Senescence‐associated secretory phenotype promotes chronic ocular graft‐vs‐host disease in mice and humans

The FASEB JournalVolume 34, Issue 8 p. 10778-10800 RESEARCH ARTICLEOpen Access Senescence-associated secretory phenotype promotes chronic ocular graft-vs-host disease in mice and humans Mio Yamane, Mio Yamane Department of Ophthalmology, Keio University School of Medicine, Tokyo, JapanSearch for more papers by this authorShinri Sato, Shinri Sato Department of Ophthalmology, Keio University School of Medicine, Tokyo, JapanSearch for more papers by this authorEisuke Shimizu, Eisuke Shimizu Department of Ophthalmology, Keio University School of Medicine, Tokyo, JapanSearch for more papers by this authorShinsuke Shibata, Shinsuke Shibata Electron Microscope Laboratory, Keio University School of Medicine, Tokyo, JapanSearch for more papers by this authorMotoshi Hayano, Motoshi Hayano Department of Ophthalmology, Keio University School of Medicine, Tokyo, JapanSearch for more papers by this authorTomonori Yaguchi, Tomonori Yaguchi Division of Cellular Signaling, Institute for Advanced Medical Research, Keio University School of Medicine, Tokyo, JapanSearch for more papers by this authorHajime Kamijuku, Hajime Kamijuku Division of Cardiology, Department of Internal Medicine, Keio University School of Medicine, Tokyo, JapanSearch for more papers by this authorMamoru Ogawa, Mamoru Ogawa Department of Ophthalmology, Keio University School of Medicine, Tokyo, JapanSearch for more papers by this authorTakanori Suzuki, Takanori Suzuki Department of Ophthalmology, Keio University School of Medicine, Tokyo, JapanSearch for more papers by this authorShin Mukai, Shin Mukai Center for Interdisciplinary Cardiovascular Sciences, Brigham and Women's Hospital, Harvard Medical School, Boston, MA, USASearch for more papers by this authorShigeto Shimmura, Shigeto Shimmura Department of Ophthalmology, Keio University School of Medicine, Tokyo, JapanSearch for more papers by this authorHideyuki Okano, Hideyuki Okano Department of Physiology, Keio University School of Medicine, Tokyo, JapanSearch for more papers by this authorTsutomu Takeuchi, Tsutomu Takeuchi Division of Rheumatology, Department of Internal Medicine, Keio University School of Medicine, Tokyo, JapanSearch for more papers by this authorYutaka Kawakami, Yutaka Kawakami Division of Cellular Signaling, Institute for Advanced Medical Research, Keio University School of Medicine, Tokyo, JapanSearch for more papers by this authorYoko Ogawa, Corresponding Author Yoko Ogawa yoko@z7.keio.jp Department of Ophthalmology, Keio University School of Medicine, Tokyo, Japan Correspondence Yoko Ogawa and Kazuo Tsubota, Department of Ophthalmology, Keio University School of Medicine, 35 Shinanomachi, Shinjuku-ku, Tokyo 160-8582, Japan. Email: yoko@z7.keio.jp; tsubota@z3.keio.jpSearch for more papers by this authorKazuo Tsubota, Corresponding Author Kazuo Tsubota tsubota@z3.keio.jp Department of Ophthalmology, Keio University School of Medicine, Tokyo, Japan Correspondence Yoko Ogawa and Kazuo Tsubota, Department of Ophthalmology, Keio University School of Medicine, 35 Shinanomachi, Shinjuku-ku, Tokyo 160-8582, Japan. Email: yoko@z7.keio.jp; tsubota@z3.keio.jpSearch for more papers by this author Mio Yamane, Mio Yamane Department of Ophthalmology, Keio University School of Medicine, Tokyo, JapanSearch for more papers by this authorShinri Sato, Shinri Sato Department of Ophthalmology, Keio University School of Medicine, Tokyo, JapanSearch for more papers by this authorEisuke Shimizu, Eisuke Shimizu Department of Ophthalmology, Keio University School of Medicine, Tokyo, JapanSearch for more papers by this authorShinsuke