Elevated soluble CD226 in Takayasu arteritis is useful for differentiation from giant cell arteritis and is associated with the disease activity and prognosis

Elevated soluble CD226 in Takayasu arteritis is useful for differentiation from giant cell arteritis and is associated with the disease activity and prognosis | 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 Research Article Elevated soluble CD226 in Takayasu arteritis is useful for differentiation from giant cell arteritis and is associated with the disease activity and prognosis Miki Nakano, Masahiro Ayano, Shoichi Fukui, Nozomi Iwanaga, Tomofumi Tatsutani, and 7 more This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-4115713/v1 This work is licensed under a CC BY 4.0 License Status: Posted Version 1 posted You are reading this latest preprint version Abstract Background Takayasu arteritis (TAK) is characterized by vascular injury, in which endothelial cells and immune cells, such as natural killer (NK) cells, have key roles. CD226 is an activating receptor expressed on the cell membrane of NK cells and T cells, and the soluble form of CD226 (sCD226) is increased in diseases involving these cells. Therefore, we investigated the utility of serum sCD226 as a biomarker for TAK. Methods Serum sCD226 levels were measured using an enzyme-linked immunosorbent assay in 34 patients with TAK and 21 patients with giant cell arteritis (GCA). The associations between sCD226 levels and the angiographic classification, disease activity, and prognosis of TAK were analysed. Results Serum sCD226 levels were significantly higher in TAK patients than in GCA patients. In patients with TAK, serum sCD226 levels were significantly elevated in the group of type Ⅴ, which had the most extensive lesions, compared with the group of other types excluding type Ⅴ. Serum sCD226 levels were also elevated in patients with active TAK and in those with poor responses to corticosteroids. Regarding prognosis, the cumulative probability of relapse was higher in patients with high sCD226 levels than in those with low sCD226 levels. Conclusions Serum sCD226 levels differentiated TAK from GCA and were associated with disease activity and relapse of TAK. Serum sCD226 may be a useful biomarker for the management of TAK. Takayasu arteritis giant cell arteritis soluble CD226 disease activity relapse biomarker Figures Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 Background Takayasu arteritis (TAK) and giant cell arteritis (GCA) are defined as large vessel vasculitis (LVV) [ 1 ]. Although TAK and GCA are classified as different diseases based on age, clinical symptoms, and vascular imaging [ 2 , 3 ], they share some similarities, including constitutional symptoms, headache, ischemic manifestations, and elevated erythrocyte sedimentation rate and C-reactive protein levels [ 4 , 5 ]. Although the onset of TAK was thought to be younger than that of GCA, late-onset TAK has been recognized recently [ 5 , 6 ]. These things often make it difficult to distinguish between TAK and GCA. Furthermore, the management of TAK is also challenging. Diagnosis of TAK can be difficult because of its non-specific symptoms in the early phase [ 7 ] and the lack of characteristic autoantibodies or biomarkers [ 5 , 8 ]. As the disease progresses, TAK frequently relapses [ 5 ], often without adequate dose reduction of corticosteroids, leading to arterial stenosis and occlusion, resulting in life-threatening ischemic symptoms [ 9 ]. Therefore, the correct and early diagnosis of TAK is essential, along with monitoring disease activity and predicting prognosis. However, proper biomarkers have not yet been established [ 5 , 10 – 12 ]. Although the pathogenesis of TAK is not fully understood, it is characterized by vascular injury, in which endothelial cells and immune cells play key roles [ 5 , 12 – 14 ]. Genetic factors are also involved in pathogenesis [ 5 , 12 – 14 ], and recent genome-wide association studies (GWAS) have revealed the importance of CD8 + T cells and natural killer (NK) cells [ 15 ]. Because NK cells mainly influence vascular injury in TAK [ 12 – 14 , 16 – 18 ], they play a pivotal role in TAK. CD226 is expressed on the surface of NK cells and T cells, acting as an activating receptor [ 19 – 22 ]. The CD226 ligands CD155 and CD112 are expressed on endothelial cells and epithelial cells [ 20 ]. CD226 is involved in various immune functions in NK cells and endothelial cells, and the interactions between these cells [ 19 – 23 ]. The soluble form of CD226 (sCD226), which is shed from the membrane type of CD226 (mCD226) by a certain protease [ 24 ], has been reported to be a useful biomarker for cancers [ 25 , 26 ] and acute graft-versus-host disease [ 24 , 27 ]. In addition, increased serum sCD226 levels were accompanied by decreased mCD226 on peripheral blood mononuclear cells [ 25 ], NK cells, and CD8 + T cells and reflected disease activity [ 26 ], indicating that sCD226 may be a biomarker reflecting the immune response in diseases in which NK cells and CD8 + T cells play key roles. Therefore, sCD226 can be a valuable biomarker for TAK. This study aimed to clarify the utility of sCD226 as a biomarker for TAK by measuring serum sCD226 levels via enzyme-linked immunosorbent assay (ELISA) in patients with TAK. Methods Study population We investigated 34 TAK and 21 GCA patients treated at Kyushu University Hospital, Saga University Hospital, Nagasaki University Hospital, and NHO Nagasaki Medical Center, between 2004 and 2020. Patients who met the 2022 American College of Rheumatology (ACR)/ European League Against Rheumatism (EULAR) classification criteria for TAK [ 2 ] and GCA [ 3 ] were enrolled in this study; those with infection or cancer at the time of serum sample collection were excluded. Some of these patients were treated with corticosteroids, immunosuppressive agents, and biological agents, either as monotherapy or in combination. This study was approved by the ethics committees of Kyushu University Hospital (approval number 30–282) and Nagasaki University Hospital (approval number 15072753) in accordance with the Helsinki Declaration. All participants gave written informed consent. Data collection The following information was obtained from the medical records of the patients: demographic data, clinical manifestations, laboratory findings, medications at baseline and after treatment, and relapse after treatment. Patients with TAK were classified into types Ⅰ–Ⅴ using contrast-enhanced computed tomography based on the angiographic classification of Hata et al. [ 28 ]. Disease activity of TAK was evaluated using the National Institutes of Health (NIH) criteria: with active TAK defined as having an NIH score of ≥ 2 [ 29 ]. Relapse of TAK was defined as the worsening of an NIH score of ≥ 2 [ 29 ]. We defined patients who achieved an inactive state only with corticosteroids as having a good response to corticosteroids and others as having a poor response. Enzyme-linked immunosorbent assay Serum sCD226 levels were measured using sandwich ELISA according to previous reports [ 24 , 30 ]. Briefly, 96-well plates were coated with purified anti-human CD226 (DNAM-1) antibody (TX25; BioLegend, San Diego, CA, USA) (8 µg/ml, 100 µl/well) for 2 hours at room temperature. The plates were washed with washing buffer (0.05% Tween 20), and then blocked using a blocking buffer (1% BSA in PBS, 100 µl/well) for 2 hours at room temperature. After washing, recombinant human DNAM-1/CD226 Fc chimera protein (as a standard) (R&D Systems, Minneapolis, MN, USA) and serum samples were added at 100 µl/well and incubated overnight