 3 may be the result of tofacitinib’s effects on both epithelial and inflammatory cells. Overall, these findings are consistent with those of previous studies demonstrating that tofacitinib can suppress innate immune responses.19,27 Experimental DED was used to assess the effects of topical Jak inhibition on ocular surface inflammation and immunity.3 Hyperosmolarity-induced ocular surface inflammation is thought to precipitate the infiltration and maturation of APCs that subsequently prime an ocular surface-specific T-cell response.3 The corneal infiltration of CD11b+ APCs has been shown to parallel the clinical severity of DED.10,11 Immunohistochemical staining revealed that tofacitinib treatment twice per day decreased the corneal infiltration of CD11b+ APCs. Further, tofacitinib twice per day improved the corneal and conjunctival cytokine profiles as compared with those in the untreated and vehicle-treated groups. Interestingly, there were no intergroup differences in the conjunctival expression of IFN-g. Previous studies have shown that IFN-g primarily contributes to the induction of DED, suggesting that day 15 may not be early enough to assess the effects of Jak inhibition on IFN-g expression.28 However, the conjunctival expression of IL-17A was significantly decreased in both the tofacitinib-treated groups. This is noteworthy given that IL-17 has been shown to promote corneal epithelial barrier dysfunction after desiccating stress.29,30 IL-17 can be produced by a variety of inflammatory cells, including T helper (Th) 17 cells and neutrophils. Because IL-23 is important for Th17 cell differentiation, the decreased expression of IL-17A may have been related to the decreased expression of IL-23 in addition to the direct effects of Jak1 and Jak3 inhibition.27 The expression of Treg-associated transcription factor FoxP3 was significantly elevated in the conjunctivae of eyes treated with tofacitinib twice per day, suggesting that Jak inhibition increased Treg activity. Treg dysfunction has been linked to the immunopathogenesis of DED, and increased Treg activity could help explain the decrease in disease severity.30 These results corroborate previous findings indicating that Jak inhibition suppresses the activity of effector T cells (eg, Th17 cells) while preserving the function of Tregs.31 Overall, these results are consistent with the reduction in corneal fluorescein staining severity observed with twice daily tofacitinib treatment. Topical ophthalmic tofacitinib treatment once per day did not suppress ocular surface inflammation as effectively as twice daily treatment. Ocular surface inflammation can cause a variety of sight-threatening complications. Safe and effective steroidal-
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and nonsteroidal antiinflammatory drug-sparing treatments are needed to treat conditions that involve dysregulated ocular surface inflammation. The data presented here suggest that Jak inhibition may be an effective treatment modality for pathologic ocular surface inflammation. This study demonstrated that topical tofacitinib, at the concentration and duration used, effectively suppressed ocular surface inflammation in experimental models of corneal thermocautery and DED. The therapeutic effects associated with topical vehicle treatment were most likely the result of ocular surface hydration and lubrication. Overall, these findings correlate well with those in the literature regarding the clinical use of topical ophthalmic tofacitinib for the treatment of DED.32,33 Patients with DED treated with topical tofacitinib for 8 weeks (0.003% twice per day or 0.005% once per day) exhibited reduced conjunctival expression of human leukocyte antigen-DR, decreased corneal staining, improved tear production, and ameliorated dry eye symptoms. Additionally, proinflammatory cytokines, including IL-1b, IL-17, and IL-12, were reduced from baseline, whereas IL-1RA showed an upward trend in the tears of patients treated with 0.005% tofacitinib once per day.32,33 Further study will be necessary to determine the optimal concentration and dosing regimen, and long-term safety and efficacy of topical ophthalmic tofacitinib. REFERENCES 1. Clements JL, Dana R. Inflammatory corneal neovascularization: etiopathogenesis. Semin Ophthalmol. 2011;26:235–245. 2. Sadrai Z, Hajrasouliha AR, Chauhan S, et al. Effect of topical azithromycin on corneal innate immune responses. Invest Ophthalmol Vis Sci. 2011;52:2525–2531. 3. Stevenson W, Chauhan SK, Dana R. Dry eye disease: an immunemediated ocular surface disorder. Arch Ophthalmol. 2012;130:90–100. 4. McGhee CN, Dean S, Danesh-Meyer H. Locally administered ocular corticosteroids: benefits and risks. Drug Saf. 2002;25:33–55. 5. Pesu M, Laurence A, Kishore N, et al. Therapeutic targeting of Janus kinases. Immunol Rev. 2008;223:132–142. 6. Kisseleva T, Bhattacharya S, Braunstein J, et al. Signaling through the JAK/STAT pathway, recent advances and future challenges. Gene. 2002; 285:1–24. 7. Vijayakrishnan L, Venkataramanan R, Gulati P. Treating inflammation with the Janus kinase inhibitor CP-690,550. Trends Pharmacol Sci. 2011;32:25–34. 8. Dekaris I, Zhu SN, Dana MR. TNF-alpha regulates corneal Langerhans cell migration. J Immunol. 1999;162:4235–4239. 9. Barabino S, Shen L, Chen L, et al. The controlled-environment chamber: a new mouse model of dry eye. Invest Ophthalmol Vis Sci. 2005;46: 2766–2771. 10. Goyal S, Chauhan SK, Zhang Q, et al. Amelioration of murine dry eye disease by topical antagonist to chemokine receptor 2. Arch Ophthalmol. 2009;127:882–887. 11. Sadrai Z, Stevenson W, Okanobo A, et al. PDE4 inhibition suppresses IL-17-associated immunity in dry eye disease. Invest Ophthalmol Vis Sci. 2012;53:3584–3591. 12. Lemp MA. Report of the National Eye Institute/Industry Workshop on clinical trials in dry eyes. CLAO J. 1995;21:221–232. 13. Cannon JG. Inflammatory cytokines in nonpathological states. News Physiol Sci. 2000;15:298–303. 14. Aaronson DS, Horvath CM. A road map for those who don’t know JAKSTAT. Science. 2002;296:1653–1655. 15. Rodig SJ, Meraz MA, White JM, et al. Disruption of the Jak1 gene demonstrates obligatory and nonredundant roles of the Jaks in cytokineinduced biologic responses. Cell. 1998;93:373–383. 16. Noguchi M, Yi H, Rosenblatt HM, et al. Interleukin-2 receptor gamma chain mutation results in X-linked severe combined immunodeficiency in humans. Cell. 1993;73:147–157.
