The IL-33/ST2 Axis Promotes Post-COVID-19 Pulmonary Fibrosis Through β-catenin/PPAR-γ-mediated Epithelial-Mesenchymal Transition

The IL-33/ST2 Axis Promotes Post-COVID-19 Pulmonary Fibrosis Through β-catenin/PPAR-γ-mediated Epithelial-Mesenchymal Transition | 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 IL-33/ST2 Axis Promotes Post-COVID-19 Pulmonary Fibrosis Through β-catenin/PPAR-γ-mediated Epithelial-Mesenchymal Transition Anqi Li, Xiaojian Xiong, Chuang Nie, Zucan Luo, Xiaoling Deng, and 4 more This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-7024312/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 Post-COVID-19 pulmonary fibrosis (PCPF) presents an increasingly significant public health challenge. Although fibrotic mechanisms in PCPF are extensively studied, their exact pathogenesis remains unresolved. Interleukin-33 (IL-33), a critical alarmin in viral infections, including COVID-19, has been reported to drive fibrotic processes across multiple organs, yet its role in PCPF remains undefined. This study aims to investigate the specific contribution of IL-33 to PCPF. Methods Serum cytokine profiles (IL-1β, IL-6, IL-8, IL-10, IL-33, ST2) were analyzed across healthy controls, COVID-19 patients, and PCPF cohorts. In vitro , SARS-CoV-2 spike protein was applied to A549 alveolar epithelial cells to model viral infection, observing its impact on IL-33/ST2 signaling and epithelial-mesenchymal transition (EMT). Morphological changes and migration capacity were evaluated. Functional validation was performed using exogenous recombinant human IL-33 (rhIL-33), IL-33-overexpressing, and IL-33-knockdown A549 models to determine the role of IL-33 in EMT and fibrotic phenotypes. Mechanistic studies employed PPAR-γ agonism (rosiglitazone) in conjunction with spike stimulation to assess the regulation of the pathway. Results Serum levels of IL-33 and ST2 were remarkably elevated in PCPF patients compared to control groups and COVID-19 patients. In A549 cells, treatment with Spike protein (1000 ng/mL, 48 h) upregulated both extracellular and intracellular IL-33 and significantly increased the expression of IL-33 and ST2 at both mRNA and protein levels. Spike protein treatment further induced a spindle-shaped morphology, enhanced cell migration, and promoted EMT, as evidenced by decreased E-Cadherin and increased N-Cadherin, Vimentin, and α-SMA expression. Similar pro-EMT phenotype and migratory effects were observed upon exogenous rIL-33 administration or IL-33 overexpression. Conversely, IL-33 knockdown attenuated Spike protein-induced alterations in EMT markers. Mechanistically, Spike protein upregulated β-catenin while suppressing PPAR-γ protein expression—effects reversed by IL-33 knockdown. Co-treatment with rosiglitazone partially inhibited Spike protein-induced upregulation of N-Cadherin, Vimentin, and α-SMA and restored E-Cadherin expression. Conclusions Our findings demonstrate remarkable upregulation of IL-33 in PCPF. SARS-CoV-2 Spike protein drives alveolar EMT via IL-33/ST2-dependent β-catenin activation and PPAR-γ suppression, unveiling novel therapeutic targets for PCPF intervention. COVID-19 PCPF IL-33 Epithelial mesenchymal transformation (EMT) β-catenin/PPAR-γ Figures Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 Figure 6 Figure 7 Introduction SARS-CoV-2 infection can lead to persistent pulmonary and extrapulmonary symptoms, collectively referred to as post-acute sequelae of SARS-CoV-2 [ 1 ] . Fibrotic alterations with impaired lung function—designated post-COVID-19 pulmonary fibrosis (PCPF)—affect approximately 44.9% of COVID-19 survivors [ 2 , 3 ] . Patients typically present with dry cough, wheezing, chest pain, and dyspnea, which substantially impact patient outcomes and quality of life [ 4 ] . Currently, available antifibrotic therapies, including pirfenidone and nintedanib, offer limited efficacy and are associated with considerable side effects [ 5 , 6 ] . While research indicates that PCPF pathogenesis involves a combination of direct viral injury, immune dysregulation, and abnormal pulmonary repair [ 7 – 9 ] , the specific molecular mechanisms driving persistent fibrosis remain elusive, highlighting an urgent need for mechanistic insights and novel therapeutic targets. Interleukin-33 (IL-33), a member of the IL-1 cytokine family, exhibits dual functions in both physiological and pathological contexts [ 10 , 11 ] . It is released in response to cellular damage or viral infection and functions as an alarmin [ 12 ] . IL-33 binds its specific receptor, suppression of tumorigenicity 2 (ST2), and co-receptor IL-1 receptor accessory protein (IL-1RAcP), forming a trimeric complex that activates downstream pathways, such as NF-κB and MAPK, to promote disease progression [ 11 , 13 ] . Emerging evidence continues to shed light on the role of IL-33 in COVID-19 and its complications. Burke et al. [ 14 ] identified elevated serum IL-33 as a critical predictor of adverse outcomes in patients with COVID-19. Additionally, IL-33 is targeted by SARS-CoV-2-encoded microRNAs and co-expressed with ACE2 receptors in alveolar epithelium, further underscoring its pathogenic relevance [ 15 , 16 ] . The IL-33/ST2 axis promotes tissue remodeling through inflammation-regeneration imbalance [ 17 ] and has been shown to drive fibrosis in multiple organs, including the lungs, liver, and skin [ 18 ] . Notably, PCPF lung tissues demonstrate elevated IL-33 levels and increased abundance of type II alveolar epithelial cells compared to healthy controls, IPF patients, and COVID-19 patients [ 19 ] , suggesting that these cells may instigate fibrotic repair through IL-33 secretion. During the fibrotic progression, epithelial-mesenchymal transition (EMT) describes the partial or complete transformation of epithelial cells into mesenchymal phenotypes in response to cellular or environmental cues [ 20 ] . Substantial evidence confirms that alveolar epithelial EMT activation significantly contributes to the development of pulmonary fibrosis [ 21 ] . Indeed, SARS-CoV-2 spike protein has been shown to induce EMT in macrophages, fibroblasts, and bronchial epithelium [ 22 – 24 ] . Nevertheless, the precise mechanism by which SARS-CoV-2 influences EMT in pulmonary epithelial cells remains undefined. Of particular relevance, the IL-33/ST2 axis promotes EMT and collagen deposition in experimental fibrosis through multi-pathway activation [ 18 , 25 ] , positioning it as a plausible mediator of SARS-CoV-2-driven fibrogenesis. Therefore, we hypothesize that SARS-CoV-2 infection may facilitate the development of PCPF by inducing EMT in type II alveolar epithelial cells through the IL-33/ST2 axis. In this study, we investigate the role and molecular mechanisms of the IL-33/ST2 axis in PCPF through clinical serum analysis and in vitro approaches. We observed significantly elevated levels of IL-33 and ST2 in PCPF patients compared to healthy controls and COVID-19 patients. In vitro , SARS-CoV-2 spike protein activated IL-33/ST2 signaling and promoted EMT in A549 alveolar epithelial cells. Further analysis revealed that both exogenous and overexpressed IL-33 induced a pro-EMT phenotype, whereas IL-33 knockdown attenuated this effect. Mechanistically, we demonstrated that the IL-33/ST2 was mediated through β-catenin upregulation and PPAR-γ suppression. Rosiglitazone-activated PPAR-γ partially reversed spike-induced EMT. Thereby, our findings establish IL-33 as a novel target for PCPF, providing new theoretical insights for the treatment of this condition. Materials and Methods Clinical Samples collection A total of 79 serum samples were retrospectively collected from residual blood remaining after routine clinical tests at The First Affiliated Hospital of Nanchang University between May and July 2023. No additional blood draws were performed. This study received ethics approval (No. (2024)CDYFYYLK(08–074)) with specific waiver of informed consent granted for the retrospective use of de-identified specimens. The inclusion criteria are: 1) Confirmed COVID-19 diagnosis with or without pulmonary fibrosis. 2) Stable condition with normal organ and bone marrow function. Exclusion criteria: 1) Severe systemic organ dysfunction at sample collection, such as severe respiratory failure, heart failure, or liver/kidney impairment. 2) History of desmosis or malignant tumors. Withdrawal criteria: 1) Subsequent withdrawal of sample usage authorization. 2) Study termination for medical reasons. Cell culture A549 cells, which are epithelial-derived and possess stable alveolar epithelial cell characteristics, are commonly used as a model for type II alveolar epithelial cells. A549 cells in this study were obtained from Wuhan Pricella Biotechnology Co., Ltd. The cells were confirmed to maintain normal morphology and growth kinetics and were free from pathogen contamination. These cells were cultured in the complete DMEM medium containing 10% fetal bovine serum (FBS) and 1% penicillin-streptomycin at 37°C with 5% CO 2 in a humidified incubator. Cell viability assay A549 cells were seeded at a density of 1×10 4 cells/well in a 96-well plate overnight and incubated with escalating doses of Spike protein or exogenous recombinant human protein IL-33 (rhIL-33) for 24 h, 48h, or 72h. Cells without treatment were used as controls. The medium was then replaced with CCK8 reagent containing the complete medium, followed by incubation at 37°C for 2 hours. Absorbance at 450 nm was then measured using a multifunctional microplate reader (Thermo Fisher Scientific, Waltham, USA). The values were blank-subtracted and normalized to the untreated cell values to give relative cell viability. Enzyme‑linked immunosorbent assay (ELISA) Concentrations of cytokines, including IL-33, IL-1β, and IL-6, in human serum samples were determined using a commercial ELISA system according to the manufacturer's instructions (Elabscience, Wuhan, China). Concentrations of IL-33 and ST2 in cell supernatants and lysates were determined using commercial ELISA kits (Beyotime, Shanghai, China) according to the manufacturer's instructions. The optical density (OD) was measured at 450 nm with a multifunctional microplate reader (Thermo Fisher Scientific, Waltham, USA). Colony formation assay A549 cells were seeded in six-well plates (500 cells/well). Following adherence, cells were treated with exogenous Spike proteins at varying concentrations in culture medium for 1–2 weeks until macroscopic colonies formed. Colonies were fixed with 4% paraformaldehyde, stained with crystal violet solution, and air-dried upright at room temperature. Blue-violet colonies were photographed, and quantification was performed using Image J software. Scratch wound-healing assay A549 cells were seeded at a density of 1.2 × 10 ^5 cells per well and allowed to reach 90% confluence. A sterile pipette tip was used to make a straight scratch in the cell monolayer, followed by three PBS washes to remove debris and detached cells. The cells were then incubated with serum-free medium containing different concentrations of exogenouSpike proteins and cultured at 37°C for 24 or 48 hours. Images of the scratches were captured, and wound closure percentages were quantified using Image J software (Image Pro Plus 6.0, Media Cybernetics, USA). The migration rate, calculated as (initial scratch area - remaining scratch area)/initial scratch area×100%, reflected cell migration speed. Transwell migration assay To assess cell migration, transwells were placed in a 24-well plate. A549 Cells (4×10⁴/well) were seeded in the upper chamber in the serum-free medium containing varying concentrations of exogenous Spike proteins, while the complete medium supplemented with 20% FBS was placed in the lower chamber. Following 24- or 48-hour incubation, transwells were removed, washed twice with PBS, and fixed in 4% paraformaldehyde for 30 minutes at room temperature. Membranes were then stained with 1% crystal violet for 30 minutes. After three PBS washes, non-migrated cells