Anti-inflammatory and anti-fibrotic effects of topical pan-JAK inhibitor in a chronic graft-versus- host disease model mouse

Anti-inflammatory and anti-fibrotic effects of topical pan-JAK inhibitor in a chronic graft-versus- host disease model mouse | Research Square window.SnipcartSettings = { analytics: { enabled: false } }; (function() { var accessVector = localStorage.getItem('access_vector') || ''; window.dataLayer = window.dataLayer || []; if (accessVector) { window.dataLayer.push({ user: { profile: { profileInfo: { snid: accessVector } } } }); } })(); (function(w,d,s,l,i){w[l]=w[l]||[];w[l].push({'gtm.start':new Date().getTime(),event:'gtm.js'});var f=d.getElementsByTagName(s)[0],j=d.createElement(s),dl=l!='dataLayer'?'&l='+l:'';j.async=true;j.src='https://www.googletagmanager.com/gtm.js?id='+i+dl;f.parentNode.insertBefore(j,f);})(window,document,'script','dataLayer','GTM-K279D39R'); Browse Preprints In Review Journals COVID-19 Preprints AJE Video Bytes Research Tools Research Promotion AJE Professional Editing AJE Rubriq About Preprint Platform In Review Editorial Policies Our Team Advisory Board Help Center Sign In Submit a Preprint Cite Share Download PDF Article Anti-inflammatory and anti-fibrotic effects of topical pan-JAK inhibitor in a chronic graft-versus- host disease model mouse Shinri Sato, Kazuki Asai, Yoko Ogawa, Eisuke Shimizu, Shota Shimizu, and 5 more This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-4252479/v1 This work is licensed under a CC BY 4.0 License Status: Published Journal Publication published 02 Jan, 2025 Read the published version in Scientific Reports → Version 1 posted 10 You are reading this latest preprint version Abstract Systemic administration of Janus kinase (JAK) inhibitors is effective in treating chronic graft-versus-host disease (cGVHD) but is associated with side effects. Topical drug administration is an effective approach in minimizing these effects. We aimed to demonstrate the efficacy of topical delgocitinib administration in a cGVHD mouse model. Allogenic bone-marrow transplantation (BMT) was performed from B10.D2. to BALB/c mice, leading to cGVHD. cGVHD mice were treated with delgocitinib eye drops or ointments; their samples were analyzed 4 weeks post-BMT. Topical delgocitinib ointment and eye-drop administration significantly increased the meibomian-gland (MG) area and attenuated corneal epithelial damage. Pathological and immunohistochemical analyses revealed a substantial reduction in inflammation and pathological fibrosis of the skin and eyelids in delgocitinib-treated cGVHD mice. Signal transducer and activator of transcription (STAT)1, STAT3, and STAT5A phosphorylation was significantly increased in the back skin and eyelids of vehicle-treated cGVHD mice; topical delgocitinib administration significantly reduced the expression of these phosphorylated STAT molecules. Delgocitinib eye drops significantly attenuated corneal epithelial damage, MG acinar depletion, and inflammatory cells infiltration in cGVHD mouse corneas. The JAK/STAT signaling pathway was significantly upregulated in cGVHD mice. In summary, a topical delgocitinib administration attenuated cGVHD phenotype severity in the skin and eyes of cGVHD mice. Health sciences/Diseases Health sciences/Diseases/Eye diseases Health sciences/Diseases/Eye diseases/Eyelid diseases graft-versus-host disease meibomian gland dysfunction pan-Janus kinase inhibitor delgocitinib Figures Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 Figure 6 Figure 7 Figure 8 Figure 9 Figure 10 Introduction Based on the onset of the disease, GVHD is classified into acute (aGVHD) and chronic (cGVHD) GVHD, occurring approximately 100 days after transplantation. However, the current classification is based on clinical signs and pathological and histological findings, regardless of the onset period 1 . While the symptoms of aGVHD are characterized by skin conditions, jaundice, and diarrhea, cGVHD affects various organs, such as the nails, skin, digestive tract, liver, lungs, reproductive organs, oral cavity, and eyes 2 . Cutaneous cGVHD can be clinically classified as sclerotic or non-sclerotic. According to the National Institutes of Health Consensus Development Project, the following skin manifestations are diagnostic for chronic GVHD: poikiloderma, lichen planus-like eruptions, lichen sclerosus-like lesions, morphea-like sclerosis, and deep sclerosis/fasciitis 3 . Ocular GVHD affects approximately half of patients with cGVHD and primarily manifests as keratoconjunctivitis sicca 4 . Advanced ocular complications include corneal epithelial defects, corneal ulcers, secondary infections, perforations, stromal scarring, fornix shortening, symblepharon, and loss of vision 5 . Meibomian gland dysfunction (MGD) is also commonly observed in patients with ocular GVHD, characterized by abnormalities in the morphology of meibomian glands (MGs), inflammatory changes in the lid margin, terminal duct obstruction, and/or quantitative and qualitative changes in glandular secretion 6,7 . While fibrotic ocular cGVHD are not directly life-threatening, their widespread involvement leads to considerable functional disability. The first-line treatment for cGVHD is steroid therapy; however, if sufficient improvement is not achieved or symptoms recur, secondary therapy becomes necessary. Janus kinases (JAKs) are non-receptor tyrosine kinases comprising four enzymes (JAK1, JAK2, JAK3, and Tyk2) 8 . They activate signal transducer and activator of transcription (STAT) proteins, transduce signals from multiple type I and type II cytokine receptors, and mediate various inflammatory responses 9,10 . STATs are latent cytoplasmic proteins that mediate extracellular signals into the nucleus upon stimulation with ligands, including many hormones, interferons (IFNs), interleukins (ILs), and colony stimulating factors 10,11 . Various JAK-STAT signaling pathway-related cytokines, such as IL-2, IL-6, and IFN-γ, are involved in the pathophysiology of cGVHD 12 . JAK inhibitors block the JAK-STAT signaling pathway, which is involved in the signaling of hematopoietic factors and cytokines 13 . Several small-molecule JAK inhibitors are currently under clinical development for the treatment of autoimmune diseases and chronic dermatitis 14,15 . Systemic administration of the JAK1/2 blocker, ruxolunitib, is effective for refractory aGVHD and cGVHD 16,17 but has side effects like thrombocytopenia and/or anemia. Topical administration of drugs, especially for the skin and eyes, may be a good alternative to avoid these side effects. Delgocitinib (JTE-052) is a novel JAK inhibitor that has been shown to inhibit JAK1, JAK2, JAK3, and Tyk2 18 . Topical administration of delgocitinib has been effective in ameliorating the severity of chronic dermatitis in rodent disease models 19 . In this study, we aimed to demonstrate the efficacy of topical administration of the pan-JAK inhibitor, delgocitinib, in a cGVHD mouse model using eye drops and ointments. Results Administration of 0.5% delgocitinib ointment preserves the morphology of MGs and attenuates the severity of the corneal epithelial damage To assess the efficacy of topical administration of a pan-JAK inhibitor for ocular GVHD, we applied delgocitinib ointment to the eyelids of an established sclerodermatous cGVHD mouse model 20,21 (Fig. 1 a). Compared to the syngeneic control group, systemic GVHD scores were significantly increased in the vehicle-treated cGVHD mice, but there was no significant difference between the vehicle-treated and delgocitinib ointment-treated cGVHD mice (Fig. 1 b). In contrast to vehicle-treated cGVHD mice, delgocitinib ointment-treated cGVHD mice and syngeneic controls exhibited no blepharitis (Fig. 1 c). The topographical results showed that the MG area in cGVHD mice was significantly decreased than that in syngeneic control mice, and delgocitinib ointment-treated cGVHD mice retained a significantly larger MG area (Fig. 1 d, e). The corneal fluorescein staining (CFS) score was significantly more severe in vehicle-treated cGVHD mice than in syngeneic control and delgocitinib ointment-treated cGVHD mice (Fig. 1 f, g). In summary, these results indicate that delgocitinib ointment attenuated the severity of ocular GVHD. Administration of 0.5% delgocitinib ointment alleviates inflammatory cell infiltration and fibrosis in the eyelids of cGVHD mice We assessed the eyelids by dividing them into three segments: the MGs, eyelid margin, and eyelid skin (Fig. 2 a). To examine inflammatory cell infiltration, we performed immunohistochemistry for the pan-leukocyte marker cluster of differentiation (CD) 45 and the T cell marker CD4. Increased number of CD45 + inflammatory cells and CD4 + T cells infiltrated the eyelid margins of vehicle-treated cGVHD mice than those of syngeneic and delgocitinib ointment-treated mice (Fig. 2 b-d). The fibrotic areas in the eyelid skin and around the MG acini of cGVHD mice were analyzed using Mallory staining. The fibrotic area per field in vehicle-treated cGVHD mice was significantly larger than that in syngeneic control mice, and pathological fibrosis was significantly alleviated in delgocitinib ointment-treated mice (Fig. 2 e-g). These results suggested that delgocitinib ointment alleviated inflammatory cell infiltration and fibrosis in the eyelids of cGVHD mice. Administration of 0.5% delgocitinib ointment alleviates inflammatory cell infiltration and fibrosis in the skin of cGVHD mice The blue fibrotic area in Mallory staining of the skin of vehicle-treated cGVHD mice was significantly larger than that in syngeneic control mice, and this pathological fibrosis was significantly alleviated in delgocitinib-treated mice (Fig. 3 a). Increased number of CD45 + inflammatory cells and CD4 + T cells infiltrated the skin of vehicle-treated cGVHD mice than syngeneic mice and delgocitinib-treated mice (Fig. 3 c-d). Myofibroblast marker α-smooth muscle actin (α-SMA) + area per fields in the skin of vehicle-treated cGVHD mice was significantly larger than that of syngeneic control mice and delgocitinib-treated mice (Fig. 3 . d). These results suggested that delgocitinib ointment alleviated inflammatory cell infiltration and fibrosis in the skin of cGVHD mice. 0.5% delgocitinib ointment attenuates the expression of phosphorylated STATs The eyelids were assessed by dividing them into three segments: the MGs, eyelid margin, and eyelid skin. Increased number of pSTAT1 positive cells were observed in the interstitial space of these three segments of the eyelids in vehicle-treated cGVHD mice than in the syngeneic controls and delgocitinib-treated cGVHD mice (Fig. 4 a). In addition to cells in the interstitial space, pSTAT3 positive cells in vehicle-treated cGVHD mice included epithelial cells of the eyelid skin, eyelid margin, and MG acini (Fig. 4 b). pSTAT3 positive area was significantly increased in these three segments of the eyelids in vehicle-treated cGVHD mice compared with syngeneic controls and delgocitinib ointment-treated cGVHD mice (Fig. 4 b). Similarly, pSTAT5A positive area were detected in both epithelial and interstitial cells and were significantly larger in the three segments of the eyelids in vehicle-treated cGVHD mice than those in the syngeneic controls and delgocitinib ointment-treated cGVHD mice (Fig. 4 c). Subsequently, we focused on the expression of activated STAT1, STAT3, and STAT5A in the skin of the cGVHD mice. Similar to the eyelids, the expression of phosphorylated STAT1, STAT3, and STAT5A was significantly upregulated in the vehicle-treated cGVHD mice than that of the syngeneic controls, with delgocitinib-treatment significantly suppressing the expression of these molecules (Fig. 5 a-c). Infiltration of JAK/STAT signaling pathway activating leukocytes are attenuated in the eyelids of delgocitinib-treated cGVHD mice The JAK/STAT signaling pathway is activated in various types of leukocytes associated with T cells and macrophages 8,22,23 . To elucidate whether the JAK/STAT signaling pathway is promoted in leukocytes infiltrating the eyelids of cGVHD mice, we double-stained for the pan-leukocyte marker CD45 or the T cell marker CD4 and pSTAT1, pSTAT3, or pSTAT5A. Compared with the syngeneic controls, both CD45 + pSTAT1 + , CD45 + pSTAT3 + and CD45 + pSTAT5A + cells infiltrated significantly more in the eyelids of vehicle-treated cGVHD mice (Fig. 6 a-c). The numbers of CD45 + pSTAT1 + , CD45 + pSTAT3 + and CD45 + pSTAT5A + cells in the eyelids of cGVHD mice treated with delgocitinib ointment were significantly lower than those in vehicle-treated cGVHD mice. Furthermore, the numbers of CD4 + pSTAT3 + and CD4 + pSTAT5A + in the eyelids of vehicle-treated cGVHD mice were significantly higher than those in syngeneic control and delgocitinib ointment-treated cGVHD mice (Fig. 7 a-b). These