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17. Macchi P, Villa A, Giliani S, et al. Mutations of Jak-3 gene in patients with autosomal severe combined immune deficiency (SCID). Nature. 1995;377:65–68. 18. Russell SM, Tayebi N, Nakajima H, et al. Mutation of Jak3 in a patient with SCID: essential role of Jak3 in lymphoid development. Science. 1995;270:797. 19. Meyer DM, Jesson MI, Li X, et al. Anti-inflammatory activity and neutrophil reductions mediated by the JAK1/JAK3 inhibitor, CP-690,550, in rat adjuvant-induced arthritis. J Inflamm (Lond). 2010;7:41. 20. Kremer JM, Bloom BJ, Breedveld FC, et al. The safety and efficacy of a JAK inhibitor in patients with active rheumatoid arthritis: results of a double-blind, placebo-controlled phase IIa trial of three dosage levels of CP-690,550 versus placebo. Arthritis Rheum. 2009;60:1895–1905. 21. Busque S, Leventhal J, Brennan DC, et al. Calcineurin-inhibitor-free immunosuppression based on the JAK inhibitor CP-690,550: a pilot study in de novo kidney allograft recipients. Am J Transplant. 2009;9: 1936–1945. 22. Boy MG, Wang C, Wilkinson BE, et al. Double-blind, placebo-controlled, dose-escalation study to evaluate the pharmacologic effect of CP-690,550 in patients with psoriasis. J Invest Dermatol. 2009;129:2299–2302. 23. Hamrah P, Dana MR. Corneal antigen-presenting cells. Chem Immunol Allergy. 2007;92:58–70. 24. Hamrah P, Liu Y, Zhang Q, et al. Alterations in corneal stromal dendritic cell phenotype and distribution in inflammation. Arch Ophthalmol. 2003; 121:1132–1140.
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25. Dana MR, Dai R, Zhu S, et al. Interleukin-1 receptor antagonist suppresses Langerhans cell activity and promotes ocular immune privilege. Invest Ophthalmol Vis Sci. 1998;39:70–77. 26. Dana R. Comparison of topical interleukin-1 vs tumor necrosis factor-alpha blockade with corticosteroid therapy on murine corneal inflammation, neovascularization, and transplant survival (an American Ophthalmological Society thesis). Trans Am Ophthalmol Soc. 2007;105:330–343. 27. Ghoreschi K, Jesson MI, Li X, et al. Modulation of innate and adaptive immune responses by tofacitinib (CP-690,550). J Immunol. 2011;186: 4234–4243. 28. Chen Y, Chauhan SK, Saban DR, et al. Interferon-g-secreting NK cells promote induction of dry eye disease. J Leukoc Biol. 2011;89:965–972. 29. De Paiva CS, Chotikavanich S, Pangelinan SB, et al. IL-17 disrupts corneal barrier following desiccating stress. Mucosal Immunol. 2009;2:243–253. 30. Chauhan SK, El Annan J, Ecoiffier T, et al. Autoimmunity in dry eye is due to resistance of Th17 to Treg suppression. J Immunol. 2009;182:1247–1252. 31. Sewgobind VD, Quaedackers ME, van der Laan LJ, et al. The Jak inhibitor CP-690,550 preserves the function of CD4CD25FoxP3 regulatory T cells and inhibits effector T cells. Am J Transplant. 2010;10:1785–1795. 32. Liew SH, Nichols KK, Klamerus KJ, et al. Tofacitinib (CP-690,550), a Janus kinase inhibitor for dry eye disease: results from a phase 1/2 trial. Ophthalmology. 2012;119:1328–1335. 33. Huang JF, Yafawi R, Zhang M, et al. Immunomodulatory effect of the topical ophthalmic Janus kinase inhibitor tofacitinib (CP-690,550) in patients with dry eye disease. Ophthalmology. 2012;119:e43–e50.
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Effects of Topical Janus Kinase Inhibition on Ocular Surface Inflammation and Immunity William Stevenson, MD,* Zahra Sadrai, MD,* Jing Hua, MD,* Shilpa Kodati, MD,* Jing-Feng Huang, PhD,† Sunil K. Chauhan, PhD, DVM,* and Reza Dana, MD, MPH, MSc*
Purpose: To determine the effects of topical Janus kinase inhibition on ocular surface inflammation and immunity.
