Targeting CXCR4–JNK/c-Jun–CTSB Signaling Axis Attenuates Silicosis Fibrosis in Mice

Targeting CXCR4–JNK/c-Jun–CTSB Signaling Axis Attenuates Silicosis Fibrosis in Mice | 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 Targeting CXCR4–JNK/c-Jun–CTSB Signaling Axis Attenuates Silicosis Fibrosis in Mice Fei Wang, Yifei Zhu, Ruiqing Yan, Xia Li, Zihao Xie, Liying Wang, and 4 more This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-8497777/v1 This work is licensed under a CC BY 4.0 License Status: Under Review Version 1 posted 4 You are reading this latest preprint version Abstract Silicosis is a severe occupational lung disease characterized by progressive and irreversible pulmonary fibrosis. CXCR4 (C-X-C chemokine receptor type 4) has been implicated in the pathogenesis of silicosis, yet the specific molecular mechanism through which CXCR4 signaling promotes fibrotic progression remain unclear. In this study, we systematically investigated the role of CXCR4 in silica-induced lung inflammation and fibrosis using pharmacological inhibition with AMD3100 and conditional Cxcr4 knockout mouse models, supported by complementary in vitro mechanistic studies. Silica exposure significantly upregulated CXCR4 expression in the lungs, which was accompanied by activation of the JNK/c-Jun pathway and enhanced transcription of cathepsin B (CTSB). Both AMD3100 treatment and genetic deletion of Cxcr4 markedly reduced inflammatory cell infiltration, collagen deposition, and lung function impairment, while also suppressing the expression of CTSB, TGF-β1, α-SMA, and fibronectin. Mechanistically, CXCR4 activation promoted JNK-dependent phosphorylation of c-Jun, enabling c-Jun to bind directly to the Ctsb promoter and drive its transcription. The subsequent increase in CTSB levels facilitated TGF-β1 activation, thereby amplifying downstream profibrotic signaling. Notably, CTSB was predominantly expressed in macrophages, where it co-localized with C1QC and was positively regulated by CXCR4 signaling. Taken together, our findings reveal a previously unrecognized CXCR4–JNK/c-Jun–CTSB signaling axis in macrophages that drives silica-induced pulmonary fibrosis, highlighting CXCR4 as a promising therapeutic target for silicosis. Silicosis CXCR4 signaling Cathepsin B JNK/c-Jun pathway TGF-β1 Figures Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 Introduction Pulmonary fibrosis is characterized by excessive extracellular matrix deposition and a progressive decline in lung function, representing the debilitating endpoint of various chronic pulmonary diseases, including silicosis[1, 2]. Inhalation of crystalline silica particles triggers a persistent inflammatory response within the alveoli, leading to the activation of immune cells, particularly macrophages, that reside in or are recruited to the lungs[3]. This chronic injury-repair cycle ultimately results in irreversible fibrotic scarring[4]. Despite extensive evidence implicating oxidative stress and inflammation in silicosis, the molecular mechanisms that link silica-induced macrophage activation to progressive fibrosis remain incompletely understood. CXCR4, a seven-transmembrane G protein-coupled receptor, is widely expressed in epithelial cells, endothelial cells, macrophages, and hematopoietic stem cells, among others[5]. Together with its ligand stromal cell-derived factor-1 (SDF-1/CXCL12), it forms a crucial chemotactic axis regulating immune cell recruitment, cell survival, and tissue remodeling[6, 7]. Increasing evidence suggests that CXCR4 signaling is upregulated in fibrotic lung diseases; however, its functional role and downstream molecular programs in macrophage-driven, silica-induced fibrosis remain poorly defined. In particular, whether CXCR4 activation in macrophages merely facilitates cell recruitment or actively orchestrates profibrotic signaling cascades has yet to be clarified. Cathepsin B (CTSB), a lysosomal cysteine protease, has emerged as a key mediator of inflammasome activation and extracellular matrix degradation[8, 9]. Under physiological conditions, CTSB serves as an autophagy regulator, modulating autophagy and lysosome-mediated protein degradation processes[10]. In pathological states characterized by lysosomal membrane destabilization, CTSB can be aberrantly activated and released into the cytosol or extracellular space, where it exerts pleiotropic effects on inflammation and tissue remodeling[11, 12]. Notably, CTSB has been shown to promote the activation of latent transforming growth factor-β1 (TGF-β1), a central driver of fibrosis, and genetic or pharmacological disruption of CTSB impairs TGF-β1 activation[13, 14]. Despite these observations, the upstream signals governing CTSB transcriptional regulation in macrophages during silicosis remain unknown. Here, we hypothesized that silica exposure activates a CXCR4-dependent signaling program in macrophages that transcriptionally regulates CTSB and drives fibrotic progression. Using a combination of pharmacological inhibition with AMD3100, conditional Cxcr4 knockout mouse models, and in vitro mechanistic studies, we demonstrate that CXCR4 activation promotes JNK-mediated phosphorylation of c-Jun, enabling c-Jun to directly bind to the Ctsb promoter and enhance its transcription. Elevated CTSB is required for efficient activation of TGF-β1 and the subsequent profibrotic cascade (Scheme 1 ). Collectively, our study identifies a previously unrecognized CXCR4–JNK/c-Jun–CTSB signaling axis in macrophages that links silica-induced inflammation to pulmonary fibrosis, highlighting CXCR4 as a potential therapeutic target for silicosis. Silica exposure via intranasal instillation induces persistent lung injury and inflammatory cell infiltration, leading to the accumulation and activation of macrophages in the alveolar microenvironment. In response to silica stimulation, CXCR4 is upregulated in macrophages, promoting downstream signaling through the G protein–coupled receptor pathway. CXCR4 activation triggers JNK signaling, resulting in c-Jun phosphorylation and nuclear translocation. Activated c-Jun directly binds to the promoter region of the Ctsb gene, enhancing CTSB transcription. Elevated CTSB promotes lysosomal destabilization and facilitates the activation of latent TGF-β1, thereby driving fibroblast activation and extracellular matrix deposition. Pharmacological inhibition of CXCR4 with AMD3100 or genetic ablation of Cxcr4 attenuates macrophage activation, suppresses CTSB expression, and reduces TGF-β1–mediated fibrotic remodeling, ultimately alleviating silica-induced pulmonary fibrosis. Material and methods Reagents Silica particles (Sigma-Aldrich) subjected to high pressure were suspended in sterile 0.9% saline solution under sterile conditions, and the suspension was sonicated for 30 minutes before use. Plerixafor, the CTSB inhibitor CA-074, the TGF-β1 inhibitor SD-208, and tamoxifen were all purchased from MedChemExpress. Mice model of silicosis C57BL/6 wild-type male mice, 8 weeks old, were purchased from Henan Sikebes Biotechnology Co., Ltd. (License No: SCXK (Yu) 2020-0005), and C57BL/6 gene knockdown male mice, 8 weeks old, were purchased from Nanmo Biology. Prior to the experiment, the mice were acclimatized to the new environment for 1–2 weeks, with a room temperature of 22–26°C, humidity of 45–50%, and natural light/dark cycle. Mice were provided with ad libitum food and water. All procedures adhered to the "Regulations on the Administration of Experimental Animals" (published in 1988 and revised in 2011 and 2017) and were approved by the Institutional Animal Care and Use Committee of Anhui University of Science and Technology (Ethical approval number: 2023022601). After the environmental acclimatization period, 55 C57BL/6 wild-type male mice and 10 C57BL/6 gene knockdown male mice were randomly assigned to the experiment. Fifteen wild-type mice were randomly divided into three groups (n = 5 per group): vehicle control (Veh), silica-exposed (Sil), and silica-exposed mice treated with AMD3100 (Sil + AMD3100). Thirty wild-type mice were randomly divided into six groups (n = 5 per group): vehicle control, silica-exposed, Sil + CA-074Me group, Sil + SD-208 group, CA-074Me + SD-208 group, and Sil + CA-074Me + SD-208 group. Gene knockdown mice were randomly divided into two groups (n = 5 per group): control group and silica exposure group. The initial body weight of each mouse was recorded before the experiment. Anesthesia was induced using isoflurane inhalation, and once the mice reached a deep anesthetic state (determined by the disappearance of the toe pinch reflex and the absence of spontaneous rolling when positioned laterally), the treatments were administered via nasal drip: 60 µl of 0.9% saline was given to the control group, while 60 µl of silica suspension (200 mg/ml) was administered to the silica and treatment groups. The 15 wild type and 10 gene knockdown mice in the Sil + AMD3100 group began intraperitoneal injections of Plerixafor (AMD3100, 5 mg/kg) 12 hours after silica suspension administration, once daily for 7 consecutive days. In the 30 wild-type mice, the Sil + CA-074Me group, Sil + SD-208 group, CA-074Me + SD-208 group, and Sil + CA-074Me + SD-208 group began intraperitoneal injections of CTSB inhibitor CA-074Me (10 mg/kg), TGF-β1 inhibitor SD-208 (30 mg/kg), or a combination of both CA-074Me and SD-208 12 hours after silica suspension administration. The total experimental period lasted 14 days. At the end of the experiment, all mice were euthanized in a humane manner, and lung tissue samples were collected, either frozen at -80°C or fixed in 4% formaldehyde for subsequent histological and molecular biological analyses. Histopathological staining After perfusion to remove blood, the left lung of the mice was carefully excised and fixed in 4% formaldehyde solution for 48–72 hours. After fixation, the tissues were dehydrated, cleared, embedded in paraffin, and sectioned into 5 µm thick slices. The slices were placed in a 60°C oven and baked for 2 hours, followed by deparaffinization with xylene and rehydration through graded ethanol (100%, 95%, 85%, and 75%). The tissue sections were stained with hematoxylin and eosin (H&E) to observe pathological changes, and Masson’s trichrome staining was performed to assess collagen deposition and the degree of fibrosis. Stained sections were imaged using a BX53 + DP74 microscope. The collagen deposition area related to fibrosis was quantified using ImageJ software, and the final result was expressed as the percentage of collagen area relative to the total area. Cell culture The mouse monocyte macrophage cell line (Raw264.7) was cultured in complete DMEM medium containing 10% fetal bovine serum and 1% antibiotics (0.1 mg/ml penicillin, 0.1 mg/ml streptomycin). The culture conditions were 37°C and 5% CO2. After exposing Raw264.7 cells to silica particles for 12 hours, the cells were treated with or without CTSB inhibitor for 24 hours, and a monotherapy group was included. Western blot Western blot Mouse lung tissues or Raw264.7 cells were lysed using radioimmunoprecipitation assay (RIPA) lysis buffer. The lysate was then centrifuged at 12,000 rpm for 15 minutes at 4°C, and the supernatant was collected as the total protein sample. Protein concentration was quantified using a BCA protein assay kit. Prior to loading, protein samples were denatured by heating at 100°C for 10 minutes in a metal bath. Equal amounts of protein were separated by electrophoresis using 8% or 10% sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE), followed by transfer to a polyvinylidene fluoride (PVDF) membrane via wet transfer. After transfer, the PVDF membrane was blocked at room temperature with 5% bovine serum albumin (BSA) or non-fat dry milk for 1 hour. The membrane was then incubated overnight at 4°C with the following primary antibodies: C1QC (1:5000), CTSB (1:1000), CXCR4 (1:2000), JNK (1:1000), p-JNK (1:1000), c-Jun (1:1000), p-c-Jun (1:1000),α-SMA (1:5000), TGF-β1 (1:1000), and Fibronectin (1:5000). The following day, the membrane was washed and incubated with the appropriate horseradish peroxidase (HRP)-conjugated secondary antibodies (including goat anti-rabbit and rabbit anti-mouse) at room temperature for 1 hour. Target protein bands were visualized using enhanced chemiluminescence (ECL) reagent. The intensity of the bands was quantified using ImageJ software and normalized to GAPDH as the internal reference protein. Antibody information is provided in Table S1 . Multiplex immunofluorescence staining Mouse lung tissue paraffin sections were baked in a 60°C oven for 6 hours, followed by deparaffinization using xylene and rehydration through a graded ethanol series (100%, 95%, 85%, 75%). Antigen retrieval was performed using an antigen retrieval solution for heat-mediated antigen unmasking. After retrieval, the sections were permeabilized at room temperature with 0.3% Triton X-100 solution for 10 minutes. Endogenous peroxidase activity was quenched by incubating the sections with 3% hydrogen peroxide (H₂O₂) solution at room temperature for 10 minutes. The sections were then blocked with PBS containing 5% bovine serum albumin (BSA) at room temperature for 1 hour. Subsequently, the primary antibodies (C1QC, 1:200, 16889-1-AP; CTSB, 1:1000, 3178S; CXCR4, 1:200, 60042-1-Ig; TGF-β1, 1:500, bs-0086R) were incubated overnight at 4°C. The following day, the sections were washed 3 times with PBS, each wash lasting 5 minutes. The sections were then incubated with HRP-conjugated secondary antibodies at room temperature for 1 hour. After thorough washing with PBS, fluorescence signal amplification was achieved by incubating with TSA-488, TSA-555, and TSA-647 fluorescent staining reagents. Finally, the sections were mounted with an anti-fluorescence quenching mounting medium and images were acquired using an Olympus fluorescence microscope. Dual-luciferase reporter gene assay To construct the pcDNA3.1-Jun and pGL3-Basic-Ctsb recombinant plasmids, the following molecular cloning