Lack of alpha1,2-fucosylation protects mice from bleomycin-induced lung fibrosis

Lack of alpha1,2-fucosylation protects mice from bleomycin-induced lung fibrosis | 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 Lack of alpha1,2-fucosylation protects mice from bleomycin-induced lung fibrosis Chenxi Zhu, Xinjia Mai, Yicheng Jiang, Zhaohui Ji, Gulberdiyev Abdylla, and 1 more This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-6044497/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 : The mechanism responsible for lung fibrosis remain unknown. This purpose of this study is to investigate the impact of alpha1,2-fucosylation on bleomycin-induced pulmonary fibrosis in a murine model. Methods : Wild-type and knockout mice deficient of alpha1,2 fucose (DFTKO) were treated by bleomycin and lung fibrosis was studied. Bronchoalveolar lavage fluid was collected on Day 7 and Day 14 for Tandem Mass Tag-labeled(TMT) mass spectrometry proteomic analysis. Results : Lung fibrosis is less severe in mice lacking alpha1,2 fucose. Multiple bronchoalveolar lavage proteins were elevated 7 days after bleomycin treatment, including 1) proteins involved in lipid metabolism, antimicrobial defense and inflammation: Bpifa2, Apoa1, Apoa2, C1qtnf5, Serpina3n; 2) proteins involved in TGF-beta and extracellular matrix signaling: Fst, Bgn, Timp1, Vcan, Ltbp1, Sparcl1, Mmp2; 3) Collagens: Col5a1, Col5a2. Several proteins involved in detoxification of reactive oxygen species (ROS) were found to be decreased 7 days after bleomycin treatment: Hspa1a, Selenbp1, Glrx5, Uqcrc1, Npc1, Ifi30, Hadh, Prdx6. When wild-type and knockout mice deficient of alpha1,2 fucose were compared 7 days after bleomycin treatment, multiple proteins were elevated in knockout mice: 1) proteins involved in DNA damage repair and maintenance of genome stability: H3c1, Ssbp1, Hmga1; 2) proteins involved in inflammation: S100a8, S100a9; 3) proteins involved in signaling pathways of wound healing and tissue remodeling: Hdgfl3, Plekhf2, Ceacam1. Conclusions : Lack of alpha1,2 fucosylated structures are found to play protective roles by upregulating components of three critical pathways, while exact mechanisms will be focus of our future study. Identification of alpha1,2 fucosylated structures as facilitators of lung fibrosis also provide an interesting target for therapeutic interventions for lung fibrosis. Idiopathic pulmonary fibrosis Bleomycin Fucosyltransferase Proteomic analysis Figures Figure 1 Figure 2 Figure 3 Figure 4 Introduction Idiopathic pulmonary fibrosis (IPF) is a chronic and progressive lung disease characterized by the development of irreversible fibrosis within the lung, leading to a gradual loss of pulmonary function 1 . The exact etiology of IPF remains unclear, but it is likely multifactorial, involving environmental factors such as smoking and asbestos exposure, genetic predisposition, and autoimmune responses 2 , 3 . Bleomycin, an antibiotic used in the treatment of various malignant tumors, can induce interstitial pneumonia, which can subsequently lead to fibrosis 4 . The symptoms of pulmonary fibrosis include dyspnea, cough, fatigue, and weight loss, and in severe cases, it can progress to respiratory failure and death 5 – 7 . Current treatment methods, such as antifibrotic drugs (such as pirfenidone 8 , 9 and nintedanib 10 ), can only slow the progression of the disease and cannot cure it, and long-term use can cause side effects. Therefore, the search for new treatment methods and potential therapeutic targets is crucial for improving the prognosis of patients with pulmonary fibrosis. Recent studies have shown that glycosylation modifications play an important role in the pathogenesis and development of pulmonary fibrosis 11 – 13 . Fucose is an important glycosylation modification that interacts with proteins and other biomolecules to participate in various biological processes, including cell adhesion, signal transduction, and immune responses. α1,2-Fucosylation is one of the main types of fucose glycosylation, and its deficiency is associated with the development of various diseases, including cancer 14 , inflammation 15 , and neurodegenerative diseases 16 . Studies have shown that α1,2-fucosylation deficiency can affect cell adhesion 17 , signal transduction 18 , and immune responses 15 . However, the impact of α1,2-fucosylation deficiency on pulmonary fibrosis remains unclear. This study aims to investigate the effect of α1,2-fucosylation deficiency on bleomycin-induced pulmonary fibrosis and reveal its potential mechanism through proteomics analysis, that may contribute to a deeper understanding of the molecular pathways involved in IPF. Method Bleomycin-Induced Pulmonary Fibrosis in mice Six-to-eight-week-old wild-type C57 mice and C57 background alpha1,2-fucosylation-deficient (DFTKO) mice were used 19 . The mice were housed in specific pathogen-free (SPF) facilities. The procedures for bleomycin infusion and collection of bronchoalveolar lavage fluid (BALF) cells were performed as previously described 20 . Briefly, mice were anesthetized with a single dose of pentobarbital sodium via intraperitoneal injection. We employed a widely used model of intratracheal instillation of a single dose of bleomycin. Bleomycin (Selleck, S1214) in 25 μL of PBS was instilled intranasally into the trachea of the mice. The dose of bleomycin for all mice was 15 mg/kg 21 . Animals in the control group received physiological saline only at day 0 before bleomycin treatment. Mice were euthanized on days 7 and 14, and lung tissue and BALF were collected for experimentation. To collect BALF, 1 ml of ice-cold PBS was injected in fractions into the trachea of the mice using a 19-G needle. After gentle aspiration four times, the BALF was centrifuged at 1000 rpm for 10 minutes at 4°C to separate the supernatant and cells. The left lung was fixed in 4% formaldehyde solution for histopathological section analysis. Paraffin sections of lung tissue specimens from each group were prepared and subjected to routine hematoxylin and eosin (HE) and Masson’s trichrome staining. Sample preparation for mass spectrometry analysis The protein concentration of the samples was determined using the bicinchoninic acid assay. For each sample, 100 μg of protein was precipitated with acetone. The reduced disulfide bonds were incubated with 5 mM dithiothreitol (DTT) at 55°C with oscillation for 10 minutes, followed by reaction with 10 mM iodoacetamide (IAA) in the dark for 15 minutes to alkylate the reduced disulfide bonds. Subsequently, protein digestion was performed using trypsin at a concentration of 0.5 mg/ml (with a trypsin to protein ratio of 1:50) (Promega, Madison, WI, USA), and incubated with oscillation at 37°C overnight. Peptides were labeled using the TMT Isobaric Label Reagent Set (Thermo Scientific, Rockford, USA), and equal volumes of labeled samples from each group were mixed. TFA was added to the mixed samples (final concentration 2%, pH < 2) to remove SDC. The peptides were then desalted through a C18 column. The desalted peptides were vacuum-dried and subsequently stored at -80°C for liquid chromatography-mass spectrometry (LC-MS/MS) analysis. nanoLC-MS/MS analysis Each sample containing 1 μg of total peptides was separated and analyzed using a nano-UPLC (EASY-nLC1200) coupled to a Q Exactive HF-X Orbitrap mass spectrometer (Thermo Fisher Scientific) equipped with a nano-electrospray ion source. Peptide separation was achieved on a reversed-phase column (100 μm ID × 15 cm, Reprosil Pur 120 C18 AQ, 1.9 μm, Dr. Maisch) with a mobile phase gradient of 90 minutes at a flow rate of 300 nL/min, consisting of H 2 O with 0.1% FA and 2% ACN (phase A) and 80% ACN with 0.1% FA (phase B). The gradient program was as follows: 2-5% B for 2 min, 5-22% B for 68 min, 22-45% B for 16 min, 45-95% B for 2 min, and 95% B for 2 min. Data-dependent acquisition (DDA) was conducted in profile and positive mode using an Orbitrap analyzer with a resolution of 120,000 (@200 m/z) for MS1 scans in the m/z range of 350-1600. For MS2, the resolution was set to 45,000 with a fixed first mass of 110 m/z. The AGC target was 3E6 for MS1 with a max IT of 30 ms, and 1E5 for MS2 with a max IT of 96 ms. The top 20 most intense ions were selected for higher-energy collisional