BR Biomedical Reports 2049-9434 2049-9442 D.A. Spandidos BR-23-6-02065 10.3892/br.2025.2065 Articles Proteomic analysis identifies novel molecular signatures and immune-metabolic pathways in rheumatoid arthritis-associated interstitial lung disease CuiWei 1 ZhangYan 2 YeQing 3 YanBing 4 YangQunzhi 4 ZhangGe 4 Department of Clinical Laboratory, Beijing Haidian Hospital, Beijing Haidian Section of Peking University Third Hospital, Beijing 100080, P.R. China Rheumatology and Immunology Department, Beijing Shunyi Hospital, Beijing 101300, P.R. China Respiratory Department, Beijing Haidian Hospital, Beijing Haidian Section of Peking University Third Hospital, Beijing 100080, P.R. China Rheumatology and Immunology Department, Beijing Haidian Hospital, Beijing Haidian Section of Peking University Third Hospital, Beijing 100080, P.R. China Correspondence to: Dr Ge Zhang, Rheumatology and Immunology Department, Beijing Haidian Hospital, Beijing Haidian Section of Peking University Third Hospital, 29 Zhongguancun Street, Haidian, Beijing 100080, P.R. China 18612035789@163.com

Abbreviations: RA, rheumatoid arthritis; RA-ILD, RA-associated interstitial lung disease; HRCT, high-resolution computed tomography; PCA, principal component analysis; GO, Gene Ontology; KEGG, Kyoto encyclopedia of genes and genomes; PPI, protein-protein interaction

 122025 02102025 23 6 187 03 05 2025 05 09 2025 Copyright: © 2025 Cui et al. 2025 This is an open access article distributed under the terms of the Creative Commons Attribution-NonCommercial-NoDerivs License, which permits use and distribution in any medium, provided the original work is properly cited, the use is non-commercial and no modifications or adaptations are made.

Rheumatoid arthritis (RA)-associated interstitial lung disease (RA-ILD) is a severe extra-articular manifestation of RA characterized by complex pathogenesis and limited therapeutic options. The present study aimed to identify circulating serum proteins that may reveal novel molecular mechanisms underlying RA-ILD and inform the development of disease-modifying strategies. A multi-center cohort study was conducted including patients with RA-ILD (n=40), patients with RA but without ILD (n=40) and healthy controls (n=7). Pooled serum samples were analyzed using a high-throughput antibody array targeting 440 proteins. Differentially expressed proteins were defined by statistical criteria (P<0.05) and a fold change >1.2 or <0.83. Functional enrichment and protein-protein interaction (PPI) analyses were performed to explore associated biological pathways. A total of 20 proteins that showed a stepwise increase in expression were identified: Levels were significantly higher in patients with RA compared with healthy controls and further elevated in patients with RA-ILD relative to RA alone. Hierarchical clustering and principal component analysis revealed distinct protein expression profiles across groups. Gene Ontology analysis indicated enrichment in pathways related to immune cell activation, proliferation and cytokine production. Kyoto Encyclopedia of Genes and Genomes pathway analysis highlighted cytokine-cytokine receptor interactions and PI3K-Akt signaling. PPI network analysis identified insulin as a central hub interacting with IGF-1R, IL-7, and other profibrotic mediators. Several proteins (for example, CA9, EDA-A2, Gas1, CRTAM, IL-2Rb and IL-31) emerged as novel candidates not previously linked to RA-ILD. The present study identified a panel of 20 dysregulated serum proteins in RA-ILD, implicating immune dysregulation, fibrotic processes and metabolic signaling-particularly the insulin/IGF-1R-PI3K-Akt axis-in disease pathogenesis. These findings provide potential therapeutic targets for RA-ILD that warrant further validation.

 RA ILD cytokines inflammation fibrosis Funding: The present study was supported by the 2024 Haidian Health Development Research, Cultivation Plan Project (grant no. HP2024-30-101004) and the Beijing Shunyi District Hospital Research and Development Special Fund (grant no. 2025Y01). Introduction

Rheumatoid arthritis-associated interstitial lung disease (RA-ILD) is a serious extra-articular manifestation of RA that significantly increases morbidity and mortality. Epidemiological studies indicate that 10-40% of patients with RA develop ILD, with mortality rates up to 3-fold higher than those with RA without pulmonary involvement (1,2). RA-ILD is marked by progressive pulmonary fibrosis and inflammation, ultimately leading to respiratory failure and reduced survival. It accounts for 10-20% of RA-related deaths (3,4). The high prevalence and poor prognosis highlight the urgent need for improved understanding of its pathogenesis and more effective management strategies.

The pathogenesis of RA-ILD remains poorly understood. Diagnostic delays are common due to nonspecific early symptoms and the absence of validated screening protocols, which often result in treatment initiation only after irreversible lung damage has occurred (5-7). Current evidence points to a multifactorial etiology involving genetic susceptibility (for example, HLA-DRB1 alleles), environmental exposures (for example, smoking), and autoimmune dysregulation (8,9). Molecular mechanisms driving RA-ILD include abnormal immune activation (for example, TNF-α and IL-6), fibroblast proliferation and dysregulated extracellular matrix deposition-pathways that parallel those in idiopathic pulmonary fibrosis (IPF) (10-12). Emerging evidence implicates neutrophil extracellular traps (NETs), YKL-40 and KL-6 in lung injury, while signaling pathways such as AKT/TMEM175 and JAK-STAT may contribute to fibrosis progression (13-16). However, the exact molecular drivers linking RA to ILD remain unclear, and currently available biomarkers (for example, KL-6 and YKL-40) lack sufficient specificity for broad clinical application (13). Management primarily relies on immunosuppressive agents (for example, methotrexate and rituximab) and antifibrotic therapies (for example, nintedanib and pirfenidone), but their effectiveness is limited by heterogeneous patient responses and the lack of RA-ILD-specific targeted treatments (12,17). For instance, antifibrotic agents have demonstrated benefit in IPF, their effectiveness in RA-ILD has been inconsistent, likely due to distinct underlying molecular pathways (18). These challenges highlight the urgent need for precision medicine strategies tailored to the unique pathogenesis of RA-ILD (19,20).

