Umbilical cord-derived mesenchymal stromal cells prime monocytes to enhance eosinophils-mediated defense against Klebsiella pneumoniae

Umbilical cord-derived mesenchymal stromal cells prime monocytes to enhance eosinophils-mediated defense against Klebsiella pneumoniae | 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 Umbilical cord-derived mesenchymal stromal cells prime monocytes to enhance eosinophils-mediated defense against Klebsiella pneumoniae Quanming Zou, Yue Ming, Yu Gong, Yue Yuan, Ke Wang, Jiang Li, and 9 more This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-9059506/v1 This work is licensed under a CC BY 4.0 License Status: Under Review Version 1 posted 7 You are reading this latest preprint version Abstract The increasing prevalence of antibiotic-resistant Klebsiella pneumoniae (KP) necessitates novel preventive strategies. Although human mesenchymal stromal cells (MSCs) exhibit immunomodulatory and antimicrobial properties, their clinical utility against bacterial infection remains limited. Here, we examined the protective efficacy and underlying mechanism of prophylaxis using human umbilical cord-derived MSCs (hUC-MSCs) in a murine model of KP lung infection. Pretreatment with hUC-MSCs significantly protected both young and aged mice from KP pneumonia, as evidenced by improved survival, reduced pulmonary bacterial loads, and attenuated lung injury. Mechanistically, hUC-MSCs reprogrammed monocytes toward an immunoregulatory phenotype that enhanced bacterial clearance and drove CCL24-dependent recruitment of eosinophils (EOS) to infected lungs. EOS were subsequently activated by epithelial-derived IL-33 and exhibited potent antimicrobial activity. Disruption of the monocyte-EOS axis abolished hUC-MSC-mediated protection. Clinically, higher circulating EOS level was associated with better clinical outcomes in KP-infected patients. Thus, we identify a novel hUC-MSC-monocyte-EOS protective axis that balances anti-inflammatory and antimicrobial responses, providing a mechanistic basis for hUC-MSC-based prophylaxis against severe bacterial pneumonia. Biological sciences/Immunology/Antimicrobial responses Biological sciences/Immunology/Infection Figures Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 Figure 6 Figure 7 Figure 8 Full Text Additional Declarations (Not answered) Supplementary Files ExtendedDataFig.4.jpg Extended Data Fig.4 ExtendedDataFig.3.jpg Extended Data Fig.3 ExtendedDataFig.5.jpg Extended Data Fig.5 ExtendedDataFigureLegends.docx Extended Data Figure Legends ExtendedDataFig.8.jpg Extended Data Fig.8 TableS2.xlsx Table S2 ExtendedDataFig.7.jpg Extended Data Fig.7 ExtendedDataFig.2.jpg Extended Data Fig.2 TableS3.xlsx Table S3 TableS1.xlsx Table S1 ExtendedDataFig.6.jpg Extended Data Fig.6 ExtendedDataFig.1.jpg Extended Data Fig.1 SourceFiguredata.pdf Source Figure data Cite Share Download PDF Status: Under Review Version 1 posted Review # 1 received at journal 01 Apr, 2026 Reviewer # 1 agreed at journal 20 Mar, 2026 Reviewers invited by journal 20 Mar, 2026 Editor assigned by journal 18 Mar, 2026 Submission checks completed at journal 18 Mar, 2026 First submitted to journal 18 Mar, 2026 Unknown event 08 Mar, 2026 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. 