Shibata, Shinsuke Shibata Electron Microscope Laboratory, Keio University School of Medicine, Tokyo, JapanSearch for more papers by this authorMotoshi Hayano, Motoshi Hayano Department of Ophthalmology, Keio University School of Medicine, Tokyo, JapanSearch for more papers by this authorTomonori Yaguchi, Tomonori Yaguchi Division of Cellular Signaling, Institute for Advanced Medical Research, Keio University School of Medicine, Tokyo, JapanSearch for more papers by this authorHajime Kamijuku, Hajime Kamijuku Division of Cardiology, Department of Internal Medicine, Keio University School of Medicine, Tokyo, JapanSearch for more papers by this authorMamoru Ogawa, Mamoru Ogawa Department of Ophthalmology, Keio University School of Medicine, Tokyo, JapanSearch for more papers by this authorTakanori Suzuki, Takanori Suzuki Department of Ophthalmology, Keio University School of Medicine, Tokyo, JapanSearch for more papers by this authorShin Mukai, Shin Mukai Center for Interdisciplinary Cardiovascular Sciences, Brigham and Women's Hospital, Harvard Medical School, Boston, MA, USASearch for more papers by this authorShigeto Shimmura, Shigeto Shimmura Department of Ophthalmology, Keio University School of Medicine, Tokyo, JapanSearch for more papers by this authorHideyuki Okano, Hideyuki Okano Department of Physiology, Keio University School of Medicine, Tokyo, JapanSearch for more papers by this authorTsutomu Takeuchi, Tsutomu Takeuchi Division of Rheumatology, Department of Internal Medicine, Keio University School of Medicine, Tokyo, JapanSearch for more papers by this authorYutaka Kawakami, Yutaka Kawakami Division of Cellular Signaling, Institute for Advanced Medical Research, Keio University School of Medicine, Tokyo, JapanSearch for more papers by this authorYoko Ogawa, Corresponding Author Yoko Ogawa yoko@z7.keio.jp Department of Ophthalmology, Keio University School of Medicine, Tokyo, Japan Correspondence Yoko Ogawa and Kazuo Tsubota, Department of Ophthalmology, Keio University School of Medicine, 35 Shinanomachi, Shinjuku-ku, Tokyo 160-8582, Japan. Email: yoko@z7.keio.jp; tsubota@z3.keio.jpSearch for more papers by this authorKazuo Tsubota, Corresponding Author Kazuo Tsubota tsubota@z3.keio.jp Department of Ophthalmology, Keio University School of Medicine, Tokyo, Japan Correspondence Yoko Ogawa and Kazuo Tsubota, Department of Ophthalmology, Keio University School of Medicine, 35 Shinanomachi, Shinjuku-ku, Tokyo 160-8582, Japan. Email: yoko@z7.keio.jp; tsubota@z3.keio.jpSearch for more papers by this author First published: 03 July 2020 https://doi.org/10.1096/fj.201900218RCitations: 16 Mio Yamane, Shinri Sato, Eisuke Shimizu are contributed equally to this study. AboutSectionsPDF ToolsRequest permissionExport citationAdd to favoritesTrack citation ShareShare Give accessShare full text accessShare full-text accessPlease review our Terms and Conditions of Use and check box below to share full-text version of article.I have read and accept the Wiley Online Library Terms and Conditions of UseShareable LinkUse the link below to share a full-text version of this article with your friends and colleagues. Learn more.Copy URL Share a linkShare onFacebookTwitterLinkedInRedditWechat Abstract Chronic graft-vs-host disease (cGVHD) is a multifactorial inflammatory disease that affects patients undergoing hematopoietic stem cell transplantation. Multiple organs, including the lacrimal glands (LGs), are negatively affected by cGVHD and lose function due to the resultant fibrosis. An abnormal immune response is thought to be a major factor in the development of chronic ocular GVHD, which is currently treated primarily with immunosuppressive