dissociation (HCD) with a normalized collision energy of 32% and an isolation window of 0.7 m/z. Dynamic exclusion was set for 45 s, excluding single-charged ions and those with charges exceeding 6 from the DDA process 22 . Database search and quantification Raw MS files were processed using Proteome Discoverer (PD) software (version 2.4.0.305) with the built-in Sequest HT search engine. MS spectra were searched against the species-specific UniProt FASTA database (uniprot-Mus+musculus-10090-2020-10.fasta), with Carbamidomethyl [C] , TMT 6 plex(K) and TMT 6-plex (N-term) as a fixed modification and Oxidation (M) and Acetyl (Protein N-term) as variable modifications. Trypsin was used as protease. A maximum of 2 missed cleavage(s) was allowed. The false discovery rate (FDR) was set to 0.01 for both peptide-spectrum match (PSM) and peptide levels. Peptide identification was performed with an initial precursor mass deviation of up to 10 ppm and a fragment mass deviation of 0.02 Da. Unique peptide and Razor peptide were used for protein quantification and total peptide amount for normalization. All other parameters were set to default values. Result Lack of alpha1,2 fucosylation protects mice from bleomycin-induced fibrosis We compared the lung fibrosis in wild type versus knockout mice deficient of alpha1,2 fucose. The survival rate of WT mice was significantly lower than that of DFTKO mice (Figure 1B). Histopathological evaluation of lung fibrosis revealed that by day 7, WT mice displayed marked pulmonary inflammation and evident fibrosis, characterized by substantial immune cell infiltration—mainly lymphocytes and macrophages—into interstitial and alveolar spaces. Notable collagen deposition signaled the onset of fibrotic changes with disrupted lung architecture. By day 14, inflammation in WT mice lessened, but fibrosis intensified, with increased collagen accumulation and a more organized fibrotic pattern, accompanied by greater alveolar structure loss and interstitial thickening. In contrast, DFTKO mice exhibited a milder inflammatory response and less fibrosis at both time points. Day 7 lung sections from DFTKO mice showed only mild immune cell infiltration and limited collagen deposition, indicating a mitigated fibrotic process. On day 14, fibrosis progressed but remained significantly milder than in WT mice, with preserved alveolar architecture and decreased interstitial collagen buildup (Figure 1C and D). Proteins involved in pro-fibrotic pathways are elevated in bronchoalveolar lavage fluid in bleomycin-induced lung fibrosis In this experiment, a total of 3,957 proteins (groups) and 26,476 peptides were identified. For missing value filtering, only proteins detected in all samples were retained for the TMT experiment. A filter was applied based on the number of unique peptides per protein, retaining those with one or more unique peptides. After preprocessing, 2,933 proteins were retained. Differentially expressed proteins were identified using statistical methods, with the criteria for differential expression being a P-value< 0.05 from Student’s t-test for the TMT experiment, and a Fold Change of either 1.2. In a comparison between wild-type mice induced by bleomycin on day 7 and the day 0 group, 1,105 differentially expressed proteins were identified in the BALF, with 168 proteins upregulated and 937 proteins downregulated. Among the upregulated proteins (Figure 2, Supplementary Table 1), those with the most significantly increased expression included proteins involved in lipid metabolism, antimicrobial defense and inflammation (Bpifa2, C1qtnf5); proteins involved in TGF-beta and extracellular matrix signaling (Fst, Bgn, Timp1, Vcan, Ltbp1, Sparcl1, Mmp2); and collagens (Col5a1, Col5a2). These proteins have played significant roles in immune response, inflammatory response, extracellular matrix structure and remodeling, lipid metabolism, tissue repair, and fibrosis. Proteins involved in ROS detoxification are downregulated in bleomycin-induced lung fibrosis The downregulated proteins (Figure 3, Supplementary Table 2) with the most significant changes in expression included those involved in detoxification of reactive oxygen species (Hspa1a, Selenbp1, Glrx5, Uqcrc1, Npc1, Ifi30, Hadh, Prdx6). The downregulation of these proteins indicates the occurrence of cellular stress, metabolic disorders, gene expression dysregulation, and impaired energy metabolism. Lack of alpha1,2-fucosylated structures are linked with increased protective pathways in bleomycin-induced lung fibrosis On day 7 of bleomycin induction, compared to wild-type mice, the BALF from DFTKO mice exhibited 198 proteins upregulated and 190 proteins downregulated. Upregulated proteins include those involved in DNA damage repair and maintenance of genome stability (H3c1, Ssbp1, Hmga1); those involved in inflammation (S100a8, S100a9); and those involved in signaling pathways of wound healing and tissue remodeling (Hdgfl3, Plekhf2, Ceacam1). (Figure 4, Supplementary Table 3). Conversely, proteins such as Rreb1, Nectin3, Aldh1a7, Ndufs6, Cnpy2, Ehd2, Cnn3, Ces1, Gsta4, Uckl1, and Ddt were down-regulated in the DFTKO mice’s BALF compared to WT mice (Supplementary Table 4). These proteins play crucial roles that may be part of pro-fibrosis pathways, including cell signaling, metabolism, inflammatory response, DNA repair, maintenance of chromatin structure, cell proliferation, and differentiation. Discussion The exact mechanisms responsible for pulmonary fibrosis remain unclear. Wolters et al. proposed the primary role of lung epithelial in IPF pathogenesis 23 , that the molecular changes within lung epithelial cells are sufficient to promote lung remodeling and fibrosis. Numerous mediators including TGF-beta and platelet-derived growth factor-beta are generated to activate mesenchymal cells, while suppressive mediators as represented by prostaglandin E2 are reduced in dysfunctional epithelium. As the disease progresses, multiple matrix molecules are expressed that cause lung remodeling and activation of profibrotic-signaling pathways in mesenchymal cells. The fibroblasts further invade matrix and deposit collagens leading to chronic lung remodeling. In our proteomic analysis, we identified several components of inflammation and pro-fibrotic pathways. Signaling molecules in inflammation that are upregulated in lung fibrosis In this study, we first discovered Bpifa2 is elevated 25-fold in lung fibrosis. Bpifa2 was reported as an antimicrobial peptide, which was elevated in acute kidney injury and fatal radiation injury 24-26 . Apoa1 was previously reported as a candidate biomarker for lung fibrosis 27,28 . Serpina3n can widely inhibit the activity of serine peptidases 29 . Knockdown of Serpina3n ameliorates bleomycin-induced pulmonary fibrosis. C1qtnf5 was proposed as a candidate biomarker for systemic sclerosis, while its upregulation in lung fibrosis was first discovered in our study 30 . Components of TGF-beta signaling and profibrotic pathways that are upregulated in lung fibrosis Our study discovered several protein components of TGF-beta signaling and profibrotic pathway in bronchoalveolar lavage fluid. Fst was previously identified in bleomycin-induced fibrosis as TGF-beta antagonist 31 . We found it was upregulated 14-fold on day 7 after bleomycin injury. Bgn and Vcan were reported to be upregulated in bleomycin-treated lung fibroblasts, which were increased 13.5-fold and 9-fold respectively in our study 32 . Timp1 was reported as a key factor of fibrogenic response in bleomycin-induced lung fibrosis 33 ; it was increased 10-fold in our study. Latent transforming growth factor (TGF)-β binding protein-1 (LTBP1) was reported to bind to Fbln1c and induce TGF-β activation 34 ; it was increased 7.6-fold in our study. Sparcl1 was detected as a potential biomarker for airway remodeling in severe asthma patients 35 ; it was increased 7.5-fold in our study. MMP2, a well-known collagenase involved in tissue remodeling 36 , was increased 7-fold in our study. Col5a1 and Col5a2 were previously reported to be upregulated in IPF 37,38 , which were increased 9.6-fold and 7-fold respectively in our study. Detoxification pathway of reactive oxygen species (ROS) is suppressed in lung fibrosis. Several proteins involved in ROS detoxification were reduced