Key terms “systemic sclerosis AND pulmonary fibrosis” and species “Homo sapiens” were used to search the NCBI GEO database ( https://www.ncbi.nlm.nih.gov/geo/ ), yielding a dataset (GSE215841) comparing whole transcriptome expression profiles between normal lung fibroblasts and systemic sclerosis patient derived fibroblasts. Detailed dataset information is provided in Data Sheet 3. Differential expression analysis was performed using GEO2R ( https://www.ncbi.nlm.nih.gov/geo/geo2r/?acc=GSE21584 ) with criteria of |logFC| > 1 and adjusted P<0.05, followed by visualization of results via a volcano plot. Pathway enrichment analysis of differentially expressed genes was conducted using Metascape ( http://metascape.org ). Genes with significant upregulation in disease states were selected for further evaluation. PubMed ( https://pubmed.ncbi.nlm.nih.gov/ ) was queried to assess the feasibility and novelty of candidate genes (Data Sheet 4).

A total of 97 CTD without ILD patients and 169 CTD-ILD patients were enrolled between December 31, 2023, and December 31, 2024, at the Minda Hospital of Hubei Minzu University, alongside 113 healthy controls (HC) recruited via a physical examination center. CTD diagnoses followed the 2013 American College of Rheumatology (ACR)/European League Against Rheumatism (EULAR) criteria (33–37) while CTD-ILD patients met radiological criteria for ILD on high resolution computed tomography, including diffuse ground glass opacities, reticular opacities, traction bronchiectasis, or honeycombing (38). The extent of ILD was assessed using a validated semi-quantitative HRCT scoring system, which categorized the involvement into four grades: Grade 1 (0-25%), Grade 2 (26-50%), Grade 3 (51-75%), and Grade 4 (76-100%) (39). To minimize inter-observer variability, HRCT scoring was independently performed by two radiologists and two senior respiratory physicians, with final scores calculated as the mean of the four assessments.

Peripheral serum samples were acquired from healthy volunteers, CTD-ILD patients, and CTD patients without ILD. Comprehensive clinical data were systematically recorded for all participants. Exclusion criteria for the disease group: (1) other pulmonary diseases (e.g., tumors, bronchiectasis, COPD, tuberculosis); (2) non-CTD-ILD (e.g., pneumoconiosis, radiation pneumonitis); (3) severe organ dysfunction (cardiac/renal failure); (4) prior malignancy; (5) pregnancy/lactation; (6) infectious diseases (hepatitis, syphilis, HIV); (7) neuropsychiatric disorders or refusal to consent; (8) smoking history. Healthy controls, matched for age and sex, were recruited from a physical examination center, with inclusion criteria including age between 18 and 80 years, absence of underlying diseases, no smoking history, and normal blood counts, liver function, and kidney function. Exclusion criteria included age <18 or >80 years, pregnancy, psychiatric disorders, or recent medication use. The inclusion criteria for CTD-ILD complicated by infection were a confirmed diagnosis of CTD-ILD, fever (temperature >37.5°C, non-drug-induced), new-onset or worsening cough with purulent sputum, new pulmonary rales or wheezing, white blood cell count (WBC) >10×10⁹/L or <4×10⁹/L with neutrophilia, C-reactive protein (CRP) >10 mg/L, and procalcitonin (PCT) >0.5 ng/mL. BALF was collected from CTD-ILD patients with pulmonary infections and from individuals with isolated pulmonary infections. Exclusion criteria included acute exacerbation of ILD without evidence of infection, pulmonary embolism, and drug-induced lung injury. In this study, patients with pulmonary infection (rather than ILD) were selected as the BALF control group. The primary purpose of this selection is to provide a reference baseline that can match the inflammatory background of ILD complicated with infection.

Human lung tissue samples were sourced from paraffin blocks of patients with CTD-ILD who underwent lung tissue biopsy for other diseases and whose samples were preserved in the hospital. Healthy lung tissue was obtained from paraffin blocks of lung nodules that were surgically resected, with the final diagnosis being benign nodules, and the tissue was taken from the area adjacent to the nodules. The study protocol was approved by the Ethics Committee of Minda Hospital, Hubei Minzu University (Approval No. 2024004), and written informed consent was obtained from all participants.

Eight-week-old male C57BL/6 mice (body weight 20-22g) were purchased from Chengdu Kemeixin Biotechnology (Chengdu, China) and housed under specific pathogen-free conditions. After 1 week of acclimation with ad libitum access to food and water, mice were randomized into two groups: bleomycin (BLM)-treated (4 mg/kg intratracheal instillation) and saline control (isovolumetric saline), with 6-8 animals per group. Mice were anesthetized with isoflurane. After cervical incision, BLM (4 mg/kg dissolved in saline) or saline alone was instilled into the trachea. Wounds were sutured, and animals were placed on a heating pad until recovery. At specified time points (day 0, 7, 14, 21, and 28 post-instillation), mice were euthanized under deep isoflurane anesthesia. Peripheral blood and lung tissues were collected for analysis. All animal experiments were performed in accordance with guidelines from the Institutional Animal Care and Use Committee, with protocols approved by West China Hospital, Sichuan University (Approval No. 20230427002).

