Capns1 Specific Deletion in Macrophage Attenuates Skin Inflammation and Fibrosis in Systemic Sclerosis by Suppressing SHP2

Capns1 Specific Deletion in Macrophage Attenuates Skin Inflammation and Fibrosis in Systemic Sclerosis by Suppressing SHP2 | 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 Capns1 Specific Deletion in Macrophage Attenuates Skin Inflammation and Fibrosis in Systemic Sclerosis by Suppressing SHP2 Dianyu Cao, Zhe Zheng, Feifei Hu, Haiying Zeng, Zheng Li, Ruizhen Chen, and 2 more This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-8004677/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: In systemic sclerosis (SSc), Capns1 is largely up-regulated in dermal tissue to control calpain activities, thus providing pro-inflammatory and pro-fibrotic effect. However, the mechanisms under this process still remain unknown. We aimed at investigating how Capns1 participates in SSc inflammation and fibrosis progression. Methods: This study includes clinical skin biopsy samples from 7 SSc patients and 5 healthy controls to detect the expression pattern of Capns1 and its downstreaming regulator SHP2. In vitro , bone marrow-derived macrophage (BMDM) and Raw264.7 cell line are utilized to clarify the relation between Capns1 and SHP2-mediated inflammatory response. We further explore the macrophage-fibroblast interaction and its signaling pathway targets after SHP2 knockout. In vivo , we breed myeloid-specific Capns1 deletion mice to evaluate its effect, and the therapeutic function of SHP2 inhibition is investigated by systemic administration of its pharmacological inhibitor with different intervention regimens. Results: Clinical skin samples show that Capns1 and SHP2 are both amplified in SSc dermis and co-express in CD68 + macrophages. In vivo , myeloid-specific Capns1 deletion mice show significant lower levels of SHP2 expression in dermal tissue, attenuating dermal thickness and subcutaneous fat loss. In vitro , when incubating fibroblasts with macrophage supernatant, the Capns1 deletion group indicates less amount of fibrotic protein production[1] . Additionally, SHP2 knockdown can downregulate NF-κB activation and inflammatory cytokines secretion in Raw264.7, such as interleukin-1β (IL-1β). Conversely, pharmacological SHP2 inhibitor provides significant mitigation in a intervention time-dependent manner. Conclusions: Collectively, our results indicate that Capns1-SHP2 axis is over-activated to regulate macrophage inflammatory response in SSc, which activates nearby fibroblasts to further drive skin fibrosis. Macrophage Capns1 deficiency and pharmacological SHP2 inhibition can significantly provide the anti-fibrotic protection against disease progression. Capns1 SHP2 NF-κB macrophage skin systemic sclerosis Figures Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 Figure 6 Figure 7 Full Text Additional Declarations No competing interests reported. Supplementary Files SupplementaryTable1.Clinincalinformationaboutpatientsandvolunteers.docx SupplementaryTable2.Sequencesforlentivirusconstruction.docx SupplementaryTable3.PrimaryantibodiesforIFandIHCstaning.docx SupplementaryTable4.PrimersequencesforqPCR.docx SupplementaryTable5.Primaryantibodiesforwesternblotting.docx SupplementaryFigure.doc SupFig1Bcalpain1.tif SupFig1Bactb.tif SupFig1Bcalpain2.tif SupFig1BCAPN4.tif Fig1Dactbfibroblast.tif Fig1Dcapn4BMDM.tif Fig1DactbBMDM.tif Fig1Dcapn4fibroblast.tif Fig2Eactb.tif Fig2Ecalpain1.tif Fig2ESHP2.tif Fig2ECAPN4.tif Fig2Ecalpain2.tif SupFig3Ashp2.tif SupFig3Acapn42.png SupFig3A0112actb5Chemiluminescence.tif Fig3Dshp2.tif Fig3Dactb.tif SupFig4ACAPN4.tif SupFig4Aactb6Chemiluminescence.tif Fig4DFAP.tif Fig4Dcol3.tif Fig4DaSMA.tif Fig4Dcol1a.tif Fig4Dcol3.tif Fig4Dcol1.tif Fig4DaSMA.tif Fig4DFAP.tif Fig5FSma.tif Fig5Fcol12.tif Fig5Fcol3.tif Fig5FFAP.tif Fig5Ffap8Chemiluminescence.png Fig5FSMA.png Fig5Fcol3Chemiluminescence.tif Fig5Fcol1.tif Fig6p65.png Fig6pp65.tif Fig6laminb1.tif Fig6shp26Chemiluminescence.tif Fig6IkBa.tif Fig6p65nu.tif Fig60110actbkoChemiluminescence.tif Fig6shp23Chemiluminescence.tif Fig6actb.tif Fig6laminb1.tif Fig6p65.tif Fig7Aactb.tif Fig7Ashp2.tif 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-8004677","acceptedTermsAndConditions":true,"allowDirectSubmit":true,"archivedVersions":[],"articleType":"Research Article","associatedPublications":[],"authors":[{"id":550081005,"identity":"202f2ab9-733e-4ebb-b464-2426968500ca","order_by":0,"name":"Dianyu