strategy was employed. First, genomic DNA was extracted from mouse lung tissue, and the target fragment was amplified using high-fidelity DNA polymerase (PrimeSTAR GXL DNA Polymerase). After separation of the PCR products by agarose gel electrophoresis, the expected DNA fragment was excised and purified. The Jun fragment and the pcDNA3.1 vector were digested with EcoR I and BamH I, respectively, while the Ctsb fragment and the pGL3-Basic vector were digested with Hind III and Xho I. After purification of the digested products, the Jun fragment was ligated into the pcDNA3.1 vector and the Ctsb fragment into the pGL3-Basic vector using T4 DNA ligase to generate recombinant plasmids. The ligation products were then transformed into competent cells (e.g., DH5α). The transformed bacterial cultures were plated on LB agar plates containing the appropriate antibiotics and incubated overnight at 37°C. Single colonies were selected and used as templates for colony PCR with specific primers for the Jun and Ctsb genes. Colonies with the correct band size were then sent for sequencing verification. After sequencing confirmed the correct constructs, the positive clones were inoculated into liquid LB medium for further culture. Subsequently, the recombinant plasmids were extracted using a plasmid purification kit. Finally, the recombinant plasmids and reporter plasmid were co-transfected into 293T cells using the Lipo8000™ transfection reagent. After 48 hours of transfection, luciferase activity was measured using the Dual-Luciferase® Reporter Assay System (Promega). The Renilla luciferase activity was used as an internal control, and the ratio of firefly to Renilla luciferase activity (RLUfirefly / RLUrenilla) was calculated to assess the transcriptional activity of the target gene. Flow cytometry To obtain bone marrow-derived cells and perform phenotypic analysis, the following standardized protocol was employed in this study. First, after euthanizing the experimental mice, both femurs were isolated, and surrounding soft tissues were removed. The epiphyses at both ends of the femurs were then excised to expose the bone marrow cavity. Using a syringe, the bone marrow cavity was repeatedly washed with cold phosphate-buffered saline (PBS, 4°C). The collected cell suspension was centrifuged at 300 × g for 10 minutes at 4°C, and the supernatant was discarded. The cell pellet was resuspended in an appropriate amount of red blood cell lysis buffer and incubated on ice for 3 minutes. This process was repeated twice. Subsequently, the cells were resuspended in flow cytometry buffer (PBS containing 3% fetal bovine serum) and counted. When the cell concentration reached 1×10⁶ – 1×10⁷ cells, the cells were treated with anti-CD16/32 antibody (Fc receptor blocker) for blocking, followed by incubation at 4°C for 10–15 minutes. The cells were then incubated with fluorescently labeled antibodies against surface markers CD11b and CXCR4 at 4°C, protected from light, for 30 minutes for immunostaining. After staining, the cells were resuspended in flow cytometry buffer and filtered through a 70 µm cell strainer into a flow cytometry tube. Finally, data was collected using a flow cytometer and analyzed using the corresponding software. RT-qPCR Total RNA was extracted from mouse lung tissues using TRIzol reagent, following the manufacturer's recommended protocol. Then, cDNA was synthesized from 1 µg of total RNA using the Epizyme (Shanghai) Reverse Transcription Kit in a 20 µL reaction volume. For quantitative real-time PCR (RT-qPCR) analysis, the Biosharp (Beijing) SYBR qPCR Mix was used as the detection system. The total reaction volume was 10 µL, which included 1 µL of cDNA template. Each sample was analyzed in triplicate to ensure result consistency and reliability. The qPCR reaction program consisted of an initial denaturation at 95°C for 2 minutes, followed by 40 cycles of 95°C for 10 seconds, 60°C for 30 seconds for annealing and extension, and a final melting curve analysis to confirm amplification specificity. The expression levels of target genes were relative quantified using the 2 − ΔΔCt method, with Gapdh as the reference gene. Final data are presented as the mean ± standard error of the mean (SEM), and statistical analysis and graphing were performed using GraphPad Prism 9.5.0 software. Between-group comparisons were conducted using one-way analysis of variance (ANOVA), with P < 0.05 considered statistically significant. The primers used are shown in Table S2. ELISA Whole blood samples were allowed to stand at room temperature for 0.5 to 2 hours until they naturally clot, after which the serum was separated by centrifugation for further analysis. The sample activation procedure was as follows: first, 30 µl of TGF-β1 activator I was added to the pre-diluted serum sample and thoroughly mixed, followed by incubation at room temperature for 1 hour. Next, 30 µl of TGF-β1 activator II was added, mixed, and the activation process was completed. The standard was prepared using recombinant mouse TGF-β1 protein, with stock solutions of 10 ng/mL and 1,000 pg/ml prepared. The 1,000 pg/mL standard was serially diluted using sample dilution buffer to obtain eight concentration points, including 1,000, 500, 250, 125, 62.5, 31.25, 15.62, and 0 pg/ml. The activated samples and the standard solutions at various concentrations were each added at 100 µl per well to the appropriate wells of a microplate. The wells were sealed with sealing film and incubated at 37°C for 90 minutes. After incubation, the liquid in the wells was discarded, and the microplate was inverted and gently tapped on absorbent paper to remove any residual liquid. Then, 100 µl of biotinylated anti-mouse TGF-β1 antibody working solution, 100 µl of diluted enzyme complex, and 100 µl of TMB substrate solution were added sequentially to the wells. After each addition, the wells were sealed and incubated as required. Finally, 100 µl of stop solution was added to terminate the reaction, and the absorbance value of each well was immediately measured using a microplate reader. A four-parameter logistic (4-PL) curve fitting method was used to plot a standard curve with standard concentrations on the x-axis and corresponding absorbance values on the y-axis, using specialized analysis software. The concentrations of the samples were calculated based on their measured absorbance values using this standard curve. Statistical analysis Statistical analysis was performed using GraphPad Prism 9.5.0 software. Numerical data are expressed as mean ± standard deviation. Data were analyzed using a one-way analysis of variance (ANOVA). A P -value of < 0.05 was considered statistically significant. Results CXCR4 Inhibition Mitigates Silica-Induced Pulmonary Fibrosis in Mice: A Histopathological and Functional Assessment Based on our previous findings showing elevated CXCR4 expression in the lungs of silica-exposed mice[15], we investigated whether pharmacological inhibition of CXCR4 could alleviate silica-induced pulmonary fibrosis. To this end, wild-type C57BL/6 mice were randomly assigned to three groups: vehicle control (Veh), silica exposure (Sil), and silica exposure with AMD3100 treatment (Sil + AMD3100) (Fig. 1 a). Following silica exposure, mice in the Sil group exhibited a rapid loss of body weight, which gradually recovered after day 5 (Fig. 1 b). Notably, mice treated with AMD3100 showed a significantly accelerated recovery of body weight compared with the Sil group, reaching baseline levels by day 14, suggesting an overall improvement in systemic condition following CXCR4 inhibition. Histological analysis of lung tissue further revealed marked structural damage in silica-exposed mice. Hematoxylin and eosin (H&E) staining demonstrated severe disruption of alveolar architecture accompanied by extensive inflammatory cell infiltration in the Sil group. Consistently, Masson’s trichrome staining showed pronounced collagen deposition within the lung parenchyma, indicative of fibrotic remodeling ( Fig. 1 c-d ) . In contrast, the AMD3100-treated group showed a significant reduction in inflammatory infiltration and collagen deposition, preserving alveolar structure and attenuating fibrotic lesions. To assess whether these histological improvements translated into functional benefits, pulmonary function tests were performed. Compared with control mice, silica-exposed mice displayed significant reductions in peak inspiratory flow (PIF), peak expiratory flow (PEF), and minute ventilation (MV), reflecting restrictive ventilatory dysfunction ( Fig. 1 e ) . Importantly, AMD3100 administration significantly improved all measured pulmonary function parameters relative to the Sil group, indicating partial restoration of lung ventilatory capacity. Collectively, these results demonstrate that pharmacological inhibition of CXCR4 effectively mitigates silica-induced lung injury and fibrosis at both histopathological and functional levels, supporting a critical role for CXCR4 signaling in the development of silicosis-associated pulmonary fibrosis. CXCR4 Regulates CTSB Expression and Localization in lung Macrophages During Silica-Induced Pulmonary Fibrosis To identify the cellular populations and molecular programs associated with CXCR4 signaling during silica-induced pulmonary fibrosis, we analyzed single-cell RNA sequencing (scRNA-seq) data from lung tissues. Unsupervised clustering revealed a distinct macrophage subset characterized by high expression of complement component C1qc and cathepsin B (Ctsb) in silica-exposed lungs (Fig. 2 a-b). Notably, C1qc and Ctsb exhibited highly overlapping expression patterns within this macrophage population, and correlation analysis demonstrated a significant positive association between their transcript levels, indicating coordinated regulation in silica-induced macrophages[16]. We next examined whether CXCR4 signaling regulates Ctsb expression in vivo. Compared with control mice, Ctsb mRNA levels were significantly increased in the lungs of silica-exposed mice, whereas pharmacological inhibition of CXCR4 with AMD3100 markedly reduced Ctsb transcription (Fig. 2 c). These results suggest that CXCR4 activity is required for the upregulation of Ctsb during silica-induced lung injury. To validate these findings at the protein level and determine their cellular localization, immunofluorescence staining and Western blot analyses were performed. Immunofluorescence analysis demonstrated prominent colocalization of C1QC, CXCR4, and CTSB within lung macrophages in silica-exposed mice, accompanied by increased fluorescence intensity of all three proteins (Fig. 2 d-e). In contrast, AMD3100 treatment substantially attenuated the expression of CXCR4 and CTSB and reduced their colocalization within macrophages. Consistently, Western blot analysis confirmed significant upregulation of C1QC, CXCR4, and CTSB protein levels in the silica-exposed group, which was markedly suppressed following CXCR4 inhibition (Fig. 2 f–g). Collectively, these data demonstrate that CTSB is predominantly expressed in lung macrophages during silica-induced pulmonary fibrosis and that its expression and subcellular distribution are positively regulated by CXCR4 signaling. Inhibition of CXCR4 disrupts the coordinated upregulation of C1QC and CTSB in macrophages, supporting a macrophage-intrinsic CXCR4–CTSB regulatory axis in the fibrotic lung. CXCR4 Activates JNK/c-Jun Signaling to Transcriptionally Regulate CTSB Expression Based on our findings that CTSB expression in lung macrophages is positively regulated by CXCR4, we next investigated the downstream signaling mechanisms linking CXCR4 activation to CTSB transcription. Analysis of single-cell RNA sequencing data revealed that silica exposure significantly increased the expression of Jun, which was markedly attenuated by AMD3100 treatment (Fig. 3 a). These results suggest that CXCR4 signaling may regulate Ctsb expression through a c-Jun–dependent transcriptional program. Previous literature has shown that Jun is a key downstream effector molecule of the c-Jun N-terminal kinase (JNK, MAPK8/9) signaling pathway, and the activation of c-Jun usually relies on JNK-mediated phosphorylation modification [17, 18]. Protein–protein interaction (PPI) network analysis indicated that JNK and c-Jun are closely connected within a known MAPK signaling module (Fig. 3 b), consistent with their established upstream–downstream relationship in stress-responsive signaling pathways. We then examined the activation status of the JNK/c-Jun signaling pathway. Western blot analysis showed that silica exposure markedly increased the phosphorylation levels of JNK and c-Jun, whereas pharmacological inhibition of CXCR4 with AMD3100 significantly suppressed the phosphorylation of both proteins (Fig. 3 c-d). These data indicate that CXCR4 activation promotes c-Jun phosphorylation via JNK signaling in silica-induced lung injury. To determine whether c-Jun directly regulates Ctsb transcription, a luciferase reporter construct containing the Ctsb promoter region (− 2000 bp to + 100 bp) was generated (Fig. 3 e). Dual-luciferase reporter assays demonstrated that overexpression of c-Jun significantly enhanced Ctsb promoter activity (Fig. 3 f). To further validate direct binding of c-Jun to the Ctsb promoter, chromatin immunoprecipitation (ChIP) followed by qPCR was performed. Agarose gel electrophoresis confirmed specific amplification of the expected promoter fragment (Fig. 3 g), and quantitative analysis revealed significant enrichment of c-Jun at the Ctsb promoter compared with IgG controls (Fig. 3 h). Collectively, these results demonstrate that CXCR4 activation induces JNK-dependent phosphorylation of c-Jun, which directly binds to the Ctsb promoter and enhances its transcription in silica-induced pulmonary fibrosis. CTSB Promotes TGF-β1 Activation and Fibrotic Remodeling in Silica-Induced Pulmonary Fibrosis To determine whether CTSB contributes to TGF-β1 activation during silica-induced pulmonary fibrosis, we first examined their spatial