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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-9059506","acceptedTermsAndConditions":true,"allowDirectSubmit":false,"archivedVersions":[],"articleType":"Article","associatedPublications":[],"authors":[{"id":609217610,"identity":"f72d85e3-1077-4051-bf65-e4260a2fee38","order_by":0,"name":"Quanming Zou","email":"data:image/png;base64,iVBORw0KGgoAAAANSUhEUgAAAZAAAAAyAQMAAABI0h/eAAAABlBMVEX///8AAABVwtN+AAAACXBIWXMAAA7EAAAOxAGVKw4bAAAAyElEQVRIie3QMQrCMBTG8VcEXR7F8ZVCbiBEAk5Sr9JQaFdHR0shLl5A8BzOlYBdOovQpcFZ6AlEV1FIRof85u8/vAfgeX9rQ7hqytIM7km7ZNDqSpBzEahcwK1QU3RZh9fG3HGsZXkwCggSNttakqhLM4GoZRVL1a8hE4vakvAuvcRIWqpY7jhBLU/25D1GruU+OitCtyQbx5jmgihwTKIuH82P9ZJxlO8nc4dbwq4w/eNJyCeNMcMmYdYEPgfcNv9OPM/zvB9emgNDk/l3Y28AAAAASUVORK5CYII=","orcid":"https://orcid.org/0000-0002-6971-0786","institution":"Third Military Medical University","correspondingAuthor":true,"prefix":"","firstName":"Quanming","middleName":"","lastName":"Zou","suffix":""},{"id":609217611,"identity":"46387871-3f9c-4447-b0d0-bab8ccac5a0f","order_by":1,"name":"Yue Ming","email":"","orcid":"","institution":"Army Medical University","correspondingAuthor":false,"prefix":"","firstName":"Yue","middleName":"","lastName":"Ming","suffix":""},{"id":609217612,"identity":"01cabcb5-8a2b-433f-b8b9-3fac371441e2","order_by":2,"name":"Yu Gong","email":"","orcid":"","institution":"University-Town Hospital of Chongqing Medical University","correspondingAuthor":false,"prefix":"","firstName":"Yu","middleName":"","lastName":"Gong","suffix":""},{"id":609217613,"identity":"f57215bd-2281-495b-bb5f-6710bd596fa7","order_by":3,"name":"Yue Yuan","email":"","orcid":"","institution":"Army Medical University","correspondingAuthor":false,"prefix":"","firstName":"Yue","middleName":"","lastName":"Yuan","suffix":""},{"id":609217614,"identity":"b1040d50-0f4f-4242-8719-b7470613429c","order_by":4,"name":"Ke Wang","email":"","orcid":"","institution":"Army Medical University","correspondingAuthor":false,"prefix":"","firstName":"Ke","middleName":"","lastName":"Wang","suffix":""},{"id":609217615,"identity":"bebf6116-cdbd-4712-9719-606b9bc6553e","order_by":5,"name":"Jiang Li","email":"","orcid":"","institution":"The Third Hospital Affiliated to the Third Military Medical University","correspondingAuthor":false,"prefix":"","firstName":"Jiang","middleName":"","lastName":"Li","suffix":""},{"id":609217616,"identity":"744f4051-bec7-4bf7-908b-31bb0bc7063d","order_by":6,"name":"Liusheng Peng","email":"","orcid":"","institution":"Army Medical University","correspondingAuthor":false,"prefix":"","firstName":"Liusheng","middleName":"","lastName":"Peng","suffix":""},{"id":609217617,"identity":"1d06dbe7-7cfb-43e0-9aed-e5fdfa66b32d","order_by":7,"name":"Bin Wang","email":"","orcid":"","institution":"The Second Affiliated Hospital of Army Medical University","correspondingAuthor":false,"prefix":"","firstName":"Bin","middleName":"","lastName":"Wang","suffix":""},{"id":609217618,"identity":"792efebf-56c6-4e8c-8568-ae758ad63610","order_by":8,"name":"Xiao Li","email":"","orcid":"","institution":"Army Medical University","correspondingAuthor":false,"prefix":"","firstName":"Xiao","middleName":"","lastName":"Li","suffix":""},{"id":609217619,"identity":"aba68344-6e71-4740-8a20-f4f1bf70ea30","order_by":9,"name":"Xi Zeng","email":"","orcid":"","institution":"Army Medical University","correspondingAuthor":false,"prefix":"","firstName":"Xi","middleName":"","lastName":"Zeng","suffix":""},{"id":609217620,"identity":"fd6812df-3cf7-47c3-acfa-1a462749d66b","order_by":10,"name":"Yong He","email":"","orcid":"https://orcid.org/0000-0002-9404-798X","institution":"Department of Respiratory Disease, Daping Hospital, Army Medical University, Chongqing, China.","correspondingAuthor":false,"prefix":"","firstName":"Yong","middleName":"","lastName":"He","suffix":""},{"id":609217621,"identity":"bdd8af72-0161-4752-8107-d5c8ed9a61d8","order_by":11,"name":"Rui Yang","email":"","orcid":"","institution":"People's Liberation Army General Hospital of Central Theater Command","correspondingAuthor":false,"prefix":"","firstName":"Rui","middleName":"","lastName":"Yang","suffix":""},{"id":609217622,"identity":"df8c027f-8f36-43b2-9bf5-a436bd8478a2","order_by":12,"name":"Hao