Single nuclei/cell transcriptomics reveal DMD driven cell dynamics and mechanisms of fibroblast inflammatory tissue priming in human dystrophic muscle

Single nuclei/cell transcriptomics reveal DMD driven cell dynamics and mechanisms of fibroblast inflammatory tissue priming in human dystrophic muscle | 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 Resource Single nuclei/cell transcriptomics reveal DMD driven cell dynamics and mechanisms of fibroblast inflammatory tissue priming in human dystrophic muscle M. Carrie Miceli, Kevin Chesmore, Deirdre Scripture-Adams, Florian BARTHELEMY, and 11 more This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-4934147/v1 This work is licensed under a CC BY 4.0 License Status: Under Review Version 1 posted You are reading this latest preprint version Abstract Single cell/nuclei technologies have revolutionized our understanding of the remodeling of complex multi-cellular tissue that accompanies injury, regeneration, and disease. Duchenne muscular dystrophy (DMD) is a fatal genetic disease of childhood characterized by progressive skeletal muscle weakness resulting from mutation of DMD and loss of functional dystrophin. Here we report, at single nuclei/cell resolution, on intramuscular cell and gene expression dynamics within a broad cohort of needle muscle biopsies obtained from DMD individuals with varying degrees of severity, including a subset with low levels of dystrophin. We report a strong negative correlation between expression of dystrophin and disease severity and report substantial differences in cellularity and cell type-specific gene expression in DMD severity groups versus healthy muscle. Expression signatures indicate that DMD myofibers become immunologically alert, upregulating innate and adaptive immune sensors, including TLR4, IL15, TNF family receptors and MHC and costimulators. In this cohort, dystrophic muscle was remodeled with 50% fewer myofibers with expansion and diversification of fibroblasts and myeloid cells. We identify a DMD-specific TNFα-responsive Thy-1+/C3+ fibroblast subpopulation which we propose are inflammatory tissue priming fibroblasts and three DMD-specific myeloid populations which express signatures of innate immune memory. There is an 8-fold increase in CD8+GZMK+/GZMB+ T cells (Tek), with characteristics of both adaptive and innate immune activity. We propose that these non-myofiber muscle resident cells interact and epigenetically instill long-term tissue memory to perpetuate and amplify a hyper-inflammatory state in DMD muscle, contributing to impaired regeneration, myofiber death and fibrosis. This compendium of single/cell nuclei serves as a valuable reference and has immediate impact for biomarker discovery, clinical trial design, identification of barriers to dystrophin replacement therapies and novel druggable cell mechanisms operating in DMD. Health sciences/Pathogenesis/Inflammation/Chronic inflammation Biological sciences/Immunology/Innate immunity/Pattern recognition receptors/Toll-like receptors Biological sciences/Biological techniques/Sequencing/RNA sequencing Full Text Additional Declarations There is NO Competing Interest. Supplementary Files ExtendedFigure24.06.262.19.pdf Extended Figure 2 ExtendedFigure3.Fibrobolasts3.20.26.pdf Extended Figure 3 ExtendedFigure5.Myeloid3.20.26.pdf Extended Figure 5 ExtendedFigure7.Lymphoidcells3.20.26.pdf Extended Figure 7 SupplementalFigure14.06.26.pdf Supplemental Figure 1 SupplementalFigure23.20.26.pdf Supplemental Figure 2 Supptable1NatImm12.12.25.xlsx Supplemental Table 1 Supptable2NatImm4.06.26.xlsx Supplemental Table 2 Cite Share Download PDF Status: Under Review 