Elucidating the molecular mechanisms underlying RA-ILD is crucial for the discovery of novel therapeutic targets. Emerging evidence implicates that dysregulated immune pathways (such as IL-36 and NETs), aberrant fibroblast activation and interactions between the gut and lungs play a role in disease progression (11,21). For example, targeting NETs may help mitigate lung injury, while modulation of the gut microbiota could attenuate systemic inflammation (9,16). Despite these advances, our understanding of RA-ILD pathogenesis remains incomplete, limiting the development of effective targeted therapies. The present study aimed to identify novel molecular mechanisms that could inform the design of disease-modifying strategies for RA-ILD.

 Materials and methods <sec> <title>Study design and population

The present study was conducted as part of a multi-center cohort that includes participants from Beijing Haidian Hospital and Beijing Shunyi Hospital. The study population consisted of three groups: (i) Patients diagnosed with RA-ILD, (ii) patients with RA but without ILD and (iii) healthy individuals. All patients with RA met the 2010 American College of Rheumatology/European League Against Rheumatism classification criteria for RA (22), while patients with RA-ILD also met the diagnostic criteria for ILD established by the American Thoracic Society/European Respiratory Society (23). Health controls were selected from individuals without inflammatory or rheumatic diseases. All participants underwent pulmonary function tests and chest high-resolution computed tomography (HRCT). Patients with RA but without ILD and healthy controls showing pulmonary symptoms or abnormal HRCT findings were excluded to ensure specificity. The study protocol was approved by the Research Ethics Committees of Beijing Haidian Hospital (approval no. 2024-006; Beijing, China) and Beijing Shunyi Hospital (approval no. 2023k-021; Beijing, China). Written informed consent was obtained from all participants prior to enrollment. Blood samples were collected for further analysis.

 Antibody array assay

Serum proteins were analyzed in three cohorts: Healthy controls (n=7), RA (n=40, with 10 subjects pooled), and RA-ILD (n=40, with 10 subjects pooled). The clinical characteristics of the participants are summarized in Table I. Serum cytokine profiling was performed using the Human Cytokine Antibody Array (cat. no. GSH-CAA-440; RayBiotech, Inc.), a high-throughput platform with 11 non-overlapping arrays for simultaneous detection of 440 cytokines. Peripheral blood serum samples were diluted 1:2 with blocking buffer and incubated overnight in array chambers coated with cytokine-specific capture antibodies. After washing away unbound proteins, a biotin-conjugated anti-cytokine antibody cocktail was added to form antibody-cytokine-antibody sandwich complexes. Cy3-conjugated streptavidin was then applied to amplify fluorescent signals via biotin-streptavidin binding. All incubation steps were conducted with 100 µl of reagents per well. Fluorescence intensity was measured with an InnoScan 300 Microarray Scanner (Innopsys) at optimized 532 nm excitation/emission wavelengths for Cy3. Raw signal values were normalized against internal positive and negative controls for assay reproducibility.

 Enzyme-linked immunosorbent assays (ELISAs)

Insulin and IL-31 were validated by ELISAs using commercially available kits (Human Insulin ELISA Kit; cat. no. ELH-Insulin; Human IL-31 ELISA Kit; cat. no. ELH-IL31; RayBiotech, Inc.) with an expanded cohort consisting of 64 patients with RA-ILD, 64 patients with RA and 40 healthy controls (demographics provided in Table II). Briefly, serum samples were incubated in antibody-precoated wells overnight at 4˚C. Following washing steps, biotin-conjugated detection antibodies were added and incubated for 2 h. Horseradish peroxidase-conjugated streptavidin was then applied to bind the biotinylated complexes for 45 min. The enzymatic reaction was developed using tetramethylbenzidine substrate form 30 min. After the reaction was stopped, absorbance was measured at 450 nm using an ELx800NB microplate reader (BioTek; Agilent Technologies, Inc.).

 Statistical analysis

Statistical comparisons between experimental groups were conducted using a one-way ANOVA followed by Bonferroni's post hoc test in SPSS version 20.0 (IBM Corp.). Differences were considered statistically significant when meeting two criteria: (i) P<0.05; (ii) a fold change threshold of <0.83 (downregulation) or >1.2 (upregulation). Continuous data were expressed as the mean ± standard deviation (SD). Correlation analyses between serum biomarkers (IL-31 and insulin) and clinical parameters of RA-ILD severity were performed using GraphPad Prism 9.0 (GraphPad Software; Dotmatics). Pearson's correlation coefficient (r) was calculated to assess linear relationships between biomarkers and clinical parameters. P<0.05 was considered to indicate a statistically significant difference.

 Results <sec> <title>Proteomic screening identifies 20 proteins associated with RA and RA-ILD progression

A panel of 20 proteins demonstrated sequential upregulation, significantly elevated in patients with RA compared with healthy controls (P<0.05), and showing even greater increases in patients with RA-ILD compared with patients with RA (P<0.05) (Fig. 1). These proteins included carbonic anhydrase IX (CA9), IL-1F9, ectodysplasin A2 (EDA-A2), growth arrest-specific gene 1 (Gas1), IL-2Rb, IL-7, amphiregulin (AR), sonic hedgehog N-terminal (Shh-N), IGF-1R, MMP-7, insulin, Eotaxin, EpCAM, tissue factor pathway inhibitor (TFPI), MIP-3b, CRTAM, IL-31, tissue factor (TF), syndecan-1 and cathepsin L. The information of these 20 proteins is summarized in Table III.

