The CD163 + tissue-infiltrating macrophages regulate ferroptosis in thyroid-associated ophthalmopathy orbital fibroblasts via the TGF-β/Smad2/3 signaling pathway.

{"sci_version": "1.2", "schema": "https://corpus.bntrg.co.uk/sci/1.0/sci.schema.json", "id": "pmid:40211281", "kind": "science-record", "revision": 4, "content_hash": "sha256:06ce99211e9ac6290fea613442bd35bd2068831bdbc36d48bf6ea776c0f26b8b", "created_at": "2026-11-01T20:30:22Z", "updated_at": "2026-11-01T22:56:20Z", "generator": {"name": "corpus-assay-engine", "run_id": "reading-sci-2026-11-01"}, "paper": {"state": "complete", "title": "The CD163 + tissue-infiltrating macrophages regulate ferroptosis in thyroid-associated ophthalmopathy orbital fibroblasts via the TGF-β/Smad2/3 signaling pathway.", "doi": "10.1186/s12967-025-06443-0", "pmid": "40211281", "authors": ["Xuemei Li", "Siyi Wang", "Hanwen Cao", "Simin Xu", "Chao Xiong", "Jinhai Yu", "Yunxiu Chen", "Zhangjun Ren", "Min Li", "Ying Hu", "Puying Gan", "Qihua Xu", "Yaohua Wang", "Hongfei Liao"], "journal": "Journal of translational medicine", "year": 2025, "abstract": "BACKGROUND: Thyroid-associated ophthalmopathy (TAO) is a thyroid function-related, organ-specific autoimmune disease that primarily leads to specific reactive changes and tissue remodeling in the periocular region. The exact pathogenesis of TAO remains unclear.\nMETHODS: High-throughput gene expression datasets related to TAO were comprehensively retrieved from the Gene Expression Omnibus (GEO) database, selecting GSE174139 and GSE158464 for analysis. Differentially expressed genes (DEGs) between TAO patients and healthy controls were identified, and ferroptosis-related genes (FRGs) were obtained from the FerrDb database. The intersection of DEGs and FRGs yielded ferroptosis-related genes associated with TAO.The transcriptional expression of FRGs was validated using real-time quantitative polymerase chain reaction (RT-qPCR) on orbital adipose tissue samples from TAO patients and healthy controls. Single-cell sequencing of six human tissue samples further analyzed changes in cellular subpopulations within the TAO microenvironment.Additionally, a co-culture model of CD163 + macrophages and TAO orbital fibroblasts, along with an in vitro TGF-β1-induced orbital fibroblast (OF) model, was constructed to validate the role of the TGF-β1/SMAD2/3 axis in ferroptosis regulation. Finally, potential clinical drugs targeting CD163 + macrophages with high ferroptosis activity in TAO were predicted using the Random Walk with Restart (RWR) algorithm combined with the DGIdb database.\nRESULTS: We first utilized TAO-related datasets from the GEO database, combined with the FerrDb ferroptosis database, to identify changes in iron metabolism genes during TAO progression through differential expression analysis, screening 7 key ferroptosis-related proteins. In vitro validation revealed that all but AOPQ and LGMN, which were upregulated, exhibited downregulated expression.Single-cell sequencing of orbital connective tissue from 4 TAO patients and 2 healthy controls identified 16,364 cells spanning 18 cell types. Analysis of the 7 key ferroptosis-related proteins revealed that fibroblasts and macrophages displayed elevated ferroptosis signaling during TAO progression. Subcluster analysis of macrophages identified 4 distinct subpopulations, with the C2 subpopulation-characterized by high expression of CD163 and CCL18-exhibiting prominent ferroptosis activation signals.Further validation using clinical tissue samples, a co-culture model of CD163 + macrophages and TAO orbital fibroblasts, and an in vitro TGF-β1-induced orbital fibroblast (OF) model confirmed aberrant activation of the TGF-β1/SMAD2/3 pathway as a key regulator of ferroptosis. Hub gene analysis of C2 subpopulation marker genes, combined with the DGIdb database, predicted potential clinical drugs targeting the C2 macrophages.\nCONCLUSION: This study, integrating single-cell RNA-Seq and bulk transcriptome analysis, revealed the involvement of CD163 + tissue-infiltrating macrophages in regulating ferroptosis of orbital fibroblasts during TAO progression and identified therapeutic candidates targeting macrophage ferroptosis signaling in TAO. Furthermore, in vitro experiments demonstrated that activation of the TGF-β1/SMAD2/3 axis promotes ferroptosis in TAO orbital fibroblasts, highlighting a novel pathway for potential therapeutic intervention.", "claims": [{"id": "claim-method", "statement": "Differentially expressed genes (DEGs) were identified using DESeq2 with a threshold of p.adj < 0.05 and |log2FoldChange| > 0.5.", "kind": "methodological", "target": null, "evidence": [{"asset": "source:pmid:40211281", "locator": "sections[4] \"Differential gene expression analysis\" paragraph 2", "quote": "Differentially expressed genes (DEGs) were identified using DESeq2 with a threshold of p.adj < 0.05 and |log2FoldChange| > 0.5."}], "state": "complete"}, {"id": "claim-data-access", "statement": "The primary data of the current study are available from the corresponding author on reasonable request.", "kind": "methodological", "target": null, "evidence": [{"asset": "source:pmid:40211281", "locator": "sections[29] \"notes\" paragraph 1", "quote": "The primary data of the current study are available from the corresponding author on reasonable request."