on the upper membrane surface were gently removed. Once dried, membranes were imaged under a microscope with five random fields captured per group. Migrated cells were quantified using Image J software. Western Blotting (WB) Cells were lysed in RIPA buffer containing 1× protease inhibitor cocktail and then centrifuged at 12,000 g for 15 min at 4°C to collect the supernatants. The Bicinchoninic acid assay (BCA) kit (Absin, Shanghai, China) was used to determine the total protein concentrations. An equal volume of the samples was loaded and separated by 8–15% sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE). The target bands were transferred to a polyvinylidene difluoride (PVDF) membrane (Millipore, USA), which was then blocked with 5% non-fat milk and incubated with the corresponding primary antibodies overnight at 4°C. After that, the membranes were incubated with Horseradish peroxidase (HRP)-conjugated secondary antibodies (Santa Cruz Biotechnology, USA) for one hour at room temperature. Bands were visualized using an enhanced chemiluminescence kit (Proteintech, Wuhan, China) and imaged with a gel imaging system (Amersham ImageQuant™ 800, Cytiva, China). The WB images were analyzed using ImageJ software (ImageJ 1.52a, National Institutes of Health, USA). Real‑time polymerase chain reaction (RT-qPCR) Total RNA was extracted from A549 cells with TRIzol reagent. The RNA concentration was spectrophotometrically measured. Complementary DNA (cDNA) was reverse transcribed from an appropriate amount of RNA using a PCR Thermal Cycler (BioRad, USA) with Hifair® III 1st Strand cDNA Synthesis SuperMix for qPCR kit (Yeasen Biotechnology, Shanghai, China). Real-time PCR was performed using Hieff UNICON® Universal Blue qPCR SYBR Green Master Mix (Yeasen Biotechnology, Shanghai, China) on QuantStudio Dx Real-Time PCR instrument (Thermo Fisher Scientific, Waltham, USA) with the appropriate primer pairs. The mean cycle threshold (Ct) of the gene of interest was calculated from triplicate measurements and normalized to GAPDH, serving as the housekeeping standard. Fold changes of target mRNAs were analyzed using the 2 −∆∆CT method. The PCR primer sequences were as follows: GAPDH forward, 5’-ACCCAGAAGACTGTGGATGG − 3’ and reverse, 5’-TCAGCTCAGGGATGACCTTG-3’ and N-cadherin forward, 5’-AATCGTGTCTCAGGCTCCAA-3’ and reverse, 5’-TGGGATTGCCTTCCATGTCT-3’, E-cadherin forward, 5’-ACGCATTGCCACATACACTC-3’ and reverse, 5’-AGAGGTTCCTGGAAGAGCAC-3’, Vimentin forward, 5’-TTGCAGGAGGAGATGCTTCA-3’ and reverse, 5’-CCACTTTGCGTTCAAGGTCA-3’, α-SMA forward, 5’-CAGCCCTCCTTCATCGGTAT-3’ and reverse, 5’-TGATCTTCATGGTGCTGGGT-3’, IL-33 forward, 5’-TCCCAACAGAAGGCCAAAGA-3’ and reverse, 5’-AAAGGCAAAGCACTCCACAG-3’, and ST2 forward, 5’-TCCTGTGGCAGCTTAATGGA-3’ and reverse, 5’-ATGCAAATTCAGGGCCAGAC-3’. Lentivirus packaging and production To culture HEK-293T cells in 10-cm dishes until reaching 70–80% confluence for transfection. Lentiviral packaging plasmids (8 µg pLP1, 4 µg pLP2, 2 µg VSV-G) and the transfer plasmid (14 µg Sh- IL33 ) were complexed, and the transfection reagent mixture was prepared following the Lipofectamine 3000 protocol. After mixing, the solution was added dropwise to the HEK-293T cultures. After six hours, the medium was replaced with the complete medium, followed by incubation for 48 to 72 hours. The supernatant was collected and filtered through a 0.22 µm membrane, and the lentiviral stock was used directly for target cell infection. A549 cells were seeded in 10-cm dishes until attaining 60–70% confluence. Lentiviral infection was performed using a mixture of growth medium, lentiviral stock, and polybrene. After 72 h, the selection was initiated with 2 µg/mL puromycin. Statistical analysis GraphPad Prism 9 was used for statistical analysis and graph generation. A Student's t-test was applied for comparisons between two groups, and a one-way ANOVA was used for comparisons among multiple groups. Data are presented as the mean ± standard error of the mean (SEM), with p < 0.05 indicating statistical significance. Each experiment was repeated three times. Results High expression of IL‑33 in the serum of patients with PCPF To investigate the precise role of IL-33 in PCPF development, serum samples were collected from 25 clinically confirmed healthy individuals, 28 COVID-19 patients, and 26 PCPF patients (Table S1 ). Expression of IL-1β, IL-8, IL-6, IL-10, and IL-33 was analyzed by ELISA. As shown in Fig. 1 A&B, no significant differences in serum IL-1β and IL-8 levels were found among the three groups. Compared to the healthy control group, serum IL-6 levels were elevated in both COVID-19 and PCPF groups (Fig. 1 C), with no difference between the latter two. Serum IL-10 levels were substantially higher in COVID-19 and PCPF groups than in the healthy group (Fig. 1 D) and further increased in PCPF patients compared to COVID-19 patients. Notably, serum IL-33 levels were significantly higher in PCPF patients than in the other two groups (Fig. 1 E), and this elevation was specifically observed in PCPF patients. We then measured the levels of the IL-33-specific receptor ST2. As shown in Fig. 1 F, ST2 levels followed a similar pattern, being significantly higher in the PCPF group than in the other groups, which did not differ from each other. These results suggest that IL-33 may play a key role in the development of PCPF and serve as an independent risk factor for PCPF. SARS-CoV-2 Spike protein induced IL-33 and ST2 expression and secretion in vitro Having observed elevated IL-33 and ST2 levels in PCPF patients, we next investigated whether their expression is associated with SARS-CoV-2 infection. A549 cells were exposed to a concentration gradient of Spike protein for 24 and 48 hours, and cell viability was evaluated using the CCK-8 assay to determine optimal treatment conditions. Results demonstrated that only 1000 ng/mL of Spike protein, applied for 48 h, significantly reduced A549 cell viability (Fig. 2 A), suggesting that this dosage and duration effectively induced cellular damage. Since SARS-CoV-2 infection is known to injure alveolar epithelial cells and trigger pulmonary fibrosis directly, we adopted a 1000 ng/mL Spike protein treatment for 48 hours in subsequent studies. To investigate the role of IL-33 in PCPF, we assessed IL-33 expression in A549 cells following treatment with the Spike protein. ELISA revealed that after 48-hour exposure to the Spike protein, IL-33 levels in both cellular supernatants and lysates were significantly elevated compared to the PBS control group (P < 0.01) (Fig. 2 B and C). RT-qPCR and western blotting further confirmed that Spike protein treatment upregulated both IL-33 and its receptor ST2 at the mRNA and protein levels, respectively (Fig. 2 D and E). Collectively, these findings indicate that in vitro treatment with the SARS-CoV-2 Spike protein potently enhances the expression and secretion of IL-33 and ST2. SARS-CoV-2 Spike protein triggered EMT in A549 cells. We have observed that the Spike protein upregulates the expression and secretion of IL-33 and ST2 in vitro , but whether it induces pulmonary fibrosis remains unclear. Therefore, we further investigated the effects of the Spike protein on A549 cell morphology, biological functions, and the epithelial-mesenchymal transition (EMT) pathway, a hallmark signaling cascade in fibrosis. We first examined the morphological changes in A549 cells after treatment with the Spike protein. As shown in Fig. 3 A, cells treated with 1000 ng/mL Spike protein for 48 h exhibited increased intercellular spacing, reduced cell-cell contacts, and partial adoption of elongated spindle-shaped, stellate, or irregular morphologies. These alterations indicate that the Spike protein modifies A549 cell morphology. Next, transwell migration and wound-healing assays were conducted to assess the impact of the Spike protein on the biological functions of A549 cells. The results (Fig. 3 B&C) demonstrated that, compared to the PBS control group, Spike protein-treated A549 cells showed a significant increase in the number of migrated cells and the area of wound closure, suggesting promoted migration. Given the critical role of the EMT pathway in organ fibrosis, we used RT-qPCR and WB to analyze the Spike protein's effect on EMT-related molecules. As depicted in Fig. 3 D and E, Spike protein treatment upregulated both mRNA and protein expression levels of N-cadherin, Vimentin, and alpha-smooth muscle actin (α-SMA) in A549 cells while downregulating the mRNA and protein expression levels of E-cadherin. These results demonstrate that the SARS-CoV-2 Spike protein upregulates α-SMA expression and induces EMT in A549 cells. IL-33/ST2 axis activation promoted EMT To explore the potential link between Spike protein-induced EMT and IL-33 upregulation, we assessed the effects of rhIL-33 protein and IL-33 overexpression on EMT in A549 cells, aiming to determine whether IL-33 can replicate the phenotypic changes induced by the Spike protein. A549 cells were exposed to escalating doses of rhIL-33 (0, 5, 10, 20 ng/mL) for 48 h. We observed no differences in clonogenic capacity (Figure S1 A) or proliferation kinetics (Figure S1 B) versus controls. However, wound-healing assays showed that IL-33 treatment dose-dependently enhanced cell migration (Fig. 4 A). This indicates that IL-33 potentiates the migratory capacity of cells without affecting their proliferation. Subsequent analysis of EMT markers demonstrated that IL-33 treatment upregulated mesenchymal markers (ST2, N-cadherin, Vimentin, α-SMA) while downregulating epithelial marker E-cadherin in a dose-dependent manner at both transcriptional and translational levels (Fig. 4 B-C). To delve deeper into the relationship between IL-33 and EMT, the IL-33 sequence was delivered to A549 by a lentiviral vector GV492 to establish stable overexpressing cell lines. After lentiviral infection and single-clone selection, heightened IL-33 expression was validated via RT-qPCR, ELISA, and WB, which unveiled markedly elevated IL-33 mRNA and protein levels in the high-expression cohort relative to controls (Fig. 5 A-C). In these engineered cells, IL-33 overexpression significantly elevated the expression of its receptor, ST2, and mesenchymal markers (N-cadherin, Vimentin, α-SMA) while suppressing E-cadherin at both the transcriptional and protein levels (Fig. 5 D-F). These findings confirm that activation of the IL-33/ST2 axis markedly promotes EMT in A549 cells. SARS-CoV-2 Spike protein promoted EMT in vitro via the IL-33/ST2 axis. Our findings indicate that SARS-CoV-2 might cause pulmonary fibrosis by upregulating IL-33 to trigger EMT. To clarify the role of IL-33 in PCPF, we generated IL-33-knockdown A549 cell lines using lentiviral transduction and short hairpin RNA (shRNA) technology. shRNA-IL-33C group achieved the most pronounced IL-33 knockdown in A549 cells, as validated by RT-qPCR, ELISA, and WB (Fig. 6 A-B and Figure. S2). These cells, termed IL33-KD, along with shRNA-NC-transfected control cells, were used in subsequent studies. We then treated IL33-KD and control A549 cells with the Spike protein and assessed the expression of EMT-related proteins. As presented in Fig. 6 C-E, IL33-KD cells exhibited reduced expression of IL-33, ST2, N-Cadherin, Vimentin, and α-SMA, along with upregulated E-Cadherin compared to control cells. In control cells, treatment with the Spike protein resulted in an upregulation of these markers and a downregulation of E-cadherin. Interestingly, in IL33-KD cells, Spike protein treatment did not significantly alter the expression of N-Cadherin, α-SMA, or E-Cadherin compared to untreated cells, implying that knocking down IL-33 can partially reverse the Spike protein-induced EMT process. Overall, these findings suggest that the Spike protein stimulates EMT in A549 cells through the IL-33/ST2 signaling axis. SARS-CoV-2 Spike protein-induced EMT may involve IL-33/ST2-mediated regulation of β-catenin/PPAR-γ signaling To uncover the downstream pathways and mechanisms by