Aging-Associated Microbiota Drives Treg Dysfunction via TNF Signaling

Aging-Associated Microbiota Drives Treg Dysfunction via TNF Signaling | 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 Aging-Associated Microbiota Drives Treg Dysfunction via TNF Signaling Ari Waisman, Jefferson Leite, Zeynep Ergün, Emmanouil Stylianakis, and 14 more This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-7064776/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 Aging is associated with a chronic, low-grade inflammatory state referred to as inflammaging, which contributes to impaired immune regulation and increased susceptibility to disease. While regulatory T (Treg) cells are key mediators of immune homeostasis, their role in the context of age-related inflammation remains poorly understood. Here we demonstrate that age-related changes in the microbiota promote impaired Treg cell function, resulting in the differentiation of inflammatory T cells. In agreement, we find that aged germ-free (GF) mice exhibited a more balanced immune profile, where the Treg cells are functional and pro-inflammatory mediators are reduced, suggesting that microbial exposure is essential for the establishment of inflammaging. Furthermore, we show that the use of old microbiota in young animals was sufficient to induce pro-inflammatory T cell responses and impaired mucosal Treg cell proliferation, while young microbiota restored Treg cell function in old animals. Mechanistically, we show that exposure to aged microbiota was associated with sustained TNF signaling, elevated oxidative stress, DNA damage, and increased expression of senescence markers such as γH2AX and p16 in Treg cells. These findings uncover a microbiota-TNF- dependent mechanism by which age-associated microbial dysbiosis drives Treg cell dysfunction and promotes immune aging, highlighting the therapeutic potential of microbiota-targeted strategies to restore immune homeostasis in the elderly. Biological sciences/Immunology/Adaptive immunity/Immune tolerance/Peripheral tolerance Biological sciences/Immunology/Cytokines/Tumour-necrosis factors Aging Inflammaging Regulatory T cells Microbiota TNF signaling Full Text Additional Declarations There is NO Competing Interest. Supplementary Files SupFigure1.pdf Supplementary Figure 1: Microbiota transfer modulates Treg proliferation and inflammatory profile in germ-free mice. (A) Representative dot plots showing Foxp3+ Treg frequencies in the spleen of germ-free (GF) control mice, GF + young FMT, and GF + aged FMT recipients. The adjacent bar graph quantifies Treg cell frequencies (%). (B) Quantification of Ki67+ proliferating Treg cells in the mesenteric lymph nodes (mLNs) and large intestinal lamina propria (LI-LP) of GF control, GF + young FMT, and GF + aged FMT groups. (C) Heatmap showing Z-score normalized expression of TNF signaling-related genes in Treg cells from GF + young FMT and GF + aged FMT mice. Increased expression of inflammatory mediators is observed in aged FMT recipients. Color scale: red (high expression), blue (low expression). (D) Representative dot plots and quantification of c-Maf expression (%) in Foxp3+ Treg cells from FMT-treated mice (aged control, young + aged FMT, aged + young FMT). (E) Quantification of Foxp3+c-Maf+ Treg frequencies (%) and Foxp3+c-Maf - Treg frequencies (%) in FMT-treated mice (aged control, young + aged FMT, aged + young FMT). (F, G) Quantification of γH2AX and p16 expression (MFI) in Foxp3+ Treg cells from young and aged control and after FMT. (H) Representative histograms and quantification of γH2AX and p16 expression (MFI) in Foxp3+ Treg cells isolated from the lamina propria (LP) of the colon from control mice and Foxp3 cre Maf flox/flox mice. Data shown are representative of two to three independent experiments (n = 4–5 mice per group) and presented as mean ± SEM. Statistical analysis was performed using ANOVA with post-hoc test or unpaired t-test. *p < 0.05; **p < 0.01. Each symbol represents one mouse. 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-7064776","acceptedTermsAndConditions":true,"allowDirectSubmit":false,"archivedVersions":[],"articleType":"Article","associatedPublications":[],"authors":[{"id":487587002,"identity":"0bc6731a-0f66-405c-b011-b2478f2d3ef7","order_by":0,"name":"Ari 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13:17:35","extension":"pdf","order_by":1,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":6025257,"visible":true,"origin":"","legend":"","description":"","filename":"Manuscriptfinalversion.pdf","url":"https://assets-eu.researchsquare.com/files/rs-7064776/v1_covered_40fc4225-6432-48d3-aac8-b0657eb451cb.pdf"},{"id":97228113,"identity":"4a96eb78-2e83-401c-847a-90bf0feade33","added_by":"auto","created_at":"2025-12-02 08:52:34","extension":"pdf","order_by":1,"title":"","display":"","copyAsset":false,"role":"supplement","size":981714,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eSupplementary Figure 1: Microbiota transfer modulates