 Classification capacity of the 20 proteins

To assess the discriminatory value of the 20 proteins, hierarchical clustering and principal component analysis (PCA) were performed. Using normalized expression data from the pooled analysis, hierarchical clustering of these proteins unveiled unique expression patterns and demonstrated 100% accuracy in classifying RA-ILD from RA and the healthy groups (Fig. 2). PCA effectively segregated RA, RA-ILD, and healthy controls into separate clusters within the reduced-dimensional space (Fig. 3).

 Functional enrichment analysis

Gene Ontology (GO) enrichment analysis revealed critical biological processes associated with the 20 proteins, including T cell differentiation, T cell activation, lymphocyte differentiation, mononuclear cell proliferation, leukocyte proliferation and positive regulation of cytokine production. Further analysis using the Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway implicated two key pathways cytokine-cytokine receptor interaction and the PI3K-Akt signaling pathway (Fig. 4).

 Protein-protein interaction (PPI) analysis

PPI network analysis identified insulin as the highest-degree hub protein (node degree=6), showing direct interactions with multiple profibrotic and immunomodulatory mediators: IGF-1R, Shh-N, AR, TF, IL-7 and EpCAM (Fig. 5). This indicates its central role in coordinating molecular crosstalk within the RA-ILD proteomic landscape.

 ELISA validation and clinical value analysis

The independent validation of insulin and IL-31 with an expanded cohort by ELISA demonstrated strong concordance with the initial antibody array data (Fig. 6). The diagnostic potential of these two biomarkers showed excellent discriminatory capacity with area under the curve values of >75% (Fig. 7). Notably, the correlation analysis revealed a strong positive correlation between these two biomarkers and the Scleroderma Lung Study I score of HRCT images (Fig. 8, r>0.6). This finding suggests that insulin and IL-31 may play a role in the disease activity of RA-ILD.

 Discussion

RA-ILD is a severe extra-articular manifestation of RA, characterized by progressive pulmonary fibrosis and high morbidity. Its etiology is multifactorial and complex, involving genetic predisposition (for example, MUC5B promoter variants), autoimmune-driven inflammation, aberrant fibroblast activation, and environmental exposures such as smoking. The interplay of these factors leads to dysregulated immune responses and irreversible lung remodeling (10). However, the precise molecular mechanisms triggering these immune responses remain elusive, limiting the development of effective targeted therapies for RA-ILD. In the present study, 20 proteins including CA9, IL-1F9, EDA-A2, Gas1, IL-2Rb, IL-7, AR, Shh-N, IGF-1R, MMP-7, insulin, Eotaxin, EpCAM, TFPI, MIP-3b, CRTAM, IL-31, TF, Syndecan-1 and Cathepsin L were identified, which were significantly elevated in patients with RA compared with healthy controls, with further upregulation in RA-ILD relative to RA alone. The stepwise increase in these proteins suggests their contribution to RA pathogenesis and progression to ILD, the most common and serious pulmonary complication of RA.

GO analysis revealed that these 20 proteins were enriched in immune-related processes, including T cell differentiation, T cell activation and cytokine production. These findings align with prior studies implicating aberrant T cell responses and cytokine dysregulation in RA-ILD pathogenesis (24-26). KEGG pathway analysis highlighted the cytokine-cytokine receptor interaction pathway and PI3K-Akt signaling pathway, both of which play key roles in immune cell proliferation and differentiation in ILD (27). The PI3K-Akt pathway is known to drive fibroblast activation and fibrosis in ILD (28). Moreover, cytokine-receptor interactions -such as IL-7/IL-7R, IGF-1R/insulin-may perpetuate chronic inflammation and fibrogenesis (29,30). Notably, Wu et al (31) reported that the AhR/IGF1R axis contributes to the development of IPF through activation of the TGF-β/Smad/STAT signaling cascade. In the present study, KEGG pathway analysis revealed that IGF-1R and insulin were involved in the PI3K-Akt signaling pathway, suggesting a potential role for the insulin/IGF-1R axis in the progression of RA to ILD through this pathway. It was hypothesized that the insulin/IGF1R axis could be a promising therapeutic target for the treatment of RA-ILD. PPI analysis revealed that insulin is a central hub protein that interacts with IGF-1R, Shh-N, AR, TF, IL-7 and EpCAM. These interactions suggest that insulin and its network partners may collectively regulate immune and metabolic pathways in RA-ILD, potentially exacerbating disease progression. While no previous studies have directly linked insulin to RA-ILD, its network prominence and functional interactions generate a strong interest in investigating the modulation of the insulin pathway as a potential therapeutic strategy. Further validation is needed to confirm the potential involvement of insulin in RA-ILD.

Furthermore, among these 20 proteins, several have well-established roles in RA or pulmonary fibrosis. For example, IL-7 promotes T cell survival and Th17 differentiation (29), and its elevation has been linked to the exacerbation of IPF (32). MMP-7 serves as a predictive biomarker of disease progression and mediates extracellular matrix remodeling in IPF (33). IL-1F9 acts as a proinflammatory cytokine in lung disease by enhancing chemokine production and inflammatory cell recruitment (34). Eotaxin is associated with increased pulmonary infiltration of eosinophils and neutrophils, as well as the production of profibrotic cytokines contributing to pulmonary fibrosis (35). Both cathepsin L and tissue factor have been implicated in the pathogenesis of IPF and ILD (36,37). AR is elevated in inflammatory lung disease associated with RA (38), and EpCAM, TFPI and MIP-3b are upregulated in IPF (39-41). Shh-N promotes pulmonary fibrosis through the hedgehog signaling pathway (42), and Syndecan-1 shedding exacerbates the transition from inflammation to fibrosis by releasing heparan sulfate-bound growth factors (43).