which the IL-33/ST2 axis promotes EMT, we conducted a literature review and analyzed transcriptomic datasets from other research groups. Specifically, we examined data from lung cells treated with the SARS-CoV-2 Spike protein and from preadipocytes with IL-33 knockdown compared to their respective controls [ 23 , 26 ] . The analysis revealed that the differentially expressed genes from both the Spike protein-treated and IL-33-knockdown models were significantly enriched in the peroxisome proliferator-activated receptor (PPAR) signaling pathway [ 23 , 26 ] , which strongly suggests that the PPAR pathway may be regulated by IL-33 and potentially involved in SARS-CoV-2-related pathological processes. To investigate whether IL-33 mediates the effects of the Spike protein through the β-catenin/PPAR-γ pathway, we assessed the expression of β-catenin and PPAR-γ in IL-33-knockdown A549 cells treated with the Spike protein. As shown in Fig. 7 A, compared to the PBS control group, β-catenin expression was significantly upregulated, and PPAR-γ expression was downregulated in the Spike protein-treated group. However, following IL-33-knockdown, the Spike protein-induced upregulation of β-catenin was attenuated, and PPAR-γ expression was restored. Subsequently, we employed the PPAR-γ agonist rosiglitazone (20 µmol/L) to investigate further whether IL-33 mediates the Spike protein's effects through the β-catenin/PPAR-γ pathway. After pretreating A549 cells with rosiglitazone, we observed that the combined treatment of Spike protein and rosiglitazone partially reversed the expression of EMT markers compared to Spike protein treatment alone. Specifically, N-cadherin, Vimentin, and α-SMA levels were decreased. At the same time, E-cadherin expression was increased (Fig. 7 B). These results suggest that the Spike protein likely promotes EMT in A549 cells through the IL-33/ST2 axis by modulating the β-catenin/PPAR-γ pathway, thereby contributing to pulmonary fibrosis progression. Discussion PCPF imposes a substantial burden on global healthcare systems. While existing research has elucidated various mechanisms of COVID-19-induced pulmonary injury, the pathogenesis of PCPF remains poorly defined. Here, we analyzed cytokine in 79 clinical serum samples (healthy controls, COVID-19 patients, and PCPF patients), identifying IL-33 as the only cytokine specifically elevated in PCPF patients. Levels of its specific receptor ST2 mirrored this increase, suggesting a pivotal role for IL-33 in PCPF pathogenesis. To model SARS-CoV-2 infection in vitro , we treated A549 cells (surrogates for alveolar epithelial type II cells—the primary viral targets during early infection) with the SARS-CoV-2 Spike protein. Spike protein stimulation significantly upregulated both the expression and secretion of IL-33 and ST2 in A549 cells, further supporting the involvement of IL-33 in PCPF development. Concurrently, while the Spike protein has been shown to trigger inflammatory responses and activate EMT in macrophages, fibroblasts, and bronchial epithelial cells, its EMT-inducing effects on alveolar epithelial type II cells remain understudied [ 22 , 23 ] . Our results demonstrate that Spike protein robustly induces EMT in A549 cells. Critically, rhIL-33 treatment or IL-33 overexpression potentiated EMT, whereas IL-33 knockdown partially reversed Spike protein-induced EMT, which highly indicates that Spike protein promotes EMT in A549 cells primarily through IL-33-mediated signaling, thereby driving PCPF progression. To delineate the mechanism underlying IL-33-mediated EMT induction, we integrated a literature review with a re-analysis of published transcriptome datasets (lung cells ± Spike protein; preadipocytes ± IL-33 knockdown). Both IL-33 knockdown and Spike protein treatment consistently altered the PPAR signaling pathway, suggesting that IL-33 mediates the regulation of the PPAR pathway during PCPF pathogenesis. PPARs (PPAR-α, PPAR-β/δ, PPAR-γ), which are ligand-activated receptors in the nuclear hormone receptor family, have garnered significant interest, particularly PPAR-γ [ 27 , 28 ] . Recent studies indicate that SARS-CoV-2 downregulates PPAR-γ expression, disrupting PPAR signaling and inducing metabolic reprogramming that exacerbates pulmonary inflammation in infected lung epithelium [ 29 ] . Synthetic PPAR-γ agonists (e.g., rosiglitazone, pioglitazone) ameliorate SARS-CoV-2-induced hyperinflammation [ 30 ] . Notably, PPAR-γ demonstrates antifibrotic activity across multiple organs (liver, kidney, skin, lung) [ 31 – 34 ] . In PCPF, PPAR-γ activation modulates macrophage polarization toward the M2 phenotype during infection [ 35 ] . Its activator, cannabidiol, reduces cytokine secretion, inflammation, and pulmonary fibrosis [ 36 ] . β-catenin, a potent negative regulator of PPAR-γ, governs cell proliferation, differentiation, and adhesion; its dysregulation drives fibrogenesis in multiple organs [ 37 ] . Emerging evidence suggests that SARS-CoV-2-induced β-catenin hyperactivation suppresses PPAR-γ, thereby promoting organ fibrosis [ 38 ] . These findings prompted our focus on the β-catenin/PPAR-γ axis. Our study reveals that the Spike protein upregulates β-catenin while downregulating PPAR-γ expression in A549 cells. Strikingly, IL-33 knockdown reversed these effects. Furthermore, pretreatment with the PPAR-γ agonist rosiglitazone attenuated Spike protein-induced EMT, confirming the involvement of the β-catenin/PPAR-γ pathway. Collectively, we demonstrate that SARS-CoV-2 Spike protein activates β-catenin via the IL-33/ST2 axis, suppresses PPAR-γ, and consequently induces EMT to promote pulmonary fibrosis. Despite the advancements presented herein, certain limitations warrant consideration. First, biosafety constraints (BSL-2 compliance) precluded direct experimentation with live SARS-CoV-2; consequently, we utilized its Spike protein—the primary mediator of viral entry—as a biologically relevant surrogate. Second, while our cellular models robustly demonstrated IL-33/ST2 axis involvement in Spike protein-induced EMT, validation in animal models remains imperative to establish physiological relevance. Conclusions In summary, this study demonstrates that the SARS-CoV-2 Spike protein upregulates IL-33 in alveolar epithelium and orchestrates EMT through the IL-33/ST2-mediated modulation of the β-catenin/PPAR-γ signaling axis, thereby driving PCPF pathogenesis. Our findings nominate IL-33 as a novel therapeutic target for PCPF, providing a mechanistic foundation for antifibrotic strategy. Abbreviations PCPF post-COVID-19 pulmonary fibrosis EMT epithelial-mesenchymal transition IL-33 Interleukin-33 rhIL-33 recombinant human IL-33 ST2 suppression of tumorigenicity 2 RT-qPCR Real‑time polymerase chain reaction PPAR peroxisome proliferator-activated receptor Declarations Ethics approval and consent to participate This study was conducted in strict accordance with the Declaration of Helsinki and national ethical regulations (China's Ethical Review Measures for Biomedical Research Involving Human Subjects). All actions regarding clinical data collection and serum sample acquisition have been approved by the Ethics Committee of the First Affiliated Hospital of Nanchang University (Approval No.: (2024)CDYFYYLK(08-074)). Specific waiver of informed consent was granted in accordance with the following provisions: 1) Samples were collected retrospectively from anonymized residual biological material. 2) The research posed no additional risk to patients. 3) Patient privacy was strictly protected through de-identification procedures. Clinical trial number Not applicable. Consent for publication Not applicable. Availability of data and materials Not applicable. Competing interests The authors declare that they have no competing interests. Funding The work was supported by the Major Project of Technology Innovation 2030-the "Research on Prevention and Treatment of Cancer, Cardio-cerebrovascular, Respiratory and Metabolic Diseases" (Project No. 2023ZD0506200; Sub-project No. 2023ZD0506203), the National Natural Science Foundation of China (82360130), the Jiangxi Key Research and Development Program (20232BBG70020), the Jiangxi Science and Technology Cooperation Project (20244BDF60008) and the Jiangxi Provincial Key Laboratory of Prevention and Treatment of Infectious Diseases (2024SSY06031). Authors' contributions Anqi Li and Xiaojian Xiong conducted the experiments and drafted the manuscript. Chuang Nie and Zucan Luo contributed to the methodology and discussion of the results. Xiaoling Deng performed transcription data analysis. Yujie Wang was responsible for collecting clinical samples. Xiuhua Kang provided critical clinical data interpretation. Tianxin Xiang and Ying Ying designed the study, provided overall scientific supervision, and revised the manuscript. All the authors reviewed the draft and approved the final manuscript before submission. Acknowledgments We would like to thank the research team that shared their transcriptome sequencing data on the platform. References NALBANDIAN A, SEHGAL K, GUPTA A, et al. Post-acute COVID-19 syndrome [J]. Nat Med. 2021;27(4):601–15. GEORGE P M, WELLS A U, JENKINS RG. Pulmonary fibrosis and COVID-19: the potential role for antifibrotic therapy [J]. Lancet Respir Med. 2020;8(8):807–15. HAMA AMIN B J, KAKAMAD F H, AHMED GS, et al. Post COVID-19 pulmonary fibrosis; a meta-analysis study [J]. Ann Med Surg (Lond). 2022;77:103590. LEE JH, KOH J, JEON Y K, et al. An Integrated Radiologic-Pathologic Understanding of COVID-19 Pneumonia [J]. Radiology. 2023;306(2):e222600. BERMUDO-PELOCHE G, DEL RIO B, VICENS-ZYGMUNT V et al. Pirfenidone in post-COVID-19 pulmonary fibrosis (FIBRO-COVID): a phase 2 randomised clinical trial [J]. Eur Respir J, 2025, 65(4). LASSAN S, TESAR T, TISONOVA J, et al. Pharmacological approaches to pulmonary fibrosis following COVID-19 [J]. Front Pharmacol. 2023;14:1143158. HIRAWAT R, SAIFI M A GODUGUC. Targeting inflammatory cytokine storm to fight against COVID-19 associated severe complications [J]. Life Sci. 2021;267:118923. HUANG W J. TANG X X. Virus infection induced pulmonary fibrosis [J]. J Transl Med. 2021;19(1):496. JOHN A E, JOSEPH C, JENKINS G, et al. COVID-19 and pulmonary fibrosis: A potential role for lung epithelial cells and fibroblasts [J]. Immunol Rev. 2021;302(1):228–40. LIEW F Y, GIRARD J P TURNQUISTHR. Interleukin-33 in health and disease [J]. Nat Rev Immunol. 2016;16(11):676–89. CAYROL C, GIRARD JP. Interleukin-33 (IL-33): A nuclear cytokine from the IL-1 family [J]. Immunol Rev. 2018;281(1):154–68. MARTIN N T, MARTIN M U. Interleukin 33 is a guardian of barriers and a local alarmin [J]. Nat Immunol. 2016;17(2):122–31. SCHMITZ J, OWYANG A, OLDHAM E, et al. IL-33 is an interleukin-1-like cytokine that signals via the IL-1 receptor-related protein ST2 and induces T helper type 2-associated cytokines [J]. Immunity. 2005;23(5):479–90. BURKE H, FREEMAN A, CELLURA D C, et al. Inflammatory phenotyping predicts clinical outcome in COVID-19 [J]. Respir Res. 2020;21(1):245. AYDEMIR M N, AYDEMIR H B, KORKMAZ E M, et al. Computationally predicted SARS-COV-2 encoded microRNAs target NFKB, JAK/STAT and TGFB signaling pathways [J]. Gene Rep. 2021;22:101012. WRUCK W. SARS-CoV-2 receptor ACE2 is co-expressed with genes related to transmembrane serine proteases, viral entry, immunity and cellular stress [J]. Sci Rep. 2020;10(1):21415. GRIESENAUER B, PACZESNY S. The ST2/IL-33 Axis in Immune Cells during Inflammatory Diseases [J]. Front Immunol. 2017;8:475. KOTSIOU O S, GOURGOULIANIS K I, ZAROGIANNIS S G.. IL-33/ST2 Axis in Organ Fibrosis [J]. Front Immunol. 2018;9:2432. GAURAV R, ANDERSON D R, RADIO S J, et al. IL-33 Depletion in COVID-19 Lungs [J]. Chest. 2021;160(5):1656–9. LAMOUILLE S, XU J. Molecular mechanisms of epithelial-mesenchymal transition [J]. Nat Rev Mol Cell Biol. 2014;15(3):178–96. WANG XC, SONG K, TU B, et al. New aspects of the epigenetic regulation of EMT related to pulmonary fibrosis [J]. Eur J Pharmacol. 