therapies. However, all the treatments yield unsatisfactory outcomes, and additional treatment strategies are needed. To meet this unmet medical need, we aimed to elucidate an additional pathway of chronic ocular GVHD. Our findings suggest a potential association between chronic ocular GVHD pathogenesis and stress-induced cellular senescence through the senescence-associated secretory phenotype (SASP). Senescent cells produce cytokines and chemokines, such as IL-6 and CXCL9. Indeed, senescent cell accumulation was presumably associated with cGVHD development in LGs, as evidenced by the improvement in LGs after the selective elimination of senescent cells (senolysis) with ABT-263. Results in the sclerodermatous cGVHD mouse model suggest that inhibiting the major components of the SASP, including IL-6 and CXCL9, with senolytics is a potential novel strategy for treating cGVHD-affected LGs. Taken together, our results indicate a potential association between the SASP and cGVHD development in LGs and suggest that targeted senolytic treatment may be a new therapeutic option for this disease. Abbreviations 53BP1 p53-binding protein 1 α-SMA α-smooth muscle actin BMT bone marrow transplantation CD cluster of differentiation cGVHD chronic graftvshost disease CXCL chemokine (C-X-C motif) ligand DDR DNA damage response ELISA enzyme-linked immunosorbent assay GFP green fluorescent protein GVHD graft-vs-host disease HSCT hematopoietic stem cell transplantation H2A.X H2A histone family member X HSP47 heat shock protein 47 IL interleukin IP intraperitoneal LG lacrimal gland miHA minor histocompatibility antigen OPN osteopontin PCR polymerase chain reaction RNA ribonucleic acid RPMI Roswell Park Memorial Institute SASP senescence-associated secretory phenotype TEM transmission electron microscopy TBI total body irradiation 1 INTRODUCTION Hematopoietic stem cell transplantation (HSCT) is an established treatment for life-threatening hematologic malignancies. The major complication of allogeneic HSCT is graft-vs-host disease (GVHD).1, 2 Chronic GVHD (cGVHD) is a multifactorial disease characterized by immune system activation and progressive fibrosis3 and decreases the success of allogeneic HSCT due to increased risks of death and disability.4 Fibrosis in exocrine glands, including the lacrimal and salivary glands, as well as in the skin, lung, liver, and gastrointestinal tract is observed in cGVHD4-6 and distinct from acute GVHD.7 One of the most common complications is chronic ocular GVHDi.e. cGVHD-related dry eye disease, which occurs in more than 50% of cGVHD patients after HSCT and decreases quality of life.8-11 Progress in treating chronic ocular GVHD is hampered by the complex pathogenic mechanisms. Current treatments for chronic ocular GVHD include the local and/or systemic administration of glucocorticoids and immunosuppressants and topical lubricants, since the immune response is a major component of the pathophysiology of cGVHD. However, the response to immunosuppression remains unsatisfactory, with an overall response rate of 43%-86%.5, 12 cGVHD is observed in 10%-19% of patients after T cell-depleted HSCT.13 Thus, effective treatment options for chronic ocular GVHD and systemic cGVHD are needed.5, 14 Although T cells are known to play important roles in the initiation of chronic ocular GVHD,12 we hypothesize the involvement of other pathways related to stress-induced senescence.15, 16 Previous experimental studies have explained the pathophysiology of cGVHD as regulatory T cell deficiency, thymic dysfunction, B cell activation, and cellular and cytokine network interactions.17 Murine minor histocompatibility antigen (miHA)-disparate models have been used to