Notably, CA9, EDA-A2, Gas1, CRTAM, IL-2Rb and IL-31 have emerged as novel candidates, with no prior studies linking them to RA-ILD or fibrotic diseases. However, chronic inflammation is a well-established precursor to fibrotic tissue remodeling (44), suggesting that these proteins may contribute indirectly to fibrosis through sustained inflammatory signaling. Among these novel targets, EDA-A2 activates the inflammatory responses through NF-κB signaling by binding to the EDA receptor (45). CRTAM promotes STAT signaling via STAT1 phosphorylation (46). IL-2Rb contributes to immune microenvironment disorder by disrupting the Th1/Th2 cell differentiation balance (47), and IL-31 drives inflammation primarily through JAK-STAT pathway activation (48). Gas1 may play a critical role in fibrotic diseases, including RA-ILD through the Gas1/Axl axis, analogous to the pro-fibrotic Gas6/Axl signaling pathway (49,50). In addition, CA9, a hypoxia-inducible protein, may promote hypoxic-associated fibrosis (51). These findings indicate the potential of these novel candidates to drive RA-ILD progression through their roles in inflammatory and hypoxic signaling, warranting further investigation.

However, the present study has several limitations. First, the cohort included a relatively small number of healthy controls (n=7), which limited the statistical power to detect biologically relevant differences between healthy individuals and patient groups. Second, the use of pooled samples for the antibody array analysis-implemented due to cost constraints associated with high-throughput proteomic screening-represents a significant methodological limitation. Third, the present study only validated insulin (the highest-degree hub protein) and IL-31 (a novel candidate) using independent methods due to budget limitations. Fourth, the functional roles of the identified protein signatures were not experimentally validated. Future studies should prioritize individual sample analysis, larger cohort sizes, and rigorous validation of all these candidate proteins as potential biomarkers. In addition, in vitro and in vivo studies are warranted to elucidate the functional relevance of these proteins, particularly those with novel associations, and to explore their potential as therapeutic targets for RA-ILD.

In conclusion, our study highlights the complex molecular interplay underlying RA-ILD pathogenesis. A total of 20 dysregulated proteins that collectively drive immune dysregulation and fibrotic progression were identified. These proteins are functionally enriched in critical pathways, including T cell differentiation, cytokine-cytokine receptor interactions (such as IL-7/IL-7R and IGF-1R/Insulin), and the PI3K-Akt signaling pathway. The PPI network identifies insulin as a central hub, interacting with multiple profibrotic mediators (IGF-1R, Shh-N and AR) and immune modulators (IL-7, EpCAM), suggesting its pivotal role in orchestrating profibrotic and inflammatory responses in RA-ILD. While several proteins (MMP-7, IL-1F9 and Cathepsin L) have established roles in pulmonary fibrosis, our identification of novel candidates points to additional mechanisms involving sustained inflammatory signaling and hypoxia-responsive pathways. These findings provide new insight into RA-ILD pathogenesis and suggest that targeting the insulin/IGF1R-PI3K-Akt axis may represent a promising therapeutic strategy to disrupt the immune-fibrotic cascade in this disease. Further validation and mechanistic studies are warranted to explore these potential targets.

 Acknowledgements

Not applicable.

 Availability of data and materials

The data generated in the present study may be found in zenodo database under accession number 15852072 or at the following URL: (https://zenodo.org/records/15852072).

 Authors' contributions

WC conducted all experiments and wrote the first draft of the manuscript. YZ and QY contributed to sample collection and processing. BY and QY conducted statistical analyses. GZ contributed to conception and design of the present study, and revised the manuscript. WC and GZ confirm the authenticity of all the raw data. All authors read and approved the final version of the manuscript.

 Ethics approval and consent to participate

Approval was obtained from the Research Ethics Committees of Beijing Haidian Hospital (approval no. 2024-006; Beijing, China) and Beijing Shunyi Hospital (approval no. 2023-k-021; Beijing, China). All participants provided informed consent to participate in the study.

 Patient consent for publication

Not applicable.

 Competing interests

The authors declare that they have no competing interests.