Lung tissue sections were dewaxed in xylene, hydrated through graded ethanol washes, and rinsed with PBS buffer. Antigen retrieval was performed via microwave boiling followed by natural cooling to room temperature and PBS rinsing. Endogenous peroxidase activity was quenched with 3% hydrogen peroxide, and sections were again rinsed with PBS. Non-specific binding sites were blocked with goat serum for 20-30 minutes at room temperature. Sections were then incubated overnight with MFAP5 primary antibody (1:200 dilution, Abcam: ab232846). After washing, biotinylated secondary antibody (1:200) was applied, followed by streptavidin-peroxidase complex incubation for 30 minutes at room temperature. Diaminobenzidine (DAB) chromogen was used for visualization, with reactions terminated by distilled water rinsing. Sections were counterstained with hematoxylin, dehydrated, and mounted with neutral resin. ILD severity was individually graded using Ashcroft’s semi-quantitative system (40).

Lung tissue sections were sequentially dewaxed in xylene, hydrated through graded ethanol washes, and rinsed with PBS buffer. Antigen retrieval was performed via microwave boiling followed by natural cooling to room temperature and PBS rinsing. Sections were blocked with immunofluorescence blocking solution (Beyotime, P0102) for 1 hour at room temperature. Primary antibodies (MFAP5 1:200 dilution, Abcam: ab232846, ab203828) were applied and incubated overnight at 4°C. After PBS rinsing, sections were incubated with Alexa Fluor™ 488-conjugated goat anti-rabbit secondary antibody (1:200 dilution, Invitrogen) for 1 hour at room temperature. Nuclei were counterstained with DAPI for 5 minutes, and sections were mounted with antifade mounting medium and coverslips, sealed with neutral resin.

Lung tissue sections were sequentially dewaxed in xylene, hydrated through graded ethanol, rinsed with PBS buffer, and stained with Masson’s Trichrome Stain kit. Nuclear staining was performed with Weigert’s Hematoxylin Staining Solution for 5-10 minutes. Sections were differentiated in 1% hydrochloric acid in ethanol for 5-15 seconds, followed by bluing in 1% lithium carbonate solution for 1 minute. Masson’s ponceau-acid fuchsin solution was applied for 5-10 minutes, and slides were rinsed in 2% glacial acetic acid for 1 minute. Differentiation was carried out in 1% phosphomolybdic acid aqueous solution for 3-5 minutes, followed by another 1-minute rinse in 2% glacial acetic acid. Counterstaining with 1% light green aqueous solution was performed for 1-2 minutes. After a final 0.2% glacial acetic acid rinse, sections were dehydrated in ethanol, cleared in xylene, and mounted with neutral resin.

Total mRNA was extracted from cells and mouse lung tissues using Trizol reagent (Vazyme, R401-01). Complementary DNA (cDNA) was synthesized via reverse transcription with a Takara kit, followed by cDNA amplification using SYBR Green Master Mix (Takara). Primers used in qPCR are listed in Data Sheet 1 and were synthesized by Tsingke Biotech. Relative target gene expression was normalized to 18S ribosomal RNA levels and calculated using the 2(-△△CT) method.

Mouse lung tissues were lysed with RIPA lysis buffer containing protease/phosphatase inhibitors (Beyotime) for 30 minutes, followed by centrifugation to collect supernatants. Protein concentrations were determined using a BCA protein assay kit (Beyotime). Lysates were boiled in loading buffer for 10 minutes, separated via SDS-PAGE (20-30μg protein per lane), and transferred to PVDF membranes (Millipore). Membranes were blocked with 5% non-fat milk at room temperature for 1 hour, incubated with primary antibodies overnight at 4°C, washed three times with TBST, and probed with species-specific HRP-conjugated secondary antibodies for 1 hour at room temperature. Protein bands were visualized using a Bio-Rad imaging system and quantified with ImageJ 1.8.0 software. Antibodies used included: anti-MFAP5 (1:1000 dilution, Abcam: ab232846) and β-Tubulin (1:5000 dilution, HUABIO: ET1602-4) (Data Sheet 2).

Human MFAP5 levels were measured using a commercial ELISA kit (E20250102-RX100335H, RUIXIN Biotech, Fujian, China). Test samples were added to the solid-phase carrier pre-coated with anti-MFAP5 antibodies, allowing target molecules to bind. After washing, enzyme-conjugated secondary antibodies were added to specifically recognize bound MFAP5. Unbound conjugates were removed via washing, followed by the addition of chromogenic substrate solution. Enzymatic reactions were visualized by color development, and optical density values were measured at 450 nm using a microplate reader.