findings showed that the infiltration of JAK/STAT signaling pathway activating leukocytes was promoted in the eyelids of cGVHD mice and the pan-JAK inhibitor delgocitinib attenuated this pathological finding. Expressions of pro-inflammatory cytokines attenuated in the eyelids of delgocitinib-treated cGVHD mice We focused on the proinflammatory cytokines IL-2, IL-6, and IFN-γ, which activate the JAK/STAT signaling pathway to transduce intracellular signals and contribute to cGVHD pathogenesis 24 25 . The expression of IL-2, IL-6 and IFN-γ were significantly elevated in the eyelids of cGVHD mice treated with vehicle than those of syngeneic control mice, with delgocitinib significantly attenuating the expression of these molecules (Fig. 8 a-c). Additionally, we focused on IL-1β, which does not activate JAK/STAT signaling pathway directly but enhances the effects of JAK/STAT-dependent proinflammatory cytokines by repressing the suppressor of cytokine signaling 3 (SOCS3) 26 . The expression of IL-1β was significantly elevated in the eyelids of vehicle-treated cGVHD mice than those of syngeneic control and delgocitinib-treated cGVHD mice (Fig. 8 d). Expressions of pro-inflammatory cytokines attenuated in the eyelids of delgocitinib-treated cGVHD mice We hypothesized that delgocitinib eye drops would suppress inflammation on the ocular surface of cGVHD mice. After 28 days of BMT, there was no significant difference in the systemic GVHD clinical score between the vehicle-treated cGVHD mice and the delgocitinib eye-drop-treated cGVHD mice (Fig. 9 a-b). The topographical results showed that the MG area in cGVHD mice was significantly decreased than that in syngeneic control mice, and delgocitinib eye drop-treated cGVHD mice retained a significantly larger MG area (Fig. 9 c-d). Furthermore, the CFS score in the vehicle-treated cGVHD mice was significantly higher than that in the syngeneic controls and delgocitinib eye drop-treated cGVHD mice (Fig. 9 e,f). In summary, these results indicated that delgocitinib eye drops attenuated the severity of ocular GVHD. Inflammatory cell infiltration is attenuated in the corneas of delgocitinib eye drops-treated cGVHD mice We analyzed the dynamics of inflammatory cell infiltration in the cornea using whole-mount immunostaining. Compared with syngeneic control mice, significantly increased number of CD45 + leukocytes infiltrated the corneas of vehicle-treated cGVHD mice. In the corneas of 0.5% delgocitinib eye drop-treated cGVHD mice, the infiltration of CD45 + cells was significantly attenuated compared with that in vehicle-treated cGVHD mice (Fig. 10 a). Subsequently, we analyzed the dynamics of the macrophages that reside in the homeostatic cornea and infiltrate the corneas from the blood vessels of the limbus and tears when inflammation occurs 34,27,28 . F4/80 positive macrophages infiltrated significantly in the corneas of the vehicle-treated cGVHD mice than in the syngeneic control and the cGVHD mice treated with delgocitinib. Thus, treatment with delgocitinib significantly attenuated macrophage infiltration (Fig. 10 b). Discussion In this study, we demonstrated that the JAK/STAT signaling pathway was upregulated in the eyelids and skin of a cGVHD mouse model. Moreover, we found that administering the pan-JAK inhibitor, delgocitinib, locally suppressed the severity of cGVHD in the eyes and skin. Our results also showed that delgocitinib ointment had beneficial effects on inflammation and sclerotic changes in the eyelids, as well as on MGD and corneal epithelial damage. We previously reported that this sclerotic cGVHD mouse model mimics MGD and blepharitis with the depletion of MG acini, pathological fibrosis, and infiltration of inflammatory cells, as observed in patients with cGVHD 29,30 . MGD causes abnormalities in the lipid components of tear fluid, leading to evaporative dry eye, and approximately half of the patients with ocular GVHD develop MGD 6,7 . Current treatments for MGD include antimicrobial eye drops, topical corticosteroid eye drops, eyelid hygiene, and warming 31 . However, none of these treatments align with the pathophysiological mechanisms of blepharitis and MGD in ocular GVHD, where significant infiltration of inflammatory cells and fibrosis occur in the eyelids. Local administration of JAK inhibitors may be a promising treatment option for eyelid inflammation and MGD associated with GVHD. In this study, the expression of phosphorylated STAT1, STAT3, and STAT5A was significantly elevated in the eyelids and skin of cGVHD mice and attenuated in delgocitinib-treated cGVHD mice (Figs. 4 and 5 ). As the proinflammatory cytokines are related to JAK/STAT signaling pathway, we also focused on the expression of IL-2, IL-6 and IFN-γ and observed that expression of these cytokines was higher in the eyelids of cGVHD mice, suggesting their role in cGVHD pathogenesis. IL-2 in tear is elevated in patients with systemic cGVHD than in patients without systemic cGVHD 32 and its level correlates with the severity of ocular cGVHD 33 . Although IL-2 has been shown to activate several STAT family members, including STAT1, STAT3, and STAT5A, STAT5A is the predominant IL-2 signaling molecule 34–36 . IL-2 signaling through the phosphorylation of STAT5A increases the proliferation of T cells 29 and delgocitinib (JTE-052) inhibits the IL-2-induced proliferation of T cells, as observed in in vitro experiments 37 . In this study, topical administration of the pan-JAK inhibitor, delgocitinib, significantly attenuated the expression of phosphorylated STAT5A (Fig. 4 c), and the number of CD4 positive cells significantly decreased in the eyelid margins and skin of delgocitinib-treated cGVHD mice (Fig. 2 b). Additionally, IL-2 signaling through STAT5 promotes the proliferation and differentiation to effector T cells, such as Th1 cells, which produce IFN-γ 38–40 . Indeed, our data confirmed the significant attenuation of CD4 + STAT5A + cell infiltration and IFN-γ expression in the eyelids of delgocitinib-treated cGVHD mice (Fig. 7 b and Fig. 8 c). Previously, we reported that IL-6 expression is upregulated in the targeted organs of the mouse model, including the lacrimal glands, liver, and lungs 41 . STAT3 plays a central role in transmitting IL-6 intracellular signals from the membrane to the nucleus 42 . In our study, IL-6 expression was significantly elevated, and phosphorylated STAT3 was observed in the eyelids of cGVHD mice (Fig. 4 b and Fig. 8 b). Dendritic cells (DCs) and macrophages are the principal sources of IL-6 dysregulation after hematopoietic stem cell transplantation (HSCT), and IL-6-dependent GVHD pathogenesis is driven by IL-6R in donor T cells 43,44 . Some pSTAT3 + CD4 + cells that infiltrate the eyelids of cGVHD mice (Fig. 7 a) may be T cells activated by IL-6 secreted from DCs and macrophages. Elevated pSTAT3 is also observed in the ocular surface epithelium of a dry-eye mouse model induced by benzalkonium chloride, and STAT3 inhibition has therapeutic effects in dry eye 13 . Suppressing IL-6 signaling may have the potential to treat various ocular surface diseases. IFN-γ, a soluble cytokine secreted by immune and mucosal epithelial cells, plays a role in the development of cGVHD. It binds to the interferon-γ receptor (IFNγR)1/2 to activate JAK1/2 and STAT proteins, primarily STAT1 45 . The expression of IFN-γ and pSTAT1 was significantly upregulated in the eyelids and skin of cGVHD mice (Fig. 4 a and Fig. 5 a). The previous report shows that IFN-γ secreted from CD8 positive T cells act on keratinocytes, which produce transforming growth factor-β and promote the development of sclerodermatous changes observed in cGVHD 46 . Inhibiting IFN-γ and its intracellular signaling may hold the potential for effectively suppressing pathological fibrosis. Subsequently, we evaluated the expression of IL-1β. The intracellular signaling of IL-1β through IL-1R does not directly involve the JAK/STAT pathway; however, IL-1β enhances the effect of phosphorylated STAT3 by repressing SOCS3, known as a negative regulator of JAK/STAT signaling pathway, thereby ultimately leading to the promotion of Th17 cell differentiation and enhancement of the effects of STAT3-dependent proinflammatory cytokines such as IL-6 26 . The elevated expression of IL-1β in the eyelids of cGVHD mice (Fig. 8 d) might enhance the effects of pSTAT3 by repressing SOCS3. In corneal whole-mount immunostaining using CD45, with dendritic-shaped immune cells, globular-shaped immune cells significantly infiltrated the corneas of vehicle-treated cGVHD mice than that of syngeneic control and delgocitinib-treated cGVHD mice (Fig. 10 a). According to the observation using in vivo confocal microscopy, globular-shaped cells are rarely observed in the corneas of syngeneic control and wild-type mice 47 . Furthermore, single-cell analysis using the corneas of wild-type mice revealed that myeloid cells comprise over 90% of the immune cells in the central part of the cornea of wild-type mice, with the population of lymphocytes being almost absent 48,49 . Therefore, the globular-shaped immune cells observed in our corneal whole-mount immunostaining may potentially include lymphocytes, such as T cells. In future, we intend to analyze the expression of markers specific to T cells and their subtypes. In the early post-HSCT phase, cytotoxic therapy, irradiation, and aGVHD trigger soluble inflammatory mediators, which lead to increased antigen presentation in myeloid cells, including macrophages 12 . The corneal whole-mount immunostaining analysis demonstrated a significantly higher number of F4/80-positive macrophages in cGVHD model mice than in the syngeneic control and delgocitinib-treated cGVHD mice (Fig. 10 b). Oral administration of the JAK1/2 inhibitor, ruxilitinib, to cGVHD model mice significantly suppresses CD11b positive macrophages/mononuclear cells infiltrating in the skin by suppressing IFN-γ secretion from T cells and subsequent monocyte chemotactic protein 1 secretion from macrophages 50 . These findings suggested that delgocitinib eye drops have a suppressive effect on the infiltration of inflammatory cells in the corneas of cGVHD mice. This study has some limitations. Local administration of delgocitinib reduces the severity of chronic skin and ocular GVHD. However, since the drug was administered before the onset of chronic skin and ocular cGVHD in this study, it is unclear if it has a preventive or therapeutic effect. The precise mechanism by which topical delgocitinib suppressed the inflammatory response remains unclear. While STAT1, STAT3, and STAT5A are activated in diverse cell types, including epithelial and infiltrating inflammatory cells, the impact of cell type-specific activation of this pathway on cGVHD remains to be established. In future studies, we aim to explore the underlying mechanism linking cGVHD and the JAK/STAT signaling pathway by conducting experiments in a cell type-specific manner. In summary, the JAK/STAT signaling pathway is activated in the eyelids and skin of cGVHD mice, and topical administration of the pan-JAK inhibitor, delgocitinib, significantly alleviated the severity of chronic skin and ocular GVHD phenotypes. These findings highlight the potential of targeting the JAK/STAT signaling pathway as an effective approach to prevent chronic skin and ocular GVHD development. Methods Mice B10.D2/nSnSlc and BALB/cCrSlc mice (7–9 weeks old) were purchased from Sankyo Laboratory Inc. (Tokyo, Japan). All experimental procedures were conducted in accordance with the Association for Research in Vision and Ophthalmology Statement and the Institutional Guidelines on Animal Experimentation at Keio University. All animal and genetically modified mouse experimental protocols were approved by the Keio University Institutional Animal Care and Use Committee (# A2022-178). Whole Bone Marrow Transplantation To create the cGVHD mouse model, allogeneic BMT was performed using 7–9-week-old male B10.D2/nSnSlc and female BALB/cCrSlc mice as transplant donors and recipients, respectively, as previously reported, representing major histocompatibility complex-compatible, miHA-mismatched BMT 21,51 . As a non-cGVHD control, syngeneic BMT was conducted by transplanting donor cells from male BALB/cCrSlc mice into female BALB/cCrSlc mice, which were irradiated with 7 Gy X-rays using a Gammacell 137 Cs source (Hitachi Medico Ltd., Tokyo, Japan). Donor cells (1 × 10 6 bone marrow cells/mouse and 2 × 10 6 spleen cells/mouse) were suspended separately in 100 μL of RPMI1640 medium, and then, combined. This 200-μL suspension was injected into the recipient via the tail vein. The recipient animals were maintained in sterile cages and provided with autoclaved food and acidified water. Mice were monitored for characteristic signs of GVHD by clinical assessment using a standard