at 4°C. The plates were washed and incubated with human DNAM-1/CD226 biotinylated antibody (R&D Systems) (0.6 µg/ml, 100 µl/well) for 1 hour at room temperature, and then washed. Streptavidin–horseradish peroxidase (R&D Systems) (1:200 in a washing buffer, 100 µl/well) was added and incubated for 30 minutes at room temperature. The plates were washed and reacted with the 3,3′,5,5′-tetramethylbenzidine substrate reagent set (BD Biosciences, San Jose, CA, USA) (100 µl/well) for 20 minutes at room temperature. The reaction was terminated using H 2 SO 4 (2N) (50 µl/well). Absorbance was measured at 450 nm using a microtiter plate reader (Thermo Fisher Scientific, Waltham, MA, USA). All values were determined in duplicate. The assay range was 0.1–20.0 ng/ml. Statistical analysis The data are expressed as median and interquartile range unless otherwise stated. Differences between the two groups were evaluated using Student’s t-test for normally distributed continuous variables or using the Mann–Whitney U test for non-normally distributed variables. Relationships between two continuous variables were evaluated using Spearman’s rank correlation. The cumulative probability of relapse was analysed using the Kaplan–Meier method and log-rank test. All tests were two-tailed, with P -values < 0.05 considered statistically significant. All analyses were performed using JMP software, version 17 (SAS Institute, Cary, NC, USA). Results Serum sCD226 levels are elevated in patients with TAK To investigate the association between sCD226 and LVV, serum sCD226 levels were measured using ELISA in 34 patients with TAK and 21 patients with GCA. Although no significant differences were observed between TAK and GCA patients in terms of gender, TAK patients were significantly younger than GCA patients because of the classification criteria [ 2 , 3 ]. The baseline characteristics of patients with TAK and GCA are presented in Table 1. Serum sCD226 levels were significantly elevated in patients with TAK than in patients with GCA (Fig. 1 ). Serum sCD226 levels are elevated in patients with active TAK and reflect disease activity To assess the association between sCD226 levels and the extent of vascular lesions, the relationship between serum sCD226 levels and the angiographic classification of TAK was evaluated [ 28 ]. Serum sCD226 levels were significantly increased in the group of type Ⅴ, which had the most extensive lesions, compared with the group of other types excluding type Ⅴ (Fig. 2 ). Next, the relationship between serum sCD226 levels and TAK disease activity was assessed. Serum sCD226 levels were elevated in patients with active TAK (Fig. 3 ). There were 22 drug-naive patients with active TAK in this study. To examine the relationship between sCD226 levels and response to therapy, we compared serum sCD226 levels between patients with good and poor responses to corticosteroids, revealing elevations in those with poor responses (Fig. 4 ). Serum sCD226 levels can predict disease relapse Relapse after treatment was observed in 6 out of 22 drug-naive active TAK patients. We defined a median sCD226 level of 3.3 ng/ml among patients with TAK as the cut-off value to classify patients as having high or low sCD226 levels. The cumulative probability of relapse was higher in patients with high sCD226 levels (Fig. 5 , shown in solid lines) than in those with low sCD226 levels (Fig. 5 , shown in dashed lines). Discussion Our results revealed that serum sCD226 levels were higher in TAK patients than in GCA patients, significantly increased in active TAK patients, and associated with disease activity and prognosis. In this study, we aimed to investigate the association between sCD226 and LVV and found that serum sCD226 levels were significantly elevated in patients with TAK compared with GCA. Previous reports on cancer patients showed an increase in serum sCD226 levels accompanied by a decrease in mCD226 on immune cells, such as NK cells [ 25 , 26 ]. Regarding autoimmune diseases, our previous study demonstrated that serum sCD226 levels were increased in patients with active systemic lupus erythematosus (SLE) [ 30 ], whereas Huang et al. showed that expression of mCD226 on NK cells was decreased in patients with active SLE [ 31 ]. These findings suggest the involvement of CD226 and the association of serum sCD226 levels and mCD226 on NK cells in SLE, although further analyses are needed. The present study showed that serum sCD226 levels were elevated in TAK patients but not in GCA patients. Despite their similarities in pathogenesis, TAK and GCA have key differences [ 4 , 14 ], particularly in their roles in NK cells [ 12 – 18 ]. Our results may reflect alterations in NK cells, which are important in the pathogenesis of TAK. However, the present study did not examine immune cells, including NK cells. The mechanism underlying the elevation of serum sCD226 levels in TAK needs to be further studied in the future. Our study showed that serum sCD226 levels were elevated in patients with active TAK and those with poor responses to corticosteroids and that the cumulative probability of relapse was higher in patients with high sCD226 levels. Monitoring disease activity in patients with LVV is recommended by the 2018 EULAR recommendations [ 10 ] and the 2021 ACR guidelines [ 11 ], and imaging modalities have been advanced recently [ 32 ]. However, appropriate biomarkers have not yet been established [ 5 , 10 – 12 ]. In our study, serum sCD226 levels were associated with disease activity and prognosis. Thus, sCD226 may be a useful biomarker for TAK. Our results suggest an association between CD226 and TAK although the mechanism remains unknown. Other autoimmune diseases, such as SLE [ 30 , 31 , 33 – 37 ], rheumatoid arthritis (RA) [ 36 , 38 – 42 ], and systemic sclerosis (SSc) [ 43 – 45 ], have been associated with CD226, specifically in terms of genetic factors and immune responses. Several GWAS have reported associations between the nonsynonymous rs763361 polymorphism in CD226 and SLE [ 33 – 36 ], RA [ 36 , 38 – 40 ], and SSc [ 43 , 44 ]. In addition, some studies on immune cells, including our group, have suggested the involvement of CD226 in autoimmune diseases [ 30 , 31 , 37 , 41 , 45 ]. Regarding the association between CD226 and LVV, there are few GWAS and no immunological studies; only one Spanish study showed that CD226 gene variants are not involved in GCA [ 46 ], and the association between CD226 gene variants and TAK is not revealed. In our analysis, serum sCD226 levels were increased in the group of type Ⅴ, which had the most extensive lesions, indicating that sCD226 may reflect the extent of vascular lesions and affect endothelial cells as well. Given that mCD226 expression on CD8 + T cells was associated with endothelial injury [ 45 ] and sCD226 could directly affect cells expressing the ligand, such as cancer cells [ 25 , 47 ], sCD226 may be involved in the pathogenesis of TAK. Based on this result, further GWAS and immunological studies are required. Our study had several limitations. First, this study had a small sample size with only Japanese patients. Since the incidence of TAK varies according to ancestry [ 5 , 12 ], further studies should include a larger sample size and/or other ancestries. Second, the functions of sCD226 remain unknown, and these need to be clarified in further investigations. Finally, our study was retrospective. To ensure the association between sCD226 levels with the disease activity and prognosis of TAK, a prospective study with longitudinal assessments should be conducted. Conclusions Serum sCD226 levels were elevated