Key Words: ocular surface inflammation, corneal thermocautery, dry eye disease, Janus kinase, tofacitinib (CP-690,550) (Cornea 2014;33:177–183)
Methods: Ophthalmic 0.003% tofacitinib (CP-690,550) was administered topically to inhibit Janus kinase activation at the ocular surface. Male BALB/c mice 6 to 8 weeks of age were subjected to corneal thermocautery and randomized to receive tofacitinib, vehicle, or no treatment. Corneas were subsequently excised for fluorescenceactivated cell sorting and quantitative real-time reverse transcription polymerase chain reaction. Female C57BL/6 mice 6 to 8 weeks of age were exposed to desiccating stress to induce experimental dry eye disease and randomized to receive tofacitinib, tofacitinib and vehicle, vehicle, or no treatment. Corneal fluorescein staining was performed to evaluate clinical disease severity. The corneas and conjunctivae were harvested for immunohistochemical staining and quantitative real-time reverse transcription polymerase chain reaction.
Results: After corneal thermocautery, it was found that tofacitinib treatment decreased the corneal infiltration of CD45+, Gr-1+, and CD11b+ cells on days 1 and 3. Transcripts encoding interleukin (IL)-1b and IL-6 were significantly decreased by tofacitinib treatment at post-thermocautery day 3. In experimental dry eye disease, tofacitinib treatment twice per day significantly decreased corneal fluorescein staining on days 12 and 15. The corneal infiltration of CD11b+ cells was significantly decreased by tofacitinib treatment twice per day. Tofacitinib treatment twice per day significantly increased the corneal expression of IL-1RA, and significantly decreased the corneal expression of tumor necrosis factor and IL-23. Further, tofacitinib treatment twice per day significantly decreased the conjunctival expression of IL-17A and significantly increased the conjunctival expression of FoxP3. Conclusions: Topical ophthalmic tofacitinib, a Janus kinase inhibitor, suppressed ocular surface inflammation and immunity in experimental corneal thermocautery and dry eye disease. Received for publication April 17, 2013; revision received September 9, 2013; accepted September 26, 2013. Published online ahead of print December 16, 2013. From the *Schepens Eye Research Institute, Massachusetts Eye and Ear Infirmary, Department of Ophthalmology, Harvard Medical School, Boston, MA; and †Pfizer, Inc, Medicine Development Group, San Diego, CA. This research was supported by NIH Grant EY-20889 and Pfizer, Inc. No authors received compensation for the development of this manuscript. J.-F. Huang was an employee of Pfizer, Inc at the time of this study. The authors have no other funding or conflicts of interest to disclose. W. Stevenson and Z. Sadrai contributed equally to this manuscript and should be viewed as co–first authors. Reprints: Reza Dana, Schepens Eye Research Institute, 20 Staniford St, Boston, MA 02114 912-0117 (e-mail: [email protected]). Copyright © 2013 by Lippincott Williams & Wilkins
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T
he ocular surface provides physical, chemical, and immunological barriers that insulate the eye from the external environment. Intermittent exposure to potential irritants (eg, pathogens or allergens) entails that low-level inflammation is a relatively common occurrence at the ocular surface. Inflammation is an important physiologic response to potentially injurious stimuli; however, conditions such as ocular trauma, infection, allergy, and autoimmune disease have the potential to cause irreversible inflammatory damage in the form of sight-threatening corneal opacification, scarring, or neovascularization.1 A variety of experimental models are available to investigate the innate and adaptive immune responses that mediate ocular surface inflammation. Corneal thermocautery provides an experimental model of acute, antigenindependent ocular surface inflammation characterized by proinflammatory cytokine expression and leukocyte infiltration.2 Experimental dry eye disease (DED) involves innate and adaptive immune responses as evidenced by proinflammatory cytokine expression, antigen-presenting cell (APC) maturation and migration to regional lymphatic tissues, and pathogenic T-cell priming and trafficking to the ocular surface.3 The antiinflammatory medications currently available for ophthalmic use (eg, corticosteroids and nonsteroidal antiinflammatory agents) are associated with a variety of deleterious side effects including cataract, ocular hypertension, and corneal thinning.4 Novel therapeutic targets are needed to address the dearth of safe and effective treatments for pathologic ocular surface inflammation.5 Janus kinase– signal transducer and activator of transcription (Jak-STAT) signaling facilitates the intracellular transmission of extracellular polypeptide signals. Jak1 and Jak3 mediate cellular responses to a variety of immunologically relevant cytokines.6 Tofacitinib (CP-690,550), a small molecule inhibitor of Jak1 and Jak3, has been shown to modulate immune responses in experimental and clinical trials involving conditions such as rheumatoid arthritis and kidney allotransplantation.7 Here, ophthalmic tofacitinib was used to evaluate the effects of topical Jak inhibition on ocular surface inflammation and immunity in experimental corneal thermocautery and DED. www.corneajrnl.com |
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MATERIALS AND METHODS Animals Male BALB/c mice (Charles River Laboratories; Wilmington, MA) 6 to 8 weeks of age were subjected to corneal thermocautery. Female C57BL/6 mice (Charles River Laboratories) 6 to 8 weeks of age underwent DED induction. Mice were housed in a secure, pathogen-free environment at the Schepens Eye Research Institute Animal Care Facility. All the procedures and protocols were approved by the Schepens Eye Research Institute Animal Care and Use Committee. All the animals were treated in accordance with the Association for Research in Vision and Ophthalmology Statement for the Use of Animals in Ophthalmic and Visual Research.