investigate the pathogenesis of cGVHD, including chronic ocular GVHD.17-19 miHA-mismatched bone marrow stromal/stem cells,19 the tissue renin-angiotensin system,20 and endoplasmic reticulum stress21 are involved in the immune-mediated fibrosis in cGVHD in mice. In an animal model, macrophages in the lacrimal glands (LGs), which are affected by cGVHD, express senescence and oxidative stress markers. The senescence-related product lipofuscin accumulates in the LGs of cGVHD mice and chronologically aged mice, suggesting that cGVHD LGs undergo changes indicative of senescence.22 Age-related changes, including the development of eyebrow and eyelash poliosis or skin wrinkles, can progress rapidly in patients with severe cGVHD.22 Based on these findings, we hypothesized that chronic ocular GVHD is related to senescence.23 Cellular senescence is a stress response induced by external or internal chemical and physical insults that leads to malignant tumorigenesis by affecting checkpoint activation and inducing a stable cell cycle arrest.24-27 Depending on the degree of stress, temporarily arrested cells can (1) transition into senescent growth arrest, (2) retain permanent damage and resume proliferation, or (3) successfully repair damage and resume normal cell cycle progression.26 Senescent cells not only can live for a long time, but also irreversibly arrest the cell cycle and secrete substances, including inflammatory cytokines and chemokines, that promote chronic inflammation; this condition is termed the senescence-associated secretory phenotype (SASP).25, 27 SASP is the major downstream mediator of cellular senescence.25, 28 Interleukin (IL)-1β, IL-6, IL-8, CXCL1, and CXCL9 are reported to be major components of the SASP.25, 27, 29 Moreover, IL-6 is known to play a critical role in cGVHD progression.19, 30, 31 In the cGVHD mouse model, the mice undergo total body irradiation (TBI) and allogeneic BMT, both of which may accelerate cellular senescence in these animals compared with syngeneic controls beyond that expected from conventional immune responses alone.22 This study aimed to determine whether cGVHD-affected LGs show signs of stress-induced senescence and release cytokines and chemokines, the major components of the SASP. In addition, we aimed to confirm that the selective elimination of senescent cells (senolysis) by ABT-26328, 32, 33 improves LGs affected by cGVHD. 2 MATERIALS AND METHODS 2.1 Mice B10.D2/nSnSlc and BALB/cCrSlc mice (7-9 weeks old) were purchased from Sankyo Laboratory, Inc (Tokyo, Japan). GFP mice were obtained by backcrossing B10.D2/nSnSlc mice with C57BL/6 GFP mice (Japan SLC Ltd, Shizuoka, Japan). Progeny of the 10th generation of backcrossed B10.D2/nSnSlc GFP were used for the experiments. 2.2 Whole bone marrow transplantation (BMT) To generate the cGVHD mouse model, allogeneic BMT was performed with 7- to 9-week-old male B10.D2/nSnSlc and female BALB/cCrSlc mice as transplant donors and recipients, respectively, as previously reported, representing MHC-compatible, miHA-mismatched BMT. 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 of X-ray using a Gammacell 137Cs source (Hitachi Medico, Ltd, Tokyo, Japan). Donor cells (1 × 106 bone marrow cells/mouse and 2 × 106 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.18 The recipient animals were maintained in sterile cages and given autoclaved food and acidified water. The cGVHD and non-cGVHD control mice were used for experiments at 2, 3, 4, or 8 weeks after BMT. 2.3 Treatment of allogeneic BMT recipient mice with ABT-263 Allogeneic BMT recipient mice were divided into 2 groups; one was treated with ABT-263 (Navitoclax, S1001, Selleck Chemicals, Houston, TX), and