2023;956:175959. KHAN S, SHAFIEI M S, LONGORIA C et al. SARS-CoV-2 spike protein induces inflammation via TLR2-dependent activation of the NF-kappaB pathway [J]. Elife, 2021, 10. CAI J, MA W, WANG X, et al. The spike protein of SARS-CoV-2 induces inflammation and EMT of lung epithelial cells and fibroblasts through the upregulation of GADD45A [J]. Open Med (Wars). 2023;18(1):20230779. PI P, ZENG Z. Molecular mechanisms of COVID-19-induced pulmonary fibrosis and epithelial-mesenchymal transition [J]. Front Pharmacol. 2023;14:1218059. XU J, ZHENG J, SONG P, et al. IL–33/ST2 pathway in a bleomycin–induced pulmonary fibrosis model [J]. Mol Med Rep. 2016;14(2):1704–8. XU D, ZHUANG S, CHEN H, et al. IL-33 regulates adipogenesis via Wnt/beta-catenin/PPAR-gamma signaling pathway in preadipocytes [J]. J Transl Med. 2024;22(1):363. BERGER J, MOLLER D E. The mechanisms of action of PPARs [J]. Annu Rev Med. 2002;53:409–35. HERNANDEZ-QUILES M, BROEKEMA M F KALKHOVENE. PPARgamma in Metabolism, Immunity, and Cancer: Unified and Diverse Mechanisms of Action [J]. Front Endocrinol (Lausanne). 2021;12:624112. HEFFERNAN K S, RANADIVE S M, JAE S Y. Exercise as medicine for COVID-19: On PPAR with emerging pharmacotherapy [J]. Med Hypotheses. 2020;143:110197. CIAVARELLA C, MOTTA I. VALENTE S, Pharmacological (or Synthetic) and Nutritional Agonists of PPAR-gamma as Candidates for Cytokine Storm Modulation in COVID-19 Disease [J]. Molecules, 2020, 25(9). BONILLA-MARTINEZ R BOATENGE, AHLEMEYER B, et al. It takes two peroxisome proliferator-activated receptors (PPAR-beta/delta and PPAR-gamma) to tango idiopathic pulmonary fibrosis [J]. Respir Res. 2024;25(1):345. SON M, KIM G Y, YANG Y et al. PPAR Pan Agonist MHY2013 Alleviates Renal Fibrosis in a Mouse Model by Reducing Fibroblast Activation and Epithelial Inflammation [J]. Int J Mol Sci, 2023, 24(5). LIU X, XU J, ROSENTHAL S, et al. Identification of Lineage-Specific Transcription Factors That Prevent Activation of Hepatic Stellate Cells and Promote Fibrosis Resolution [J]. Gastroenterology. 2020;158(6):1728–e4414. RUZEHAJI N, FRANTZ C, PONSOYE M, et al. Pan PPAR agonist IVA337 is effective in prevention and treatment of experimental skin fibrosis [J]. Ann Rheum Dis. 2016;75(12):2175–83. BATABYAL R, FREISHTAT N. Metabolic dysfunction and immunometabolism in COVID-19 pathophysiology and therapeutics [J]. Int J Obes (Lond). 2021;45(6):1163–9. ESPOSITO G, PESCE M. The potential of cannabidiol in the COVID-19 pandemic [J]. Br J Pharmacol. 2020;177(21):4967–70. NUSSE R. Wnt/beta-Catenin Signaling, Disease, and Emerging Therapeutic Modalities [J]. Cell. 2017;169(6):985–99. VALLEE A, LECARPENTIER Y, VALLEE JN. Interplay of Opposing Effects of the WNT/beta-Catenin Pathway and PPARgamma and Implications for SARS-CoV2 Treatment [J]. Front Immunol. 2021;12:666693. Additional Declarations No competing interests reported. Supplementary Files Additionalfiles20250705.docx Additional file 1: Table. S1 Baseline characteristics of patients providing serum samples. Figure. S1 IL-33 does not alter the proliferative capacity of A549 cells. (A) Colony formation assay following 48 h treatment with graded concentrations of exogenous rhIL-33. (B) Cell viability was assessed by CCK-8 assay. ns = not significant (P>0.05, one-way ANOVA). Figure. S2shRNA-mediated stable knockdown A549 cell lines were established. (A) IL-33 quantified by ELISA. (B) Quantification of IL-33 protein expression. Significance denoted as *P<0.05, **P<0.01. Cite Share Download PDF Status: Posted Version 1 posted You are reading this latest preprint version Research Square lets you share your work early, gain feedback from the community, and start making changes to your manuscript prior to peer review in a journal. As a division of Research Square Company, we’re committed to making research communication faster, fairer, and more useful. We do this by developing innovative software and high quality services for the global research community. Our growing team is made up of researchers and industry professionals working together to solve the most critical problems facing scientific publishing. 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-7024312","acceptedTermsAndConditions":true,"allowDirectSubmit":true,"archivedVersions":[],"articleType":"Research Article","associatedPublications":[],"authors":[{"id":509498186,"identity":"29475802-fdf2-46a6-bf9a-686bb73538fe","order_by":0,"name":"Anqi Li","email":"","orcid":"","institution":"Nanchang University","correspondingAuthor":false,"prefix":"","firstName":"Anqi","middleName":"","lastName":"Li","suffix":""},{"id":509498187,"identity":"3d318299-e63f-400e-ad2f-cfd3fc1ef914","order_by":1,"name":"Xiaojian Xiong","email":"","orcid":"","institution":"Nanchang University","correspondingAuthor":false,"prefix":"","firstName":"Xiaojian","middleName":"","lastName":"Xiong","suffix":""},{"id":509498188,"identity":"1b548c55-4ceb-4f71-a13e-73a2c03f7456","order_by":2,"name":"Chuang Nie","email":"","orcid":"","institution":"Nanchang University","correspondingAuthor":false,"prefix":"","firstName":"Chuang","middleName":"","lastName":"Nie","suffix":""},{"id":509498189,"identity":"a824051a-f85b-4092-bd36-c426609161fe","order_by":3,"name":"Zucan Luo","email":"","orcid":"","institution":"Nanchang University","correspondingAuthor":false,"prefix":"","firstName":"Zucan","middleName":"","lastName":"Luo","suffix":""},{"id":509498190,"identity":"39c51c9e-dec5-4ede-94b8-91dd9d0a2e9c","order_by":4,"name":"Xiaoling Deng","email":"","orcid":"","institution":"Nanchang University","correspondingAuthor":false,"prefix":"","firstName":"Xiaoling","middleName":"","lastName":"Deng","suffix":""},{"id":509498191,"identity":"e7a5f468-d367-4747-8c40-0302564c34a1","order_by":5,"name":"Yujie Wang","email":"","orcid":"","institution":"Nanchang University","correspondingAuthor":false,"prefix":"","firstName":"Yujie","middleName":"","lastName":"Wang","suffix":""},{"id":509498192,"identity":"bb7c2263-23e9-4adc-b971-d5aff59352bd","order_by":6,"name":"Xiuhua Kang","email":"","orcid":"","institution":"Nanchang University","correspondingAuthor":false,"prefix":"","firstName":"Xiuhua","middleName":"","lastName":"Kang","suffix":""},{"id":509498193,"identity":"f969a4fc-fcf9-4cb3-b66b-ec5de5fd0652","order_by":7,"name":"Ying Ying","email":"","orcid":"","institution":"Nanchang University","correspondingAuthor":false,"prefix":"","firstName":"Ying","middleName":"","lastName":"Ying","suffix":""},{"id":509498194,"identity":"4e844516-a0f8-4315-889a-034b160f5843","order_by":8,"name":"Tianxin Xiang","email":"data:image/png;base64,iVBORw0KGgoAAAANSUhEUgAAAZAAAAAyAQMAAABI0h/eAAAABlBMVEX///8AAABVwtN+AAAACXBIWXMAAA7EAAAOxAGVKw4bAAAA8klEQVRIiWNgGAWjYFCCBIYPDAY2cvbtzQcgAgcIa2GcwVCRZmzAcyyBFC1nDidukMgxIE6LOXuOYTNvGzPjdp4z3x78bGOQ47uRwPi5AI8Wy543IC1szJbtvdsNe9sYjCVvJDBLz8CjxeBGjvlj3jYeNoYzZ7dJM7YxJG64kcDGzINfC8gWCR6GGznPQFrqidPCc8ZAAshgA2lJMCCo5cyzwsY5FQkGkj3HzCR7zkkYzjzzsFkar5bjyRsb3hj8r+9nb34m8aPMRp7vePLBz/i0MDBwGDAhKZAAYsYGvBoYGNgfMP4goGQUjIJRMApGOAAAEDRRbC51sa4AAAAASUVORK5CYII=","orcid":"","institution":"Nanchang University","correspondingAuthor":true,"prefix":"","firstName":"Tianxin","middleName":"","lastName":"Xiang","suffix":""}],"badges":[],"createdAt":"2025-07-02 02:23:07","currentVersionCode":1,"declarations":"","doi":"10.21203/rs.3.rs-7024312/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-7024312/v1","draftVersion":[],"editorialEvents":[],"editorialNote":"","failedWorkflow":false,"files":[{"id":90908619,"identity":"568b84d8-03bf-4e73-bdcb-3d4a239f8aa9","added_by":"auto","created_at":"2025-09-09 13:27:22","extension":"jpeg","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":149733,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eSerum cytokine levels across clinical cohorts.\u003c/strong\u003e (A) IL-1β. (B) IL-8. (C) IL-6. (D) IL-10. (E) IL-33. (F) ST2 receptor. Significance denoted as *\u003cem\u003eP\u003c/em\u003e\u0026lt;0.05, **\u003cem\u003eP\u003c/em\u003e\u0026lt;0.01, ***\u003cem\u003eP\u003c/em\u003e\u0026lt;0.001, ****\u003cem\u003eP\u003c/em\u003e\u0026lt;0.0001.\u003c/p\u003e","description":"","filename":"floatimage1.jpeg","url":"https://assets-eu.researchsquare.com/files/rs-7024312/v1/b785adafbfcc300ef4442cf1.jpeg"},{"id":90908621,"identity":"1acab1e5-2328-4f69-aac9-b01b7dae8de4","added_by":"auto","created_at":"2025-09-09 13:27:22","extension":"png","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":190305,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eSARS-CoV-2 Spike protein upregulates the expression of IL-33 and ST2 in A549 cells. \u003c/strong\u003e(A) Cell viability following 24 h or 48 h treatment with graded Spike protein concentrations. (B) Secreted IL-33 levels in supernatants. (C) Total IL-33 levels in cell lysates. (D) mRNA expression of IL-33 and ST2. (E) Protein expression of IL-33 and ST2. Data in B-E from cells treated with 1000 ng/mL Spike protein for 48 h. Significance denoted as *\u003cem\u003eP\u003c/em\u003e\u0026lt;0.05, **\u003cem\u003eP\u003c/em\u003e\u0026lt;0.01.\u003c/p\u003e","description":"","filename":"floatimage26.png","url":"https://assets-eu.researchsquare.com/files/rs-7024312/v1/db7f67cc77d9769e6bd71523.png"},{"id":90910116,"identity":"6dfcc795-cc7b-4f46-8cc1-b78856b80cd0","added_by":"auto","created_at":"2025-09-09 13:35:22","extension":"jpeg","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":507308,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eSARS-CoV-2 Spike protein triggers EMT in A549 cells.\u003c/strong\u003e (A) Morphological alterations (scale bar: 50 mm). (B, C) Cell migration by transwell assay (B) and scratch wound assay (C). (D) mRNA expression of EMT-related markers. (E) Protein expression of EMT-related markers. Cells were treated with 1000 ng/mL Spike protein for 48 h in all panels. Significance denoted as *\u003cem\u003eP\u003c/em\u003e\u0026lt;0.05, **\u003cem\u003eP\u003c/em\u003e\u0026lt;0.01.\u003c/p\u003e","description":"","filename":"floatimage3.jpeg","url":"https://assets-eu.researchsquare.com/files/rs-7024312/v1/8c78138c96fa9d6a488bcc4d.jpeg"},{"id":90908625,"identity":"8ddae7a6-c244-4976-8ff4-35564d1ed83c","added_by":"auto","created_at":"2025-09-09 13:27:22","extension":"jpeg","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":528681,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eExogenous rhIL-33 promotes migration and EMT in A549 cells. \u003c/strong\u003e(A) Cell migration by scratch assay after 24 h and 48 h treatment with graded concentrations of rIL-33. (B) mRNA expression of ST2, N-cadherin, E-cadherin, Vimentin, and α-SMA by RT-qPCR. (C, D) Immunoblots and quantification of protein expression. *\u003cem\u003eP\u003c/em\u003e\u0026lt;0.05, **\u003cem\u003eP\u003c/em\u003e\u0026lt;0.01, ***\u003cem\u003eP\u003c/em\u003e\u0026lt;0.001.\u003c/p\u003e","description":"","filename":"floatimage4.jpeg","url":"https://assets-eu.researchsquare.com/files/rs-7024312/v1/8c89fbe59ec89ded01d66469.jpeg"},{"id":90910117,"identity":"e375bbf0-1a55-4a57-b6af-7c779106eb8c","added_by":"auto","created_at":"2025-09-09 13:35:23","extension":"jpeg","order_by":5,"title":"Figure 5","display":"","copyAsset":false,"role":"figure","size":389753,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eIL-33 overexpression promotes EMT in A549 cells. \u003c/strong\u003e(A) IL-33 mRNA expression post-transfection. (B) IL-33 protein levels post-transfection by immunoblotting. (C) IL-33 protein levels quantified by ELISA. (D)mRNA expression of ST2, N-cadherin, E-cadherin, Vimentin, and α-SMA in IL-33-overexpression A549 cells. (E, F) Immunoblots and quantification of protein expression in IL-33-overexpression A549 cells. *\u003cem\u003eP\u003c/em\u003e\u0026lt;0.05, **\u003cem\u003eP\u003c/em\u003e\u0026lt;0.01, ***\u003cem\u003eP\u003c/em\u003e\u0026lt;0.001, ****\u003cem\u003eP\u003c/em\u003e\u0026lt;0.0001.