in our study. HSPA1 (rs1043618) polymorphisms were reported as associated with a decreased risk of IPF in Mexican patients 39 . Prdx6 has been reported as a target to attenuate lung inflammation and fibrosis in silicosis 40 . Our study also first found the decrease of Selenbp1, Glrx5, Uqcrc1, Npc1, Ifi30, and Hadh in bronchoalveolar fluid of lung fibrosis, suggesting their potential value as therapeutic targets. Lack of alpha1,2 fucosylated structures attenuate bleomycin-induced lung fibrosis In the past decade, alpha1,2 fucosylated glycans have been found to be critical for inflammation signaling pathways. Epithelial fucose is used as a dietary carbohydrate by gut symbionts in a mouse model, while disruption of intestinal fucosylation led to increased susceptibility to infection by Salmonella typhimurium 41,42 . In the mouse model, alpha1,2 fucosylated glycans were reported to be critical for complement activation, which exaggerates airway inflammation 43 . The exact mechanism by which DFTKO mice displayed attenuated lung fibrosis remains unclear. Previously, pathogenic bacteria species among lung symbionts have been reported to exaggerate the lung inflammation through activating IL-17R signaling 21 . Our current research is focused on whether the fucosylated glycans influence the colonization of lung symbionts. In summary, our study first identified the genetic link between lack of alpha1,2 fucosylated glycans and bleomycin-induced lung fibrosis. Genetic editing of alpha1,2 fucosyltransferases may be achieved by current antisense oligonucleotides technologies, to deplete the alpha1,2 glycan structures in vivo. Chemically synthesized enzyme inhibitors may also suppress the synthesis of alpha1,2 fucose in vivo. The novel protein targets discovered in bronchoalveolar lavage fluid provide critical clues for drug development. The unknown role of alpha1,2 fucosylated glycans indicates that self or microbial lectins might be involved in etiology of this intriguing clinical disease. Abbreviations IPF Idiopathic Pulmonary Fibrosis BALF Bronchoalveolar Lavage Fluid ROS Reactive Oxygen Species TGF-β Transforming Growth Factor Beta DFTKO Alpha1,2 Fucose-Deficient Knockout Mice WT Wild-Type HE Hematoxylin and Eosin SDC Sodium Deoxycholate APOA1 Apolipoprotein A1 APOA2 Apolipoprotein A2 BPIFA2 BPI Fold Containing Family A Member 2 C1QTNF5 C1q Tumor Necrosis Factor-Related Protein 5 COL5A1 Collagen Type V Alpha 1 Chain COL5A2 Collagen Type V Alpha 2 Chain HSPA1A Heat Shock Protein Family A (Hsp70) Member 1A HMGA1 High-Mobility Group AT-Hook 1 LTBP1 Latent Transforming Growth Factor Beta Binding Protein 1 MMP2 Matrix Metalloproteinase 2 PRDX6 Peroxiredoxin 6 SERPINA3N Serpin Family A Member 3N SSBP1 Single-Stranded DNA Binding Protein 1 S100A8 S100 Calcium-Binding Protein A8 S100A9 S100 Calcium-Binding Protein A9 BGN Biglycan VCAN Versican TIMP1 Tissue Inhibitor of Metalloproteinases 1 FN14 Fibroblast Growth Factor-Inducible 14 SPARCL1 Sparc-Related Modular Calcium Binding 1 H3C1 Histone H3.1 PLEKHF2 Pleckstrin Homology Domain-Containing Family F Member 2 HDGFL3 Hepatoma-Derived Growth Factor-Like 3 CEACAM1 Carcinoembryonic Antigen-Related Cell Adhesion Molecule 1 MS Mass Spectrometry LC-MS/MS Liquid Chromatography-Mass Spectrometry TMT Tandem Mass Tag DDA Data-Dependent Acquisition HCD Higher-Energy Collisional Dissociation MS/MS Tandem Mass Spectrometry FDR False Discovery Rate PSM Peptide-Spectrum Match PD Proteome Discoverer Declarations Ethics approval and consent to participate This study was approved by institutional board of Tongji University. No human subjects were involved in this study. Consent for publication Not applicable. Availability of data and materials The authors confirm that the data supporting the findings of this study are available within the article and its supplementary materials. Competing interests The authors declare no competing interests. Funding The authors are supported by National Key Research and Development Plan grants 2021YFE0200500, Fundamental Research Funds for the Central Universities 22120200163, National Natural Science Foundation of China grant 31870972, Sino-German Scientific Research Program M0693, Major Program of National Natural Science Foundation of China 9235920, and Shanghai Science and Technology Commission grant 20410713500. Authors' contributions C.Z., Conceptualization, Data curation, Formal analysis, Investigation, Methodology, Validation, Writing-original draft, Writing-review and editing. X.M., Investigation, Supervision, Methodology. Y.J., Investigation, Data curation. Z.J., Investigation, Methodology. G.A., Data curation, Revision. D.Z., Conceptualization, Data curation, Formal analysis, Funding acquisition, Investigation, Methodology, Project administration, Supervision, Validation, Writing-original draft, Writing-review and editing. All authors have read and agreed to the published version of the manuscript. All the authors are giving consent to publish. Corresponding authors Correspondence to Dapeng Zhou, [email protected] Acknowledgements We thank Yingpeng Zhou and Xiaojing Zhou of Shanghai BioTree Biotech Co., Ltd for mass spectrometry analysis. Authors' information Authors and Affiliations Department of Immunology and Pathogen Biology, Key Laboratory of Pathogen and Host-Interactions, Ministry of Education, Tongji University School of Medicine, 500 Zhennan Road, Shanghai 200331, China Chenxi Zhu, Xinjia Mai, Yicheng Jiang, Zhaohui Ji, Gulberdiyev Abdylla, Dapeng Zhou References Moss, B. J., Ryter, S. W. & Rosas, I. O. 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Analysis of genetic diversity of the heat shock protein 70 gene on the basis of abundant sequence polymorphisms in chicken breeds. Genet Mol Res 14 , 1538-1545, doi:10.4238/2015.March.6.1 (2015). Bian, Y. et al. The glycyl-l-histidyl-l-lysine-Cu(2+) tripeptide complex attenuates lung inflammation and fibrosis in silicosis by targeting peroxiredoxin 6. Redox Biol 75 , 103237, doi:10.1016/j.redox.2024.103237 (2024). Goto, Y., Uematsu, S. & Kiyono, H. Epithelial glycosylation in gut homeostasis and inflammation. Nat Immunol 17 , 1244-1251, doi:10.1038/ni.3587 (2016). Goto, Y. et al. Innate lymphoid cells regulate intestinal epithelial cell glycosylation. Science 345 , 1254009, doi:10.1126/science.1254009 (2014). Saku, A. et al. Fucosyltransferase 2 induces lung epithelial fucosylation and exacerbates house dust mite-induced airway inflammation. J Allergy Clin Immunol 144 , 698-709 e699, doi:10.1016/j.jaci.2019.05.010 (2019). Additional Declarations No competing interests reported. Supplementary Files SupplementaryTable14.docx Supplementary Table 1. Top 20 up-regulated proteins in wild type mice, 7 days after bleomycin treatment. Supplementary Table 2. Top 20 down-regulated proteins in wild type mice, 7 days after bleomycin treatment. Supplementary Table 3. Top 20 up-regulated proteins in DFTKO mice as compared to wild type mice. Data were 7 days after bleomycin treatment. Supplemental Table 4. Top 20 down-regulated proteins in DFTKO mice as compared to wild type mice. Data were 7 days after bleomycin treatment. SupplementalTable5DataofTMTlabeledmassspectrometryproteomicanalysisforwildtypemiceandDFTKOmice.xlsx Supplemental Table 5. Data of TMT-labeled mass spectrometry proteomic analysis for wildtype mice and DFTKO mice. 