The Role of IL-33 and RUNX2 in the Pathogenesis of Idiopathic Pulmonary Fibrosis

The Role of IL-33 and RUNX2 in the Pathogenesis of Idiopathic Pulmonary 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 The Role of IL-33 and RUNX2 in the Pathogenesis of Idiopathic Pulmonary Fibrosis Shuai Liu, Lixin Wang, Yinyan Yue, Zhao Zhang, Fang Li, Dongdong Wu, and 2 more This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-7626166/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: Idiopathic Pulmonary Fibrosis (IPF) is a chronic, progressive lung disease characterized by abnormal fibrosis of the lung tissue, leading to a gradual decline in lung function. The exact cause of this disease is not well understood, hence the term "idiopathic." Although the incidence of IPF is not high, it significantly impacts the quality of life and life expectancy of patients. The molecular mechanisms underlying IPF, including immune responses and fibroblast activation, remain insufficiently understood. This study explores the roles of interleukin-33 (IL-33) and RUNX2 in the pathogenesis of pulmonary fibrosis, aiming to identify novel therapeutic targets. Methods: A comprehensive approach combining bioinformatics analysis, molecular docking, single-cell RNA sequencing, and both in vitro and in vivo experimental models was used to investigate the interaction between IL-33 and RUNX2 in IPF. Gene expression analysis, KEGG pathway enrichment, and co-immunoprecipitation assays were performed to validate. In vitro inflammation and apoptosis assays were conducted using BEAS-2B lung epithelial cells, and in vivo fibrosis models were established in mice. Results: Bioinformatics analysis revealed the upregulation of IL-33 and RUNX2 in pulmonary fibrosis, with a significant correlation between these two molecules. In vitro , RUNX2 overexpression exacerbated BLM-induced inflammation and apoptosis, while knockdown of RUNX2 attenuated these effects. Co-immunoprecipitation confirmed the physical interaction between IL-33 and RUNX2. In vivo , IL-33 knockout mice exhibited exacerbated fibrosis, highlighting a complex dual role of IL-33 in the disease process. Conclusions: This study identifies the RUNX2-IL-33 axis as a key mediator in pulmonary fibrosis. Our findings provide new insights into the molecular mechanisms of IPF and suggest that targeting the RUNX2-IL-33 interaction could represent a promising therapeutic strategy for fibrosis-related diseases. Pulmonary fibrosis Innate lymphoid cells (ILC2s) Interleukin-33 ST2 Runt-related transcription factor 2 Figures Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 Introduction Idiopathic pulmonary fibrosis (IPF) is a progressive, irreversible lung disease characterized by excessive extracellular matrix (ECM) deposition and structural damage to lung tissue, ultimately leading to impaired pulmonary function 1 . The median survival of IPF is 3–5 years after diagnosis 2 . The pathophysiology of IPF is multifactorial, involving chronic inflammation, fibroblast activation, and epithelial-mesenchymal transition (EMT), which together contribute to excessive collagen deposition and fibrosis 3 . Current antifibrotic therapies, such as pirfenidone and nintedanib, are only effective in slowing disease progression, without providing curative or reversing effects 4 . This underscores the urgent need for novel therapeutic targets and strategies. Over the past few years, the role of immune-mediated mechanisms in the pathogenesis of pulmonary fibrosis has garnered increasing attention 5 . Interleukin-33 (IL-33), a member of the IL-1 cytokine family, plays a pivotal role in fibrosis. As an “alarmin,” IL-33 is released upon cell injury or stress and exerts its biological effects via the ST2 receptor (also known as IL1RL1) 6 . The ST2 receptor exists in two forms: a membrane-bound variant (ST2L) that mediates intracellular signaling, and a soluble form (sST2) that acts as a decoy receptor 7 , 8 . The IL-33/ST2 signaling pathway regulates immune responses, including type 2 inflammation, by activating type 2 innate lymphoid cells (ILC2s), eosinophils, and Th2 cells. ILC2s are tissue-resident, non-T, non-B innate immune cells that play a critical role in type 2 immunity 9 . Upon IL-33 activation, ILC2s rapidly produce Th2 cytokines, such as IL-5 and IL-13, which promote eosinophilic inflammation, airway remodeling, and fibroblast activation. Increasing evidence suggests that ILC2s are key regulators of fibrosis in various tissues, including the lung. In pulmonary fibrosis, the expansion of ILC2s correlates with increased ECM deposition and fibroblast proliferation. However, the specific downstream mediators linking ILC2 activation to fibroblast function remain largely unexplored. RUNX2 (Runt-related transcription factor 2) has also emerged as a critical regulator in the fibrotic process. Recent studies indicate that RUNX2 plays an important role in the conversion of alveolar fibroblasts to pathological fibroblasts, which are the key source of fibrosis 10 . In IPF patients, the expression of RUNX2 is significantly elevated in pathological fibroblasts, and its inhibition reduces ECM deposition and fibroblast activation. These findings suggest that RUNX2 could be a potential therapeutic target for alleviating fibrosis. WNT1-inducible signaling pathway protein 1 (WISP1), an extracellular matrix protein belonging to the CCN family, is involved in tissue remodeling, fibrosis, and tumorigenesis 11 , 12 . WISP1 is upregulated in fibrotic lung tissue and has been shown to promote fibroblast proliferation, myofibroblast differentiation, and collagen deposition 13 . It interacts with integrins and receptor tyrosine kinases to activate pro-fibrotic signaling pathways, such as TGF-β/SMAD and PI3K/AKT 14,15 . While WISP1 is implicated in pulmonary fibrosis, the regulatory mechanisms controlling its expression remain unclear. In this study, we integrate bioinformatics analysis, molecular docking, single-cell RNA sequencing, and both in vitro and in vivo experimental models to comprehensively explore the roles of IL-33 and RUNX2 in the pathogenesis of pulmonary fibrosis. Our in vitro results demonstrate that overexpression of RUNX2 significantly exacerbates BLM-induced inflammation and apoptosis in BEAS-2B cells, whereas knockdown of RUNX2 attenuates these effects. However, in vivo experiments reveal a discrepancy with in vitro findings: in IL-33 knockout (KO) mice, lung fibrosis is exacerbated, suggesting a complex, dual role for IL-33 in pulmonary fibrosis 16 . This observation suggests that the mechanisms by which IL-33 and RUNX2 contribute to fibrosis are more complex than previously recognized, necessitating further investigation. By delving into the interactions between IL-33, RUNX2, and ILC2s, this study aims to provide new insights into the molecular mechanisms of pulmonary fibrosis and identify potential therapeutic targets for this debilitating disease. Materials and Methods Cell Culture BEAS-2B human normal lung epithelial cells were obtained from the Shanghai Institute of Life Sciences (Shanghai, China) and cultured in Dulbecco's Modified Eagle Medium (DMEM, Gibco) supplemented with 10% fetal bovine serum (FBS, Gibco) and 1% penicillin-streptomycin (Gibco) at 37°C in a 5% CO₂ incubator. For inflammation induction, cells were treated with Bleomycin (BLM) at a final concentration of 15 µg/mL for 24 hours. Plasmid Transfection and Knockdown RUNX2 overexpression was achieved by transfecting BEAS-2B cells with a plasmid expressing RUNX2 (OriGene) using Lipofectamine 3000 reagent (Thermo Fisher) according to the manufacturer’s instructions. RUNX2 knockdown was performed using small interfering RNA (siRNA) targeting RUNX2 (Santa Cruz Biotechnology), transfected into BEAS-2B cells using Lipofectamine RNAiMAX (Thermo Fisher). Co-Culture System For co-culture experiments, BEAS-2B cells transfected with RUNX2 plasmid or siRNA were co-cultured with ILC2 cells under inflammatory conditions induced by BLM. The supernatants were collected for cytokine analysis, and cells were harvested for subsequent assays. UMAP Analysis The single-cell RNA sequencing dataset GSE213017 was obtained from the publicly accessible Gene Expression Omnibus (GEO) database. UMAP (Uniform Manifold Approximation and Projection) was performed using the Seurat package in R to visualize the distribution and clustering of cell types based on gene expression profiles. Molecular Docking Molecular docking simulations were performed using AlphaFold3 to predict the potential binding sites and interactions between IL-33 and RUNX2. Western Blotting (WB) Cells were lysed using RIPA buffer (Thermo Fisher) supplemented with protease inhibitors (Sigma-Aldrich). Protein concentration was determined using the BCA Protein Assay Kit (Thermo Fisher). Equal amounts of protein (30 µg) were separated by SDS-PAGE, transferred to PVDF membranes (Millipore), and incubated with primary antibodies against IL-33 (#88513, CST), RUNX2 (#12556, CST), ST2 (12-9338-42, ThermoFisher), α-SMA (sc-53015, SantaCruz), and COL1A1 (Abcam). Protein bands were visualized using an ECL detection system (Bio-Rad). Enzyme-Linked Immunosorbent Assay (ELISA) IL-33, IL-1β, IL-6, TNF-α, and WISP1 levels in cell culture supernatants or lung tissue homogenates were measured using ELISA kits (R&D Systems) according to the manufacturer’s instructions. Absorbance was measured at 450 nm using a microplate reader (BioTek). Flow Cytometry Apoptosis was measured using the Annexin V-FITC/PI Apoptosis Detection Kit (BD Biosciences) according to the manufacturer’s protocol. Briefly, cells were harvested, stained with Annexin V and propidium iodide (PI), and analyzed by flow cytometry (BD Accuri C6). The apoptotic cell population was quantified as the percentage of PI-positive cells. Animal Models All animal experiments were conducted according to the guidelines of the Institutional Animal Care and Use Committee (IACUC) of The First Affiliated Hospital of Zhengzhou University (Approval No. 2025-KY-0823-001). C57BL/6N mice (6–8 weeks old, 213) were purchased from Vital River. Pulmonary fibrosis was induced using the cecal ligation and puncture (CLP) method followed by mechanical ventilation (MTV, 4 hours, 10 mL/kg) to exacerbate lung injury and