relationship in lung tissues. Immunofluorescence analysis revealed prominent colocalization of CTSB and TGF-β1 within lung macrophages in silica-exposed mice (Fig. 4 a-b). Notably, pharmacological inhibition of CXCR4 with AMD3100 markedly reduced both the fluorescence intensity and colocalization of CTSB and TGF-β1. Consistent with these observations, Western blot analysis of lung tissue revealed that AMD3100 treatment significantly downregulated the expression levels of TGF-β1, as well as downstream fibrotic markers, including α-smooth muscle actin (α-SMA), and fibronectin, compared with silica-exposed mice (Fig. 4 c-d). To directly assess the role of CTSB in regulating TGF-β1 expression, silica-stimulated macrophages were treated with the CTSB-specific inhibitor CA-074Me in vitro. Cells were lysed for protein extraction after 24 hours. The remaining results are presented in Figure S1 . Inhibition of CTSB activity resulted in a marked reduction in TGF-β1 protein levels, as determined by Western blot analysis (Fig. 4 e-f). We next evaluated the functional significance of the CTSB–TGF-β1 axis in vivo. Silica-exposed mice were treated with the CTSB inhibitor CA-074Me, the TGF-β receptor inhibitor SD-208, or a combination of both inhibitors. Histological analysis using H&E and Masson’s trichrome staining showed that inflammatory infiltration and collagen deposition were substantially attenuated in inhibitor-treated groups compared with the silica-only group (Fig. 4 g-h). In parallel, RT–qPCR analysis revealed that silica exposure markedly upregulated the mRNA expression of Tgfb1, Acta2 (α-SMA), and Fn1, whereas treatment with CA-074Me or SD-208 significantly reduced their expression (Fig. 4 i). Consistently, ELISA analysis of lung tissue homogenates confirmed that elevated TGF-β1 protein levels induced by silica exposure were markedly diminished following CTSB or TGF-β signaling inhibition (Fig. 4 j). Collectively, these results demonstrate that CTSB promotes TGF-β1 activation and downstream fibrotic responses during silica-induced pulmonary fibrosis and pharmacological targeting of the CTSB–TGF-β1 axis effectively attenuates lung fibrosis. Genetic ablation of CXCR4 in macrophages attenuates silica-induced pulmonary fibrosis. Homozygous Cxcr4-floxed mice (Cxcr fl/fl) were crossed with Lyz2CreERT2 mice to generate Lyz2CreERT2; Cxcr fl/fl mice, which harbor a myeloid cell-specific inducible Cre recombinase system. Cre-positive CXCR4fl/fl mice were identified by PCR genotyping and used for subsequent experiments (see Figure S2). To induce macrophage-specific Cxcr4 deletion, mice received intraperitoneal injections of tamoxifen (100 mg/kg) once daily for five consecutive days prior to silica exposure (Fig. 5 a). Histological analysis revealed that inflammatory cell infiltration was significantly reduced in the lungs of Cxcr4-deficient mice, as shown by H&E staining (Fig. 5 b). Consistently, Masson’s trichrome staining demonstrated a substantial decrease in collagen deposition and fibrosis scores in Cxcr4 knockdown mice compared with control silicosis mice (Fig. 5 b-c). To determine whether genetic ablation of Cxcr4 affects CTSB expression in macrophages, immunofluorescence staining was performed. In wild-type silicosis mice, CXCR4 and CTSB exhibited prominent colocalization within lung macrophages, whereas this colocalization was markedly diminished in Cxcr4-deficient mice (Figs. 5 d-e). At the protein level, Western blot analysis further confirmed a significant reduction in CTSB expression in lung tissues from Cxcr4 knockdown mice (Fig. 5 f–g). In parallel, key downstream profibrotic markers, including TGF-β1, α-smooth muscle actin (α-SMA), and fibronectin, were significantly downregulated following macrophage-specific deletion of Cxcr4 (Fig. 5 g). These findings indicate that loss of CXCR4 in macrophages disrupts CTSB expression and suppresses activation of downstream fibrotic signaling pathways. Collectively, these genetic data demonstrate that macrophage-intrinsic CXCR4 is required for CTSB upregulation and fibrotic remodeling in silica-induced pulmonary fibrosis, providing in vivo evidence that the CXCR4–JNK/c-Jun–CTSB signaling axis is a critical driver of silicosis pathogenesis. Discussion Silicosis is an occupational pulmonary fibrosis disease caused by the prolonged inhalation of crystalline silica dust, and its global incidence continues to rise, becoming a significant public health burden[19, 20]. The pathological progression of this disease typically begins with acute and chronic inflammation in the lungs, eventually leading to irreversible alveolar destruction and abnormal extracellular matrix deposition[4]. In recent years, the role of C-X-C chemokine receptor 4 (CXCR4) and its ligand CXCL12 in tissue repair and fibrosis has gained increasing attention. As a classical G protein-coupled receptor, CXCR4 plays a crucial regulatory role in a variety of pathological and physiological processes[21]. CXCR4 is involved not only in the migration and homing of immune cells but also in regulating epithelial-to-mesenchymal transition and extracellular matrix remodeling in various organ fibrosis models[22, 23]. Upon binding to its ligand CXCL12 (SDF-1α), CXCR4 activates downstream signaling pathways, including PI3K/Akt, MAPK/ERK, and JAK/STAT, thereby regulating cellular processes such as proliferation, migration, survival, and differentiation[24]. Notably, in silica-induced pulmonary fibrosis, CXCR4 may serve as a key molecular hub linking environmental exposure to fibrotic responses. This study establishes, for the first time, a direct molecular link between CXCR4 and cathepsin B (CTSB) in a silica-induced pulmonary fibrosis model and elucidates the key mediating role of the JNK/c-Jun signaling axis in this process. Using single-cell RNA sequencing technology, we observed co-expression of Ctsb with the macrophage marker gene C1qc. Through protein interaction network analysis, dual-luciferase reporter assays, and chromatin immunoprecipitation (ChIP), we confirmed that silica exposure upregulates CXCR4 expression, thereby activating the downstream JNK/c-Jun signaling pathway. Activation of JNK (evidenced by elevated phosphorylated p-JNK levels) directly catalyzes the phosphorylation of the transcription factor c-Jun at Ser63 and Ser73. Phosphorylated c-Jun (p-c-Jun) significantly enhances its DNA-binding capacity and transcriptional activity. Importantly, activated c-Jun specifically recognizes and binds to the AP-1 response element within the Ctsb gene promoter region, directly driving the upregulation of CTSB transcription, leading to a marked increase in its mRNA and protein expression levels. This result is consistent with previous studies, where CXCR4 regulates the transcriptional activity of multiple target genes through downstream MAPK/JNK signaling modules, thereby affecting the expression of inflammation- and fibrosis-related molecules[25]. Cathepsin B (CTSB) is an important lysosomal cysteine protease[26]. When overexpressed and secreted into the extracellular microenvironment, CTSB can precisely cleave the latency-associated peptide (LAP) within the precursor complex of transforming growth factor-β1 (TGF-β1), thereby releasing biologically active mature TGF-β1 [27]. In this study, through immunofluorescence co-localization and Western blot analysis, we revealed the high consistency between the protein expression and cellular localization of CTSB and TGF-β1, further linking protease-mediated matrix degradation to classic pro-fibrotic signaling pathways. Activated TGF-β1 subsequently acts in an autocrine or paracrine manner to bind to the TGF-β receptor II/I complex on the cell surface, initiating the canonical Smad2/3 signaling pathway as well as non-canonical pathways such as MAPK and PI3K-Akt[28, 29]. The convergence of these downstream signals ultimately induces the transformation of fibroblasts into myofibroblasts, promoting the excessive synthesis and deposition of extracellular matrix proteins, such as α-smooth muscle actin (α-SMA) and fibronectin[30, 31], which constitute the core pathological processes of pulmonary fibrosis. In conclusion, this study integrates multiple layers of evidence, including animal models, single-cell transcriptomics, molecular interaction validation, and genetic intervention, to unveil a novel signaling axis composed of CXCR4/JNK/c-Jun/CTSB/TGF-β1. This finding provides new insights into the pathogenesis of chronic fibrotic diseases induced by environmental factors. The results of this study both corroborate and extend previous findings. Numerous studies have confirmed the critical role of CXCR4 in organ fibrosis, with the JNK/c-Jun pathway often involved in oxidative stress responses[32]. However, previous research has predominantly focused on the role of CXCR4 in cell migration, survival, or inflammatory cytokine secretion[33, 34], with limited attention given to its regulation of protease expression. Our study bridges CXCR4 signaling with CTSB, an important lysosomal protease, providing a fresh perspective on its role in extracellular matrix remodeling. Furthermore, while the mechanism of CTSB-mediated activation of TGF-β1 has been described in cancer and liver fibrosis[13, 35], functional studies in environment-exposure related pulmonary diseases, such as silicosis, remain scarce. This study, through in vivo and in vitro inhibitor experiments and genetic intervention models, establishes the indispensable role of the CTSB/TGF-β1 axis in silicosis fibrosis, thus filling a gap in this area of research. Although this study has made significant findings, several limitations remain. The experiments were primarily conducted using mouse models, which exhibit species differences in the pathological progression of silicosis compared to humans. Therefore, further validation of the conservation and relevance of this signaling pathway in clinical samples or organoid models is necessary. Moreover, while this study focused on the CXCR4/CTSB axis in macrophages, it remains unclear whether other cell types, such as fibroblasts or epithelial cells, contribute to fibrosis through similar or parallel mechanisms. Additionally, silica particle-induced oxidative stress may directly activate kinases such as JNK. Although we have demonstrated that CXCR4 acts as an important upstream regulator, the direct regulatory relationship between CXCR4 and JNK, the potential existence of other parallel pathways, and how TGF-β1 is further regulated in CXCR4 knockdown mice, still require further investigation. From a therapeutic perspective, CXCR4 antagonists (such as AMD3100) have shown promising antifibrotic effects in various disease models. Their mechanisms of action are not limited to blocking inflammatory cell recruitment but may also involve inhibiting fibroblast activation, reducing extracellular matrix (ECM) deposition, and modulating the immune microenvironment. However, the functional heterogeneity of CXCR4 across different cell types, its interaction with the homologous receptor CXCR7, and its dynamic changes at different stages of the disease remain unresolved key scientific issues that must be addressed in future research. CXCR4 plays a multifaceted regulatory role in oxidative stress-related fibrotic diseases [36]. A deeper understanding of the spatiotemporal expression patterns of CXCR4 and the dynamic changes in its downstream signaling networks in specific disease environments will provide an essential theoretical foundation for the development of targeted therapeutic strategies. Based on the findings and limitations of this study, future research could involve clinical investigations, such as the collection of bronchoalveolar lavage fluid or lung biopsy samples from silicosis patients, to assess the expression levels of CXCR4, CTSB, and phosphorylated c-Jun, and analyze their correlation with disease stage, pulmonary function parameters, and prognosis, thereby advancing the translational medicine research on this mechanism. At the mechanistic level, cell-specific knockout models can be employed to dissect the contributions of different pulmonary cell types in the CXCR4/CTSB axis, and to explore the causal relationship between silica-induced reactive oxygen species (ROS) generation and the upregulation of CXCR4 expression, thereby providing a more complete picture of the signaling network from environmental exposure to fibrosis formation. Additionally, the therapeutic potential of small-molecule inhibitors targeting this pathway (such as JNK inhibitors and CTSB inhibitors) or combination therapies in advanced fibrosis models could be evaluated, providing preclinical evidence for the development of new anti-fibrotic drugs. By integrating multi-omics approaches, a comprehensive study of the global impact of this signaling axis on extracellular matrix components and immune microenvironment remodeling will help to fully elucidate its pathophysiological significance in the progression of pulmonary fibrosis. In conclusion, the CXCR4/JNK/c-Jun/CTSB/TGF-β1 signaling pathway revealed in this study provides a novel framework for understanding the molecular mechanisms of silica-induced pulmonary fibrosis. This study not only expands our knowledge of the role of chemokine receptors in oxidative stress-related diseases but also lays a crucial experimental foundation for the future development of intervention strategies targeting key nodes of this pathway. Conclusion In summary, this study identifies CXCR4 as a critical mediator in silica-induced pulmonary fibrosis. Pharmacological inhibition of CXCR4 with AMD3100 and conditional genetic deletion of Cxcr4 in mice both effectively attenuated the JNK/c-Jun/CTSB pathway and subsequent fibrotic responses. These results not only clarify the molecular underpinnings of silicosis but also establish a theoretical and experimental rationale for targeting CXCR4 as a novel therapeutic strategy for pulmonary fibrosis, offering new directions for the development of targeted silicosis