}], "state": "complete"}, {"id": "claim-target-result", "statement": "DESeq2 analysis of TAO-related RNA-seq data from six public GEO samples identified 334 differentially expressed genes and seven ferroptosis-associated genes, indicating abnormal activation of ferroptosis signaling during TAO progression.", "kind": "quantitative", "target": {"value": 2.0, "unit": "fold change", "tolerance": null, "source": "Based on RNA-seq data related to TAO from the public GEO database, including 6 samples, differential gene expression analysis was performed using DESeq2 with the criteria padj < 0.05 and |log2FoldChange| > 0.5, identifying a total of 334 differentially expressed genes (Fig. 1A)."}, "evidence": [{"asset": "source:pmid:40211281", "locator": "sections[19] \"Identification of ferroptosis signaling activation in TAO based on bulk data\" paragraph 2", "quote": "Based on RNA-seq data related to TAO from the public GEO database, including 6 samples, differential gene expression analysis was performed using DESeq2 with the criteria padj < 0.05 and |log2FoldChange| > 0.5, identifying a total of 334 differentially expressed genes (Fig. 1A)."}, {"asset": "source:pmid:40211281", "locator": "sections[19] \"Identification of ferroptosis signaling activation in TAO based on bulk data\" paragraph 2", "quote": "A comparative analysis with the ferroptosis-related database FerrDb revealed seven ferroptosis-associated genes: the ferroptosis driver genes AQP3 and LGMN, and the suppressor genes CP, FTL, MGST1, NQO1, and TMSB4X (Fig. 1B)."}, {"asset": "source:pmid:40211281", "locator": "sections[19] \"Identification of ferroptosis signaling activation in TAO based on bulk data\" paragraph 3", "quote": "These findings suggest abnormal activation of the ferroptosis signaling pathway during TAO progression."}], "state": "complete"}], "gaps": []}, "experiment": {"state": "complete", "objective": "DESeq2 analysis of TAO-related RNA-seq data from six public GEO samples identified 334 differentially expressed genes and seven ferroptosis-associated genes, indicating abnormal activation of ferroptosis signaling during TAO progression.", "steps": [{"id": "read_published_estimate", "order": 1, "name": "Read the published estimate from the source", "kind": "other", "state": "complete", "blocked_on": null, "description": "The claim, method and evidence span published with the source article.", "duration_hours": null, "parameters": {}, "inputs": ["source:pmid:40211281"], "outputs": ["the published estimate and its evidence"], "instruments": [], "reagents": []}, {"id": "obtain_source_data", "order": 2, "name": "Obtain the source data from the named repository", "kind": "data-preparation", "state": "complete", "blocked_on": null, "description": "The GEO series accession resolves and its sample accessions are listed. 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CONCLUSION: This study, integrating single-cell RNA-Seq and bulk transcriptome analysis, revealed th", "gnaling during TAO progression, we performed single-cell RNA sequencing on eye tissues from 2 normal volunteers and"], "order_basis": "paper_text_order:2967", "id": "assay:2"}, {"role": "data_generation", "definition_id": "rna_seq", "definition_version": "99e1195c7d6f", "definition_digest": null, "required_operations": [], "support_state": "partial", "source_hashes": {"assay_lexicon": "99e1195c7d6f"}, "confidence": 0.99, "evidence": ["CLUSION: This study, integrating single-cell RNA-Seq and bulk transcriptome analysis, revealed th", "ng TAO progression, we performed single-cell RNA sequencing on eye tissues from 2 normal volunteers and", "dy, integrating single-cell RNA-Seq and bulk transcriptome analysis, revealed the involvement of CD163", "Analysis of cell proportions in single-cell transcriptome data was performed to show changes in cell p"], "order_basis": "paper_text_order:2979", "id": "assay:3"}, {"role": "data_generation", "definition_id": "ihc", "definition_version": "99e1195c7d6f", "definition_digest": null, "required_operations": [], "support_state": "partial", "source_hashes": {"assay_lexicon": "99e1195c7d6f"}, "confidence": 0.93, "evidence": ["y terms displayed We validated, using tissue immunofluorescence, that the infiltration of CD163 + macrophage"], "order_basis": "paper_text_order:8892", "id": "assay:4"}, {"role": "data_generation", "definition_id": "flow_cytometry", "definition_version": "99e1195c7d6f", "definition_digest": null, "required_operations": [], "support_state": "partial", "source_hashes": {"assay_lexicon": "99e1195c7d6f"}, "confidence": 0.99, "evidence": ["ther validation was conducted via RT-PCR and flow cytometry (Fig. 5D, E, G-J). RT-PCR results showed a s", "3 + macrophage marker CD163 mRNA expression. Flow cytometry analysis revealed a rightward shift in the f"], "order_basis": "paper_text_order:9238", "id": "assay:5"}, {"role": "data_generation", "definition_id": "cell_culture", "definition_version": "99e1195c7d6f", "definition_digest": null, "required_operations": [], "support_state": "partial", "source_hashes": {"assay_lexicon": "99e1195c7d6f"}, "confidence": 0.77, "evidence": ["sue. (A-C) Morphology of CD163 + macrophages cultured in vitro.(D, G, H) Identification of M0 macroph"], "order_basis": "paper_text_order:9892", "id": "assay:6"}, {"role": "analysis", "definition_id": "differential_expression", "definition_version": "f2132e96ae6c7a4e", "definition_digest": null, "required_operations": ["normalise_counts", "differential_expression_fit", "fdr_bh"], "support_state": "supported", "source_hashes": {"dsl_catalogue": "f2132e96ae6c7a4e"}, "confidence": 1.0, "evidence": ["Differentially expressed genes (DEGs) were identified using DESeq2 with a threshold of p.adj < 0.05 and |log2FoldChange| > 0.5."], "order_basis": "consumes_earlier_output", "id": "assay:7"}]}