Zeng","email":"","orcid":"","institution":"Army Medical University","correspondingAuthor":false,"prefix":"","firstName":"Hao","middleName":"","lastName":"Zeng","suffix":""},{"id":609217623,"identity":"bf029786-8fc3-4fef-a933-40a40197fd09","order_by":13,"name":"Jin Zhang","email":"","orcid":"","institution":"Army Medical University","correspondingAuthor":false,"prefix":"","firstName":"Jin","middleName":"","lastName":"Zhang","suffix":""},{"id":609217624,"identity":"b30b7377-c176-47db-9d6e-d582c46cbd6f","order_by":14,"name":"Ting Yu","email":"","orcid":"","institution":"Army Medical University","correspondingAuthor":false,"prefix":"","firstName":"Ting","middleName":"","lastName":"Yu","suffix":""}],"badges":[],"createdAt":"2026-03-07 15:35:17","currentVersionCode":1,"declarations":"","doi":"10.21203/rs.3.rs-9059506/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-9059506/v1","draftVersion":[],"editorialEvents":[],"editorialNote":"","failedWorkflow":false,"files":[{"id":105320999,"identity":"eb25b69d-282e-4ca8-9279-44dacde5e922","added_by":"auto","created_at":"2026-03-24 17:19:46","extension":"jpg","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":3079695,"visible":true,"origin":"","legend":"\u003cp\u003ehUC-MSCs pretreatment confers protection against KP pneumonia in mice. \u0026nbsp;(A) Bioluminescence imaging tracks the in vivo biodistribution and clearance of \u0026nbsp;luciferase-expressing hUC-MSCs after i.t. or i.v. administration. Shown are \u0026nbsp;representative whole-body and ex vivo lung images (left) and quantification of total \u0026nbsp;lung flux (right) (n = 3–10 per group per time point). (B) Experimental design (top) and \u0026nbsp;survival curves of mice pretreated with hUC-MSCs or PBS at indicated time point (1 8 days) before KP challenge (n = 14–17; combined data from two independent \u0026nbsp;experiments). (C) Lung bacterial burden quantified at 24 and 48 hours post infection \u0026nbsp;(hpi) (n = 5). (D) Representative H\u0026amp;E-stained lung sections at 24 hpi (left; Scale bars: \u0026nbsp;upper 2 mm; lower 200 µm) and corresponding histopathological injury scores (right) \u0026nbsp;(n = 5). (E) Levels of pro-inflammatory cytokines in lung homogenates and BALF from \u0026nbsp;uninfected versus KP-infected mice at 24 hpi (n = 3–5). (F) Flow cytometric analysis \u0026nbsp;of innate immune cell populations following KP infection. Representative plots (left), \u0026nbsp;percentages (middle) and absolute numbers (right) of lung neutrophils \u0026nbsp;(CD45+CD11b+Ly6G+), AMs (CD45+CD11b⁻ Ly6G⁻CD11c+SiglecF+) and \u0026nbsp;monocyte/macrophages (CD45+Ly6G⁻CD11c⁻SiglecF⁻CD11b+F4/80+) in hUC-MSC- \u0026nbsp;versus PBS-pretreated mice at indicated time points post infection (n = 6–7). (G) \u0026nbsp;Heatmap of differentially expressed genes in lungs at 24 hpi from hUC-MSC- versus \u0026nbsp;PBS-pretreated mice (n = 4). Data are presented as mean ± SD. Significance was \u0026nbsp;determined by Student’s t test in (C), (F), one-way ANOVA with Tukey’s post hoc test \u0026nbsp;in (D and E); * P \u0026lt; 0.05; ** P \u0026lt; 0.01; *** P \u0026lt; 0.001; **** P \u0026lt; 0.0001. ns, not significant.