treatments. Declarations Author contributions F.W., Y.F.Z, R.Q.Y., L.Y.W.: Performed the experiments and drafted the initial manuscript. F.W., Y.F.Z, X.L., Z.H.X.: Conducted correlation analysis, processed experimental data, and generated figures and tables. M.M., J.H.W., W.F.W., X.R.T.: Reviewed and revised the manuscript. M.M., J.H.W., W.F.W.: Supervised the research project. This manuscript was collaboratively prepared by all authors. All authors have reviewed and approved the final version of the manuscript. Institutional review board statement Animal research procedures adhere to the guidelines outlined in the NIH Guide for the Care and Use of Laboratory Animals (NIH Publication No. 8023, revised 1978), and the Institutional Animal Care and Use Committee of Anhui University of Science and Technology approved the animal protocol for this study (No. 2023028). Approved by the Ethics Committee of the First Affiliated Hospital of Anhui University of Science and Technology (batch number: 2023-KY-B110-001). Funding This work was supported by Natural Science Foundation of China (82304112)；Introduce talent research start-up fund, Anhui University of Science and Technology(2023yjrc63); 2023 Medical Specialized Cultivation Project, Anhui University of Science and Technology (YZ2023H1A001), Research Funds of Joint Research Center for Occupational Medicine and Health of IHM (NO. OMH-2023-01, OMH-2023-03) Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper. Data Availability All generated and analyzed data are included in this published article and its supplementary information material. References Scheme 1 Scheme 1 is available in the Supplementary Files section. Additional Declarations No competing interests reported. Supplementary Files SupplementaryMaterial.docx Appendix A. Supplementary data Supplementary details are provided in accompanying files. Scheme.tif Scheme 1. The role of CXCR4 and Cathepsin B in macrophages during silica-induced pulmonary fibrosis. Cite Share Download PDF Status: Under Review Version 1 posted Reviewers invited by journal 29 Jan, 2026 Editor assigned by journal 08 Jan, 2026 Submission checks completed at journal 06 Jan, 2026 First submitted to journal 01 Jan, 2026 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-8497777","acceptedTermsAndConditions":true,"allowDirectSubmit":false,"archivedVersions":[],"articleType":"Research Article","associatedPublications":[],"authors":[{"id":583587130,"identity":"bfe6b3fc-2c9b-421c-9570-c1c9b25f167b","order_by":0,"name":"Fei Wang","email":"","orcid":"","institution":"Anhui University of Science and Technology","correspondingAuthor":false,"prefix":"","firstName":"Fei","middleName":"","lastName":"Wang","suffix":""},{"id":583587131,"identity":"af067a52-ff0f-4b9e-a6a9-feede928d139","order_by":1,"name":"Yifei Zhu","email":"","orcid":"","institution":"Anhui University of Science and Technology","correspondingAuthor":false,"prefix":"","firstName":"Yifei","middleName":"","lastName":"Zhu","suffix":""},{"id":583587132,"identity":"ee38d207-5d05-4813-8da8-02082c1fdaf3","order_by":2,"name":"Ruiqing Yan","email":"","orcid":"","institution":"Anhui University of Science and Technology","correspondingAuthor":false,"prefix":"","firstName":"Ruiqing","middleName":"","lastName":"Yan","suffix":""},{"id":583587133,"identity":"51f6065d-795b-41c8-826b-08d52fbb6cca","order_by":3,"name":"Xia Li","email":"","orcid":"","institution":"Anhui University of Science and Technology","correspondingAuthor":false,"prefix":"","firstName":"Xia","middleName":"","lastName":"Li","suffix":""},{"id":583587134,"identity":"4a409b9a-7388-4d12-a068-9eb8e70f7af2","order_by":4,"name":"Zihao Xie","email":"","orcid":"","institution":"Anhui University of Science and Technology","correspondingAuthor":false,"prefix":"","firstName":"Zihao","middleName":"","lastName":"Xie","suffix":""},{"id":583587135,"identity":"ff08f7f5-8c42-4220-b060-648c1a59f4bc","order_by":5,"name":"Liying Wang","email":"","orcid":"","institution":"Anhui University of Science and Technology","correspondingAuthor":false,"prefix":"","firstName":"Liying","middleName":"","lastName":"Wang","suffix":""},{"id":583587136,"identity":"7f8f8aaa-06c5-4d45-b065-fc4429d04821","order_by":6,"name":"Xinrong Tao","email":"","orcid":"","institution":"Anhui University of Science and Technology","correspondingAuthor":false,"prefix":"","firstName":"Xinrong","middleName":"","lastName":"Tao","suffix":""},{"id":583587137,"identity":"57f58270-acd2-4cbe-97b5-386beedcaf7f","order_by":7,"name":"Wenfeng Wang","email":"","orcid":"","institution":"Fudan University","correspondingAuthor":false,"prefix":"","firstName":"Wenfeng","middleName":"","lastName":"Wang","suffix":""},{"id":583587138,"identity":"02af7333-f1e7-4c84-a06b-52c086de9543","order_by":8,"name":"Jianhua Wang","email":"","orcid":"","institution":"Fudan University","correspondingAuthor":false,"prefix":"","firstName":"Jianhua","middleName":"","lastName":"Wang","suffix":""},{"id":583587139,"identity":"f355b23c-aa5d-4904-8bd6-632e107152c1","order_by":9,"name":"Min Mu","email":"data:image/png;base64,iVBORw0KGgoAAAANSUhEUgAAAZAAAAAyAQMAAABI0h/eAAAABlBMVEX///8AAABVwtN+AAAACXBIWXMAAA7EAAAOxAGVKw4bAAAAr0lEQVRIiWNgGAWjYJACCSCWY2NvPkCaFmM+nmMJpGlJnCeRo0Cccv72swdv8+YcTm9jyGFg+FGxjQgbzuQlW/NuO5zbxnD2AGPPmduEtRgw5JhJg7Uw9iUwM7YRo4X/DVhLOhszjwGRWiQgtiSwsRGrReLGG2PLudvSDdt42BIOEuUX/v4cwxtvt1nLy89/fPDBjwoitIAAEw+UcYA49UDA+INopaNgFIyCUTAiAQBbiDeZUbygRgAAAABJRU5ErkJggg==","orcid":"","institution":"Anhui University of Science and Technology","correspondingAuthor":true,"prefix":"","firstName":"Min","middleName":"","lastName":"Mu","suffix":""}],"badges":[],"createdAt":"2026-01-02 03:23:12","currentVersionCode":1,"declarations":"","doi":"10.21203/rs.3.rs-8497777/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-8497777/v1","draftVersion":[],"editorialEvents":[],"editorialNote":"","failedWorkflow":false,"files":[{"id":101690263,"identity":"66a0c7fe-116a-4ae9-801d-47b9da5b3e99","added_by":"auto","created_at":"2026-02-02 15:58:02","extension":"png","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":8243800,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eCXCR4 inhibition attenuates silica-induced lung inflammation and fibrosis in mice.\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e(a) Schematic illustration of the silica-induced silicosis model and AMD3100 treatment protocol in wild-type C57BL/6 mice. Mice were assigned to vehicle control (Veh), silica exposure (Sil), or silica exposure with AMD3100 treatment (Sil+AMD3100).\u003c/p\u003e\n\u003cp\u003e(b) Body weight changes in mice following silica exposure with or without AMD3100 treatment.\u003c/p\u003e\n\u003cp\u003e(c) Representative histological images of lung sections stained with hematoxylin and eosin (H\u0026amp;E) and Masson’s trichrome. Silica exposure resulted in marked alveolar destruction, inflammatory cell infiltration, and collagen deposition, whereas AMD3100 treatment attenuated these pathological changes. Polarized light microscopy revealed comparable silica deposition in the lungs of Sil and Sil+AMD3100 groups. Scale bar, 100 μm.\u003c/p\u003e\n\u003cp\u003e(d) Quantification of collagen deposition based on Masson’s trichrome staining.\u003c/p\u003e\n\u003cp\u003e(e) Pulmonary function parameters, including peak inspiratory flow (PIF), peak expiratory flow (PEF), and minute ventilation (MV). Data are presented as mean ± SD, (n = 3 mice per group).\u003c/p\u003e\n\u003cp\u003eStatistical significance was determined by one-way ANOVA with post hoc analysis. *P \u0026lt; 0.05, **P \u0026lt; 0.01.\u003c/p\u003e","description":"","filename":"Fig1.png","url":"https://assets-eu.researchsquare.com/files/rs-8497777/v1/cc62fb6761bdd3eaa6ebf1d9.png"},{"id":101690262,"identity":"15c5fbfe-7fb3-40a7-8ba4-94642a0b8c1b","added_by":"auto","created_at":"2026-02-02 15:58:02","extension":"png","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":7644209,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eCTSB is predominantly expressed in lung macrophages and is downregulated following CXCR4 inhibition.\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e(a) Violin plots showing the expression of C1qc across major lung cell populations identified by single-cell RNA sequencing.\u003c/p\u003e\n\u003cp\u003e(b) Violin plots showing the expression of Ctsb across major lung cell populations. Both C1qc and Ctsb were enriched in a macrophage subset.\u003c/p\u003e\n\u003cp\u003e(c) Relative Ctsb mRNA expression levels in lung tissues from vehicle control (Veh), silica-exposed (Sil), and silica-exposed mice treated with AMD3100 (Sil+AMD3100).\u003c/p\u003e\n\u003cp\u003e(d) Representative immunofluorescence images showing the localization of C1QC, CXCR4, and CTSB in lung macrophages at day 14 after silica exposure. Nuclei were counterstained with DAPI (blue).\u003c/p\u003e\n\u003cp\u003e(e) Merged immunofluorescence images illustrating colocalization of C1QC, CXCR4, and CTSB. Arrowheads indicate regions of colocalization. Scale bar, 20 μm.\u003c/p\u003e\n\u003cp\u003e(f) Representative Western blot images showing the protein expression of C1QC, CXCR4, and CTSB in lung tissues from each group.\u003c/p\u003e\n\u003cp\u003e(g) Quantification of Western blot band intensities normalized to loading controls. Data are presented as mean ± SD, (n = 3 mice per group).\u003c/p\u003e\n\u003cp\u003eStatistical significance was determined by one-way ANOVA with post hoc analysis. *P \u0026lt; 0.05, **P \u0026lt; 0.01.\u003c/p\u003e","description":"","filename":"Fig2.png","url":"https://assets-eu.researchsquare.com/files/rs-8497777/v1/10aecf10d86ae62a7a207df0.png"},{"id":101690266,"identity":"355e4da8-a226-42ce-a929-400b81a3cdf1","added_by":"auto","created_at":"2026-02-02 15:58:04","extension":"png","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":3603748,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eCXCR4 activates JNK/c-Jun signaling and promotes transcription of Ctsb.\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e(a) Relative Jun mRNA expression levels in lung tissues from vehicle control (Veh), silica-exposed (Sil), and silica-exposed mice treated with AMD3100 (Sil+AMD3100).\u003c/p\u003e\n\u003cp\u003e(b) Protein–protein interaction (PPI) network analysis of core signaling molecules associated with the MAPK/JNK pathway. JNK and c-Jun are shown as closely connected nodes within the same signaling module.\u003c/p\u003e\n\u003cp\u003e(c) Representative Western blot images showing total and phosphorylated forms of JNK and c-Jun in lung tissues at day 14 after silica exposure.\u003c/p\u003e\n\u003cp\u003e(d) Quantification of phosphorylated JNK (p-JNK) and phosphorylated c-Jun (p–c-Jun) normalized to their respective total protein levels.\u003c/p\u003e\n\u003cp\u003e(e) Schematic illustration of the construction of c-Jun overexpression plasmid (pcDNA3.1-Jun) and Ctsb promoter luciferase reporter plasmid (pGL3-Basic-Ctsb).\u003c/p\u003e\n\u003cp\u003e(f) Dual-luciferase reporter assay showing Ctsb promoter activity following c-Jun overexpression in 293T cells.\u003c/p\u003e\n\u003cp\u003e(g) Agarose gel electrophoresis showing specific amplification of the Ctsb promoter fragment (~200 bp) following chromatin immunoprecipitation (ChIP).\u003c/p\u003e\n\u003cp\u003e(h) ChIP–qPCR analysis showing enrichment of c-Jun binding at the Ctsb promoter region compared with IgG controls. Data are presented as mean ±SD (n = 3 independent experiments).\u003c/p\u003e\n\u003cp\u003eStatistical significance was determined by one-way ANOVA or Student’s t-test, as appropriate. *P \u0026lt; 0.05, **P \u0026lt; 0.01, ***P \u0026lt; 0.001.\u003c/p\u003e","description":"","filename":"Fig3.png","url":"https://assets-eu.researchsquare.com/files/rs-8497777/v1/a3c0458cd81ccf3b1035b5f7.png"},{"id":101690265,"identity":"2a3b0e97-b0a4-4864-9e1c-82395deefc88","added_by":"auto","created_at":"2026-02-02 15:58:04","extension":"png","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":13545818,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eCTSB inhibition attenuates TGF-β1–associated fibrotic responses in silica-induced pulmonary fibrosis.\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e(a) Representative immunofluorescence images showing the localization of C1QC, TGF-β1, and CTSB in lung macrophages from vehicle control (Veh), silica-exposed (Sil), and inhibitor-treated mice. Nuclei were counterstained with DAPI (blue).\u003c/p\u003e\n\u003cp\u003e(b) Merged images illustrating colocalization of C1QC, TGF-β1, and CTSB. Arrowheads indicate regions of colocalization. Scale bar, 20 μm.\u003c/p\u003e\n\u003cp\u003e(c) Representative Western blot images showing the expression of TGF-β1 and fibrotic markers (α-SMA and fibronectin) in lung tissues from the indicated groups.\u003c/p\u003e\n\u003cp\u003e(d) Quantification of Western blot band intensities normalized to loading controls.\u003c/p\u003e\n\u003cp\u003e(e) Representative Western blot images showing TGF-β1 expression in silica-stimulated macrophages treated with the CTSB inhibitor CA-074Me.\u003c/p\u003e\n\u003cp\u003e(f) Quantification of CTSB and TGF-β1 protein levels in macrophages.\u003c/p\u003e\n\u003cp\u003e(g) Representative H\u0026amp;E and Masson’s trichrome staining of lung sections from silica-exposed mice treated with CA-074Me, the TGF-β receptor inhibitor SD-208, or both inhibitors.\u003c/p\u003e\n\u003cp\u003e(h) Quantification of collagen deposition based on Masson’s trichrome staining. Scale bar, 100 μm.\u003c/p\u003e\n\u003cp\u003e(i) RT–qPCR analysis of fibrosis-related gene expression (Tgfb1, Acta2, and Fn1) in lung tissues from the indicated groups.\u003c/p\u003e\n\u003cp\u003e(j) ELISA quantification of TGF-β1 protein levels in lung tissue homogenates. Data are presented as mean ±SD (n = 3 mice per group).\u003c/p\u003e\n\u003cp\u003eStatistical significance was determined by one-way ANOVA with post hoc analysis. *P \u0026lt; 0.05, **P \u0026lt; 0.01.