Statistical analyses were performed using GraphPad Prism 9.5 (GraphPad Software, Inc., CA, USA) and SPSS 26.0 (IBM Corp., NY, USA). Normally distributed continuous variables were compared using independent samples t-tests (two groups) or one-way analysis of variance (ANOVA) with post-hoc testing (multiple groups). Non-normally distributed data were presented as median and interquartile range (IQR), analyzed via Mann-Whitney U tests. Correlations were evaluated using Spearman rank correlation coefficients. Study participants were divided into positive and negative groups, which included three comparison pairs: healthy control group vs. CTD-non-ILD group, healthy control group vs. CTD-ILD group, and CTD-ILD group vs. CTD-non-ILD group. The Area Under the ROC Curve (AUC) was used to quantify the overall discriminative efficacy, with the following interpretive criteria: an AUC of 0.5 indicates no discriminative value, an AUC between 0.7 and 0.8 (inclusive of 0.7, exclusive of 0.8) indicates good discriminative efficacy, and an AUC between 0.8 and 0.9 (inclusive of 0.8, exclusive of 0.9) indicates excellent discriminative efficacy. Based on the “Thresholds” table output by SPSS, the optimal diagnostic threshold was determined using the method of maximizing the Youden’s Index (Youden’s Index= Sensitivity+Specificity-1). Meanwhile, the sensitivity and specificity corresponding to this optimal threshold were extracted. Statistical significance was defined as two-tailed P<0.05, denoted as: *P<0.05, **P<0.01, ***P <0.001, with “ns” indicating non-significance.

We first analyzed mRNA expression profiles between SSc-PF samples and normal controls from public datasets. The GSE215841 dataset included 23 samples: 12 lung fibroblasts from SSc-PF patients and 11 from healthy donors. GEO2R analysis of transcriptome sequencing data identified 1,6248 genes, with 364 upregulated and 336 downregulated ( Figure 1A ). Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway enrichment analysis of these DEGs highlighted significant enrichment in PI3K-AKT and TGF-β signaling pathways ( Figure 1B ). The PI3K-AKT pathway regulates core cellular processes, including growth, proliferation, and survival (41), The TGF-β/SMAD signaling pathway plays a pivotal role in pulmonary fibrosis pathogenesis (42), with excessive TGF-β production driving pathological scarring and extracellular matrix deposition (43). Crosstalk between TGF-β and PI3K/AKT signaling further promotes fibrogenesis (44), providing a mechanistic foundation for downstream pathway analysis. Based on literature review, Gene Cards analysis, and novelty assessment, 10 candidate genes were shortlisted (Data Sheet 4). These were validated in a BLM-induced ILD mouse model via qPCR. Among them, MFAP5 showed the most significant differential expression, leading to its selection as the target molecule ( Figure 1C ).

The study included 97 CTD patients, 169 CTD-ILD patients (sub-grouped as 82 RA-ILD, 20 PSS-ILD, 35 SSc-ILD, 12 SLE-ILD, 12 undifferentiated CTD-ILD, and 7 idiopathic inflammatory myopathy-ILD (IIM-ILD), Additionally, BALF samples were collected from 18 patients with pulmonary infection, 9 of whom had concurrent CTD-ILD. Demographics were balanced across groups: median age (interquartile range) was 62 (56,69) years for CTD, 56.5 (51,63) years for CTD-ILD, and 53 (46,61) years for HC. Statistical analysis showed no significant differences in median age or sex distribution (female proportion, P > 0.05). between CTD and HC groups, confirming demographic comparability ( Table 1 ). Serum MFAP5 levels were significantly higher in CTD-ILD patients [2.82 (2.27, 5.27) pg/mL] than in CTD patients without ILD [1.99 (1.69, 2.30) pg/mL] (P<0.001), which, in turn, were markedly elevated compared with healthy controls [1.52 (1.025, 2.05) pg/mL] (P<0.001) ( Figure 1D ). No significant difference in MFAP5 expression was observed across CTD-ILD subtypes ( Figure 1E ). CTD-ILD patients exhibited significantly higher levels of Age, MFAP5, WBC, ALB, LDH, HBDH, D-dimer, CRP, ESR, and PCT compared to CTD-non-ILD patients (P < 0.05). with significantly higher expression in BALF of CTD-ILD patients with pulmonary infection ( Figure 1F ). MFAP5 positively correlated with degree of ILD involvement ( Figure 1G ) and serum CRP ( Figure 1H ).