the other was given solvent vehicle. Mice were treated daily for 7 consecutive days starting 10 days after BMT by gavage with either 50 mg/kg, 25 mg/kg, 12.5 mg/kg of ABT-263 or vehicle (ethanol [Sigma-Aldrich, St. Louis, MO], polyethylene glycol 400 [HR2-603, Hampton Research, Aliso Viejo, CA], and Phosal 50 PG [H. Holstein, Hamburg, Germany] at a percentage ratio of 10:30:60).28, 32, 34 Mice were analyzed 28 days after BMT. 2.4 Treatment of allogeneic BMT recipient mice with anti-mouse IL-6 receptor (anti-IL-6R) monoclonal antibody (mAb) Allogeneic BMT recipient mice were divided into 2 groups; one was treated with MR16-1 (Tocilizumab, Chugai, Tokyo, Japan), a humanized mAb specific for IL-6R, and the other was given vehicle (PBS, pH 7.4). The anti-IL-6R mAb or PBS was administered by IP injection at 2 mg/mouse 1 day before and at 0.5 mg/mouse 10, 17, and 24 days after BMT.35 The mice were analyzed 28 days after BMT. 2.5 In vivo imaging To detect myeloperoxidase activity of activated phagocytes such as macrophages in deep tissues in vivo, mice were anesthetized and received an IP injection of XenoLight RediJect Inflammation Probe (760535, PerkinElmer, Waltham, MA), a chemiluminescent reagent for monitoring inflammation, at 200 mg/kg 10 minutes before beginning the photon recording. The mice were placed in a dark box, and luminescence images were acquired using an electron multiplying CCD camera (C9100-13, Hamamatsu Photonics KK, Hamamatsu, Japan).36 2.6 Histological analysis Mice were sacrificed by cervical dislocation 3, 4, or 8 weeks after BMT. The LGs and skin were harvested and fixed with 20% neutralized buffered formalin overnight, embedded in paraffin wax, and processed for H&E and Mallory staining.19, 20 To quantify the fibrotic areas20, 37 in the LGs and skin in tissue sections, images were acquired at 400 times magnification, and 3 images of the LGs and skin from each Mallory-stained section were examined. These images were analyzed using Color Deconvolution, a plugin for Image J, with which we separated the colors of each image into blue, green, and red. The area of blue color was measured using the same threshold.38 The average value obtained from the 3 images from each sample was regarded as the value for the fibrotic area. 2.7 Immunohistochemistry using whole-mount lacrimal glands Mice were sacrificed, after which the LGs were removed using sterile technique and minced into small pieces using a scalpel. The tissue pieces were washed, incubated in BD CellFIX (340181, BD Pharmingen, San Diego, CA) for 1 hour at room temperature, and permeabilized with 0.2% of Triton X-100 (3501-02, Nacalai tesque, Kyoto, Japan) for 10 minutes. The specimens were then blocked with 10% of normal goat serum (50062Z, Thermo Fisher Scientific, Waltham, MA) for 30 minutes and incubated with optimally diluted primary antibodies specific to CD4 (14-0042-81, RM4-5, eBioscience, San Diego, CA) and CD153 (14-1531-85, RM153, eBioscience) overnight at 4°C, and then, with an Alexa Fluor 488-conjugated goat anti-rat IgG secondary antibody (A11006, Thermo Fisher Scientific) and DAPI (D21490, Thermo Fisher Scientific) for 45 minutes at room temperature to stain the nuclei, as described previously.39 Isotype antibodies including rat IgG2a, κ (14-4321-82, eBR2a, eBioscience) for CD4 and rat IgG2b, κ (14-4031-82, eB149/10H5, eBioscience) for CD153 were used as negative controls. The samples were mounted with Fluorescent Mounting Medium (S3023, Agilent Technologies, Santa Clara, CA) on a glass slide and covered with a cover glass (C024241, Matsunami Glass, Osaka, Japan). Fluorescence images were captured with an LSM 700 or LSM 710 confocal microscope (Carl Zeiss, Jena, Germany). 