\u003c/p\u003e","description":"","filename":"Fig4.png","url":"https://assets-eu.researchsquare.com/files/rs-8497777/v1/c0f45947852065e159bbee93.png"},{"id":101690260,"identity":"139e9f8b-8fc0-4284-b120-b88733b0dd1b","added_by":"auto","created_at":"2026-02-02 15:57:59","extension":"png","order_by":5,"title":"Figure 5","display":"","copyAsset":false,"role":"figure","size":16677690,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eMacrophage-specific deletion of Cxcr4 attenuates silica-induced pulmonary fibrosis and reduces CTSB expression.\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e(a) Schematic illustration of the silica-induced silicosis model in macrophage-specific Cxcr4 conditional knockout mice. Tamoxifen was administered intraperitoneally for five consecutive days to induce Cre-mediated deletion of Cxcr4, followed by a two-week washout period prior to intranasal silica administration.\u003c/p\u003e\n\u003cp\u003e(b) Representative H\u0026amp;E and Masson’s trichrome staining of lung sections from control and macrophage-specific Cxcr4 knockout mice following silica exposure. Dashed boxes indicate representative areas selected for higher-magnification views in the corresponding panels.\u003c/p\u003e\n\u003cp\u003e(c) Representative Masson’s trichrome staining showing collagen deposition in lung tissues. Scale bar, 100 μm.\u003c/p\u003e\n\u003cp\u003e(d) Immunofluorescence staining showing the localization of C1QC, CXCR4, and CTSB in lung macrophages. Nuclei were counterstained with DAPI (blue).\u003c/p\u003e\n\u003cp\u003e(e) Merged immunofluorescence images illustrating colocalization of C1QC, CXCR4, and CTSB. Arrowheads indicate regions of colocalization. Scale bar, 20 μm.\u003c/p\u003e\n\u003cp\u003e(f) Representative Western blot images showing protein expression of C1QC, CXCR4, CTSB, and fibrotic markers in lung tissues.\u003c/p\u003e\n\u003cp\u003e(g) Quantification of Western blot band intensities for CTSB, TGF-β1, α-SMA, and fibronectin normalized to loading controls. Data are presented as mean ±SD (n = 3 mice per group).\u003c/p\u003e\n\u003cp\u003eStatistical significance was determined by one-way ANOVA with post hoc analysis. *P \u0026lt; 0.05, **P \u0026lt; 0.01.\u003c/p\u003e","description":"","filename":"Fig5.png","url":"https://assets-eu.researchsquare.com/files/rs-8497777/v1/75a2fe8f4b5cee8a7178f8f3.png"},{"id":101754166,"identity":"f3883f68-1201-47ff-89b3-0a6022b30988","added_by":"auto","created_at":"2026-02-03 10:41:48","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":45964700,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-8497777/v1/5ba7a617-baf2-44be-84af-b5f2ac4f0276.pdf"},{"id":101690264,"identity":"820b2bee-34eb-4f5c-b2dc-ce11617abf98","added_by":"auto","created_at":"2026-02-02 15:58:03","extension":"docx","order_by":1,"title":"","display":"","copyAsset":false,"role":"supplement","size":862949,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eAppendix A. Supplementary data\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eSupplementary details are provided in accompanying files.\u003c/p\u003e","description":"","filename":"SupplementaryMaterial.docx","url":"https://assets-eu.researchsquare.com/files/rs-8497777/v1/d80afce5b0553b3773c34586.docx"},{"id":101690239,"identity":"b8b3c6a7-1a0a-4587-8cc1-ad7448733f99","added_by":"auto","created_at":"2026-02-02 15:57:55","extension":"tif","order_by":4,"title":"","display":"","copyAsset":false,"role":"supplement","size":931522,"visible":true,"origin":"","legend":"\u003cp\u003eScheme 1. The role of CXCR4 and Cathepsin B in macrophages during silica-induced pulmonary fibrosis.\u003c/p\u003e","description":"","filename":"Scheme.tif","url":"https://assets-eu.researchsquare.com/files/rs-8497777/v1/efc135f9991dfff5c6e751df.tif"}],"financialInterests":"No competing interests reported.","formattedTitle":"\u003cp\u003e\u003cstrong\u003eTargeting CXCR4–JNK/c-Jun–CTSB Signaling Axis Attenuates Silicosis Fibrosis in Mice\u003c/strong\u003e\u003c/p\u003e","fulltext":[{"header":"Introduction","content":"\u003cp\u003ePulmonary fibrosis is characterized by excessive extracellular matrix deposition and a progressive decline in lung function, representing the debilitating endpoint of various chronic pulmonary diseases, including silicosis[1, 2]. Inhalation of crystalline silica particles triggers a persistent inflammatory response within the alveoli, leading to the activation of immune cells, particularly macrophages, that reside in or are recruited to the lungs[3]. This chronic injury-repair cycle ultimately results in irreversible fibrotic scarring[4]. Despite extensive evidence implicating oxidative stress and inflammation in silicosis, the molecular mechanisms that link silica-induced macrophage activation to progressive fibrosis remain incompletely understood.\u003c/p\u003e \u003cp\u003eCXCR4, a seven-transmembrane G protein-coupled receptor, is widely expressed in epithelial cells, endothelial cells, macrophages, and hematopoietic stem cells, among others[5]. Together with its ligand stromal cell-derived factor-1 (SDF-1/CXCL12), it forms a crucial chemotactic axis regulating immune cell recruitment, cell survival, and tissue remodeling[6, 7]. Increasing evidence suggests that CXCR4 signaling is upregulated in fibrotic lung diseases; however, its functional role and downstream molecular programs in macrophage-driven, silica-induced fibrosis remain poorly defined. In particular, whether CXCR4 activation in macrophages merely facilitates cell recruitment or actively orchestrates profibrotic signaling cascades has yet to be clarified.\u003c/p\u003e \u003cp\u003eCathepsin B (CTSB), a lysosomal cysteine protease, has emerged as a key mediator of inflammasome activation and extracellular matrix degradation[8, 9]. Under physiological conditions, CTSB serves as an autophagy regulator, modulating autophagy and lysosome-mediated protein degradation processes[10]. In pathological states characterized by lysosomal membrane destabilization, CTSB can be aberrantly activated and released into the cytosol or extracellular space, where it exerts pleiotropic effects on inflammation and tissue remodeling[11, 12]. Notably, CTSB has been shown to promote the activation of latent transforming growth factor-β1 (TGF-β1), a central driver of fibrosis, and genetic or pharmacological disruption of CTSB impairs TGF-β1 activation[13, 14]. Despite these observations, the upstream signals governing CTSB transcriptional regulation in macrophages during silicosis remain unknown.\u003c/p\u003e \u003cp\u003eHere, we hypothesized that silica exposure activates a CXCR4-dependent signaling program in macrophages that transcriptionally regulates CTSB and drives fibrotic progression. Using a combination of pharmacological inhibition with AMD3100, conditional Cxcr4 knockout mouse models, and in vitro mechanistic studies, we demonstrate that CXCR4 activation promotes JNK-mediated phosphorylation of c-Jun, enabling c-Jun to directly bind to the Ctsb promoter and enhance its transcription. Elevated CTSB is required for efficient activation of TGF-β1 and the subsequent profibrotic cascade (Scheme \u003cspan refid=\"Sch1\" class=\"InternalRef\"\u003e1\u003c/span\u003e). Collectively, our study identifies a previously unrecognized CXCR4\u0026ndash;JNK/c-Jun\u0026ndash;CTSB signaling axis in macrophages that links silica-induced inflammation to pulmonary fibrosis, highlighting CXCR4 as a potential therapeutic target for silicosis.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eSilica exposure via intranasal instillation induces persistent lung injury and inflammatory cell infiltration, leading to the accumulation and activation of macrophages in the alveolar microenvironment. In response to silica stimulation, CXCR4 is upregulated in macrophages, promoting downstream signaling through the G protein\u0026ndash;coupled receptor pathway. CXCR4 activation triggers JNK signaling, resulting in c-Jun phosphorylation and nuclear translocation. Activated c-Jun directly binds to the promoter region of the Ctsb gene, enhancing CTSB transcription. Elevated CTSB promotes lysosomal destabilization and facilitates the activation of latent TGF-β1, thereby driving fibroblast activation and extracellular matrix deposition. Pharmacological inhibition of CXCR4 with AMD3100 or genetic ablation of Cxcr4 attenuates macrophage activation, suppresses CTSB expression, and reduces TGF-β1\u0026ndash;mediated fibrotic remodeling, ultimately alleviating silica-induced pulmonary fibrosis.\u003c/p\u003e"},{"header":"Material and methods","content":"\u003cdiv id=\"Sec3\" class=\"Section2\"\u003e \u003ch2\u003eReagents\u003c/h2\u003e \u003cp\u003eSilica particles (Sigma-Aldrich) subjected to high pressure were suspended in sterile 0.9% saline solution under sterile conditions, and the suspension was sonicated for 30 minutes before use. Plerixafor, the CTSB inhibitor CA-074, the TGF-β1 inhibitor SD-208, and tamoxifen were all purchased from MedChemExpress.\u003c/p\u003e \u003c/div\u003e\n\u003ch3\u003eMice model of silicosis\u003c/h3\u003e\n\u003cp\u003eC57BL/6 wild-type male mice, 8 weeks old, were purchased from Henan Sikebes Biotechnology Co., Ltd. (License No: SCXK (Yu) 2020-0005), and C57BL/6 gene knockdown male mice, 8 weeks old, were purchased from Nanmo Biology. Prior to the experiment, the mice were acclimatized to the new environment for 1\u0026ndash;2 weeks, with a room temperature of 22\u0026ndash;26\u0026deg;C, humidity of 45\u0026ndash;50%, and natural light/dark cycle. Mice were provided with ad libitum food and water. All procedures adhered to the \"Regulations on the Administration of Experimental Animals\" (published in 1988 and revised in 2011 and 2017) and were approved by the Institutional Animal Care and Use Committee of Anhui University of Science and Technology (Ethical approval number: 2023022601).\u003c/p\u003e \u003cp\u003eAfter the environmental acclimatization period, 55 C57BL/6 wild-type male mice and 10 C57BL/6 gene knockdown male mice were randomly assigned to the experiment. Fifteen wild-type mice were randomly divided into three groups (n\u0026thinsp;=\u0026thinsp;5 per group): vehicle control (Veh), silica-exposed (Sil), and silica-exposed mice treated with AMD3100 (Sil\u0026thinsp;+\u0026thinsp;AMD3100). Thirty wild-type mice were randomly divided into six groups (n\u0026thinsp;=\u0026thinsp;5 per group): vehicle control, silica-exposed, Sil\u0026thinsp;+\u0026thinsp;CA-074Me group, Sil\u0026thinsp;+\u0026thinsp;SD-208 group, CA-074Me\u0026thinsp;+\u0026thinsp;SD-208 group, and Sil\u0026thinsp;+\u0026thinsp;CA-074Me\u0026thinsp;+\u0026thinsp;SD-208 group. Gene knockdown mice were randomly divided into two groups (n\u0026thinsp;=\u0026thinsp;5 per group): control group and silica exposure group. The initial body weight of each mouse was recorded before the experiment. Anesthesia was induced using isoflurane inhalation, and once the mice reached a deep anesthetic state (determined by the disappearance of the toe pinch reflex and the absence of spontaneous rolling when positioned laterally), the treatments were administered via nasal drip: 60 \u0026micro;l of 0.9% saline was given to the control group, while 60 \u0026micro;l of silica suspension (200 mg/ml) was administered to the silica and treatment groups. The 15 wild type and 10 gene knockdown mice in the Sil\u0026thinsp;+\u0026thinsp;AMD3100 group began intraperitoneal injections of Plerixafor (AMD3100, 5 mg/kg) 12 hours after silica suspension administration, once daily for 7 consecutive days. In the 30 wild-type mice, the Sil\u0026thinsp;+\u0026thinsp;CA-074Me group, Sil\u0026thinsp;+\u0026thinsp;SD-208 group, CA-074Me\u0026thinsp;+\u0026thinsp;SD-208 group, and Sil\u0026thinsp;+\u0026thinsp;CA-074Me\u0026thinsp;+\u0026thinsp;SD-208 group began intraperitoneal injections of CTSB inhibitor CA-074Me (10 mg/kg), TGF-β1 inhibitor SD-208 (30 mg/kg), or a combination of both CA-074Me and SD-208 12 hours after silica suspension administration. The total experimental period lasted 14 days. At the end of the experiment, all mice were euthanized in a humane manner, and lung tissue samples were collected, either frozen at -80\u0026deg;C or fixed in 4% formaldehyde for subsequent histological and molecular biological analyses.\u003c/p\u003e\n\u003ch3\u003eHistopathological staining\u003c/h3\u003e\n\u003cp\u003eAfter perfusion to remove blood, the left lung of the mice was carefully excised and fixed in 4% formaldehyde solution for 48\u0026ndash;72 hours. After fixation, the tissues were dehydrated, cleared, embedded in paraffin, and sectioned into 5 \u0026micro;m thick slices. The slices were placed in a 60\u0026deg;C oven and baked for 2 hours, followed by deparaffinization with xylene and rehydration through graded ethanol (100%, 95%, 85%, and 75%). The tissue sections were stained with hematoxylin and eosin (H\u0026amp;E) to observe pathological changes, and Masson\u0026rsquo;s trichrome staining was performed to assess collagen deposition and the degree of fibrosis. Stained sections were imaged using a BX53\u0026thinsp;+\u0026thinsp;DP74 microscope. The collagen deposition area related to fibrosis was quantified using ImageJ software, and the final result was expressed as the percentage of collagen area relative to the total area.\u003c/p\u003e\n\u003ch3\u003eCell culture\u003c/h3\u003e\n\u003cp\u003eThe mouse monocyte macrophage cell line (Raw264.7) was cultured in complete DMEM medium containing 10% fetal bovine serum and 1% antibiotics (0.1 mg/ml penicillin, 0.1 mg/ml streptomycin). The culture conditions were 37\u0026deg;C and 5% CO2. After exposing Raw264.7 cells to silica particles for 12 hours, the cells were treated with or without CTSB inhibitor for 24 hours, and a monotherapy group was included.