fibrosis. Mice were sacrificed after 3 days for histological analysis. Histological Analysis Lung tissues were fixed in 4% paraformaldehyde, embedded in paraffin, and sectioned. Hematoxylin and eosin (H&E) and Masson’s trichrome staining were performed to assess lung injury and fibrosis. Statistical Analysis Data are expressed as mean ± standard deviation (SD). Statistical significance was assessed using one-way ANOVA followed by Tukey’s post hoc test. A p-value of less than 0.05 was considered statistically significant. All statistical analyses were performed using GraphPad Prism (version 8.0). Results 1. Role of ILC2 Cells in Pulmonary Fibrosis Group 2 innate lymphoid cells (ILC2s) are widely distributed in the lungs, particularly enriched in pulmonary tissues and the submucosa of the respiratory tract. These cells play a crucial role in maintaining pulmonary immune homeostasis, mediating inflammatory responses, and contributing to the process of fibrosis 17-19 . Data analysis from the GEO database (GSE267904) revealed differential gene expression between fibrotic lung tissue and normal lung tissue (Fig. 1A). KEGG pathway enrichment analysis highlighted significant upregulation of the cytokine-cytokine receptor interaction pathway in the context of lung fibrosis (Fig. 1B). Among the differentially expressed genes, we identified five genes associated with ILC2 cells that exhibited the most significant changes in expression. By intersecting these genes with known cytokine-cytokine receptor interaction genes, two key genes were identified: IL33 and IL13 (Fig. 1C). Notably, IL33 showed a stronger association with ILC2 cells. These findings indicate that ILC2 cells, along with their associated genes, may play a crucial role in the progression of lung fibrosis, particularly highlighting the strong correlation between IL-33 and ILC2 cells. This underscores the potential of IL33 as a critical player in the pathogenesis of pulmonary fibrosis. Further investigation into the mechanisms of IL33 in lung fibrosis and its potential as a therapeutic target is warranted. 2. Interaction between IL33 and RUNX2 in Pulmonary Fibrosis Based on our previous findings, we further examined genes associated with IL33 in the differentially expressed genes (DEGs) of IPF. This analysis led to the identification of five key genes: FOXA2, GATA2, JUN, MAFK, and RUNX2 (Fig. 2A). Volcano plot analysis revealed that among these genes, only RUNX2 showed a significant upregulation in the context of pulmonary fibrosis (Fig. 2B). Molecular docking predictions using AlphaFold3 indicated potential binding sites between IL33 and RUNX2, specifically at IL33’s GLY174 and RUNX2’s SER196, as well as IL33’s ASP175 and RUNX2’s LYS218 (Fig. 2C). To further explore the specific roles of IL33 and RUNX2 in pulmonary fibrosis, we conducted UMAP dimensionality reduction analysis of single-cell sequencing dataset GSE213017. This technique projects high-dimensional data into two or three-dimensional space, clustering similar samples together (Fig. 2D). The results revealed co-localization of RUNX2 (Fig. 2E) and IL33 (Fig. 2F) in lung epithelial cells (Fig. 2G). Further bubble plot analysis demonstrated that IL33 is primarily expressed in fibroblasts, epithelial cells, and vascular endothelial cells in the lung (Fig. 2H), whereas RUNX2 is expressed across various lung cell types, with the highest expression observed in lung epithelial cells (Fig. 2I). To validate the interaction between IL33 and RUNX2, we performed co-immunoprecipitation (Co-IP) assays on BEAS-2B lung epithelial cells. After crosslinking, immunoprecipitation targeting either RUNX2 or IL33 confirmed their physical interaction. Specifically, RUNX2 immunoprecipitation resulted in the detection of IL33 in the eluate (Fig. 2J), and vice versa, immunoprecipitating IL33 allowed for the detection of RUNX2 (Fig. 2K). These results validate the molecular docking and UMAP co-localization findings, demonstrating a direct physical interaction between RUNX2 and IL33 in BEAS-2B cells. In summary, this study emphasizes the interaction between IL-33 and RUNX2 in pulmonary fibrosis, offering new insights into the molecular mechanisms underlying the disease. Further investigations are warranted to explore the specific roles of IL33 and RUNX2 in the pathogenesis of pulmonary fibrosis. 3. RUNX2-IL-33 Axis Induces Apoptosis and Inflammatory Response in BEAS-2B Cells To investigate the role of the RUNX2-IL-33 axis in lung epithelial cells, we first confirmed their interaction in BEAS-2B cells. Upon BLM treatment, a significant upregulation of RUNX2 expression was observed (Fig. 3A). In parallel, the expression levels of classic inflammatory cytokines, including IL-1β, IL-6, and TNF-α, were also elevated, and IL-33 expression significantly increased (Fig. 3B). These findings suggest that the interaction between RUNX2 and IL-33 is activated under inflammatory conditions. Further, to explore the functional implications of RUNX2 overexpression, we transfected BEAS-2B cells with a RUNX2 overexpression plasmid and subjected them to BLM-induced inflammation (Fig. 3C). Flow cytometry analysis revealed a significant increase in PI-positive cells in the RUNX2-overexpressing group, indicating enhanced apoptosis (Fig. 3D). Additionally, compared to the control group (BLM-treated cells transfected with the NC plasmid), cells overexpressing RUNX2 exhibited further increases in IL-1β, IL-6, TNF-α, and IL-33 levels (Fig. 3E), suggesting that RUNX2 overexpression exacerbates both the inflammatory response and apoptosis. To verify that these changes were indeed driven by RUNX2, we knocked down RUNX2 expression in BEAS-2B cells and induced inflammation with BLM (Fig. 3F). Knockdown of RUNX2 led to a significant reduction in the proportion of PI-positive cells (Fig. 3G), and the expression of IL-1β, IL-6, and TNF-α was notably decreased (Fig. 3H). These results confirm that RUNX2 mediates BLM-induced inflammation and apoptosis in lung epithelial cells. In summary, our findings demonstrate that the RUNX2-IL-33 axis contributes to the induction of apoptosis and inflammation in BEAS-2B cells, providing insight into its role in the fibrotic process in the lung. Further studies are warranted to explore the downstream targets of this signaling axis in the context of lung fibrosis. 4. RUNX2 Modulates ST2 Activation in ILC2 via IL-33 Previous studies have demonstrated that IL-33 exerts its biological functions by forming a heterodimeric complex with its receptor ST2 and the co-receptor IL-1RAcP 20 , with ST2 being expressed in ILC2 cells (Fig. 4A). To investigate the role of RUNX2 in regulating ST2 activation, BEAS-2B cells overexpressing RUNX2 were co-cultured with ILC2 cells under inflammatory conditions induced by BLM. WB analysis revealed an upregulation of ST2 expression in ILC2 cells (Fig. 4B). Furthermore, when IL-33 signaling was neutralized using the anti-IL-33 antibody (Tozorakimab) under the same conditions, co-culture with ILC2 resulted in a significant reduction in ST2 expression (Fig. 4C). These findings suggest that RUNX2 in lung epithelial cells can regulate the secretion of IL-33, thereby modulating ST2 activation in ILC2 cells across cellular interactions. Reducing the levels of IL-33 in the microenvironment effectively inhibits the activation of ST2. This section reveals the mechanism by which RUNX2 regulates ST2 activation in ILC2 cells via IL-33, further elucidating the interaction between lung epithelial cells and ILC2 cells and their role in the inflammatory response. Future studies will focus on investigating the role of this mechanism in in vivo models of lung fibrosis. 5. IL-33 Drives Pulmonary Fibrosis in Sepsis Model To investigate the role of IL-33 in mediating pulmonary fibrosis under inflammatory conditions in vivo , we first established a bacterial infection-induced inflammatory model in mice via cecal ligation and puncture (CLP) to induce lung injury 21 . Given the clinical evidence suggesting that patients with prolonged mechanical ventilation often exhibit more severe pulmonary fibrosis, we subjected the mice to mechanical ventilation (MTV, 4 hours, 10 mL/kg) following sepsis-induced lung injury to further induce fibrosis 22 . All groups were sacrificed after 3 days of treatment for histological analysis using H&E and Masson’s trichrome staining. Normal mice showed a loose and porous alveolar structure, while CLP-treated mice displayed an increase in lung parenchyma, with Masson’s trichrome staining revealing elevated collagen deposition (blue). In the CLP+MTV group, alveolar spaces were further compressed, and collagen accumulation was significantly increased (Fig. 5A). These results demonstrate that CLP and CLP+MTV treatments progressively transition the lung tissue from a healthy to a fibrotic state, with the latter showing more severe fibrosis. We also observed a progressive increase in total cell and neutrophil counts in the bronchoalveolar lavage fluid (BALF) of treated mice, which followed the order of sham, CLP, and CLP+MTV treatment groups (Fig. 5B). This suggests a positive correlation between the degree of pulmonary fibrosis and immune cell recruitment. α-SMA, a cytoskeletal protein expressed in smooth muscle cells and certain types of fibroblasts, is a marker of myofibroblast differentiation, and COL1A1, which encodes the α1 chain of type I collagen, is a major component of collagen in fibrotic tissue. We assessed the expression levels of RUNX2, ST2, α-SMA, and COL1A1 via WB analysis. In the sham group, baseline expression levels of these proteins were low, while in the CLP-induced lung injury model, their expression was significantly upregulated. Further mechanical ventilation treatment led to a further increase in RUNX2, ST2, α-SMA, and COL1A1 expression (Fig. 5C), suggesting a positive correlation between RUNX2 and ST2 expression with fibrotic markers. The levels of classical inflammatory cytokines IL-1β, IL-6, and TNF-α also increased with the progression of fibrosis, indicating a positive correlation between inflammation and fibrosis (Fig. 5D). This further highlights the role of inflammatory factors, including IL-33, in the fibrotic process (Fig. 5E). Additionally, WISP1, a protein secreted by ILC2 cells that can act on