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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-6044497","acceptedTermsAndConditions":true,"allowDirectSubmit":true,"archivedVersions":[],"articleType":"Research Article","associatedPublications":[],"authors":[{"id":418195673,"identity":"d7b8f1f1-b17c-4a0e-9d41-e66cb2dd23af","order_by":0,"name":"Chenxi Zhu","email":"","orcid":"","institution":"Tongji University School of Medicine","correspondingAuthor":false,"prefix":"","firstName":"Chenxi","middleName":"","lastName":"Zhu","suffix":""},{"id":418195674,"identity":"176f4440-4aa4-43be-a1f0-feacdee7b52e","order_by":1,"name":"Xinjia Mai","email":"","orcid":"","institution":"Tongji University School of Medicine","correspondingAuthor":false,"prefix":"","firstName":"Xinjia","middleName":"","lastName":"Mai","suffix":""},{"id":418195675,"identity":"e9810e09-b7f9-42aa-a7c4-e49c143dca55","order_by":2,"name":"Yicheng Jiang","email":"","orcid":"","institution":"Tongji University School of Medicine","correspondingAuthor":false,"prefix":"","firstName":"Yicheng","middleName":"","lastName":"Jiang","suffix":""},{"id":418195676,"identity":"80f5755e-e11c-4f02-b70d-c85c503ee6e9","order_by":3,"name":"Zhaohui Ji","email":"","orcid":"","institution":"Tongji University School of Medicine","correspondingAuthor":false,"prefix":"","firstName":"Zhaohui","middleName":"","lastName":"Ji","suffix":""},{"id":418195677,"identity":"68e2cc92-1b18-4fd2-89d3-69e57fdf2ae2","order_by":4,"name":"Gulberdiyev Abdylla","email":"","orcid":"","institution":"Tongji University School of Medicine","correspondingAuthor":false,"prefix":"","firstName":"Gulberdiyev","middleName":"","lastName":"Abdylla","suffix":""},{"id":418195678,"identity":"0c8f3238-f443-4f61-a9ee-9cb12d3ae90c","order_by":5,"name":"Dapeng Zhou","email":"data:image/png;base64,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","orcid":"","institution":"Tongji University School of Medicine","correspondingAuthor":true,"prefix":"","firstName":"Dapeng","middleName":"","lastName":"Zhou","suffix":""}],"badges":[],"createdAt":"2025-02-17 05:23:09","currentVersionCode":1,"declarations":"","doi":"10.21203/rs.3.rs-6044497/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-6044497/v1","draftVersion":[],"editorialEvents":[],"editorialNote":"","failedWorkflow":false,"files":[{"id":78504850,"identity":"c436be3e-09b8-4485-bb74-73b1fec360a1","added_by":"auto","created_at":"2025-03-14 07:41:38","extension":"png","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":1888160,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eDFTKO mice lack of alpha1,2 fucose showed disease attenuation in the IPF mouse model induced by bleomycin.\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e(A) Experimental scheme of IPF model. (B) Survival rate of C57BL/6 wildtype(n=16) or DFTKO (n=12) mice treated with bleomycin (15 mg/kg) intranasally. (C) HE staining (D) Masson’s trichrome staining of the representative lungs from the mice on day 7 or day 14 of the fibrosis model as in (A). Scale=200μm.\u003c/p\u003e","description":"","filename":"1.png","url":"https://assets-eu.researchsquare.com/files/rs-6044497/v1/81fb63df2ae0b04617f3ebc7.png"},{"id":78504854,"identity":"3d002abd-fd52-4918-9898-0ce6e7e1bdf7","added_by":"auto","created_at":"2025-03-14 07:41:38","extension":"png","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":151744,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eMultiple bronchoalveolar lavage proteins showed significant increases 7 days after bleomycin treatment.\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e(A) Proteins involved in lipid metabolism, antimicrobial defense, and inflammation. (B) Proteins involved in TGF-beta signaling and extracellular matrix. (C) Collagens.\u003c/p\u003e","description":"","filename":"2.png","url":"https://assets-eu.researchsquare.com/files/rs-6044497/v1/bdc030e9850cd6e6123dfac2.png"},{"id":78505074,"identity":"c3615d45-7538-445b-accf-b7bf9e4ad82b","added_by":"auto","created_at":"2025-03-14 07:49:38","extension":"png","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":89904,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eProteins involved in detoxification of ROS were significantly decreased 7 days after bleomycin treatment.\u003c/strong\u003e\u003c/p\u003e","description":"","filename":"3.png","url":"https://assets-eu.researchsquare.com/files/rs-6044497/v1/c4b53aad050faa46d21d5551.png"},{"id":78504226,"identity":"fe738f38-537c-4363-bb26-97d5072ecd10","added_by":"auto","created_at":"2025-03-14 07:33:38","extension":"png","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":105989,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eProteins involved in pathways that attenuate disease in alpha1,2 fucose-deficient knockout mice.\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e(A) Proteins involved in DNA damage repair and maintenance of genome stability. (B) Proteins involved in inflammation. (C) Proteins involved in signaling pathways of wound healing and tissue remodeling.\u003c/p\u003e","description":"","filename":"4.png","url":"https://assets-eu.researchsquare.com/files/rs-6044497/v1/41c26b0594e20f443c18a3a8.png"},{"id":79384165,"identity":"7606d0ba-1bb7-4b65-93b3-ee3203a30133","added_by":"auto","created_at":"2025-03-27 17:23:29","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":2634611,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-6044497/v1/cad42362-70c4-44d1-bcc0-40d3485ae39b.pdf"},{"id":78504852,"identity":"39acf2f9-c39e-40cf-ad81-9f64afafc868","added_by":"auto","created_at":"2025-03-14 07:41:38","extension":"docx","order_by":1,"title":"","display":"","copyAsset":false,"role":"supplement","size":51858,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eSupplementary Table 1. Top 20 up-regulated proteins in wild type mice, 7 days after bleomycin treatment.\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eSupplementary Table 2. Top 20 down-regulated proteins in wild type mice, 7 days after bleomycin treatment.\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eSupplementary Table 3. Top 20 up-regulated proteins in DFTKO mice as compared to wild type mice. \u003c/strong\u003eData were 7 days after bleomycin treatment.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eSupplemental Table 4. Top 20 down-regulated proteins in DFTKO mice as compared to wild type mice. \u003c/strong\u003eData were 7 days after bleomycin treatment.\u003c/p\u003e","description":"","filename":"SupplementaryTable14.docx","url":"https://assets-eu.researchsquare.com/files/rs-6044497/v1/f0c0356049974287c9f225a8.docx"},{"id":78505075,"identity":"16c952fb-6e11-4ecf-99f9-1fa11d1da9c5","added_by":"auto","created_at":"2025-03-14 07:49:39","extension":"xlsx","order_by":2,"title":"","display":"","copyAsset":false,"role":"supplement","size":1588849,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eSupplemental Table 5. Data of TMT-labeled mass spectrometry proteomic analysis for wildtype mice and DFTKO mice.\u003c/strong\u003e\u003c/p\u003e","description":"","filename":"SupplementalTable5DataofTMTlabeledmassspectrometryproteomicanalysisforwildtypemiceandDFTKOmice.xlsx","url":"https://assets-eu.researchsquare.com/files/rs-6044497/v1/7af3c572a63572d50d1851f8.xlsx"}],"financialInterests":"No competing interests reported.","formattedTitle":"Lack of alpha1,2-fucosylation protects mice from bleomycin-induced lung fibrosis","fulltext":[{"header":"Introduction","content":"\u003cp\u003eIdiopathic pulmonary fibrosis (IPF) is a chronic and progressive lung disease characterized by the development of irreversible fibrosis within the lung, leading to a gradual loss of pulmonary function\u003csup\u003e\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e\u003c/sup\u003e. The exact etiology of IPF remains unclear, but it is likely multifactorial, involving environmental factors such as smoking and asbestos exposure, genetic predisposition, and autoimmune responses\u003csup\u003e\u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e,\u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e\u003c/sup\u003e. Bleomycin, an antibiotic used in the treatment of various malignant tumors, can induce interstitial pneumonia, which can subsequently lead to fibrosis\u003csup\u003e\u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e4\u003c/span\u003e\u003c/sup\u003e. The symptoms of pulmonary fibrosis include dyspnea, cough, fatigue, and weight loss, and in severe cases, it can progress to respiratory failure and death\u003csup\u003e\u003cspan additionalcitationids=\"CR6\" citationid=\"CR5\" class=\"CitationRef\"\u003e5\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e\u003c/sup\u003e.\u003c/p\u003e \u003cp\u003eCurrent treatment methods, such as antifibrotic drugs (such as pirfenidone\u003csup\u003e\u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e8\u003c/span\u003e,\u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e9\u003c/span\u003e\u003c/sup\u003e and nintedanib\u003csup\u003e\u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e10\u003c/span\u003e\u003c/sup\u003e), can only slow the progression of the disease and cannot cure it, and long-term use can cause side effects. Therefore, the search for new treatment methods and potential therapeutic targets is crucial for improving the prognosis of patients with pulmonary fibrosis.