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-4934147","acceptedTermsAndConditions":true,"allowDirectSubmit":false,"archivedVersions":[],"articleType":"Resource","associatedPublications":[],"authors":[{"id":618900352,"identity":"f2ee47ee-8986-42df-bdcb-915f28e19204","order_by":0,"name":"M. Carrie Miceli","email":"data:image/png;base64,iVBORw0KGgoAAAANSUhEUgAAAZAAAAAyAQMAAABI0h/eAAAABlBMVEX///8AAABVwtN+AAAACXBIWXMAAA7EAAAOxAGVKw4bAAAAuklEQVRIiWNgGAWjYFACxgYGhgIJOQYGZiCDgZlYLQYSxhAGcVpAwIAhsYFoLfzTDjd++GBgkb7h+MHGDwwV1okNhLRI3E5slpxhIJG74UxiswTDmXTCWgykE9uYeUBabjA2SDC2HSZSyx8DiXSDG4zNPxj/EasFGGIJQC1tEowNRGgB+6XHQMJw5pnENouEY+nGBLXwz05/+OFHRZ083/HDh298qLGWJagFFSSQpnwUjIJRMApGAS4AAET0PNIxD22QAAAAAElFTkSuQmCC","orcid":"","institution":"UCLA","correspondingAuthor":true,"prefix":"","firstName":"M.","middleName":"Carrie","lastName":"Miceli","suffix":""},{"id":618900353,"identity":"4a70ca5c-4054-4b88-95d7-f48247f6626a","order_by":1,"name":"Kevin Chesmore","email":"","orcid":"https://orcid.org/0000-0002-5351-5724","institution":"University of California Los Angeles","correspondingAuthor":false,"prefix":"","firstName":"Kevin","middleName":"","lastName":"Chesmore","suffix":""},{"id":618900355,"identity":"453b7d9e-891a-49c0-96c4-3ae42b3dfa2c","order_by":2,"name":"Deirdre Scripture-Adams","email":"","orcid":"https://orcid.org/0000-0002-3676-1771","institution":"University of California, Los Angeles","correspondingAuthor":false,"prefix":"","firstName":"Deirdre","middleName":"","lastName":"Scripture-Adams","suffix":""},{"id":618900356,"identity":"e2d1b07c-eabf-488e-b49e-63905359b602","order_by":3,"name":"Florian BARTHELEMY","email":"","orcid":"https://orcid.org/0000-0002-9896-9551","institution":"ucla","correspondingAuthor":false,"prefix":"","firstName":"Florian","middleName":"","lastName":"BARTHELEMY","suffix":""},{"id":618900358,"identity":"707df191-b99e-446a-ae8e-942f49836963","order_by":4,"name":"Isaiah Little","email":"","orcid":"https://orcid.org/0000-0002-3073-553X","institution":"University of California Los Angeles","correspondingAuthor":false,"prefix":"","firstName":"Isaiah","middleName":"","lastName":"Little","suffix":""},{"id":618900359,"identity":"7eb313f2-df30-424c-aecb-88ffcca22702","order_by":5,"name":"Richard Wang","email":"","orcid":"https://orcid.org/0000-0002-2483-3205","institution":"University of California Los Angeles","correspondingAuthor":false,"prefix":"","firstName":"Richard","middleName":"","lastName":"Wang","suffix":""},{"id":618900360,"identity":"4bb53106-b009-468e-ae8d-470937dea919","order_by":6,"name":"Shirley Nieves-Rodriguez","email":"","orcid":"https://orcid.org/0000-0002-2329-4879","institution":"University of California, Los Angeles","correspondingAuthor":false,"prefix":"","firstName":"Shirley","middleName":"","lastName":"Nieves-Rodriguez","suffix":""},{"id":618900363,"identity":"10c3c129-2b9e-4383-b145-f5b89de29172","order_by":7,"name":"Rosario Corona","email":"","orcid":"","institution":"University of California, Los Angeles","correspondingAuthor":false,"prefix":"","firstName":"Rosario","middleName":"","lastName":"Corona","suffix":""},{"id":618900366,"identity":"b2daa88c-9a02-4c90-a80f-3b93ecc0d53b","order_by":8,"name":"Nicole Steady","email":"","orcid":"","institution":"University of California, Los Angeles","correspondingAuthor":false,"prefix":"","firstName":"Nicole","middleName":"","lastName":"Steady","suffix":""},{"id":618900369,"identity":"302056ab-b73d-406e-ad2d-704aaca1f2ef","order_by":9,"name":"Artemis Yip","email":"","orcid":"","institution":"University of California, Los Angeles","correspondingAuthor":false,"prefix":"","firstName":"Artemis","middleName":"","lastName":"Yip","suffix":""},{"id":618900371,"identity":"72124a3c-2fa4-4ef2-8ce2-f153721edefb","order_by":10,"name":"Emilie