Cao","email":"","orcid":"","institution":"Zhongshan Hospital","correspondingAuthor":false,"prefix":"","firstName":"Dianyu","middleName":"","lastName":"Cao","suffix":""},{"id":550081006,"identity":"65b873a2-efa1-4a28-ac0f-83315c4771ae","order_by":1,"name":"Zhe Zheng","email":"","orcid":"","institution":"Zhongshan 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SHP2\u003c/p\u003e","fulltext":[],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":false,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":false,"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":"Capns1, SHP2, NF-κB, macrophage, skin, systemic sclerosis ","lastPublishedDoi":"10.21203/rs.3.rs-8004677/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-8004677/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003cp\u003e\u003cstrong\u003eBackground:\u003c/strong\u003e\u0026nbsp;In systemic sclerosis (SSc), Capns1 is largely up-regulated in dermal tissue to control calpain activities, thus providing pro-inflammatory and pro-fibrotic effect. However, the mechanisms under this process still remain unknown. We aimed at investigating how Capns1 participates in SSc inflammation and fibrosis progression.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eMethods:\u003c/strong\u003e\u0026nbsp;This study includes clinical skin biopsy samples from 7 SSc patients and 5 healthy controls to detect the expression pattern of Capns1 and its downstreaming regulator SHP2.\u0026nbsp;\u003cem\u003eIn vitro\u003c/em\u003e, bone marrow-derived macrophage (BMDM) and Raw264.7 cell line are utilized to clarify the relation between Capns1 and SHP2-mediated inflammatory response. We further explore the macrophage-fibroblast interaction and its signaling pathway targets after SHP2 knockout.\u0026nbsp;\u003cem\u003eIn vivo\u003c/em\u003e, we breed myeloid-specific\u0026nbsp;\u003cem\u003eCapns1\u003c/em\u003e\u0026nbsp;deletion mice to evaluate its effect, and the therapeutic function of SHP2 inhibition is investigated by systemic administration of its pharmacological inhibitor with different intervention regimens.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eResults:\u003c/strong\u003e\u0026nbsp;Clinical skin samples show that Capns1 and SHP2 are both amplified in SSc dermis and co-express in CD68\u003csup\u003e+\u003c/sup\u003e\u0026nbsp;macrophages.\u0026nbsp;\u003cem\u003eIn vivo\u003c/em\u003e, myeloid-specific\u0026nbsp;\u003cem\u003eCapns1\u003c/em\u003e\u0026nbsp;deletion mice show significant lower levels of SHP2 expression in dermal tissue, attenuating dermal thickness and subcutaneous fat loss.\u0026nbsp;\u003cem\u003eIn vitro\u003c/em\u003e, when incubating fibroblasts with macrophage supernatant, the\u0026nbsp;\u003cem\u003eCapns1\u003c/em\u003e\u0026nbsp;deletion group indicates less amount of fibrotic protein production[1] . Additionally, SHP2 knockdown can downregulate NF-κB activation and inflammatory cytokines secretion in Raw264.7, such as interleukin-1β (IL-1β). Conversely, pharmacological SHP2 inhibitor provides significant mitigation in a intervention time-dependent manner.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eConclusions:\u003c/strong\u003e\u0026nbsp;Collectively, our results indicate that Capns1-SHP2 axis is over-activated to regulate macrophage inflammatory response in SSc, which activates nearby fibroblasts to further drive skin fibrosis. Macrophage\u0026nbsp;\u003cem\u003eCapns1\u003c/em\u003e\u0026nbsp;deficiency and pharmacological SHP2 inhibition can significantly provide the anti-fibrotic protection against disease progression.\u0026nbsp;\u003c/p\u003e","manuscriptTitle":"Capns1 Specific Deletion in Macrophage Attenuates Skin Inflammation and Fibrosis in Systemic Sclerosis by Suppressing SHP2","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2025-11-26 16:32:45","doi":"10.21203/rs.3.rs-8004677/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":"efbb24c2-78d2-41a8-a780-6f09d1dd4a84","owner":[],"postedDate":"November 26th, 2025","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"posted","subjectAreas":[],"tags":[],"updatedAt":"2025-12-14T10:38:56+00:00","versionOfRecord":[],"versionCreatedAt":"2025-11-26 16:32:45","video":"","vorDoi":"","vorDoiUrl":"","workflowStages":[]},"version":"v1","identity":"rs-8004677","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-8004677","identity":"rs-8004677","version":["v1"]},"buildId":"8U1c8b4HqxoKbykW_rLl7","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}