 References ZhangMYinJZhangXFactors associated with interstitial lung disease in patients with rheumatoid arthritis: A systematic review and meta-analysisPLoS One18e028619120233735217410.1371/journal.pone.0286191 FarquharHJBeckertLEdwardsALMattesonELFramptonCMAGanlyEYettonRThiessenRHaslettJBucknallDStampLKRheumatoid interstitial lung disease in Canterbury, Aotearoa New Zealand-A retrospective cohort studySemin Arthritis Rheum6415235920243815776110.1016/j.semarthrit.2023.152359 WangHFWangYYLiZYHePJLiuSLiQSThe prevalence and risk factors of rheumatoid arthritis-associated interstitial lung disease: A systematic review and meta-analysisAnn Med56233240620243854753710.1080/07853890.2024.2332406 JugePAWemeauLOttavianiSDesjeuxGZhuoJVannier-MoreauVFlipoRMCrestaniBDieudéPIncreased mortality in patients with RA-associated interstitial lung disease: Data from a French administrative healthcare databaseRMD Open9e00349120233805346110.1136/rmdopen-2023-003491 SullivanDIAschermanDPRheumatoid arthritis-associated interstitial lung disease (RA-ILD): Update on prevalence, risk factors, pathogenesis, and therapyCurr Rheumatol Rep2643144920243932042710.1007/s11926-024-01155-8 NarváezJMoving forward in Rheumatoid arthritis-associated interstitial lung disease screeningJ Clin Med13538520243933687310.3390/jcm13185385 KoduriGSolomonJJIdentification, monitoring, and management of rheumatoid arthritis-associated interstitial lung diseaseArthritis Rheumatol752067207720233739572510.1002/art.42640 LiuMJiangZLiuMNiHLiYFangJDuQDongYSLAMF1 as a novel molecule mediating the causal association between rheumatoid arthritis and interstitial lung disease: A Mendelian randomization study combined with transcriptomics and in vivo validationInt Immunopharmacol142 (Pt A)11308220243926030810.1016/j.intimp.2024.113082 ZhongXWangXXuLZhangJYuWJiLHuangJZhongXZhangJLongLAlterations in gut microbiota in Rheumatoid arthritis patients with interstitial lung Disease: A Comparative studyHum Immunol8611123920253998366310.1016/j.humimm.2025.111239 KimYYangHIKimKSEtiology and pathogenesis of rheumatoid arthritis-interstitial lung diseaseInt J Mol Sci241450920233783395710.3390/ijms241914509 ZhengWHuXZouMHuNSongWWangRLiuYHouQLiuYChenXChengZPlasma IL-36α and IL-36γ as potential biomarkers in interstitial lung disease associated with rheumatoid arthritis: A pilot study in the Chinese populationInflammation4628529620233604409910.1007/s10753-022-01733-x BesCKöybaşıGİçaçanOCYalçın MutluMYıldırımFAntifibrotic therapies in rheumatoid arthritis associated interstitial lung diseaseEur J Rheumatol917617920223515663610.5152/eurjrheum.2021.21102 LiangBZhangYKeDYanRJiangMNLiLZhangLXZhaoXGYuanGPXuBLiuXMSerum YKL-40 and Serum Krebs von den lungen-6 as potential predictive biomarkers for rheumatoid arthritis-associated interstitial lung diseaseImmunol Invest53989100020243890004510.1080/08820139.2024.2366966 LiuNFanXShaoYChenSWangTYaoTChenXResveratrol attenuates inflammation and fibrosis in rheumatoid arthritis-associated interstitial lung disease via the AKT/TMEM175 pathwayJ Transl Med2245720243874520410.1186/s12967-024-05228-1 LiuHYangYZhangJLiXBaricitinib improves pulmonary fibrosis in mice with rheumatoid arthritis-associated interstitial lung disease by inhibiting the Jak2/Stat3 signaling pathwayAdv Rheumatol634520233764110610.1186/s42358-023-00325-z VenetsanopoulouAINtinopoulouMPapagianniEKoletsosNVoulgariPVChrysanthopoulouANeutrophil extracellular traps as immunofibrotic mediators in RA-ILD; pilot evaluation of the nintedanib therapyFront Immunol15148059420243950754010.3389/fimmu.2024.1480594 ShodaTKotaniTMitsuhiroKYoshikawaAWadaYMakinoHOsugaKTakeuchiTThe therapeutic efficacy of abatacept for rheumatoid arthritis-associated interstitial lung disease: Insights from a 12-month trial using semi-quantitative chest high-resolution computed tomography imagingJ Clin Med13587120243940793110.3390/jcm13195871 WangSLiuMLiXZhangJWangFZhangCRodenARyuJHWarringtonKJSunJCanonical and noncanonical regulatory roles for JAK2 in the pathogenesis of rheumatoid arthritis-associated interstitial lung disease and idiopathic pulmonary fibrosisFASEB J36e2233620223552224310.1096/fj.202101436R Rodríguez PortalJABrito GarcíaNDíaz Del Campo FontechaPValenzuelaCOrtizAMNietoMAMena-VázquezNCano-JiménezECastellvíIAburtoMSER-SEPAR recommendations for the management of rheumatoid arthritis-related interstitial lung disease. Part 1: Epidemiology, risk factors and prognosisReumatol Clin (Engl Ed)1844345220223608519610.1016/j.reumae.2022.02.004 AlbrechtKStrangfeldAMarschallUCallhoffJInterstitial lung disease in rheumatoid arthritis: Incidence, prevalence and related drug prescriptions between 2007 and 2020RMD Open9e00277720233666983010.1136/rmdopen-2022-002777 HuangZWuTLuRZhouHZhangYHuangLGanYHeHPrevalence and clinical significance of anti-neutrophil cytoplasmic antibodies in rheumatoid arthritis-associated interstitial lung diseaseBMC Pulm Med2517720254022974210.1186/s12890-025-03644-7 AletahaDNeogiTSilmanAJFunovitsJFelsonDTBinghamCO IIIBirnbaumNSBurmesterGRBykerkVPCohenMD2010 Rheumatoid arthritis classification criteria: An American college of rheumatology/European league against rheumatism collaborative initiativeArthritis Rheum622569258120102069924110.1136/ard.2010.138461 RaghuGRemy-JardinMMyersJLRicheldiLRyersonCJLedererDJBehrJCottinVDanoffSKMorellFDiagnosis of idiopathic pulmonary fibrosis. An Official ATS/ERS/JRS/ALAT clinical practice guidelineAm J Respir Crit Care Med198e44e6820183016875310.1164/rccm.201807-1255ST KonoMNew insights into the metabolism of Th17 cellsImmunol Med46152420233632675410.1080/25785826.2022.2140503 ZhangYZhuJXiaoKLiuHDuKWuDZouQSingle-cell sequencing of PBMC characterizes the transformation of T cell subsets in the inflammatory