Jak Inhibition Prevents Bleomycin-Induced Fibrosis in Mice and Is Effective in Patients with Morphea
2020; Elsevier BV; Volume: 140; Issue: 7 Linguagem: Inglês
10.1016/j.jid.2019.12.019
ISSN1523-1747
AutoresWilliam Damsky, Dhrumil Patel, Colton J. Garelli, Madhuri Garg, Alice Wang, Karen Dresser, April Deng, John E. Harris, Jillian M. Richmond, Brett King,
Tópico(s)Inflammatory Myopathies and Dermatomyositis
ResumoMorphea is an autoimmune disease that causes fibrosis of the skin that may result in significant morbidity. Eosinophilic fasciitis (EF) may be considered a deeper form of morphea, and features of both morphea and EF can be present in individual patients. Morphea and EF have been postulated to have pathogenic features that overlap with systemic sclerosis (Laxer and Zulian, 2006Laxer R.M. Zulian F. Localized scleroderma.Curr Opin Rheumatol. 2006; 18: 606-613Crossref PubMed Scopus (252) Google Scholar). In these diseases, dysregulated immune responses are thought to induce fibrosis (Torok et al., 2019Torok K.S. Li S.C. Jacobe H.M. Taber S.F. Stevens A.M. Zulian F. et al.Immunopathogenesis of pediatric localized scleroderma.Front Immunol. 2019; 10: 908Crossref PubMed Scopus (38) Google Scholar). Treatment of these diseases with nonspecific immunosuppressants such as corticosteroids is often ineffective and associated with toxicity. IL-4/IL-13, IL-12, and IFN-γ have been implicated in autoimmune fibrosis (Torok et al., 2019Torok K.S. Li S.C. Jacobe H.M. Taber S.F. Stevens A.M. Zulian F. et al.Immunopathogenesis of pediatric localized scleroderma.Front Immunol. 2019; 10: 908Crossref PubMed Scopus (38) Google Scholar). These cytokines signal via the janus kinase–signal transducer and activator of transcription (Jak-STAT) pathway, suggesting inhibition of this pathway might be therapeutic in this group of diseases. Additionally, transforming growth factor-β, a cytokine with well-characterized profibrotic effects, has been reported to activate Jak-STAT in the context of fibrosis (Chakraborty et al., 2017Chakraborty D. Šumová B. Mallano T. Chen C.W. Distler A. Bergmann C. et al.Activation of STAT3 integrates common profibrotic pathways to promote fibroblast activation and tissue fibrosis.Nat Commun. 2017; 8: 1130Crossref PubMed Scopus (167) Google Scholar). STAT4 and IRF5 polymorphisms are associated with scleroderma (Tsou and Sawalha, 2017Tsou P.S. Sawalha A.H. Unfolding the pathogenesis of scleroderma through genomics and epigenomics.J Autoimmun. 2017; 83: 73-94Crossref PubMed Scopus (55) Google Scholar), further implicating Jak-STAT signaling. We previously reported successful treatment of two patients, one with generalized morphea and one with EF, with the Jak inhibitor tofacitinib (Kim et al., 2018Kim S.R. Charos A. Damsky W. Heald P. Girardi M. King B.A. Treatment of generalized deep morphea and eosinophilic fasciitis with the Janus kinase inhibitor tofacitinib.JAAD Case Rep. 2018; 4: 443-445Abstract Full Text Full Text PDF PubMed Scopus (40) Google Scholar). Here, four additional patients with generalized morphea (totaling six consecutive patients) were treated with tofacitinib (inhibits Jak1/3>2) (n = 3) or baricitinib (inhibits Jak1/2) (n = 1), and long-term follow-up of the previously reported patients is presented. Verbal informed consent for this off-label treatment with a Jak inhibitor was obtained. These six patients had previously failed multiple therapies typically used for severe morphea, including methotrexate, prednisone, psoralen plus UVA, and extracorporeal photopheresis (Table 1). In some patients, Jak inhibitors were initially used in combination with either prednisone or methotrexate and/or extracorporeal photopheresis.Table 1Patients with Morphea and EF Treated with TofacitinibAge (y) /SexDiagnosisSkin biopsy findingsDuration of disease before Jak inhibitorTherapies