Douine-Barthelemy","email":"","orcid":"","institution":"Cedars-Sinai","correspondingAuthor":false,"prefix":"","firstName":"Emilie","middleName":"","lastName":"Douine-Barthelemy","suffix":""},{"id":618900373,"identity":"09592bce-8283-4afd-b456-d424ef4c1233","order_by":11,"name":"Stanley Nelson","email":"","orcid":"","institution":"University of California, Los Angeles","correspondingAuthor":false,"prefix":"","firstName":"Stanley","middleName":"","lastName":"Nelson","suffix":""},{"id":618900376,"identity":"0049f1df-cc1e-4efd-8c57-783d487c515d","order_by":12,"name":"Sophie Rengarajan","email":"","orcid":"","institution":"University of California Los Angeles","correspondingAuthor":false,"prefix":"","firstName":"Sophie","middleName":"","lastName":"Rengarajan","suffix":""},{"id":618900379,"identity":"ba75a45b-fd09-4b14-889c-d64636112d3d","order_by":13,"name":"Daniela Nasif","email":"","orcid":"","institution":"University of California Los Angeles","correspondingAuthor":false,"prefix":"","firstName":"Daniela","middleName":"","lastName":"Nasif","suffix":""},{"id":618900380,"identity":"3a44fddf-ccd8-404f-9f69-d040be2250b3","order_by":14,"name":"Annabel Sen","email":"","orcid":"","institution":"University of California Los Angeles","correspondingAuthor":false,"prefix":"","firstName":"Annabel","middleName":"","lastName":"Sen","suffix":""}],"badges":[],"createdAt":"2024-08-18 16:05:16","currentVersionCode":1,"declarations":"","doi":"10.21203/rs.3.rs-4934147/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-4934147/v1","draftVersion":[],"editorialEvents":[],"editorialNote":"","failedWorkflow":false,"files":[{"id":107015944,"identity":"bd56a703-f2df-4b5d-875d-d96a105957a7","added_by":"auto","created_at":"2026-04-15 19:37:59","extension":"pdf","order_by":1,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":10747670,"visible":true,"origin":"","legend":"","description":"","filename":"NatImmsnRNASEQMainMethods04062026finish.pdf","url":"https://assets-eu.researchsquare.com/files/rs-4934147/v1_covered_f00f768b-aa4a-4594-8c7e-3e3742922b00.pdf"},{"id":106379779,"identity":"2ff5f7b8-e7a9-40d9-b487-7fa83a5713df","added_by":"auto","created_at":"2026-04-08 04:48:04","extension":"pdf","order_by":1,"title":"","display":"","copyAsset":false,"role":"supplement","size":317209,"visible":true,"origin":"","legend":"Extended Figure 2","description":"","filename":"ExtendedFigure24.06.262.19.pdf","url":"https://assets-eu.researchsquare.com/files/rs-4934147/v1/9b48590374e57609263aa00d.pdf"},{"id":106404395,"identity":"b0a52dc4-ffdb-49e2-933c-835e56ef0d40","added_by":"auto","created_at":"2026-04-08 09:15:56","extension":"pdf","order_by":2,"title":"","display":"","copyAsset":false,"role":"supplement","size":245425,"visible":true,"origin":"","legend":"Extended Figure 3","description":"","filename":"ExtendedFigure3.Fibrobolasts3.20.26.pdf","url":"https://assets-eu.researchsquare.com/files/rs-4934147/v1/066a3bd1f85f7b9d37b38cbd.pdf"},{"id":106404024,"identity":"26cf80d3-7c4b-47cb-bf0b-30dbddb25e5a","added_by":"auto","created_at":"2026-04-08 09:15:23","extension":"pdf","order_by":3,"title":"","display":"","copyAsset":false,"role":"supplement","size":74217,"visible":true,"origin":"","legend":"Extended Figure 5","description":"","filename":"ExtendedFigure5.Myeloid3.20.26.pdf","url":"https://assets-eu.researchsquare.com/files/rs-4934147/v1/2d1155198de19866d5a711a0.pdf"},{"id":106404034,"identity":"e29321c3-1a94-4ab1-bc2a-685074083071","added_by":"auto","created_at":"2026-04-08 09:15:24","extension":"pdf","order_by":4,"title":"","display":"","copyAsset":false,"role":"supplement","size":235892,"visible":true,"origin":"","legend":"Extended