\u003c/p\u003e\n\u003ch3\u003eWestern blot\u003c/h3\u003e\n\u003cdiv class=\"Heading\"\u003eWestern blot\u003c/div\u003e \u003cp\u003eMouse lung tissues or Raw264.7 cells were lysed using radioimmunoprecipitation assay (RIPA) lysis buffer. The lysate was then centrifuged at 12,000 rpm for 15 minutes at 4\u0026deg;C, and the supernatant was collected as the total protein sample. Protein concentration was quantified using a BCA protein assay kit. Prior to loading, protein samples were denatured by heating at 100\u0026deg;C for 10 minutes in a metal bath. Equal amounts of protein were separated by electrophoresis using 8% or 10% sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE), followed by transfer to a polyvinylidene fluoride (PVDF) membrane via wet transfer. After transfer, the PVDF membrane was blocked at room temperature with 5% bovine serum albumin (BSA) or non-fat dry milk for 1 hour. The membrane was then incubated overnight at 4\u0026deg;C with the following primary antibodies: C1QC (1:5000), CTSB (1:1000), CXCR4 (1:2000), JNK (1:1000), p-JNK (1:1000), c-Jun (1:1000), p-c-Jun (1:1000),α-SMA (1:5000), TGF-β1 (1:1000), and Fibronectin (1:5000). The following day, the membrane was washed and incubated with the appropriate horseradish peroxidase (HRP)-conjugated secondary antibodies (including goat anti-rabbit and rabbit anti-mouse) at room temperature for 1 hour. Target protein bands were visualized using enhanced chemiluminescence (ECL) reagent. The intensity of the bands was quantified using ImageJ software and normalized to GAPDH as the internal reference protein. Antibody information is provided in Table \u003cspan refid=\"MOESM1\" class=\"InternalRef\"\u003eS1\u003c/span\u003e.\u003c/p\u003e \u003cdiv id=\"Sec8\" class=\"Section2\"\u003e \u003ch2\u003eMultiplex immunofluorescence staining\u003c/h2\u003e \u003cp\u003eMouse lung tissue paraffin sections were baked in a 60\u0026deg;C oven for 6 hours, followed by deparaffinization using xylene and rehydration through a graded ethanol series (100%, 95%, 85%, 75%). Antigen retrieval was performed using an antigen retrieval solution for heat-mediated antigen unmasking. After retrieval, the sections were permeabilized at room temperature with 0.3% Triton X-100 solution for 10 minutes. Endogenous peroxidase activity was quenched by incubating the sections with 3% hydrogen peroxide (H₂O₂) solution at room temperature for 10 minutes. The sections were then blocked with PBS containing 5% bovine serum albumin (BSA) at room temperature for 1 hour. Subsequently, the primary antibodies (C1QC, 1:200, 16889-1-AP; CTSB, 1:1000, 3178S; CXCR4, 1:200, 60042-1-Ig; TGF-β1, 1:500, bs-0086R) were incubated overnight at 4\u0026deg;C. The following day, the sections were washed 3 times with PBS, each wash lasting 5 minutes. The sections were then incubated with HRP-conjugated secondary antibodies at room temperature for 1 hour. After thorough washing with PBS, fluorescence signal amplification was achieved by incubating with TSA-488, TSA-555, and TSA-647 fluorescent staining reagents. Finally, the sections were mounted with an anti-fluorescence quenching mounting medium and images were acquired using an Olympus fluorescence microscope.\u003c/p\u003e \u003c/div\u003e\n\u003ch3\u003eDual-luciferase reporter gene assay\u003c/h3\u003e\n\u003cp\u003eTo construct the pcDNA3.1-Jun and pGL3-Basic-Ctsb recombinant plasmids, the following molecular cloning strategy was employed. First, genomic DNA was extracted from mouse lung tissue, and the target fragment was amplified using high-fidelity DNA polymerase (PrimeSTAR GXL DNA Polymerase). After separation of the PCR products by agarose gel electrophoresis, the expected DNA fragment was excised and purified. The Jun fragment and the pcDNA3.1 vector were digested with EcoR I and BamH I, respectively, while the Ctsb fragment and the pGL3-Basic vector were digested with Hind III and Xho I. After purification of the digested products, the Jun fragment was ligated into the pcDNA3.1 vector and the Ctsb fragment into the pGL3-Basic vector using T4 DNA ligase to generate recombinant plasmids. The ligation products were then transformed into competent cells (e.g., DH5α). The transformed bacterial cultures were plated on LB agar plates containing the appropriate antibiotics and incubated overnight at 37\u0026deg;C. Single colonies were selected and used as templates for colony PCR with specific primers for the Jun and Ctsb genes. Colonies with the correct band size were then sent for sequencing verification. After sequencing confirmed the correct constructs, the positive clones were inoculated into liquid LB medium for further culture. Subsequently, the recombinant plasmids were extracted using a plasmid purification kit. Finally, the recombinant plasmids and reporter plasmid were co-transfected into 293T cells using the Lipo8000\u0026trade; transfection reagent. After 48 hours of transfection, luciferase activity was measured using the Dual-Luciferase\u0026reg; Reporter Assay System (Promega). The Renilla luciferase activity was used as an internal control, and the ratio of firefly to Renilla luciferase activity (RLUfirefly / RLUrenilla) was calculated to assess the transcriptional activity of the target gene.\u003c/p\u003e\n\u003ch3\u003eFlow cytometry\u003c/h3\u003e\n\u003cp\u003eTo obtain bone marrow-derived cells and perform phenotypic analysis, the following standardized protocol was employed in this study. First, after euthanizing the experimental mice, both femurs were isolated, and surrounding soft tissues were removed. The epiphyses at both ends of the femurs were then excised to expose the bone marrow cavity. Using a syringe, the bone marrow cavity was repeatedly washed with cold phosphate-buffered saline (PBS, 4\u0026deg;C). The collected cell suspension was centrifuged at 300 \u0026times; g for 10 minutes at 4\u0026deg;C, and the supernatant was discarded. The cell pellet was resuspended in an appropriate amount of red blood cell lysis buffer and incubated on ice for 3 minutes. This process was repeated twice. Subsequently, the cells were resuspended in flow cytometry buffer (PBS containing 3% fetal bovine serum) and counted. When the cell concentration reached 1\u0026times;10⁶ \u0026ndash; 1\u0026times;10⁷ cells, the cells were treated with anti-CD16/32 antibody (Fc receptor blocker) for blocking, followed by incubation at 4\u0026deg;C for 10\u0026ndash;15 minutes. The cells were then incubated with fluorescently labeled antibodies against surface markers CD11b and CXCR4 at 4\u0026deg;C, protected from light, for 30 minutes for immunostaining. After staining, the cells were resuspended in flow cytometry buffer and filtered through a 70 \u0026micro;m cell strainer into a flow cytometry tube. Finally, data was collected using a flow cytometer and analyzed using the corresponding software.\u003c/p\u003e \u003cdiv id=\"Sec11\" class=\"Section2\"\u003e \u003ch2\u003eRT-qPCR\u003c/h2\u003e \u003cp\u003eTotal RNA was extracted from mouse lung tissues using TRIzol reagent, following the manufacturer's recommended protocol. Then, cDNA was synthesized from 1 \u0026micro;g of total RNA using the Epizyme (Shanghai) Reverse Transcription Kit in a 20 \u0026micro;L reaction volume. For quantitative real-time PCR (RT-qPCR) analysis, the Biosharp (Beijing) SYBR qPCR Mix was used as the detection system. The total reaction volume was 10 \u0026micro;L, which included 1 \u0026micro;L of cDNA template. Each sample was analyzed in triplicate to ensure result consistency and reliability. The qPCR reaction program consisted of an initial denaturation at 95\u0026deg;C for 2 minutes, followed by 40 cycles of 95\u0026deg;C for 10 seconds, 60\u0026deg;C for 30 seconds for annealing and extension, and a final melting curve analysis to confirm amplification specificity. The expression levels of target genes were relative quantified using the 2\u0026thinsp;\u0026minus;\u0026thinsp;ΔΔCt method, with Gapdh as the reference gene. Final data are presented as the mean\u0026thinsp;\u0026plusmn;\u0026thinsp;standard error of the mean (SEM), and statistical analysis and graphing were performed using GraphPad Prism 9.5.0 software. Between-group comparisons were conducted using one-way analysis of variance (ANOVA), with P\u0026thinsp;\u0026lt;\u0026thinsp;0.05 considered statistically significant. The primers used are shown in Table S2.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec12\" class=\"Section2\"\u003e \u003ch2\u003eELISA\u003c/h2\u003e \u003cp\u003eWhole blood samples were allowed to stand at room temperature for 0.5 to 2 hours until they naturally clot, after which the serum was separated by centrifugation for further analysis. The sample activation procedure was as follows: first, 30 \u0026micro;l of TGF-β1 activator I was added to the pre-diluted serum sample and thoroughly mixed, followed by incubation at room temperature for 1 hour. Next, 30 \u0026micro;l of TGF-β1 activator II was added, mixed, and the activation process was completed. The standard was prepared using recombinant mouse TGF-β1 protein, with stock solutions of 10 ng/mL and 1,000 pg/ml prepared. The 1,000 pg/mL standard was serially diluted using sample dilution buffer to obtain eight concentration points, including 1,000, 500, 250, 125, 62.5, 31.25, 15.62, and 0 pg/ml. The activated samples and the standard solutions at various concentrations were each added at 100 \u0026micro;l per well to the appropriate wells of a microplate. The wells were sealed with sealing film and incubated at 37\u0026deg;C for 90 minutes. After incubation, the liquid in the wells was discarded, and the microplate was inverted and gently tapped on absorbent paper to remove any residual liquid. Then, 100 \u0026micro;l of biotinylated anti-mouse TGF-β1 antibody working solution, 100 \u0026micro;l of diluted enzyme complex, and 100 \u0026micro;l of TMB substrate solution were added sequentially to the wells. After each addition, the wells were sealed and incubated as required. Finally, 100 \u0026micro;l of stop solution was added to terminate the reaction, and the absorbance value of each well was immediately measured using a microplate reader. A four-parameter logistic (4-PL) curve fitting method was used to plot a standard curve with standard concentrations on the x-axis and corresponding absorbance values on the y-axis, using specialized analysis software. The concentrations of the samples were calculated based on their measured absorbance values using this standard curve.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec13\" class=\"Section2\"\u003e \u003ch2\u003eStatistical analysis\u003c/h2\u003e \u003cp\u003eStatistical analysis was performed using GraphPad Prism 9.5.0 software. Numerical data are expressed as mean\u0026thinsp;\u0026plusmn;\u0026thinsp;standard deviation. Data were analyzed using a one-way analysis of variance (ANOVA). A \u003cem\u003eP\u003c/em\u003e-value of \u0026lt;\u0026thinsp;0.05 was considered statistically significant.\u003c/p\u003e \u003c/div\u003e"},{"header":"Results","content":"\u003cdiv id=\"Sec15\" class=\"Section2\"\u003e \u003ch2\u003eCXCR4 Inhibition Mitigates Silica-Induced Pulmonary Fibrosis in Mice: A Histopathological and Functional Assessment\u003c/h2\u003e \u003cp\u003eBased on our previous findings showing elevated CXCR4 expression in the lungs of silica-exposed mice[15], we investigated whether pharmacological inhibition of CXCR4 could alleviate silica-induced pulmonary fibrosis. To this end, wild-type C57BL/6 mice were randomly assigned to three groups: vehicle control (Veh), silica exposure (Sil), and silica exposure with AMD3100 treatment (Sil\u0026thinsp;+\u0026thinsp;AMD3100) (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003ea). Following silica exposure, mice in the Sil group exhibited a rapid loss of body weight, which gradually recovered after day 5 (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eb). Notably, mice treated with AMD3100 showed a significantly accelerated recovery of body weight compared with the Sil group, reaching baseline levels by day 14, suggesting an overall improvement in systemic condition following CXCR4 inhibition.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eHistological analysis of lung tissue further revealed marked structural damage in silica-exposed mice. Hematoxylin and eosin (H\u0026amp;E) staining demonstrated severe disruption of alveolar architecture accompanied by extensive inflammatory cell infiltration in the Sil group. Consistently, Masson\u0026rsquo;s trichrome staining showed pronounced collagen deposition within the lung parenchyma, indicative of fibrotic remodeling \u003cb\u003e(\u003c/b\u003eFig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003ec-d\u003cb\u003e)\u003c/b\u003e. In contrast, the AMD3100-treated group showed a significant reduction in inflammatory infiltration and collagen deposition, preserving alveolar structure and attenuating fibrotic lesions. To assess whether these histological improvements translated into functional benefits, pulmonary function tests were performed. Compared with control mice, silica-exposed mice displayed significant reductions in peak inspiratory flow (PIF), peak expiratory flow (PEF), and minute ventilation (MV), reflecting restrictive ventilatory dysfunction \u003cb\u003e(\u003c/b\u003eFig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003ee\u003cb\u003e)\u003c/b\u003e. Importantly, AMD3100 administration significantly improved all measured pulmonary function parameters relative to the Sil group, indicating partial restoration of lung ventilatory capacity. Collectively, these results demonstrate that pharmacological inhibition of CXCR4 effectively mitigates silica-induced lung injury and fibrosis at both histopathological and functional levels, supporting a critical role for CXCR4 signaling in the development of silicosis-associated pulmonary fibrosis.