utilized before Jak inhibitorJak inhibitor and doseTime to: initial response (1), significant clinical and symptomatic improvement (2), maximal response (3)Therapies weaned off while on Jak inhibitorTotal time on Jak inhibitorAdverse effects69, femaleEosinophilic fasciitisDermal fibrosis, fascial thickening12 monthsPrednisoneTofacitinib5 mg b.i.d1 month (1)9 months (2)26 months (3)Prednisone30 monthsNone59, femaleGeneralized deep morphea (postradiation)Dermal fibrosis10 monthsPrednisoneECPTofacitinib 5–10 mg b.i.d1 month (1)7 months (2)16 months (3)PrednisoneECPMethotrexate40 monthsNone69, femaleGeneralized morpheaDermal fibrosis18 monthsPrednisoneMethotrexateTofacitinib 5 mg b.i.d2 months (1)5 months (2)11 months (3)Methotrexate16 monthsNone84, femaleGeneralized morphea with ulcerationDermal fibrosis40 monthsPrednisoneMethotrexateILTTofacitinib 5 mg b.i.d1 month (1)7 months (2)11 months (3)Methotrexate17 monthsNone54, maleGeneralized morpheaDermal fibrosis30 monthsPrednisonePUVAECPBaricitinib2 mg daily2 months (1)6 months (2)NA (3)ECP12 monthsNone73, femaleGeneralized morpheaDermal fibrosis48 monthsMethotrexateMycophenolate mofetilTofacitinib 5–10 mg b.i.d3 months (1)4 months (2)4 months (3)N/A10 monthsNoneAbbreviations: ECP, extracorporeal photopheresis; EF, eosinophilic fasciitis; ILT, intralesional triamcinolone; PUVA, psoralen and UVA phototherapy.Previously published (Kim et al., 2018Kim S.R. Charos A. Damsky W. Heald P. Girardi M. King B.A. Treatment of generalized deep morphea and eosinophilic fasciitis with the Janus kinase inhibitor tofacitinib.JAAD Case Rep. 2018; 4: 443-445Abstract Full Text Full Text PDF PubMed Scopus (40) Google Scholar) with updated follow-up data provided herein. Open table in a new tab Abbreviations: ECP, extracorporeal photopheresis; EF, eosinophilic fasciitis; ILT, intralesional triamcinolone; PUVA, psoralen and UVA phototherapy. Previously published (Kim et al., 2018Kim S.R. Charos A. Damsky W. Heald P. Girardi M. King B.A. Treatment of generalized deep morphea and eosinophilic fasciitis with the Janus kinase inhibitor tofacitinib.JAAD Case Rep. 2018; 4: 443-445Abstract Full Text Full Text PDF PubMed Scopus (40) Google Scholar) with updated follow-up data provided herein. All six patients treated with Jak inhibitors experienced improvement in signs/symptoms of disease, such as erythema, induration, range-of-motion, and strength. The time to initial response ranged from 1 to 3 months, and the time to meaningful clinical improvement ranged from 5 to 9 months. Patients continued to improve up to 30 months after initiation of Jak inhibitor treatment (Table 1) (Figure 1a and c). Objective improvement in morphea/EF was observed using a morphea severity scale (Cunningham et al., 1998Cunningham B.B. Landells I.D. Langman C. Sailer D.E. Paller A.S. Topical calcipotriene for morphea/linear scleroderma.J Am Acad Dermatol. 1998; 39: 211-215Abstract Full Text Full Text PDF PubMed Scopus (141) Google Scholar) (Supplementary Material). All patients consented to publication of their clinical images. Patient 1 had resolution of the prayer sign with tofacitinib treatment, and both patients 1 and 2 had been intermittently requiring a cane for ambulation because of restricted range-of-motion involving the lower extremities (Kim et al., 2018Kim S.R. Charos A. Damsky W. Heald P. Girardi M. King B.A. Treatment of generalized deep morphea and eosinophilic fasciitis with the Janus kinase inhibitor tofacitinib.JAAD Case Rep. 2018; 4: 443-445Abstract Full Text Full Text PDF PubMed Scopus (40) Google Scholar), and the latter resolved in both patients with tofacitinib treatment. Patient 4 had ulcerative morphea and had complete resolution of painful ulcerations with tofacitinib (Figure 1a); stopping tofacitinib led to recurrence of painful ulcerations (Supplementary Figure S1) and restarting