\u003c/p\u003e","description":"","filename":"Fig1.jpg","url":"https://assets-eu.researchsquare.com/files/rs-9059506/v1/f25809050b39b0b142c45830.jpg"},{"id":105321005,"identity":"974b3564-21b1-4e87-b0f2-5363b650a5a6","added_by":"auto","created_at":"2026-03-24 17:19:46","extension":"jpg","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":1962910,"visible":true,"origin":"","legend":"\u003cp\u003eMonocytes are essential mediators of hUC-MSC-induced protection against \u0026nbsp;KP pneumonia. (A) Representative flow cytometry plots showing lung neutrophils, \u0026nbsp;monocytes/macrophages, and AMs that have engulfed GFP-MSCs at 24 hours after i.t. \u0026nbsp;administration. Numbers indicate the percentage of each gated subset within \u0026nbsp;CD45+GFP+ cells. (B) Lung bacterial burden at 24 hpi following selective depletion of \u0026nbsp;monocytes or AMs in PBS- or hUC-MSC-pretreated mice. Empty liposomes served as \u0026nbsp;negative controls (NC) (n = 5). (C) Uniform Manifold Approximation and Projection \u0026nbsp;(UMAP) visualization of immune cell heterogeneity based on scRNA-seq of lung \u0026nbsp;CD45+ cells from KP-infected mice (n = 5). (D) Dot plot showing the representative \u0026nbsp;marker genes used to annotate immune cell clusters identified by scRNA-seq. (E) \u0026nbsp;KEGG pathway enrichment analysis of monocyte/macrophage cluster from KP infected mice with or without hUC-MSCs pretreatment, highlighting upregulated \u0026nbsp;microbicidal pathways (left) and downregulated inflammatory signaling pathways \u0026nbsp;(right). (F) Flow cytometric analysis of lung monocyte/macrophage subsets 24 hpi, \u0026nbsp;revealing an increase in Ly6C⁻MHCII+ mature macrophages with hUC-MSCs \u0026nbsp;pretreatment (n = 5). (G) Flow cytometric assessment of lung monocyte/macrophage \u0026nbsp;polarization at 24 hpi (n = 5). Data are presented as mean ± SD. Significance was \u0026nbsp;determined by Student’s t test; * P \u0026lt; 0.05. ns, not significant.\u003c/p\u003e","description":"","filename":"Fig2.jpg","url":"https://assets-eu.researchsquare.com/files/rs-9059506/v1/0501a9c32fb8d11beb24d600.jpg"},{"id":105565060,"identity":"031de587-2e54-4896-a6f9-b124d2c472c5","added_by":"auto","created_at":"2026-03-27 12:51:45","extension":"jpg","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":2315114,"visible":true,"origin":"","legend":"\u003cp\u003ehUC-MSCs pretreatment enhances pulmonary EOS recruitment via a \u0026nbsp;monocyte-CCL24 axis. (A) Heatmap of chemokine expression in lung homogenates rom PBS- or hUC-MSC-pretreated mice at 24 hpi; with CCL24 protein levels \u0026nbsp;quantified at right (n = 5). (B) Validation of CCL24 protein levels by ELISA in a \u0026nbsp;separate cohort of mice, comparing uninfected and KP-infected conditions with or \u0026nbsp;without hUC-MSCs pretreatment (n = 5). (C) Representative flow cytometry plots (left) \u0026nbsp;and quantification (right) of pulmonary EOS (CD45+ Ly6G⁻CD11c⁻SiglecF+) at \u0026nbsp;indicated time points following KP infection in PBS- or hUC-MSC-pretreated mice (n \u0026nbsp;= 5–7). (D) Immunofluorescence staining of eosinophil cationic protein (ECP; red) in \u0026nbsp;lung sections from PBS- or hUC-MSC-pretreated mice at 24 hpi. Nuclei counterstained \u0026nbsp;with DAPI (blue). Quantification of ECP+ cell number per mm2 lung area shown at \u0026nbsp;right (n = 3 animals, with 4 random fields analyzed per mouse). Scale bars: 200 μm. (E) \u0026nbsp;Representative flow cytometry plot and quantification of pulmonary EOS infiltration \u0026nbsp;24 h after i.t. administration of recombinant CCL24. (F) Lung bacterial burden at 24 \u0026nbsp;hpi in mice treated with recombinant CCL24 or PBS control (n = 5). (G) UMAP \u0026nbsp;visualization of monocyte/macrophage subsets from lung scRNA-seq data, with \u0026nbsp;overlay of CCL24 transcript expression. (H) Dot plot showing expression of selected \u0026nbsp;marker genes across monocyte/macrophage subsets identified by scRNA-seq. (I) \u0026nbsp;UMAP feature plot illustrating enrichment of CCL24 expression within Ly6Chi \u0026nbsp;classical monocytes. (J) Lung CCL24 protein levels at 24 hpi in PBS- or hUC-MSC pretreated mice following monocytes depletion via i.v. delivery of clodronate liposomes \u0026nbsp;(n = 5). (K) Quantification of pulmonary EOS at 24 hpi from PBS- or hUC-MSC pretreated mice following selective depletion of monocytes (n = 3–4). Data are \u0026nbsp;presented as mean ± SD. Student’s t test in (A), (C), (E and F), one-way ANOVA with \u0026nbsp;Tukey’s post hoc test for (B), (D), (J and K). * P \u0026lt; 0.05; ** P \u0026lt; 0.01; *** P \u0026lt; 0.001, \u0026nbsp;**** P \u0026lt; 0.0001. ns, not significant.