scoring system, as described previously 52 . This system assessed seven systemic clinical traits: activity, posture, fur texture, weight loss, skin integrity, presence of diarrhea, and degree of alopecia. Each clinical parameter was awarded a score from 0–2, with a total available score of 0–14. The cGVHD and syngeneic control mice were used for experiments 28 days after BMT. Treatment of allogeneic BMT recipient mice with delgocitinib The JAK inhibitor, delgocitinib (JTE-052), was synthesized by Japan Tobacco (JT) (Osaka, Japan). Delgocitinib inhibits JAK1, JAK2, JAK3 and Tyk2 with IC50 values of 2.8, 2.6, 13, and 58 nM, respectively 18 . JT provided 0.5% delgocitinib ointment and solvent vehicle ointments. To prepare 0.5% delgocitinib eye drops, delgocitinib was mixed with 0.9% sodium chloride solution and adjusted with hydrochloric acid to achieve a pH of 5.0–6.0. A 0.9% sodium chloride solution with pH of 5.0–6.0 was used as vehicle-eye drops. To assess the efficacy of topical administration of a pan-JAK inhibitor for ocular GVHD, allogeneic BMT recipients were treated with either 0.5% delgocitinib ointment or solvent vehicle ointment applied to the eyelids twice a day for 21 consecutive days, starting from 7 to 28 days after BMT (Fig. 1a). Syngeneic mice were used as the controls. Another group of allogeneic BMT recipients were treated with either 0.5% delgocitinib ointment or solvent vehicle ointment applied to the skin of the back twice a day for 21 consecutive days, starting from 7 to 28 days after BMT. To observe the effects of delgocitinib eye drops, allogeneic BMT recipients were treated with either 0.5% delgocitinib eye drops, or the solvent vehicle eye drops twice daily for 21 consecutive days, starting from 7 to 28 days after BMT. Eyelid imaging After 28 days of BMT, the eyelids were collected from the right eye of each mouse. The MG area was analyzed using a topographical method, as previously reported 29,30,53 . After harvesting, photographs of the upper eyelid were taken immediately under a microscope (SZ61, Olympus, Japan) with white light. The percentage of the MG area was analyzed using ImageJ software. The central 3 mm eyelid area, including the eyelid margin and the furthest endpoint of the MGs, was measured (area A) using the freehand tool. The depletion area of the MG was semiautomatically selected and measured (area B) using the threshold tool. The percentage of MG area was calculated using the following formula: MG Area% = (area A-B)/area A × 100%. Histological analysis The mice were euthanized by cervical dislocation 28 days after BMT. The eyeballs with eyelids were harvested and fixed with 10% neutralized buffered formalin for 12 hours at room temperature, embedded in paraffin wax, and processed for hematoxylin and eosin (H&E) and Mallory staining 20,54 . To quantify the fibrotic areas in the eyelids in the tissue sections, images were acquired at 400 × magnification, and three images of the Mallory-stained sections were examined. These images were analyzed using Color Deconvolution, a plugin for ImageJ (NIH Image, Bethesda, MD, USA), in which the colors of each image were separated into blue, green, and red. The blue area was measured using the same threshold. The average value obtained from the three images of each sample was considered the value of the fibrotic area 41 . Immunohistochemistry Immunohistochemical analyses were performed on formalin-fixed and paraffin-embedded sections as described previously 55 . Following deparaffinization and dehydration, the target antigens were unmasked using the microwave method at 100℃ for 10 min. This process was applied to antibodies against interleukin (IL)-1β (AF-401-NA, polyclonal, R&D Systems, Minneapolis, MN, USA), IL-6 (ab6672, Abcam, Cambridge, United Kingdom), Allophycocyanin (APC)-labeled CD45 (103112, 30-F11, Biolegend, San Diego, CA, USA), or through autoclave techniques at 120°C for 20 min for antibodies, α-smooth muscle actin (α-SMA) (ab7817, 1A4, Abcam). Antigens were processed in a target retrieval solution (S169984; Agilent Technologies, Santa Clara, CA, USA). Subsequently, the sections were blocked with 10% normal goat serum (012-000-120; Jackson ImmunoResearch Laboratories, West Grove, PA, USA) for 30 min at room temperature and then incubated overnight at 4°C with appropriately diluted primary antibodies. This was followed by 45-min incubation at room temperature with 4',6-diamidino-2-phenylindole, dihydrochloride (DAPI ) and secondary antibodies, including (1) Alexa Fluor 488-conjugated goat anti-rabbit IgG secondary antibody (A11034, Thermo Fisher Scientific, Waltham, MA, USA ) for IL-6,pSTAT1, pSTAT3, and pSTAT5A; (2) Alexa Fluor 488-conjugated goat anti-mouse IgG secondary antibody (A11029, Thermo Fisher Scientific) for α-SMA; and (3) Alexa Fluor 488-conjugated rabbit anti-goat IgG secondary antibody (A11078, Thermo Fisher Scientific) for IL-1β. To demonstrate activation of the JAK/STAT signaling pathway in the skin and eyelids of cGVHD mice, we performed immunobiological analysis with the sections treated with pSTAT1, pSTAT3, and pSTAT5A antibodies were incubated with Horseradish peroxidase (HRP)-coupled antibodies, followed by diaminobenzidine development and H&E staining using the Dako Real Envision Detection system (K5007, Agilent Technologies). Isotype-matched antibodies used as negative controls included, (1) rabbit IgG antibody (2729, Cell Signaling Technology,Danver, MA, USA) for IL-6, pSTAT1, pSTAT3, and pSTAT5A; (2) mouse IgG2a, κ antibody (ab18415, abcam) for α-SMA; and (3) Goat IgG antibody (02-6202, Thermo Fisher Scientific) for IL-1β. Subsequently, we captured 1–3 images of each eyelid margin, MG, and eyelid skin area from each stained section, using a digital slide scanner (Nanozoomer S60, Hamamatsu, Japan) and THUNDER Imager 3D Cell Culture (Leica, Wetzlar, Germany). The positive areas and cells in each field were then quantified manually or automatically using ImageJ. Corneal whole-mount staining Whole-mount corneal staining was performed. Eyeballs were fixed in a solution consisting of 1% formaldehyde (133-10311, Fuji Film, Tokyo, Japan), 2mM MgCl2 (95812-85, Nakarai, Kyoto, Japan), 5mM Ethylene Glycol Bis (2-aminoethyl ether)- N , N , N ', N '-tetraacetic a cid (37346-05, Nakarai), and 0.02% Nonidet p40 (NP 40) (25223-04, Nakarai) in phosphate-buffered saline (PBS) at 4℃ for 75 min. After washing by 0.02% NP40 in PBS at 23℃ for 15 min, eyeballs were fixed in 80% methanol diluted with Dimethyl sulfoxide (DMSO) (13407-45, Nakarai) at -20℃ for 2 h. After fixation, the cornea was removed from the eyeballs using forceps and scissors. Corneas were permeabilized by incubation in 25% Triton X-100 (35501-02, Nakarai) diluted with methanol for 15 min, 50% Triton X-100 for 15 min, and 75% Triton X-100 for 10 min. After washing with PBS three times at 23℃ for 30 min, corneas were incubated in the blocking solution consisting of 10% normal goat serum (50062Z, Thermo Fisher Scientific) and 0.1% Triton X-100 at 23℃ for two hours. Corneas were incubated in APC-labeled CD45 (103112, 30-F11, Biolegend) and PE-labeled F4/80 (157303, QA17A29, BioLegend) antibodies diluted in PBS (1:200) at 4℃ for 12 h. After washing five time with by 0.02% tween 20 in PBS at 23℃ five times for an hour, corneas were incubated in DAPI (62247, Thermo Fisher Scientific) diluted with PBS for 5 min. After washing three times with PBS at 23℃, corneas were mounted using Fluorescence Mounting Medium (S302380-2, Agilent Technologies) on slide glass (MAS-01, Matsunami, Osaka, Japan). Corneal fluorescein staining After 4 weeks (28 days) post-BMT, the recipient mice were evaluated for corneal epithelitis. One microliter of 0.5% fluorescein sodium solution (Fluorescite, 877290; Novartis Pharma, Tokyo, Japan) was instilled into the temporal conjunctival sac without anesthesia. The ocular surface was observed under cobalt blue light using a Smart Eye Camera (13B2X10198030101; OUI Inc., Tokyo, Japan) 41,56 . CFS score was used to assess corneal epithelial damage and was calculated using a 4-point scale: 0, absent; 1, slightly punctate staining with 30 spots but does not diffuse; 3, severe diffuse staining but no positive plaques; and 4, positive fluorescein plaques 57 . The scores for the three segments including upper, middle, and lower parts of the cornea were summed to produce a final grade (0 to 12 points). The average of the total score for the left and right eyes was used as the CFS score for that individual. Statistical analysis Statistical analyses were performed using the GraphPad Prism software (GraphPad Software, USA). One-way analysis of variance with the Tukey–Kramer post-hoc test was used for statistical analyses. Statistical significance was set at P < 0.05. Declarations Acknowledgments This study was supported by grants from the Japanese Ministry of Education, Science, Sports, Culture, and Technology (22K16982) awarded to S. Sato. The authors thank Erina Igarashi at the Keio University School of Medicine and the Collaborative Research Resources of the Keio University of Medicine for their technical assistance. Author Contributions S. Sato and E.S. designed the experiments. S. Sato, K. A., E. S., T. O., S. Shimizu, and H. T. conducted the experiments. S. Sato, K.A., Y.O., E.S., T.O., S. Shimmura., and M.H. discussed and analyzed the data. S. Sato., K.A., E. S., and Y. O. wrote the manuscript. All the authors have read and agreed to the final version of the manuscript. Y.O., S. Shimmura., K.N., and M.H. supervised the study. Data Availability Statement The data that support the findings of this study are available within the article or from the corresponding author upon reasonable request. Additional Information Competing Interests Statement: S. Sato and E. S. received delgocitinib, 0.5% delgocitinib ointment and solvent vehicle ointments from JT, and the preparation method of delgocitinib eye drops was provided by ROHTO Pharmaceutical Co., Ltd. S. Shimizu, H. T., K. A., T.O., M. H., S. Shimmura, K. N., and Y. O. declare neither financial nor nonfinancial conflicts of interest associated with this manuscript. References Wolff, D. et al. National Institutes of Health Consensus Development Project on Criteria for Clinical Trials in Chronic Graft-versus-Host Disease: IV. The 2020 Highly morbid forms report. Transplant Cell Ther 27, 817-835, doi:10.1016/j.jtct.2021.06.001 (2021). Ferrara, J. L., Levine, J. E., Reddy, P. & Holler, E. Graft-versus-host disease. 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Cite Share Download PDF Status: Published Journal Publication published 02 Jan, 2025 Read the published version in Scientific Reports → Version 1 posted Editorial decision: Revision requested 13 May, 2024 Reviews received at journal 06 May, 2024 Reviews received at journal 02 May, 2024 Reviewers agreed at journal 25 Apr, 2024 Reviewers agreed at journal 25 Apr, 2024 Reviewers invited by journal 16 Apr, 2024 Editor assigned by journal 16 Apr, 2024 Editor invited by journal 16 Apr, 2024 Submission checks completed at journal 16 Apr, 2024 First submitted to journal 11 Apr, 2024 You are reading this latest preprint version Research Square lets you share your work early, gain feedback from the community, and start making changes to your manuscript prior to peer review in a journal. As a division of Research Square Company, we’re committed to making research communication faster, fairer, and more useful. 