it again led to improvement. Patient 5 is the only patient treated with baricitinib; resolution of erythema was noted after 2 months of treatment, and subjective improvement in mobility was reported after 6 months. Patients 1 and 3 have been able to discontinue treatment without disease recurrence (Table 1), patient 2 is stably improved on low-dose tofacitinib, and the other three patients remain stably controlled on either tofacitinib or baricitinib monotherapy. To better understand the activity of Jak inhibition, we used a bleomycin-induced murine model of fibrosis with features of human scleroderma/morphea, including a similar inflammatory infiltrate, increase in collagen production, and thickening of the dermis (Yamamoto et al., 2000bYamamoto T. Kuroda M. Nishioka K. Animal model of sclerotic skin. III: histopathological comparison of bleomycin-induced scleroderma in various mice strains.Arch Dermatol Res. 2000; 292: 535-541Crossref PubMed Scopus (78) Google Scholar). In this model, mice received injections of bleomycin into flank skin for 12 days to induce a morphea-like phenotype (Supplementary Material) characterized by skin thickening and increased collagen production (Figure 1b). Mice were treated with either tofacitinib (30 mg/kg/day) or ruxolitinib (10 mg/kg/day). Mice treated with these inhibitors were protected from dermal thickening (Figure 1b and e). The effect was more apparent in female mice than male mice (Supplementary Figure S2). Given the beneficial response to Jak inhibition observed in both humans and mice, we evaluated patterns of Jak-STAT activation in fibrotic tissue using immunohistochemistry for phosphorylated (activated) STAT1 and STAT3. An archival series of seven biopsies from different patients with morphea was used (Supplementary Table S1). In all human morphea cases, STAT1 was phosphorylated in lymphocytes and occasionally spindled fibroblasts, and STAT3 was phosphorylated in both lymphocytes and spindled fibroblasts (Figure 1d and g). In murine bleomycin-treated skin, negligible STAT1 phosphorylation was observed; however, STAT3 phosphorylation was common in spindled cells and was reduced with Jak inhibitor treatment (Figure 1f and i). In patients with scleroderma/morphea, it has been shown that COL1A1 production is increased through both transcriptional and translational mechanisms and is thought to play a role in fibrosis. Given both the improvement in fibrosis and induration in our patients with morphea with Jak inhibitor treatment and, in murine bleomycin-treated skin, the reduction in STAT activation in fibroblasts during Jak inhibitor treatment, we wondered whether Jak inhibitors might directly inhibit excess collagen production by fibroblasts. Consistent with prior reports (Yamamoto et al., 2000aYamamoto T. Eckes B. Krieg T. Bleomycin increases steady-state levels of type I collagen, fibronectin and decorin mRNAs in human skin fibroblasts.Arch Dermatol Res. 2000; 292: 556-561Crossref PubMed Scopus (43) Google Scholar), we found that normal cultured human fibroblasts upregulated COL1A1 in response to bleomycin. We found that this effect could be prevented with Jak inhibition (Figure 1h and Supplementary Table S2). In murine bleomycin-treated skin, reduced Col1a1 expression was also observed with Jak inhibitor treatment (Figure 1j). In summary, Jak inhibition leads to decreased inflammation in patients with recalcitrant morphea and EF; in some patients, even reversal of fibrosis is observed. We found that both tofacitinib and baricitinib were effective. Our observations in morphea and EF are consistent with what has been reported in patients with chronic graft-versus-host-disease (which can have fibrotic manifestations in the skin) (Zeiser et al., 2015Zeiser R. Burchert A. Lengerke C. Verbeek M. Maas-Bauer K. Metzelder S.K. et al.Ruxolitinib in corticosteroid-refractory graft-versus-host disease after allogeneic stem cell transplantation: a multicenter survey.Leukemia. 