CXCL12-ACKR3 Signaling Drives Fibrosis and Inflammation in Frozen Shoulder: A Single-Cell Guided Therapeutic Discovery

CXCL12-ACKR3 Signaling Drives Fibrosis and Inflammation in Frozen Shoulder: A Single-Cell Guided Therapeutic Discovery | Research Square window.SnipcartSettings = { analytics: { enabled: false } }; (function() { var accessVector = localStorage.getItem('access_vector') || ''; window.dataLayer = window.dataLayer || []; if (accessVector) { window.dataLayer.push({ user: { profile: { profileInfo: { snid: accessVector } } } }); } })(); (function(w,d,s,l,i){w[l]=w[l]||[];w[l].push({'gtm.start':new Date().getTime(),event:'gtm.js'});var f=d.getElementsByTagName(s)[0],j=d.createElement(s),dl=l!='dataLayer'?'&l='+l:'';j.async=true;j.src='https://www.googletagmanager.com/gtm.js?id='+i+dl;f.parentNode.insertBefore(j,f);})(window,document,'script','dataLayer','GTM-K279D39R'); Browse Preprints In Review Journals COVID-19 Preprints AJE Video Bytes Research Tools Research Promotion AJE Professional Editing AJE Rubriq About Preprint Platform In Review Editorial Policies Our Team Advisory Board Help Center Sign In Submit a Preprint Cite Share Download PDF Article CXCL12-ACKR3 Signaling Drives Fibrosis and Inflammation in Frozen Shoulder: A Single-Cell Guided Therapeutic Discovery Jian Xu, Hongpu He, Guangxun Yuan, Shuai Li, Zhongding Meng, Pengju Liu, and 7 more This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-6289785/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 Frozen shoulder, a debilitating condition marked by chronic joint stiffness and pain, remains poorly understood due to unclear molecular drivers of its hallmark fibrosis and inflammation. Although fibroblast dysfunction contributes to FS pathology, specific pathogenic subsets remain unidentified. Through single-cell RNA sequencing of human synovial tissues, we discovered a distinct CXCL12-enriched fibroblast population (F1) driving disease progression via ACKR3 signaling. This axis exacerbated metalloproteinase (MMP)/tissue inhibitor of metalloproteinase (TIMP) dysregulation, promoting collagen overproduction and fibrotic niche formation. In vitro interventions demonstrated that both CXCL12 neutralization (Lit-927) and ACKR3 knockout effectively reduced pro-fibrotic responses by mitigating MMP/TIMP imbalance. Rat FS models recapitulated these findings, showing attenuated fibrosis following CXCL12 blockade. Our study establishes CXCL12-ACKR3 signaling as the central mechanism connecting fibroblast heterogeneity to extracellular matrix dysregulation in FS pathogenesis. These findings provide dual therapeutic strategies targeting ligand-receptor interactions for a condition currently managed only through symptomatic relief. Health sciences/Rheumatology/Musculoskeletal system Health sciences/Diseases/Immunological disorders Full Text Additional Declarations There is NO Competing Interest. Supplementary Files datafileS1.xlsx Dataset 1 datafileS2.xlsx Dataset 2 sm.pdf Supplementary Materials for CXCL12-ACKR3 Signaling Drives Fibrosis and Inflammation in Frozen Shoulder: A Single-Cell Guided Therapeutic Discovery Cite Share Download PDF Status: Posted Version 1 posted You are reading this latest preprint version Research Square lets you share your work early, gain feedback from the community, and start making changes to your manuscript prior to peer review in a journal. As a division of Research Square Company, we’re committed to making research communication faster, fairer, and more useful. We do this by developing innovative software and high quality services for the global research community. Our growing team is made up of researchers and industry professionals working together to solve the most critical problems facing scientific publishing. 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-6289785","acceptedTermsAndConditions":true,"allowDirectSubmit":true,"archivedVersions":[],"articleType":"Article","associatedPublications":[],"authors":[{"id":440749783,"identity":"63561523-4ab0-48ce-80cd-8283cc99cda9","order_by":0,"name":"Jian Xu","email":"data:image/png;base64,iVBORw0KGgoAAAANSUhEUgAAAZAAAAAyAQMAAABI0h/eAAAABlBMVEX///8AAABVwtN+AAAACXBIWXMAAA7EAAAOxAGVKw4bAAAA3UlEQVRIie3RMQrCMBSA4VcCdYl0TRHqFSIFF928SIpgJ8GpOKYInXqAiINn8AYpAafQ2cHBI3Szi6LVwTFxE8y/Bd5HQh6Ay/WD+QhVF7aeRgCyO1qQoFfMaaMXsT0JSz0Ot4VK+PtSC0JPjA6wr9J9rik0mYJgx42ExRiny5xr6olaATlLI5FzTCbLDWiK+oUCSpiJJFxhilK/IzcbEpbKywWbMdwRz4Y8PxlBIxcjAcdVVdYpJicD8VFwbZP7dDgU6nBps0kUCAP5RORrmdh2vnsi/2LY5XK5/qoHr4tGlizc1tEAAAAASUVORK5CYII=","orcid":"","institution":"The