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec16\" class=\"Section2\"\u003e \u003ch2\u003eCXCR4 Regulates CTSB Expression and Localization in lung Macrophages During Silica-Induced Pulmonary Fibrosis\u003c/h2\u003e \u003cp\u003eTo identify the cellular populations and molecular programs associated with CXCR4 signaling during silica-induced pulmonary fibrosis, we analyzed single-cell RNA sequencing (scRNA-seq) data from lung tissues. Unsupervised clustering revealed a distinct macrophage subset characterized by high expression of complement component C1qc and cathepsin B (Ctsb) in silica-exposed lungs (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003ea-b). Notably, C1qc and Ctsb exhibited highly overlapping expression patterns within this macrophage population, and correlation analysis demonstrated a significant positive association between their transcript levels, indicating coordinated regulation in silica-induced macrophages[16].\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eWe next examined whether CXCR4 signaling regulates Ctsb expression in vivo. Compared with control mice, Ctsb mRNA levels were significantly increased in the lungs of silica-exposed mice, whereas pharmacological inhibition of CXCR4 with AMD3100 markedly reduced Ctsb transcription (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003ec). These results suggest that CXCR4 activity is required for the upregulation of Ctsb during silica-induced lung injury.\u003c/p\u003e \u003cp\u003eTo validate these findings at the protein level and determine their cellular localization, immunofluorescence staining and Western blot analyses were performed. Immunofluorescence analysis demonstrated prominent colocalization of C1QC, CXCR4, and CTSB within lung macrophages in silica-exposed mice, accompanied by increased fluorescence intensity of all three proteins (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003ed-e). In contrast, AMD3100 treatment substantially attenuated the expression of CXCR4 and CTSB and reduced their colocalization within macrophages. Consistently, Western blot analysis confirmed significant upregulation of C1QC, CXCR4, and CTSB protein levels in the silica-exposed group, which was markedly suppressed following CXCR4 inhibition (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003ef\u0026ndash;g).\u003c/p\u003e \u003cp\u003eCollectively, these data demonstrate that CTSB is predominantly expressed in lung macrophages during silica-induced pulmonary fibrosis and that its expression and subcellular distribution are positively regulated by CXCR4 signaling. Inhibition of CXCR4 disrupts the coordinated upregulation of C1QC and CTSB in macrophages, supporting a macrophage-intrinsic CXCR4\u0026ndash;CTSB regulatory axis in the fibrotic lung.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec17\" class=\"Section2\"\u003e \u003ch2\u003eCXCR4 Activates JNK/c-Jun Signaling to Transcriptionally Regulate CTSB Expression\u003c/h2\u003e \u003cp\u003eBased on our findings that CTSB expression in lung macrophages is positively regulated by CXCR4, we next investigated the downstream signaling mechanisms linking CXCR4 activation to CTSB transcription. Analysis of single-cell RNA sequencing data revealed that silica exposure significantly increased the expression of Jun, which was markedly attenuated by AMD3100 treatment (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003ea). These results suggest that CXCR4 signaling may regulate Ctsb expression through a c-Jun\u0026ndash;dependent transcriptional program. Previous literature has shown that Jun is a key downstream effector molecule of the c-Jun N-terminal kinase (JNK, MAPK8/9) signaling pathway, and the activation of c-Jun usually relies on JNK-mediated phosphorylation modification [17, 18]. Protein\u0026ndash;protein interaction (PPI) network analysis indicated that JNK and c-Jun are closely connected within a known MAPK signaling module (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eb), consistent with their established upstream\u0026ndash;downstream relationship in stress-responsive signaling pathways.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eWe then examined the activation status of the JNK/c-Jun signaling pathway. Western blot analysis showed that silica exposure markedly increased the phosphorylation levels of JNK and c-Jun, whereas pharmacological inhibition of CXCR4 with AMD3100 significantly suppressed the phosphorylation of both proteins (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003ec-d). These data indicate that CXCR4 activation promotes c-Jun phosphorylation via JNK signaling in silica-induced lung injury.\u003c/p\u003e \u003cp\u003eTo determine whether c-Jun directly regulates Ctsb transcription, a luciferase reporter construct containing the Ctsb promoter region (\u0026minus;\u0026thinsp;2000 bp to +\u0026thinsp;100 bp) was generated (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003ee). Dual-luciferase reporter assays demonstrated that overexpression of c-Jun significantly enhanced Ctsb promoter activity (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003ef). To further validate direct binding of c-Jun to the Ctsb promoter, chromatin immunoprecipitation (ChIP) followed by qPCR was performed. Agarose gel electrophoresis confirmed specific amplification of the expected promoter fragment (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eg), and quantitative analysis revealed significant enrichment of c-Jun at the Ctsb promoter compared with IgG controls (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eh).\u003c/p\u003e \u003cp\u003eCollectively, these results demonstrate that CXCR4 activation induces JNK-dependent phosphorylation of c-Jun, which directly binds to the Ctsb promoter and enhances its transcription in silica-induced pulmonary fibrosis.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec18\" class=\"Section2\"\u003e \u003ch2\u003eCTSB Promotes TGF-β1 Activation and Fibrotic Remodeling in Silica-Induced Pulmonary Fibrosis\u003c/h2\u003e \u003cp\u003eTo determine whether CTSB contributes to TGF-β1 activation during silica-induced pulmonary fibrosis, we first examined their spatial relationship in lung tissues. Immunofluorescence analysis revealed prominent colocalization of CTSB and TGF-β1 within lung macrophages in silica-exposed mice (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003ea-b). Notably, pharmacological inhibition of CXCR4 with AMD3100 markedly reduced both the fluorescence intensity and colocalization of CTSB and TGF-β1. Consistent with these observations, Western blot analysis of lung tissue revealed that AMD3100 treatment significantly downregulated the expression levels of TGF-β1, as well as downstream fibrotic markers, including α-smooth muscle actin (α-SMA), and fibronectin, compared with silica-exposed mice (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003ec-d).\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eTo directly assess the role of CTSB in regulating TGF-β1 expression, silica-stimulated macrophages were treated with the CTSB-specific inhibitor CA-074Me in vitro. Cells were lysed for protein extraction after 24 hours. The remaining results are presented in Figure \u003cspan refid=\"MOESM1\" class=\"InternalRef\"\u003eS1\u003c/span\u003e. Inhibition of CTSB activity resulted in a marked reduction in TGF-β1 protein levels, as determined by Western blot analysis (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003ee-f). We next evaluated the functional significance of the CTSB\u0026ndash;TGF-β1 axis in vivo. Silica-exposed mice were treated with the CTSB inhibitor CA-074Me, the TGF-β receptor inhibitor SD-208, or a combination of both inhibitors. Histological analysis using H\u0026amp;E and Masson\u0026rsquo;s trichrome staining showed that inflammatory infiltration and collagen deposition were substantially attenuated in inhibitor-treated groups compared with the silica-only group (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eg-h). In parallel, RT\u0026ndash;qPCR analysis revealed that silica exposure markedly upregulated the mRNA expression of Tgfb1, Acta2 (α-SMA), and Fn1, whereas treatment with CA-074Me or SD-208 significantly reduced their expression (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003ei). Consistently, ELISA analysis of lung tissue homogenates confirmed that elevated TGF-β1 protein levels induced by silica exposure were markedly diminished following CTSB or TGF-β signaling inhibition (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003ej).\u003c/p\u003e \u003cp\u003eCollectively, these results demonstrate that CTSB promotes TGF-β1 activation and downstream fibrotic responses during silica-induced pulmonary fibrosis and pharmacological targeting of the CTSB\u0026ndash;TGF-β1 axis effectively attenuates lung fibrosis.\u003c/p\u003e \u003cp\u003e \u003cb\u003eGenetic ablation of CXCR4 in macrophages attenuates silica-induced pulmonary fibrosis.\u003c/b\u003e \u003c/p\u003e \u003cp\u003eHomozygous Cxcr4-floxed mice (Cxcr fl/fl) were crossed with Lyz2CreERT2 mice to generate Lyz2CreERT2; Cxcr fl/fl mice, which harbor a myeloid cell-specific inducible Cre recombinase system. Cre-positive CXCR4fl/fl mice were identified by PCR genotyping and used for subsequent experiments (see Figure S2). To induce macrophage-specific Cxcr4 deletion, mice received intraperitoneal injections of tamoxifen (100 mg/kg) once daily for five consecutive days prior to silica exposure (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003ea). Histological analysis revealed that inflammatory cell infiltration was significantly reduced in the lungs of Cxcr4-deficient mice, as shown by H\u0026amp;E staining (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eb). Consistently, Masson\u0026rsquo;s trichrome staining demonstrated a substantial decrease in collagen deposition and fibrosis scores in Cxcr4 knockdown mice compared with control silicosis mice (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eb-c).\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eTo determine whether genetic ablation of Cxcr4 affects CTSB expression in macrophages, immunofluorescence staining was performed. In wild-type silicosis mice, CXCR4 and CTSB exhibited prominent colocalization within lung macrophages, whereas this colocalization was markedly diminished in Cxcr4-deficient mice (Figs.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003ed-e). At the protein level, Western blot analysis further confirmed a significant reduction in CTSB expression in lung tissues from Cxcr4 knockdown mice (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003ef\u0026ndash;g). In parallel, key downstream profibrotic markers, including TGF-β1, α-smooth muscle actin (α-SMA), and fibronectin, were significantly downregulated following macrophage-specific deletion of Cxcr4 (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eg). These findings indicate that loss of CXCR4 in macrophages disrupts CTSB expression and suppresses activation of downstream fibrotic signaling pathways.\u003c/p\u003e \u003cp\u003eCollectively, these genetic data demonstrate that macrophage-intrinsic CXCR4 is required for CTSB upregulation and fibrotic remodeling in silica-induced pulmonary fibrosis, providing in vivo evidence that the CXCR4\u0026ndash;JNK/c-Jun\u0026ndash;CTSB signaling axis is a critical driver of silicosis pathogenesis.\u003c/p\u003e \u003c/div\u003e"},{"header":"Discussion","content":"\u003cp\u003eSilicosis is an occupational pulmonary fibrosis disease caused by the prolonged inhalation of crystalline silica dust, and its global incidence continues to rise, becoming a significant public health burden[19, 20]. The pathological progression of this disease typically begins with acute and chronic inflammation in the lungs, eventually leading to irreversible alveolar destruction and abnormal extracellular matrix deposition[4].\u003c/p\u003e \u003cp\u003eIn recent years, the role of C-X-C chemokine receptor 4 (CXCR4) and its ligand CXCL12 in tissue repair and fibrosis has gained increasing attention. As a classical G protein-coupled receptor, CXCR4 plays a crucial regulatory role in a variety of pathological and physiological processes[21]. CXCR4 is involved not only in the migration and homing of immune cells but also in regulating epithelial-to-mesenchymal transition and extracellular matrix remodeling in various organ fibrosis models[22, 23]. Upon binding to its ligand CXCL12 (SDF-1α), CXCR4 activates downstream signaling pathways, including PI3K/Akt, MAPK/ERK, and JAK/STAT, thereby regulating cellular processes such as proliferation, migration, survival, and differentiation[24]. Notably, in silica-induced pulmonary fibrosis, CXCR4 may serve as a key molecular hub linking environmental exposure to fibrotic responses.\u003c/p\u003e \u003cp\u003eThis study establishes, for the first time, a direct molecular link between CXCR4 and cathepsin B (CTSB) in a silica-induced pulmonary fibrosis model and elucidates the key mediating role of the JNK/c-Jun signaling axis in this process. Using single-cell RNA sequencing technology, we observed co-expression of Ctsb with the macrophage marker gene C1qc. Through protein interaction network analysis, dual-luciferase reporter assays, and chromatin immunoprecipitation (ChIP), we confirmed that silica exposure upregulates CXCR4 expression, thereby activating the downstream JNK/c-Jun signaling pathway. Activation of JNK (evidenced by elevated phosphorylated p-JNK levels) directly catalyzes the phosphorylation of the transcription factor c-Jun at Ser63 and Ser73. Phosphorylated c-Jun (p-c-Jun) significantly enhances its DNA-binding capacity and transcriptional activity. Importantly, activated c-Jun specifically recognizes and binds to the AP-1 response element within the Ctsb gene promoter region, directly driving the upregulation of CTSB transcription, leading to a marked increase in its mRNA and protein expression levels. This result is consistent with previous studies, where CXCR4 regulates the transcriptional activity of multiple target genes through downstream MAPK/JNK signaling modules, thereby affecting the expression of inflammation- and fibrosis-related molecules[25].