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Also discoverable on Platform About Our Team In Review Editorial Policies Advisory Board Help Center Resources Author Services Accessibility API Access RSS feed Manage Cookie Preferences © Research Square 2026 | ISSN 2693-5015 (online) Privacy Policy Terms of Service Do Not Sell My Personal Information {"props":{"pageProps":{"initialData":{"identity":"rs-4252479","acceptedTermsAndConditions":true,"allowDirectSubmit":false,"archivedVersions":[],"articleType":"Article","associatedPublications":[],"authors":[{"id":292937638,"identity":"af1cab80-eba0-4a1e-90ab-ae85187ff456","order_by":0,"name":"Shinri Sato","email":"data:image/png;base64,iVBORw0KGgoAAAANSUhEUgAAAZAAAAAyAQMAAABI0h/eAAAABlBMVEX///8AAABVwtN+AAAACXBIWXMAAA7EAAAOxAGVKw4bAAAA+UlEQVRIie3RMWvCQBTA8XcEzuXANYdgvsILAaEQzFe5IKSrXUrGQOAcXQ869Cvo5nhyEJegayHdAp3t5tBB29RFMNGtlPtPj4MfvLsDsNn+Yi7JAPBnJPUhDRGc79ObCAUngDK5hZwHCnRApMHOvbyXXNbpFKL+a64HT3IX9HuOhofVdYLv65lfIsSqoCJQ22rEcyqAly3EjSXPEAQUDAV7rkI0DIHLlsVUQyLvRDSj2zDqIvDWELIomJ8zqUfodBBsiBsvi2RCVDkJXEOFbruLpx4/ePYVRkNjNod9Ovbnc7OuecuL/XbxdYZnneQi8nk3sdlstn/cEZKbTIUbxLJ9AAAAAElFTkSuQmCC","orcid":"","institution":"Keio University School of Medicine","correspondingAuthor":true,"prefix":"","firstName":"Shinri","middleName":"","lastName":"Sato","suffix":""},{"id":292937639,"identity":"5876a7a0-786b-4c2c-a90a-94f1b1718a87","order_by":1,"name":"Kazuki Asai","email":"","orcid":"","institution":"Keio University School of Medicine","correspondingAuthor":false,"prefix":"","firstName":"Kazuki","middleName":"","lastName":"Asai","suffix":""},{"id":292937640,"identity":"d71ade70-1b55-4b41-b6ce-4f9649c96d7e","order_by":2,"name":"Yoko Ogawa","email":"","orcid":"","institution":"Keio University School of Medicine","correspondingAuthor":false,"prefix":"","firstName":"Yoko","middleName":"","lastName":"Ogawa","suffix":""},{"id":292937641,"identity":"95209654-92f9-47bf-9fe3-a095c2fad14f","order_by":3,"name":"Eisuke Shimizu","email":"","orcid":"","institution":"Keio University School of Medicine","correspondingAuthor":false,"prefix":"","firstName":"Eisuke","middleName":"","lastName":"Shimizu","suffix":""},{"id":292937642,"identity":"93603ab8-8586-4dbe-b896-916dbc1ce665","order_by":4,"name":"Shota Shimizu","email":"","orcid":"","institution":"Keio University School of Medicine","correspondingAuthor":false,"prefix":"","firstName":"Shota","middleName":"","lastName":"Shimizu","suffix":""},{"id":292937643,"identity":"41b7161a-2a58-45f0-9cfd-8265ea7c2306","order_by":5,"name":"Hiroko Taniguchi","email":"","orcid":"","institution":"Keio University School of Medicine","correspondingAuthor":false,"prefix":"","firstName":"Hiroko","middleName":"","lastName":"Taniguchi","suffix":""},{"id":292937644,"identity":"ebaefccf-9279-408d-9f52-a310b55e9d87","order_by":6,"name":"Takahiro Okazaki","email":"","orcid":"","institution":"Keio University School of Medicine","correspondingAuthor":false,"prefix":"","firstName":"Takahiro","middleName":"","lastName":"Okazaki","suffix":""},{"id":292937645,"identity":"0b9514d6-6f49-45fe-9a7f-9bfb9ee9a058","order_by":7,"name":"Shigeto Shimmura","email":"","orcid":"","institution":"Fujita Medical Innovation Center Tokyo, Fujita Health University","correspondingAuthor":false,"prefix":"","firstName":"Shigeto","middleName":"","lastName":"Shimmura","suffix":""},{"id":292937646,"identity":"7633a6e8-251f-49df-b0e9-5afd4b297bf6","order_by":8,"name":"Kazuno Negishi","email":"","orcid":"","institution":"Keio University School of Medicine","correspondingAuthor":false,"prefix":"","firstName":"Kazuno","middleName":"","lastName":"Negishi","suffix":""},{"id":292937647,"identity":"33268b84-631b-4d69-b941-d194baee0195","order_by":9,"name":"Masatoshi Hirayama","email":"","orcid":"","institution":"Keio University School of Medicine","correspondingAuthor":false,"prefix":"","firstName":"Masatoshi","middleName":"","lastName":"Hirayama","suffix":""}],"badges":[],"createdAt":"2024-04-11 12:38:16","currentVersionCode":1,"declarations":"","doi":"10.21203/rs.3.rs-4252479/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-4252479/v1","draftVersion":[],"editorialEvents":[{"content":"https://doi.org/10.1038/s41598-024-84380-6","type":"published","date":"2025-01-02T15:57:16+00:00"}],"editorialNote":"","failedWorkflow":false,"files":[{"id":54977414,"identity":"374cf3c7-4524-45f6-b6b5-67bd8bcb8136","added_by":"auto","created_at":"2024-04-19 13:08:04","extension":"png","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":1962182,"visible":true,"origin":"","legend":"\u003cp\u003eOcular clinical phenotypes of chronic graft-versus-host disease (cGVHD) mice. Scheme of allogeneic and syngeneic bone marrow transplantation and schematic of the experimental treatment protocol for delgocitinib ointment administration (A). Clinical GVHD scores of syngeneic control mice and cGVHD mice treated with delgocitinib ointment or vehicle (n = 5–7 per group) (B). Ocular findings of syngeneic control mice and cGVHD mice treated with vehicle or delgocitinib ointment (C). The vehicle-treated cGVHD mice exhibited blepharitis (C, middle, red arrow heads). Topographical results of meibomian glands (MGs) in syngeneic control mice and cGVHD mice treated with vehicle or delgocitinib ointment (D). MG area % analyzed by topographical results (n = 5–7 per group) (E). Corneal photographs of fluorescein staining (F). The vehicle-treated cGVHD mice exhibited corneal epithelial damages (F, middle, red arrows). Corneal fluorescein staining (CFS) score (n = 5–7 per group) (G). Data are presented as the mean ± standard error of the mean. *\u003cem\u003eP\u003c/em\u003e \u0026lt;.05, **\u003cem\u003eP \u0026lt;\u003c/em\u003e.01, ***\u003cem\u003eP\u003c/em\u003e \u0026lt;.001, ****\u003cem\u003eP\u003c/em\u003e\u0026lt;.0001. One-way analysis of variance with Tukey–Kramer’s post-hoc test was used for the analysis. cGVHD, chronic graft-versus-host disease; BMT, bone marrow transplantation; CFS, corneal fluorescein staining; MG, meibomian gland.\u003c/p\u003e","description":"","filename":"floatimage1.png","url":"https://assets-eu.researchsquare.com/files/rs-4252479/v1/2d05aced55ced2707c03435f.png"},{"id":54976721,"identity":"1bbd539d-7a0f-4265-bdaa-4d62e5de50db","added_by":"auto","created_at":"2024-04-19 13:00:03","extension":"png","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":2213536,"visible":true,"origin":"","legend":"\u003cp\u003ePathological findings and immunohistochemical analyses of the inflammatory cell infiltration in the eyelids of chronic graft-versus-host disease (cGVHD) mice: Mallory staining of eyelids from and cGVHD mice (A). Tissue sections of the eyelid margins from syngeneic control mice and cGVHD mice treated with vehicle or delgocitinib were stained for a leukocyte marker, CD45 (red) (B, upper, C, n = 5–6 per group) and a T cell marker, CD4 (green) (B, lower, D, n = 5-6 per group). Blue areas of fibrosis identified by Mallory staining (E) were measured by ImageJ software in the eyelid and skin (E, n = 5-7 per group) and meibomian glands (E, n = 5–6 per group). Data are presented as the mean ± standard error of the mean. *\u003cem\u003eP\u003c/em\u003e\u0026lt;.05, **\u003cem\u003eP \u0026lt;\u003c/em\u003e.01, ***\u003cem\u003eP\u003c/em\u003e \u0026lt;.001, ****\u003cem\u003eP\u003c/em\u003e \u0026lt;.0001. One-way analysis of variance with Tukey–Kramer’s post-hoc test was used for the analysis. Scale bar, 100 μm.\u003c/p\u003e","description":"","filename":"floatimage2.png","url":"https://assets-eu.researchsquare.com/files/rs-4252479/v1/fd78a121a6c9f214690e178b.png"},{"id":54976720,"identity":"6194ccae-01d2-434b-bd2f-877896e6a0e3","added_by":"auto","created_at":"2024-04-19 13:00:03","extension":"png","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":1608787,"visible":true,"origin":"","legend":"\u003cp\u003ePathological findings and immunohistochemical analyses of the inflammatory cell infiltration and a myofibroblast marker positive area in the skin of chronic graft-versus-host disease (cGVHD) mice: Mallory staining of the back skin from syngeneic control mice and cGVHD mice treated with vehicle or delgocitinib (A). Blue areas of fibrosis identified by Mallory staining were measured by ImageJ software (A, right, n = 5–7 per group). Tissue sections of eyelids from syngeneic control mice and cGVHD mice treated with vehicle or delgocitinib were stained for a pan-leukocyte marker, CD45 (red) (B, n = 5–6 per group); a T cell marker, CD4 (green) (C, n = 5–6 per group); and a myofibroblast marker α-smooth muscle actin, α-SMA (green) (D, n = 5–7 per group). Data are presented as the mean ± standard error of the mean. *\u003cem\u003eP\u003c/em\u003e \u0026lt;.05, **\u003cem\u003eP \u0026lt;\u003c/em\u003e.01, ***\u003cem\u003eP\u003c/em\u003e \u0026lt;.001, ****\u003cem\u003eP\u003c/em\u003e \u0026lt;.0001. One-way analysis of variance with Tukey–Kramer’s post-hoc test was used for the analysis. Scale bar, 100 μm. α-SMA, α-smooth muscle actin.\u003c/p\u003e","description":"","filename":"floatimage3.png","url":"https://assets-eu.researchsquare.com/files/rs-4252479/v1/e2a25dea1abd17fd20266d7e.png"},{"id":54976729,"identity":"952de37d-1c42-4471-86ae-cf6b480c5a93","added_by":"auto","created_at":"2024-04-19 13:00:05","extension":"png","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":3058320,"visible":true,"origin":"","legend":"\u003cp\u003eImmunohistochemical analyses of the signal transducer and activator of transcription (STAT) molecules in the eyelids. Tissue sections of the eyelids from syngeneic control mice and cGVHD mice treated with vehicle or delgocitinib were stained for phosphorylated STAT1 (pSTAT1) (A), pSTAT3 (B), and pSTAT5A (C) (n = 5–7, 5 fields per sample). Data are presented as the mean ± standard error of the mean. *\u003cem\u003eP\u003c/em\u003e\u0026lt;.05, **\u003cem\u003eP \u0026lt;\u003c/em\u003e.01, ***\u003cem\u003eP\u003c/em\u003e \u0026lt;.001, ****\u003cem\u003eP\u003c/em\u003e \u0026lt;.0001. One-way analysis of variance with Tukey–Kramer’s post-hoc test was used for the analysis. Scale bar, 50 μm.\u003c/p\u003e","description":"","filename":"floatimage4.png","url":"https://assets-eu.researchsquare.com/files/rs-4252479/v1/97b848c8c993d952c72dd677.png"},{"id":54976722,"identity":"020ce3ca-62ba-4878-95dc-5bccc0d11e07","added_by":"auto","created_at":"2024-04-19 13:00:04","extension":"png","order_by":5,"title":"Figure 5","display":"","copyAsset":false,"role":"figure","size":2633726,"visible":true,"origin":"","legend":"\u003cp\u003eImmunohistochemical findings of the phosphorylated signal transducer and activator of transcription (pSTAT) molecules in the back skin of chronic graft-versus-host disease (cGVHD) mice. Tissue sections of the skin from syngeneic control mice and cGVHD mice treated with vehicle or delgocitinib -ointment were stained for pSTAT1 (A), pSTAT3 (B), and pSTAT5 (C) (n = 5–6, per group). Data are presented as the mean ± standard error of the mean. *\u003cem\u003eP\u003c/em\u003e\u0026lt;.05, **\u003cem\u003eP \u0026lt;\u003c/em\u003e.01, ***\u003cem\u003eP\u003c/em\u003e \u0026lt;.001, ****\u003cem\u003eP\u003c/em\u003e \u0026lt;.0001. One-way analysis of variance with Tukey–Kramer’s post-hoc test was used for the analysis., Scale bar, 50 μm.\u003c/p\u003e","description":"","filename":"floatimage5.png","url":"https://assets-eu.researchsquare.com/files/rs-4252479/v1/d7b2d0329fab592874fc3cb5.png"},{"id":54977415,"identity":"62206e38-fb82-4ace-8c9d-81b79b6fb4dc","added_by":"auto","created_at":"2024-04-19 13:08:04","extension":"png","order_by":6,"title":"Figure 6","display":"","copyAsset":false,"role":"figure","size":1650869,"visible":true,"origin":"","legend":"\u003cp\u003eImmunohistochemical findings of proinflammatory cytokines and phosphorylated signal transducer and activator of transcriptions (pSTATs)-positive leukocytes in the eyelids of chronic graft-versus-host disease (cGVHD) mice. Tissue sections of the eyelids from syngeneic control mice and cGVHD mice treated with vehicle or delgocitinib ointment were double-stained for pSTAT1 (red) and CD45 (green) (A), pSTAT3 (red) and CD45 (green) (B), and pSTAT5A (red) and CD45 (green) (C) (n = 5–6 per group). Data are presented as the mean ± standard error of the mean. *\u003cem\u003eP\u003c/em\u003e \u0026lt;.05, **\u003cem\u003eP \u0026lt;\u003c/em\u003e.01, ***\u003cem\u003eP\u003c/em\u003e \u0026lt;.001, ****\u003cem\u003eP\u003c/em\u003e \u0026lt;.0001. One-way analysis of variance with Tukey–Kramer’s post-hoc test was used for the analysis. Scale bar, 50 μm. H, hair follicle.\u003c/p\u003e","description":"","filename":"floatimage6.png","url":"https://assets-eu.researchsquare.com/files/rs-4252479/v1/5af0ab1f2bc87105d93da60d.png"},{"id":54976723,"identity":"97132c9f-6521-49d8-b283-36e50f47c4e1","added_by":"auto","created_at":"2024-04-19 13:00:04","extension":"png","order_by":7,"title":"Figure 7","display":"","copyAsset":false,"role":"figure","size":1294787,"visible":true,"origin":"","legend":"\u003cp\u003eImmunohistochemical findings of proinflammatory cytokines and phosphorylated signal transducer and activator of transcriptions (pSTATs)-positive CD4\u003csup\u003e+\u003c/sup\u003e T cells in the eyelids of chronic graft-versus-host disease (cGVHD) mice. Tissue sections of the eyelids from syngeneic control mice and cGVHD mice treated with vehicle or delgocitinib ointment were double-stained for pSTAT3 (red) and CD4 (green) (A), pSTAT5A (red) and CD4 (green) (B) (n = 5–7 per group). Data are presented as the mean ± standard error of the mean. *\u003cem\u003eP\u003c/em\u003e \u0026lt;.05, **\u003cem\u003eP \u0026lt;\u003c/em\u003e.01, ***\u003cem\u003eP\u003c/em\u003e \u0026lt;.001, ****\u003cem\u003eP\u003c/em\u003e \u0026lt;.0001. One-way analysis of variance with Tukey–Kramer’s post-hoc test was used for the analysis. Scale bar, 50 μm. H, hair follicle.