fibroblasts, showed an increase in parallel with inflammation (Fig. 5F), suggesting a potential mechanism through which IL-33 mediates pulmonary fibrosis via ILC2. Next, we employed IL-33 knockout (KO) mice to assess the role of IL-33 in sepsis and mechanical ventilation-induced pulmonary fibrosis. Contrary to our expectations, in IL-33 KO mice, the lung parenchyma still increased, and Masson’s trichrome staining revealed even greater fibrosis compared to wild-type (WT) mice (Fig. 5G). Recruitment of lymphocytes in the BALF was not reduced, and neutrophils made up an even larger proportion of the total cells (Fig. 5H). Although the upregulation of RUNX2, ST2, α-SMA, and COL1A1 in response to CLP and CLP+MTV treatment was less pronounced in the IL-33 KO mice, it remained significant (Fig. 5I). However, the levels of classic immune factors IL-1β, IL-6, and TNF-α in IL-33 KO mice were almost identical to those in WT mice (Fig. 5J). Similarly, WISP1 secretion in IL-33 KO mice did not show a decrease compared to WT mice (Fig. 5K). This section presents in vivo findings that contrast with the cell culture results, indicating that IL-33 may have a mitigatory role in sepsis-induced pulmonary fibrosis. These findings suggest that, in vivo , the interaction between IL-33 and other immune and fibrotic factors forms a complex network, which is likely influenced by the broader tissue context, differing from the more controlled conditions of in vitro models. Future studies should refine experimental models and explore alternative mechanisms to better understand the role of IL-33 in pulmonary fibrosis, particularly its dual function under different pathological conditions. Discussion In this study, we employed a comprehensive approach combining bioinformatics analysis, molecular docking, and both in vitro and in vivo experimental models to systematically investigate the role of ILC2 cells, IL-33, and RUNX2 in the pathogenesis of pulmonary fibrosis and to explore the underlying molecular mechanisms. Bioinformatics analysis revealed an upregulation of IL-33 and RUNX2 in pulmonary fibrosis, suggesting their interaction within lung epithelial cells. Further in vitro experiments confirmed that this interaction could mediate inflammatory responses and cell apoptosis in cultured cells. However, discrepancies were observed between the results from in vivo and in vitro experiments in the animal models, IL-33 KO did not effectively block the inflammatory response. Although the upregulation of fibroblast markers under inflammatory stress was slightly reduced at the molecular level, collagen deposition increased. Previous studies have demonstrated that IL-33-deficient mice exhibit heightened susceptibility to severe lung injury, as evidenced by increased levels of inflammatory cytokines and immune cell infiltration 16 . Similar observations were made in our experiments, and we hypothesize that the absence of IL-33, a key “early warning” molecule, impairs the initial recruitment of immune cells, thus hindering the early immune response to injury and infection. This delay in immune response exacerbates tissue damage, leading to the activation of other inflammatory factors that drive a more pronounced fibrotic response as a compensatory mechanism. In addition, several factors may account for the discrepancies observed between in vitro and in vivo results. First, the complex immune environment in vivo may alter the effects of IL-33. While IL-33 mediates inflammation and fibrosis in a simplified signaling context, its role in the presence of other potent inflammatory and fibrotic factors could lead to competitive inhibition or "dragging effects," where other fibrotic mediators might become more prominent after IL-33 deletion. Second, IL-33 may function within a feedback mechanism that regulates inflammation. As an inflammatory cytokine, IL-33 likely initiates downstream feedback processes that limit excessive inflammation to prevent further tissue damage. In the absence of IL-33, this negative regulatory mechanism may be disrupted, resulting in exacerbated fibrosis. Third, the splice variants of the ST2 receptor, a known downstream receptor for IL-33, may also play a crucial role. It is said that the soluble decoy receptor ST2 can sequester excess IL-33, and we observed that overexpression of IL-33 upregulates ST2 in BEAS-2B cells. It suggests a possible mechanism that IL-33 expression may influence the splicing of ST2, thereby modulating the balance between ST2 activation and IL-33 neutralization, which process still needs to be further verified in the future. Although IL-33 is the only known ligand for ST2, it is possible that, in the absence of IL-33, other factors could activate membrane-bound ST2. Bioinformatics analysis using gene expression data from the GEO database identified IL33 and IL13 as key genes associated with ILC2 cells in pulmonary fibrosis. KEGG pathway analysis further revealed a strong association between cytokine-cytokine receptor interactions and fibrosis progression. Based on these findings, we focused on the interaction between IL33 and RUNX2, and verified their colocalization and physical interaction in lung epithelial cells through immunoprecipitation experiments. In vitro results demonstrated that overexpression of RUNX2 exacerbated BLM-induced inflammation and apoptosis in BEAS-2B cells, while RUNX2 knockdown significantly reduced these effects. Moreover, we found that RUNX2 regulates IL-33 secretion, which in turn activates ST2 in ILC2 cells, thereby promoting fibrosis. Through in vivo sepsis models, we observed that IL-33 might have a bidirectional regulatory effect in mediating inflammation-induced pulmonary fibrosis. Our findings are in line with current understanding of pulmonary fibrosis, while also extending it. Previous studies have established the importance of IL-33 and ILC2 cells in fibrosis, particularly in their roles in promoting inflammation and tissue remodeling. However, our study introduces RUNX2 as a critical regulator of IL-33 secretion and its interaction with lung epithelial cells, providing new insights into the molecular mechanisms underlying fibrosis. Nonetheless, our study has several limitations. First, we were unable to reconcile the results between in vitro and in vivo experiments. Future studies should optimize experimental models or delve deeper into understanding the role of IL-33 in the fibrosis process from a broader perspective. Additionally, while this study primarily focused on the role of ILC2 cells, we did not extensively explore the contributions of other immune cells, such as macrophages or T cells, which are also closely linked to fibrosis. Future research should further investigate the interactions among these cell types and their roles in the fibrotic process. The results of this study encourage a reevaluation of pulmonary fibrosis as a disease driven by dysregulated molecular signaling and immune cell interactions. Identifying the RUNX2-IL-33 axis as a key driver of fibrosis suggests that targeting this pathway could represent a promising therapeutic strategy. Furthermore, our findings underscore the importance of considering intercellular interactions and cross-cell signaling in fibrosis development, which may lead to more effective and targeted therapeutic approaches. Beyond pulmonary fibrosis, fibrosis is a common pathological feature of many chronic inflammatory diseases affecting organs such as the liver, heart, and kidneys. The role of IL-33 and RUNX2 in pulmonary fibrosis suggests that similar mechanisms may be at play in other fibrotic diseases. For instance, in cardiac fibrosis, IL-33 has been shown to promote fibroblast activation and collagen deposition 23 , while RUNX2 is known to regulate osteoblastic differentiation and extracellular matrix remodeling 24 , 25 . Thus, therapeutic strategies such as targeting the RUNX2-IL-33 axis or modulating ST2 activation may be applicable to other fibrotic diseases. In conclusion, this study provides valuable insights into the potential molecular and cellular mechanisms of pulmonary fibrosis and offers new directions for future therapeutic strategies. Declarations Disclosure statement/ Competing interests The authors report no declarations of interest. Ethics Approval and Consent to Participate All animal experiments were conducted according to the guidelines of the Institutional Animal Care and Use Committee (IACUC) of The First Affiliated Hospital of Zhengzhou University (Approval No. 2025-KY-0823-001) and were performed in accordance with the National Institutes of Health Guidelines for the Care and Use of Laboratory Animals. Consent for publication Not applicable. Data availability The data analyzed during the current study are available from the corresponding author on reasonable request. Funding Study funded financially by National Natural Science Foundation of China (82100092), Natural Science Foundation of Henan Province of China (232300420255), Henan Province Medical Science and Technology Research Plan (LHGJ20200380, LHGJ20200341) Authors' contributions Shuai Liu and Qiuge Wu conceived, designed and supervised the whole study; Shuai Liu, Lixin Wang, Yinyan Yue, Zhao Zhang, Fang Li, Dongdong Wu and Hui Zhang operated the experiment, performed the analyses and audited the data; Shuai Liu wrote the manuscript; Qiuge Wu revised the manuscript. All authors provided critical comments and approved the final manuscript. Acknowledgments Not applicable. References Bridges, J. P. et al. Progressive lung fibrosis: reprogramming a genetically vulnerable bronchoalveolar epithelium. J Clin Invest 135 , doi:10.1172/jci183836 (2025). Raghu, G. et al. Diagnosis of Idiopathic Pulmonary Fibrosis. An Official ATS/ERS/JRS/ALAT Clinical Practice Guideline. 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Integrin β3-PKM2 pathway-mediated aerobic glycolysis contributes to mechanical ventilation-induced pulmonary fibrosis. Theranostics 12 , 6057-6068, doi:10.7150/thno.72328 (2022). Chen, W. Y. et al. Group 2 innate lymphoid cells contribute to IL-33-mediated alleviation of cardiac fibrosis. Theranostics 11 , 2594-2611, doi:10.7150/thno.51648 (2021). Dong, Q. et al. Nuclear farnesoid X receptor protects against bone loss by driving osteoblast differentiation through stabilizing RUNX2. Bone research 13 , 20, doi:10.1038/s41413-024-00394-w (2025). Zhang, Y. et al. RUNX2 Phase Separation Mediates Long-Range Regulation Between Osteoporosis-Susceptibility Variant and XCR1 to Promote Osteoblast Differentiation. Adv Sci (Weinh) 12 , e2413561, doi:10.1002/advs.202413561 (2025). Additional Declarations No competing interests reported. 