Inhibition of PADI2-mediated vimentin citrullination alleviates silica-induced pulmonary fibrosis in mice

Abstract

Silicosis remains a critical occupational health concern worldwide, lacking effective treatments due to unclear mechanisms. In this study, we investigated the citrullinated proteomic profile and its effects in mice exposed to silica. Our findings demonstrated elevated levels of citrullinated peptides and citrullinated vimentin (Cit-Vim) in silicotic mice and silica-treated macrophages, regulated by peptidylarginine deiminase (PADI2). Unlike vimentin, Cit-Vim amplified the production of tumor necrosis factor-α (TNF-α), Interleukin-6 (IL-6), and IL-1β in silica-treated macrophages through interaction with Toll-like receptor 4 (TLR4) signaling. RNA sequencing revealed that early growth response protein 1 (EGR1) is a target of PADI2, with Cit-Vim inducing lung inflammation via EGR1 signaling. Pharmacological inhibition or genetic knockout of Padi2 attenuated silica-induced lung inflammation and fibrosis. These findings suggest that targeting PADI2 may represent a novel therapeutic strategy of silicosis.

Supplementary Information

The online version contains supplementary material available at 10.1186/s12989-026-00671-y.

Introduction

Silicosis remains a major occupational health challenge, responsible for 88.3% new cases, 75.9% of deaths, 85.4% of disability-adjusted life years (DALYs) related to pneumoconiosis in China in 2019 [1, 2]. This disease imposes a significant global burden, and despite ongoing efforts, there is still an urgent need to develop effective treatments to improve the quality of life (QOL) for those affected [3–5].

Citrullination, a post-translational modification catalyzed by peptidylarginine deiminases (PADIs), plays a central role in pathogenesis of rheumatoid arthritis and has been implicated in the early stages of idiopathic pulmonary fibrosis (IPF) [6]. Padi4 knockout models have shown attenuation of bleomycin (BLM)-induced neutrophil extracellular traps (NETs), fibrotic gene expression, and lung fibrosis in mice [7]. Additionally, citrullinated vimentin (Cit-Vim), produced by lung macrophages, acts as a damage-associated molecular pattern molecule (DAMP) regulated by PADI2 and promotes lung fibrosis [8]. Despite these findings, the role of citrullination, particularly in silicosis, remains largely unexplored.

In this study, we present for the first time a citrullinated proteomic profile in silica-exposed mice, further validated in silicotic rodent models. We demonstrate that Cit-Vim, a key mediator of silicosis-induced lung inflammation, can be attenuated through pharmacological inhibition or gene knockout of Padi2, offering potential therapeutic insights into silicosis.

Materials and methods

Establishment of the mouse and rat models

All the experimental rodents were housed under specific pathogen-free conditions, maintained on a 12-h light/dark cycle with unrestricted access to food and water (SYXK-Hebei-2020-007). The experimental protocol was approved by the Ethics Committee for Animal Experimentation of North China University of Science and Technology, which complies with the US National Institutes of Health Guide for the Care and Use of Laboratory Animals (Approval No. 2022025). Wistar rats (aged 3 weeks, male) were obtained from the Beijing HFK Bioscience Co., Ltd. Male C57BL/6 J mice (8 weeks old), were obtained from Vital River Laboratory Animal Technology (Beijing, China).

Rats were exposed to silica via inhalation for 24 weeks and divided into two groups: control rats exposed to ambient air and experimental rats exposed to silica (n = 10 per group) [9, 10].

A macrophage depletion model was generated as described in prior studies [11, 12]. Male C57BL/6 J male mice (8 weeks old) were divided into two groups (n = 10 per group): silica + control liposome and silica + clodronate liposome.

Forty mice were randomly assigned to four groups (n = 10 per group): (1) Control group: Intratracheal instillation of 50 µL 0.9% saline. (2) AFM-30a group: Intraperitoneal instillation of AFM-30a (CAY10723, Cayman Chemical, Ann Arbor, MI, USA) at a dose of 20 mg/kg in 100 µL [13, 14] for 2 weeks, followed by 2 weeks of saline treatment. (3) Silica group: Intratracheal instillation of 10 mg silica (s5631, Sigma-Aldrich, St.Louis, MO, USA [15–17] suspended in 50 µL saline. (4) Silica + AFM-30a group: Intraperitoneal injection of AFM-30a (20 mg/kg, 100 µL) for 2 weeks, after 2 weeks of silica exposure.