microenvironment of RA-ILD. Research Square: https://doi.org/10.21203/rs.3.rs-3990097/v1. JeongEHongHLeeYAKimKSPotential rheumatoid arthritis-associated interstitial lung disease treatment and computational approach for future drug developmentInt J Mol Sci25268220243847392810.3390/ijms25052682 LiuCGeYImmune-related genes associated with interstitial lung disease in dermatomyositisInt J Gen Med175261527120243956378710.2147/IJGM.S490294 YangZHanRYinHLiJCaoYGuoRShengYSongLZhangYMechanism of Lycopodii herba for RA-ILD using integrated metabolomics and network pharmacologyAnal Biochem64811467920223536721710.1016/j.ab.2022.114679 WangCKongLKimSLeeSOhSJoSJangIKimTDThe role of IL-7 and IL-7R in cancer pathophysiology and immunotherapyInt J Mol Sci231041220223614232210.3390/ijms231810412 KnueverJWillenborgSDingXAkyüzMDPartridgeLNiessenCMBrüningJCEmingSAMyeloid cell-restricted insulin/IGF-1 receptor deficiency protects against skin inflammationJ Immunol1955296530820152651953010.4049/jimmunol.1501237 WuSMTsaiJJPanHCArbiserJLEliaLSheuMLAggravation of pulmonary fibrosis after knocking down the aryl hydrocarbon receptor in the insulin-like growth factor 1 receptor pathwayBr J Pharmacol1793430345120223508373810.1111/bph.15806 LiuZPengZLinHZhouKLiangLCaoJHuangZMeiJIdentifying potential drug targets for idiopathic pulmonary fibrosis: A mendelian randomization study based on the druggable genesRespir Res2521720243878323610.1186/s12931-024-02848-5 BauerYWhiteESde BernardSCornelissePLeconteIMorgantiARouxSNaylerOMMP-7 is a predictive biomarker of disease progression in patients with idiopathic pulmonary fibrosisERJ Open Res300074201620172843584310.1183/23120541.00074-2016 RamadasRAEwartSLMedoffBDLeVineAMInterleukin-1 family member 9 stimulates chemokine production and neutrophil influx in mouse lungsAm J Respir Cell Mol Biol4413414520102029954010.1165/rcmb.2009-0315OC HuauxFGharaee-KermaniMLiuTMorelVMcGarryBUllenbruchMKunkelSLWangJXingZPhanSHRole of Eotaxin-1 (CCL11) and CC chemokine receptor 3 (CCR3) in bleomycin-induced lung injury and fibrosisAm J Pathol1671485149620051631446410.1016/S0002-9440(10)61235-7 YuanLZouCGeWLiuYHuBWangJLinBLiYMaEA novel cathepsin L inhibitor prevents the progression of idiopathic pulmonary fibrosisBioorg Chem9410341720203174460010.1016/j.bioorg.2019.103417 NovelliFNeriTTavantiLArmaniCNoceCFalaschiFBartoliMLMartinoFPallaACeliAPaggiaroPProcoagulant, tissue factor-bearing microparticles in bronchoalveolar lavage of interstitial lung disease patients: An observational studyPLoS One9e9501320142477700010.1371/journal.pone.0095013 PooleJAThieleGMJanikeKNelsonAJDuryeeMJRentfroKEnglandBRRombergerDJCarringtonJMWangDCombined collagen-induced arthritis and organic dust-induced airway inflammation to model inflammatory lung disease in rheumatoid arthritisJ Bone Miner Res341733174320193099534410.1002/jbmr.3745 SchuligaMKanwalAReadJBloklandKECBurgessJKPrêleCMMutsaersSEGraingeCThomsonCJamesAA cGAS-dependent response links DNA damage and senescence in alveolar epithelial cells: A potential drug target in IPFAm J Physiol Lung Cell Mol Physiol321L859L87120213452491210.1152/ajplung.00574.2020 CellaGCiprianiATommasiniARampinESbaraiARocconiRMazzaroGLuzzattoGTissue factor pathway inhibitor (TFPI) antigen plasma level in patients with interstitial lung disease before and after heparin administrationSemin Thromb Hemost2345491997915641010.1055/s-2007-996069 RussoRCRyffelBThe chemokine system as a key regulator of pulmonary fibrosis: Converging pathways in human idiopathic pulmonary fibrosis (IPF) and the bleomycin-induced lung fibrosis model in miceCells13205820243976815010.3390/cells13242058 HeCHLvJMKhanGJDuanHWangWZhaiKFZouGAAisaHATotal flavonoid extract from Dracocephalum moldavica L. improves pulmonary fibrosis by reducing inflammation and inhibiting the hedgehog signaling pathwayPhytother Res372745275820233679439110.1002/ptr.7771 FengFWangLJLiJCChenTTLiuLRole of heparanase in ARDS through autophagy and exosome pathway (review)Front Pharmacol14120078220233736122710.3389/fphar.2023.1200782 LiYZhaoJYinYLiKZhangCZhengYThe role of IL-6 in fibrotic diseases: Molecular and cellular mechanismsInt J Biol Sci185405541420223614745910.7150/ijbs.75876 CaiZDengXJiaJWangDYuanGEctodysplasin A/Ectodysplasin A receptor system and their roles in multiple diseasesFront Physiol1278841120213493820510.3389/fphys.2021.788411 ZhengSYangBLiLChenMZhangLChiWShaoZMXiuBChiYWuJRTAM promotes antitumor immune response in triple negative breast cancer by enhancing CD8+ T cell infiltrationInt Immunopharmacol12911162520243835450910.1016/j.intimp.2024.111625 LiaoYKeBLongXXuJWuYUpregulated expression of IL2RB causes disorder of immune microenvironment in patients with Kawasaki diseaseBiomed Res Int2022211469920223592426910.1155/2022/2114699 Pastor BandeiraIde Almeida FranzoiAEMurillo WollmannGde Medeiros JuniorWLGNogueira BrandãoWSchatzmann PeronJPBeckerJNascimentoOJMMagno GonçalvesMVInterleukin-31 and soluble CD40L: New candidate serum biomarkers that predict therapeutic response in multiple sclerosisNeurol Sci436271627820223584919910.1007/s10072-022-06276-5 ChenCCChenCYYehCTLiuYTLeuYLChuangWYShihYHChouLFShiehTMWangTHCorylin attenuates CCl4-induced liver fibrosis in mice by regulating the GAS6/AXL signaling pathway in hepatic stellate cellsInt J Mol Sci241693620233806925910.3390/ijms242316936 FiebelerAParkJKMullerDNLindschauCMengelMMerkelSBanasBLuftFCHallerHGrowth arrest specific protein 6/Axl signaling in human inflammatory renal diseasesAm J Kidney Dis4328629520041475009410.1053/j.ajkd.2003.10.016 CaoZLiJHuWXuJZhaoFWangYQinSLiuMWangPDuanJNear-infrared imaging agent ABSi-148 alleviates CA IX-mediated hypoxic fibrosis in inflammation-cancer transitionAdv Healthc Mater18e240493520254009942010.1002/adhm.202404935