The results of Spearman correlation analysis indicated that serum MFAP5 levels were significantly associated with disease activity in patients with CTD-ILD. Specifically, positive correlations were observed between MFAP5 levels and WBC (r = 0.274, P < 0.001), CRP (r = 0.718, P<0.001), RF (r = 0.224, P=0.012), C1q (r = 0.176, P=0.032), ESR (r = 0.169, P=0.030), IL6 (r = 0.357, P<0.001), and PCT (r = 0.381, P <0.001). Conversely, a negative correlation was found with ALB (r = -0.223, P=0.004) ( Table 2 ). ROC curve analysis further demonstrated the predictive value of MFAP5 for CTD-ILD. For discriminating CTD-non-ILD, the cutoff value was 1.525 pg/mL, with AUCof 0.717, sensitivity of 86.6%, and specificity of 51.3%. For discriminating CTD-ILD, the cutoff value was 2.095 pg/mL, with an AUC of 0.895, sensitivity of 82.2%, and specificity of 85.8%. Additionally, for distinguishing CTD-ILD from CTD-non-ILD, the cutoff value was 2.480 pg/mL, with an AUC of 0.811, sensitivity of 66.9%, and specificity of 89.5% ( Figures 2A–C ). These findings underscore the high specificity of MFAP5 in discriminating CTD-ILD and CTD-non-ILD, highlighting it potential as a valuable biomarker in this context.

Chest CT scans revealed symmetrical reticular or honeycomb changes in the posterior basal segments of both lower lobes in RA, SSC, and DM-ILD patients, with intralobular interstitial thickening, interlobular septal thickening (“grid-like” patterns), focal/diffuse ground-glass opacities, and fibrotic bands ( Figure 3A ). Histological analysis by hematoxylin-eosin (H&E) staining showed disrupted alveolar architecture, diffuse alveolar septal thickening, chronic inflammatory cell infiltration (lymphocytes, plasma cells), thickened small vessel walls with luminal stenosis, and perivascular fibrous encapsulation ( Figure 3B ). Masson trichrome staining demonstrated collagen fibers deposited in bundles, sheets, or reticular patterns within alveolar septa and bronchovascular bundles, with red fibrinoid exudates in alveolar spaces. Organizing lesions were observed in DM-ILD lungs, with blue-green collagen encasing small vessels and their surroundings ( Figure 3C ). MFAP5 protein expression was significantly higher in all three ILD subtypes compared to healthy controls, with the lowest levels in RA-ILD, moderate elevation in SSC-ILD, and maximal expression in DM-ILD, correlating with disease severity ( Figure 3D ). Immunofluorescence co-localization studies in ILD tissues showed MFAP5 expression in fibroblasts ( Figure 3E ), co-localization with adipose fibroblast (ADRP) subsets ( Figure 3F ), suggesting functional interplay between these cell types.

To validate MFAP5 expression in vivo and its role in ILD, the BLM-induced ILD mouse model was established via intratracheal instillation ( Figure 4A ). Protein and mRNA levels were analyzed in lung tissues from BLM-treated and saline control mice. Results showed MFAP5 protein levels were significantly higher in lung tissues of BLM-treated mice compared to saline controls ( Figures 4B, C ), indicating upregulation during BLM-induced ILD. mRNA analysis revealed increased expression of extracellular matrix (ECM) genes Collagen type Ia (Col1a), Fibronectin (Fn), and α-smooth muscle actin (α-SMA) in BLM-treated lungs ( Figure 4D ), The successful induction of the model was confirmed, as evidenced by the significantly higher MFAP5 mRNA levels in the bleomycin BLM-treated mice compared to those in the saline group ( Figure 4E ), corroborating the association between MFAP5 and ILD markers. Histological analysis showed alveolar damage, inflammatory cell infiltration, and thickened alveolar septa in BLM-treated lungs via H&E staining, accompanied by elevated Ashcroft scores ( Figures 4F, G ). Masson trichrome staining confirmed extensive collagen deposition in BLM-treated tissues, consistent with H&E findings ( Figures 4F, H ). Immunohistochemistry revealed that MFAP5 was primarily localized to the extracellular matrix, with significantly higher expression in BLM-treated lungs compared to saline controls ( Figures 4F, I ), further supporting its role in ILD. To investigate the dynamic changes of MFAP5 during fibrogenesis, lung tissues were collected at multiple time points from BLM-induced ILD mice ( Figure 4J ) and analyzed via immunofluorescence. Results showed that MFAP5 expression began increasing by day 7, peaked at day 21, and declined by day 28 post-BLM instillation ( Figures 4K, L ), indicating early activation and maximal expression during peak fibrosis. These findings demonstrate that MFAP5 is robustly upregulated in BLM-induced ILD, with expression levels tightly correlated with histological severity. Collectively, these data demonstrate a significant association between MFAP5 and ILD progression, suggesting its potential role and providing a foundation for future mechanistic studies.