\u003c/p\u003e","description":"","filename":"floatimage7.png","url":"https://assets-eu.researchsquare.com/files/rs-4252479/v1/f9c951a9dbc261e5b421ba60.png"},{"id":54976724,"identity":"f4c33a80-491b-4c57-8eb2-4845c2b3b82e","added_by":"auto","created_at":"2024-04-19 13:00:04","extension":"png","order_by":8,"title":"Figure 8","display":"","copyAsset":false,"role":"figure","size":3779734,"visible":true,"origin":"","legend":"\u003cp\u003eImmunohistochemical findings of proinflammatory cytokines in the eyelids of a chronic graft-versus-host disease (cGVHD) mice. Tissue sections of eyelids from syngeneic control mice and cGVHD mice treated with vehicle or delgocitinib were stained for IL-2 (red) (A), IL-6 (green) (B), IFN-γ (green) (C, and IL-1β (green) (D). n = 5-7 per group, Data are presented as the mean ± standard error of the mean. *\u003cem\u003eP\u003c/em\u003e \u0026lt;.05, **\u003cem\u003eP \u0026lt;\u003c/em\u003e.01, ***\u003cem\u003eP\u003c/em\u003e \u0026lt;.001, ****\u003cem\u003eP\u003c/em\u003e \u0026lt;.0001. One-way analysis of variance with Tukey–Kramer’s post-hoc test was used for the analysis, Scale bar, 50 μm. IL, interleukin; IFN, interferon; H, hair follicle.\u003c/p\u003e","description":"","filename":"floatimage8.png","url":"https://assets-eu.researchsquare.com/files/rs-4252479/v1/09ae1dde136170a7421ba272.png"},{"id":54976728,"identity":"e188d55a-a4e1-4e0b-a37b-5d6878a73e45","added_by":"auto","created_at":"2024-04-19 13:00:05","extension":"png","order_by":9,"title":"Figure 9","display":"","copyAsset":false,"role":"figure","size":1431997,"visible":true,"origin":"","legend":"\u003cp\u003eOcular clinical phenotypes of chronic graft-versus-host disease (cGVHD) mice. Scheme of allogeneic and syngeneic bone marrow transplantation (BMT) and schematic of the experimental treatment protocol for 0.5% delgocitinib eye drops administration (A). Clinical GVHD scores of syngeneic control mice and cGVHD mice treated with delgocitinib eye drops or vehicle (n = 5–6 per group) (B). Topographical results of meibomian glands (MGs) in syngeneic control mice and cGVHD mice treated with vehicle or delgocitinib eye drops (C). MG area % analyzed by topographical results (D) (n = 5–6 per group). Corneal photographs of fluorescein staining (E). The vehicle-treated cGVHD mice exhibited corneal epithelial damages (E, middle, red arrows). Corneal fluorescein staining scores (n = 5–7 per group) (E). Data are presented as the mean ± standard error of the mean. *\u003cem\u003eP\u003c/em\u003e \u0026lt;.05, **\u003cem\u003eP \u0026lt;\u003c/em\u003e.01, ***\u003cem\u003eP\u003c/em\u003e \u0026lt;.001, ****\u003cem\u003eP\u003c/em\u003e\u0026lt;.0001. One-way analysis of variance with Tukey–Kramer’s post-hoc test was used for the analysis. cGVHD, chronic graft-versus-host disease; BMT, bone marrow transplantation; CFS, corneal fluorescein staining; MG, meibomian gland.\u003c/p\u003e","description":"","filename":"floatimage9.png","url":"https://assets-eu.researchsquare.com/files/rs-4252479/v1/d5654d37d24b52b101c1e20e.png"},{"id":54976725,"identity":"dc01ee34-9206-49e8-ae5c-357fd54e0ee5","added_by":"auto","created_at":"2024-04-19 13:00:04","extension":"png","order_by":10,"title":"Figure 10","display":"","copyAsset":false,"role":"figure","size":2741605,"visible":true,"origin":"","legend":"\u003cp\u003eWhole-mount immunohistochemistry of the corneas. The corneas from syngeneic control mice and chronic graft-versus-host disease (cGVHD) mice treated with vehicle or delgocitinib were stained for a leukocyte marker, CD45 (A) (n = 5–6 per group) and a macrophage marker, F4/80 (B) (n = 5–7 per group). Data are presented as the mean ± standard error of the mean. *\u003cem\u003eP\u003c/em\u003e \u0026lt;.05, **\u003cem\u003eP \u0026lt;\u003c/em\u003e.01, ***\u003cem\u003eP\u003c/em\u003e \u0026lt;.001, ****\u003cem\u003eP\u003c/em\u003e \u0026lt;.0001. One-way analysis of variance with Tukey–Kramer’s post-hoc test was used for the analysis, Scale bar, 1000 μm.\u003c/p\u003e","description":"","filename":"floatimage10.png","url":"https://assets-eu.researchsquare.com/files/rs-4252479/v1/d4be2c4baf968fd269bb5453.png"},{"id":73093272,"identity":"2553dc00-570a-4aae-9833-746ed76a09eb","added_by":"auto","created_at":"2025-01-06 16:12:33","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":29148490,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-4252479/v1/574a131a-5a6f-4957-8fcd-94aa4f3f2a45.pdf"}],"financialInterests":"No competing interests reported.","formattedTitle":"Anti-inflammatory and anti-fibrotic effects of topical pan-JAK inhibitor in a chronic graft-versus- host disease model mouse","fulltext":[{"header":"Introduction","content":"\u003cp\u003eBased on the onset of the disease, GVHD is classified into acute (aGVHD) and chronic (cGVHD) GVHD, occurring approximately 100 days after transplantation. However, the current classification is based on clinical signs and pathological and histological findings, regardless of the onset period \u003csup\u003e1\u003c/sup\u003e. While the symptoms of aGVHD are characterized by skin conditions, jaundice, and diarrhea, cGVHD affects various organs, such as the nails, skin, digestive tract, liver, lungs, reproductive organs, oral cavity, and eyes \u003csup\u003e2\u003c/sup\u003e. Cutaneous cGVHD can be clinically classified as sclerotic or non-sclerotic. According to the National Institutes of Health Consensus Development Project, the following skin manifestations are diagnostic for chronic GVHD: poikiloderma, lichen planus-like eruptions, lichen sclerosus-like lesions, morphea-like sclerosis, and deep sclerosis/fasciitis \u003csup\u003e3\u003c/sup\u003e. Ocular GVHD affects approximately half of patients with cGVHD and primarily manifests as keratoconjunctivitis sicca \u003csup\u003e4\u003c/sup\u003e. Advanced ocular complications include corneal epithelial defects, corneal ulcers, secondary infections, perforations, stromal scarring, fornix shortening, symblepharon, and loss of vision \u003csup\u003e5\u003c/sup\u003e. Meibomian gland dysfunction (MGD) is also commonly observed in patients with ocular GVHD, characterized by abnormalities in the morphology of meibomian glands (MGs), inflammatory changes in the lid margin, terminal duct obstruction, and/or quantitative and qualitative changes in glandular secretion \u003csup\u003e6,7\u003c/sup\u003e. While fibrotic ocular cGVHD are not directly life-threatening, their widespread involvement leads to considerable functional disability.\u003c/p\u003e \u003cp\u003eThe first-line treatment for cGVHD is steroid therapy; however, if sufficient improvement is not achieved or symptoms recur, secondary therapy becomes necessary. Janus kinases (JAKs) are non-receptor tyrosine kinases comprising four enzymes (JAK1, JAK2, JAK3, and Tyk2) \u003csup\u003e8\u003c/sup\u003e. They activate signal transducer and activator of transcription (STAT) proteins, transduce signals from multiple type I and type II cytokine receptors, and mediate various inflammatory responses \u003csup\u003e9,10\u003c/sup\u003e. STATs are latent cytoplasmic proteins that mediate extracellular signals into the nucleus upon stimulation with ligands, including many hormones, interferons (IFNs), interleukins (ILs), and colony stimulating factors \u003csup\u003e10,11\u003c/sup\u003e. Various JAK-STAT signaling pathway-related cytokines, such as IL-2, IL-6, and IFN-γ, are involved in the pathophysiology of cGVHD \u003csup\u003e12\u003c/sup\u003e. JAK inhibitors block the JAK-STAT signaling pathway, which is involved in the signaling of hematopoietic factors and cytokines \u003csup\u003e13\u003c/sup\u003e. Several small-molecule JAK inhibitors are currently under clinical development for the treatment of autoimmune diseases and chronic dermatitis \u003csup\u003e14,15\u003c/sup\u003e. Systemic administration of the JAK1/2 blocker, ruxolunitib, is effective for refractory aGVHD and cGVHD \u003csup\u003e16,17\u003c/sup\u003ebut has side effects like thrombocytopenia and/or anemia. Topical administration of drugs, especially for the skin and eyes, may be a good alternative to avoid these side effects. Delgocitinib (JTE-052) is a novel JAK inhibitor that has been shown to inhibit JAK1, JAK2, JAK3, and Tyk2 \u003csup\u003e18\u003c/sup\u003e. Topical administration of delgocitinib has been effective in ameliorating the severity of chronic dermatitis in rodent disease models \u003csup\u003e19\u003c/sup\u003e. In this study, we aimed to demonstrate the efficacy of topical administration of the pan-JAK inhibitor, delgocitinib, in a cGVHD mouse model using eye drops and ointments.\u003c/p\u003e"},{"header":"Results","content":"\u003cp\u003e \u003cb\u003eAdministration of 0.5% delgocitinib ointment preserves the morphology of MGs and attenuates the severity of the corneal epithelial damage\u003c/b\u003e \u003c/p\u003e \u003cp\u003eTo assess the efficacy of topical administration of a pan-JAK inhibitor for ocular GVHD, we applied delgocitinib ointment to the eyelids of an established sclerodermatous cGVHD mouse model \u003csup\u003e20,21\u003c/sup\u003e (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003ea). Compared to the syngeneic control group, systemic GVHD scores were significantly increased in the vehicle-treated cGVHD mice, but there was no significant difference between the vehicle-treated and delgocitinib ointment-treated cGVHD mice (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eb). In contrast to vehicle-treated cGVHD mice, delgocitinib ointment-treated cGVHD mice and syngeneic controls exhibited no blepharitis (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003ec). The topographical results showed that the MG area in cGVHD mice was significantly decreased than that in syngeneic control mice, and delgocitinib ointment-treated cGVHD mice retained a significantly larger MG area (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003ed, e). The corneal fluorescein staining (CFS) score was significantly more severe in vehicle-treated cGVHD mice than in syngeneic control and delgocitinib ointment-treated cGVHD mice (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003ef, g). In summary, these results indicate that delgocitinib ointment attenuated the severity of ocular GVHD.\u003c/p\u003e \u003cp\u003e \u003cb\u003eAdministration of 0.5% delgocitinib ointment alleviates inflammatory cell infiltration and fibrosis in the eyelids of cGVHD mice\u003c/b\u003e \u003c/p\u003e \u003cp\u003eWe assessed the eyelids by dividing them into three segments: the MGs, eyelid margin, and eyelid skin (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003ea). To examine inflammatory cell infiltration, we performed immunohistochemistry for the pan-leukocyte marker cluster of differentiation (CD) 45 and the T cell marker CD4. Increased number of CD45\u003csup\u003e+\u003c/sup\u003e inflammatory cells and CD4\u003csup\u003e+\u003c/sup\u003e T cells infiltrated the eyelid margins of vehicle-treated cGVHD mice than those of syngeneic and delgocitinib ointment-treated mice (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eb-d). The fibrotic areas in the eyelid skin and around the MG acini of cGVHD mice were analyzed using Mallory staining. The fibrotic area per field in vehicle-treated cGVHD mice was significantly larger than that in syngeneic control mice, and pathological fibrosis was significantly alleviated in delgocitinib ointment-treated mice (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003ee-g). These results suggested that delgocitinib ointment alleviated inflammatory cell infiltration and fibrosis in the eyelids of cGVHD mice.\u003c/p\u003e \u003cp\u003e \u003cb\u003eAdministration of 0.5% delgocitinib ointment alleviates inflammatory cell infiltration and fibrosis in the skin of cGVHD mice\u003c/b\u003e \u003c/p\u003e \u003cp\u003eThe blue fibrotic area in Mallory staining of the skin of vehicle-treated cGVHD mice was significantly larger than that in syngeneic control mice, and this pathological fibrosis was significantly alleviated in delgocitinib-treated mice (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003ea). Increased number of CD45\u003csup\u003e+\u003c/sup\u003e inflammatory cells and CD4\u003csup\u003e+\u003c/sup\u003e T cells infiltrated the skin of vehicle-treated cGVHD mice than syngeneic mice and delgocitinib-treated mice (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003ec-d). Myofibroblast marker α-smooth muscle actin (α-SMA)\u003csup\u003e+\u003c/sup\u003e area per fields in the skin of vehicle-treated cGVHD mice was significantly larger than that of syngeneic control mice and delgocitinib-treated mice (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003e. d). These results suggested that delgocitinib ointment alleviated inflammatory cell infiltration and fibrosis in the skin of cGVHD mice.