\u003c/p\u003e","description":"","filename":"Fig3.jpg","url":"https://assets-eu.researchsquare.com/files/rs-9059506/v1/ff980c892e6ad1e08974388f.jpg"},{"id":105565546,"identity":"4adc8b76-9071-4f92-9cc3-bf3f49d68494","added_by":"auto","created_at":"2026-03-27 12:53:33","extension":"jpg","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":2611488,"visible":true,"origin":"","legend":"\u003cp\u003ePulmonary EOS are essential effectors of host defense against KP \u0026nbsp;pneumonia. (A to D) Lung bacterial burden (A and C) and inflammatory cytokine \u0026nbsp;levels (B and D) at 24 hpi in mice adoptively transferred with lung EOS (1×105 cells) \u0026nbsp;or BmEOS (5×105 cells) prior to KP infection (n=7–8 mice per group; combined data \u0026nbsp;from two independent experiments). (E) Representative H\u0026amp;E-stained lung sections \u0026nbsp;from mice receiving PBS or BmEOS, with or without KP infection (left; scale bars, \u0026nbsp;upper: 2 mm; lower: 200 μm) and corresponding histopathological injury scores (right) \u0026nbsp;(n = 5). (F) Representative flow cytometry plots and quantification of pulmonary EOS \u0026nbsp;at 24 hpi in mice treated with anti-IL-5 or isotype control antibody before KP infection. \u0026nbsp;(G) Lung bacterial burden at 24 hpi in mice treated with anti-IL-5 antibody or isotype \u0026nbsp;control (n = 5). (H) Survival curves of PHIL mice and wild-type (WT) littermates \u0026nbsp;following KP infection (n = 8). (I) Lung bacterial burden at 24 hpi in PHIL mice and \u0026nbsp;WT littermates (n = 4–5). (J) Representative H\u0026amp;E-stained lung sections from PHIL \u0026nbsp;mice and WT littermates at 24 hpi (left; scale bars: upper 2 mm; lower 100 µm), and \u0026nbsp;corresponding histopathological injury scores (right) (n = 4). (K) Representative flow \u0026nbsp;cytometry plots (upper) and quantification (lower) of pulmonary EOS at 24 hpi in mice \u0026nbsp;treated with anti-CCR3 or isotype control antibody before KP infection. (L) Survival \u0026nbsp;curves of mice pretreated with PBS or hUC-MSCs and then administered respective \u0026nbsp;anti-CCR3 or isotype control antibody prior to KP infection (n = 10). (M) Lung \u0026nbsp;bacterial burden of mice pretreated with PBS or hUC-MSCs and administered by anti CCL24 or isotype control antibody prior to KP infection (n = 5). Data are presented as \u0026nbsp;mean ± SD. Student’s t test for (A–D), (F and G), (I–K). One-way ANOVA with Tukey’s \u0026nbsp;post hoc test for (E), (M). * P \u0026lt; 0.05; ** P \u0026lt; 0.01. ns, not significant.\u003c/p\u003e","description":"","filename":"Fig4.jpg","url":"https://assets-eu.researchsquare.com/files/rs-9059506/v1/fd0c4c7d0a4f45587c2d533c.jpg"},{"id":105321014,"identity":"edba1607-63b4-4c25-a376-40cffcf0af99","added_by":"auto","created_at":"2026-03-24 17:19:47","extension":"jpg","order_by":5,"title":"Figure 5","display":"","copyAsset":false,"role":"figure","size":2070107,"visible":true,"origin":"","legend":"\u003cp\u003eActivated pulmonary EOS exert potent anti-KP ability. (A) Flow cytometric \u0026nbsp;quantification of activation marker expression (CD11c, CD11b, CD101 and SiglecF) \u0026nbsp;on pulmonary EOS at indicated time points