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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-7626166","acceptedTermsAndConditions":true,"allowDirectSubmit":true,"archivedVersions":[],"articleType":"Research Article","associatedPublications":[],"authors":[{"id":528162004,"identity":"34ee33ad-afd2-48ec-90c0-84f81ab0e49b","order_by":0,"name":"Shuai Liu","email":"","orcid":"","institution":"Department of Pulmonary and Critical Care Medicine, The First Affiliated Hospital of Zhengzhou University","correspondingAuthor":false,"prefix":"","firstName":"Shuai","middleName":"","lastName":"Liu","suffix":""},{"id":528162005,"identity":"e22fc754-3ac0-4568-8eb6-916c86feaa6e","order_by":1,"name":"Lixin 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1","display":"","copyAsset":false,"role":"figure","size":256716,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eGene expression analysis and pathway enrichment in pulmonary fibrosis.\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e(A) Volcano plot depicting the differential gene expression analysis in pulmonary fibrosis based on the GSE267904 dataset. The x-axis represents the log2 fold change in gene expression, while the y-axis shows the negative log10 of the corrected p-value (-log10 FDR). Differentially expressed genes (DEGs) are highlighted in red (upregulated) and blue (downregulated).\u003cbr\u003e\n(B) Bubble plot illustrating the potential pathways identified through KEGG pathway enrichment analysis of the differentially expressed genes. The y-axis lists the pathway names, and the x-axis represents the negative log10 of the corrected p-value. The size of each bubble corresponds to the number of genes involved in each pathway.\u003cbr\u003e\n(C) Venn diagram showing the intersection of the five most significantly differentially expressed genes associated with ILC2 and genes related to cytokine-cytokine receptor interactions during pulmonary fibrosis. Two genes, IL33 and IL13, are identified in the intersection. A table provides detailed information about these genes and their roles in ILC2 and cytokine-cytokine receptor interactions during pulmonary fibrosis.\u003c/p\u003e","description":"","filename":"floatimage2.png","url":"https://assets-eu.researchsquare.com/files/rs-7626166/v1/0d24f4ec8f33423fe92d4a12.png"},{"id":93575064,"identity":"8934ffb1-d581-49a6-aeb0-0efdb135005b","added_by":"auto","created_at":"2025-10-15 09:21:43","extension":"png","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":585788,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eInteraction between IL-33 and RUNX2 in pulmonary fibrosis.\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e(A) Venn diagram showing the intersection of differentially expressed genes (DEGs) associated with pulmonary fibrosis and IL-33-related genes, containing five genes (top). A table below lists the IL-33-related DEGs, including FOXA2, GATA2, JUN, MAFK, and RUNX2, along with relevant information.\u003cbr\u003e\n(B) Volcano plot displaying gene expression changes in pulmonary fibrosis. The x-axis represents the log2 fold change in gene expression, while the y-axis shows the negative log10 of the corrected p-value (-log10 FDR). Differentially expressed genes are marked in red (upregulated) and blue (downregulated).\u003cbr\u003e\n(C) AlphaFold3 molecular docking prediction showing potential hydrogen bond interactions between IL-33 (GLY174) and RUNX2 (SER196), as well as IL-33 (ASP175) and RUNX2 (LYS218). These binding sites provide structural evidence for the interaction between IL-33 and RUNX2.\u003cbr\u003e\n(D) UMAP plot depicting single-cell RNA sequencing data from pulmonary fibrosis patients, revealing the distribution and clustering features of different cell types.\u003cbr\u003e\n(E) UMAP plot showing that IL-33-expressing cells (red) are highly enriched in lung epithelial cells in pulmonary fibrosis samples, suggesting specific expression of IL-33 in lung epithelial cells.\u003cbr\u003e\n(F) UMAP plot showing that RUNX2-expressing cells (blue) are highly enriched in lung epithelial cells in pulmonary fibrosis samples, indicating specific expression of RUNX2 in lung epithelial cells.\u003cbr\u003e\n(G) UMAP plot illustrating that cells co-expressing IL-33 and RUNX2 (purple) are highly enriched in lung epithelial cells in pulmonary fibrosis samples, suggesting co-expression of both genes in these cells.\u003cbr\u003e\n(H) Bubble plot showing the expression of IL-33 across different cell types. The x-axis represents cell types, and the y-axis represents the IL-33 gene. Bubble color indicates average expression levels (dark red for high expression, light for low expression), and bubble size represents the percentage of cells expressing IL-33 in each cell type. Endothelial cells exhibit the highest IL-33 expression, both in terms of average expression and the proportion of expressing cells.\u003cbr\u003e\n(I) Bubble plot displaying RUNX2 expression across different cell types. The x-axis represents cell types, and the y-axis represents the RUNX2 gene. Bubble color reflects average expression levels, and bubble size represents the percentage of cells expressing RUNX2 in each cell type.\u003cbr\u003e\n(J) WB results showing that targeting RUNX2 in BEAS-2B cells results in co-immunoprecipitation of IL-33, confirming the interaction between RUNX2 and IL-33.\u003cbr\u003e\n(K) WB results showing that targeting IL-33 in BEAS-2B cells leads to co-immunoprecipitation of RUNX2, further confirming the interaction between IL-33 and RUNX2.\u003c/p\u003e","description":"","filename":"floatimage3.png","url":"https://assets-eu.researchsquare.com/files/rs-7626166/v1/8539dbf7babcc0d973f63bf3.png"},{"id":93576293,"identity":"d6d62594-53bd-4c61-a2c7-53c66f719b78","added_by":"auto","created_at":"2025-10-15 09:29:43","extension":"png","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":368168,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eEffects of RUNX2 expression modulation on inflammation and apoptosis.\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e(A) WB results showing that \u0026nbsp;\u0026nbsp;treatment with Bleomycin (BLM, 15 μg/mL) significantly upregulates RUNX2 \u0026nbsp;\u0026nbsp;expression in BEAS-2B cells.\u003cbr\u003e\n \u0026nbsp;(B) Enzyme-linked immunosorbent assay (ELISA) results demonstrating \u0026nbsp;\u0026nbsp;significant changes in the secretion levels of interleukin-1β (IL-1β), \u0026nbsp;\u0026nbsp;interleukin-6 (IL-6), tumor necrosis factor-α (TNF-α), and IL-33 in the \u0026nbsp;\u0026nbsp;culture supernatants of BEAS-2B cells after BLM treatment.\u003cbr\u003e\n \u0026nbsp;(C) WB results showing that overexpression of RUNX2 significantly increases \u0026nbsp;\u0026nbsp;its protein expression levels in BEAS-2B cells.\u003cbr\u003e\n \u0026nbsp;(D) Flow cytometry analysis showing that overexpression of RUNX2 \u0026nbsp;\u0026nbsp;significantly increases the apoptosis rate in BEAS-2B cells.\u003cbr\u003e\n \u0026nbsp;(E) ELISA results indicating that overexpression of RUNX2 significantly \u0026nbsp;\u0026nbsp;upregulates the secretion of IL-1β, IL-6, TNF-α, and IL-33 in the culture \u0026nbsp;\u0026nbsp;supernatants of BEAS-2B cells after BLM treatment.\u003cbr\u003e\n \u0026nbsp;(F) WB results showing that knockdown of RUNX2 significantly reduces its \u0026nbsp;\u0026nbsp;protein expression levels in BEAS-2B cells.\u003cbr\u003e\n \u0026nbsp;(G) Flow cytometry analysis demonstrating that knockdown of RUNX2 \u0026nbsp;\u0026nbsp;significantly reduces the apoptosis rate in BEAS-2B cells.\u003cbr\u003e\n \u0026nbsp;(H) ELISA results showing that knockdown of RUNX2 significantly downregulates \u0026nbsp;\u0026nbsp;the secretion levels of IL-1β, IL-6, and TNF-α in the culture supernatants of \u0026nbsp;\u0026nbsp;BEAS-2B cells after BLM treatment.\u003cbr\u003e\n \u0026nbsp;*P \u0026lt; 0.05; **P \u0026lt; 0.01; ***P \u0026lt; 0.001.\u003c/p\u003e","description":"","filename":"floatimage4.png","url":"https://assets-eu.researchsquare.com/files/rs-7626166/v1/e99a713923b6d2d13b6a6d40.png"},{"id":93573992,"identity":"e921692f-3d6d-447f-a81b-63b760ee56b6","added_by":"auto","created_at":"2025-10-15 09:13:43","extension":"png","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":223407,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eRegulation \u0026nbsp;\u0026nbsp;of ST2 expression by RUNX2 in BEAS-2B cells.\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e(A) Dot plot illustrating the expression of ST2 (IL1RL1) in different cell types.\u003cbr\u003e\n(B) WB results showing that overexpression of RUNX2 significantly upregulates ST2 expression in BEAS-2B cells.\u003cbr\u003e\n(C) WB results demonstrating that treatment with neutralizing IL-33 antibody (anti-IL33) inhibits the RUNX2-induced upregulation of ST2 in BEAS-2B cells.\u003cbr\u003e\n**P \u0026lt; 0.01; ***P \u0026lt; 0.001.\u003c/p\u003e","description":"","filename":"floatimage5.png","url":"https://assets-eu.researchsquare.com/files/rs-7626166/v1/4081ecb5ffee882bab29f6fe.png"},{"id":93573986,"identity":"1cc10702-f5d8-4ce9-bc28-66b1d2933a65","added_by":"auto","created_at":"2025-10-15 09:13:43","extension":"png","order_by":5,"title":"Figure 5","display":"","copyAsset":false,"role":"figure","size":683594,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eEffects of CLP and MTV treatments on lung injury and fibrosis in mice.\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e(A) H\u0026amp;E and Masson staining showing the impact of CLP and MTV treatments on lung injury and fibrosis in mice.\u003cbr\u003e\n(B) BALF analysis demonstrating the effects of CLP and MTV treatments on the recruitment of neutrophils and other immune cells in mouse lungs.\u003cbr\u003e\n(C) WB results showing the effects of different treatments on the expression levels of RUNX2, ST2, α-SMA, and COL1A1 in mouse lungs.