Treg proliferation and inflammatory profile in germ-free mice. \u003c/strong\u003e(A) Representative dot plots showing Foxp3+ Treg frequencies in the spleen of germ-free (GF) control mice, GF + young FMT, and GF + aged FMT recipients. The adjacent bar graph quantifies Treg cell frequencies (%). (B) Quantification of Ki67+ proliferating Treg cells in the mesenteric lymph nodes (mLNs) and large intestinal lamina propria (LI-LP) of GF control, GF + young FMT, and GF + aged FMT groups. (C) Heatmap showing Z-score normalized expression of TNF signaling-related genes in Treg cells from GF + young FMT and GF + aged FMT mice. Increased expression of inflammatory mediators is observed in aged FMT recipients. Color scale: red (high expression), blue (low expression). (D) Representative dot plots and quantification of c-Maf expression (%) in Foxp3+ Treg cells from FMT-treated mice (aged control, young + aged FMT, aged + young FMT). (E) Quantification of Foxp3+c-Maf+ Treg frequencies (%) and Foxp3+c-Maf\u003csup\u003e-\u003c/sup\u003e Treg frequencies (%) in FMT-treated mice (aged control, young + aged FMT, aged + young FMT). (F, G) Quantification of γH2AX and p16 expression (MFI) in Foxp3+ Treg cells from young and aged control and after FMT. (H) Representative histograms and quantification of γH2AX and p16 expression (MFI) in Foxp3+ Treg cells isolated from the lamina propria (LP) of the colon from control mice and Foxp3\u003csup\u003ecre\u003c/sup\u003eMaf\u003csup\u003eflox/flox\u003c/sup\u003e mice. Data shown are representative of two to three independent experiments (n = 4–5 mice per group) and presented as mean ± SEM. Statistical analysis was performed using ANOVA with post-hoc test or unpaired t-test. *p \u0026lt; 0.05; **p \u0026lt; 0.01. Each symbol represents one mouse.\u003c/p\u003e","description":"","filename":"SupFigure1.pdf","url":"https://assets-eu.researchsquare.com/files/rs-7064776/v1/be65219d325bb5fb2d04f609.pdf"}],"financialInterests":"There is \u003cb\u003eNO\u003c/b\u003e Competing Interest.","formattedTitle":"Aging-Associated Microbiota Drives Treg Dysfunction via TNF Signaling","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":"Aging, Inflammaging, Regulatory T cells, Microbiota, TNF signaling","lastPublishedDoi":"10.21203/rs.3.rs-7064776/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-7064776/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"Aging is associated with a chronic, low-grade inflammatory state referred to as\r\ninflammaging, which contributes to impaired immune regulation and increased\r\nsusceptibility to disease. While regulatory T (Treg) cells are key mediators of immune\r\nhomeostasis, their role in the context of age-related inflammation remains poorly\r\nunderstood. Here we demonstrate that age-related changes in the microbiota promote\r\nimpaired Treg cell function, resulting in the differentiation of inflammatory T cells. In\r\nagreement, we find that aged germ-free (GF) mice exhibited a more balanced immune\r\nprofile, where the Treg cells are functional and pro-inflammatory mediators are reduced,\r\nsuggesting that microbial exposure is essential for the establishment of inflammaging.\r\nFurthermore, we show that the use of old microbiota in young animals was sufficient to\r\ninduce pro-inflammatory T cell responses and impaired mucosal Treg cell proliferation,\r\nwhile young microbiota restored Treg cell function in old animals. Mechanistically, we\r\nshow that exposure to aged microbiota was associated with sustained TNF signaling,\r\nelevated oxidative stress, DNA damage, and increased expression of senescence markers\r\nsuch as γH2AX and p16 in Treg cells. These findings uncover a microbiota-TNF-\r\ndependent mechanism by which age-associated microbial dysbiosis drives Treg cell\r\ndysfunction and promotes immune aging, highlighting the therapeutic potential of\r\nmicrobiota-targeted strategies to restore immune homeostasis in the elderly.","manuscriptTitle":"Aging-Associated Microbiota Drives Treg Dysfunction via TNF Signaling","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2025-12-02 08:52:30","doi":"10.21203/rs.3.rs-7064776/v1","editorialEvents":[],"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":"e4be5e64-7310-4b36-ae0a-bcfef8414a03","owner":[],"postedDate":"December 2nd, 2025","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"posted","subjectAreas":[{"id":51769515,"name":"Biological sciences/Immunology/Adaptive immunity/Immune tolerance/Peripheral tolerance"},{"id":51769516,"name":"Biological sciences/Immunology/Cytokines/Tumour-necrosis factors"}],"tags":[],"updatedAt":"2025-12-02T08:52:30+00:00","versionOfRecord":[],"versionCreatedAt":"2025-12-02 08:52:30","video":"","vorDoi":"","vorDoiUrl":"","workflowStages":[]},"version":"v1","identity":"rs-7064776","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-7064776","identity":"rs-7064776","version":["v1"]},"buildId":"8U1c8b4HqxoKbykW_rLl7","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}