Medication Formulation Ophthalmic 0.003% tofacitinib (CP-690,550), a small molecule inhibitor of Jak1 and Jak3, was supplied in a masked fashion along with its corresponding vehicle by Pfizer, Inc (San Diego, CA).
Murine Corneal Thermocautery Each mouse was anesthetized with an intraperitoneal injection of ketamine (120 mg/kg) and xylazine (20 mg/kg) before corneal thermocautery. The tip of a hand-held thermocautery unit (Aaron Medical Industries, Saint Petersburg, FL) was used to create 6 light burns in the central 50% of the cornea as previously described.2,8 Vetropolycin ophthalmic ointment containing bacitracin–neomycin–polymyxin (Dechra Veterinary Products; Overland Park, KS) was subsequently applied to the affected area. After corneal thermocautery, mice were randomly divided into 3 groups: (1) topical 0.003% tofacitinib treated, (2) topical vehicle treated, and (3) untreated. Experimental groups received 3 mL of their respective treatments via topical ocular instillation twice per day beginning immediately after corneal thermocautery and continuing throughout the study period. Mice were killed using carbon dioxide inhalation on postthermocautery days 1 and 3, and the affected corneas were excised for cellular and molecular analysis.
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tofacitinib once per day and topical vehicle treated once per day, (3) those treated with a topical vehicle twice per day, and (4) those that were untreated (control). Experimental groups received 3 mL of their respective treatments bilaterally by topical ocular instillation from days 3 to 15. Carbon dioxide inhalation was used to euthanize the mice on day 15 before tissue harvesting for cellular and molecular analysis.
Corneal Fluorescein Staining
Corneal fluorescein staining was used to evaluate corneal epitheliopathy in experimental DED (n = 4–5 mice per group). The National Eye Institute’s standardized grading system was used to score punctuate staining.12 Corneal fluorescein staining was performed in a masked fashion on day 0 (before beginning dry eye induction), day 3 (baseline measure before treatment initiation), day 12, and day 15 (final measurement). Corneal fluorescein staining was performed by administering 0.6 mL of 2.5% fluorescein (Sigma–Aldrich Corporation) to the inferior lateral conjunctival sac as was previously described.9–11 Corneal staining was examined 3 minutes later using slit-lamp biomicroscopy under cobalt blue light. The average corneal fluorescein score was calculated for each individual mouse.
Fluorescence-Activated Cell Sorting Corneas were harvested and pooled (n = 7–10 corneas per group) on post-thermocautery days 1 and 3. The corneas were digested with 2 mg/mL of collagenase D (Sigma–Aldrich Corporation) for 50 minutes at 37°C. The suspension was filtered through a 70-mm cell strainer (Becton-Dickinson and Company; Franklin Lakes, NJ), and nonspecific staining was blocked with anti-FcR CD16/CD32 antibody (Becton-Dickinson and Company). The cells were subsequently labeled with CY5-conjugated anti–CD-45 (eBioscience; San Diego, CA), AlexaFluor 488 anti-CD-11b (Becton-Dickinson and Company), and fluorescein isothiocyanate–conjugated anti–Gr-1 (eBioscience) for 45 minutes at 4°C. The cells were then analyzed using a flow cytometer (Beckman Coulter; Fullerton, CA). The percentage of stained cells out of the total corneal cells was calculated with respect to isotype control staining.
Experimental Dry Eye Disease
Real-Time Polymerase Chain Reaction
Mice were housed in a controlled environment chamber that allowed for the continuous regulation of airflow (15 L/min), relative humidity (15%–20%), and temperature (21–23°C) as previously described with minor modifications.9–11 Atropine sulfate, 1.0% (Bausch & Lomb; Rochester, NY) was administered via topical ocular instillation twice per day for the first 3 days of DED induction.10 Subcutaneous injections of 0.1 mL of 5.0 mg/mL of scopolamine hydrobromide (Sigma– Aldrich Corporation; St Louis, MO) were administered 3 times per day for the duration of the experiment.10,11 After 3 days of dry eye induction, topical atropine treatment was discontinued, and the mice were randomly divided into the following experimental groups: (1) those treated with topical 0.003% tofacitinib twice per day, (2) those treated with topical 0.003%
Total RNA was isolated from corneas (n = 2–4 corneas per group) or conjunctivae (n = 3–4 conjunctivae per group) using a commercially available kit (RNeasy; Qiagen; Valencia, CA). The first strand of complementary DNA was synthesized by reverse transcriptase using random hexamers (SuperScript III; Life Technologies; Carlsbad, CA). Real-time polymerase chain reaction (PCR) was performed in duplicate or triplicate using Taqman Universal PCR Mastermix and dye-labeled, predesigned primers for interleukin (IL)-1b (Mm00434228_m1), IL-6 (Mm00446190_m1), IL-1Ra (Mm00446186_m1), TNF (Mm99999068_m1), IL-23 (Mm00518984_m1), interferon (IFN)-g (Mm01168134_m1), IL-17A (Mm00439619_m1), FoxP3 (Mm00475156_m1), and GAPDH (Mm99999915_g1) (Applied Biosystems; Foster
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City, CA). The glyceraldehyde-3-phosphate dehydrogenase gene was used as the endogenous reference for each reaction, and the results were normalized to the untreated groups.