2.8 Immunostaining of frozen tissue sections Immunohistochemical analyses were performed as described previously.19 The LG tissues were embedded in optimal cutting temperature (OCT) compound (4583, Sakura Finetek, Torrance, CA) in precooled isopentane and stored at −80°C until cutting into 6-µm-thick sections. The sections were fixed in acetone for 20 minutes at room temperature, rehydrated in PBS, and blocked with 10% of normal goat serum for 30 minutes at room temperature. In some experiments for single staining, the sections were incubated overnight at 4°C with optimally diluted primary antibodies. The primary antibodies used for the LGs from human samples were 53BP1 (SC-22760, H-300, Santa Cruz Biotechnology, Dallas, TX), IL-8 (ab18672, 807, Abcam, Cambridge, United Kingdom), and IL-6 (ab6672, Abcam). Next, the sections were incubated for 45 minutes at room temperature with DAPI and the secondary antibodies. Each step was followed by three washes with PBS. In some experiments using mouse LGs, the tissue sections were double stained with Bax (active monomer) (ALX-804-224-C100, 6A7, Enzo Life Science, Farmingdale, NY) or Cleaved Caspase-3 (9661, Cell Signaling Technology, Danvers, MA). In some experiments using mouse LGss, acetone-fixed the tissue sections were double stained with(1) purified p16 (sc-1207, M-156, Santa Cruz Biotechnology) and CXCL1 (ab17882, polyclonal, Abcam) or (2) CXCL9 (ab137792, polyclonal, Abcam) and APC-labeled CD68 (137007, FA-11, BioLegend, San Diego, CA) overnight at 4°C. After being washed, the sections were incubated for 45 minutes at room temperature with DAPI and the secondary antibodies. For double staining using fluorophore-conjugated primary antibodies, PE-labeled MHC class II (12-5321, M5/114.15.2, eBioscience) or PE-labeled IL-6(554401, MP5-20F3, BD Pharmingen) and either APC-labeled CD3 (17-0031, 145-2C11, eBioscience), APC-labeled CD68, or APC-labeled CD154 (17-1541, MR1, eBioscience) were co-stained over night at 4°C. After being washed, the sections were incubated for 45 minutes with DAPI. For triple staining, the tissue sections were stained with Osteopontin (OPN) (O7635, polyclonal, Sigma-Aldrich), APC-labeled CD279 (PD-1) (109111, RMP1-30, eBioscience), and either CD4 (14-0042-81, RM4-5, eBioscience) or PE-labeled CD153(137007, FA-11, BioLegend) over night at 4°C. After being washed, the sections were incubated with DAPI and secondary antibodies for 45 minutes at room temperature. In other experiments for double staining using same species for two primary antibodies, acetone-fixed tissue sections were stained with PE-labeled MHC class II and CD40 (124601, 3/23, BioLegend) in combination with Alexa Fluor 647-conjugated goat anti-rat IgG secondary antibody (A21247, Thermo Fisher Scientific) by two steps. The sections were incubated overnight at 4°C with optimally diluted the conjugated primary antibody as a first reaction, and then, for two hours at room temperature with optimally diluted purified primary antibodies as a second reaction. Next, the sections were incubated for 45 minutes with DAPI and the secondary antibodies.40 The secondary antibodies used were (1) Alexa Fluor 488-conjugated goat anti-rabbit IgG secondary antibody (A11034, Thermo Fisher Scientific) for p16, CXCL1, CXCL9, 53BP1, and IL-8; (2) Alexa Fluor 488-conjugated goat anti-mouse IgG secondary antibody (A11029, Thermo Fisher Scientific) for IL-8; (3) Alexa Fluor 568-conjugated goat anti-rat IgG secondary antibody (A11077, Thermo Fisher Scientific) for CD68; (4) Alexa Fluor 647-conjugated goat anti-hamster IgG secondary antibody (127-605-160, Jackson ImmunoResearch Laboratories, West Grove, PA) for CD3 and CD154; (5) Alexa