\u003cbr\u003e\n(D) ELISA results indicating the effects of different treatments on the secretion levels of inflammatory cytokines IL-1β, IL-6, and TNF-α in mouse lungs.\u003cbr\u003e\n(E) ELISA results showing the effects of different treatments on IL-33 secretion levels in mouse lungs.\u003cbr\u003e\n(F) ELISA results demonstrating the effects of different treatments on WISP1 secretion levels in mouse lungs.\u003cbr\u003e\n(G) H\u0026amp;E and Masson staining showing the impact of CLP and MTV treatments on lung injury and fibrosis in IL-33 KO mice.\u003cbr\u003e\n(H) BALF analysis showing the effects of CLP and MTV treatments on the recruitment of neutrophils and other immune cells in IL-33 KO mouse lungs.\u003cbr\u003e\n(I) WB results showing the effects of different treatments on the expression levels of RUNX2, ST2, α-SMA, and COL1A1 in IL-33 KO mouse lungs.\u003cbr\u003e\n(J) ELISA results indicating the effects of different treatments on the secretion levels of inflammatory cytokines IL-1β, IL-6, and TNF-α in IL-33 KO mouse lungs.\u003cbr\u003e\n(K) ELISA results showing the effects of different treatments on WISP1 secretion levels in IL-33 KO mouse lungs.\u003cbr\u003e\n*P \u0026lt; 0.05; **P \u0026lt; 0.01; ***P \u0026lt; 0.001.\u003c/p\u003e","description":"","filename":"floatimage6.png","url":"https://assets-eu.researchsquare.com/files/rs-7626166/v1/f7608e842de3745cdc520924.png"},{"id":102397293,"identity":"b78c2e1f-94db-4075-af81-cec5a0fab9e1","added_by":"auto","created_at":"2026-02-11 10:15:04","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":2983952,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-7626166/v1/125be76b-76f0-4492-a36e-264934233a9d.pdf"},{"id":93575063,"identity":"41970808-92fd-4841-b9e8-20a0afadf215","added_by":"auto","created_at":"2025-10-15 09:21:43","extension":"docx","order_by":1,"title":"","display":"","copyAsset":false,"role":"supplement","size":575486,"visible":true,"origin":"","legend":"","description":"","filename":"GraphicalAbstract.docx","url":"https://assets-eu.researchsquare.com/files/rs-7626166/v1/da833071e08c3a65751bda98.docx"}],"financialInterests":"No competing interests reported.","formattedTitle":"The Role of IL-33 and RUNX2 in the Pathogenesis of Idiopathic Pulmonary Fibrosis","fulltext":[{"header":"Introduction","content":"\u003cp\u003eIdiopathic pulmonary fibrosis (IPF) is a progressive, irreversible lung disease characterized by excessive extracellular matrix (ECM) deposition and structural damage to lung tissue, ultimately leading to impaired pulmonary function\u003csup\u003e\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e\u003c/sup\u003e. The median survival of IPF is 3\u0026ndash;5 years after diagnosis\u003csup\u003e\u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e\u003c/sup\u003e. The pathophysiology of IPF is multifactorial, involving chronic inflammation, fibroblast activation, and epithelial-mesenchymal transition (EMT), which together contribute to excessive collagen deposition and fibrosis\u003csup\u003e\u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e\u003c/sup\u003e. Current antifibrotic therapies, such as pirfenidone and nintedanib, are only effective in slowing disease progression, without providing curative or reversing effects\u003csup\u003e\u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e4\u003c/span\u003e\u003c/sup\u003e. This underscores the urgent need for novel therapeutic targets and strategies.\u003c/p\u003e\u003cp\u003eOver the past few years, the role of immune-mediated mechanisms in the pathogenesis of pulmonary fibrosis has garnered increasing attention\u003csup\u003e\u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e5\u003c/span\u003e\u003c/sup\u003e. Interleukin-33 (IL-33), a member of the IL-1 cytokine family, plays a pivotal role in fibrosis. As an \u0026ldquo;alarmin,\u0026rdquo; IL-33 is released upon cell injury or stress and exerts its biological effects via the ST2 receptor (also known as IL1RL1)\u003csup\u003e\u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e6\u003c/span\u003e\u003c/sup\u003e. The ST2 receptor exists in two forms: a membrane-bound variant (ST2L) that mediates intracellular signaling, and a soluble form (sST2) that acts as a decoy receptor\u003csup\u003e\u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e,\u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e8\u003c/span\u003e\u003c/sup\u003e. The IL-33/ST2 signaling pathway regulates immune responses, including type 2 inflammation, by activating type 2 innate lymphoid cells (ILC2s), eosinophils, and Th2 cells. ILC2s are tissue-resident, non-T, non-B innate immune cells that play a critical role in type 2 immunity\u003csup\u003e\u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e9\u003c/span\u003e\u003c/sup\u003e. Upon IL-33 activation, ILC2s rapidly produce Th2 cytokines, such as IL-5 and IL-13, which promote eosinophilic inflammation, airway remodeling, and fibroblast activation. Increasing evidence suggests that ILC2s are key regulators of fibrosis in various tissues, including the lung. In pulmonary fibrosis, the expansion of ILC2s correlates with increased ECM deposition and fibroblast proliferation. However, the specific downstream mediators linking ILC2 activation to fibroblast function remain largely unexplored.\u003c/p\u003e\u003cp\u003eRUNX2 (Runt-related transcription factor 2) has also emerged as a critical regulator in the fibrotic process. Recent studies indicate that RUNX2 plays an important role in the conversion of alveolar fibroblasts to pathological fibroblasts, which are the key source of fibrosis\u003csup\u003e\u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e10\u003c/span\u003e\u003c/sup\u003e. In IPF patients, the expression of RUNX2 is significantly elevated in pathological fibroblasts, and its inhibition reduces ECM deposition and fibroblast activation. These findings suggest that RUNX2 could be a potential therapeutic target for alleviating fibrosis.\u003c/p\u003e\u003cp\u003eWNT1-inducible signaling pathway protein 1 (WISP1), an extracellular matrix protein belonging to the CCN family, is involved in tissue remodeling, fibrosis, and tumorigenesis\u003csup\u003e\u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e,\u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e12\u003c/span\u003e\u003c/sup\u003e. WISP1 is upregulated in fibrotic lung tissue and has been shown to promote fibroblast proliferation, myofibroblast differentiation, and collagen deposition\u003csup\u003e\u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e13\u003c/span\u003e\u003c/sup\u003e. It interacts with integrins and receptor tyrosine kinases to activate pro-fibrotic signaling pathways, such as TGF-β/SMAD and PI3K/AKT\u003csup\u003e14,15\u003c/sup\u003e. While WISP1 is implicated in pulmonary fibrosis, the regulatory mechanisms controlling its expression remain unclear.\u003c/p\u003e\u003cp\u003eIn this study, we integrate bioinformatics analysis, molecular docking, single-cell RNA sequencing, and both \u003cem\u003ein vitro\u003c/em\u003e and \u003cem\u003ein vivo\u003c/em\u003e experimental models to comprehensively explore the roles of IL-33 and RUNX2 in the pathogenesis of pulmonary fibrosis. Our \u003cem\u003ein vitro\u003c/em\u003e results demonstrate that overexpression of RUNX2 significantly exacerbates BLM-induced inflammation and apoptosis in BEAS-2B cells, whereas knockdown of RUNX2 attenuates these effects. However, \u003cem\u003ein vivo\u003c/em\u003e experiments reveal a discrepancy with \u003cem\u003ein vitro\u003c/em\u003e findings: in IL-33 knockout (KO) mice, lung fibrosis is exacerbated, suggesting a complex, dual role for IL-33 in pulmonary fibrosis\u003csup\u003e\u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e16\u003c/span\u003e\u003c/sup\u003e. This observation suggests that the mechanisms by which IL-33 and RUNX2 contribute to fibrosis are more complex than previously recognized, necessitating further investigation.\u003c/p\u003e\u003cp\u003eBy delving into the interactions between IL-33, RUNX2, and ILC2s, this study aims to provide new insights into the molecular mechanisms of pulmonary fibrosis and identify potential therapeutic targets for this debilitating disease.\u003c/p\u003e"},{"header":"Materials and Methods","content":"\u003ch4\u003e\u003cstrong\u003eCell Culture\u003c/strong\u003e\u003c/h4\u003e\n\u003cp\u003eBEAS-2B human normal lung epithelial cells were obtained from the Shanghai Institute of Life Sciences (Shanghai, China) and cultured in Dulbecco\u0026apos;s Modified Eagle Medium (DMEM, Gibco) supplemented with 10% fetal bovine serum (FBS, Gibco) and 1% penicillin-streptomycin (Gibco) at 37\u0026deg;C in a 5% CO₂ incubator. For inflammation induction, cells were treated with Bleomycin (BLM) at a final concentration of 15 \u0026micro;g/mL for 24 hours.\u003c/p\u003e\n\u003ch4\u003e\u003cstrong\u003ePlasmid Transfection and Knockdown\u003c/strong\u003e\u003c/h4\u003e\n\u003cp\u003eRUNX2 overexpression was achieved by transfecting BEAS-2B cells with a plasmid expressing RUNX2 (OriGene) using Lipofectamine 3000 reagent (Thermo Fisher) according to the manufacturer\u0026rsquo;s instructions. RUNX2 knockdown was performed using small interfering RNA (siRNA) targeting RUNX2 (Santa Cruz Biotechnology), transfected into BEAS-2B cells using Lipofectamine RNAiMAX (Thermo Fisher).\u003c/p\u003e\n\u003ch4\u003e\u003cstrong\u003eCo-Culture System\u003c/strong\u003e\u003c/h4\u003e\n\u003cp\u003eFor co-culture experiments, BEAS-2B cells transfected with RUNX2 plasmid or siRNA were co-cultured with ILC2 cells under inflammatory conditions induced by BLM. The supernatants were collected for cytokine analysis, and cells were harvested for subsequent assays.\u003c/p\u003e\n\u003ch4\u003e\u003cstrong\u003eUMAP Analysis\u003c/strong\u003e\u003c/h4\u003e\n\u003cp\u003eThe single-cell RNA sequencing dataset GSE213017 was obtained from the publicly accessible Gene Expression Omnibus (GEO) database. UMAP (Uniform Manifold Approximation and Projection) was performed using the Seurat package in R to visualize the distribution and clustering of cell types based on gene expression profiles.\u003c/p\u003e\n\u003ch4\u003e\u003cstrong\u003eMolecular Docking\u003c/strong\u003e\u003c/h4\u003e\n\u003cp\u003eMolecular docking simulations were performed using AlphaFold3 to predict the potential binding sites and interactions between IL-33 and RUNX2.