\u003c/p\u003e \u003cp\u003eRecent studies have shown that glycosylation modifications play an important role in the pathogenesis and development of pulmonary fibrosis\u003csup\u003e\u003cspan additionalcitationids=\"CR12\" citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e13\u003c/span\u003e\u003c/sup\u003e. Fucose is an important glycosylation modification that interacts with proteins and other biomolecules to participate in various biological processes, including cell adhesion, signal transduction, and immune responses. α1,2-Fucosylation is one of the main types of fucose glycosylation, and its deficiency is associated with the development of various diseases, including cancer\u003csup\u003e\u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e14\u003c/span\u003e\u003c/sup\u003e, inflammation\u003csup\u003e\u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e15\u003c/span\u003e\u003c/sup\u003e, and neurodegenerative diseases\u003csup\u003e\u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e16\u003c/span\u003e\u003c/sup\u003e. Studies have shown that α1,2-fucosylation deficiency can affect cell adhesion\u003csup\u003e\u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e17\u003c/span\u003e\u003c/sup\u003e, signal transduction\u003csup\u003e\u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e18\u003c/span\u003e\u003c/sup\u003e, and immune responses\u003csup\u003e\u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e15\u003c/span\u003e\u003c/sup\u003e. However, the impact of α1,2-fucosylation deficiency on pulmonary fibrosis remains unclear.\u003c/p\u003e \u003cp\u003eThis study aims to investigate the effect of α1,2-fucosylation deficiency on bleomycin-induced pulmonary fibrosis and reveal its potential mechanism through proteomics analysis, that may contribute to a deeper understanding of the molecular pathways involved in IPF.\u003c/p\u003e"},{"header":"Method","content":"\u003cp\u003e\u003cem\u003eBleomycin-Induced Pulmonary Fibrosis in mice\u003c/em\u003e\u003c/p\u003e\n\u003cp\u003eSix-to-eight-week-old wild-type C57 mice and C57 background alpha1,2-fucosylation-deficient (DFTKO) mice were used\u003csup\u003e19\u003c/sup\u003e. The mice were housed in specific pathogen-free (SPF) facilities. The procedures for bleomycin infusion and collection of bronchoalveolar lavage fluid (BALF) cells were performed as previously described\u003csup\u003e20\u003c/sup\u003e. Briefly, mice were anesthetized with a single dose of pentobarbital sodium via intraperitoneal injection. We employed a widely used model of intratracheal instillation of a single dose of bleomycin. Bleomycin (Selleck, S1214) in 25 \u0026mu;L of PBS was instilled intranasally into the trachea of the mice. The dose of bleomycin for all mice was 15 mg/kg\u003csup\u003e21\u003c/sup\u003e. Animals in the control group received physiological saline only at day 0 before bleomycin treatment. Mice were euthanized on days 7 and 14, and lung tissue and BALF were collected for experimentation.\u003c/p\u003e\n\u003cp\u003eTo collect BALF, 1 ml of ice-cold PBS was injected in fractions into the trachea of the mice using a 19-G needle. After gentle aspiration four times, the BALF was centrifuged at 1000 rpm for 10 minutes at 4\u0026deg;C to separate the supernatant and cells. The left lung was fixed in 4% formaldehyde solution for histopathological section analysis. Paraffin sections of lung tissue specimens from each group were prepared and subjected to routine hematoxylin and eosin (HE) and Masson\u0026rsquo;s trichrome staining.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cem\u003eSample preparation for mass spectrometry analysis\u003c/em\u003e\u003c/p\u003e\n\u003cp\u003eThe protein concentration of the samples was determined using the bicinchoninic acid assay. For each sample, 100 \u0026mu;g of protein was precipitated with acetone. The reduced disulfide bonds were incubated with 5 mM dithiothreitol (DTT) at 55\u0026deg;C with oscillation for 10 minutes, followed by reaction with 10 mM iodoacetamide (IAA) in the dark for 15 minutes to alkylate the reduced disulfide bonds. Subsequently, protein digestion was performed using trypsin at a concentration of 0.5 mg/ml (with a trypsin to protein ratio of 1:50) (Promega, Madison, WI, USA), and incubated with oscillation at 37\u0026deg;C overnight. Peptides were labeled using the TMT Isobaric Label Reagent Set (Thermo Scientific, Rockford, USA), and equal volumes of labeled samples from each group were mixed. TFA was added to the mixed samples (final concentration 2%, pH \u0026lt; 2) to remove SDC. The peptides were then desalted through a C18 column. The desalted peptides were vacuum-dried and subsequently stored at -80\u0026deg;C for liquid chromatography-mass spectrometry (LC-MS/MS) analysis.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cem\u003enanoLC-MS/MS analysis\u003c/em\u003e\u003c/p\u003e\n\u003cp\u003eEach sample containing\u0026nbsp;1 \u0026mu;g of total peptides was separated and analyzed using a nano-UPLC (EASY-nLC1200) coupled to a Q Exactive HF-X Orbitrap mass spectrometer (Thermo Fisher Scientific) equipped with a nano-electrospray ion source. Peptide separation was achieved on a reversed-phase column (100 \u0026mu;m ID \u0026times; 15 cm, Reprosil Pur 120 C18 AQ, 1.9 \u0026mu;m, Dr. Maisch) with a mobile phase gradient of 90 minutes at a flow rate of 300 nL/min, consisting of H\u003csub\u003e2\u003c/sub\u003eO with 0.1% FA and 2% ACN (phase A) and 80% ACN with 0.1% FA (phase B). The gradient program was as follows: 2-5% B for 2 min, 5-22% B for 68 min, 22-45% B for 16 min, 45-95% B for 2 min, and 95% B for 2 min. Data-dependent acquisition (DDA) was conducted in profile and positive mode using an Orbitrap analyzer with a resolution of 120,000 (@200 m/z) for MS1 scans in the m/z range of 350-1600. For MS2, the resolution was set to 45,000 with a fixed first mass of 110 m/z. The AGC target was 3E6 for MS1 with a max IT of 30 ms, and 1E5 for MS2 with a max IT of 96 ms. The top 20 most intense ions were selected for higher-energy collisional dissociation (HCD) with a normalized collision energy of 32% and an isolation window of 0.7 m/z. Dynamic exclusion was set for 45 s, excluding single-charged ions and those with charges exceeding 6 from the DDA process\u003csup\u003e22\u003c/sup\u003e.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cem\u003eDatabase search and quantification\u003c/em\u003e\u003c/p\u003e\n\u003cp\u003eRaw MS files were processed using Proteome Discoverer (PD) software (version 2.4.0.305) with the built-in Sequest HT search engine. MS spectra were searched against the species-specific UniProt FASTA database (uniprot-Mus+musculus-10090-2020-10.fasta), with Carbamidomethyl [C] , TMT 6 plex(K) and TMT 6-plex (N-term) as a fixed modification and Oxidation (M) and Acetyl (Protein N-term) as variable modifications. Trypsin was used as protease. A maximum of 2 missed cleavage(s) was allowed. The false discovery rate (FDR) was set to 0.01 for both peptide-spectrum match (PSM) and peptide levels. Peptide identification was performed with an initial precursor mass deviation of up to 10 ppm and a fragment mass deviation of 0.02 Da. Unique peptide and Razor peptide were used for protein quantification and total peptide amount for normalization. All other parameters were set to default values.\u003c/p\u003e"},{"header":"Result","content":"\u003cp\u003e\u003cem\u003eLack of alpha1,2 fucosylation protects mice from bleomycin-induced fibrosis\u003c/em\u003e\u003c/p\u003e\n\u003cp\u003eWe compared the lung fibrosis in wild type versus knockout mice deficient of alpha1,2 fucose. The survival rate of WT mice was significantly lower than that of DFTKO mice (Figure 1B).