Padi2^{flox/flox; Lyz2−cre} mice were generated and obtained from Cyagen Biosciences Inc. Conditional macrophage-specific Padi2 knockout (Padi2^{flox/flox; Lyz2−cre}) mice were produced by crossing Padi2^flox/flox mice with Lyz2-cre transgenic mice. Padi2^flox/flox were used as controls. The groups (n = 10 each group) were as follows: (1) WTC group: Padi2^flox/flox mice treated with intratracheal instillation of 50 µL saline. (2) KOC group: Padi2^{flox/flox; Lyz2−cre} mice treated with 50 µL saline. (3) WTS group: Padi2^flox/flox mice treated with 10 mg/50 µL silica suspension. (4) KOS group: Padi2^{flox/flox; Lyz2−cre} mice treated with 10 mg/50 µL silica suspension.

Citrulline proteomics

Proteomic analysis of citrullinated proteins was conducted by Hangzhou Jingjie PTM BioLab Co., Ltd. Lung tissues were harvested from two groups of mice, snap-frozen in liquid nitrogen, and subjected to protein extraction. Protein concentration was measured, and citrullinated peptides were enriched, digested, and analyzed using liquid chromatography-tandem mass spectrometry (LC-MS/MS) [18].

High-throughput transcriptome sequencing

RNA sequencing was performed by Guangzhou RiboBio Co., Ltd. Lung tissue from four groups of mice and immediately frozen in liquid nitrogen. Total RNA was extracted using TRIzol reagent. RNA purity and concentration were assessed. High-throughput sequencing was carried out after RNA fragmentation and cDNA library preparation [19].

Cell culture

RAW 264.7 murine macrophages were cultured in Ham’s F-12 K medium (J120703, Shanghai BasalMedia Technologies Co., Ltd. Shanghai, China). Cells were treated with silica (50, 100–200 µg/mL), or transfected with siRNAs targeting scavenger receptor B 1 (Srb1, NO 327; RIBOBIO, Guangzhou, China), Padi2 (sc-61282; Santa cruz, CA, USA) or Early Growth Response Protein 1 (Egr1, sc-35267; Santa cruz, CA, USA). Additional treatments included 10 nmol/L of toll-like receptor 4 (TLR4) inhibitor (C34, 18512; Cayman Chemical, Ann Arbor, MI, USA), 2 µg/mL of biotinylated Vimentin (37622; Cayman Chemical), 2 µg/mL of Cit-Vim (37624; Cayman Chemical), and 5 µmol/L of AFM-30a.

Pulmonary function measurement

Pulmonary function in conscious mice was measured using the FinePointe WBP system (Buxco Research Systems, Wilmington, NC, USA) according to the manufacturer’s protocol. Measurements were recorded at 2-second intervals. Key indices included tidal volume (TVb), minute volume (MVb), expiratory flow (EF50), peak expiratory flow (PEF), and inspiratory time (Ti), normalized to body weight.

Micro-CT imaging

High-resolution CT scans of mouse lungs were acquired using the NEMO Micro CT (NMC-100) machine (PINGSENG Healthcare Inc., Kunshan, China). Mice were anesthetized with isoflurane, and the 3D lung images were reconstructed using the AVATAR 1.5.0 software.

Histological staining

For H&E staining, paraffin-embedded lung sections were deparaffinized, rehydrated, and stained with hematoxylin and eosin to assess tissue morphology. For Van Gieson staining, deparaffinized sections were stained with hematoxylin and Van Giesson’s solution (BA4084, BaSO, Zhuhai, China) to visualize collagen. Silicon nodules and collagen fiber areas were quantified using Image-Pro Plus 6.0 software and normalized by the total tissue area.

Immunofluorescence staining

Paraffin-embedded sections and cell slides were hydrated and incubated overnight with primary antibodies against Cit-Vim, TNF-α, and EGR1 at 1:100 dilution. This was followed by incubation with fluorescent secondary antibodies at 37 °C for 40 min. Nuclei were counterstained with DAPI, and positive cell counts were normalized using Image-Pro-Plus 6.0 software.

Immunohistochemical staining

Tissue sections were deparaffinized and rehydrated, and endogenous peroxidase activity was quenched with 3% hydrogen peroxide. Samples were incubated overnight at 4 °C with anti-PADI2 antibodies (1:200 dilution), followed by secondary antibody incubation (PV- 6000; Beijing Zhongshan Jinqiao Biotechnology Co. Ltd., Beijing, China) at 37 °C for 20 min. DAB staining (ZLI-9018; Beijing Zhongshan Jinqiao Biotechnology Co. Ltd., Beijing, China) was used to visualize immunoreactivity.