in patients with TAK and were associated with disease activity and disease relapse. Serum sCD226 may be a useful biomarker for managing TAK, and its monitoring can help in the precise management of TAK. Abbreviations TAK: Takayasu arteritis; GCA: giant cell arteritis; LVV: large vessel vasculitis; GWAS: genome-wide association studies; NK: natural killer; sCD226: soluble CD226; mCD226: membrane CD226; ELISA: enzyme-linked immunosorbent assay; ACR: American College of Rheumatology; EULAR: European League Against Rheumatism; NIH: National Institutes of Health; SLE: systemic lupus erythematosus; RA: rheumatoid arthritis; SSc: systemic sclerosis. Declarations Ethics approval and consent to participate This study was approved by the ethics committees of Kyushu University Hospital (approval number 30–282) and Nagasaki University Hospital (approval number 15072753) in accordance with the Helsinki Declaration. All participants gave written informed consent. Consent for publication Not applicable. Availability of data and materials All data generated or analysed during this study are included in this published article. Competing interests The authors declare that they have no competing interests. Funding This work was supported by Japan Society for the Promotion of Science [grant number JSPS KAKENHI 19K17887 and JSPS KAKENHI JP23K06868]. Authors' contributions MN and MAy participated in study conception and design. MN, MAy, SF, NI, ATK, KM, and YT participated in data acquisition. MN and TT performed the ELISA experiments. MN and MAy participated in data analysis. 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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-4115713","acceptedTermsAndConditions":true,"allowDirectSubmit":true,"archivedVersions":[],"articleType":"Research Article","associatedPublications":[],"authors":[{"id":281373668,"identity":"2f60e418-69a7-4d81-aaad-7d7a4eaef628","order_by":0,"name":"Miki Nakano","email":"","orcid":"","institution":"Kyushu University Graduate School of Medical Sciences","correspondingAuthor":false,"prefix":"","firstName":"Miki","middleName":"","lastName":"Nakano","suffix":""},{"id":281373669,"identity":"1424d952-8dfa-421c-a128-5f1fbcc7c35d","order_by":1,"name":"Masahiro Ayano","email":"data:image/png;base64,iVBORw0KGgoAAAANSUhEUgAAAZAAAAAyAQMAAABI0h/eAAAABlBMVEX///8AAABVwtN+AAAACXBIWXMAAA7EAAAOxAGVKw4bAAABTklEQVRIie2RP0vEMBiH3xKoS/QcI+L1K6QU7gb/fZWUwt1SxEkKglaEczm4taDYr5Dp5isFb8k5B+rgdNMNddDFxaToXXuIOArmGdKQvk9+bxIAg+Fvg2GixraNkPrqKVn+mvyoeFsbNvu9ovHvW5iuKw1aSTB/LmHsODezLI8i8GyEXzmI3E9vhxRhOHLAmtVjiOx13QQKl4sTlgmhz7I5liBznz8JrQRuDI+sHiNZZxdDYXEIaXY10ClaKZVCwu47BqTKBa0Zjuy/aeU4HS0qxR8gPK+UNAl1yuW6QmVYpfixDJeKXTWmV5SSryuuWJztJLQIuFQpsSCqMbsjmeh7XPZOrTs6dQfNs7Sn/TEpo+IwHYXeSxwdtJ1RPpflw/5emgQcFtG50yKNG4NtvcEq9/MtGFx/dQ5gE9F8mMYGKy7qNcPvawwGg+Gf8AG7JIBQWcBkSQAAAABJRU5ErkJggg==","orcid":"","institution":"Kyushu University Graduate School of Medical Sciences","correspondingAuthor":true,"prefix":"","firstName":"Masahiro","middleName":"","lastName":"Ayano","suffix":""},{"id":281373670,"identity":"c6c9f591-ff43-496a-9772-c7d20c6ce6e6","order_by":2,"name":"Shoichi Fukui","email":"","orcid":"","institution":"Nagasaki University Graduate School of Biomedical Sciences","correspondingAuthor":false,"prefix":"","firstName":"Shoichi","middleName":"","lastName":"Fukui","suffix":""},{"id":281373671,"identity":"3fe65d0b-6be3-479e-9706-91a3bb5beae3","order_by":3,"name":"Nozomi Iwanaga","email":"","orcid":"","institution":"NHO Nagasaki Medical Center","correspondingAuthor":false,"prefix":"","firstName":"Nozomi","middleName":"","lastName":"Iwanaga","suffix":""},{"id":281373674,"identity":"e199462f-416f-4997-bfb1-28fad49f4658","order_by":4,"name":"Tomofumi Tatsutani","email":"","orcid":"","institution":"Kyushu University Graduate School of Medical Sciences","correspondingAuthor":false,"prefix":"","firstName":"Tomofumi","middleName":"","lastName":"Tatsutani","suffix":""},{"id":281373677,"identity":"fc4dd523-5693-4935-806d-4195cc762165","order_by":5,"name":"Ayako Takaki-Kuwahara","email":"","orcid":"","institution":"Kyushu University Graduate School of Medical Sciences","correspondingAuthor":false,"prefix":"","firstName":"Ayako","middleName":"","lastName":"Takaki-Kuwahara","suffix":""},{"id":281373678,"identity":"8ac13010-cba9-4910-9be2-f0c6f01ee51a","order_by":6,"name":"Yasutaka Kimoto","email":"","orcid":"","institution":"Kyushu University Graduate School of Medical Sciences","correspondingAuthor":false,"prefix":"","firstName":"Yasutaka","middleName":"","lastName":"Kimoto","suffix":""},{"id":281373681,"identity":"160cfb22-8344-4346-84e6-2060d79d649b","order_by":7,"name":"Mitsuteru Akahoshi","email":"","orcid":"","institution":"Saga University","correspondingAuthor":false,"prefix":"","firstName":"Mitsuteru","middleName":"","lastName":"Akahoshi","suffix":""},{"id":281373684,"identity":"f0f53ac1-cf6a-4be9-8a84-13792924150e","order_by":8,"name":"Kiyoshi Migita","email":"","orcid":"","institution":"Fukushima Medical University School of Medicine","correspondingAuthor":false,"prefix":"","firstName":"Kiyoshi","middleName":"","lastName":"Migita","suffix":""},{"id":281373687,"identity":"2ed01c5b-ceed-4073-affd-da640d8e1872","order_by":9,"name":"Atsushi Kawakami","email":"","orcid":"","institution":"Nagasaki University Graduate School of Biomedical Sciences","correspondingAuthor":false,"prefix":"","firstName":"Atsushi","middleName":"","lastName":"Kawakami","suffix":""},{"id":281373689,"identity":"11ae6c00-aef8-4e2d-8549-15aae829818b","order_by":10,"name":"Yoshifumi Tada","email":"","orcid":"","institution":"Saga University","correspondingAuthor":false,"prefix":"","firstName":"Yoshifumi","middleName":"","lastName":"Tada","suffix":""},{"id":281373691,"identity":"846115bd-0816-4ae8-8859-8353a420dc96","order_by":11,"name":"Hiroaki Niiro","email":"","orcid":"","institution":"Kyushu University Graduate School of Medical Sciences","correspondingAuthor":false,"prefix":"","firstName":"Hiroaki","middleName":"","lastName":"Niiro","suffix":""}],"badges":[],"createdAt":"2024-03-17 06:44:18","currentVersionCode":1,"declarations":"","doi":"10.21203/rs.3.rs-4115713/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-4115713/v1","draftVersion":[],"editorialEvents":[],"editorialNote":"","failedWorkflow":false,"files":[{"id":53196729,"identity":"0de0de0d-a7df-40df-a006-89c28f4e59fa","added_by":"auto","created_at":"2024-03-21 18:34:44","extension":"png","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":18742,"visible":true,"origin":"","legend":"\u003cp\u003eSerum\u003cstrong\u003e \u003c/strong\u003esCD226 levels in patients with Takayasu arteritis and giant cell arteritis\u003c/p\u003e\n\u003cp\u003eSerum sCD226 levels were compared between patients with TAK and GCA. Each data point represents a single subject. The horizontal lines show the median. Statistical differences among groups were evaluated using the Mann–Whitney \u003cem\u003eU\u003c/em\u003e test. sCD226: soluble CD226; TAK: Takayasu arteritis; GCA: giant cell arteritis.\u003c/p\u003e","description":"","filename":"figure1TAK.png","url":"https://assets-eu.researchsquare.com/files/rs-4115713/v1/9c1fafd729051f4637331675.png"},{"id":53196727,"identity":"bf9f46c4-5cdb-42de-a5d3-f6bd371f7d14","added_by":"auto","created_at":"2024-03-21 18:34:44","extension":"png","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":18283,"visible":true,"origin":"","legend":"\u003cp\u003eSerum\u003cstrong\u003e \u003c/strong\u003esCD226 levels in patients with Takayasu arteritis\u003c/p\u003e\n\u003cp\u003eSerum sCD226 levels were compared between the group of type Ⅰ–Ⅳ and type Ⅴ in TAK patients. Each data point represents a single subject. The horizontal lines show the median. Statistical differences among groups were evaluated using the Mann–Whitney \u003cem\u003eU\u003c/em\u003e test. sCD226: soluble CD226; TAK: Takayasu arteritis.