Immunohistochemistry Whole-mount corneas were harvested on day 15 of the DED experiment and stained for CD11b+ cells, as previously described.10 Immunostained corneas were mounted using Vector Shield mounting media with 4’, 6-diamidino-2-phenylindole (Vector Laboratories; Burlingame, CA). Using a confocal microscope (Leica TCS–SP5; Leica Microsystems; Buffalo Grove, IL) at 40· magnification and Z-stack images were taken through the entire thickness of the cornea. CD11b+ cells were enumerated in a masked fashion in 3 areas in the periphery (0.5 mm from the limbus) and 2 areas in the center (central 2 mm) of each cornea.10 To account for differences in stromal thickness, cell counts were converted to CD11b+ cells per cubic millimeters of cornea, after which they were averaged.
Statistical Analysis
The 2-tailed t test was used for data analysis, and P # 0.05 was considered statistically significant. Because of the exploratory nature of the analyses, P values were not adjusted for multiple endpoints/comparisons. Representative results from 2 to 3 trials are presented as the mean 6 SEM.
Jak Inhibition in Ocular Surface Inflammation
RESULTS Topical Jak Inhibition Decreased Leukocyte Infiltration After Corneal Thermocautery Corneal thermocautery is a well-described murine model of acute corneal inflammation and immunity.2,8 Fluorescenceactivated cell sorting (FACS) was performed on postthermocautery days 1 and 3 to evaluate the corneal infiltration of CD45+, GR-1+, and CD11b+ cells (n = 7–10 corneas per group). On postthermocautery day 1, there was a marked reduction in the corneal infiltration of CD45+, GR-1+, and CD11b+ cells in the tofacitinib-treated group as compared with that in the vehicle-treated and untreated groups (Fig. 1A). The differences were less pronounced on postthermocautery day 3 when comparing the tofacitinib-treated with the vehicletreated groups (Fig. 1B).
Topical Jak Inhibition Suppressed Proinflammatory Cytokine Expression After Corneal Thermocautery Corneal thermocautery has been shown to upregulate the expression of numerous proinflammatory cytokines, including IL-1 and IL-6.2 These cytokines are produced by a variety of cell types (eg, epithelial and inflammatory cells) in response to injurious stimuli.13 Real-time PCR was used to
FIGURE 1. Leukocyte infiltration after corneal thermocautery. FACS (n = 7–10 corneas per group) results on (A) postthermocautery day 1 and (B) postthermocautery day 3 revealed reduced corneal infiltration of CD45+ and GR-1+ cells in the tofacitinib-treated group. Values represent labeled cells as a percentage of the total cell population (BID = twice daily). 2013 Lippincott Williams & Wilkins
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quantify the corneal expression of transcripts encoding IL-1b and IL-6 on days 1 and 3 postthermocautery (n = 2–3 corneas per group). On postthermocautery day 1, intergroup comparisons did not reveal any significant differences in the corneal expression of IL-1b and IL-6 (data not shown). However, on postthermocautery day 3, there were significant reductions in the expression of IL-1b (P = 0.02) and IL-6 (P = 0.02) in the tofacitinib-treated group as compared with that in the vehicletreated group (Fig. 2).
Topical Jak Inhibition Decreased Corneal Fluorescein Staining in Experimental DED
Corneal fluorescein staining was used to evaluate corneal epitheliopathy. The desiccating challenge began on day 0, and mice were randomized to 4 distinct groups on day 3, including (1) topical 0.003% tofacitinib treatment twice per day, (2) topical 0.003% tofacitinib treatment once per day and topical vehicle-treatment once per day, (3) topical vehicle treatment twice per day, and (4) no treatment (control; n = 4–5 mice per group). On day 12, there were significant differences (P , 0.05) when comparing the corneal fluorescein staining scores of both tofacitinib-treated groups with the corneal fluorescein staining scores of the vehicle-treated and untreated groups (Fig. 3). However, by day 15, only the group that received tofacitinib treatment twice per day exhibited a reduction in corneal fluorescein staining from baseline (246.0 6 15.2%), and this was significant (P , 0.05) when compared with all the other groups.
Topical Jak Inhibition Decreased Corneal Infiltration of CD11b+ Cells in Experimental DED The cell surface glycoprotein CD11b is expressed by a variety of leukocytes including monocytes, macrophages, granulocytes, and natural killer cells. Immunohistochemical
FIGURE 2. Proinflammatory cytokine expression after corneal thermocautery. Real-time PCR results (n = 2–3 corneas per group) from postthermocautery day 3 demonstrated reduced IL-1b and IL-6 mRNA expression in the tofacitinib-treated group relative to the vehicle-treated and untreated groups. Results were normalized to the untreated group (*P , 0.05; BID = twice daily).
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FIGURE 3. Corneal fluorescein staining in experimental DED. Corneal fluorescein staining (n = 4–5 mice per group) demonstrated that tofacitinib treatment twice per day (BID) reduced average corneal fluorescein staining scores on days 12 and 15 (*P , 0.05 as compared with that for vehicle-treated and untreated groups; BID = twice daily; SID = once daily).
staining was used to quantify CD11b+ cells in DED corneas on day 15. Treatment with topical tofacitinib twice per day significantly reduced the number of CD11b+ cells in the peripheral and central cornea as compared with the vehicletreated (P = 0.029 and 0.015, respectively) and untreated (P = 0.015 and 0.032, respectively) groups (Fig. 4). Although tofacitinib treatment once per day also decreased the corneal infiltration of CD11b+ cells relative to the vehicle-treated and untreated groups, these differences were not statistically significant (P . 0.05).