\u003c/p\u003e \u003cdiv id=\"Sec3\" class=\"Section2\"\u003e \u003ch2\u003e0.5% delgocitinib ointment attenuates the expression of phosphorylated STATs\u003c/h2\u003e \u003cp\u003eThe eyelids were assessed by dividing them into three segments: the MGs, eyelid margin, and eyelid skin. Increased number of pSTAT1 positive cells were observed in the interstitial space of these three segments of the eyelids in vehicle-treated cGVHD mice than in the syngeneic controls and delgocitinib-treated cGVHD mice (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003ea). In addition to cells in the interstitial space, pSTAT3 positive cells in vehicle-treated cGVHD mice included epithelial cells of the eyelid skin, eyelid margin, and MG acini (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eb). pSTAT3 positive area was significantly increased in these three segments of the eyelids in vehicle-treated cGVHD mice compared with syngeneic controls and delgocitinib ointment-treated cGVHD mice (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eb). Similarly, pSTAT5A positive area were detected in both epithelial and interstitial cells and were significantly larger in the three segments of the eyelids in vehicle-treated cGVHD mice than those in the syngeneic controls and delgocitinib ointment-treated cGVHD mice (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003ec). Subsequently, we focused on the expression of activated STAT1, STAT3, and STAT5A in the skin of the cGVHD mice. Similar to the eyelids, the expression of phosphorylated STAT1, STAT3, and STAT5A was significantly upregulated in the vehicle-treated cGVHD mice than that of the syngeneic controls, with delgocitinib-treatment significantly suppressing the expression of these molecules (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003ea-c).\u003c/p\u003e \u003cp\u003e \u003cb\u003eInfiltration of JAK/STAT signaling pathway activating leukocytes are attenuated in the eyelids of delgocitinib-treated cGVHD mice\u003c/b\u003e \u003c/p\u003e \u003cp\u003eThe JAK/STAT signaling pathway is activated in various types of leukocytes associated with T cells and macrophages \u003csup\u003e\u003cb\u003e8,22,23\u003c/b\u003e\u003c/sup\u003e. To elucidate whether the JAK/STAT signaling pathway is promoted in leukocytes infiltrating the eyelids of cGVHD mice, we double-stained for the pan-leukocyte marker CD45 or the T cell marker CD4 and pSTAT1, pSTAT3, or pSTAT5A. Compared with the syngeneic controls, both CD45\u003csup\u003e+\u003c/sup\u003epSTAT1\u003csup\u003e+\u003c/sup\u003e, CD45\u003csup\u003e+\u003c/sup\u003epSTAT3\u003csup\u003e+\u003c/sup\u003e and CD45\u003csup\u003e+\u003c/sup\u003epSTAT5A\u003csup\u003e+\u003c/sup\u003e cells infiltrated significantly more in the eyelids of vehicle-treated cGVHD mice (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003ea-c). The numbers of CD45\u003csup\u003e+\u003c/sup\u003epSTAT1\u003csup\u003e+\u003c/sup\u003e, CD45\u003csup\u003e+\u003c/sup\u003epSTAT3\u003csup\u003e+\u003c/sup\u003e and CD45\u003csup\u003e+\u003c/sup\u003epSTAT5A\u003csup\u003e+\u003c/sup\u003e cells in the eyelids of cGVHD mice treated with delgocitinib ointment were significantly lower than those in vehicle-treated cGVHD mice. Furthermore, the numbers of CD4\u003csup\u003e+\u003c/sup\u003epSTAT3\u003csup\u003e+\u003c/sup\u003e and CD4\u003csup\u003e+\u003c/sup\u003epSTAT5A\u003csup\u003e+\u003c/sup\u003e in the eyelids of vehicle-treated cGVHD mice were significantly higher than those in syngeneic control and delgocitinib ointment-treated cGVHD mice (Fig.\u0026nbsp;\u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e7\u003c/span\u003ea-b). These findings showed that the infiltration of JAK/STAT signaling pathway activating leukocytes was promoted in the eyelids of cGVHD mice and the pan-JAK inhibitor delgocitinib attenuated this pathological finding.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec4\" class=\"Section2\"\u003e \u003ch2\u003eExpressions of pro-inflammatory cytokines attenuated in the eyelids of delgocitinib-treated cGVHD mice\u003c/h2\u003e \u003cp\u003eWe focused on the proinflammatory cytokines IL-2, IL-6, and IFN-γ, which activate the JAK/STAT signaling pathway to transduce intracellular signals and contribute to cGVHD pathogenesis \u003csup\u003e24 25\u003c/sup\u003e. The expression of IL-2, IL-6 and IFN-γ were significantly elevated in the eyelids of cGVHD mice treated with vehicle than those of syngeneic control mice, with delgocitinib significantly attenuating the expression of these molecules (Fig.\u0026nbsp;\u003cspan refid=\"Fig8\" class=\"InternalRef\"\u003e8\u003c/span\u003ea-c). Additionally, we focused on IL-1β, which does not activate JAK/STAT signaling pathway directly but enhances the effects of JAK/STAT-dependent proinflammatory cytokines by repressing the suppressor of cytokine signaling 3 (SOCS3) \u003csup\u003e26\u003c/sup\u003e. The expression of IL-1β was significantly elevated in the eyelids of vehicle-treated cGVHD mice than those of syngeneic control and delgocitinib-treated cGVHD mice (Fig.\u0026nbsp;\u003cspan refid=\"Fig8\" class=\"InternalRef\"\u003e8\u003c/span\u003ed).\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec5\" class=\"Section2\"\u003e \u003ch2\u003eExpressions of pro-inflammatory cytokines attenuated in the eyelids of delgocitinib-treated cGVHD mice\u003c/h2\u003e \u003cp\u003eWe hypothesized that delgocitinib eye drops would suppress inflammation on the ocular surface of cGVHD mice. After 28 days of BMT, there was no significant difference in the systemic GVHD clinical score between the vehicle-treated cGVHD mice and the delgocitinib eye-drop-treated cGVHD mice (Fig.\u0026nbsp;\u003cspan refid=\"Fig9\" class=\"InternalRef\"\u003e9\u003c/span\u003ea-b). The topographical results showed that the MG area in cGVHD mice was significantly decreased than that in syngeneic control mice, and delgocitinib eye drop-treated cGVHD mice retained a significantly larger MG area (Fig.\u0026nbsp;\u003cspan refid=\"Fig9\" class=\"InternalRef\"\u003e9\u003c/span\u003ec-d). Furthermore, the CFS score in the vehicle-treated cGVHD mice was significantly higher than that in the syngeneic controls and delgocitinib eye drop-treated cGVHD mice (Fig.\u0026nbsp;\u003cspan refid=\"Fig9\" class=\"InternalRef\"\u003e9\u003c/span\u003ee,f). In summary, these results indicated that delgocitinib eye drops attenuated the severity of ocular GVHD.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec6\" class=\"Section2\"\u003e \u003ch2\u003eInflammatory cell infiltration is attenuated in the corneas of delgocitinib eye drops-treated cGVHD mice\u003c/h2\u003e \u003cp\u003eWe analyzed the dynamics of inflammatory cell infiltration in the cornea using whole-mount immunostaining. Compared with syngeneic control mice, significantly increased number of CD45\u003csup\u003e+\u003c/sup\u003e leukocytes infiltrated the corneas of vehicle-treated cGVHD mice. In the corneas of 0.5% delgocitinib eye drop-treated cGVHD mice, the infiltration of CD45\u003csup\u003e+\u003c/sup\u003e cells was significantly attenuated compared with that in vehicle-treated cGVHD mice (Fig.\u0026nbsp;\u003cspan refid=\"Fig10\" class=\"InternalRef\"\u003e10\u003c/span\u003ea). Subsequently, we analyzed the dynamics of the macrophages that reside in the homeostatic cornea and infiltrate the corneas from the blood vessels of the limbus and tears when inflammation occurs \u003csup\u003e34,27,28\u003c/sup\u003e. F4/80 positive macrophages infiltrated significantly in the corneas of the vehicle-treated cGVHD mice than in the syngeneic control and the cGVHD mice treated with delgocitinib. Thus, treatment with delgocitinib significantly attenuated macrophage infiltration (Fig.\u0026nbsp;\u003cspan refid=\"Fig10\" class=\"InternalRef\"\u003e10\u003c/span\u003eb).\u003c/p\u003e \u003c/div\u003e"},{"header":"Discussion","content":"\u003cp\u003eIn this study, we demonstrated that the JAK/STAT signaling pathway was upregulated in the eyelids and skin of a cGVHD mouse model. Moreover, we found that administering the pan-JAK inhibitor, delgocitinib, locally suppressed the severity of cGVHD in the eyes and skin. Our results also showed that delgocitinib ointment had beneficial effects on inflammation and sclerotic changes in the eyelids, as well as on MGD and corneal epithelial damage. We previously reported that this sclerotic cGVHD mouse model mimics MGD and blepharitis with the depletion of MG acini, pathological fibrosis, and infiltration of inflammatory cells, as observed in patients with cGVHD\u003csup\u003e29,30\u003c/sup\u003e. MGD causes abnormalities in the lipid components of tear fluid, leading to evaporative dry eye, and approximately half of the patients with ocular GVHD develop MGD \u003csup\u003e6,7\u003c/sup\u003e. Current treatments for MGD include antimicrobial eye drops, topical corticosteroid eye drops, eyelid hygiene, and warming \u003csup\u003e31\u003c/sup\u003e. However, none of these treatments align with the pathophysiological mechanisms of blepharitis and MGD in ocular GVHD, where significant infiltration of inflammatory cells and fibrosis occur in the eyelids. Local administration of JAK inhibitors may be a promising treatment option for eyelid inflammation and MGD associated with GVHD.\u003c/p\u003e \u003cp\u003eIn this study, the expression of phosphorylated STAT1, STAT3, and STAT5A was significantly elevated in the eyelids and skin of cGVHD mice and attenuated in delgocitinib-treated cGVHD mice (Figs.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003e and \u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003e). As the proinflammatory cytokines are related to JAK/STAT signaling pathway, we also focused on the expression of IL-2, IL-6 and IFN-γ and observed that expression of these cytokines was higher in the eyelids of cGVHD mice, suggesting their role in cGVHD pathogenesis.\u003c/p\u003e \u003cp\u003eIL-2 in tear is elevated in patients with systemic cGVHD than in patients without systemic cGVHD \u003csup\u003e32\u003c/sup\u003e and its level correlates with the severity of ocular cGVHD \u003csup\u003e33\u003c/sup\u003e. Although IL-2 has been shown to activate several STAT family members, including STAT1, STAT3, and STAT5A, STAT5A is the predominant IL-2 signaling molecule \u003csup\u003e34\u0026ndash;36\u003c/sup\u003e. IL-2 signaling through the phosphorylation of STAT5A increases the proliferation of T cells \u003csup\u003e29\u003c/sup\u003e and delgocitinib (JTE-052) inhibits the IL-2-induced proliferation of T cells, as observed in \u003cem\u003ein vitro\u003c/em\u003e experiments \u003csup\u003e37\u003c/sup\u003e. In this study, topical administration of the pan-JAK inhibitor, delgocitinib, significantly attenuated the expression of phosphorylated STAT5A (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003ec), and the number of CD4 positive cells significantly decreased in the eyelid margins and skin of delgocitinib-treated cGVHD mice (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eb). Additionally, IL-2 signaling through STAT5 promotes the proliferation and differentiation to effector T cells, such as Th1 cells, which produce IFN-γ \u003csup\u003e38\u0026ndash;40\u003c/sup\u003e. Indeed, our data confirmed the significant attenuation of CD4\u003csup\u003e+\u003c/sup\u003eSTAT5A\u003csup\u003e+\u003c/sup\u003e cell infiltration and IFN-γ expression in the eyelids of delgocitinib-treated cGVHD mice (Fig.\u0026nbsp;\u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e7\u003c/span\u003eb and Fig.\u0026nbsp;\u003cspan refid=\"Fig8\" class=\"InternalRef\"\u003e8\u003c/span\u003ec).\u003c/p\u003e \u003cp\u003ePreviously, we reported that IL-6 expression is upregulated in the targeted organs of the mouse model, including the lacrimal glands, liver, and lungs\u003csup\u003e41\u003c/sup\u003e. STAT3 plays a central role in transmitting IL-6 intracellular signals from the membrane to the nucleus \u003csup\u003e42\u003c/sup\u003e. In our study, IL-6 expression was significantly elevated, and phosphorylated STAT3 was observed in the eyelids of cGVHD mice (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eb and Fig.\u0026nbsp;\u003cspan refid=\"Fig8\" class=\"InternalRef\"\u003e8\u003c/span\u003eb). Dendritic cells (DCs) and macrophages are the principal sources of IL-6 dysregulation after hematopoietic stem cell transplantation (HSCT), and IL-6-dependent GVHD pathogenesis is driven by IL-6R in donor T cells \u003csup\u003e43,44\u003c/sup\u003e. Some pSTAT3\u003csup\u003e+\u003c/sup\u003eCD4\u003csup\u003e+\u003c/sup\u003e cells that infiltrate the eyelids of cGVHD mice (Fig.\u0026nbsp;\u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e7\u003c/span\u003ea) may be T cells activated by IL-6 secreted from DCs and macrophages. Elevated pSTAT3 is also observed in the ocular surface epithelium of a dry-eye mouse model induced by benzalkonium chloride, and STAT3 inhibition has therapeutic effects in dry eye \u003csup\u003e13\u003c/sup\u003e. Suppressing IL-6 signaling may have the potential to treat various ocular surface diseases.