2015; 29: 2062-2068Crossref PubMed Scopus (343) Google Scholar) treated with ruxolitinib and in a single patient with systemic sclerosis treated with tofacitinib (Deverapalli and Rosmarin, 2018Deverapalli S.C. Rosmarin D. The use of JAK inhibitors in the treatment of progressive systemic sclerosis.J Eur Acad Dermatol Venereol. 2018; 32: e328Crossref PubMed Scopus (21) Google Scholar). We also demonstrate that Jak inhibition prevents fibrosis in a bleomycin-induced mouse model of morphea and directly decreases collagen production by fibroblasts. Our murine data suggest that ruxolitinib may have slightly more potent antifibrotic effects than tofacitinib. Wang et al., 2019Wang W. Bhattacharyya S. Marangoni R.G. Carns M. Dennis-Aren K. Yeldandi A. et al.The JAK/STAT pathway is activated in systemic sclerosis and is effectively targeted by tofacitinib.J Scleroderma Relat Disord. 2020; 5: 40-50Crossref Scopus (19) Google Scholar recently reported that tofacitinib can decrease bleomycin-induced fibrosis in mice but did not evaluate the effects of ruxolitinib, nor did they evaluate efficacy in patients. These data suggest that Jak inhibitors are targeted treatment for morphea and EF and highlight the need for additional study of Jak inhibitors for the treatment of fibrosing disorders in general. No new large-scale datasets were created as part of this work. William Damsky: https://orcid.org/0000-0003-0975-4071 Dhrumil Patel: https://orcid.org/0000-0003-0988-3922 Colton J. Garelli: https://orcid.org/0000-0003-0225-7910 Madhuri Garg: https://orcid.org/0000-0002-4809-542X Alice Wang: https://orcid.org/0000-0003-1771-2103 Karen Dresser: https://orcid.org/0000-0002-8599-4352 John E. Harris: https://orcid.org/0000-0002-3236-4680 Jillian Richmond: https://orcid.org/0000-0003-1589-6770 Brett King: https://orcid.org/0000-0002-4576-4616 WD has research funding from Pfizer, the manufacturer of tofacitinib; however, this was not used to support this work, which was initiated before the research funding. WD is also a consultant for Eli Lily. BK is an investigator for Concert Pharmaceuticals Inc, Eli Lilly and Company, and Pfizer Inc; he is a consultant to and/or has served on advisory boards for Aclaris Therapeutics, Arena Pharmaceuticals, Concert Pharmaceuticals Inc, Dermavant Sciences, Eli Lilly and Company, and Pfizer Inc; and he is on speakers' bureau for Pfizer Inc, Regeneron, and Sanofi Genzyme. JMR and JEH are inventors on patent application #62489191, "Diagnosis and Treatment of Vitiligo," which covers targeting IL-15 and Trm for the treatment of vitiligo. JEH is scientific founder of Villaris Therapeutics, Inc, which is focused on developing treatments for vitiligo. JMR and JEH are inventors on patent application #15/851,651, "Anti-human CXCR3 antibodies for the Treatment of Vitiligo," which covers targeting CXCR3 for the treatment of vitiligo. The authors thank M. Ahmed Refat, H. Jacobe, C. Torok, C. Feghali-Bostwick, T. Watanabe, and Z. Abbas for insightful comments and X. Fan for technical assistance. The authors thank E. Hast and D. Mekael and the Yale Dermatopathology lab. Supported by Calder Research Scholar Awards from the American Skin Association , Career Development Awards from the Dermatology Foundation (to JMR and WD), Target Identification in Lupus Award from the Lupus Research Alliance (to JMR), NIH awards AR09114 and AR07302 (to JEH), and the Ranjini and Ajay Poddar Resource Fund for Dermatologic Diseases Research (to BK). Microscopy equipment used for this study is maintained by the Morphology Core Facility, and tissue sectioning and pathology services are maintained by the University of Massachusetts Medical School Diabetes and Endocrinology Research Center Morphology