Western blot analysis

Proteins were separated via SDS-PAGE and transferred to membranes for western blotting as previously described [20, 21]. Membranes were incubated with primary antibodies against Pro-collage I (Pro-CoL I), fibronectin-1 (FN1), PADI2, PADI3, PADI4, Cit, Cit-Vim, TNF-α, IL-6, IL-1β, EGR1 and β-actin (Table S1) at a 1:1000 dilution. Secondary antibodies (1:5000 dilution) were applied, and immunoblots were visualized using Enhanced Chemiluminescence reagents.

Statistical analysis

Statistical analyses were performed using SPSS 20.0 software. Data are presented as mean ± standard deviation (SD). Group comparisons were made using Student’s t-test or one-way ANOVA followed by Tukey’s post-hoc test for multiple comparisons. Statistical significance was set at P < 0.05.

Results

Citrullination proteomic profile in silicotic mice

Using mass-spectrometry-based deep proteomic analysis, we identified citrullination sites in lung tissue of control and silicotic mice. As showed in Figs. 1A and 198 citrullination sites and 173 mice proteins were validated, with 141 citrullinated sites and 125 proteins significantly up-regulated in silicotic mice compared to controls (Table S2). Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway enrichment analyses indicated that the citrullinated proteins were mainly involved in processes related to cell development, signal transduction, and metabolism, with a prominent role in regulating immune system function (Fig. 1B–C and Figure S1A–B).

Fig. 1.

Citrullination proteomic profile in silicotic mice. A Hierarchical cluster heatmaps illustrating gene expression patterns in group and silicotic groups. B KEGG pathway analysis of upregulated proteins in the silicotic (S) group. C KEGG pathway analysis of downregulated proteins in the silicotic (S) group

Further pathway enrichment analysis revealed that the upregulated citrullinated proteins were associated with the gap junction pathway (Reactome), oxidative stress (WikiPathways), and AhpC/TSA protein domains. In contrast, downregulated citrullinated proteins were enriched in pathways related to collagen degradation (Reactome), cell adhesion (WikiPathways), and the EGF protein domain (Figure S1C-H).

Elevated expression of Cit-peptide and Cit-Vim in silicotic rodents

We next assessed the location and expression levels of Cit-peptide and Cit-Vim in silicotic rat and mouse models. As shown in Fig. 2A–F, Cit-peptide expression and Cit-Vim co-expression were detected in silicotic nodules in both rats and mice exposed to silica. Additionally, the levels of Cit-peptide and Cit-Vim were elevated in lung tissues from these rodents. Further analysis revealed that among PADIs, only PADI2 was significantly increased in lung tissues of silica-exposed rats and mice (Fig. 2 and Figure S2). In a macrophage depletion model, we observed decreased levels of PADI2, Cit-peptide, and Cit-Vim in silica-exposed mice (Fig. 2G–H).

Fig. 2.

Upregulation of PADI2, Citrullinated Peptides, and Citrullinated Vimentin in a silicosis model. A–F Immunofluorescence staining of Cit-peptide and vimentin (Vim) co-expression in silicotic rats and mice (scale bar = 50 μm). Western blot analysis showing the expression levels of PADI2, Cit-peptide, and Cit-Vim across groups (Results are presented as Mean ± SD, n = 6 per group). G–H Immunohistochemical staining of PADI2 and immunofluorescence staining of Cit-peptide and Vim co-expression in macrophage-depleted mice (scale bar = 50 μm). Western blot analysis of PADI2, Cit-peptide, and Cit-Vim expression (Results are presented as Mean ± SD, n = 3 per group)

In silica-treated RAW 264.7 cells, co-expression of Cit and Vim was detected by immunofluorescence staining, with PADI2, Cit-peptide, and Cit-Vim levels increasing in a dose- and time-dependent manner (Figure S3A-C). Treatment with siRNA-Srb1 or the TLR4 inhibitor TLR-C34 attenuated the silica-induced increase in PADI2, Cit-peptide, and Cit-Vim levels in RAW 264.7 cells (Figure S3D–G).

Inhibition of PADI2 reduced pulmonary fibrosis in silica-treated mice

Treatment with AFM-30a, a PADI2 inhibitor, or genetic knockout of Padi2 resulted in a significant reduction in the silicotic nodule area and collagen deposition, as observed through Van Gieson staining, in silica-exposed mice compared to control groups. Similarly, the levels of pro-collagen I (Pro-CoL I) and fibronectin 1 (FN1) were reduced in silicotic mice following these treatments. Micro-CT imaging and pulmonary function tests further demonstrated that PADI2 inhibition, either by AFM-30a or Padi2 gene knockout, attenuated lung injury and improved lung function in silica-exposed mice (Figs. 3 and 4; Figure S4 and Figure S5).

Fig. 3.