\u003c/p\u003e \u003cp\u003eCathepsin B (CTSB) is an important lysosomal cysteine protease[26]. When overexpressed and secreted into the extracellular microenvironment, CTSB can precisely cleave the latency-associated peptide (LAP) within the precursor complex of transforming growth factor-β1 (TGF-β1), thereby releasing biologically active mature TGF-β1 [27]. In this study, through immunofluorescence co-localization and Western blot analysis, we revealed the high consistency between the protein expression and cellular localization of CTSB and TGF-β1, further linking protease-mediated matrix degradation to classic pro-fibrotic signaling pathways. Activated TGF-β1 subsequently acts in an autocrine or paracrine manner to bind to the TGF-β receptor II/I complex on the cell surface, initiating the canonical Smad2/3 signaling pathway as well as non-canonical pathways such as MAPK and PI3K-Akt[28, 29]. The convergence of these downstream signals ultimately induces the transformation of fibroblasts into myofibroblasts, promoting the excessive synthesis and deposition of extracellular matrix proteins, such as α-smooth muscle actin (α-SMA) and fibronectin[30, 31], which constitute the core pathological processes of pulmonary fibrosis.\u003c/p\u003e \u003cp\u003eIn conclusion, this study integrates multiple layers of evidence, including animal models, single-cell transcriptomics, molecular interaction validation, and genetic intervention, to unveil a novel signaling axis composed of CXCR4/JNK/c-Jun/CTSB/TGF-β1. This finding provides new insights into the pathogenesis of chronic fibrotic diseases induced by environmental factors. The results of this study both corroborate and extend previous findings. Numerous studies have confirmed the critical role of CXCR4 in organ fibrosis, with the JNK/c-Jun pathway often involved in oxidative stress responses[32]. However, previous research has predominantly focused on the role of CXCR4 in cell migration, survival, or inflammatory cytokine secretion[33, 34], with limited attention given to its regulation of protease expression. Our study bridges CXCR4 signaling with CTSB, an important lysosomal protease, providing a fresh perspective on its role in extracellular matrix remodeling. Furthermore, while the mechanism of CTSB-mediated activation of TGF-β1 has been described in cancer and liver fibrosis[13, 35], functional studies in environment-exposure related pulmonary diseases, such as silicosis, remain scarce. This study, through in vivo and in vitro inhibitor experiments and genetic intervention models, establishes the indispensable role of the CTSB/TGF-β1 axis in silicosis fibrosis, thus filling a gap in this area of research.\u003c/p\u003e \u003cp\u003eAlthough this study has made significant findings, several limitations remain. The experiments were primarily conducted using mouse models, which exhibit species differences in the pathological progression of silicosis compared to humans. Therefore, further validation of the conservation and relevance of this signaling pathway in clinical samples or organoid models is necessary. Moreover, while this study focused on the CXCR4/CTSB axis in macrophages, it remains unclear whether other cell types, such as fibroblasts or epithelial cells, contribute to fibrosis through similar or parallel mechanisms. Additionally, silica particle-induced oxidative stress may directly activate kinases such as JNK. Although we have demonstrated that CXCR4 acts as an important upstream regulator, the direct regulatory relationship between CXCR4 and JNK, the potential existence of other parallel pathways, and how TGF-β1 is further regulated in CXCR4 knockdown mice, still require further investigation. From a therapeutic perspective, CXCR4 antagonists (such as AMD3100) have shown promising antifibrotic effects in various disease models. Their mechanisms of action are not limited to blocking inflammatory cell recruitment but may also involve inhibiting fibroblast activation, reducing extracellular matrix (ECM) deposition, and modulating the immune microenvironment. However, the functional heterogeneity of CXCR4 across different cell types, its interaction with the homologous receptor CXCR7, and its dynamic changes at different stages of the disease remain unresolved key scientific issues that must be addressed in future research.\u003c/p\u003e \u003cp\u003eCXCR4 plays a multifaceted regulatory role in oxidative stress-related fibrotic diseases [36]. A deeper understanding of the spatiotemporal expression patterns of CXCR4 and the dynamic changes in its downstream signaling networks in specific disease environments will provide an essential theoretical foundation for the development of targeted therapeutic strategies. Based on the findings and limitations of this study, future research could involve clinical investigations, such as the collection of bronchoalveolar lavage fluid or lung biopsy samples from silicosis patients, to assess the expression levels of CXCR4, CTSB, and phosphorylated c-Jun, and analyze their correlation with disease stage, pulmonary function parameters, and prognosis, thereby advancing the translational medicine research on this mechanism. At the mechanistic level, cell-specific knockout models can be employed to dissect the contributions of different pulmonary cell types in the CXCR4/CTSB axis, and to explore the causal relationship between silica-induced reactive oxygen species (ROS) generation and the upregulation of CXCR4 expression, thereby providing a more complete picture of the signaling network from environmental exposure to fibrosis formation. Additionally, the therapeutic potential of small-molecule inhibitors targeting this pathway (such as JNK inhibitors and CTSB inhibitors) or combination therapies in advanced fibrosis models could be evaluated, providing preclinical evidence for the development of new anti-fibrotic drugs. By integrating multi-omics approaches, a comprehensive study of the global impact of this signaling axis on extracellular matrix components and immune microenvironment remodeling will help to fully elucidate its pathophysiological significance in the progression of pulmonary fibrosis.\u003c/p\u003e \u003cp\u003eIn conclusion, the CXCR4/JNK/c-Jun/CTSB/TGF-β1 signaling pathway revealed in this study provides a novel framework for understanding the molecular mechanisms of silica-induced pulmonary fibrosis. This study not only expands our knowledge of the role of chemokine receptors in oxidative stress-related diseases but also lays a crucial experimental foundation for the future development of intervention strategies targeting key nodes of this pathway.\u003c/p\u003e"},{"header":"Conclusion","content":"\u003cp\u003eIn summary, this study identifies CXCR4 as a critical mediator in silica-induced pulmonary fibrosis. Pharmacological inhibition of CXCR4 with AMD3100 and conditional genetic deletion of Cxcr4 in mice both effectively attenuated the JNK/c-Jun/CTSB pathway and subsequent fibrotic responses. These results not only clarify the molecular underpinnings of silicosis but also establish a theoretical and experimental rationale for targeting CXCR4 as a novel therapeutic strategy for pulmonary fibrosis, offering new directions for the development of targeted silicosis treatments.\u003c/p\u003e"},{"header":"Declarations","content":"\u003cp\u003e\u003cstrong\u003eAuthor contributions\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eF.W., Y.F.Z, R.Q.Y., L.Y.W.:\u0026nbsp;\u003c/strong\u003ePerformed the experiments and drafted the initial manuscript. \u003cstrong\u003eF.W., Y.F.Z, X.L., Z.H.X.:\u0026nbsp;\u003c/strong\u003eConducted correlation analysis, processed experimental data, and generated figures and tables.\u003cstrong\u003e\u0026nbsp;M.M., J.H.W., W.F.W., X.R.T.:\u0026nbsp;\u003c/strong\u003eReviewed and revised the manuscript. \u003cstrong\u003eM.M., J.H.W., W.F.W.:\u0026nbsp;\u003c/strong\u003eSupervised the research project. This manuscript was collaboratively prepared by all authors. All authors have reviewed and approved the final version of the manuscript.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eInstitutional review board statement\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eAnimal research procedures adhere to the guidelines outlined in the NIH Guide for the Care and Use of Laboratory Animals (NIH Publication No. 8023, revised 1978), and the Institutional Animal Care and Use Committee of Anhui University of Science and Technology approved the animal protocol for this study (No. 2023028). Approved by the Ethics Committee of the First Affiliated Hospital of Anhui University of Science and Technology (batch number: 2023-KY-B110-001).\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eFunding\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThis work was supported by Natural Science Foundation of China (82304112)；Introduce talent research start-up fund, Anhui University of Science and Technology(2023yjrc63); 2023 Medical Specialized Cultivation Project, Anhui University of Science and Technology (YZ2023H1A001), Research Funds of Joint Research Center for Occupational Medicine and Health of IHM (NO. OMH-2023-01, OMH-2023-03)\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eDeclaration of competing interest\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper.\u003c/p\u003e\u003ch2\u003eData Availability\u003c/h2\u003e\u003cp\u003eAll generated and analyzed data are included in this published article and its supplementary information material.\u003c/p\u003e"},{"header":"References","content":"\u003cp\u003e\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003cp\u003e\u003c/p\u003e"},{"header":"Scheme 1","content":"\u003cp\u003eScheme 1 is available in the Supplementary Files section.\u003c/p\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":false,"hideJournal":false,"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":"particle-and-fibre-toxicology","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"pftx","sideBox":"Learn more about [Particle and Fibre Toxicology](http://particleandfibretoxicology.biomedcentral.com)","snPcode":"12989","submissionUrl":"https://submission.nature.com/new-submission/12989/3","title":"Particle and Fibre Toxicology","twitterHandle":"@BioMedCentral","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"em","reportingPortfolio":"BMC/SO AJ","inReviewEnabled":true,"inReviewRevisionsEnabled":true},"keywords":"Silicosis, CXCR4 signaling, Cathepsin B, JNK/c-Jun pathway, TGF-β1","lastPublishedDoi":"10.21203/rs.3.rs-8497777/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-8497777/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003cp\u003eSilicosis is a severe occupational lung disease characterized by progressive and irreversible pulmonary fibrosis. CXCR4 (C-X-C chemokine receptor type 4) has been implicated in the pathogenesis of silicosis, yet the specific molecular mechanism through which CXCR4 signaling promotes fibrotic progression remain unclear. In this study, we systematically investigated the role of CXCR4 in silica-induced lung inflammation and fibrosis using pharmacological inhibition with AMD3100 and conditional Cxcr4 knockout mouse models, supported by complementary \u003cem\u003ein vitro\u003c/em\u003e mechanistic studies. Silica exposure significantly upregulated CXCR4 expression in the lungs, which was accompanied by activation of the JNK/c-Jun pathway and enhanced transcription of cathepsin B (CTSB). Both AMD3100 treatment and genetic deletion of Cxcr4 markedly reduced inflammatory cell infiltration, collagen deposition, and lung function impairment, while also suppressing the expression of CTSB, TGF-β1, α-SMA, and fibronectin. Mechanistically, CXCR4 activation promoted JNK-dependent phosphorylation of c-Jun, enabling c-Jun to bind directly to the Ctsb promoter and drive its transcription. The subsequent increase in CTSB levels facilitated TGF-β1 activation, thereby amplifying downstream profibrotic signaling. Notably, CTSB was predominantly expressed in macrophages, where it co-localized with C1QC and was positively regulated by CXCR4 signaling. Taken together, our findings reveal a previously unrecognized CXCR4\u0026ndash;JNK/c-Jun\u0026ndash;CTSB signaling axis in macrophages that drives silica-induced pulmonary fibrosis, highlighting CXCR4 as a promising therapeutic target for silicosis.\u003c/p\u003e","manuscriptTitle":"Targeting CXCR4–JNK/c-Jun–CTSB Signaling Axis Attenuates Silicosis Fibrosis in Mice","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2026-02-02 15:56:44","doi":"10.21203/rs.3.rs-8497777/v1","editorialEvents":[{"type":"communityComments","content":0},{"type":"reviewersInvited","content":"","date":"2026-01-29T16:37:11+00:00","index":"","fulltext":""},{"type":"editorAssigned","content":"","date":"2026-01-08T06:35:52+00:00","index":"","fulltext":""},{"type":"checksComplete","content":"","date":"2026-01-06T11:03:48+00:00","index":"","fulltext":""},{"type":"submitted","content":"Particle and Fibre Toxicology","date":"2026-01-02T03:06:53+00:00","index":"","fulltext":""}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"particle-and-fibre-toxicology","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"pftx","sideBox":"Learn more about [Particle and Fibre Toxicology](http://particleandfibretoxicology.biomedcentral.com)","snPcode":"12989","submissionUrl":"https://submission.nature.com/new-submission/12989/3","title":"Particle and Fibre Toxicology","twitterHandle":"@BioMedCentral","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"em","reportingPortfolio":"BMC/SO AJ","inReviewEnabled":true,"inReviewRevisionsEnabled":true}}],"origin":"","ownerIdentity":"9030adb0-29bc-49b2-be87-84805a199006","owner":[],"postedDate":"February 2nd, 2026","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"under-review","subjectAreas":[],"tags":[],"updatedAt":"2026-02-02T15:56:44+00:00","versionOfRecord":[],"versionCreatedAt":"2026-02-02 15:56:44","video":"","vorDoi":"","vorDoiUrl":"","workflowStages":[]},"version":"v1","identity":"rs-8497777","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-8497777","identity":"rs-8497777","version":["v1"]},"buildId":"XKTyCvWXoU3ODBz1xrDgd","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}