Histogram. Serum protein levels were significantly higher in patients with RA compared with healthy controls, and even more elevated in patients with RA-ILD compared with patients with RA. The bar graph illustrates the relative expression levels of 20 differentially expressed proteins among healthy controls, patients with RA and patients with RA-ILD. The data are presented as mean ± SD. The data from patients with RA and patients with RA-ILD represent pooled samples. *P<0.05, **P<0.01 and ***P<0.001. RA, rheumatoid arthritis; RA-ILD, RA-associated interstitial lung disease; TFPI, tissue factor pathway inhibitor; CA9, carbonic anhydrase IX; AR, amphiregulin; Shh-N, sonic hedgehog N-terminal; Gas1, growth arrest-specific gene 1; EDA-A2, ectodysplasin A2; TF, tissue factor.

Heatmap. This heatmap represents hierarchical clustering analysis based on the expression patterns of 20 proteins, showing distinct molecular signatures among the three groups. The color scale indicates normalized protein expression levels (blue: low; red: high). The data from patients with RA and patients with RA-ILD represent pooled samples. RA, rheumatoid arthritis; RA-ILD, RA-associated interstitial lung disease; TFPI, tissue factor pathway inhibitor; CA9, carbonic anhydrase IX; AR, amphiregulin; Shh-N, sonic hedgehog N-terminal; Gas1, growth arrest-specific gene 1; EDA-A2, ectodysplasin A2; TF, tissue factor.

PCA. The two-dimensional PCA score plot demonstrates clear clustering of healthy controls, patients with RA, and patients with RA-ILD based on the expression patterns of 20 differentially expressed proteins. Principal components 1 and 2 (PC1 and PC2) account for 90.5 and 3% of the total variance, respectively. The ellipses on the plot represent 95% confidence intervals for each group. The data from patients with RA and patients with RA-ILD are from pooled samples. PCA, principal component analysis; RA, rheumatoid arthritis; RA-ILD, RA-associated interstitial lung disease.

Bioinformatic analysis. The bubble plot displays significantly enriched Gene Ontology terms and Kyoto encyclopedia of genes and genomes pathways. The size of bubble represents the number of enriched proteins in each term, while the color intensity indicates the -log10 (P-value).

STRING-based protein-protein interaction network of the 20 differentially expressed proteins. Lines connecting two proteins represent a biological functional correlation. The thickness of the edges indicates the combined interaction scores, which exceeded the 0.7 confidence threshold. TFPI, tissue factor pathway inhibitor; CA9, carbonic anhydrase IX; Gas1, growth arrest-specific gene 1; EDA, ectodysplasin.

Showing serum concentrations of IL-31 and insulin measured by ELISA. The bar graph compares the mean serum levels of IL-31 and insulin with error bars representing the standard deviation of the measurements. **P<0.01 and ***P<0.001. RA, rheumatoid arthritis; RA-ILD, RA-associated interstitial lung disease.

Receiver Operating Characteristic curve analysis for IL-31 and insulin. The figure compares the diagnostic performance of IL-31 (AUC=80.31%) and insulin (AUC=78.50%) in distinguishing between non-RA-ILD and RA-ILD. Higher AUC values indicate better predictive accuracy, with IL-31 demonstrating slightly superior performance over insulin in this analysis. AUC, area under the curve; RA, rheumatoid arthritis; RA-ILD, RA-associated interstitial lung disease.

Correlation between serum IL-31 and insulin levels with disease severity markers in patients with RA-ILD. The clinical parameters used to assess disease severity in RA-ILD for the correlation analysis with IL-31 and insulin levels include percentage of FVC, DLCO and FEV, and SLS I. RA, rheumatoid arthritis; RA-ILD, RA-associated interstitial lung disease; FVC, forced vital capacity; DLCO, diffusing capacity of the lung for carbon monoxide; FEV1, forced expiratory volume in the first second; SLS I, scleroderma lung study I.

Clinical information of subjects for the antibody array assay.