\u003c/p\u003e","description":"","filename":"figure2TAK.png","url":"https://assets-eu.researchsquare.com/files/rs-4115713/v1/7176348c2e5274345cd2a1cf.png"},{"id":53196732,"identity":"0c0dcc02-a64c-4604-9c01-8d2d17d08c2b","added_by":"auto","created_at":"2024-03-21 18:34:44","extension":"png","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":17815,"visible":true,"origin":"","legend":"\u003cp\u003eSerum\u003cstrong\u003e \u003c/strong\u003esCD226 levels in patients with Takayasu arteritis\u003c/p\u003e\n\u003cp\u003eSerum sCD226 levels were compared between active and inactive TAK patients. Each data point represents a single subject. The horizontal lines show the median. Statistical differences among groups were evaluated using the Mann–Whitney \u003cem\u003eU\u003c/em\u003e test. sCD226: soluble CD226; TAK: Takayasu arteritis.\u003c/p\u003e","description":"","filename":"figure3TAK.png","url":"https://assets-eu.researchsquare.com/files/rs-4115713/v1/52b2e940175436732a5d85e3.png"},{"id":53197821,"identity":"1a12923b-5562-43bd-b427-8fbf9ead9e61","added_by":"auto","created_at":"2024-03-21 18:42:44","extension":"png","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":24382,"visible":true,"origin":"","legend":"\u003cp\u003eSerum\u003cstrong\u003e \u003c/strong\u003esCD226 levels in drug-naive patients with active Takayasu arteritis\u003c/p\u003e\n\u003cp\u003eSerum sCD226 levels were compared between patients with good and poor responses to corticosteroids. Each data point represents a single subject. The horizontal lines show the median. Statistical differences among groups were evaluated using the Mann–Whitney \u003cem\u003eU\u003c/em\u003e test. sCD226: soluble CD226.\u003c/p\u003e","description":"","filename":"figure4TAK.png","url":"https://assets-eu.researchsquare.com/files/rs-4115713/v1/817547caa1f6b11d93eddaf2.png"},{"id":53197822,"identity":"a900e160-fd2b-493b-9414-aace8cb53cfa","added_by":"auto","created_at":"2024-03-21 18:42:44","extension":"png","order_by":5,"title":"Figure 5","display":"","copyAsset":false,"role":"figure","size":19890,"visible":true,"origin":"","legend":"\u003cp\u003eKaplan–Meier analysis of the cumulative probability of relapse\u003c/p\u003e\n\u003cp\u003eCurves were compared between patients with TAK having high and low sCD226 levels using log-rank tests. TAK: Takayasu arteritis; sCD226: soluble CD226.\u003c/p\u003e","description":"","filename":"figure5TAK.png","url":"https://assets-eu.researchsquare.com/files/rs-4115713/v1/44afd1e6706e4879ba8e132b.png"},{"id":53344696,"identity":"f9cf1660-93a2-467f-932e-8640d5eedc41","added_by":"auto","created_at":"2024-03-24 19:07:37","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":527235,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-4115713/v1/32b3c433-8d27-4c38-9f56-c8d88c763aea.pdf"},{"id":53196728,"identity":"0f7db05c-8613-48df-a34a-cde127d25e24","added_by":"auto","created_at":"2024-03-21 18:34:44","extension":"docx","order_by":1,"title":"","display":"","copyAsset":false,"role":"supplement","size":33639,"visible":true,"origin":"","legend":"","description":"","filename":"Table1.docx","url":"https://assets-eu.researchsquare.com/files/rs-4115713/v1/e1dde5dca212e0d2c92da2f6.docx"}],"financialInterests":"No competing interests reported.","formattedTitle":"Elevated soluble CD226 in Takayasu arteritis is useful for differentiation from giant cell arteritis and is associated with the disease activity and prognosis","fulltext":[{"header":"Background","content":"\u003cp\u003eTakayasu arteritis (TAK) and giant cell arteritis (GCA) are defined as large vessel vasculitis (LVV) [\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e]. Although TAK and GCA are classified as different diseases based on age, clinical symptoms, and vascular imaging [\u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e, \u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e], they share some similarities, including constitutional symptoms, headache, ischemic manifestations, and elevated erythrocyte sedimentation rate and C-reactive protein levels [\u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e4\u003c/span\u003e, \u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e5\u003c/span\u003e]. Although the onset of TAK was thought to be younger than that of GCA, late-onset TAK has been recognized recently [\u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e5\u003c/span\u003e, \u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e6\u003c/span\u003e]. These things often make it difficult to distinguish between TAK and GCA. Furthermore, the management of TAK is also challenging. Diagnosis of TAK can be difficult because of its non-specific symptoms in the early phase [\u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e] and the lack of characteristic autoantibodies or biomarkers [\u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e5\u003c/span\u003e, \u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e8\u003c/span\u003e]. As the disease progresses, TAK frequently relapses [\u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e5\u003c/span\u003e], often without adequate dose reduction of corticosteroids, leading to arterial stenosis and occlusion, resulting in life-threatening ischemic symptoms [\u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e9\u003c/span\u003e]. Therefore, the correct and early diagnosis of TAK is essential, along with monitoring disease activity and predicting prognosis. However, proper biomarkers have not yet been established [\u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e5\u003c/span\u003e, \u003cspan additionalcitationids=\"CR11\" citationid=\"CR10\" class=\"CitationRef\"\u003e10\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e12\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eAlthough the pathogenesis of TAK is not fully understood, it is characterized by vascular injury, in which endothelial cells and immune cells play key roles [\u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e5\u003c/span\u003e, \u003cspan additionalcitationids=\"CR13\" citationid=\"CR12\" class=\"CitationRef\"\u003e12\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e14\u003c/span\u003e]. Genetic factors are also involved in pathogenesis [\u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e5\u003c/span\u003e, \u003cspan additionalcitationids=\"CR13\" citationid=\"CR12\" class=\"CitationRef\"\u003e12\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e14\u003c/span\u003e], and recent genome-wide association studies (GWAS) have revealed the importance of CD8\u003csup\u003e+\u003c/sup\u003e T cells and natural killer (NK) cells [\u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e15\u003c/span\u003e]. Because NK cells mainly influence vascular injury in TAK [\u003cspan additionalcitationids=\"CR13\" citationid=\"CR12\" class=\"CitationRef\"\u003e12\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e14\u003c/span\u003e, \u003cspan additionalcitationids=\"CR17\" citationid=\"CR16\" class=\"CitationRef\"\u003e16\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e18\u003c/span\u003e], they play a pivotal role in TAK.