\u0026nbsp;\u003c/p\u003e\n\u003ch4\u003e\u003cstrong\u003eWestern Blotting (WB)\u003c/strong\u003e\u003c/h4\u003e\n\u003cp\u003eCells were lysed using RIPA buffer (Thermo Fisher) supplemented with protease inhibitors (Sigma-Aldrich). Protein concentration was determined using the BCA Protein Assay Kit (Thermo Fisher). Equal amounts of protein (30 \u0026micro;g) were separated by SDS-PAGE, transferred to PVDF membranes (Millipore), and incubated with primary antibodies against IL-33 (#88513, CST), RUNX2 (#12556, CST), ST2 (12-9338-42, ThermoFisher), \u0026alpha;-SMA (sc-53015, SantaCruz), and COL1A1 (Abcam). Protein bands were visualized using an ECL detection system (Bio-Rad).\u003c/p\u003e\n\u003ch4\u003e\u003cstrong\u003eEnzyme-Linked Immunosorbent Assay (ELISA)\u003c/strong\u003e\u003c/h4\u003e\n\u003cp\u003eIL-33, IL-1\u0026beta;, IL-6, TNF-\u0026alpha;, and WISP1 levels in cell culture supernatants or lung tissue homogenates were measured using ELISA kits (R\u0026amp;D Systems) according to the manufacturer\u0026rsquo;s instructions. Absorbance was measured at 450 nm using a microplate reader (BioTek).\u003c/p\u003e\n\u003ch4\u003e\u003cstrong\u003eFlow Cytometry\u003c/strong\u003e\u003c/h4\u003e\n\u003cp\u003eApoptosis was measured using the Annexin V-FITC/PI Apoptosis Detection Kit (BD Biosciences) according to the manufacturer\u0026rsquo;s protocol. Briefly, cells were harvested, stained with Annexin V and propidium iodide (PI), and analyzed by flow cytometry (BD Accuri C6). The apoptotic cell population was quantified as the percentage of PI-positive cells.\u003c/p\u003e\n\u003ch4\u003e\u003cstrong\u003eAnimal Models\u003c/strong\u003e\u003c/h4\u003e\n\u003cp\u003eAll animal experiments were conducted according to the guidelines of the Institutional Animal Care and Use Committee (IACUC) of The First Affiliated Hospital of Zhengzhou University (Approval No.\u0026nbsp;2025-KY-0823-001). C57BL/6N mice (6\u0026ndash;8 weeks old, 213) were purchased from Vital River. Pulmonary fibrosis was induced using the cecal ligation and puncture (CLP) method followed by mechanical ventilation (MTV, 4 hours, 10 mL/kg) to exacerbate lung injury and fibrosis. Mice were sacrificed after 3 days for histological analysis.\u003c/p\u003e\n\u003ch4\u003e\u003cstrong\u003eHistological Analysis\u003c/strong\u003e\u003c/h4\u003e\n\u003cp\u003eLung tissues were fixed in 4% paraformaldehyde, embedded in paraffin, and sectioned. Hematoxylin and eosin (H\u0026amp;E) and Masson\u0026rsquo;s trichrome staining were performed to assess lung injury and fibrosis.\u0026nbsp;\u003c/p\u003e\n\u003ch4\u003e\u003cstrong\u003eStatistical Analysis\u003c/strong\u003e\u003c/h4\u003e\n\u003cp\u003eData are expressed as mean \u0026plusmn; standard deviation (SD). Statistical significance was assessed using one-way ANOVA followed by Tukey\u0026rsquo;s post hoc test. A p-value of less than 0.05 was considered statistically significant. All statistical analyses were performed using GraphPad Prism (version 8.0).\u003c/p\u003e"},{"header":"Results","content":"\u003cp\u003e\u003cstrong\u003e1. Role of ILC2 Cells in Pulmonary Fibrosis\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eGroup 2 innate lymphoid cells (ILC2s) are widely distributed in the lungs, particularly enriched in pulmonary tissues and the submucosa of the respiratory tract. These cells play a crucial role in maintaining pulmonary immune homeostasis, mediating inflammatory responses, and contributing to the process of fibrosis\u003csup\u003e17-19\u003c/sup\u003e.\u003c/p\u003e\n\u003cp\u003eData analysis from the GEO database (GSE267904) revealed differential gene expression between fibrotic lung tissue and normal lung tissue (Fig. 1A). KEGG pathway enrichment analysis highlighted significant upregulation of the cytokine-cytokine receptor interaction pathway in the context of lung fibrosis (Fig. 1B).\u003c/p\u003e\n\u003cp\u003eAmong the differentially expressed genes, we identified five genes associated with ILC2 cells that exhibited the most significant changes in expression. By intersecting these genes with known cytokine-cytokine receptor interaction genes, two key genes were identified: IL33 and IL13 (Fig. 1C). Notably, IL33 showed a stronger association with ILC2 cells.\u003c/p\u003e\n\u003cp\u003eThese findings indicate that ILC2 cells, along with their associated genes, may play a crucial role in the progression of lung fibrosis, particularly highlighting the strong correlation between IL-33 and ILC2 cells. This underscores the potential of IL33 as a critical player in the pathogenesis of pulmonary fibrosis. Further investigation into the mechanisms of IL33 in lung fibrosis and its potential as a therapeutic target is warranted.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e2. Interaction between IL33 and RUNX2 in Pulmonary Fibrosis\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eBased on our previous findings, we further examined genes associated with IL33 in the differentially expressed genes (DEGs) of IPF. This analysis led to the identification of five key genes: FOXA2, GATA2, JUN, MAFK, and RUNX2 (Fig. 2A). Volcano plot analysis revealed that among these genes, only RUNX2 showed a significant upregulation in the context of pulmonary fibrosis (Fig. 2B).\u003c/p\u003e\n\u003cp\u003eMolecular docking predictions using AlphaFold3 indicated potential binding sites between IL33 and RUNX2, specifically at IL33\u0026rsquo;s GLY174 and RUNX2\u0026rsquo;s SER196, as well as IL33\u0026rsquo;s ASP175 and RUNX2\u0026rsquo;s LYS218 (Fig. 2C).\u003c/p\u003e\n\u003cp\u003eTo further explore the specific roles of IL33 and RUNX2 in pulmonary fibrosis, we conducted UMAP dimensionality reduction analysis of single-cell sequencing dataset GSE213017. This technique projects high-dimensional data into two or three-dimensional space, clustering similar samples together (Fig. 2D). The results revealed co-localization of RUNX2 (Fig. 2E) and IL33 (Fig. 2F) in lung epithelial cells (Fig. 2G). Further bubble plot analysis demonstrated that IL33 is primarily expressed in fibroblasts, epithelial cells, and vascular endothelial cells in the lung (Fig. 2H), whereas RUNX2 is expressed across various lung cell types, with the highest expression observed in lung epithelial cells (Fig. 2I).\u003c/p\u003e\n\u003cp\u003eTo validate the interaction between IL33 and RUNX2, we performed co-immunoprecipitation (Co-IP) assays on BEAS-2B lung epithelial cells. After crosslinking, immunoprecipitation targeting either RUNX2 or IL33 confirmed their physical interaction. Specifically, RUNX2 immunoprecipitation resulted in the detection of IL33 in the eluate (Fig. 2J), and vice versa, immunoprecipitating IL33 allowed for the detection of RUNX2 (Fig. 2K). These results validate the molecular docking and UMAP co-localization findings, demonstrating a direct physical interaction between RUNX2 and IL33 in BEAS-2B cells.\u003c/p\u003e\n\u003cp\u003eIn summary, this study emphasizes the interaction between IL-33 and RUNX2 in pulmonary fibrosis, offering new insights into the molecular mechanisms underlying the disease. Further investigations are warranted to explore the specific roles of IL33 and RUNX2 in the pathogenesis of pulmonary fibrosis.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e3. RUNX2-IL-33 Axis Induces Apoptosis and Inflammatory Response in BEAS-2B Cells\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eTo investigate the role of the RUNX2-IL-33 axis in lung epithelial cells, we first confirmed their interaction in BEAS-2B cells. Upon BLM treatment, a significant upregulation of RUNX2 expression was observed (Fig. 3A). In parallel, the expression levels of classic inflammatory cytokines, including IL-1\u0026beta;, IL-6, and TNF-\u0026alpha;, were also elevated, and IL-33 expression significantly increased (Fig. 3B). These findings suggest that the interaction between RUNX2 and IL-33 is activated under inflammatory conditions.\u003c/p\u003e\n\u003cp\u003eFurther, to explore the functional implications of RUNX2 overexpression, we transfected BEAS-2B cells with a RUNX2 overexpression plasmid and subjected them to BLM-induced inflammation (Fig. 3C). Flow cytometry analysis revealed a significant increase in PI-positive cells in the RUNX2-overexpressing group, indicating enhanced apoptosis (Fig. 3D). Additionally, compared to the control group (BLM-treated cells transfected with the NC plasmid), cells overexpressing RUNX2 exhibited further increases in IL-1\u0026beta;, IL-6, TNF-\u0026alpha;, and IL-33 levels (Fig. 3E), suggesting that RUNX2 overexpression exacerbates both the inflammatory response and apoptosis.\u003c/p\u003e\n\u003cp\u003eTo verify that these changes were indeed driven by RUNX2, we knocked down RUNX2 expression in BEAS-2B cells and induced inflammation with BLM (Fig. 3F). Knockdown of RUNX2 led to a significant reduction in the proportion of PI-positive cells (Fig. 3G), and the expression of IL-1\u0026beta;, IL-6, and TNF-\u0026alpha; was notably decreased (Fig. 3H). These results confirm that RUNX2 mediates BLM-induced inflammation and apoptosis in lung epithelial cells.\u003c/p\u003e\n\u003cp\u003eIn summary, our findings demonstrate that the RUNX2-IL-33 axis contributes to the induction of apoptosis and inflammation in BEAS-2B cells, providing insight into its role in the fibrotic process in the lung. Further studies are warranted to explore the downstream targets of this signaling axis in the context of lung fibrosis.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e4. RUNX2 Modulates ST2 Activation in ILC2 via IL-33\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003ePrevious studies have demonstrated that IL-33 exerts its biological functions by forming a heterodimeric complex with its receptor ST2 and the co-receptor IL-1RAcP\u003csup\u003e20\u003c/sup\u003e, with ST2 being expressed in ILC2 cells (Fig. 4A). To investigate the role of RUNX2 in regulating ST2 activation, BEAS-2B cells overexpressing RUNX2 were co-cultured with ILC2 cells under inflammatory conditions induced by BLM. WB analysis revealed an upregulation of ST2 expression in ILC2 cells (Fig. 4B).