Figure 7","description":"","filename":"ExtendedFigure7.Lymphoidcells3.20.26.pdf","url":"https://assets-eu.researchsquare.com/files/rs-4934147/v1/8231d708b7cb626039198ea3.pdf"},{"id":106404076,"identity":"16d06649-6d15-418c-b004-b166a99009c9","added_by":"auto","created_at":"2026-04-08 09:15:27","extension":"pdf","order_by":5,"title":"","display":"","copyAsset":false,"role":"supplement","size":1036741,"visible":true,"origin":"","legend":"\u003cp\u003eSupplemental Figure 1\u003c/p\u003e","description":"","filename":"SupplementalFigure14.06.26.pdf","url":"https://assets-eu.researchsquare.com/files/rs-4934147/v1/57c5cd9fed1bf393f1dc9bf9.pdf"},{"id":106379783,"identity":"21f2dd68-ea89-468e-926b-03e7447f46b1","added_by":"auto","created_at":"2026-04-08 04:48:04","extension":"pdf","order_by":6,"title":"","display":"","copyAsset":false,"role":"supplement","size":2633339,"visible":true,"origin":"","legend":"\u003cp\u003eSupplemental Figure 2\u003c/p\u003e","description":"","filename":"SupplementalFigure23.20.26.pdf","url":"https://assets-eu.researchsquare.com/files/rs-4934147/v1/6b86a8eb0b7d16d179ace765.pdf"},{"id":106404018,"identity":"40c80ddc-4fb0-4160-a495-6df2231097d4","added_by":"auto","created_at":"2026-04-08 09:15:22","extension":"xlsx","order_by":7,"title":"","display":"","copyAsset":false,"role":"supplement","size":9271014,"visible":true,"origin":"","legend":"Supplemental Table 1","description":"","filename":"Supptable1NatImm12.12.25.xlsx","url":"https://assets-eu.researchsquare.com/files/rs-4934147/v1/72369d76c8052ec58a9d519e.xlsx"},{"id":106379786,"identity":"8f9d211d-e2c0-445e-ab2d-3be699146a2b","added_by":"auto","created_at":"2026-04-08 04:48:04","extension":"xlsx","order_by":8,"title":"","display":"","copyAsset":false,"role":"supplement","size":24994939,"visible":true,"origin":"","legend":"Supplemental Table 2","description":"","filename":"Supptable2NatImm4.06.26.xlsx","url":"https://assets-eu.researchsquare.com/files/rs-4934147/v1/65fb24dac617bf05a358e753.xlsx"}],"financialInterests":"There is \u003cb\u003eNO\u003c/b\u003e Competing Interest.","formattedTitle":"\u003cp\u003eSingle nuclei/cell transcriptomics reveal DMD driven cell dynamics and mechanisms of fibroblast inflammatory tissue priming in human dystrophic muscle\u003c/p\u003e","fulltext":[],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":false,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":true,"hideJournal":false,"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":"nature-portfolio","isNatureJournal":true,"hasQc":false,"allowDirectSubmit":false,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"","title":"Nature Portfolio","twitterHandle":"","acdcEnabled":false,"dfaEnabled":false,"editorialSystem":"ejp","reportingPortfolio":"","inReviewEnabled":true,"inReviewRevisionsEnabled":false},"keywords":"","lastPublishedDoi":"10.21203/rs.3.rs-4934147/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-4934147/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"Single cell/nuclei technologies have revolutionized our understanding of the remodeling of complex multi-cellular tissue that accompanies injury, regeneration, and disease. Duchenne muscular dystrophy (DMD) is a fatal genetic disease of childhood characterized by progressive skeletal muscle weakness resulting from mutation of DMD and loss of functional dystrophin. Here we report, at single nuclei/cell resolution, on intramuscular cell and gene expression dynamics within a broad cohort of needle muscle biopsies obtained from DMD individuals with varying degrees of severity, including a subset with low