Topical Jak Inhibition Decreased the Corneal and Conjunctival Expression of Proinflammatory Cytokines in Experimental DED Real-time PCR was used to evaluate the expression of mRNA transcripts encoding a variety of cytokines in dry eye corneas and conjunctivae (n = 3–4 corneas per group). The corneal expression of IL-1b was decreased in all the treatment groups relative to that in the untreated group, but there were no significant differences in IL-1b expression between the various treatment groups (data not shown). Tofacitinib treatment twice per day significantly increased (P , 0.03) the expression of the specific receptor antagonist IL-1RA relative to all other groups (Fig. 5A). Tofacitinib treatment twice per day also decreased the corneal expression of the proinflammatory cytokines TNF and IL-23 as compared with vehicle treatment (P = 0.019 and 0.009, respectively). There were no significant intergroup differences in the conjunctival expression of IFN-g (data not shown). Both the tofacitinib treatment regimens significantly decreased (P # 0.002) the conjunctival expression of IL-17A, but only tofacitinib treatment twice per day significantly increased (P # 0.005) the conjunctival expression of the regulatory T cell (Treg)-expressed transcription factor FoxP3 as compared with that in the vehicle-treated and untreated groups (Fig. 5B). 2013 Lippincott Williams & Wilkins
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FIGURE 4. Corneal CD11b+ cell enumeration in DED. A, CD11b+ cell enumeration demonstrated that tofacitinib treatment twice per day significantly decreased the corneal infiltration of CD11b+ cells as compared with the vehicle treatment and no treatment in the peripheral and central cornea. B, Representative grayscale images of CD11b+ staining cells immediately subjacent to the corneal epithelium (*P , 0.05; BID = twice daily; SID = once daily).
DISCUSSION Jak-STAT signaling facilitates the transmission of extracellular polypeptide signals (eg, growth factors and cytokines) from receptors on the cell surface to gene promoters in the nucleus.14 In mammals, there are 4 members of the Jak family (Jak1, Jak2, Jak3, and Tyk2) and 7 members of the STAT family (STAT1—5a, 5b, and 6).6 The widely expressed tyrosine kinase Jak1 associates with the IL-2, IL-4, gp130 (eg, IL-6), type I (IFN-a/b) IFN, and type II (IFN-g) IFN receptor families.6 Jak1 knockout mice are deficient in T- and B-cell production and experience perinatal death.15 Leukocyte-expressed Jak3 associates with receptors that employ the common g-chain, that is, the IL-2, IL-4, IL-7, IL-9, IL-15, and IL-21 receptors.6 The most common form of inherited immunodeficiency in humans, X-linked severe combined immunodeficiency, is the result of a common g-chain mutation.16 Jak3-deficient patients suffer from severe combined immunodeficiency manifesting as a profound lack of T and natural killer cells, and dysfunctional B cells.17,18 The Jak inhibitor tofacitinib (CP-690,550) has demonstrated functional specificity 2013 Lippincott Williams & Wilkins
Jak Inhibition in Ocular Surface Inflammation
FIGURE 5. Corneal and conjunctival cytokine expression in DED. A, Reduced corneal mRNA expression (n = 3–4 corneas per group) of proinflammatory TNF, and IL-23, and increased corneal mRNA expression of antiinflammatory IL-1RA after tofacitinib treatment twice per day. B, Reduced conjunctival mRNA expression (n = 3–4 conjunctivae per group) of Th17associated IL-17A and increased conjunctival mRNA expression of Treg-associated FoxP3 after tofacitinib treatment twice per day. The results are normalized to the untreated group (*P , 0.05; BID = twice daily; SID = once daily).