\u003c/p\u003e \u003cp\u003eIFN-γ, a soluble cytokine secreted by immune and mucosal epithelial cells, plays a role in the development of cGVHD. It binds to the interferon-γ receptor (IFNγR)1/2 to activate JAK1/2 and STAT proteins, primarily STAT1 \u003csup\u003e45\u003c/sup\u003e. The expression of IFN-γ and pSTAT1 was significantly upregulated in the eyelids and skin of cGVHD mice (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003ea and Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003ea). The previous report shows that IFN-γ secreted from CD8 positive T cells act on keratinocytes, which produce transforming growth factor-β and promote the development of sclerodermatous changes observed in cGVHD \u003csup\u003e46\u003c/sup\u003e. Inhibiting IFN-γ and its intracellular signaling may hold the potential for effectively suppressing pathological fibrosis.\u003c/p\u003e \u003cp\u003eSubsequently, we evaluated the expression of IL-1β. The intracellular signaling of IL-1β through IL-1R does not directly involve the JAK/STAT pathway; however, IL-1β enhances the effect of phosphorylated STAT3 by repressing SOCS3, known as a negative regulator of JAK/STAT signaling pathway, thereby ultimately leading to the promotion of Th17 cell differentiation and enhancement of the effects of STAT3-dependent proinflammatory cytokines such as IL-6 \u003csup\u003e\u003cb\u003e26\u003c/b\u003e\u003c/sup\u003e. The elevated expression of IL-1β in the eyelids of cGVHD mice (Fig.\u0026nbsp;\u003cspan refid=\"Fig8\" class=\"InternalRef\"\u003e8\u003c/span\u003ed) might enhance the effects of pSTAT3 by repressing SOCS3.\u003c/p\u003e \u003cp\u003eIn corneal whole-mount immunostaining using CD45, with dendritic-shaped immune cells, globular-shaped immune cells significantly infiltrated the corneas of vehicle-treated cGVHD mice than that of syngeneic control and delgocitinib-treated cGVHD mice (Fig.\u0026nbsp;\u003cspan refid=\"Fig10\" class=\"InternalRef\"\u003e10\u003c/span\u003ea). According to the observation using in vivo confocal microscopy, globular-shaped cells are rarely observed in the corneas of syngeneic control and wild-type mice \u003csup\u003e\u003cb\u003e47\u003c/b\u003e\u003c/sup\u003e. Furthermore, single-cell analysis using the corneas of wild-type mice revealed that myeloid cells comprise over 90% of the immune cells in the central part of the cornea of wild-type mice, with the population of lymphocytes being almost absent \u003csup\u003e\u003cb\u003e48,49\u003c/b\u003e\u003c/sup\u003e. Therefore, the globular-shaped immune cells observed in our corneal whole-mount immunostaining may potentially include lymphocytes, such as T cells. In future, we intend to analyze the expression of markers specific to T cells and their subtypes. In the early post-HSCT phase, cytotoxic therapy, irradiation, and aGVHD trigger soluble inflammatory mediators, which lead to increased antigen presentation in myeloid cells, including macrophages \u003csup\u003e12\u003c/sup\u003e. The corneal whole-mount immunostaining analysis demonstrated a significantly higher number of F4/80-positive macrophages in cGVHD model mice than in the syngeneic control and delgocitinib-treated cGVHD mice (Fig.\u0026nbsp;\u003cspan refid=\"Fig10\" class=\"InternalRef\"\u003e10\u003c/span\u003eb). Oral administration of the JAK1/2 inhibitor, ruxilitinib, to cGVHD model mice significantly suppresses CD11b positive macrophages/mononuclear cells infiltrating in the skin by suppressing IFN-γ secretion from T cells and subsequent monocyte chemotactic protein 1 secretion from macrophages \u003csup\u003e\u003cb\u003e50\u003c/b\u003e\u003c/sup\u003e. These findings suggested that delgocitinib eye drops have a suppressive effect on the infiltration of inflammatory cells in the corneas of cGVHD mice.\u003c/p\u003e \u003cp\u003eThis study has some limitations. Local administration of delgocitinib reduces the severity of chronic skin and ocular GVHD. However, since the drug was administered before the onset of chronic skin and ocular cGVHD in this study, it is unclear if it has a preventive or therapeutic effect. The precise mechanism by which topical delgocitinib suppressed the inflammatory response remains unclear. While STAT1, STAT3, and STAT5A are activated in diverse cell types, including epithelial and infiltrating inflammatory cells, the impact of cell type-specific activation of this pathway on cGVHD remains to be established. In future studies, we aim to explore the underlying mechanism linking cGVHD and the JAK/STAT signaling pathway by conducting experiments in a cell type-specific manner.\u003c/p\u003e \u003cp\u003eIn summary, the JAK/STAT signaling pathway is activated in the eyelids and skin of cGVHD mice, and topical administration of the pan-JAK inhibitor, delgocitinib, significantly alleviated the severity of chronic skin and ocular GVHD phenotypes. These findings highlight the potential of targeting the JAK/STAT signaling pathway as an effective approach to prevent chronic skin and ocular GVHD development.\u003c/p\u003e"},{"header":"Methods","content":"\u003cp\u003e\u003cstrong\u003eMice\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eB10.D2/nSnSlc and BALB/cCrSlc mice (7\u0026ndash;9 weeks old) were purchased from Sankyo Laboratory Inc. (Tokyo, Japan). All experimental procedures were conducted in accordance with the Association for Research in Vision and Ophthalmology Statement and the Institutional Guidelines on Animal Experimentation at Keio University. All animal and genetically modified mouse experimental protocols were approved by the Keio University Institutional Animal Care and Use Committee (# A2022-178).\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eWhole Bone Marrow Transplantation\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eTo create the cGVHD mouse model, allogeneic BMT was performed using 7\u0026ndash;9-week-old male B10.D2/nSnSlc and female BALB/cCrSlc mice as transplant donors and recipients, respectively, as previously reported, representing major histocompatibility complex-compatible, miHA-mismatched BMT\u0026nbsp;\u003csup\u003e21,51\u003c/sup\u003e. As a non-cGVHD control, syngeneic BMT was conducted by transplanting donor cells from male BALB/cCrSlc mice into female BALB/cCrSlc mice, which were irradiated with 7 Gy X-rays using a Gammacell 137 Cs source (Hitachi Medico Ltd., Tokyo, Japan). Donor cells (1 \u0026times; 10\u003csup\u003e6\u003c/sup\u003e bone marrow cells/mouse and 2 \u0026times; 10\u003csup\u003e6\u003c/sup\u003e spleen cells/mouse) were suspended separately in 100 \u0026mu;L of RPMI1640 medium, and then, combined. This 200-\u0026mu;L suspension was injected into the recipient via the tail vein. The recipient animals were maintained in sterile cages and provided with autoclaved food and acidified water. Mice were monitored for characteristic signs of GVHD by clinical assessment using a standard scoring system, as described previously\u0026nbsp;\u003csup\u003e52\u003c/sup\u003e. This system assessed seven systemic clinical traits: activity, posture, fur texture, weight loss, skin integrity, presence of diarrhea, and degree of alopecia. Each clinical parameter was awarded a score from 0\u0026ndash;2, with a total available score of 0\u0026ndash;14. The cGVHD and syngeneic control mice were used for experiments 28 days after BMT.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eTreatment of allogeneic BMT recipient mice with delgocitinib\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe JAK inhibitor, delgocitinib (JTE-052), was synthesized by Japan Tobacco (JT) (Osaka, Japan).\u0026nbsp;Delgocitinib inhibits JAK1, JAK2, JAK3 and Tyk2 with IC50 values of 2.8, 2.6, 13, and 58\u0026nbsp;nM, respectively\u0026nbsp;\u003csup\u003e18\u003c/sup\u003e.\u0026nbsp;JT provided 0.5% delgocitinib\u0026nbsp;ointment and solvent vehicle ointments.\u0026nbsp;To prepare 0.5% delgocitinib eye drops, delgocitinib was mixed with 0.9% sodium chloride solution and adjusted with hydrochloric acid to achieve a pH of 5.0\u0026ndash;6.0. A 0.9% sodium chloride solution with pH of 5.0\u0026ndash;6.0 was used as vehicle-eye drops. To assess the efficacy of topical administration of a pan-JAK inhibitor for ocular GVHD, allogeneic BMT recipients were treated with either 0.5% delgocitinib ointment or solvent vehicle ointment applied to the eyelids twice a day for 21 consecutive days, starting from 7 to 28 days after BMT\u0026nbsp;(Fig. 1a). Syngeneic mice were used as the controls. Another group of allogeneic BMT recipients were treated with either 0.5% delgocitinib ointment or solvent vehicle ointment applied to the skin of the back twice a day for 21 consecutive days, starting from 7 to 28 days after BMT. To observe the effects of delgocitinib eye drops, allogeneic BMT recipients\u0026nbsp;were treated with either 0.5% delgocitinib eye drops, or the solvent vehicle eye drops twice daily for 21 consecutive days, starting from 7 to 28 days after BMT.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eEyelid imaging\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eAfter 28 days of BMT, the eyelids were collected from the right eye of each mouse. The MG area was analyzed using a topographical method, as previously reported \u003csup\u003e29,30,53\u003c/sup\u003e. After harvesting, photographs of the upper eyelid were taken immediately under a microscope\u0026nbsp;(SZ61, Olympus, Japan) with white light. The percentage of the MG area was analyzed using ImageJ software. The central 3 mm eyelid area, including the eyelid margin and\u0026nbsp;the furthest endpoint of\u0026nbsp;the\u0026nbsp;MGs, was measured (area A) using the freehand tool. The\u0026nbsp;depletion area of the MG\u0026nbsp;was semiautomatically selected and measured (area B) using the threshold tool. The percentage of MG\u0026nbsp;area was calculated using the following formula: MG Area% = (area A-B)/area A \u0026times; 100%.\u003c/p\u003e\n\u003cp\u003eHistological analysis\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eThe mice were euthanized by cervical dislocation 28 days after BMT. The eyeballs with eyelids were harvested and fixed with 10% neutralized buffered formalin for 12 hours at room temperature, embedded in paraffin wax, and processed for\u0026nbsp;hematoxylin and eosin\u0026nbsp;(H\u0026amp;E) and Mallory staining\u0026nbsp;\u003csup\u003e20,54\u003c/sup\u003e. To quantify the fibrotic areas\u0026nbsp;in the eyelids in the tissue sections, images were acquired at 400 \u0026times; magnification, and three images of the Mallory-stained sections were examined. These images were analyzed using Color Deconvolution, a plugin for ImageJ\u0026nbsp;(NIH Image, Bethesda, MD, USA), in which the colors of each image were separated into blue, green, and red. The blue area was measured using the same threshold.\u0026nbsp;The average value obtained from the three images of each sample was considered the value of the fibrotic area\u0026nbsp;\u003csup\u003e41\u003c/sup\u003e.\u003c/p\u003e\n\u003cp\u003eImmunohistochemistry\u003c/p\u003e\n\u003cp\u003eImmunohistochemical analyses were performed on formalin-fixed and paraffin-embedded sections as described previously\u0026nbsp;\u003csup\u003e55\u003c/sup\u003e. Following deparaffinization and dehydration, the target antigens were unmasked using the microwave method at 100℃\u0026nbsp;for 10 min. This process was applied to antibodies against interleukin (IL)-1\u0026beta; (AF-401-NA, polyclonal, R\u0026amp;D Systems, Minneapolis, MN, USA), IL-6 (ab6672, Abcam, Cambridge, United Kingdom), Allophycocyanin\u0026nbsp;(APC)-labeled CD45 (103112, 30-F11, Biolegend, San Diego, CA, USA), or through autoclave techniques at 120\u0026deg;C for 20 min for antibodies, \u0026alpha;-smooth muscle actin (\u0026alpha;-SMA) (ab7817, 1A4, Abcam). Antigens were processed in a target retrieval solution (S169984; Agilent Technologies, Santa Clara, CA, USA). Subsequently, the sections were blocked with 10% normal goat serum (012-000-120; Jackson ImmunoResearch Laboratories, West Grove, PA, USA)\u0026nbsp;for 30 min at room temperature and then incubated overnight at 4\u0026deg;C with appropriately diluted primary antibodies. This was followed by 45-min incubation at room temperature with\u0026nbsp;4\u0026apos;,6-diamidino-2-phenylindole, dihydrochloride\u0026nbsp;(DAPI\u0026nbsp;)\u0026nbsp;and secondary antibodies, including (1) Alexa Fluor 488-conjugated goat anti-rabbit IgG secondary antibody (A11034, Thermo Fisher Scientific, Waltham, MA, USA ) for IL-6,pSTAT1, pSTAT3, and pSTAT5A; (2) Alexa Fluor 488-conjugated goat anti-mouse IgG secondary antibody (A11029, Thermo Fisher Scientific) for \u0026alpha;-SMA; and (3) Alexa Fluor 488-conjugated rabbit anti-goat IgG secondary antibody (A11078, Thermo Fisher Scientific) for IL-1\u0026beta;. To demonstrate activation of the JAK/STAT signaling pathway in the skin and eyelids of cGVHD mice, we performed immunobiological analysis with the sections treated with pSTAT1, pSTAT3, and pSTAT5A antibodies were incubated with Horseradish peroxidase (HRP)-coupled antibodies, followed by\u0026nbsp;diaminobenzidine development and H\u0026amp;E staining using\u0026nbsp;the\u0026nbsp;Dako Real Envision Detection system (K5007, Agilent Technologies).