\u003c/p\u003e \u003cp\u003eCD226 is expressed on the surface of NK cells and T cells, acting as an activating receptor [\u003cspan additionalcitationids=\"CR20 CR21\" citationid=\"CR19\" class=\"CitationRef\"\u003e19\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e22\u003c/span\u003e]. The CD226 ligands CD155 and CD112 are expressed on endothelial cells and epithelial cells [\u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e20\u003c/span\u003e]. CD226 is involved in various immune functions in NK cells and endothelial cells, and the interactions between these cells [\u003cspan additionalcitationids=\"CR20 CR21 CR22\" citationid=\"CR19\" class=\"CitationRef\"\u003e19\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e23\u003c/span\u003e]. The soluble form of CD226 (sCD226), which is shed from the membrane type of CD226 (mCD226) by a certain protease [\u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e24\u003c/span\u003e], has been reported to be a useful biomarker for cancers [\u003cspan citationid=\"CR25\" class=\"CitationRef\"\u003e25\u003c/span\u003e, \u003cspan citationid=\"CR26\" class=\"CitationRef\"\u003e26\u003c/span\u003e] and acute graft-versus-host disease [\u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e24\u003c/span\u003e, \u003cspan citationid=\"CR27\" class=\"CitationRef\"\u003e27\u003c/span\u003e]. In addition, increased serum sCD226 levels were accompanied by decreased mCD226 on peripheral blood mononuclear cells [\u003cspan citationid=\"CR25\" class=\"CitationRef\"\u003e25\u003c/span\u003e], NK cells, and CD8\u003csup\u003e+\u003c/sup\u003e T cells and reflected disease activity [\u003cspan citationid=\"CR26\" class=\"CitationRef\"\u003e26\u003c/span\u003e], indicating that sCD226 may be a biomarker reflecting the immune response in diseases in which NK cells and CD8\u003csup\u003e+\u003c/sup\u003e T cells play key roles. Therefore, sCD226 can be a valuable biomarker for TAK.\u003c/p\u003e \u003cp\u003eThis study aimed to clarify the utility of sCD226 as a biomarker for TAK by measuring serum sCD226 levels via enzyme-linked immunosorbent assay (ELISA) in patients with TAK.\u003c/p\u003e"},{"header":"Methods","content":"\u003cdiv id=\"Sec3\" class=\"Section2\"\u003e \u003ch2\u003eStudy population\u003c/h2\u003e \u003cp\u003eWe investigated 34 TAK and 21 GCA patients treated at Kyushu University Hospital, Saga University Hospital, Nagasaki University Hospital, and NHO Nagasaki Medical Center, between 2004 and 2020. Patients who met the 2022 American College of Rheumatology (ACR)/ European League Against Rheumatism (EULAR) classification criteria for TAK [\u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e] and GCA [\u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e] were enrolled in this study; those with infection or cancer at the time of serum sample collection were excluded. Some of these patients were treated with corticosteroids, immunosuppressive agents, and biological agents, either as monotherapy or in combination.\u003c/p\u003e \u003cp\u003e This study was approved by the ethics committees of Kyushu University Hospital (approval number 30\u0026ndash;282) and Nagasaki University Hospital (approval number 15072753) in accordance with the Helsinki Declaration. All participants gave written informed consent.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec4\" class=\"Section2\"\u003e \u003ch2\u003eData collection\u003c/h2\u003e \u003cp\u003eThe following information was obtained from the medical records of the patients: demographic data, clinical manifestations, laboratory findings, medications at baseline and after treatment, and relapse after treatment. Patients with TAK were classified into types Ⅰ\u0026ndash;Ⅴ using contrast-enhanced computed tomography based on the angiographic classification of Hata et al. [\u003cspan citationid=\"CR28\" class=\"CitationRef\"\u003e28\u003c/span\u003e]. Disease activity of TAK was evaluated using the National Institutes of Health (NIH) criteria: with active TAK defined as having an NIH score of \u0026ge;\u0026thinsp;2 [\u003cspan citationid=\"CR29\" class=\"CitationRef\"\u003e29\u003c/span\u003e]. Relapse of TAK was defined as the worsening of an NIH score of \u0026ge;\u0026thinsp;2 [\u003cspan citationid=\"CR29\" class=\"CitationRef\"\u003e29\u003c/span\u003e]. We defined patients who achieved an inactive state only with corticosteroids as having a good response to corticosteroids and others as having a poor response.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec5\" class=\"Section2\"\u003e \u003ch2\u003eEnzyme-linked immunosorbent assay\u003c/h2\u003e \u003cp\u003eSerum sCD226 levels were measured using sandwich ELISA according to previous reports [\u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e24\u003c/span\u003e, \u003cspan citationid=\"CR30\" class=\"CitationRef\"\u003e30\u003c/span\u003e]. Briefly, 96-well plates were coated with purified anti-human CD226 (DNAM-1) antibody (TX25; BioLegend, San Diego, CA, USA) (8 \u0026micro;g/ml, 100 \u0026micro;l/well) for 2 hours at room temperature. The plates were washed with washing buffer (0.05% Tween 20), and then blocked using a blocking buffer (1% BSA in PBS, 100 \u0026micro;l/well) for 2 hours at room temperature. After washing, recombinant human DNAM-1/CD226 Fc chimera protein (as a standard) (R\u0026amp;D Systems, Minneapolis, MN, USA) and serum samples were added at 100 \u0026micro;l/well and incubated overnight at 4\u0026deg;C. The plates were washed and incubated with human DNAM-1/CD226 biotinylated antibody (R\u0026amp;D Systems) (0.6 \u0026micro;g/ml, 100 \u0026micro;l/well) for 1 hour at room temperature, and then washed. Streptavidin\u0026ndash;horseradish peroxidase (R\u0026amp;D Systems) (1:200 in a washing buffer, 100 \u0026micro;l/well) was added and incubated for 30 minutes at room temperature. The plates were washed and reacted with the 3,3\u0026prime;,5,5\u0026prime;-tetramethylbenzidine substrate reagent set (BD Biosciences, San Jose, CA, USA) (100 \u0026micro;l/well) for 20 minutes at room temperature. The reaction was terminated using H\u003csub\u003e2\u003c/sub\u003eSO\u003csub\u003e4\u003c/sub\u003e (2N) (50 \u0026micro;l/well). Absorbance was measured at 450 nm using a microtiter plate reader (Thermo Fisher Scientific, Waltham, MA, USA). All values were determined in duplicate. The assay range was 0.1\u0026ndash;20.0 ng/ml.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec6\" class=\"Section2\"\u003e \u003ch2\u003eStatistical analysis\u003c/h2\u003e \u003cp\u003eThe data are expressed as median and interquartile range unless otherwise stated. Differences between the two groups were evaluated using Student\u0026rsquo;s t-test for normally distributed continuous variables or using the Mann\u0026ndash;Whitney U test for non-normally distributed variables. Relationships between two continuous variables were evaluated using Spearman\u0026rsquo;s rank correlation. The cumulative probability of relapse was analysed using the Kaplan\u0026ndash;Meier method and log-rank test. All tests were two-tailed, with \u003cem\u003eP\u003c/em\u003e-values\u0026thinsp;\u0026lt;\u0026thinsp;0.05 considered statistically significant. All analyses were performed using JMP software, version 17 (SAS Institute, Cary, NC, USA).\u003c/p\u003e \u003c/div\u003e"},{"header":"Results","content":"\u003cdiv id=\"Sec8\" class=\"Section2\"\u003e \u003ch2\u003eSerum sCD226 levels are elevated in patients with TAK\u003c/h2\u003e \u003cp\u003eTo investigate the association between sCD226 and LVV, serum sCD226 levels were measured using ELISA in 34 patients with TAK and 21 patients with GCA. Although no significant differences were observed between TAK and GCA patients in terms of gender, TAK patients were significantly younger than GCA patients because of the classification criteria [\u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e, \u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e]. The baseline characteristics of patients with TAK and GCA are presented in Table\u0026nbsp;1. Serum sCD226 levels were significantly elevated in patients with TAK than in patients with GCA (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003e).