First Affiliated Hospital, Zhejiang University School of Medicine","correspondingAuthor":true,"prefix":"","firstName":"Jian","middleName":"","lastName":"Xu","suffix":""},{"id":440749784,"identity":"3d74299f-07ec-4521-8a1a-93e4348abdc8","order_by":1,"name":"Hongpu He","email":"","orcid":"","institution":"The First Affiliated Hospital of Zhejiang University School of Medicine","correspondingAuthor":false,"prefix":"","firstName":"Hongpu","middleName":"","lastName":"He","suffix":""},{"id":440749785,"identity":"45e31609-0111-474a-acc8-9c1e3ed987a3","order_by":2,"name":"Guangxun Yuan","email":"","orcid":"","institution":"The First Affiliated Hospital of Zhejiang University School of Medicine","correspondingAuthor":false,"prefix":"","firstName":"Guangxun","middleName":"","lastName":"Yuan","suffix":""},{"id":440749786,"identity":"cfd06d63-1e29-4029-9961-0c71fd82d955","order_by":3,"name":"Shuai Li","email":"","orcid":"https://orcid.org/0000-0001-6722-017X","institution":"The First Affiliated Hospital, Zhejiang University School of Medicine","correspondingAuthor":false,"prefix":"","firstName":"Shuai","middleName":"","lastName":"Li","suffix":""},{"id":440749787,"identity":"287d0595-9b62-4124-8f25-16a8ea214429","order_by":4,"name":"Zhongding Meng","email":"","orcid":"","institution":"GuangXi University of Chinese Medicine","correspondingAuthor":false,"prefix":"","firstName":"Zhongding","middleName":"","lastName":"Meng","suffix":""},{"id":440749788,"identity":"6dbdf4ff-9ec3-4722-84c6-c1d5dc7ec4b9","order_by":5,"name":"Pengju Liu","email":"","orcid":"https://orcid.org/0009-0008-5484-9364","institution":"The First Affiliated Hospital of Zhejiang University School of Medicine","correspondingAuthor":false,"prefix":"","firstName":"Pengju","middleName":"","lastName":"Liu","suffix":""},{"id":440749789,"identity":"6fa2ce68-d748-4e1e-923e-a05213044f20","order_by":6,"name":"Zhiqin Liu","email":"","orcid":"","institution":"Zhuzhou Central Hospital","correspondingAuthor":false,"prefix":"","firstName":"Zhiqin","middleName":"","lastName":"Liu","suffix":""},{"id":440749790,"identity":"e5bc1ef6-7212-4a3a-81f9-3d9fe0f63a14","order_by":7,"name":"Binbin Ma","email":"","orcid":"","institution":"The First Affiliated Hospital of Zhejiang University School of Medicine","correspondingAuthor":false,"prefix":"","firstName":"Binbin","middleName":"","lastName":"Ma","suffix":""},{"id":440749791,"identity":"4742a4e7-7bd9-4c63-9ff9-7a63c45c8817","order_by":8,"name":"Danmei Li","email":"","orcid":"","institution":"The First Affiliated Hospital of Zhejiang University School of Medicine","correspondingAuthor":false,"prefix":"","firstName":"Danmei","middleName":"","lastName":"Li","suffix":""},{"id":440749792,"identity":"4b43a662-7481-4051-a4cb-61ad46b6e15f","order_by":9,"name":"Xiangyu Du","email":"","orcid":"","institution":"The First Affiliated Hospital of Zhejiang University School of Medicine","correspondingAuthor":false,"prefix":"","firstName":"Xiangyu","middleName":"","lastName":"Du","suffix":""},{"id":440749793,"identity":"7cbe3788-80e2-47bd-beb1-bfc2bf0361bc","order_by":10,"name":"Yijun Zhang","email":"","orcid":"","institution":"The First Affiliated Hospital of Zhejiang University School of Medicine","correspondingAuthor":false,"prefix":"","firstName":"Yijun","middleName":"","lastName":"Zhang","suffix":""},{"id":440749794,"identity":"2ee61688-66e2-425a-a39b-424f747cf1d8","order_by":11,"name":"Chengjie Yuan","email":"","orcid":"","institution":"The First Affiliated Hospital of Zhejiang University School of Medicine","correspondingAuthor":false,"prefix":"","firstName":"Chengjie","middleName":"","lastName":"Yuan","suffix":""},{"id":440749795,"identity":"a07d7c9f-180c-4d24-8db3-bf71448af417","order_by":12,"name":"Zhenhan Deng","email":"","orcid":"https://orcid.org/0000-0003-0522-8269","institution":"Department of Orthopedics and Geriatrics Center","correspondingAuthor":false,"prefix":"","firstName":"Zhenhan","middleName":"","lastName":"Deng","suffix":""}],"badges":[],"createdAt":"2025-03-23 18:30:14","currentVersionCode":1,"declarations":"","doi":"10.21203/rs.3.rs-6289785/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-6289785/v1","draftVersion":[],"editorialEvents":[],"editorialNote":"","failedWorkflow":false,"files":[{"id":87307063,"identity":"e779f578-e2dd-4b71-9425-f5ba415affbd","added_by":"auto","created_at":"2025-07-22 14:20:16","extension":"pdf","order_by":1,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":8593198,"visible":true,"origin":"","legend":"Article