Attenuation of Silicosis-induced Fibrosis by AFM-30a treatment in mice. A Hematoxylin and eosin (H&E) and Van Gieson (VG) staining of lung tissue in AFM-30a-treated mice (scale bars = 1–50 μm) (Results are presented as Mean ± SD, n = 6 per group). B Micro CT imaging of lung tissue in AFM-30a-treated mice (Results are presented as Mean ± SD, n = 6 per group). C Western blot analysis of pro-collagen I (pro-CoL I) and fibronectin 1 (FN1) expression in AFM-30a-treated mice (Results are presented as Mean ± SD, n = 3 per group). D Pulmonary Function Assessment in AFM-30a-treated mice (Results are presented as Mean ± SD, n = 6 per group)

Fig. 4.

Attenuation of Silicosis-induced Fibrosis and protection of lung function by PADI2 inhibition. A H&E and VG staining in Padi2 knockout mice (scale bars = 1–100 μm) (Results are presented as Mean ± SD, n = 6 per group). B Micro CT imaging of lung tissue in Padi2 knockout mice (Results are presented as Mean ± SD, n = 6 per group). C Western blot analysis of pro-CoL I and FN1 expression in Padi2 knockout mice (Results are presented as Mean ± SD, n = 3 per group). D Pulmonary Function Assessment in Padi2 knockout mice (Results are presented as Mean ± SD, n = 6 per group)

Inhibition of PADI2 decreased Cit-Vim expression in silicotic mice

As expected, PADI2, Cit-peptide, and Cit-Vim levels were significantly reduced in silica-exposed mice treated with AFM-30a or in Padi2 knockout mice compared to controls (Fig. 5). Similarly, pharmacological inhibition of PADI2 or Padi2 gene silencing in silica-treated RAW 264.7 cells decreased the expression of Cit-peptide and Cit-Vim, as confirmed by immunofluorescence staining and western blotting (Figure S6A-B). Additionally, Cit-Vim levels in the culture medium of silica-treated RAW 264.7 cells were reduced following AFM-30a or siRNA-Padi2 treatment (Figure S6C).

Fig. 5.

PADI2 regulates citrullinated peptides and citrullinated vimentin expression. A Immunofluorescence staining of Cit-peptide and Vim co-expression (scale bar = 50 μm) and Western blot analysis of PADI2, Cit-peptide, and Cit-Vim expression in AFM-30a-treated mice. B Immunofluorescence staining of Cit-peptide and Vim co-expression (scale bar = 100 μm) and Western blot analysis of PADI2, Cit-peptide, and Cit-Vim expression in Padi2 knockout mice. Results are presented as Mean ± SD, n = 3 per group

Inhibition of PADI2 reduced lung inflammation via EGR1 induced by Cit-Vim

RNA-sequencing analysis revealed 954 upregulated and 279 downregulated genes in silicotic mice compared to controls (Table S3). In Padi2 knockout mice, 141 differentially expressed genes were identified, visualized in a heatmap (Fig. 6A). GO and KEGG pathway analysis suggested that Padi2 knockout modulated inflammatory pathways including IL-1 production, TNF signaling, chemokine signaling, and Th17 cell differentiation (Fig. 6B-C). Among the top 10 hub genes, Egr1 was identified as a potential target of Padi2 knockout in silicotic mice (Fig. 6D).

Fig. 6.

Transcriptome Sequencing Reveals pathways and targets of Padi2 knock in Mitigating Pulmonary Fibrosis in Silicotic Mice. A Hierarchical cluster heatmaps showing gene expression in different groups. B–C GO and KEGG pathway analysis of downregulated and upregulated genes in the Padi2 knockout (KOS) group. D Heatmaps displaying significantly downregulated genes in the KOS Group. E–F Immunofluorescence staining of TNF-α expression (scale bar = 100 μm) in RAW 264.7 cells treated with Cit-Vim. G–H Western blot analysis of EGR1, TNF-α, IL-6 and IL-1β expression in RAW 264.7 cells treated with Cit-Vim. I–J Western blot analysis of TNF-α, IL-6 and IL-1β expression in Cit-Vim and siRNA-Egr1-treated RAW 264.7 cells. Results are presented as Mean ± SD, n = 3 per group

Finally, we observed that treatment with Cit-Vim, but not Vim, led to increased expression of EGR1, TNF-α, IL-6, IL-1β in RAW 264.7 cells compared to controls (Fig. 6E–H). Silencing of Egr1 with siRNA reduced the levels of TNF-α, IL-6, IL-1β in Cit-Vim-treated RAW 264.7 cells (Fig. 6I–J). The levels of EGR1 and inflammatory cytokines TNF-α, IL-6, and IL-1β were significantly reduced in Padi2 knockout and AFM-30a-treated silicotic mice. (Fig. 7 and Figure S7).