\u003c/p\u003e","description":"","filename":"floatimage5.jpeg","url":"https://assets-eu.researchsquare.com/files/rs-7024312/v1/4c0cbbfa4220ab0539022a34.jpeg"},{"id":90911469,"identity":"7a6f66a1-2b53-4b93-ace3-ffb07388581e","added_by":"auto","created_at":"2025-09-09 13:43:23","extension":"jpeg","order_by":6,"title":"Figure 6","display":"","copyAsset":false,"role":"figure","size":373066,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eSpike protein promotes EMT through the IL-33/ST2 axis in A549 cells. \u003c/strong\u003e(A) IL-33 mRNA expression in shRNA-mediated knockdown cells. (B) Cellular IL-33 protein levels in shRNA-mediated knockdown cells by immunoblotting. (C) Protein expression profiles of IL-33, ST2, N-cadherin, E-cadherin, Vimentin, and α-SMA in control and IL-33-knockdown cells ± Spike protein (1000 ng/mL, 48 h). (D) Quantification of IL-33 /ST2 expression. (E) Quantification of EMT-related expression. Significance denoted as *\u003cem\u003eP\u003c/em\u003e\u0026lt;0.05, **\u003cem\u003eP\u003c/em\u003e\u0026lt;0.01, ***\u003cem\u003eP\u003c/em\u003e\u0026lt;0.001.\u003c/p\u003e","description":"","filename":"floatimage6.jpeg","url":"https://assets-eu.researchsquare.com/files/rs-7024312/v1/3d9336545eac0b41e02e8b00.jpeg"},{"id":90908623,"identity":"db59e7c7-f0da-4f06-beed-dacf5ac18c77","added_by":"auto","created_at":"2025-09-09 13:27:22","extension":"jpeg","order_by":7,"title":"Figure 7","display":"","copyAsset":false,"role":"figure","size":204297,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eSpike protein induces EMT through the IL-33/β-catenin/PPAR-γ axis.\u003c/strong\u003e (A) Protein expression of β-catenin and PPAR-γ in IL-33- knockdown cells. (B) EMT-related protein expression in control and IL-33-knockdown cells treated with Spike protein ± rosiglitazone (PPAR-γ agonist). Significance denoted as *\u003cem\u003eP\u003c/em\u003e\u0026lt;0.05, **\u003cem\u003eP\u003c/em\u003e\u0026lt;0.01, ***\u003cem\u003eP\u003c/em\u003e\u0026lt;0.001, ****\u003cem\u003eP\u003c/em\u003e\u0026lt;0.0001.\u003c/p\u003e","description":"","filename":"floatimage7.jpeg","url":"https://assets-eu.researchsquare.com/files/rs-7024312/v1/f0719cd2607a91f4971bd7a3.jpeg"},{"id":93163905,"identity":"ac054513-78bd-47ce-8e74-2c350ac0fef4","added_by":"auto","created_at":"2025-10-09 17:31:41","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":3320978,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-7024312/v1/2e65718b-9ce4-4e31-97eb-c666f314ae91.pdf"},{"id":90908630,"identity":"b98f6dca-f91d-4cc2-a2ad-c640cc634c73","added_by":"auto","created_at":"2025-09-09 13:27:23","extension":"docx","order_by":1,"title":"","display":"","copyAsset":false,"role":"supplement","size":1695684,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eAdditional file 1:\u003c/strong\u003e \u003cstrong\u003eTable. S1\u003c/strong\u003e Baseline characteristics of patients providing serum samples. \u003cstrong\u003eFigure. S1\u003c/strong\u003e IL-33 does not alter the proliferative capacity of A549 cells. (A) Colony formation assay following 48 h treatment with graded concentrations of exogenous rhIL-33. (B) Cell viability was assessed by CCK-8 assay. ns = not significant (P\u0026gt;0.05, one-way ANOVA). \u003cstrong\u003eFigure. S2\u003c/strong\u003eshRNA-mediated stable knockdown A549 cell lines were established. (A) IL-33 quantified by ELISA. (B) Quantification of IL-33 protein expression. Significance denoted as *P\u0026lt;0.05, **P\u0026lt;0.01.\u003c/p\u003e","description":"","filename":"Additionalfiles20250705.docx","url":"https://assets-eu.researchsquare.com/files/rs-7024312/v1/e4b2f361c86503e9a3800bf1.docx"}],"financialInterests":"No competing interests reported.","formattedTitle":"The IL-33/ST2 Axis Promotes Post-COVID-19 Pulmonary Fibrosis Through β-catenin/PPAR-γ-mediated Epithelial-Mesenchymal Transition","fulltext":[{"header":"Introduction","content":"\u003cp\u003eSARS-CoV-2 infection can lead to persistent pulmonary and extrapulmonary symptoms, collectively referred to as post-acute sequelae of SARS-CoV-2 \u003csup\u003e[\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e]\u003c/sup\u003e. Fibrotic alterations with impaired lung function\u0026mdash;designated post-COVID-19 pulmonary fibrosis (PCPF)\u0026mdash;affect approximately 44.9% of COVID-19 survivors\u003csup\u003e[\u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e, \u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e]\u003c/sup\u003e. Patients typically present with dry cough, wheezing, chest pain, and dyspnea, which substantially impact patient outcomes and quality of life \u003csup\u003e[\u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e4\u003c/span\u003e]\u003c/sup\u003e. Currently, available antifibrotic therapies, including pirfenidone and nintedanib, offer limited efficacy and are associated with considerable side effects\u003csup\u003e[\u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e5\u003c/span\u003e, \u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e6\u003c/span\u003e]\u003c/sup\u003e. While research indicates that PCPF pathogenesis involves a combination of direct viral injury, immune dysregulation, and abnormal pulmonary repair\u003csup\u003e[\u003cspan additionalcitationids=\"CR8\" citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e9\u003c/span\u003e]\u003c/sup\u003e, the specific molecular mechanisms driving persistent fibrosis remain elusive, highlighting an urgent need for mechanistic insights and novel therapeutic targets.\u003c/p\u003e\u003cp\u003eInterleukin-33 (IL-33), a member of the IL-1 cytokine family, exhibits dual functions in both physiological and pathological contexts \u003csup\u003e[\u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e10\u003c/span\u003e, \u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e]\u003c/sup\u003e. It is released in response to cellular damage or viral infection and functions as an alarmin\u003csup\u003e[\u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e12\u003c/span\u003e]\u003c/sup\u003e. IL-33 binds its specific receptor, suppression of tumorigenicity 2 (ST2), and co-receptor IL-1 receptor accessory protein (IL-1RAcP), forming a trimeric complex that activates downstream pathways, such as NF-κB and MAPK, to promote disease progression \u003csup\u003e[\u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e, \u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e13\u003c/span\u003e]\u003c/sup\u003e. Emerging evidence continues to shed light on the role of IL-33 in COVID-19 and its complications. Burke et al. \u003csup\u003e[\u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e14\u003c/span\u003e]\u003c/sup\u003e identified elevated serum IL-33 as a critical predictor of adverse outcomes in patients with COVID-19. Additionally, IL-33 is targeted by SARS-CoV-2-encoded microRNAs and co-expressed with ACE2 receptors in alveolar epithelium, further underscoring its pathogenic relevance \u003csup\u003e[\u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e15\u003c/span\u003e, \u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e16\u003c/span\u003e]\u003c/sup\u003e. The IL-33/ST2 axis promotes tissue remodeling through inflammation-regeneration imbalance\u003csup\u003e[\u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e17\u003c/span\u003e]\u003c/sup\u003e and has been shown to drive fibrosis in multiple organs, including the lungs, liver, and skin\u003csup\u003e[\u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e18\u003c/span\u003e]\u003c/sup\u003e. Notably, PCPF lung tissues demonstrate elevated IL-33 levels and increased abundance of type II alveolar epithelial cells compared to healthy controls, IPF patients, and COVID-19 patients \u003csup\u003e[\u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e19\u003c/span\u003e]\u003c/sup\u003e, suggesting that these cells may instigate fibrotic repair through IL-33 secretion.\u003c/p\u003e\u003cp\u003eDuring the fibrotic progression, epithelial-mesenchymal transition (EMT) describes the partial or complete transformation of epithelial cells into mesenchymal phenotypes in response to cellular or environmental cues\u003csup\u003e[\u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e20\u003c/span\u003e]\u003c/sup\u003e. Substantial evidence confirms that alveolar epithelial EMT activation significantly contributes to the development of pulmonary fibrosis \u003csup\u003e[\u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e21\u003c/span\u003e]\u003c/sup\u003e. Indeed, SARS-CoV-2 spike protein has been shown to induce EMT in macrophages, fibroblasts, and bronchial epithelium\u003csup\u003e[\u003cspan additionalcitationids=\"CR23\" citationid=\"CR22\" class=\"CitationRef\"\u003e22\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e24\u003c/span\u003e]\u003c/sup\u003e. Nevertheless, the precise mechanism by which SARS-CoV-2 influences EMT in pulmonary epithelial cells remains undefined. Of particular relevance, the IL-33/ST2 axis promotes EMT and collagen deposition in experimental fibrosis through multi-pathway activation\u003csup\u003e[\u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e18\u003c/span\u003e, \u003cspan citationid=\"CR25\" class=\"CitationRef\"\u003e25\u003c/span\u003e]\u003c/sup\u003e, positioning it as a plausible mediator of SARS-CoV-2-driven fibrogenesis. Therefore, we hypothesize that SARS-CoV-2 infection may facilitate the development of PCPF by inducing EMT in type II alveolar epithelial cells through the IL-33/ST2 axis.\u003c/p\u003e\u003cp\u003eIn this study, we investigate the role and molecular mechanisms of the IL-33/ST2 axis in PCPF through clinical serum analysis and \u003cem\u003ein vitro\u003c/em\u003e approaches. We observed significantly elevated levels of IL-33 and ST2 in PCPF patients compared to healthy controls and COVID-19 patients. \u003cem\u003eIn vitro\u003c/em\u003e, SARS-CoV-2 spike protein activated IL-33/ST2 signaling and promoted EMT in A549 alveolar epithelial cells. Further analysis revealed that both exogenous and overexpressed IL-33 induced a pro-EMT phenotype, whereas IL-33 knockdown attenuated this effect. Mechanistically, we demonstrated that the IL-33/ST2 was mediated through β-catenin upregulation and PPAR-γ suppression. Rosiglitazone-activated PPAR-γ partially reversed spike-induced EMT. Thereby, our findings establish IL-33 as a novel target for PCPF, providing new theoretical insights for the treatment of this condition.\u003c/p\u003e"},{"header":"Materials and Methods","content":"\u003cp\u003e\u003cb\u003eClinical Samples collection\u003c/b\u003e\u003c/p\u003e\u003cp\u003eA total of 79 serum samples were retrospectively collected from residual blood remaining after routine clinical tests at The First Affiliated Hospital of Nanchang University between May and July 2023. No additional blood draws were performed. This study received ethics approval (No. (2024)CDYFYYLK(08\u0026ndash;074)) with specific waiver of informed consent granted for the retrospective use of de-identified specimens. The inclusion criteria are: 1) Confirmed COVID-19 diagnosis with or without pulmonary fibrosis. 2) Stable condition with normal organ and bone marrow function. Exclusion criteria: 1) Severe systemic organ dysfunction at sample collection, such as severe respiratory failure, heart failure, or liver/kidney impairment. 2) History of desmosis or malignant tumors. Withdrawal criteria: 1) Subsequent withdrawal of sample usage authorization. 2) Study termination for medical reasons.\u003c/p\u003e\u003cp\u003e\u003cb\u003eCell culture\u003c/b\u003e\u003c/p\u003e\u003cp\u003eA549 cells, which are epithelial-derived and possess stable alveolar epithelial cell characteristics, are commonly used as a model for type II alveolar epithelial cells. A549 cells in this study were obtained from Wuhan Pricella Biotechnology Co., Ltd. The cells were confirmed to maintain normal morphology and growth kinetics and were free from pathogen contamination. These cells were cultured in the complete DMEM medium containing 10% fetal bovine serum (FBS) and 1% penicillin-streptomycin at 37\u0026deg;C with 5% CO\u003csub\u003e2\u003c/sub\u003e in a humidified incubator.