\u003c/p\u003e\n\u003cp\u003eFurthermore, when IL-33 signaling was neutralized using the anti-IL-33 antibody (Tozorakimab) under the same conditions, co-culture with ILC2 resulted in a significant reduction in ST2 expression (Fig. 4C). These findings suggest that RUNX2 in lung epithelial cells can regulate the secretion of IL-33, thereby modulating ST2 activation in ILC2 cells across cellular interactions. Reducing the levels of IL-33 in the microenvironment effectively inhibits the activation of ST2.\u003c/p\u003e\n\u003cp\u003eThis section reveals the mechanism by which RUNX2 regulates ST2 activation in ILC2 cells via IL-33, further elucidating the interaction between lung epithelial cells and ILC2 cells and their role in the inflammatory response. Future studies will focus on investigating the role of this mechanism in \u003cem\u003ein vivo\u003c/em\u003e models of lung fibrosis.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e5. IL-33 Drives Pulmonary Fibrosis in Sepsis Model\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eTo investigate the role of IL-33 in mediating pulmonary fibrosis under inflammatory conditions \u003cem\u003ein vivo\u003c/em\u003e, we first established a bacterial infection-induced inflammatory model in mice via cecal ligation and puncture (CLP) to induce lung injury\u003csup\u003e21\u003c/sup\u003e. Given the clinical evidence suggesting that patients with prolonged mechanical ventilation often exhibit more severe pulmonary fibrosis, we subjected the mice to mechanical ventilation (MTV, 4 hours, 10 mL/kg) following sepsis-induced lung injury to further induce fibrosis\u003csup\u003e22\u003c/sup\u003e.\u003c/p\u003e\n\u003cp\u003eAll groups were sacrificed after 3 days of treatment for histological analysis using H\u0026amp;E and Masson\u0026rsquo;s trichrome staining. Normal mice showed a loose and porous alveolar structure, while CLP-treated mice displayed an increase in lung parenchyma, with Masson\u0026rsquo;s trichrome staining revealing elevated collagen deposition (blue). In the CLP+MTV group, alveolar spaces were further compressed, and collagen accumulation was significantly increased (Fig. 5A). These results demonstrate that CLP and CLP+MTV treatments progressively transition the lung tissue from a healthy to a fibrotic state, with the latter showing more severe fibrosis.\u003c/p\u003e\n\u003cp\u003eWe also observed a progressive increase in total cell and neutrophil counts in the bronchoalveolar lavage fluid (BALF) of treated mice, which followed the order of sham, CLP, and CLP+MTV treatment groups (Fig. 5B). This suggests a positive correlation between the degree of pulmonary fibrosis and immune cell recruitment.\u003c/p\u003e\n\u003cp\u003e\u0026alpha;-SMA, a cytoskeletal protein expressed in smooth muscle cells and certain types of fibroblasts, is a marker of myofibroblast differentiation, and COL1A1, which encodes the \u0026alpha;1 chain of type I collagen, is a major component of collagen in fibrotic tissue. We assessed the expression levels of RUNX2, ST2, \u0026alpha;-SMA, and COL1A1 via WB analysis. In the sham group, baseline expression levels of these proteins were low, while in the CLP-induced lung injury model, their expression was significantly upregulated. Further mechanical ventilation treatment led to a further increase in RUNX2, ST2, \u0026alpha;-SMA, and COL1A1 expression (Fig. 5C), suggesting a positive correlation between RUNX2 and ST2 expression with fibrotic markers.\u003c/p\u003e\n\u003cp\u003eThe levels of classical inflammatory cytokines IL-1\u0026beta;, IL-6, and TNF-\u0026alpha; also increased with the progression of fibrosis, indicating a positive correlation between inflammation and fibrosis (Fig. 5D). This further highlights the role of inflammatory factors, including IL-33, in the fibrotic process (Fig. 5E). Additionally, WISP1, a protein secreted by ILC2 cells that can act on fibroblasts, showed an increase in parallel with inflammation (Fig. 5F), suggesting a potential mechanism through which IL-33 mediates pulmonary fibrosis via ILC2.\u003c/p\u003e\n\u003cp\u003eNext, we employed IL-33 knockout (KO) mice to assess the role of IL-33 in sepsis and mechanical ventilation-induced pulmonary fibrosis. Contrary to our expectations, in IL-33 KO mice, the lung parenchyma still increased, and Masson\u0026rsquo;s trichrome staining revealed even greater fibrosis compared to wild-type (WT) mice (Fig. 5G). Recruitment of lymphocytes in the BALF was not reduced, and neutrophils made up an even larger proportion of the total cells (Fig. 5H). Although the upregulation of RUNX2, ST2, \u0026alpha;-SMA, and COL1A1 in response to CLP and CLP+MTV treatment was less pronounced in the IL-33 KO mice, it remained significant (Fig. 5I). However, the levels of classic immune factors IL-1\u0026beta;, IL-6, and TNF-\u0026alpha; in IL-33 KO mice were almost identical to those in WT mice (Fig. 5J). Similarly, WISP1 secretion in IL-33 KO mice did not show a decrease compared to WT mice (Fig. 5K).\u003c/p\u003e\n\u003cp\u003eThis section presents \u003cem\u003ein vivo\u003c/em\u003e findings that contrast with the cell culture results, indicating that IL-33 may have a mitigatory role in sepsis-induced pulmonary fibrosis. These findings suggest that, \u003cem\u003ein vivo\u003c/em\u003e, the interaction between IL-33 and other immune and fibrotic factors forms a complex network, which is likely influenced by the broader tissue context, differing from the more controlled conditions of \u003cem\u003ein vitro\u003c/em\u003e models. Future studies should refine experimental models and explore alternative mechanisms to better understand the role of IL-33 in pulmonary fibrosis, particularly its dual function under different pathological conditions.\u003c/p\u003e"},{"header":"Discussion","content":"\u003cp\u003eIn this study, we employed a comprehensive approach combining bioinformatics analysis, molecular docking, and both \u003cem\u003ein vitro\u003c/em\u003e and \u003cem\u003ein vivo\u003c/em\u003e experimental models to systematically investigate the role of ILC2 cells, IL-33, and RUNX2 in the pathogenesis of pulmonary fibrosis and to explore the underlying molecular mechanisms. Bioinformatics analysis revealed an upregulation of IL-33 and RUNX2 in pulmonary fibrosis, suggesting their interaction within lung epithelial cells. Further \u003cem\u003ein vitro\u003c/em\u003e experiments confirmed that this interaction could mediate inflammatory responses and cell apoptosis in cultured cells. However, discrepancies were observed between the results from \u003cem\u003ein vivo\u003c/em\u003e and \u003cem\u003ein vitro\u003c/em\u003e experiments in the animal models, IL-33 KO did not effectively block the inflammatory response. Although the upregulation of fibroblast markers under inflammatory stress was slightly reduced at the molecular level, collagen deposition increased.\u003c/p\u003e\u003cp\u003ePrevious studies have demonstrated that IL-33-deficient mice exhibit heightened susceptibility to severe lung injury, as evidenced by increased levels of inflammatory cytokines and immune cell infiltration\u003csup\u003e\u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e16\u003c/span\u003e\u003c/sup\u003e. Similar observations were made in our experiments, and we hypothesize that the absence of IL-33, a key \u0026ldquo;early warning\u0026rdquo; molecule, impairs the initial recruitment of immune cells, thus hindering the early immune response to injury and infection. This delay in immune response exacerbates tissue damage, leading to the activation of other inflammatory factors that drive a more pronounced fibrotic response as a compensatory mechanism.\u003c/p\u003e\u003cp\u003eIn addition, several factors may account for the discrepancies observed between \u003cem\u003ein vitro\u003c/em\u003e and \u003cem\u003ein vivo\u003c/em\u003e results. First, the complex immune environment \u003cem\u003ein vivo\u003c/em\u003e may alter the effects of IL-33. While IL-33 mediates inflammation and fibrosis in a simplified signaling context, its role in the presence of other potent inflammatory and fibrotic factors could lead to competitive inhibition or \"dragging effects,\" where other fibrotic mediators might become more prominent after IL-33 deletion. Second, IL-33 may function within a feedback mechanism that regulates inflammation. As an inflammatory cytokine, IL-33 likely initiates downstream feedback processes that limit excessive inflammation to prevent further tissue damage. In the absence of IL-33, this negative regulatory mechanism may be disrupted, resulting in exacerbated fibrosis. Third, the splice variants of the ST2 receptor, a known downstream receptor for IL-33, may also play a crucial role. It is said that the soluble decoy receptor ST2 can sequester excess IL-33, and we observed that overexpression of IL-33 upregulates ST2 in BEAS-2B cells. It suggests a possible mechanism that IL-33 expression may influence the splicing of ST2, thereby modulating the balance between ST2 activation and IL-33 neutralization, which process still needs to be further verified in the future. Although IL-33 is the only known ligand for ST2, it is possible that, in the absence of IL-33, other factors could activate membrane-bound ST2.\u003c/p\u003e\u003cp\u003eBioinformatics analysis using gene expression data from the GEO database identified IL33 and IL13 as key genes associated with ILC2 cells in pulmonary fibrosis. KEGG pathway analysis further revealed a strong association between cytokine-cytokine receptor interactions and fibrosis progression. Based on these findings, we focused on the interaction between IL33 and RUNX2, and verified their colocalization and physical interaction in lung epithelial cells through immunoprecipitation experiments. \u003cem\u003eIn vitro\u003c/em\u003e results demonstrated that overexpression of RUNX2 exacerbated BLM-induced inflammation and apoptosis in BEAS-2B cells, while RUNX2 knockdown significantly reduced these effects. Moreover, we found that RUNX2 regulates IL-33 secretion, which in turn activates ST2 in ILC2 cells, thereby promoting fibrosis. Through \u003cem\u003ein vivo\u003c/em\u003e sepsis models, we observed that IL-33 might have a bidirectional regulatory effect in mediating inflammation-induced pulmonary fibrosis.