File","description":"","filename":"ms.pdf","url":"https://assets-eu.researchsquare.com/files/rs-6289785/v1_covered_bf17e56a-2656-4e52-9f60-09b72b1d33a8.pdf"},{"id":80356792,"identity":"c52c987b-edb6-4cf2-8d6e-f108f3413cc1","added_by":"auto","created_at":"2025-04-11 02:34:07","extension":"xlsx","order_by":1,"title":"","display":"","copyAsset":false,"role":"supplement","size":3602182,"visible":true,"origin":"","legend":"Dataset 1","description":"","filename":"datafileS1.xlsx","url":"https://assets-eu.researchsquare.com/files/rs-6289785/v1/a84b7d4de2e78878a1c74858.xlsx"},{"id":80356790,"identity":"de677c5d-8da8-4c3d-8a64-c7b5ba294c8d","added_by":"auto","created_at":"2025-04-11 02:34:07","extension":"xlsx","order_by":2,"title":"","display":"","copyAsset":false,"role":"supplement","size":14190,"visible":true,"origin":"","legend":"Dataset 2","description":"","filename":"datafileS2.xlsx","url":"https://assets-eu.researchsquare.com/files/rs-6289785/v1/027e57176623edd12f4b76c6.xlsx"},{"id":80356794,"identity":"4fcccbd8-d910-4bfa-9b68-48049fe217dd","added_by":"auto","created_at":"2025-04-11 02:34:07","extension":"pdf","order_by":3,"title":"","display":"","copyAsset":false,"role":"supplement","size":18561614,"visible":true,"origin":"","legend":"Supplementary Materials for CXCL12-ACKR3 Signaling Drives Fibrosis and Inflammation in Frozen Shoulder: A Single-Cell Guided Therapeutic Discovery","description":"","filename":"sm.pdf","url":"https://assets-eu.researchsquare.com/files/rs-6289785/v1/59c6fc24a51fcc312c2d7cc9.pdf"}],"financialInterests":"There is \u003cb\u003eNO\u003c/b\u003e Competing Interest.","formattedTitle":"CXCL12-ACKR3 Signaling Drives Fibrosis and Inflammation in Frozen Shoulder: A Single-Cell Guided Therapeutic Discovery","fulltext":[],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":false,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":true,"hideJournal":true,"highlight":"","institution":"","isAcceptedByJournal":false,"isAuthorSuppliedPdf":true,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":false,"isPdf":true,"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":"","lastPublishedDoi":"10.21203/rs.3.rs-6289785/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-6289785/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"Frozen shoulder, a debilitating condition marked by chronic joint stiffness and pain, remains poorly understood due to unclear molecular drivers of its hallmark fibrosis and inflammation. Although fibroblast dysfunction contributes to FS pathology, specific pathogenic subsets remain unidentified. Through single-cell RNA sequencing of human synovial tissues, we discovered a distinct CXCL12-enriched fibroblast population (F1) driving disease progression via ACKR3 signaling. This axis exacerbated metalloproteinase (MMP)/tissue inhibitor of metalloproteinase (TIMP) dysregulation, promoting collagen overproduction and fibrotic niche formation. In vitro interventions demonstrated that both CXCL12 neutralization (Lit-927) and ACKR3 knockout effectively reduced pro-fibrotic responses by mitigating MMP/TIMP imbalance. Rat FS models recapitulated these findings, showing attenuated fibrosis following CXCL12 blockade. Our study establishes CXCL12-ACKR3 signaling as the central mechanism connecting fibroblast heterogeneity to extracellular matrix dysregulation in FS pathogenesis. These findings provide dual therapeutic strategies targeting ligand-receptor interactions for a condition currently managed only through symptomatic relief.","manuscriptTitle":"CXCL12-ACKR3 Signaling Drives Fibrosis and Inflammation in Frozen Shoulder: A Single-Cell Guided Therapeutic Discovery","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2025-04-11 02:34:02","doi":"10.21203/rs.3.rs-6289785/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":"2bb99531-ae2c-4de7-8b6f-b492391d0211","owner":[],"postedDate":"April 11th, 2025","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"posted","subjectAreas":[{"id":46927714,"name":"Health sciences/Rheumatology/Musculoskeletal system"},{"id":46927715,"name":"Health sciences/Diseases/Immunological disorders"}],"tags":[],"updatedAt":"2025-07-22T14:12:03+00:00","versionOfRecord":[],"versionCreatedAt":"2025-04-11 02:34:02","video":"","vorDoi":"","vorDoiUrl":"","workflowStages":[]},"version":"v1","identity":"rs-6289785","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-6289785","identity":"rs-6289785","version":["v1"]},"buildId":"XKTyCvWXoU3ODBz1xrDgd","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}