\u0026nbsp;Isotype-matched antibodies used as negative controls included, (1) rabbit IgG antibody (2729, Cell Signaling Technology,Danver, MA, USA) for IL-6, pSTAT1, pSTAT3, and pSTAT5A; (2) mouse IgG2a, \u0026kappa; antibody (ab18415, abcam) for \u0026alpha;-SMA; and (3) Goat IgG antibody (02-6202, Thermo Fisher Scientific) for IL-1\u0026beta;. Subsequently, we captured 1\u0026ndash;3 images of each eyelid margin, MG, and eyelid skin area from each stained section, using a digital slide scanner (Nanozoomer S60, Hamamatsu, Japan) and THUNDER Imager 3D Cell Culture (Leica, Wetzlar, Germany). The positive areas\u0026nbsp;and cells in each field were then quantified manually or automatically using ImageJ.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eCorneal whole-mount staining\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eWhole-mount corneal staining was performed. Eyeballs were fixed in a solution consisting of 1% formaldehyde (133-10311, Fuji Film, Tokyo, Japan), 2mM MgCl2 (95812-85, Nakarai, Kyoto, Japan), 5mM Ethylene Glycol Bis (2-aminoethyl ether)-\u003cem\u003eN\u003c/em\u003e,\u003cem\u003eN\u003c/em\u003e,\u003cem\u003eN\u003c/em\u003e\u0026apos;,\u003cem\u003eN\u003c/em\u003e\u0026apos;-tetraacetic \u003cstrong\u003ea\u003c/strong\u003ecid (37346-05, Nakarai), and 0.02% Nonidet p40 (NP 40)\u0026nbsp;(25223-04, Nakarai) in\u0026nbsp;phosphate-buffered saline\u0026nbsp;(PBS) at 4℃\u0026nbsp;for 75 min. After washing by 0.02% NP40 in PBS at 23℃\u0026nbsp;for 15 min,\u0026nbsp;eyeballs were fixed in 80% methanol diluted with Dimethyl sulfoxide (DMSO) (13407-45, Nakarai) at -20℃ for 2 h. After fixation, the cornea was removed from the eyeballs using forceps and\u0026nbsp;scissors. Corneas were permeabilized by incubation in 25% Triton X-100 (35501-02, Nakarai) diluted with methanol for 15 min, 50% Triton X-100 for 15 min, and 75% Triton X-100 for 10 min. After washing with PBS three times at 23℃ for 30 min, corneas were incubated in the blocking solution consisting of 10% normal goat serum (50062Z, Thermo Fisher Scientific) and 0.1% Triton X-100 at 23℃ for two hours. Corneas were incubated in APC-labeled CD45 (103112, 30-F11, Biolegend) and PE-labeled F4/80 (157303, QA17A29, BioLegend) antibodies diluted in PBS (1:200) at 4℃ for 12 h. After washing five time with by 0.02% tween 20 in PBS at 23℃ five times for an hour, corneas were incubated in DAPI (62247, Thermo Fisher Scientific) diluted with PBS for 5 min. After washing three times with PBS at 23℃, corneas were mounted using Fluorescence Mounting Medium (S302380-2, Agilent Technologies) on slide glass (MAS-01, Matsunami, Osaka, Japan).\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eCorneal fluorescein staining \u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eAfter 4 weeks (28 days) post-BMT, the recipient mice were evaluated for corneal epithelitis. One microliter of 0.5% fluorescein sodium solution (Fluorescite, 877290; Novartis Pharma, Tokyo, Japan) was instilled into the temporal conjunctival sac without anesthesia. The ocular surface was observed under cobalt blue light using a Smart Eye Camera (13B2X10198030101; OUI Inc., Tokyo, Japan)\u0026nbsp;\u003csup\u003e41,56\u003c/sup\u003e. CFS score was used to assess corneal epithelial damage and\u0026nbsp;was calculated using a 4-point scale: 0, absent; 1, slightly punctate staining with \u0026lt; 30 spots; 2, punctate staining with \u0026gt; 30 spots but does not diffuse; 3, severe diffuse staining but no positive plaques; and 4, positive fluorescein plaques\u0026nbsp;\u003csup\u003e57\u003c/sup\u003e. The scores for the three segments including upper, middle, and lower parts of the cornea were summed to produce a final grade (0 to 12 points). The average of the total score for the left and right eyes was used as the CFS score for that individual.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eStatistical analysis \u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eStatistical analyses were performed using the GraphPad Prism software (GraphPad Software, USA). One-way analysis of variance with the Tukey\u0026ndash;Kramer post-hoc test was used for statistical analyses. Statistical significance was set at \u003cem\u003eP\u003c/em\u003e \u0026lt; 0.05.\u003c/p\u003e"},{"header":"Declarations","content":"\u003cp\u003e\u003cstrong\u003eAcknowledgments\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThis study was supported by grants from the Japanese Ministry of Education, Science, Sports, Culture, and Technology (22K16982) awarded to S.\u0026nbsp;Sato. The authors thank Erina Igarashi at\u0026nbsp;the Keio University School of Medicine and the Collaborative Research Resources of the Keio University of Medicine for their technical assistance.\u003cstrong\u003e\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAuthor Contributions\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eS. Sato and\u0026nbsp;E.S. designed the experiments. S. Sato,\u0026nbsp;K. A.,\u0026nbsp;E.\u0026nbsp;S., T. O., S. Shimizu,\u0026nbsp;and H.\u0026nbsp;T. conducted the experiments. S. Sato, K.A., Y.O., E.S., T.O.,\u0026nbsp;S.\u0026nbsp;Shimmura., and M.H.\u0026nbsp;discussed and analyzed the data. S. Sato., K.A., E. S., and Y.\u0026nbsp;O. wrote the manuscript. All the authors have read and agreed to the final version of the manuscript. Y.O.,\u0026nbsp;S. Shimmura., K.N., and\u0026nbsp;M.H.\u0026nbsp;supervised the study.\u003cstrong\u003e\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eData Availability Statement\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe data that support the findings of this study are available within the article or from the corresponding author upon reasonable request.\u003cstrong\u003e\u0026nbsp;\u003c/strong\u003e\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAdditional Information\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eCompeting Interests Statement:\u003c/strong\u003e S. Sato and E. S. received delgocitinib, 0.5% delgocitinib ointment and solvent vehicle ointments from JT, and the preparation method of delgocitinib eye drops was provided by ROHTO Pharmaceutical Co., Ltd. S. Shimizu, H. T., K. A., T.O., M. H., S. Shimmura, K. N., and Y. O. declare neither financial nor nonfinancial conflicts of interest associated with this manuscript.\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\n\u003cli\u003eWolff, D. et al. National Institutes of Health Consensus Development Project on Criteria for Clinical Trials in Chronic Graft-versus-Host Disease: IV. The 2020 Highly morbid forms report. Transplant Cell Ther 27, 817-835, doi:10.1016/j.jtct.2021.06.001 (2021).\u003c/li\u003e\n\u003cli\u003eFerrara, J. L., Levine, J. E., Reddy, P. \u0026amp; Holler, E. Graft-versus-host disease. Lancet 373, 1550-1561, doi:10.1016/s0140-6736(09)60237-3 (2009).\u003c/li\u003e\n\u003cli\u003eJagasia, M. H. et al. 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Mol Vis 17, 257-264 (2011).\u003c/li\u003e\n\u003c/ol\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":false,"hideJournal":false,"highlight":"","institution":"","isAcceptedByJournal":true,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":false,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"scientific-reports","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"scirep","sideBox":"Learn more about [Scientific Reports](http://www.nature.com/srep/)","snPcode":"","submissionUrl":"","title":"Scientific Reports","twitterHandle":"","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"stoa","reportingPortfolio":"Scientific Reports","inReviewEnabled":true,"inReviewRevisionsEnabled":true},"keywords":"graft-versus-host disease, meibomian gland dysfunction, pan-Janus kinase inhibitor, delgocitinib","lastPublishedDoi":"10.21203/rs.3.rs-4252479/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-4252479/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003cp\u003eSystemic administration of Janus kinase (JAK) inhibitors is effective in treating chronic graft-versus-host disease (cGVHD) but is associated with side effects. Topical drug administration is an effective approach in minimizing these effects. We aimed to demonstrate the efficacy of topical delgocitinib administration in a cGVHD mouse model. Allogenic bone-marrow transplantation (BMT) was performed from B10.D2. to BALB/c mice, leading to cGVHD. cGVHD mice were treated with delgocitinib eye drops or ointments; their samples were analyzed 4 weeks post-BMT. Topical delgocitinib ointment and eye-drop administration significantly increased the meibomian-gland (MG) area and attenuated corneal epithelial damage. Pathological and immunohistochemical analyses revealed a substantial reduction in inflammation and pathological fibrosis of the skin and eyelids in delgocitinib-treated cGVHD mice. Signal transducer and activator of transcription (STAT)1, STAT3, and STAT5A phosphorylation was significantly increased in the back skin and eyelids of vehicle-treated cGVHD mice; topical delgocitinib administration significantly reduced the expression of these phosphorylated STAT molecules. Delgocitinib eye drops significantly attenuated corneal epithelial damage, MG acinar depletion, and inflammatory cells infiltration in cGVHD mouse corneas. The JAK/STAT signaling pathway was significantly upregulated in cGVHD mice.\u003c/p\u003e \u003cp\u003eIn summary, a topical delgocitinib administration attenuated cGVHD phenotype severity in the skin and eyes of cGVHD mice.\u003c/p\u003e","manuscriptTitle":"Anti-inflammatory and anti-fibrotic effects of topical pan-JAK inhibitor in a chronic graft-versus- host disease model mouse","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2024-04-19 12:59:56","doi":"10.21203/rs.3.rs-4252479/v1","editorialEvents":[{"type":"communityComments","content":0},{"type":"decision","content":"Revision requested","date":"2024-05-13T04:25:16+00:00","index":"","fulltext":""},{"type":"editorInvitedReview","content":"","date":"2024-05-07T03:16:07+00:00","index":"hide","fulltext":""},{"type":"editorInvitedReview","content":"","date":"2024-05-02T18:18:31+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"8b9ba504-b2ed-4573-9c25-d210dc7d2ecc","date":"2024-04-25T14:28:40+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"991740e0-f10e-4fe8-a489-cc69b049fc19","date":"2024-04-25T11:53:39+00:00","index":"hide","fulltext":""},{"type":"reviewersInvited","content":"","date":"2024-04-16T21:11:44+00:00","index":"","fulltext":""},{"type":"editorAssigned","content":"","date":"2024-04-16T16:02:22+00:00","index":"","fulltext":""},{"type":"editorInvited","content":"","date":"2024-04-16T08:51:00+00:00","index":"","fulltext":""},{"type":"checksComplete","content":"","date":"2024-04-16T08:49:37+00:00","index":"","fulltext":""},{"type":"submitted","content":"Scientific Reports","date":"2024-04-11T12:34:56+00:00","index":"","fulltext":""}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"scientific-reports","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"scirep","sideBox":"Learn more about [Scientific Reports](http://www.nature.com/srep/)","snPcode":"","submissionUrl":"","title":"Scientific Reports","twitterHandle":"","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"stoa","reportingPortfolio":"Scientific Reports","inReviewEnabled":true,"inReviewRevisionsEnabled":true}}],"origin":"","ownerIdentity":"d127eeb8-4da4-45fe-88a9-11f2d1a56771","owner":[],"postedDate":"April 19th, 2024","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"published-in-journal","subjectAreas":[{"id":30869023,"name":"Health sciences/Diseases"},{"id":30869024,"name":"Health sciences/Diseases/Eye diseases"},{"id":30869025,"name":"Health sciences/Diseases/Eye diseases/Eyelid diseases"}],"tags":[],"updatedAt":"2025-01-06T16:00:55+00:00","versionOfRecord":{"articleIdentity":"rs-4252479","link":"https://doi.org/10.1038/s41598-024-84380-6","journal":{"identity":"scientific-reports","isVorOnly":false,"title":"Scientific Reports"},"publishedOn":"2025-01-02 15:57:16","publishedOnDateReadable":"January 2nd, 2025"},"versionCreatedAt":"2024-04-19 12:59:56","video":"","vorDoi":"10.1038/s41598-024-84380-6","vorDoiUrl":"https://doi.org/10.1038/s41598-024-84380-6","workflowStages":[]},"version":"v1","identity":"rs-4252479","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-4252479","identity":"rs-4252479","version":["v1"]},"buildId":"qtupq5eGEP_6zYnWcrvyt","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}