Characteristics RA-ILD RA Healthy P-value (RA-ILD vs. RA)
n 40 40 7  
Age, years 62.5±9.5 59.2±6.9 60.1±2.1 0.074
Sex (F/M) 30/10 30/10 4/3  
KL-6 (U/ml) 647.0±221.4 178.6±42.2 181.6±55.0 <0.001
DAS28 4.2±1.5 3.3±7.9 - 0.007
CRP (mg/l) 2.3±2.2 1.0±1.6 - 0.004
VAS 34.8±31.9 11.9±16.7 - <0.001
ESR (mm/h) 45.4±22.3 31.6±19.1 - 0.004
D-dimer (mg/l) 1.3±0.7 0.4±0.4 0.16±0.1 <0.001
FVC (%) 60.3±13.4 93.2±9.4 - <0.001
DLCO (%) 57.2±15.7 91.9±7.3 - <0.001
FEV1 (%) 59.4±14.0 90.2±6.3 - <0.001
SLS I 2.6±1.1 0±0 - <0.001

RA, rheumatoid arthritis; RA-ILD, RA-associated interstitial lung disease; DAS28, disease activity score in 28 joints; CRP, C-reactive protein; VAS, visual analogue scale; ESR, erythrocyte sedimentation rate; FVC, forced vital capacity; FEV1, forced expiratory volume in the first second; DLCO, diffusing capacity of the lung for carbon monoxide; SLS I, scleroderma lung study I.

Clinical information of subjects for ELISA.

Characteristics RA-ILD RA Healthy P-value (RA-ILD vs. RA)
n 64 64 40  
Age, years 65.6±9.6 62.1±8.0 57.8±9.9 0.081
Sex (F/M) 40/24 51/13 23/17  
KL-6 (U/ml) 457.3±260.2 208.0±127.6 - <0.001
DAS28 4.1±1.5 3.2±1.3 - <0.001
CRP (mg/l) 2.2±2.0 0.9±1.5 - <0.001
VAS 32.4±29.1 12.9±16.5 - <0.001
ESR (mm/h) 43.3±19.9 33.1±20.9 - <0.001
D-dimer (mg/l) 1.2±0.8 0.4±0.4 - <0.001
FVC (%) 63.3±13.5 92.1±7.7 - <0.001
DLCO (%) 61.2±15.1 91.5±6.8 - <0.001
FEV1 (%) 62.7±13.2 90.9±6.0 - <0.001
SLS I 6.5±5.6 0±0 - <0.001

RA, rheumatoid arthritis; RA-ILD, RA-associated interstitial lung disease; DAS28, Disease activity score in 28 joints; CRP, C-reactive protein; VAS, visual analogue scale; ESR, erythrocyte sedimentation rate; FVC, forced vital capacity; FEV1, forced expiratory volume in the first second; DLCO, diffusing capacity of the lung for carbon monoxide; SLS I, scleroderma lung study I.

Information on the 20 proteins.

Protein Uniport ID RA/Con RA-ILD/RA Known functions Novelty in RA-ILD
CA9 Q16790 1.4 1.8 Response to hypoxia Induces hypoxic fibrosis
IL-1F9 Q9NZH8 4.5 1.7 Interleukin-1 receptor binding A proinflammatory cytokine in the lung disease
EDA-A2 Q92838 3.5 1.5 A ligand activating the DEATH-domain containing receptors EDAR and EDA2R Activates the inflammatory pathway NF-κB signaling
Gas1 P54826 4.4 1.8 Specific growth arrest protein involved in growth suppression Gas1/Axl pathway
IL-2Rb P14784 2.5 1.8 Receptor for interleukin-2 Contributes to immune microenvironment disorder
IL-7 P13232 2.1 1.4 Regulates B cell proliferation Exacerbates IPF
AR P15514 1.7 2.3 Ligand of the EGF receptor/EGFR Involves into inflammatory lung disease
Shh-N Q15465 3.0 2.6 Displays an autoproteolysis and a cholesterol transferase activity Promotes pulmonary fibrosis
IGF-1R P08069 3.9 2.2 Receptor tyrosine kinase which mediates actions of insulin-like growth factor 1 Promotes IPF
MMP-7 P09237 4.5 1.6 Activates pro-collagenase A mediator of extracellular matrix remodeling in IPF
Insulin P01308 4.1 1.8 Decreases blood glucose concentration Participates in PI3K-Akt signaling
Eotaxin P51671 7.0 2.0 Promotes the accumulation of eosinophils Produces profibrotic cytokines contributing to pulmonary fibrosis
EpCAM P16422 14.0 2.6 Plays a role in embryonic stem cells proliferation and differentiation Involves into IPF
TFPI P10646 11.3 1.6 Inhibits VIIa/tissue factor activity Involves into IPF
MIP-3b Q99731 5.9 2.2 Chemokine activity Involves into IPF
CRTAM O95727 8.8 2.0 Mediates heterophilic cell-cell adhesion Activates STAT signaling
IL-31 Q6EBC2 18.3 2.2 Activates STAT3 Activates JAK-STAT signaling
TF P13726 57.2 2.3 Initiates blood coagulation Involves into IPF
Syndecan-1 P18827 1.9 1.8 Cell surface proteoglycan Exacerbates inflammation-to-fibrosis transitions in pulmonary fibrosis
Cathepsin L P07711 4.7 2.7 Regulates CD4+ T cell positive selection Involves into IPF

RA, rheumatoid arthritis; RA-ILD, RA-associated interstitial lung disease; IPF, idiopathic pulmonary fibrosis; TFPI, tissue factor pathway inhibitor; CA9, carbonic anhydrase IX.