\u003c/p\u003e\u003cp\u003e\u003cb\u003eCell viability assay\u003c/b\u003e\u003c/p\u003e\u003cp\u003eA549 cells were seeded at a density of 1\u0026times;10\u003csup\u003e4\u003c/sup\u003e cells/well in a 96-well plate overnight and incubated with escalating doses of Spike protein or exogenous recombinant human protein IL-33 (rhIL-33) for 24 h, 48h, or 72h. Cells without treatment were used as controls. The medium was then replaced with CCK8 reagent containing the complete medium, followed by incubation at 37\u0026deg;C for 2 hours. Absorbance at 450 nm was then measured using a multifunctional microplate reader (Thermo Fisher Scientific, Waltham, USA). The values were blank-subtracted and normalized to the untreated cell values to give relative cell viability.\u003c/p\u003e\u003cp\u003e\u003cb\u003eEnzyme‑linked immunosorbent assay (ELISA)\u003c/b\u003e\u003c/p\u003e\u003cp\u003eConcentrations of cytokines, including IL-33, IL-1β, and IL-6, in human serum samples were determined using a commercial ELISA system according to the manufacturer's instructions (Elabscience, Wuhan, China). Concentrations of IL-33 and ST2 in cell supernatants and lysates were determined using commercial ELISA kits (Beyotime, Shanghai, China) according to the manufacturer's instructions. The optical density (OD) was measured at 450 nm with a multifunctional microplate reader (Thermo Fisher Scientific, Waltham, USA).\u003c/p\u003e\u003cp\u003e\u003cb\u003eColony formation assay\u003c/b\u003e\u003c/p\u003e\u003cp\u003eA549 cells were seeded in six-well plates (500 cells/well). Following adherence, cells were treated with exogenous Spike proteins at varying concentrations in culture medium for 1\u0026ndash;2 weeks until macroscopic colonies formed. Colonies were fixed with 4% paraformaldehyde, stained with crystal violet solution, and air-dried upright at room temperature. Blue-violet colonies were photographed, and quantification was performed using Image J software.\u003c/p\u003e\u003cp\u003e\u003cb\u003eScratch wound-healing assay\u003c/b\u003e\u003c/p\u003e\u003cp\u003eA549 cells were seeded at a density of 1.2 \u0026times; 10 \u003csup\u003e^5\u003c/sup\u003e cells per well and allowed to reach 90% confluence. A sterile pipette tip was used to make a straight scratch in the cell monolayer, followed by three PBS washes to remove debris and detached cells. The cells were then incubated with serum-free medium containing different concentrations of exogenouSpike proteins and cultured at 37\u0026deg;C for 24 or 48 hours. Images of the scratches were captured, and wound closure percentages were quantified using Image J software (Image Pro Plus 6.0, Media Cybernetics, USA). The migration rate, calculated as (initial scratch area - remaining scratch area)/initial scratch area\u0026times;100%, reflected cell migration speed.\u003c/p\u003e\u003cp\u003e\u003cb\u003eTranswell migration assay\u003c/b\u003e\u003c/p\u003e\u003cp\u003eTo assess cell migration, transwells were placed in a 24-well plate. A549 Cells (4\u0026times;10⁴/well) were seeded in the upper chamber in the serum-free medium containing varying concentrations of exogenous Spike proteins, while the complete medium supplemented with 20% FBS was placed in the lower chamber. Following 24- or 48-hour incubation, transwells were removed, washed twice with PBS, and fixed in 4% paraformaldehyde for 30 minutes at room temperature. Membranes were then stained with 1% crystal violet for 30 minutes. After three PBS washes, non-migrated cells on the upper membrane surface were gently removed. Once dried, membranes were imaged under a microscope with five random fields captured per group. Migrated cells were quantified using Image J software.\u003c/p\u003e\u003cp\u003e\u003cb\u003eWestern Blotting (WB)\u003c/b\u003e\u003c/p\u003e\u003cp\u003eCells were lysed in RIPA buffer containing 1\u0026times; protease inhibitor cocktail and then centrifuged at 12,000 g for 15 min at 4\u0026deg;C to collect the supernatants. The Bicinchoninic acid assay (BCA) kit (Absin, Shanghai, China) was used to determine the total protein concentrations. An equal volume of the samples was loaded and separated by 8\u0026ndash;15% sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE). The target bands were transferred to a polyvinylidene difluoride (PVDF) membrane (Millipore, USA), which was then blocked with 5% non-fat milk and incubated with the corresponding primary antibodies overnight at 4\u0026deg;C. After that, the membranes were incubated with Horseradish peroxidase (HRP)-conjugated secondary antibodies (Santa Cruz Biotechnology, USA) for one hour at room temperature. Bands were visualized using an enhanced chemiluminescence kit (Proteintech, Wuhan, China) and imaged with a gel imaging system (Amersham ImageQuant\u0026trade; 800, Cytiva, China). The WB images were analyzed using ImageJ software (ImageJ 1.52a, National Institutes of Health, USA).\u003c/p\u003e\u003cp\u003e\u003cb\u003eReal‑time polymerase chain reaction (RT-qPCR)\u003c/b\u003e\u003c/p\u003e\u003cp\u003eTotal RNA was extracted from A549 cells with TRIzol reagent. The RNA concentration was spectrophotometrically measured. Complementary DNA (cDNA) was reverse transcribed from an appropriate amount of RNA using a PCR Thermal Cycler (BioRad, USA) with Hifair\u0026reg; III 1st Strand cDNA Synthesis SuperMix for qPCR kit (Yeasen Biotechnology, Shanghai, China). Real-time PCR was performed using Hieff UNICON\u0026reg; Universal Blue qPCR SYBR Green Master Mix (Yeasen Biotechnology, Shanghai, China) on QuantStudio Dx Real-Time PCR instrument (Thermo Fisher Scientific, Waltham, USA) with the appropriate primer pairs. The mean cycle threshold (Ct) of the gene of interest was calculated from triplicate measurements and normalized to GAPDH, serving as the housekeeping standard. Fold changes of target mRNAs were analyzed using the 2\u003csup\u003e\u0026minus;∆∆CT\u003c/sup\u003e method. The PCR primer sequences were as follows: GAPDH forward, 5\u0026rsquo;-ACCCAGAAGACTGTGGATGG \u0026minus;\u0026thinsp;3\u0026rsquo; and reverse, 5\u0026rsquo;-TCAGCTCAGGGATGACCTTG-3\u0026rsquo; and N-cadherin forward, 5\u0026rsquo;-AATCGTGTCTCAGGCTCCAA-3\u0026rsquo; and reverse, 5\u0026rsquo;-TGGGATTGCCTTCCATGTCT-3\u0026rsquo;, E-cadherin forward, 5\u0026rsquo;-ACGCATTGCCACATACACTC-3\u0026rsquo; and reverse, 5\u0026rsquo;-AGAGGTTCCTGGAAGAGCAC-3\u0026rsquo;, Vimentin forward, 5\u0026rsquo;-TTGCAGGAGGAGATGCTTCA-3\u0026rsquo; and reverse, 5\u0026rsquo;-CCACTTTGCGTTCAAGGTCA-3\u0026rsquo;, α-SMA forward, 5\u0026rsquo;-CAGCCCTCCTTCATCGGTAT-3\u0026rsquo; and reverse, 5\u0026rsquo;-TGATCTTCATGGTGCTGGGT-3\u0026rsquo;, IL-33 forward, 5\u0026rsquo;-TCCCAACAGAAGGCCAAAGA-3\u0026rsquo; and reverse, 5\u0026rsquo;-AAAGGCAAAGCACTCCACAG-3\u0026rsquo;, and ST2 forward, 5\u0026rsquo;-TCCTGTGGCAGCTTAATGGA-3\u0026rsquo; and reverse, 5\u0026rsquo;-ATGCAAATTCAGGGCCAGAC-3\u0026rsquo;.\u003c/p\u003e\u003cp\u003e\u003cb\u003eLentivirus packaging and production\u003c/b\u003e\u003c/p\u003e\u003cp\u003eTo culture HEK-293T cells in 10-cm dishes until reaching 70\u0026ndash;80% confluence for transfection. Lentiviral packaging plasmids (8 \u0026micro;g pLP1, 4 \u0026micro;g pLP2, 2 \u0026micro;g VSV-G) and the transfer plasmid (14 \u0026micro;g Sh-\u003cem\u003eIL33\u003c/em\u003e) were complexed, and the transfection reagent mixture was prepared following the Lipofectamine 3000 protocol. After mixing, the solution was added dropwise to the HEK-293T cultures. After six hours, the medium was replaced with the complete medium, followed by incubation for 48 to 72 hours. The supernatant was collected and filtered through a 0.22 \u0026micro;m membrane, and the lentiviral stock was used directly for target cell infection. A549 cells were seeded in 10-cm dishes until attaining 60\u0026ndash;70% confluence. Lentiviral infection was performed using a mixture of growth medium, lentiviral stock, and polybrene. After 72 h, the selection was initiated with 2 \u0026micro;g/mL puromycin.\u003c/p\u003e\u003cdiv id=\"Sec3\" class=\"Section2\"\u003e\u003ch2\u003eStatistical analysis\u003c/h2\u003e\u003cp\u003eGraphPad Prism 9 was used for statistical analysis and graph generation. A Student's t-test was applied for comparisons between two groups, and a one-way ANOVA was used for comparisons among multiple groups. Data are presented as the mean\u0026thinsp;\u0026plusmn;\u0026thinsp;standard error of the mean (SEM), with \u003cem\u003ep\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.05 indicating statistical significance. Each experiment was repeated three times.\u003c/p\u003e\u003c/div\u003e"},{"header":"Results","content":"\u003cp\u003e\u003cb\u003eHigh expression of IL‑33 in the serum of patients with PCPF\u003c/b\u003e\u003c/p\u003e\u003cp\u003eTo investigate the precise role of IL-33 in PCPF development, serum samples were collected from 25 clinically confirmed healthy individuals, 28 COVID-19 patients, and 26 PCPF patients (Table \u003cspan refid=\"MOESM1\" class=\"InternalRef\"\u003eS1\u003c/span\u003e). Expression of IL-1β, IL-8, IL-6, IL-10, and IL-33 was analyzed by ELISA. As shown in Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eA\u0026amp;B, no significant differences in serum IL-1β and IL-8 levels were found among the three groups. Compared to the healthy control group, serum IL-6 levels were elevated in both COVID-19 and PCPF groups (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eC), with no difference between the latter two. Serum IL-10 levels were substantially higher in COVID-19 and PCPF groups than in the healthy group (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eD) and further increased in PCPF patients compared to COVID-19 patients. Notably, serum IL-33 levels were significantly higher in PCPF patients than in the other two groups (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eE), and this elevation was specifically observed in PCPF patients. We then measured the levels of the IL-33-specific receptor ST2. As shown in Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eF, ST2 levels followed a similar pattern, being significantly higher in the PCPF group than in the other groups, which did not differ from each other. These results suggest that IL-33 may play a key role in the development of PCPF and serve as an independent risk factor for PCPF.\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003cp\u003e\u003cb\u003eSARS-CoV-2 Spike protein induced IL-33 and ST2 expression and secretion\u003c/b\u003e \u003cb\u003ein vitro\u003c/b\u003e\u003c/p\u003e\u003cp\u003eHaving observed elevated IL-33 and ST2 levels in PCPF patients, we next investigated whether their expression is associated with SARS-CoV-2 infection. A549 cells were exposed to a concentration gradient of Spike protein for 24 and 48 hours, and cell viability was evaluated using the CCK-8 assay to determine optimal treatment conditions. Results demonstrated that only 1000 ng/mL of Spike protein, applied for 48 h, significantly reduced A549 cell viability (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eA), suggesting that this dosage and duration effectively induced cellular damage. Since SARS-CoV-2 infection is known to injure alveolar epithelial cells and trigger pulmonary fibrosis directly, we adopted a 1000 ng/mL Spike protein treatment for 48 hours in subsequent studies.\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003cp\u003eTo investigate the role of IL-33 in PCPF, we assessed IL-33 expression in A549 cells following treatment with the Spike protein. ELISA revealed that after 48-hour exposure to the Spike protein, IL-33 levels in both cellular supernatants and lysates were significantly elevated compared to the PBS control group (P\u0026thinsp;\u0026lt;\u0026thinsp;0.01) (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eB and C). RT-qPCR and western blotting further confirmed that Spike protein treatment upregulated both IL-33 and its receptor ST2 at the mRNA and protein levels, respectively (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eD and E). Collectively, these findings indicate that \u003cem\u003ein vitro\u003c/em\u003e treatment with the SARS-CoV-2 Spike protein potently enhances the expression and secretion of IL-33 and ST2.