\u003c/p\u003e\u003cp\u003eOur findings are in line with current understanding of pulmonary fibrosis, while also extending it. Previous studies have established the importance of IL-33 and ILC2 cells in fibrosis, particularly in their roles in promoting inflammation and tissue remodeling. However, our study introduces RUNX2 as a critical regulator of IL-33 secretion and its interaction with lung epithelial cells, providing new insights into the molecular mechanisms underlying fibrosis.\u003c/p\u003e\u003cp\u003eNonetheless, our study has several limitations. First, we were unable to reconcile the results between \u003cem\u003ein vitro\u003c/em\u003e and \u003cem\u003ein vivo\u003c/em\u003e experiments. Future studies should optimize experimental models or delve deeper into understanding the role of IL-33 in the fibrosis process from a broader perspective. Additionally, while this study primarily focused on the role of ILC2 cells, we did not extensively explore the contributions of other immune cells, such as macrophages or T cells, which are also closely linked to fibrosis. Future research should further investigate the interactions among these cell types and their roles in the fibrotic process.\u003c/p\u003e\u003cp\u003eThe results of this study encourage a reevaluation of pulmonary fibrosis as a disease driven by dysregulated molecular signaling and immune cell interactions. Identifying the RUNX2-IL-33 axis as a key driver of fibrosis suggests that targeting this pathway could represent a promising therapeutic strategy. Furthermore, our findings underscore the importance of considering intercellular interactions and cross-cell signaling in fibrosis development, which may lead to more effective and targeted therapeutic approaches. Beyond pulmonary fibrosis, fibrosis is a common pathological feature of many chronic inflammatory diseases affecting organs such as the liver, heart, and kidneys. The role of IL-33 and RUNX2 in pulmonary fibrosis suggests that similar mechanisms may be at play in other fibrotic diseases. For instance, in cardiac fibrosis, IL-33 has been shown to promote fibroblast activation and collagen deposition\u003csup\u003e\u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e23\u003c/span\u003e\u003c/sup\u003e, while RUNX2 is known to regulate osteoblastic differentiation and extracellular matrix remodeling\u003csup\u003e\u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e24\u003c/span\u003e,\u003cspan citationid=\"CR25\" class=\"CitationRef\"\u003e25\u003c/span\u003e\u003c/sup\u003e. Thus, therapeutic strategies such as targeting the RUNX2-IL-33 axis or modulating ST2 activation may be applicable to other fibrotic diseases.\u003c/p\u003e\u003cp\u003eIn conclusion, this study provides valuable insights into the potential molecular and cellular mechanisms of pulmonary fibrosis and offers new directions for future therapeutic strategies.\u003c/p\u003e"},{"header":"Declarations","content":"\u003cp\u003e\u003cstrong\u003eDisclosure statement/ Competing interests\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe authors report no declarations of interest.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eEthics Approval and Consent to Participate\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eAll animal experiments were conducted according to the guidelines of the Institutional Animal Care and Use Committee (IACUC) of The First Affiliated Hospital of Zhengzhou University (Approval No.\u0026nbsp;2025-KY-0823-001) and were performed in accordance with the National Institutes of Health Guidelines for the Care and Use of Laboratory Animals.\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\u003eData availability\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe data analyzed during the current study are available from the corresponding author on reasonable request.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eFunding\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eStudy funded financially by National Natural Science Foundation of China (82100092), Natural Science Foundation of Henan Province of China (232300420255), Henan Province Medical Science and Technology Research Plan (LHGJ20200380, LHGJ20200341)\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAuthors\u0026apos; contributions\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e\u003cem\u003eShuai Liu and Qiuge Wu\u003c/em\u003e conceived, designed and supervised the whole study; \u003cem\u003eShuai Liu, Lixin Wang, Yinyan Yue, Zhao Zhang, Fang Li, Dongdong Wu and Hui Zhang\u003c/em\u003e operated the experiment, performed the analyses and audited the data; \u003cem\u003eShuai Liu\u003c/em\u003e wrote the manuscript; \u003cem\u003eQiuge Wu\u003c/em\u003e revised the manuscript. All authors provided critical comments and approved the final manuscript.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAcknowledgments\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eNot applicable.\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\n\u003cli\u003eBridges, J. P.\u003cem\u003e et al.\u003c/em\u003e Progressive lung fibrosis: reprogramming a genetically vulnerable bronchoalveolar epithelium. \u003cem\u003eJ Clin Invest\u003c/em\u003e \u003cstrong\u003e135\u003c/strong\u003e, doi:10.1172/jci183836 (2025).\u003c/li\u003e\n\u003cli\u003eRaghu, G.\u003cem\u003e et al.\u003c/em\u003e Diagnosis of Idiopathic Pulmonary Fibrosis. 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Y.\u003cem\u003e et al.\u003c/em\u003e Group 2 innate lymphoid cells contribute to IL-33-mediated alleviation of cardiac fibrosis. \u003cem\u003eTheranostics\u003c/em\u003e \u003cstrong\u003e11\u003c/strong\u003e, 2594-2611, doi:10.7150/thno.51648 (2021).\u003c/li\u003e\n\u003cli\u003eDong, Q.\u003cem\u003e et al.\u003c/em\u003e Nuclear farnesoid X receptor protects against bone loss by driving osteoblast differentiation through stabilizing RUNX2. \u003cem\u003eBone research\u003c/em\u003e \u003cstrong\u003e13\u003c/strong\u003e, 20, doi:10.1038/s41413-024-00394-w (2025).\u003c/li\u003e\n\u003cli\u003eZhang, Y.\u003cem\u003e et al.\u003c/em\u003e RUNX2 Phase Separation Mediates Long-Range Regulation Between Osteoporosis-Susceptibility Variant and XCR1 to Promote Osteoblast Differentiation. \u003cem\u003eAdv Sci (Weinh)\u003c/em\u003e \u003cstrong\u003e12\u003c/strong\u003e, e2413561, doi:10.1002/advs.202413561 (2025). \u003c/li\u003e\n\u003c/ol\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":false,"hideJournal":true,"highlight":"","institution":"","isAcceptedByJournal":false,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":false,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"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},"keywords":"Pulmonary fibrosis, Innate lymphoid cells (ILC2s), Interleukin-33, ST2, Runt-related transcription factor 2","lastPublishedDoi":"10.21203/rs.3.rs-7626166/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-7626166/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003cp\u003e\u003cstrong\u003eBackground:\u003c/strong\u003e Idiopathic Pulmonary Fibrosis (IPF) is a chronic, progressive lung disease characterized by abnormal fibrosis of the lung tissue, leading to a gradual decline in lung function. The exact cause of this disease is not well understood, hence the term \"idiopathic.\" Although the incidence of IPF is not high, it significantly impacts the quality of life and life expectancy of patients. The molecular mechanisms underlying IPF, including immune responses and fibroblast activation, remain insufficiently understood. This study explores the roles of interleukin-33 (IL-33) and RUNX2 in the pathogenesis of pulmonary fibrosis, aiming to identify novel therapeutic targets.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eMethods:\u003c/strong\u003e A comprehensive approach combining bioinformatics analysis, molecular docking, single-cell RNA sequencing, and both \u003cem\u003ein vitro\u003c/em\u003e and \u003cem\u003ein vivo\u003c/em\u003e experimental models was used to investigate the interaction between IL-33 and RUNX2 in IPF. Gene expression analysis, KEGG pathway enrichment, and co-immunoprecipitation assays were performed to validate. \u003cem\u003eIn vitro\u003c/em\u003e inflammation and apoptosis assays were conducted using BEAS-2B lung epithelial cells, and \u003cem\u003ein vivo\u003c/em\u003e fibrosis models were established in mice.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eResults:\u003c/strong\u003e Bioinformatics analysis revealed the upregulation of IL-33 and RUNX2 in pulmonary fibrosis, with a significant correlation between these two molecules. \u003cem\u003eIn vitro\u003c/em\u003e, RUNX2 overexpression exacerbated BLM-induced inflammation and apoptosis, while knockdown of RUNX2 attenuated these effects. Co-immunoprecipitation confirmed the physical interaction between IL-33 and RUNX2. \u003cem\u003eIn vivo\u003c/em\u003e, IL-33 knockout mice exhibited exacerbated fibrosis, highlighting a complex dual role of IL-33 in the disease process.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eConclusions:\u003c/strong\u003e This study identifies the RUNX2-IL-33 axis as a key mediator in pulmonary fibrosis. Our findings provide new insights into the molecular mechanisms of IPF and suggest that targeting the RUNX2-IL-33 interaction could represent a promising therapeutic strategy for fibrosis-related diseases.\u003c/p\u003e","manuscriptTitle":"The Role of IL-33 and RUNX2 in the Pathogenesis of Idiopathic Pulmonary Fibrosis","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2025-10-15 09:13:38","doi":"10.21203/rs.3.rs-7626166/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":"1eadeeef-b95f-4a4f-b675-00981ed6af7f","owner":[],"postedDate":"October 15th, 2025","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"posted","subjectAreas":[],"tags":[],"updatedAt":"2026-02-09T04:54:54+00:00","versionOfRecord":[],"versionCreatedAt":"2025-10-15 09:13:38","video":"","vorDoi":"","vorDoiUrl":"","workflowStages":[]},"version":"v1","identity":"rs-7626166","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-7626166","identity":"rs-7626166","version":["v1"]},"buildId":"8U1c8b4HqxoKbykW_rLl7","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}