Core. Experiments involving animals were approved by the University of Massachusetts Medical School Institutional Animal Care and Use Committee. Use of human materials was approved by the University of Massachusetts Medical School Institutional Review Board. For mouse model inquiries, correspondence should be addressed to: Dr. Jillian Richmond, UMass Medical School 364 Plantation St LRB 270E Worcester, MA 01605, [email protected] , @TCellTracker. Conceptualization: WD, JR, BK; Data Curation: WD, JR, BK; Formal Analysis: WD, JR, BK; Funding Acquisition: JH, JR, BK; Investigation: WD, DP, CG, MG, AW, KD, AD, JH, JR, BK; Resources: KD, AD, JH; Supervision: JR, BK; Writing - Original Draft Preparation: WD, JR, BK. The diagnosis of morphea or eosinophilic fasciitis (EF) was confirmed by skin biopsy in all patients, all of whom had been referred to our clinic for severe morphea and EF recalcitrant to standard therapies. These patients had severe, recalcitrant skin disease and were worsening with standard-of-care therapies; off-label use of Jak inhibitors was clinically indicated based on the judgment of the investigators. We discussed with all patients that tofacitinib or baricitinib were being used off-label for the treatment of their cutaneous disease. Only after verbal informed consent was obtained was Jak inhibitor therapy initiated at FDA-approved doses. Initial response was defined as the time to any detectable clinical improvement and includes subjective (patient history) and/or objective (physical examination) determinations. Maximal response was defined as the time required to reach the best effect (based on objective physical exam determination such as improvements in erythema and induration). Maximal responses were sustained on the medication. A historical series of biopsies from patients with morphea was assembled by searching the University of Massachusetts Medical School (UMMS) database. Research involving human biopsies was approved by the UMMS Institutional Review Board (Protocol 14848). The biopsies included in the immunohistochemical analysis are described in Supplementary Table S1. Normal skin samples included tips of ellipses from skin cancer excisions and mammoplasty and abdominoplasty specimens. All mice were housed in pathogen-free facilities at UMMS and procedures were approved by the UMMS Institutional Animal Care and Use Committee (Protocols 2266 and 2657) and in accordance with the National Institutes of Health Guide for the Care and Use of Laboratory Animals. Mice used for these studies were on the C57BL/6J (B6) background or a mixed 129 × C57BL/6 background that had been backcrossed to B6 for more than 10 generations. Age- and sex-matched mice were used, and both male and female mice of all strains were tested to avoid gender bias; sex-specific effects are reported in Supplementary Figure S2. Replicate experiments were performed two to five times. Bleomycin (Sigma-Aldrich, St. Louis, MO) solution was prepared by dissolving bleomycin in phosphate buffered saline at 1 mg/ml. Two locations on the dorsal flank were shaved and depilated for subcutaneous injections. Mice received injections of 50 μl of the bleomycin treatment solution or phosphate buffered saline vehicle at each site for a total of 100 μg/mouse for 12 days. At the end of the experiment, all the mice were killed and half of each site was frozen for collagen and RNA analysis, and the other half was prepared for histological analysis. Each mouse was pretreated with 30 mg/kg tofacitinib (Okiyama et al., 2014Okiyama N. Furumoto Y. Villarroel V.A. Linton J.T. Tsai W.L. Gutermuth J. et al.Reversal of CD8 T-cell-mediated mucocutaneous graft-versus-host-like disease by the JAK inhibitor tofacitinib.J Invest Dermatol. 