\u003c/p\u003e\u003cp\u003e\u003cb\u003eSARS-CoV-2 Spike protein triggered EMT in A549 cells.\u003c/b\u003e\u003c/p\u003e\u003cp\u003eWe have observed that the Spike protein upregulates the expression and secretion of IL-33 and ST2 \u003cem\u003ein vitro\u003c/em\u003e, but whether it induces pulmonary fibrosis remains unclear. Therefore, we further investigated the effects of the Spike protein on A549 cell morphology, biological functions, and the epithelial-mesenchymal transition (EMT) pathway, a hallmark signaling cascade in fibrosis.\u003c/p\u003e\u003cp\u003eWe first examined the morphological changes in A549 cells after treatment with the Spike protein. As shown in Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eA, cells treated with 1000 ng/mL Spike protein for 48 h exhibited increased intercellular spacing, reduced cell-cell contacts, and partial adoption of elongated spindle-shaped, stellate, or irregular morphologies. These alterations indicate that the Spike protein modifies A549 cell morphology. Next, transwell migration and wound-healing assays were conducted to assess the impact of the Spike protein on the biological functions of A549 cells. The results (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eB\u0026amp;C) demonstrated that, compared to the PBS control group, Spike protein-treated A549 cells showed a significant increase in the number of migrated cells and the area of wound closure, suggesting promoted migration. Given the critical role of the EMT pathway in organ fibrosis, we used RT-qPCR and WB to analyze the Spike protein's effect on EMT-related molecules. As depicted in Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eD and E, Spike protein treatment upregulated both mRNA and protein expression levels of N-cadherin, Vimentin, and alpha-smooth muscle actin (α-SMA) in A549 cells while downregulating the mRNA and protein expression levels of E-cadherin. These results demonstrate that the SARS-CoV-2 Spike protein upregulates α-SMA expression and induces EMT in A549 cells.\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003cp\u003e\u003cb\u003eIL-33/ST2 axis activation promoted EMT\u003c/b\u003e\u003c/p\u003e\u003cp\u003eTo explore the potential link between Spike protein-induced EMT and IL-33 upregulation, we assessed the effects of rhIL-33 protein and IL-33 overexpression on EMT in A549 cells, aiming to determine whether IL-33 can replicate the phenotypic changes induced by the Spike protein.\u003c/p\u003e\u003cp\u003eA549 cells were exposed to escalating doses of rhIL-33 (0, 5, 10, 20 ng/mL) for 48 h. We observed no differences in clonogenic capacity (Figure \u003cspan refid=\"MOESM1\" class=\"InternalRef\"\u003eS1\u003c/span\u003eA) or proliferation kinetics (Figure \u003cspan refid=\"MOESM1\" class=\"InternalRef\"\u003eS1\u003c/span\u003eB) versus controls. However, wound-healing assays showed that IL-33 treatment dose-dependently enhanced cell migration (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eA). This indicates that IL-33 potentiates the migratory capacity of cells without affecting their proliferation. Subsequent analysis of EMT markers demonstrated that IL-33 treatment upregulated mesenchymal markers (ST2, N-cadherin, Vimentin, α-SMA) while downregulating epithelial marker E-cadherin in a dose-dependent manner at both transcriptional and translational levels (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eB-C).\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003cp\u003eTo delve deeper into the relationship between IL-33 and EMT, the IL-33 sequence was delivered to A549 by a lentiviral vector GV492 to establish stable overexpressing cell lines. After lentiviral infection and single-clone selection, heightened IL-33 expression was validated via RT-qPCR, ELISA, and WB, which unveiled markedly elevated IL-33 mRNA and protein levels in the high-expression cohort relative to controls (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eA-C). In these engineered cells, IL-33 overexpression significantly elevated the expression of its receptor, ST2, and mesenchymal markers (N-cadherin, Vimentin, α-SMA) while suppressing E-cadherin at both the transcriptional and protein levels (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eD-F). These findings confirm that activation of the IL-33/ST2 axis markedly promotes EMT in A549 cells.\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003cp\u003e\u003cb\u003eSARS-CoV-2 Spike protein promoted EMT\u003c/b\u003e \u003cb\u003ein vitro\u003c/b\u003e \u003cb\u003evia the IL-33/ST2 axis.\u003c/b\u003e\u003c/p\u003e\u003cp\u003eOur findings indicate that SARS-CoV-2 might cause pulmonary fibrosis by upregulating IL-33 to trigger EMT. To clarify the role of IL-33 in PCPF, we generated IL-33-knockdown A549 cell lines using lentiviral transduction and short hairpin RNA (shRNA) technology. shRNA-IL-33C group achieved the most pronounced IL-33 knockdown in A549 cells, as validated by RT-qPCR, ELISA, and WB (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003eA-B and Figure. S2). These cells, termed IL33-KD, along with shRNA-NC-transfected control cells, were used in subsequent studies.\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003cp\u003eWe then treated IL33-KD and control A549 cells with the Spike protein and assessed the expression of EMT-related proteins. As presented in Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003eC-E, IL33-KD cells exhibited reduced expression of IL-33, ST2, N-Cadherin, Vimentin, and α-SMA, along with upregulated E-Cadherin compared to control cells. In control cells, treatment with the Spike protein resulted in an upregulation of these markers and a downregulation of E-cadherin. Interestingly, in IL33-KD cells, Spike protein treatment did not significantly alter the expression of N-Cadherin, α-SMA, or E-Cadherin compared to untreated cells, implying that knocking down IL-33 can partially reverse the Spike protein-induced EMT process. Overall, these findings suggest that the Spike protein stimulates EMT in A549 cells through the IL-33/ST2 signaling axis.\u003c/p\u003e\u003cp\u003e\u003cb\u003eSARS-CoV-2 Spike protein-induced EMT may involve IL-33/ST2-mediated regulation of β-catenin/PPAR-γ signaling\u003c/b\u003e\u003c/p\u003e\u003cp\u003e To uncover the downstream pathways and mechanisms by which the IL-33/ST2 axis promotes EMT, we conducted a literature review and analyzed transcriptomic datasets from other research groups. Specifically, we examined data from lung cells treated with the SARS-CoV-2 Spike protein and from preadipocytes with IL-33 knockdown compared to their respective controls \u003csup\u003e[\u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e23\u003c/span\u003e, \u003cspan citationid=\"CR26\" class=\"CitationRef\"\u003e26\u003c/span\u003e]\u003c/sup\u003e. The analysis revealed that the differentially expressed genes from both the Spike protein-treated and IL-33-knockdown models were significantly enriched in the peroxisome proliferator-activated receptor (PPAR) signaling pathway \u003csup\u003e[\u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e23\u003c/span\u003e, \u003cspan citationid=\"CR26\" class=\"CitationRef\"\u003e26\u003c/span\u003e]\u003c/sup\u003e, which strongly suggests that the PPAR pathway may be regulated by IL-33 and potentially involved in SARS-CoV-2-related pathological processes.\u003c/p\u003e\u003cp\u003eTo investigate whether IL-33 mediates the effects of the Spike protein through the β-catenin/PPAR-γ pathway, we assessed the expression of β-catenin and PPAR-γ in IL-33-knockdown A549 cells treated with the Spike protein. As shown in Fig.\u0026nbsp;\u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e7\u003c/span\u003eA, compared to the PBS control group, β-catenin expression was significantly upregulated, and PPAR-γ expression was downregulated in the Spike protein-treated group. However, following IL-33-knockdown, the Spike protein-induced upregulation of β-catenin was attenuated, and PPAR-γ expression was restored.\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003cp\u003eSubsequently, we employed the PPAR-γ agonist rosiglitazone (20 \u0026micro;mol/L) to investigate further whether IL-33 mediates the Spike protein's effects through the β-catenin/PPAR-γ pathway. After pretreating A549 cells with rosiglitazone, we observed that the combined treatment of Spike protein and rosiglitazone partially reversed the expression of EMT markers compared to Spike protein treatment alone. Specifically, N-cadherin, Vimentin, and α-SMA levels were decreased. At the same time, E-cadherin expression was increased (Fig.\u0026nbsp;\u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e7\u003c/span\u003eB). These results suggest that the Spike protein likely promotes EMT in A549 cells through the IL-33/ST2 axis by modulating the β-catenin/PPAR-γ pathway, thereby contributing to pulmonary fibrosis progression.\u003c/p\u003e"},{"header":"Discussion","content":"\u003cp\u003ePCPF imposes a substantial burden on global healthcare systems. While existing research has elucidated various mechanisms of COVID-19-induced pulmonary injury, the pathogenesis of PCPF remains poorly defined. Here, we analyzed cytokine in 79 clinical serum samples (healthy controls, COVID-19 patients, and PCPF patients), identifying IL-33 as the only cytokine specifically elevated in PCPF patients. Levels of its specific receptor ST2 mirrored this increase, suggesting a pivotal role for IL-33 in PCPF pathogenesis. To model SARS-CoV-2 infection \u003cem\u003ein vitro\u003c/em\u003e, we treated A549 cells (surrogates for alveolar epithelial type II cells\u0026mdash;the primary viral targets during early infection) with the SARS-CoV-2 Spike protein. Spike protein stimulation significantly upregulated both the expression and secretion of IL-33 and ST2 in A549 cells, further supporting the involvement of IL-33 in PCPF development.\u003c/p\u003e\u003cp\u003eConcurrently, while the Spike protein has been shown to trigger inflammatory responses and activate EMT in macrophages, fibroblasts, and bronchial epithelial cells, its EMT-inducing effects on alveolar epithelial type II cells remain understudied \u003csup\u003e[\u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e22\u003c/span\u003e, \u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e23\u003c/span\u003e]\u003c/sup\u003e. Our results demonstrate that Spike protein robustly induces EMT in A549 cells. Critically, rhIL-33 treatment or IL-33 overexpression potentiated EMT, whereas IL-33 knockdown partially reversed Spike protein-induced EMT, which highly indicates that Spike protein promotes EMT in A549 cells primarily through IL-33-mediated signaling, thereby driving PCPF progression.\u003c/p\u003e\u003cp\u003eTo delineate the mechanism underlying IL-33-mediated EMT induction, we integrated a literature review with a re-analysis of published transcriptome datasets (lung cells\u0026thinsp;\u0026plusmn;\u0026thinsp;Spike protein; preadipocytes\u0026thinsp;\u0026plusmn;\u0026thinsp;IL-33 knockdown). Both IL-33 knockdown and Spike protein treatment consistently altered the PPAR signaling pathway, suggesting that IL-33 mediates the regulation of the PPAR pathway during PCPF pathogenesis.