Fig. 7.

PADI2 regulates inflammatory response in silicosis. A–B Immunofluorescence staining of EGR1 and TNF-α expression (scale bar = 100 μm) and Western blot analysis of EGR1 and TNF-α, IL-6 and IL-1β expression in Padi2 knockout mice. Results are presented as Mean ± SD, n = 3 per group

Discussion

In the present study, we report for the first time a citrullination proteomic profile in silicotic mice, identifying PADI2 as a central player in Cit-Vim/EGR1-mediated lung inflammation. We demonstrated that pharmacological inhibition or gene knockout of PADI2 exerted both anti-inflammatory and anti-fibrotic effects in mice and silica-challenged RAW 264.7 macrophages. These findings suggest that PADI2 is a promising therapeutic target for silicosis.

Citrullination, mediated by peptidylarginine deiminases (PADIs), has been increasingly recognized for its role in autoimmune diseases, with specific targets like fibrinogen and vimentin being implicated in disease pathology [22–24]. PADIs catalyze the conversion of arginine to citrulline in a calcium-dependent process, which can disrupt immune tolerance and promote autoimmune responses [6]. Our study expands this understanding by establishing that PADI2-mediated citrullination is a hallmark of silicosis progression. The elevated levels of Cit-peptide and Cit-Vim in silicotic mice suggest that citrullination may drive the pathological and immune responses associated with silicosis, aligning with prior observations that silicosis predisposes individuals to autoimmune diseases [25–29].

Cit-Vim, but not Vim, was shown induce tissue remodeling via a TLR4-dependent mechanism, functioning as a damage-associated molecular pattern (DAMP) and triggering innate immune responses [8]. Previous studies have highlighted the role of Cit-Vim in various fibrotic diseases, such as carbon tetrachloride (CCl4)-induced liver fibrosis, where PADI inhibition reduced systemic Cit-Vim levels [30, 31]. In our study, we demonstrated that PADI2 inhibition, either pharmacologically or via gene knockout, reduces Cit-Vim and Cit-peptide levels in both silicotic mice and silica-exposed macrophages. These findings support the notion that Cit-Vim acts as a pro-inflammatory and pro-fibrotic agent in silicosis, engaging the TLR4/TNF-α signaling pathway to exacerbate lung inflammation and fibrosis.

We also identified Egr1 as a potential downstream target of Padi2 gene knockout in silicotic mice, with RNA-seq analysis highlighting its involvement in the inflammatory response. EGR1 has been implicated in various fibrotic processes [32], including the promotion of lung myofibroblast differentiation through Transforming Growth Factor Beta 1 signaling [33], and its upregulation has been linked to the activation of pro-fibrotic mediators such as connective tissue growth factor (CTGF) in response to mechanical stress [34]. Our study demonstrated that silica or Cit-Vim exposure increases EGR1 expression in macrophages, and EGR1 inhibition effectively reduces inflammatory cytokine levels. This suggests that EGR1 plays a key role in the amplification of silica-induced inflammation, further highlighting the potential of targeting PADI2/EGR1 signaling as a therapeutic strategy for silicosis.

Conclusion

In summary, our study reveals the critical role of citrullination in the pathogenesis of silicosis, with Cit-Vim, regulated by PADI2, acting as a key driver of inflammatory through the EGR1 signaling pathway. Targeting PADI2, either by pharmacological inhibition or gene knockout, effectively reduces inflammation and fibrosis in silicotic models. These findings propose a novel therapeutic approach for silicosis by inhibiting PADI2 to mitigate citrullination and its downstream inflammatory effects.

Author contributions

HX, HZ, YS and FJ conceived and supervised the study and wrote the manuscript; FJ, YL, TL and SL performed the experiments and analyzed the data; DX, HL, ZW, XG, NM and WC conceived the study. All authors have read and approved the final version of the manuscript, and agree with the order of presentation of the authors.

Funding

This work was funded by the National Natural Science Foundation of China (No. 82473607), the National Natural Science Foundation of Hebei Province (H2024209059), the Science and Technology Project of Hebei Education Department (No. QN2025407).

Data availability

No datasets were generated or analysed during the current study.

Declarations

Ethics approval and consent to participate

The experimental protocol was approved by the Ethics Committee for Animal Experimentation of North China University of Science and Technology, which complies with the US National Institutes of Health Guide for the Care and Use of Laboratory Animals (Approval No. 2022025).

Competing interests

The authors declare no competing interests.