for Jak1 and Jak3 in cellular assays.19 Tofacitinib shows promise in the treatment of immune-mediated conditions such as rheumatoid arthritis, kidney transplantation, and psoriasis.20–22 This study evaluated the effects of topical ophthalmic 0.003% tofacitinib on experimental corneal thermocautery and DED. Corneal thermocautery was used to assess the effects of topical Jak inhibition on acute ocular surface inflammation. The normal cornea contains a heterogeneous population of bone marrow–derived cells that includes epithelial Langerhans cells, anterior stromal dendritic cells, and posterior stromal macrophages.23 Corneal thermocautery provides an antigen-independent inflammatory stimulus that promotes the infiltration and maturation of leukocytes.2,8,24–26 FACS demonstrated that tofacitinib treatment markedly decreased the corneal infiltration of CD45+ leukocytes, and GR-1+ neutrophils and CD11b macrophages after corneal thermocautery. Numerous proinflammatory cytokines are upregulated in response to corneal thermocautery, including IL-1b and IL-6.2 On postthermocautery day 1, there were no significant www.corneajrnl.com |
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differences in the corneal expression of IL-1b and IL-6, potentially because of variable expression at this early time point. However, tofacitinib treatment significantly decreased the corneal expression of IL-1b and IL-6 mRNA as compared with the untreated and vehicle-treated corneas on postthermocautery day 3. Corneal leukocyte infiltration and proinflammatory cytokine expression are closely related; inflammatory cells are rich sources of proinflammatory cytokines, and these cytokines in turn act as chemoattractants for a variety of inflammatory cells.24–26 Moreover, other cells such as corneal epithelial cells express IL-1b and IL-6 in response to injury and inflammation.13 The decreased expression of IL-1b and IL-6 on postthermocautery day 3 may be the result of tofacitinib’s effects on both epithelial and inflammatory cells. Overall, these findings are consistent with those of previous studies demonstrating that tofacitinib can suppress innate immune responses.19,27 Experimental DED was used to assess the effects of topical Jak inhibition on ocular surface inflammation and immunity.3 Hyperosmolarity-induced ocular surface inflammation is thought to precipitate the infiltration and maturation of APCs that subsequently prime an ocular surface-specific T-cell response.3 The corneal infiltration of CD11b+ APCs has been shown to parallel the clinical severity of DED.10,11 Immunohistochemical staining revealed that tofacitinib treatment twice per day decreased the corneal infiltration of CD11b+ APCs. Further, tofacitinib twice per day improved the corneal and conjunctival cytokine profiles as compared with those in the untreated and vehicle-treated groups. Interestingly, there were no intergroup differences in the conjunctival expression of IFN-g. Previous studies have shown that IFN-g primarily contributes to the induction of DED, suggesting that day 15 may not be early enough to assess the effects of Jak inhibition on IFN-g expression.28 However, the conjunctival expression of IL-17A was significantly decreased in both the tofacitinib-treated groups. This is noteworthy given that IL-17 has been shown to promote corneal epithelial barrier dysfunction after desiccating stress.29,30 IL-17 can be produced by a variety of inflammatory cells, including T helper (Th) 17 cells and neutrophils. Because IL-23 is important for Th17 cell differentiation, the decreased expression of IL-17A may have been related to the decreased expression of IL-23 in addition to the direct effects of Jak1 and Jak3 inhibition.27 The expression of Treg-associated transcription factor FoxP3 was significantly elevated in the conjunctivae of eyes treated with tofacitinib twice per day, suggesting that Jak inhibition increased Treg activity. Treg dysfunction has been linked to the immunopathogenesis of DED, and increased Treg activity could help explain the decrease in disease severity.30 These results corroborate previous findings indicating that Jak inhibition suppresses the activity of effector T cells (eg, Th17 cells) while preserving the function of Tregs.31 Overall, these results are consistent with the reduction in corneal fluorescein staining severity observed with twice daily tofacitinib treatment. Topical ophthalmic tofacitinib treatment once per day did not suppress ocular surface inflammation as effectively as twice daily treatment. Ocular surface inflammation can cause a variety of sight-threatening complications. Safe and effective steroidal-
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and nonsteroidal antiinflammatory drug-sparing treatments are needed to treat conditions that involve dysregulated ocular surface inflammation. The data presented here suggest that Jak inhibition may be an effective treatment modality for pathologic ocular surface inflammation. This study demonstrated that topical tofacitinib, at the concentration and duration used, effectively suppressed ocular surface inflammation in experimental models of corneal thermocautery and DED. The therapeutic effects associated with topical vehicle treatment were most likely the result of ocular surface hydration and lubrication. Overall, these findings correlate well with those in the literature regarding the clinical use of topical ophthalmic tofacitinib for the treatment of DED.32,33 Patients with DED treated with topical tofacitinib for 8 weeks (0.003% twice per day or 0.005% once per day) exhibited reduced conjunctival expression of human leukocyte antigen-DR, decreased corneal staining, improved tear production, and ameliorated dry eye symptoms. Additionally, proinflammatory cytokines, including IL-1b, IL-17, and IL-12, were reduced from baseline, whereas IL-1RA showed an upward trend in the tears of patients treated with 0.005% tofacitinib once per day.32,33 Further study will be necessary to determine the optimal concentration and dosing regimen, and long-term safety and efficacy of topical ophthalmic tofacitinib. REFERENCES 1. Clements JL, Dana R. Inflammatory corneal neovascularization: etiopathogenesis. Semin Ophthalmol. 2011;26:235–245. 2. Sadrai Z, Hajrasouliha AR, Chauhan S, et al. Effect of topical azithromycin on corneal innate immune responses. Invest Ophthalmol Vis Sci. 2011;52:2525–2531. 3. Stevenson W, Chauhan SK, Dana R. Dry eye disease: an immunemediated ocular surface disorder. Arch Ophthalmol. 2012;130:90–100. 4. McGhee CN, Dean S, Danesh-Meyer H. Locally administered ocular corticosteroids: benefits and risks. Drug Saf. 2002;25:33–55. 5. Pesu M, Laurence A, Kishore N, et al. Therapeutic targeting of Janus kinases. Immunol Rev. 2008;223:132–142. 6. Kisseleva T, Bhattacharya S, Braunstein J, et al. Signaling through the JAK/STAT pathway, recent advances and future challenges. Gene. 2002; 285:1–24. 7. Vijayakrishnan L, Venkataramanan R, Gulati P. Treating inflammation with the Janus kinase inhibitor CP-690,550. Trends Pharmacol Sci. 2011;32:25–34. 8. Dekaris I, Zhu SN, Dana MR. TNF-alpha regulates corneal Langerhans cell migration. J Immunol. 1999;162:4235–4239. 9. Barabino S, Shen L, Chen L, et al. The controlled-environment chamber: a new mouse model of dry eye. Invest Ophthalmol Vis Sci. 2005;46: 2766–2771. 10. Goyal S, Chauhan SK, Zhang Q, et al. Amelioration of murine dry eye disease by topical antagonist to chemokine receptor 2. Arch Ophthalmol. 2009;127:882–887. 11. Sadrai Z, Stevenson W, Okanobo A, et al. PDE4 inhibition suppresses IL-17-associated immunity in dry eye disease. Invest Ophthalmol Vis Sci. 2012;53:3584–3591. 12. Lemp MA. Report of the National Eye Institute/Industry Workshop on clinical trials in dry eyes. CLAO J. 1995;21:221–232. 13. Cannon JG. Inflammatory cytokines in nonpathological states. News Physiol Sci. 2000;15:298–303. 14. Aaronson DS, Horvath CM. A road map for those who don’t know JAKSTAT. Science. 2002;296:1653–1655. 15. Rodig SJ, Meraz MA, White JM, et al. Disruption of the Jak1 gene demonstrates obligatory and nonredundant roles of the Jaks in cytokineinduced biologic responses. Cell. 1998;93:373–383. 16. Noguchi M, Yi H, Rosenblatt HM, et al. Interleukin-2 receptor gamma chain mutation results in X-linked severe combined immunodeficiency in humans. Cell. 1993;73:147–157.