\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec9\" class=\"Section2\"\u003e \u003ch2\u003eSerum sCD226 levels are elevated in patients with active TAK and reflect disease activity\u003c/h2\u003e \u003cp\u003eTo assess the association between sCD226 levels and the extent of vascular lesions, the relationship between serum sCD226 levels and the angiographic classification of TAK was evaluated [\u003cspan citationid=\"CR28\" class=\"CitationRef\"\u003e28\u003c/span\u003e]. Serum sCD226 levels were significantly increased in the group of type Ⅴ, which had the most extensive lesions, compared with the group of other types excluding type Ⅴ (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003e).\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eNext, the relationship between serum sCD226 levels and TAK disease activity was assessed. Serum sCD226 levels were elevated in patients with active TAK (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003e).\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eThere were 22 drug-naive patients with active TAK in this study. To examine the relationship between sCD226 levels and response to therapy, we compared serum sCD226 levels between patients with good and poor responses to corticosteroids, revealing elevations in those with poor responses (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003e).\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec10\" class=\"Section2\"\u003e \u003ch2\u003eSerum sCD226 levels can predict disease relapse\u003c/h2\u003e \u003cp\u003eRelapse after treatment was observed in 6 out of 22 drug-naive active TAK patients. We defined a median sCD226 level of 3.3 ng/ml among patients with TAK as the cut-off value to classify patients as having high or low sCD226 levels. The cumulative probability of relapse was higher in patients with high sCD226 levels (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003e, shown in solid lines) than in those with low sCD226 levels (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003e, shown in dashed lines).\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003c/div\u003e"},{"header":"Discussion","content":"\u003cp\u003eOur results revealed that serum sCD226 levels were higher in TAK patients than in GCA patients, significantly increased in active TAK patients, and associated with disease activity and prognosis.\u003c/p\u003e \u003cp\u003eIn this study, we aimed to investigate the association between sCD226 and LVV and found that serum sCD226 levels were significantly elevated in patients with TAK compared with GCA. Previous reports on cancer patients showed an increase in serum sCD226 levels accompanied by a decrease in mCD226 on immune cells, such as NK cells [\u003cspan citationid=\"CR25\" class=\"CitationRef\"\u003e25\u003c/span\u003e, \u003cspan citationid=\"CR26\" class=\"CitationRef\"\u003e26\u003c/span\u003e]. Regarding autoimmune diseases, our previous study demonstrated that serum sCD226 levels were increased in patients with active systemic lupus erythematosus (SLE) [\u003cspan citationid=\"CR30\" class=\"CitationRef\"\u003e30\u003c/span\u003e], whereas Huang et al. showed that expression of mCD226 on NK cells was decreased in patients with active SLE [\u003cspan citationid=\"CR31\" class=\"CitationRef\"\u003e31\u003c/span\u003e]. These findings suggest the involvement of CD226 and the association of serum sCD226 levels and mCD226 on NK cells in SLE, although further analyses are needed. The present study showed that serum sCD226 levels were elevated in TAK patients but not in GCA patients. Despite their similarities in pathogenesis, TAK and GCA have key differences [\u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e4\u003c/span\u003e, \u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e14\u003c/span\u003e], particularly in their roles in NK cells [\u003cspan additionalcitationids=\"CR13 CR14 CR15 CR16 CR17\" citationid=\"CR12\" class=\"CitationRef\"\u003e12\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e18\u003c/span\u003e]. Our results may reflect alterations in NK cells, which are important in the pathogenesis of TAK. However, the present study did not examine immune cells, including NK cells. The mechanism underlying the elevation of serum sCD226 levels in TAK needs to be further studied in the future.\u003c/p\u003e \u003cp\u003eOur study showed that serum sCD226 levels were elevated in patients with active TAK and those with poor responses to corticosteroids and that the cumulative probability of relapse was higher in patients with high sCD226 levels. Monitoring disease activity in patients with LVV is recommended by the 2018 EULAR recommendations [\u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e10\u003c/span\u003e] and the 2021 ACR guidelines [\u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e], and imaging modalities have been advanced recently [\u003cspan citationid=\"CR32\" class=\"CitationRef\"\u003e32\u003c/span\u003e]. However, appropriate biomarkers have not yet been established [\u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e5\u003c/span\u003e, \u003cspan additionalcitationids=\"CR11\" citationid=\"CR10\" class=\"CitationRef\"\u003e10\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e12\u003c/span\u003e]. In our study, serum sCD226 levels were associated with disease activity and prognosis. Thus, sCD226 may be a useful biomarker for TAK.\u003c/p\u003e \u003cp\u003eOur results suggest an association between CD226 and TAK although the mechanism remains unknown. Other autoimmune diseases, such as SLE [\u003cspan citationid=\"CR30\" class=\"CitationRef\"\u003e30\u003c/span\u003e, \u003cspan citationid=\"CR31\" class=\"CitationRef\"\u003e31\u003c/span\u003e, \u003cspan additionalcitationids=\"CR34 CR35 CR36\" citationid=\"CR33\" class=\"CitationRef\"\u003e33\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR37\" class=\"CitationRef\"\u003e37\u003c/span\u003e], rheumatoid arthritis (RA) [\u003cspan citationid=\"CR36\" class=\"CitationRef\"\u003e36\u003c/span\u003e, \u003cspan additionalcitationids=\"CR39 CR40 CR41\" citationid=\"CR38\" class=\"CitationRef\"\u003e38\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR42\" class=\"CitationRef\"\u003e42\u003c/span\u003e], and systemic sclerosis (SSc) [\u003cspan additionalcitationids=\"CR44\" citationid=\"CR43\" class=\"CitationRef\"\u003e43\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR45\" class=\"CitationRef\"\u003e45\u003c/span\u003e], have been associated with CD226, specifically in terms of genetic factors and immune responses. Several GWAS have reported associations between the nonsynonymous rs763361 polymorphism in CD226 and SLE [\u003cspan additionalcitationids=\"CR34 CR35\" citationid=\"CR33\" class=\"CitationRef\"\u003e33\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR36\" class=\"CitationRef\"\u003e36\u003c/span\u003e], RA [\u003cspan citationid=\"CR36\" class=\"CitationRef\"\u003e36\u003c/span\u003e, \u003cspan additionalcitationids=\"CR39\" citationid=\"CR38\" class=\"CitationRef\"\u003e38\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR40\" class=\"CitationRef\"\u003e40\u003c/span\u003e], and SSc [\u003cspan citationid=\"CR43\" class=\"CitationRef\"\u003e43\u003c/span\u003e, \u003cspan