2014; 134: 992-1000Abstract Full Text Full Text PDF PubMed Scopus (53) Google Scholar) in 200 μl of vehicle (ultrapure water with 0.5% methyl cellulose and 0.025% Tween 20) or vehicle alone via daily gavage for 5 days before use in the morphea model. Similarly, for experiments involving ruxolitinib, mice were pretreated with 10 mg/kg ruxolitinib (Shuey et al., 2016Shuey D.L. Oliver J. Zhou G. Roberts A. Results from oral gavage carcinogenicity studies of ruxolitinib in Tg.rasH2 mice and Sprague-Dawley (Crl:CD) rats.Regul Toxicol Pharmacol. 2016; 81: 305-315Crossref PubMed Scopus (6) Google Scholar) in 200 μl vehicle or vehicle alone via daily gavage for 5 days before use in morphea model. Treatments were continued throughout the model, and mice were killed on day 12 for analysis. The skin from the injection sites was fixed using 4% paraformaldehyde before paraffin embedding at the UMMS Diabetes and Endocrinology Research Center core facility. Samples were sectioned and stained with trichrome to visualize collagen deposition or hematoxylin and eosin to visualize inflammatory infiltrates. Images were taken with an Olympus BX51 microscope (Olympus, New Haven, CT) with ×10 and ×40 objective lenses, and epidermal and dermal thicknesses were measured with ImageJ software. The ratio of the dermal to epidermal thickness was calculated for each skin sample to control for variances in skin sampling. Immunohistochemistry was performed using heat mediated antigen retrieval with pH 6.0 citrate buffer. Phosphorylated STAT1 Tyr701 and phosphorylated STAT3 Tyr705 antibodies (Cell Signaling Technology, Danvers, MA) were incubated on the tissue according to the manufacturer's instructions. Signal was detected using anti-rabbit horseradish peroxidase–conjugated secondary antibody (Cell Signaling Technology) and a DAB chromogen (Vector Labs, Burlingame, CA). Slides were counterstained with hematoxylin. For quantification of mouse immunohistochemistry, 5 biologic replicates were analyzed for each phosphorylated STAT1 and phosphorylated STAT3. Three high power fields of dermis were selected at random and the number of positive cells was quantified (absolute number of cells per high power field). For quantification of human immunohistochemistry, 7 morphea biopsies were analyzed. Five high power fields of dermis were analyzed and the number of positive cells was quantified (absolute number of cells per high power field). Dermal tissue was isolated from eight-week-old (or older) mice which were age- and sex-matched as previously described. Briefly, epidermis was removed by incubating skin with 5 (tail) or 50 (ear and back) mg/ml Dispase II enzyme (Roche, Basel, Switzerland) 1 hour at 37 °C or overnight at 4 °C. Dermis tissue was digested for 45 minutes to 1 hour using 1 mg/ml collagenase IV and 2 mg/ml DNAse I (Sigma-Aldrich), and tissue was mechanically dissociated using the blunt end of a syringe. Samples were filtered through 70-μm mesh and were centrifuged at 330g for 10 minutes in FACS buffer (1% fetal bovine serum in phosphate buffered saline) containing 1 mg/ml DNAse I. For cell sorting of fibroblasts, dermis pellets were stained with anti-CD45 APC and anti-CD31 PE-Cy7, and all double negative singlets were collected by cell sorting with a FACS Aria. These cells, essentially all four phenotypes of mouse skin fibroblasts, were cultured in DMEM containing 10% fetal bovine serum and 1X penicillin/streptomycin for 5–7 days with one split. Cells were grown to 80% confluency and were used in collagen production assays. Human fibroblasts were obtained from a patient panniculectomy specimen and were isolated using 50 mg/ml Dispase II enzyme (Roche) overnight at 4 °C, and epidermis was removed with a razor blade. Dermis tissue was digested for 45 minutes to 1 hour using 1 mg/ml collagenase IV and 2 mg/ml DNAse I (Sigma-Aldrich), and tissue was mechanically dissociated using the blunt end of a syringe. Sample was filtered through 