Fluor 488-conjugated rabbit anti-goat IgG secondary antibody (A11078, Thermo Fisher Scientific) for IL-1β and OPN; (6) Alexa Fluor 568-conjugated goat anti-rabbit IgG secondary antibody (A11011, Thermo Fisher Scientific) for Cleaved Caspase-3; (7) Alexa Fluor 568-conjugated goat anti-mouse IgG secondary antibody (A11004, Thermo Fisher Scientific) for Bax (active monomer); (8) Alexa Fluor 660-conjugated goat anti-rabbit IgG secondary antibody (A21074, Thermo Fisher Scientific) for CD4. Isotype-matched antibodies used as negative controls were (1) rabbit IgG antibody (2729, Cell Signaling Technology) for p16, 53BP1, CXCL1, CXCL9, IL-8, and Cleaved Caspase-3; (2) rat IgG2a antibody (MAB006, 54447, R&D Systems, Minneapolis, MN) for CD68; (3) APC-labeled rat IgG2b, κ antibody (400611, RTK4530, BioLegend) for MHC class II; (4) APC-labeled rat IgG2a, κ antibody (400611, RTK4530, BioLegend) for CD68; (5) Armenian hamster IgG antibody (14-4888, eBioscience) for CD3 and CD154; (6) rat IgG2a, κ antibody for CD40; (7) PE-labeled Rat IgG1, λ antibody and (8) mouse IgG1, κ (14-4714-82, P3.6.2.8.1, eBioscience) for IL-8 and Bax (active monomer); (9) PE-labeled rat IgG1, κ antibody (401905, BioLegend) for IL-6. 2.9 Immunostaining of formalin-fixed paraffin-embedded tissue sections After deparaffinization and dehydration, target antigens were unmasked using microwave method at 100ºC for 10 minutes for p16 (sc-1661, F-12, Santa Cruz Biotechnology), Caspase-1 (06-503-I, polyclonal, EMD Millipore Corporation, Temecula, CA), IL-1β (AF-401-NA, polyclonal, R&D Systems), IL-8 (BS3479, polyclonal, Bioworld Technology, St. Louis Park, MN), CXCL1 (ab17882, polyclonal, Abcam), IL-6, CXCL9 (ab137792, polyclonal, Abcam), CD68, CD45 (550539, 30-F11, BD Pharmingen), OPN(O7635, polyclonal, Sigma-Aldrich), and α-SMA (ab7817, 1A4, Abcam) or autoclave methods at 120ºC for 20 minutes for γ-H2A.X (9718, 20E3, Cell Signaling Technology), 53BP1, p21 (ab2961, polyclonal, Abcam), Ki-67 (RM-9106, SP6, Abcam), and HSP47 (SPA-470, M16.10A1, Stressgen Biotechnologies, Victoria British Columbia, Canada) in target retrieval solution (S169984, Agilent Technologies). The sections were blocked with 10% of normal goat serum or normal rat serum (012-000-120, Jackson ImmunoResearch Laboratories) for 30 minutes at room temperature and incubated overnight at 4°C with optimally diluted primary antibodies. The primary antibodies used for mouse samples were γ-H2A.X; 53BP1; p16; p21; Ki-67; Caspase-1; IL-1β; IL-8; CXCL1; IL-6; CXCL9; CD68; CD45; OPN; HSP47; and α-SMA. The sections were then incubated for 45 minutes at room temperature with DAPI and the secondary antibodies including (1) Alexa Fluor 488-conjugated goat anti-rabbit IgG secondary antibody for γ-H2A.X, 53BP1, p21, Ki-67, Caspase-1, IL-8, CXCL1, IL-6, CXCL9, and Ki-67; (2) Alexa Fluor 488-conjugated goat anti-mouse IgG secondary antibody for p16, HSP47, and α-SMA; (3) Alexa Fluor 488-conjugated goat anti-rat IgG secondary antibody for CD68, and CD45; (4) Alexa Fluor 488-conjugated rabbit anti-goat IgG secondary antibody (A11078, Thermo Fisher Scientific) for IL-1β and OPN; (5) Alexa Fluor 568-conjugated goat anti-rat IgG secondary antibody for CD68; and (6) Alexa Fluor 568-conjugated goat anti-mouse IgG secondary antibody (A11031, Thermo Fisher Scientific) for α-SMA. In some experiments, the co-expression of IL-6 and CD68 or of CXCL9 and α-SMA on the LGs was examined by double staining methods as described previously.19 Isotype-matched antibodies used as negative controls were (1) rabbit IgG antibody for γ-H2A.X, 53BP1, p21, Ki-67, Caspase-1, IL-8, CXCL1, IL-6, CXCL9, and Ki-67; (2) mouse IgG2a antibody for p16; (3) rat IgG2a antibody