citationid=\"CR44\" class=\"CitationRef\"\u003e44\u003c/span\u003e]. In addition, some studies on immune cells, including our group, have suggested the involvement of CD226 in autoimmune diseases [\u003cspan citationid=\"CR30\" class=\"CitationRef\"\u003e30\u003c/span\u003e, \u003cspan citationid=\"CR31\" class=\"CitationRef\"\u003e31\u003c/span\u003e, \u003cspan citationid=\"CR37\" class=\"CitationRef\"\u003e37\u003c/span\u003e, \u003cspan citationid=\"CR41\" class=\"CitationRef\"\u003e41\u003c/span\u003e, \u003cspan citationid=\"CR45\" class=\"CitationRef\"\u003e45\u003c/span\u003e]. Regarding the association between CD226 and LVV, there are few GWAS and no immunological studies; only one Spanish study showed that CD226 gene variants are not involved in GCA [\u003cspan citationid=\"CR46\" class=\"CitationRef\"\u003e46\u003c/span\u003e], and the association between CD226 gene variants and TAK is not revealed. In our analysis, serum sCD226 levels were increased in the group of type Ⅴ, which had the most extensive lesions, indicating that sCD226 may reflect the extent of vascular lesions and affect endothelial cells as well. Given that mCD226 expression on CD8\u003csup\u003e+\u003c/sup\u003e T cells was associated with endothelial injury [\u003cspan citationid=\"CR45\" class=\"CitationRef\"\u003e45\u003c/span\u003e] and sCD226 could directly affect cells expressing the ligand, such as cancer cells [\u003cspan citationid=\"CR25\" class=\"CitationRef\"\u003e25\u003c/span\u003e, \u003cspan citationid=\"CR47\" class=\"CitationRef\"\u003e47\u003c/span\u003e], sCD226 may be involved in the pathogenesis of TAK. Based on this result, further GWAS and immunological studies are required.\u003c/p\u003e \u003cp\u003eOur study had several limitations. First, this study had a small sample size with only Japanese patients. Since the incidence of TAK varies according to ancestry [\u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e5\u003c/span\u003e, \u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e12\u003c/span\u003e], further studies should include a larger sample size and/or other ancestries. Second, the functions of sCD226 remain unknown, and these need to be clarified in further investigations. Finally, our study was retrospective. To ensure the association between sCD226 levels with the disease activity and prognosis of TAK, a prospective study with longitudinal assessments should be conducted.\u003c/p\u003e"},{"header":"Conclusions","content":"\u003cp\u003eSerum sCD226 levels were elevated in patients with TAK and were associated with disease activity and disease relapse. Serum sCD226 may be a useful biomarker for managing TAK, and its monitoring can help in the precise management of TAK.\u003c/p\u003e"},{"header":"Abbreviations","content":"\u003cp\u003eTAK: Takayasu arteritis; GCA: giant cell arteritis; LVV: large vessel vasculitis; GWAS: genome-wide association studies; NK: natural killer; sCD226: soluble CD226; mCD226: membrane CD226; ELISA: enzyme-linked immunosorbent assay; ACR: American College of Rheumatology; EULAR: European League Against Rheumatism; NIH: National Institutes of Health; SLE: systemic lupus erythematosus; RA: rheumatoid arthritis; SSc: systemic sclerosis.\u003c/p\u003e\n"},{"header":"Declarations","content":"\u003cp\u003e\u003cstrong\u003eEthics approval and consent to participate\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThis study was approved by the ethics committees of Kyushu University Hospital (approval number 30\u0026ndash;282) and Nagasaki University Hospital (approval number 15072753) in accordance with the Helsinki Declaration. All participants gave written informed consent.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e\u0026nbsp;\u003c/strong\u003e\u003cstrong\u003eConsent for publication\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eNot applicable.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e\u0026nbsp;\u003c/strong\u003e\u003cstrong\u003eAvailability of data and materials\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eAll data generated or analysed during this study are included in this published article.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e\u0026nbsp;\u003c/strong\u003e\u003cstrong\u003eCompeting interests\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe authors declare that they have no competing interests.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e\u0026nbsp;\u003c/strong\u003e\u003cstrong\u003eFunding\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThis work was supported by Japan Society for the Promotion of Science [grant number JSPS KAKENHI 19K17887 and JSPS KAKENHI JP23K06868].\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e\u0026nbsp;\u003c/strong\u003e\u003cstrong\u003eAuthors\u0026apos; contributions\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eMN and MAy participated in study conception and design. MN, MAy, SF, NI, ATK, KM, and YT participated in data acquisition. MN and TT performed the ELISA experiments. MN and MAy participated in data analysis. MN, MAy, YK, MAk, AK, YT, and HN contributed to the interpretation of results. MN was a major contributor in writing the manuscript. All authors read and approved the final manuscript.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e\u0026nbsp;\u003c/strong\u003e\u003cstrong\u003eAcknowledgements\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe authors thank Enago for the English language review.\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\n\u003cli\u003eJennette JC, Falk RJ, Bacon PA, Basu N, Cid MC, Ferrario F, et al. 2012 Revised International Chapel Hill consensus conference nomenclature of vasculitides. 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Front Cell Dev Biol. 2020;8:564.\u003c/li\u003e\n\u003cli\u003eChen L, Xie X, Zhang X, Jia W, Jian J, Song C, et al. The expression, regulation and adhesion function of a novel CD molecule, CD226, on human endothelial cells. Life Sci. 2003;73(18):2373\u0026ndash;82.\u003c/li\u003e\n\u003cli\u003eKanaya M, Shibuya K, Hirochika R, Kanemoto M, Ohashi K, Okada M, et al. Soluble DNAM-1, as a predictive biomarker for acute Graft-Versus-Host disease. PLoS One. 2016;11(6):e0154173.\u003c/li\u003e\n\u003cli\u003eXu Z, Zhang T, Zhuang R, Zhang Y, Jia W, Song C, et al. Increased levels of soluble CD226 in sera accompanied by decreased membrane CD226 expression on peripheral blood mononuclear cells from cancer patients. BMC Immunol. 2009;10:34.\u003c/li\u003e\n\u003cli\u003eTakahashi N, Sugaya M, Suga H, Oka T, Kawaguchi M, Miyagaki T, et al. Increased soluble CD226 in sera of patients with cutaneous T-cell lymphoma mediates cytotoxic activity against tumor cells via CD155. J Invest Dermatol. 2017;137(8):1766\u0026ndash;73.\u003c/li\u003e\n\u003cli\u003eGoshima Y, Nakaoka S, Ohashi K, Sakamaki H, Shibuya K, Shibuya A. A mathematical model for dynamics of soluble form of DNAM-1 as a biomarker for graft-versus-host disease. PLoS One. 2020;15(2):e0228508.\u003c/li\u003e\n\u003cli\u003eHata A, Noda M, Moriwaki R, Numano F. Angiographic findings of Takayasu arteritis: New classification. Int J Cardiol. 1996;54 Suppl:S155\u0026ndash;63.\u003c/li\u003e\n\u003cli\u003eKerr GS, Hallahan CW, Giordano J, Leavitt RY, Fauci AS, Rottem M, et al. Takayasu arteritis. Ann Intern Med. 1994;120(11):919\u0026ndash;29.\u003c/li\u003e\n\u003cli\u003eNakano M, Ayano M, Kushimoto K, Kawano S, Higashioka K, Inokuchi S, et al. Association of elevated serum soluble CD226 levels with the disease activity and flares of systemic lupus erythematosus. Sci Rep. 2021;11(1):16162.