levels of dystrophin. We report a strong negative correlation between expression of dystrophin and disease severity and report substantial differences in cellularity and cell type-specific gene expression in DMD severity groups versus healthy muscle. Expression signatures indicate that DMD myofibers become immunologically alert, upregulating innate and adaptive immune sensors, including TLR4, IL15, TNF family receptors and MHC and costimulators. In this cohort, dystrophic muscle was remodeled with 50% fewer myofibers with expansion and diversification of fibroblasts and myeloid cells. We identify a DMD-specific TNFα-responsive Thy-1+/C3+ fibroblast subpopulation which we propose are inflammatory tissue priming fibroblasts and three DMD-specific myeloid populations which express signatures of innate immune memory. There is an 8-fold increase in CD8+GZMK+/GZMB+ T cells (Tek), with characteristics of both adaptive and innate immune activity. We propose that these non-myofiber muscle resident cells interact and epigenetically instill long-term tissue memory to perpetuate and amplify a hyper-inflammatory state in DMD muscle, contributing to impaired regeneration, myofiber death and fibrosis. This compendium of single/cell nuclei serves as a valuable reference and has immediate impact for biomarker discovery, clinical trial design, identification of barriers to dystrophin replacement therapies and novel druggable cell mechanisms operating in DMD.","manuscriptTitle":"Single nuclei/cell transcriptomics reveal DMD driven cell dynamics and mechanisms of fibroblast inflammatory tissue priming in human dystrophic muscle","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2026-04-08 04:47:53","doi":"10.21203/rs.3.rs-4934147/v1","editorialEvents":[],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"nature-immunology","isNatureJournal":true,"hasQc":false,"allowDirectSubmit":false,"externalIdentity":"ni","sideBox":"Learn more about [Nature Immunology](http://www.nature.com/ni/)","snPcode":"","submissionUrl":"","title":"Nature Immunology","twitterHandle":"","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"ejp","reportingPortfolio":"Nature Research","inReviewEnabled":true,"inReviewRevisionsEnabled":false}}],"origin":"","ownerIdentity":"82719587-37a9-46ee-bc36-03dd3210e454","owner":[],"postedDate":"April 8th, 2026","published":true,"recentEditorialEvents":[{"type":"editorInvitedReview","content":"This content is not available.","date":"2026-05-12T18:46:06+00:00","index":2,"fulltext":"This content is not available."},{"type":"editorInvitedReview","content":"This content is not available.","date":"2026-05-11T00:48:38+00:00","index":3,"fulltext":"This content is not available."},{"type":"editorInvitedReview","content":"This content is not available.","date":"2026-04-30T15:18:59+00:00","index":1,"fulltext":"This content is not available."}],"rejectedJournal":[],"revision":"","amendment":"","status":"under-review","subjectAreas":[{"id":65901141,"name":"Health sciences/Pathogenesis/Inflammation/Chronic inflammation"},{"id":65901142,"name":"Biological sciences/Immunology/Innate immunity/Pattern recognition receptors/Toll-like receptors"},{"id":65901143,"name":"Biological sciences/Biological techniques/Sequencing/RNA sequencing"}],"tags":[],"updatedAt":"2026-04-15T19:38:06+00:00","versionOfRecord":[],"versionCreatedAt":"2026-04-08 04:47:53","video":"","vorDoi":"","vorDoiUrl":"","workflowStages":[]},"version":"v1","identity":"rs-4934147","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-4934147","identity":"rs-4934147","version":["v1"]},"buildId":"XKTyCvWXoU3ODBz1xrDgd","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}