after KP infection (n = 4–7). (B) Changes \u0026nbsp;in the proportions of pulmonary activated (CD101⁺) and basal (CD101⁻) EOS subsets \u0026nbsp;over time after KP infection (n = 7). (C) Survival curves of KP-infected mice following \u0026nbsp;adoptive transfer of CD101+ or CD101⁻ BmEOS (1×105 cells) compared with PBS \u0026nbsp;controls (n = 10). (D) In vitro bactericidal activity of sorted CD101⁺ and CD101⁻ \u0026nbsp;BmEOS against KP, quantified as CFU after co-culture (MOI = 100). (E) EPX levels \u0026nbsp;in supernatants from CD101+ or CD101⁻ BmEOS cultured with or without KP (MOI = \u0026nbsp;100). (F) Representative immunofluorescence images of BmEOS co-incubated with \u0026nbsp;mCherry-expressing KP at indicated time points, showing actin (green), nuclei (DAPI, \u0026nbsp;blue), and bacteria (red). Scale bars: 20 μm. (G) Flow cytometric assessment of CD101+ \u0026nbsp;EOS distribution in lung, BM, blood and spleen from naïve or KP-infected mice. (H \u0026nbsp;and I) Tracking of adoptively transferred CD45.1⁺CD101⁻ BmEOS in CD45.2⁺ \u0026nbsp;recipient mice following KP infection, showing EOS recruitment to the lung and \u0026nbsp;phenotypic transition by 24 hpi. Data are presented as mean ± SD. Significance was \u0026nbsp;determined by a one-way ANOVA with Tukey’s post hoc test; * P \u0026lt; 0.05; ** P \u0026lt; 0.01; \u0026nbsp;*** P \u0026lt; 0.001; **** P \u0026lt; 0.0001. ns, not significant.\u003c/p\u003e","description":"","filename":"Fig5.jpg","url":"https://assets-eu.researchsquare.com/files/rs-9059506/v1/8655c6ee7b6f90d1a6ea951c.jpg"},{"id":105321017,"identity":"810bb79a-fc60-4e7d-8988-91a0db55fcdc","added_by":"auto","created_at":"2026-03-24 17:19:47","extension":"jpg","order_by":6,"title":"Figure 6","display":"","copyAsset":false,"role":"figure","size":2174936,"visible":true,"origin":"","legend":"\u003cp\u003ehUC-MSCs pretreatment enhances pulmonary IL-33 production and \u0026nbsp;promotes EOS activation following KP infection. (A) Representative flow cytometry \u0026nbsp;plots (left) and quantification (right) of pulmonary CD101+ EOS at indicated time \u0026nbsp;points post infection from PBS- versus hUC-MSC-pretreated mice (n = 4–7). (B) IL 33 protein levels in lung homogenates from PBS- or hUC-MSC-pretreated mice at \u0026nbsp;indicated time points post-infection (n = 4–5). (C) Effect of IL-33 on the expression of \u0026nbsp;activation markers of KP-infected BmEOS. Flow cytometric quantification (top) and \u0026nbsp;representative histograms (bottom) show levels of CD11b, CD11c, CD69, SiglecF, and \u0026nbsp;CD101, in the presence or absence of recombinant IL-33. (D) Quantification of \u0026nbsp;activation marker expression (SiglecF, CD101, CD11b) and levels of pulmonary \u0026nbsp;CD101⁺ EOS in KP-infected mice treated with sST2 (10 μg) or vehicle control at 24 \u0026nbsp;hpi (n = 4). (E) Representative immunofluorescence images of infected lung sections \u0026nbsp;showing IL-33 (red) localized with SFTPC+ AT2 cells (green, white arrows) but not \u0026nbsp;with F4/80+ macrophages (violet, orange arrows). Nuclei are counterstained with DAPI \u0026nbsp;(blue). Scale bars: 20 μm. (F) IL-33 protein levels in lung homogenates from PBS- or \u0026nbsp;hUC-MSC-pretreated mice following selective depletion of monocytes (n = 3–5). Data \u0026nbsp;are presented as mean ± SD. Student’s t test for (A and B), one-way ANOVA with \u0026nbsp;Tukey’s post hoc test for (C and D), (F). * P \u0026lt; 0.05; ** P \u0026lt; 0.01; *** P \u0026lt; 0.001; **** \u0026nbsp;P \u0026lt; 0.0001. ns, not significant.