\u003c/p\u003e\n\u003cp\u003eHistopathological evaluation of lung fibrosis revealed that\u0026nbsp;by day 7, WT mice displayed marked pulmonary inflammation and evident fibrosis, characterized by substantial immune cell infiltration\u0026mdash;mainly lymphocytes and macrophages\u0026mdash;into interstitial and alveolar spaces. Notable collagen deposition signaled the onset of fibrotic changes with disrupted lung architecture. By day 14, inflammation in WT mice lessened, but fibrosis intensified, with increased collagen accumulation and a more organized fibrotic pattern, accompanied by greater alveolar structure loss and interstitial thickening. In contrast, DFTKO mice exhibited a milder inflammatory response and less fibrosis at both time points. Day 7 lung sections from DFTKO mice showed only mild immune cell infiltration and limited collagen deposition, indicating a mitigated fibrotic process. On day 14, fibrosis progressed but remained significantly milder than in WT mice, with preserved alveolar architecture and decreased interstitial collagen buildup (Figure 1C and D).\u003cem\u003e\u0026nbsp;\u003c/em\u003e\u003c/p\u003e\n\u003cp\u003e\u003cem\u003eProteins involved in pro-fibrotic pathways are elevated in bronchoalveolar lavage fluid in bleomycin-induced lung fibrosis\u003c/em\u003e\u003c/p\u003e\n\u003cp\u003eIn this experiment, a total of 3,957 proteins (groups) and 26,476 peptides were identified. For missing value filtering, only proteins detected in all samples were retained for the TMT experiment. A filter was applied based on the number of unique peptides per protein, retaining those with one or more unique peptides. After preprocessing, 2,933 proteins were retained. Differentially expressed proteins were identified using statistical methods, with the criteria for differential expression being a P-value\u0026lt; 0.05 from Student\u0026rsquo;s t-test for the TMT experiment, and a Fold Change of either \u0026lt; 0.83 or \u0026gt; 1.2.\u003c/p\u003e\n\u003cp\u003eIn a comparison between wild-type mice induced by bleomycin on day 7 and the day 0 group, 1,105 differentially expressed proteins were identified in the BALF, with 168 proteins upregulated and 937 proteins downregulated. Among the upregulated proteins (Figure 2, Supplementary Table 1), those with the most significantly increased expression included proteins involved in lipid metabolism, antimicrobial defense and inflammation (Bpifa2, C1qtnf5); proteins involved in TGF-beta and extracellular matrix signaling (Fst, Bgn, Timp1, Vcan, Ltbp1, Sparcl1, Mmp2); and collagens (Col5a1, Col5a2). These proteins have played significant roles in immune response, inflammatory response, extracellular matrix structure and remodeling, lipid metabolism, tissue repair, and fibrosis.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cem\u003eProteins involved in ROS detoxification are downregulated in bleomycin-induced lung fibrosis\u003c/em\u003e\u003c/p\u003e\n\u003cp\u003eThe downregulated proteins (Figure 3, Supplementary Table 2) with the most significant changes in expression included those involved in detoxification of reactive oxygen species (Hspa1a, Selenbp1, Glrx5, Uqcrc1, Npc1, Ifi30, Hadh, Prdx6). The downregulation of these proteins indicates the occurrence of cellular stress, metabolic disorders, gene expression dysregulation, and impaired energy metabolism.\u003cem\u003e\u0026nbsp;\u003c/em\u003e\u003c/p\u003e\n\u003cp\u003e\u003cem\u003eLack of alpha1,2-fucosylated structures are linked with increased protective pathways in bleomycin-induced lung fibrosis\u003c/em\u003e\u003c/p\u003e\n\u003cp\u003eOn day 7 of bleomycin induction, compared to wild-type mice, the BALF from DFTKO mice exhibited 198 proteins upregulated and 190 proteins downregulated. Upregulated proteins include those involved in DNA damage repair and maintenance of genome stability (H3c1, Ssbp1, Hmga1); those involved in inflammation (S100a8, S100a9); and those involved in signaling pathways of wound healing and tissue remodeling (Hdgfl3, Plekhf2, Ceacam1). (Figure 4, Supplementary Table 3). Conversely, proteins such as Rreb1, Nectin3, Aldh1a7, Ndufs6, Cnpy2, Ehd2, Cnn3, Ces1, Gsta4, Uckl1, and Ddt were down-regulated in the DFTKO mice\u0026rsquo;s BALF compared to WT mice (Supplementary Table 4). These proteins play crucial roles that may be part of pro-fibrosis pathways, including cell signaling, metabolism, inflammatory response, DNA repair, maintenance of chromatin structure, cell proliferation, and differentiation.\u003c/p\u003e"},{"header":"Discussion","content":"\u003cp\u003eThe exact mechanisms responsible for pulmonary fibrosis remain unclear. Wolters et al. proposed the primary role of lung epithelial in IPF pathogenesis\u003csup\u003e23\u003c/sup\u003e, that the molecular changes within lung epithelial cells are sufficient to promote lung remodeling and fibrosis. Numerous mediators including TGF-beta and platelet-derived growth factor-beta are generated to activate mesenchymal cells, while suppressive mediators as represented by prostaglandin E2 are reduced in dysfunctional epithelium. As the disease progresses, multiple matrix molecules are expressed that cause lung remodeling and activation of profibrotic-signaling pathways in mesenchymal cells. The fibroblasts further invade matrix and deposit collagens leading to chronic lung remodeling.\u003c/p\u003e\n\u003cp\u003eIn our proteomic analysis, we identified several components of inflammation and pro-fibrotic pathways.\u003cem\u003e\u0026nbsp;\u003c/em\u003e\u003c/p\u003e\n\u003cp\u003e\u003cem\u003eSignaling molecules in inflammation that are upregulated in lung fibrosis\u003c/em\u003e\u003c/p\u003e\n\u003cp\u003eIn this study, we first discovered Bpifa2 is elevated 25-fold in lung fibrosis. Bpifa2 was reported as an antimicrobial peptide, which was elevated in acute kidney injury and fatal radiation injury\u003csup\u003e24-26\u003c/sup\u003e. Apoa1 was previously reported as a candidate biomarker for lung fibrosis\u003csup\u003e27,28\u003c/sup\u003e. Serpina3n can widely inhibit the activity of serine peptidases\u003csup\u003e29\u003c/sup\u003e. Knockdown of Serpina3n ameliorates bleomycin-induced pulmonary fibrosis.\u0026nbsp;C1qtnf5 was proposed as a candidate biomarker for systemic sclerosis, while its upregulation in lung fibrosis was first discovered in our study\u003csup\u003e30\u003c/sup\u003e. \u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cem\u003eComponents of TGF-beta signaling and profibrotic pathways that are upregulated in lung fibrosis\u003c/em\u003e\u003c/p\u003e\n\u003cp\u003eOur study discovered several protein components of TGF-beta signaling and profibrotic pathway in bronchoalveolar lavage fluid. Fst was previously identified in bleomycin-induced fibrosis as TGF-beta antagonist\u003csup\u003e31\u003c/sup\u003e. We found it was upregulated 14-fold on day 7 after bleomycin injury.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eBgn and Vcan were reported to be upregulated in bleomycin-treated lung fibroblasts, which were increased 13.5-fold and 9-fold respectively in our study\u003csup\u003e32\u003c/sup\u003e. Timp1 was reported as a key factor of fibrogenic response in bleomycin-induced lung fibrosis\u003csup\u003e33\u003c/sup\u003e; it was increased 10-fold in our study. Latent transforming growth factor (TGF)-\u0026beta; binding protein-1 (LTBP1)\u0026nbsp;was reported to bind to Fbln1c and induce TGF-\u0026beta; activation\u003csup\u003e34\u003c/sup\u003e; it was increased 7.6-fold in our study. Sparcl1 was detected as a potential biomarker for airway remodeling in severe asthma patients\u003csup\u003e35\u003c/sup\u003e; it was increased 7.5-fold in our study. MMP2, a well-known collagenase involved in tissue remodeling\u003csup\u003e36\u003c/sup\u003e, was increased 7-fold in our study. Col5a1 and Col5a2 were previously reported to be upregulated in IPF\u003csup\u003e37,38\u003c/sup\u003e, which were increased 9.6-fold and 7-fold respectively in our study.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cem\u003eDetoxification pathway of reactive oxygen species (ROS) is suppressed in lung fibrosis.\u003c/em\u003e\u003c/p\u003e\n\u003cp\u003eSeveral proteins involved in ROS detoxification were reduced in our study. HSPA1 (rs1043618) polymorphisms were reported as associated with a decreased risk of IPF in Mexican patients\u003csup\u003e39\u003c/sup\u003e. Prdx6 has been reported as a target to attenuate lung inflammation and fibrosis in silicosis\u003csup\u003e40\u003c/sup\u003e.\u003c/p\u003e\n\u003cp\u003eOur study also first found the decrease of Selenbp1, Glrx5, Uqcrc1, Npc1, Ifi30, and Hadh in bronchoalveolar fluid of lung fibrosis, suggesting their potential value as therapeutic targets.