70-μm mesh and centrifuged at 330g for 10 minutes in FACS buffer (1% fetal bovine serum in phosphate buffered saline) containing 1 mg/ml DNAse I. The resulting cell suspension was plated and passaged three times using 10% fetal bovine serum and 1% penicillin/streptomycin in DMEM (cDMEM, Sigma-Aldrich) so that only fibroblasts remained in culture. Two additional human donor fibroblast specimens were obtained from ATCC (cat# PCS-201-012 lot# 80124171 and #70023966) and were cultured in cDMEM. Cells were grown to 80% confluency and were used in collagen production assays. For collagen mRNA assays, 1.8 μg/ml bleomycin (Sigma-Aldrich) (Nicolay et al., 2016Nicolay N.H. Ruhle A. Perez R.L. Trinh T. Sisombath S. Weber K.J. et al.Mesenchymal stem cells are sensitive to bleomycin treatment.Sci Rep. 2016; 6: 26645Crossref PubMed Scopus (30) Google Scholar) with 10 μM ruxolitinib or tofacitinib (LC Laboratories, Woburn, MA) (Heine et al., 2013Heine A. Held S.A. Daecke S.N. Wallner S. Yajnanarayana S.P. Kurts C. et al.The JAK-inhibitor ruxolitinib impairs dendritic cell function in vitro and in vivo.Blood. 2013; 122: 1192-1202Crossref PubMed Scopus (242) Google Scholar) or vehicle control (water) were added to wild-type fibroblasts for 3–4 hours, and cells were harvested by trypsinization and centrifugation. Pellets were snap frozen and were assessed RNA analysis as described subsequently. RNA was isolated from mouse back skin or fibroblast cell pellets using RNeasy kits (Qiagen, Hilden, Germany). cDNA was prepared using iScript kits (Bio-Rad, Hercules, CA), and quantitative PCR was performed with SYBR green kits (Bio-Rad). All statistical analyses were performed with GraphPad Prism software. Pairwise comparisons were analyzed using an unpaired Student's t-test. P-values < 0.05 were considered significant.Supplementary Figure S2Sex-specific effects of bleomycin and JAKi treatment in mice. Two outlier values are outside the axis of the data displayed for male vehicle (87.2 and 51.7). JAKi, Jak inhibitor; Ruxo, ruxolitinib; Tofa, tofacitinib; Veh, vehicle.View Large Image Figure ViewerDownload Hi-res image Download (PPT)Supplementary Table S1Histologic Case Series of Other Patients with MorpheaCase NumberAgeSexAnatomic siteDiagnosis based on clinicopathologic correlation149FLeft breastMorphea237FRight abdomenMorphea344FLeft breastMorphea446FBackMorphea524FLeft upper armMorphea666FRight upper abdomenMorphea723FLeft upper armMorpheaAbbreviation: F, female. Open table in a new tab Supplementary Table S2Primers Used for PCRPrimer nameSequenceMouse primers Col1a1-FGAGCGGAGAGTACTGGATCG Col1a1-RGCTTCTTTTCCTTGGGGTTC (Ito et al., 2010Ito Y. Toriuchi N. Yoshitaka T. Ueno-Kudoh H. Sato T. Yokoyama S. et al.The Mohawk homeobox gene is a critical regulator of tendon differentiation.Proc Natl Acad Sci USA. 2010; 107: 10538-10542Crossref PubMed Scopus (189) Google Scholar) B-actin-FGGCTGTATTCCCCTCCATCG B-actin-RCCAGTTGGTAACAATGCCATGT (Rashighi et al., 2014Rashighi M. Agarwal P. Richmond J.M. Harris T.H. Dresser K. Su M.W. et al.CXCL10 is critical for the progression and maintenance of depigmentation in a mouse model of vitiligo.Sci Transl Med. 2014; 6: 223ra23Crossref PubMed Scopus (260) Google Scholar) B2M-FCCGAACATACTGAACTGCTACGTAA B2M-RCCCGTTCTTCAGCATTTGGA (Groom et al., 2012Groom J.R. Richmond J. Murooka T.T. Sorensen E.W. Sung J.H. Bankert K. et al.CXCR3 chemokine receptor-ligand interactions in the lymph node optimize CD4+ T helper 1 cell differentiation.Immunity. 2012; 37: 1091-1103Abstract Full Text Full Text PDF PubMed Scopus (266) Google Scholar)Human primers COL1A1-FAGACAGTGATTGAATACAAAACCA COL1A1-RGGAGTTTACAGGAAGCAGACA HPRT1-FAGGGATTTGAATCATGTTTGTG HPRT1-RGGCTTTGTATTTTGCTTTTCCAAbbreviations: F, forward; R, reverse. Open table in a new tab Abbreviation: F, female. Abbreviations: F, forward; R, reverse.
Referência(s)