for CD68; (4) goat IgG antibody for IL-1β and OPN; (5) rat IgG2b, κ antibody for CD45; (6) mouse IgG2b antibody for HSP47; and (7) mouse IgG2a, κ antibody for α-SMA. The number of target cells per field was quantified by averaging at least five non-overlapping fields for each section. To estimate the histological architecture and staining, each slide was observed three times by two independent observers (MY and YO). Image J was used to analyze the CXCL9- and OPN-positive areas in vessels at 200 times magnification.38 Five images of the LGs for each sample were analyzed, and the colors of each image were separated into blue, green, and red. The area of green color was measured using the same threshold. The mean value obtained from 5 images for each sample was regarded as the value for the CXCL9- and OPN-positive areas. 2.10 Apoptosis assay Apoptosis was assayed with a fluometric TUNEL system (G3250, Promega, Madison, WI) as described previously.41 Each step was followed by three washes with PBS. The frozen sections were fixed by immersing slides into freshly prepared 4% of methanol-free formaldehyde in PBS for 25 minutes at 4°C and permeabilized by immersing the slides into 0.2% of Triton X-100 in PBS for 5 minutes. The frozen sections were covered with 100 µL Equilibration Buffer and equilibrated at room temperature for 10 minutes. For the experimental reactions, an incubation buffer was prepared by combining Equilibration Buffer (45 µL), Nucleotide Mix (5 µL), and rTdT Enzyme (1 µL). Cleaning wipes (Kimberly-Clark Corp., Irvine, TX) were used to blot the areas around the equilibrated tissues, and 50 µL incubation buffer was added to a 5 cm2 area of the frozen sections. The slides were incubated at 37°C for 60 minutes in a humidifying light-blocking chamber. The reaction was stopped by immersing the slides into 2× saline sodium citrate buffer for 15 minutes at room temperature. The sections were incubated with DAPI at room temperature for 15 minutes. 2.11 Immunocytochemistry of cultured fibroblasts derived from lacrimal glands in mice LG fibroblasts from cGVHD and syngeneic control recipient mice were cultured as described previously.42 Briefly, LG tissue was minced with sterilized scissors into small pieces, and transferred to plates containing DMEM supplemented with penicillin (200 U/mL), streptomycin (200 U/mL), and 10% of fetal bovine serum for fibroblast growth. Cultures were maintained at 37°C in a humidified atmosphere of 5% of CO2 and 95% of air. The medium was replaced with fresh medium every 3 days. Confluent cells were detached by incubation with 0.05% of trypsin for 1.5 minutes and centrifuged (400 ×g for 10 minutes at 4°C). The cell pellet was then resuspended in DMEM. LG fibroblast cultures were performed 3 times. The LG fibroblast cultures were used between the third and seventh passages. In addition, fibroblasts were cultured on fibronectin-coated chamber slides (354631, Corning, Corning, NY), fixed on the slide with 10% of neutral-buffered formalin (20211, Muto Pure Chemicals, Tokyo, Japan) for 30 minutes at room temperature, and incubated for 2 hours at room temperature with optimally diluted primary antibodies. Primary antibodies used for cultured fibroblasts were 53BP1, p16, Ki-67, and HSP47. The negative controls used were as described previously.19 The chambers were then incubated for 45 minutes at room temperature with DAPI and the secondary antibodies including (1) Alexa Fluor 660-conjugated goat anti-rabbit IgG secondary antibody (A21074, Thermo Fisher Scientific) for 53BP1; (2) Alexa Fluor 488-conjugated goat anti-rabbit IgG secondary antibody for p16; (3) Alexa Fluor 488-conjugated goat anti-rabbit IgG secondary antibo