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17. Macchi P, Villa A, Giliani S, et al. Mutations of Jak-3 gene in patients with autosomal severe combined immune deficiency (SCID). Nature. 1995;377:65–68. 18. Russell SM, Tayebi N, Nakajima H, et al. Mutation of Jak3 in a patient with SCID: essential role of Jak3 in lymphoid development. Science. 1995;270:797. 19. Meyer DM, Jesson MI, Li X, et al. Anti-inflammatory activity and neutrophil reductions mediated by the JAK1/JAK3 inhibitor, CP-690,550, in rat adjuvant-induced arthritis. J Inflamm (Lond). 2010;7:41. 20. Kremer JM, Bloom BJ, Breedveld FC, et al. The safety and efficacy of a JAK inhibitor in patients with active rheumatoid arthritis: results of a double-blind, placebo-controlled phase IIa trial of three dosage levels of CP-690,550 versus placebo. Arthritis Rheum. 2009;60:1895–1905. 21. Busque S, Leventhal J, Brennan DC, et al. Calcineurin-inhibitor-free immunosuppression based on the JAK inhibitor CP-690,550: a pilot study in de novo kidney allograft recipients. Am J Transplant. 2009;9: 1936–1945. 22. Boy MG, Wang C, Wilkinson BE, et al. Double-blind, placebo-controlled, dose-escalation study to evaluate the pharmacologic effect of CP-690,550 in patients with psoriasis. J Invest Dermatol. 2009;129:2299–2302. 23. Hamrah P, Dana MR. Corneal antigen-presenting cells. Chem Immunol Allergy. 2007;92:58–70. 24. Hamrah P, Liu Y, Zhang Q, et al. Alterations in corneal stromal dendritic cell phenotype and distribution in inflammation. Arch Ophthalmol. 2003; 121:1132–1140.
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25. Dana MR, Dai R, Zhu S, et al. Interleukin-1 receptor antagonist suppresses Langerhans cell activity and promotes ocular immune privilege. Invest Ophthalmol Vis Sci. 1998;39:70–77. 26. Dana R. Comparison of topical interleukin-1 vs tumor necrosis factor-alpha blockade with corticosteroid therapy on murine corneal inflammation, neovascularization, and transplant survival (an American Ophthalmological Society thesis). Trans Am Ophthalmol Soc. 2007;105:330–343. 27. Ghoreschi K, Jesson MI, Li X, et al. Modulation of innate and adaptive immune responses by tofacitinib (CP-690,550). J Immunol. 2011;186: 4234–4243. 28. Chen Y, Chauhan SK, Saban DR, et al. Interferon-g-secreting NK cells promote induction of dry eye disease. J Leukoc Biol. 2011;89:965–972. 29. De Paiva CS, Chotikavanich S, Pangelinan SB, et al. IL-17 disrupts corneal barrier following desiccating stress. Mucosal Immunol. 2009;2:243–253. 30. Chauhan SK, El Annan J, Ecoiffier T, et al. Autoimmunity in dry eye is due to resistance of Th17 to Treg suppression. J Immunol. 2009;182:1247–1252. 31. Sewgobind VD, Quaedackers ME, van der Laan LJ, et al. The Jak inhibitor CP-690,550 preserves the function of CD4CD25FoxP3 regulatory T cells and inhibits effector T cells. Am J Transplant. 2010;10:1785–1795. 32. Liew SH, Nichols KK, Klamerus KJ, et al. Tofacitinib (CP-690,550), a Janus kinase inhibitor for dry eye disease: results from a phase 1/2 trial. Ophthalmology. 2012;119:1328–1335. 33. Huang JF, Yafawi R, Zhang M, et al. Immunomodulatory effect of the topical ophthalmic Janus kinase inhibitor tofacitinib (CP-690,550) in patients with dry eye disease. Ophthalmology. 2012;119:e43–e50.
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