\u003c/p\u003e","description":"","filename":"Fig6.jpg","url":"https://assets-eu.researchsquare.com/files/rs-9059506/v1/0325839b1ff8bd4d002a9375.jpg"},{"id":105564426,"identity":"22e0bcc1-dd3f-42e3-91db-29f46c373361","added_by":"auto","created_at":"2026-03-27 12:49:31","extension":"jpg","order_by":7,"title":"Figure 7","display":"","copyAsset":false,"role":"figure","size":999036,"visible":true,"origin":"","legend":"\u003cp\u003eRetrospective analysis of blood EOS levels and clinical outcomes in patients \u0026nbsp;with KP infection. ADM: admission (within 24h of hospitalization); DIS: discharge \u0026nbsp;(within 24 hours before discharge or declaration of death). (A) Flow diagram of patient \u0026nbsp;selection for the retrospective cohort analysis. Among patients diagnosed with KP \u0026nbsp;infection between 2022 and 2024, non-KP-related mortality was excluded, yielding 12 \u0026nbsp;nonsurvivors (KP pneumonia, n = 5; KP sepsis, n = 7) and 43 survivors included for \u0026nbsp;analysis. All patients underwent routine blood testing. (B) Comparison of peripheral \u0026nbsp;blood EOS percentage and absolute counts between survivors and nonsurvivors at \u0026nbsp;admission and discharge. (C) Correlation analyses between peripheral blood EOS \u0026nbsp;percentage and monocyte percentage at admission and discharge in survivors and \u0026nbsp;nonsurvivors. Statistical significance was assessed using two-way ANOVA with \u0026nbsp;Šidák’s multiple comparisons test (B) or Pearson’s chi-squared test (C). *** P \u0026lt; 0.001; \u0026nbsp;**** P \u0026lt; 0.0001. ns, not significant.\u003c/p\u003e","description":"","filename":"Fig7.jpg","url":"https://assets-eu.researchsquare.com/files/rs-9059506/v1/e751c342365faef0a59f8ddf.jpg"},{"id":105564464,"identity":"6106d127-b4e6-4147-a453-5cc4f2c8269c","added_by":"auto","created_at":"2026-03-27 12:49:40","extension":"jpg","order_by":8,"title":"Figure 8","display":"","copyAsset":false,"role":"figure","size":538726,"visible":true,"origin":"","legend":"\u003cp\u003eSchematic illustration of the hUC-MSC-monocyte-EOS protective axis in \u0026nbsp;KP pneumonia. Upon i.t. delivery, hUC-MSCs accumulate transiently in the lung and \u0026nbsp;are phagocytosed by monocytes. This interaction reprograms monocytes toward an \u0026nbsp;anti-inflammatory phenotype and induces secretion of the EOS-specific \u0026nbsp;chemoattractant CCL24. Circulating EOS are recruited to the infected lung along the \u0026nbsp;CCL24 gradient. Concurrently, hUC-MSC-primed monocytes indirectly upregulated \u0026nbsp;AT2 cell-derived IL-33 expression, promoting phenotypic transformation of CD101⁻ \u0026nbsp;resting EOS into CD101⁺ activated EOS. These activated EOS release cytotoxic \u0026nbsp;granules (e.g., EPX), mediating direct bacterial killing. 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pneumoniae","fulltext":[],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":false,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":false,"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":"cellular-and-molecular-immunology","isNatureJournal":false,"hasQc":false,"allowDirectSubmit":false,"externalIdentity":"cmi","sideBox":"Learn more about [Cellular \u0026 Molecular Immunology](http://www.nature.com/cmi/)","snPcode":"41423","submissionUrl":"https://mts-cmi.nature.com/cgi-bin/main.plex","title":"Cellular \u0026 Molecular Immunology","twitterHandle":"","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"ejp","reportingPortfolio":"Nature AJ","inReviewEnabled":true,"inReviewRevisionsEnabled":false},"keywords":"","lastPublishedDoi":"10.21203/rs.3.rs-9059506/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-9059506/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"The increasing prevalence of antibiotic-resistant Klebsiella pneumoniae (KP) necessitates novel preventive strategies. Although human