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cem\u003eLack of alpha1,2 fucosylated structures attenuate bleomycin-induced lung fibrosis\u003c/em\u003e\u003c/p\u003e\n\u003cp\u003eIn the\u0026nbsp;past decade, alpha1,2 fucosylated glycans have been found to be critical for inflammation signaling pathways. Epithelial fucose is used as a dietary carbohydrate by gut symbionts in a mouse model, while disruption of intestinal fucosylation led to increased susceptibility to infection by \u003cem\u003eSalmonella typhimurium\u003c/em\u003e\u003csup\u003e41,42\u003c/sup\u003e. In the mouse model, alpha1,2 fucosylated glycans were reported to be critical for complement activation, which exaggerates airway inflammation\u003csup\u003e43\u003c/sup\u003e.\u003c/p\u003e\n\u003cp\u003eThe exact mechanism by which DFTKO mice displayed attenuated lung fibrosis remains unclear. Previously, pathogenic bacteria species among lung symbionts have been reported to exaggerate the lung inflammation through activating IL-17R signaling\u003csup\u003e21\u003c/sup\u003e. Our current research is focused on whether the fucosylated glycans influence the colonization of lung symbionts.\u003c/p\u003e\n\u003cp\u003eIn summary, our study first identified the genetic link between lack of alpha1,2 fucosylated glycans and bleomycin-induced lung fibrosis. Genetic editing of alpha1,2 fucosyltransferases may be achieved by current antisense oligonucleotides technologies, to deplete the alpha1,2 glycan structures in vivo. Chemically synthesized enzyme inhibitors may also suppress the synthesis of alpha1,2 fucose in vivo. The novel protein targets discovered in bronchoalveolar lavage fluid provide critical clues for drug development. The unknown role of alpha1,2 fucosylated glycans indicates that self or microbial lectins might be involved in etiology of this intriguing clinical disease.\u003c/p\u003e"},{"header":"Abbreviations","content":"\u003ctable border=\"0\" cellspacing=\"0\" cellpadding=\"0\" width=\"590\"\u003e\n \u003ctbody\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 132px;\"\u003e\n \u003cp\u003eIPF\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 459px;\"\u003e\n \u003cp\u003eIdiopathic Pulmonary Fibrosis\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 132px;\"\u003e\n \u003cp\u003eBALF\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 459px;\"\u003e\n \u003cp\u003eBronchoalveolar Lavage Fluid\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 132px;\"\u003e\n \u003cp\u003eROS\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 459px;\"\u003e\n \u003cp\u003eReactive Oxygen Species\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 132px;\"\u003e\n \u003cp\u003eTGF-\u0026beta;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 459px;\"\u003e\n \u003cp\u003eTransforming Growth Factor Beta\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 132px;\"\u003e\n \u003cp\u003eDFTKO\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 459px;\"\u003e\n \u003cp\u003eAlpha1,2 Fucose-Deficient Knockout Mice\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 132px;\"\u003e\n \u003cp\u003eWT\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 459px;\"\u003e\n \u003cp\u003eWild-Type\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 132px;\"\u003e\n \u003cp\u003eHE\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 459px;\"\u003e\n \u003cp\u003eHematoxylin and Eosin\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 132px;\"\u003e\n \u003cp\u003eSDC\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 459px;\"\u003e\n \u003cp\u003eSodium Deoxycholate\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 132px;\"\u003e\n \u003cp\u003eAPOA1\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 459px;\"\u003e\n \u003cp\u003eApolipoprotein A1\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 132px;\"\u003e\n \u003cp\u003eAPOA2\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 459px;\"\u003e\n \u003cp\u003eApolipoprotein A2\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 132px;\"\u003e\n \u003cp\u003eBPIFA2\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 459px;\"\u003e\n \u003cp\u003eBPI Fold Containing Family A Member 2\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 132px;\"\u003e\n \u003cp\u003eC1QTNF5\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 459px;\"\u003e\n \u003cp\u003eC1q Tumor Necrosis Factor-Related Protein 5\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 132px;\"\u003e\n \u003cp\u003eCOL5A1\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 459px;\"\u003e\n \u003cp\u003eCollagen Type V Alpha 1 Chain\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 132px;\"\u003e\n \u003cp\u003eCOL5A2\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 459px;\"\u003e\n \u003cp\u003eCollagen Type V Alpha 2 Chain\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 132px;\"\u003e\n \u003cp\u003eHSPA1A\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 459px;\"\u003e\n \u003cp\u003eHeat Shock Protein Family A (Hsp70) Member 1A\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 132px;\"\u003e\n \u003cp\u003eHMGA1\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 459px;\"\u003e\n \u003cp\u003eHigh-Mobility Group AT-Hook 1\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 132px;\"\u003e\n \u003cp\u003eLTBP1\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 459px;\"\u003e\n \u003cp\u003eLatent Transforming Growth Factor Beta Binding Protein 1\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 132px;\"\u003e\n \u003cp\u003eMMP2\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 459px;\"\u003e\n \u003cp\u003eMatrix Metalloproteinase 2\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 132px;\"\u003e\n \u003cp\u003ePRDX6\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 459px;\"\u003e\n \u003cp\u003ePeroxiredoxin 6\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 132px;\"\u003e\n \u003cp\u003eSERPINA3N\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 459px;\"\u003e\n \u003cp\u003eSerpin Family A Member 3N\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 132px;\"\u003e\n \u003cp\u003eSSBP1\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 459px;\"\u003e\n \u003cp\u003eSingle-Stranded DNA Binding Protein 1\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 132px;\"\u003e\n \u003cp\u003eS100A8\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 459px;\"\u003e\n \u003cp\u003eS100 Calcium-Binding Protein A8\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 132px;\"\u003e\n \u003cp\u003eS100A9\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 459px;\"\u003e\n \u003cp\u003eS100 Calcium-Binding Protein A9\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 132px;\"\u003e\n \u003cp\u003eBGN\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 459px;\"\u003e\n \u003cp\u003eBiglycan\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 132px;\"\u003e\n \u003cp\u003eVCAN\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 459px;\"\u003e\n \u003cp\u003eVersican\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 132px;\"\u003e\n \u003cp\u003eTIMP1\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 459px;\"\u003e\n \u003cp\u003eTissue Inhibitor of Metalloproteinases 1\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 132px;\"\u003e\n \u003cp\u003eFN14\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 459px;\"\u003e\n \u003cp\u003eFibroblast Growth Factor-Inducible 14\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 132px;\"\u003e\n \u003cp\u003eSPARCL1\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 459px;\"\u003e\n \u003cp\u003eSparc-Related Modular Calcium Binding 1\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 132px;\"\u003e\n \u003cp\u003eH3C1\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 459px;\"\u003e\n \u003cp\u003eHistone H3.1\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 132px;\"\u003e\n \u003cp\u003ePLEKHF2\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 459px;\"\u003e\n \u003cp\u003ePleckstrin Homology Domain-Containing Family F Member 2\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 132px;\"\u003e\n \u003cp\u003eHDGFL3\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 459px;\"\u003e\n \u003cp\u003eHepatoma-Derived Growth Factor-Like 3\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 132px;\"\u003e\n \u003cp\u003eCEACAM1\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 459px;\"\u003e\n \u003cp\u003eCarcinoembryonic Antigen-Related Cell Adhesion Molecule 1\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 132px;\"\u003e\n \u003cp\u003eMS\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 459px;\"\u003e\n \u003cp\u003eMass Spectrometry\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 132px;\"\u003e\n \u003cp\u003eLC-MS/MS\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 459px;\"\u003e\n \u003cp\u003eLiquid Chromatography-Mass Spectrometry\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 132px;\"\u003e\n \u003cp\u003eTMT\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 459px;\"\u003e\n \u003cp\u003eTandem Mass Tag\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 132px;\"\u003e\n \u003cp\u003eDDA\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 459px;\"\u003e\n \u003cp\u003eData-Dependent Acquisition\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 132px;\"\u003e\n \u003cp\u003eHCD\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 459px;\"\u003e\n \u003cp\u003eHigher-Energy Collisional Dissociation\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 132px;\"\u003e\n \u003cp\u003eMS/MS\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 459px;\"\u003e\n \u003cp\u003eTandem Mass Spectrometry\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 132px;\"\u003e\n \u003cp\u003eFDR\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 459px;\"\u003e\n \u003cp\u003eFalse Discovery Rate\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 132px;\"\u003e\n \u003cp\u003ePSM\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 459px;\"\u003e\n \u003cp\u003ePeptide-Spectrum Match\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 132px;\"\u003e\n \u003cp\u003ePD\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 459px;\"\u003e\n \u003cp\u003eProteome Discoverer\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003c/tbody\u003e\n\u003c/table\u003e"},{"header":"Declarations","content":"\u003cp\u003e\u003cstrong\u003eEthics approval and consent to participate\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThis study was approved by institutional board of Tongji University.\u003c/p\u003e\n\u003cp\u003eNo human subjects were involved in this study.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eConsent for publication\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eNot applicable.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAvailability of data and materials\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe authors confirm that the data supporting the findings of this study are available within the article and its supplementary materials.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eCompeting interests\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe authors declare no competing interests.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eFunding\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe authors are supported by National Key Research and Development Plan grants 2021YFE0200500, Fundamental Research Funds for the Central Universities 22120200163, National Natural Science Foundation of China grant 31870972, Sino-German Scientific Research Program M0693, Major Program of National Natural Science Foundation of China 9235920, and Shanghai Science and Technology Commission grant 20410713500.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAuthors\u0026apos; contributions\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eC.Z., Conceptualization, Data curation, Formal analysis, Investigation, Methodology, Validation, Writing-original draft, Writing-review and editing.\u003c/p\u003e\n\u003cp\u003eX.M., Investigation, Supervision, Methodology.\u003c/p\u003e\n\u003cp\u003eY.J., Investigation, Data curation.\u003c/p\u003e\n\u003cp\u003eZ.J., Investigation, Methodology.\u003c/p\u003e\n\u003cp\u003eG.A., Data curation, Revision.\u003c/p\u003e\n\u003cp\u003eD.Z., Conceptualization, Data curation, Formal analysis, Funding acquisition, Investigation, Methodology, Project administration, Supervision, Validation, Writing-original draft, Writing-review and editing.\u003c/p\u003e\n\u003cp\u003eAll authors have read and agreed to the published version of the manuscript. All the authors are giving consent to publish.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eCorresponding authors\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eCorrespondence to Dapeng Zhou, [email protected]\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAcknowledgements\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eWe thank Yingpeng Zhou and Xiaojing Zhou of Shanghai BioTree Biotech Co., Ltd for mass spectrometry analysis.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAuthors\u0026apos; information\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eAuthors and Affiliations\u003c/p\u003e\n\u003cp\u003eDepartment of Immunology and Pathogen Biology, Key Laboratory of Pathogen and Host-Interactions, Ministry of Education,\u0026nbsp;Tongji University School of Medicine,\u0026nbsp;500 Zhennan Road, Shanghai 200331, China\u003c/p\u003e\n\u003cp\u003eChenxi Zhu, Xinjia Mai, Yicheng Jiang, Zhaohui Ji, Gulberdiyev Abdylla, Dapeng Zhou\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\n\u003cli\u003eMoss, B. 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This purpose of this study is to investigate the impact of alpha1,2-fucosylation on bleomycin-induced pulmonary fibrosis in a murine model.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eMethods\u003c/strong\u003e: Wild-type and knockout mice deficient of alpha1,2 fucose (DFTKO) were treated by bleomycin and lung fibrosis was studied. Bronchoalveolar lavage fluid was collected on Day 7 and Day 14 for Tandem Mass Tag-labeled(TMT) mass spectrometry proteomic analysis.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eResults\u003c/strong\u003e: Lung fibrosis is less severe in mice lacking alpha1,2 fucose. Multiple bronchoalveolar lavage proteins were elevated 7 days after bleomycin treatment, including 1) proteins involved in lipid metabolism, antimicrobial defense and inflammation: Bpifa2, Apoa1, Apoa2, C1qtnf5, Serpina3n; 2) proteins involved in TGF-beta and extracellular matrix signaling: Fst, Bgn, Timp1, Vcan, Ltbp1, Sparcl1, Mmp2; 3) Collagens: Col5a1, Col5a2. Several proteins involved in detoxification of reactive oxygen species (ROS) were found to be decreased 7 days after bleomycin treatment: Hspa1a, Selenbp1, Glrx5, Uqcrc1, Npc1, Ifi30, Hadh, Prdx6. When wild-type and knockout mice deficient of alpha1,2 fucose were compared 7 days after bleomycin treatment, multiple proteins were elevated in knockout mice: 1) proteins involved in DNA damage repair and maintenance of genome stability: H3c1, Ssbp1, Hmga1; 2) proteins involved in inflammation: S100a8, S100a9; 3) proteins involved in signaling pathways of wound healing and tissue remodeling: Hdgfl3, Plekhf2, Ceacam1.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eConclusions\u003c/strong\u003e: Lack of alpha1,2 fucosylated structures are found to play protective roles by upregulating components of three critical pathways, while exact mechanisms will be focus of our future study. Identification of alpha1,2 fucosylated structures as facilitators of lung fibrosis also provide an interesting target for therapeutic interventions for lung fibrosis.\u003c/p\u003e","manuscriptTitle":"Lack of alpha1,2-fucosylation protects mice from bleomycin-induced lung fibrosis","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2025-03-14 07:33:34","doi":"10.21203/rs.3.rs-6044497/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":"744c13cf-89c5-4f6d-bb97-f7e05cd20324","owner":[],"postedDate":"March 14th, 2025","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"posted","subjectAreas":[],"tags":[],"updatedAt":"2025-03-27T17:23:12+00:00","versionOfRecord":[],"versionCreatedAt":"2025-03-14 07:33:34","video":"","vorDoi":"","vorDoiUrl":"","workflowStages":[]},"version":"v1","identity":"rs-6044497","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-6044497","identity":"rs-6044497","version":["v1"]},"buildId":"XKTyCvWXoU3ODBz1xrDgd","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}