\u003c/li\u003e\n\u003cli\u003eHuang Z, Fu B, Zheng SG, Li X, Sun R, Tian Z, et al. 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Identification of ST3AGL4, MFHAS1, CSNK2A2 and CD226 as loci associated with systemic lupus erythematosus (SLE) and evaluation of SLE genetics in drug repositioning. Ann Rheum Dis. 2018;77(7):1078\u0026ndash;84.\u003c/li\u003e\n\u003cli\u003eMaiti AK, Kim-Howard X, Viswanathan P, Guill\u0026eacute;n L, Qian X, Rojas-Villarraga A, et al. Non-synonymous variant (Gly307Ser) in CD226 is associated with susceptibility to multiple autoimmune diseases. Rheumatology (Oxford). 2010;49(7):1239\u0026ndash;44.\u003c/li\u003e\n\u003cli\u003eNakano M, Ayano M, Kushimoto K, Kawano S, Higashioka K, Inokuchi S, et al. Increased Proportion of CD226\u003csup\u003e+\u003c/sup\u003e B Cells Is Associated With the Disease Activity and Prognosis of Systemic Lupus Erythematosus. Front Immunol. 2021;12:713225.\u003c/li\u003e\n\u003cli\u003eHafler JP, Maier LM, Cooper JD, Plagnol V, Hinks A, Simmonds MJ, et al. CD226 Gly307Ser association with multiple autoimmune diseases. Genes Immun. 2009;10(1):5\u0026ndash;10.\u003c/li\u003e\n\u003cli\u003eSuzuki T, Ikari K, Kawaguchi Y, Yano K, Iwamoto T, Kawamoto M, et al. Non-synonymous variant (Gly307Ser) in CD226 is associated with susceptibility in Japanese rheumatoid arthritis patients. Mod Rheumatol. 2013;23(1):200\u0026ndash;2.\u003c/li\u003e\n\u003cli\u003eLee YH, Bae SC, Song GG. Association between the CTLA-4, CD226, FAS polymorphisms and rheumatoid arthritis susceptibility: A meta-analysis. Hum Immunol. 2015;76(2\u0026ndash;3):83\u0026ndash;9.\u003c/li\u003e\n\u003cli\u003eFasth AER, Bj\u0026ouml;rkstr\u0026ouml;m NK, Anthoni M, Malmberg KJ, Malmstr\u0026ouml;m V. Activating NK-cell receptors co-stimulate CD4\u003csup\u003e+\u003c/sup\u003eCD28\u003csup\u003e\u0026minus;\u003c/sup\u003e T cells in patients with rheumatoid arthritis. Eur J Immunol. 2010;40(2):378\u0026ndash;87.\u003c/li\u003e\n\u003cli\u003eMosaad YM, El-Toraby EE, Tawhid ZM, Abdelsalam AI, Enin AF, Hasson AM, et al. Association between CD226 polymorphism and soluble levels in rheumatoid arthritis: Relationship with clinical activity. Immunol Invest. 2018;47(3):264\u0026ndash;78.\u003c/li\u003e\n\u003cli\u003eDieud\u0026eacute; P, Guedj M, Truchetet ME, Wipff J, Revillod L, Riemekasten G, et al. Association of the CD226 Ser307 variant with systemic sclerosis: evidence of a contribution of costimulation pathways in systemic sclerosis pathogenesis. Arthritis Rheum. 2011;63(4):1097\u0026ndash;105.\u003c/li\u003e\n\u003cli\u003eJin J, Chou C, Lima M, Zhou D, Zhou X. Systemic sclerosis is a complex disease associated mainly with immune regulatory and inflammatory genes. Open Rheumatol J. 2014;8:29\u0026ndash;42.\u003c/li\u003e\n\u003cli\u003eAyano M, Tsukamoto H, Kohno K, Ueda N, Tanaka A, Mitoma H, et al. Increased CD226 Expression on CD8\u003csup\u003e+\u003c/sup\u003e T Cells Is Associated With Upregulated Cytokine Production and Endothelial Cell Injury in Patients With Systemic Sclerosis. J Immunol. 2015;195(3):892\u0026ndash;900.\u003c/li\u003e\n\u003cli\u003eSerrano A, Carmona FD, Miranda-Filloy JA, Casta\u0026ntilde;eda S, Rodr\u0026iacute;guez-Rodr\u0026iacute;guez L, Morado IC, et al. Autoimmune disease-associated CD226 gene variants are not involved in giant cell arteritis susceptibility in the Spanish population. Clin Exp Rheumatol. 2012;30(1 Suppl 70):S29\u0026ndash;33.\u003c/li\u003e\n\u003cli\u003eHou S, Zheng X, Wei H, Tian Z, Sun R. Recombinant soluble CD226 protein directly inhibits cancer cell proliferation in vitro. Int Immunopharmacol. 2014;19(1):119\u0026ndash;26.\u003c/li\u003e\n\u003c/ol\u003e"},{"header":"Table 1","content":"\u003cp\u003eTable 1 is available in the Supplementary Files section.\u003c/p\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":false,"hideJournal":true,"highlight":"","institution":"","isAcceptedByJournal":false,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":false,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"researchsquare","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":true,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"/submission","title":"Research Square","twitterHandle":"researchsquare","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"","reportingPortfolio":"","inReviewEnabled":false,"inReviewRevisionsEnabled":true},"keywords":"Takayasu arteritis, giant cell arteritis, soluble CD226, disease activity, relapse, biomarker","lastPublishedDoi":"10.21203/rs.3.rs-4115713/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-4115713/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003ch2\u003eBackground\u003c/h2\u003e \u003cp\u003eTakayasu arteritis (TAK) is characterized by vascular injury, in which endothelial cells and immune cells, such as natural killer (NK) cells, have key roles. CD226 is an activating receptor expressed on the cell membrane of NK cells and T cells, and the soluble form of CD226 (sCD226) is increased in diseases involving these cells. Therefore, we investigated the utility of serum sCD226 as a biomarker for TAK.\u003c/p\u003e\u003ch2\u003eMethods\u003c/h2\u003e \u003cp\u003eSerum sCD226 levels were measured using an enzyme-linked immunosorbent assay in 34 patients with TAK and 21 patients with giant cell arteritis (GCA). The associations between sCD226 levels and the angiographic classification, disease activity, and prognosis of TAK were analysed.\u003c/p\u003e\u003ch2\u003eResults\u003c/h2\u003e \u003cp\u003eSerum sCD226 levels were significantly higher in TAK patients than in GCA patients. In patients with TAK, serum sCD226 levels were significantly elevated in the group of type Ⅴ, which had the most extensive lesions, compared with the group of other types excluding type Ⅴ. Serum sCD226 levels were also elevated in patients with active TAK and in those with poor responses to corticosteroids. Regarding prognosis, the cumulative probability of relapse was higher in patients with high sCD226 levels than in those with low sCD226 levels.\u003c/p\u003e\u003ch2\u003eConclusions\u003c/h2\u003e \u003cp\u003eSerum sCD226 levels differentiated TAK from GCA and were associated with disease activity and relapse of TAK. Serum sCD226 may be a useful biomarker for the management of TAK.\u003c/p\u003e","manuscriptTitle":"Elevated soluble CD226 in Takayasu arteritis is useful for differentiation from giant cell arteritis and is associated with the disease activity and prognosis","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2024-03-21 18:34:39","doi":"10.21203/rs.3.rs-4115713/v1","editorialEvents":[{"type":"communityComments","content":0}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"researchsquare","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":true,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"/submission","title":"Research Square","twitterHandle":"researchsquare","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"","reportingPortfolio":"","inReviewEnabled":false,"inReviewRevisionsEnabled":true}}],"origin":"","ownerIdentity":"dbb64d1b-fd00-490d-b53c-865135511ed6","owner":[],"postedDate":"March 21st, 2024","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"posted","subjectAreas":[],"tags":[],"updatedAt":"2024-03-24T18:59:27+00:00","versionOfRecord":[],"versionCreatedAt":"2024-03-21 18:34:39","video":"","vorDoi":"","vorDoiUrl":"","workflowStages":[]},"version":"v1","identity":"rs-4115713","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-4115713","identity":"rs-4115713","version":["v1"]},"buildId":"8U1c8b4HqxoKbykW_rLl7","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}