mesenchymal stromal cells (MSCs) exhibit immunomodulatory and antimicrobial properties, their clinical utility against bacterial infection remains limited. Here, we examined the protective efficacy and underlying mechanism of prophylaxis using human umbilical cord-derived MSCs (hUC-MSCs) in a murine model of KP lung infection. Pretreatment with hUC-MSCs significantly protected both young and aged mice from KP pneumonia, as evidenced by improved survival, reduced pulmonary bacterial loads, and attenuated lung injury. Mechanistically, hUC-MSCs reprogrammed monocytes toward an immunoregulatory phenotype that enhanced bacterial clearance and drove CCL24-dependent recruitment of eosinophils (EOS) to infected lungs. EOS were subsequently activated by epithelial-derived IL-33 and exhibited potent antimicrobial activity. Disruption of the monocyte-EOS axis abolished hUC-MSC-mediated protection. Clinically, higher circulating EOS level was associated with better clinical outcomes in KP-infected patients. Thus, we identify a novel hUC-MSC-monocyte-EOS protective axis that balances anti-inflammatory and antimicrobial responses, providing a mechanistic basis for hUC-MSC-based prophylaxis against severe bacterial pneumonia.","manuscriptTitle":"Umbilical cord-derived mesenchymal stromal cells prime monocytes to enhance eosinophils-mediated defense against Klebsiella pneumoniae","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2026-03-24 17:19:41","doi":"10.21203/rs.3.rs-9059506/v1","editorialEvents":[{"type":"communityComments","content":0},{"type":"editorInvitedReview","content":"This content is not available.","date":"2026-04-01T06:59:32+00:00","index":1,"fulltext":"This content is not available."},{"type":"reviewerAgreed","content":"This content is not available.","date":"2026-03-20T07:04:32+00:00","index":1,"fulltext":"This content is not available."},{"type":"reviewersInvited","content":"","date":"2026-03-20T04:18:13+00:00","index":"","fulltext":""},{"type":"editorAssigned","content":"","date":"2026-03-19T01:54:06+00:00","index":"","fulltext":""},{"type":"checksComplete","content":"","date":"2026-03-19T00:53:39+00:00","index":"","fulltext":""},{"type":"submitted","content":"Cellular \u0026 Molecular Immunology","date":"2026-03-18T14:34:05+00:00","index":"","fulltext":""},{"type":"checksFailed","content":"","date":"2026-03-09T03:30:57+00:00","index":"","fulltext":""}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"cellular-and-molecular-immunology","isNatureJournal":false,"hasQc":false,"allowDirectSubmit":false,"externalIdentity":"cmi","sideBox":"Learn more about [Cellular \u0026 Molecular Immunology](http://www.nature.com/cmi/)","snPcode":"41423","submissionUrl":"https://mts-cmi.nature.com/cgi-bin/main.plex","title":"Cellular \u0026 Molecular Immunology","twitterHandle":"","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"ejp","reportingPortfolio":"Nature AJ","inReviewEnabled":true,"inReviewRevisionsEnabled":false}}],"origin":"","ownerIdentity":"d470f5ff-081c-4336-901d-599c32155bd0","owner":[],"postedDate":"March 24th, 2026","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"under-review","subjectAreas":[{"id":64825003,"name":"Biological sciences/Immunology/Antimicrobial responses"},{"id":64825004,"name":"Biological sciences/Immunology/Infection"}],"tags":[],"updatedAt":"2026-03-24T17:19:41+00:00","versionOfRecord":[],"versionCreatedAt":"2026-03-24 17:19:41","video":"","vorDoi":"","vorDoiUrl":"","workflowStages":[]},"version":"v1","identity":"rs-9059506","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-9059506","identity":"rs-9059506","version":["v1"]},"buildId":"XKTyCvWXoU3ODBz1xrDgd","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}