\u003c/p\u003e\u003cp\u003ePPARs (PPAR-α, PPAR-β/δ, PPAR-γ), which are ligand-activated receptors in the nuclear hormone receptor family, have garnered significant interest, particularly PPAR-γ\u003csup\u003e[\u003cspan citationid=\"CR27\" class=\"CitationRef\"\u003e27\u003c/span\u003e, \u003cspan citationid=\"CR28\" class=\"CitationRef\"\u003e28\u003c/span\u003e]\u003c/sup\u003e. Recent studies indicate that SARS-CoV-2 downregulates PPAR-γ expression, disrupting PPAR signaling and inducing metabolic reprogramming that exacerbates pulmonary inflammation in infected lung epithelium \u003csup\u003e[\u003cspan citationid=\"CR29\" class=\"CitationRef\"\u003e29\u003c/span\u003e]\u003c/sup\u003e. Synthetic PPAR-γ agonists (e.g., rosiglitazone, pioglitazone) ameliorate SARS-CoV-2-induced hyperinflammation\u003csup\u003e[\u003cspan citationid=\"CR30\" class=\"CitationRef\"\u003e30\u003c/span\u003e]\u003c/sup\u003e. Notably, PPAR-γ demonstrates antifibrotic activity across multiple organs (liver, kidney, skin, lung)\u003csup\u003e[\u003cspan additionalcitationids=\"CR32 CR33\" citationid=\"CR31\" class=\"CitationRef\"\u003e31\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR34\" class=\"CitationRef\"\u003e34\u003c/span\u003e]\u003c/sup\u003e. In PCPF, PPAR-γ activation modulates macrophage polarization toward the M2 phenotype during infection \u003csup\u003e[\u003cspan citationid=\"CR35\" class=\"CitationRef\"\u003e35\u003c/span\u003e]\u003c/sup\u003e. Its activator, cannabidiol, reduces cytokine secretion, inflammation, and pulmonary fibrosis \u003csup\u003e[\u003cspan citationid=\"CR36\" class=\"CitationRef\"\u003e36\u003c/span\u003e]\u003c/sup\u003e. β-catenin, a potent negative regulator of PPAR-γ, governs cell proliferation, differentiation, and adhesion; its dysregulation drives fibrogenesis in multiple organs\u003csup\u003e[\u003cspan citationid=\"CR37\" class=\"CitationRef\"\u003e37\u003c/span\u003e]\u003c/sup\u003e. Emerging evidence suggests that SARS-CoV-2-induced β-catenin hyperactivation suppresses PPAR-γ, thereby promoting organ fibrosis \u003csup\u003e[\u003cspan citationid=\"CR38\" class=\"CitationRef\"\u003e38\u003c/span\u003e]\u003c/sup\u003e. These findings prompted our focus on the β-catenin/PPAR-γ axis. Our study reveals that the Spike protein upregulates β-catenin while downregulating PPAR-γ expression in A549 cells. Strikingly, IL-33 knockdown reversed these effects. Furthermore, pretreatment with the PPAR-γ agonist rosiglitazone attenuated Spike protein-induced EMT, confirming the involvement of the β-catenin/PPAR-γ pathway. Collectively, we demonstrate that SARS-CoV-2 Spike protein activates β-catenin via the IL-33/ST2 axis, suppresses PPAR-γ, and consequently induces EMT to promote pulmonary fibrosis.\u003c/p\u003e\u003cp\u003eDespite the advancements presented herein, certain limitations warrant consideration. First, biosafety constraints (BSL-2 compliance) precluded direct experimentation with live SARS-CoV-2; consequently, we utilized its Spike protein\u0026mdash;the primary mediator of viral entry\u0026mdash;as a biologically relevant surrogate. Second, while our cellular models robustly demonstrated IL-33/ST2 axis involvement in Spike protein-induced EMT, validation in animal models remains imperative to establish physiological relevance.\u003c/p\u003e"},{"header":"Conclusions","content":"\u003cp\u003eIn summary, this study demonstrates that the SARS-CoV-2 Spike protein upregulates IL-33 in alveolar epithelium and orchestrates EMT through the IL-33/ST2-mediated modulation of the β-catenin/PPAR-γ signaling axis, thereby driving PCPF pathogenesis. Our findings nominate IL-33 as a novel therapeutic target for PCPF, providing a mechanistic foundation for antifibrotic strategy.\u003c/p\u003e"},{"header":"Abbreviations","content":"\u003cp\u003ePCPF \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp;\u0026nbsp; \u0026nbsp;\u0026nbsp;\u0026nbsp;\u0026nbsp;\u0026nbsp; \u0026nbsp; \u0026nbsp;post-COVID-19 pulmonary fibrosis\u003c/p\u003e\n\u003cp\u003eEMT \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp;epithelial-mesenchymal transition\u003c/p\u003e\n\u003cp\u003eIL-33 \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp;\u0026nbsp;\u0026nbsp;Interleukin-33\u003c/p\u003e\n\u003cp\u003erhIL-33 \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; recombinant human IL-33\u003c/p\u003e\n\u003cp\u003eST2 \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; suppression of tumorigenicity 2\u003c/p\u003e\n\u003cp\u003eRT-qPCR \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; Real‑time polymerase chain reaction\u003c/p\u003e\n\u003cp\u003ePPAR \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp;peroxisome proliferator-activated receptor\u003c/p\u003e"},{"header":"Declarations","content":"\u003cp\u003e\u003cstrong\u003eEthics approval and consent to participate\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThis study was conducted in strict accordance with the Declaration of Helsinki and national ethical regulations (China\u0026apos;s Ethical Review Measures for Biomedical Research Involving Human Subjects).\u0026nbsp;All actions regarding clinical data collection and serum sample acquisition have been approved by the Ethics Committee of the First Affiliated Hospital of Nanchang University\u0026nbsp;(Approval No.: (2024)CDYFYYLK(08-074)). Specific waiver of informed consent was granted in accordance with the following provisions: 1) Samples were collected retrospectively from anonymized residual biological material. 2) The research posed no additional risk to patients. 3) Patient privacy was strictly protected through de-identification procedures.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eClinical trial number\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eNot applicable.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eConsent for publication\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e\u0026nbsp;Not applicable.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAvailability of data and materials\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eNot applicable.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eCompeting interests\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe authors declare that they have no competing interests.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eFunding\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe work was supported by the Major Project of Technology Innovation 2030-the \u0026quot;Research on Prevention and Treatment of Cancer, Cardio-cerebrovascular, Respiratory and Metabolic Diseases\u0026quot; (Project No. 2023ZD0506200; Sub-project No. 2023ZD0506203), the National Natural Science Foundation of China (82360130), the Jiangxi Key Research and Development Program (20232BBG70020), the Jiangxi Science and Technology Cooperation Project (20244BDF60008) and the Jiangxi Provincial Key Laboratory of Prevention and Treatment of Infectious Diseases (2024SSY06031).\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAuthors\u0026apos; contributions\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eAnqi Li and Xiaojian Xiong conducted the experiments and drafted the manuscript. Chuang Nie and Zucan Luo contributed to the methodology and discussion of the results. Xiaoling Deng performed transcription data analysis. Yujie Wang was responsible for collecting clinical samples. Xiuhua Kang provided critical clinical data interpretation.\u0026nbsp;Tianxin Xiang and Ying Ying designed the study, provided overall scientific supervision, and revised the manuscript. All the authors reviewed the draft and approved the final manuscript before submission.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAcknowledgments\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eWe would like to thank the research team that shared their transcriptome sequencing data on the platform.\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\u003cli\u003e\u003cspan\u003eNALBANDIAN A, SEHGAL K, GUPTA A, et al. Post-acute COVID-19 syndrome [J]. Nat Med. 2021;27(4):601\u0026ndash;15.\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eGEORGE P M, WELLS A U, JENKINS RG. 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Wnt/beta-Catenin Signaling, Disease, and Emerging Therapeutic Modalities [J]. Cell. 2017;169(6):985\u0026ndash;99.\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eVALLEE A, LECARPENTIER Y, VALLEE JN. Interplay of Opposing Effects of the WNT/beta-Catenin Pathway and PPARgamma and Implications for SARS-CoV2 Treatment [J]. Front Immunol. 2021;12:666693.\u003c/span\u003e\u003c/li\u003e\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":"COVID-19, PCPF, IL-33, Epithelial mesenchymal transformation (EMT), β-catenin/PPAR-γ","lastPublishedDoi":"10.21203/rs.3.rs-7024312/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-7024312/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003ch2\u003eBackground\u003c/h2\u003e\u003cp\u003ePost-COVID-19 pulmonary fibrosis (PCPF) presents an increasingly significant public health challenge. Although fibrotic mechanisms in PCPF are extensively studied, their exact pathogenesis remains unresolved. Interleukin-33 (IL-33), a critical alarmin in viral infections, including COVID-19, has been reported to drive fibrotic processes across multiple organs, yet its role in PCPF remains undefined. This study aims to investigate the specific contribution of IL-33 to PCPF.\u003c/p\u003e\u003ch2\u003eMethods\u003c/h2\u003e\u003cp\u003eSerum cytokine profiles (IL-1β, IL-6, IL-8, IL-10, IL-33, ST2) were analyzed across healthy controls, COVID-19 patients, and PCPF cohorts. \u003cem\u003eIn vitro\u003c/em\u003e, SARS-CoV-2 spike protein was applied to A549 alveolar epithelial cells to model viral infection, observing its impact on IL-33/ST2 signaling and epithelial-mesenchymal transition (EMT). Morphological changes and migration capacity were evaluated. Functional validation was performed using exogenous recombinant human IL-33 (rhIL-33), IL-33-overexpressing, and IL-33-knockdown A549 models to determine the role of IL-33 in EMT and fibrotic phenotypes. Mechanistic studies employed PPAR-γ agonism (rosiglitazone) in conjunction with spike stimulation to assess the regulation of the pathway.\u003c/p\u003e\u003ch2\u003eResults\u003c/h2\u003e\u003cp\u003eSerum levels of IL-33 and ST2 were remarkably elevated in PCPF patients compared to control groups and COVID-19 patients. In A549 cells, treatment with Spike protein (1000 ng/mL, 48 h) upregulated both extracellular and intracellular IL-33 and significantly increased the expression of IL-33 and ST2 at both mRNA and protein levels. Spike protein treatment further induced a spindle-shaped morphology, enhanced cell migration, and promoted EMT, as evidenced by decreased E-Cadherin and increased N-Cadherin, Vimentin, and α-SMA expression. Similar pro-EMT phenotype and migratory effects were observed upon exogenous rIL-33 administration or IL-33 overexpression. Conversely, IL-33 knockdown attenuated Spike protein-induced alterations in EMT markers. Mechanistically, Spike protein upregulated β-catenin while suppressing PPAR-γ protein expression\u0026mdash;effects reversed by IL-33 knockdown. Co-treatment with rosiglitazone partially inhibited Spike protein-induced upregulation of N-Cadherin, Vimentin, and α-SMA and restored E-Cadherin expression.\u003c/p\u003e\u003ch2\u003eConclusions\u003c/h2\u003e\u003cp\u003eOur findings demonstrate remarkable upregulation of IL-33 in PCPF. SARS-CoV-2 Spike protein drives alveolar EMT via IL-33/ST2-dependent β-catenin activation and PPAR-γ suppression, unveiling novel therapeutic targets for PCPF intervention.\u003c/p\u003e","manuscriptTitle":"The IL-33/ST2 Axis Promotes Post-COVID-19 Pulmonary Fibrosis Through β-catenin/PPAR-γ-mediated Epithelial-Mesenchymal Transition","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2025-09-09 13:27:18","doi":"10.21203/rs.3.rs-7024312/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":"834f8854-8d67-4f44-afdf-24150c941d49","owner":[],"postedDate":"September 9th, 2025","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"posted","subjectAreas":[],"tags":[],"updatedAt":"2025-10-09T17:23:34+00:00","versionOfRecord":[],"versionCreatedAt":"2025-09-09 13:27:18","video":"","vorDoi":"","vorDoiUrl":"","workflowStages":[]},"version":"v1","identity":"rs-7024312","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-7024312","identity":"rs-7024312","version":["v1"]},"buildId":"8U1c8b4HqxoKbykW_rLl7","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}