Abstract
Background
Bleomycin (BLM), a widely used antitumor drug, has been demonstrated to induce pulmonary toxicity in both chemotherapy patients and experimental animals, leading to acute lung injury (ALI). However, the lack of effective treatment options limits its clinical application. While peroxiredoxin 1 (Prdx1), a novel damage-associated molecular pattern (DAMP), has been shown to exacerbate acute liver and kidney injury by promoting inflammatory responses, its role in BLM-induced ALI remains unclear.
Methods
An ALI mouse model was established via intratracheal instillation of BLM (5 mg/kg). Prdx1 gene-knockout mice, recombinant murine Prdx1 protein (rPrdx1), and Prdx1-neutralizing monoclonal antibody were utilized to investigate the role of Prdx1 in BLM-induced ALI. Further mechanistic insights were explored through single-cell RNA sequencing analysis.
Results
BLM-induced damage to bronchial epithelial cells triggered Prdx1 release, which subsequently activated the NOD1/NF-κB signaling pathway in macrophages, promoting the release of inflammatory cytokines and exacerbating pulmonary inflammation and pathological damage. These findings were confirmed by single-cell RNA sequencing. Genetic knockout of Prdx1 or administration of Prdx1-neutralizing monoclonal antibody protected mice from BLM-induced ALI, and this protective effect was attenuated by introducing rPrdx1.
Conclusion
These findings identify Prdx1 as a potential therapeutic target for BLM-induced ALI, offering a strategy to mitigate its pulmonary toxicity and facilitate the broader clinical application of BLM.
Supplementary Information
The online version contains supplementary material available at 10.1186/s12931-026-03533-5.
Keywords: Bleomycin, Acute lung ingury, DAMPs, Peroxiredoxin 1, Macrophage, NOD1/NF-kB pathway
Introduction
Bleomycin (BLM), a glycopeptide antitumor antibiotic widely used in clinical practice, is commonly employed in the treatment of patients with germ cell tumors, squamous cell carcinoma, and lymphoma [1]. However, the broad application of BLM as an anticancer agent is limited by its dose-dependent and cumulative pulmonary toxicity. Up to 46% of patients receiving bleomycin therapy exhibit pulmonary toxicity (termed bleomycin-induced pneumonitis (BIP), encompassing acute lung injury (ALI) and pulmonary fibrosis), with 3% experiencing fatal outcomes [2–4]. Consequently, BLM is frequently utilized to induce experimental ALI and pulmonary interstitial fibrosis in animal models [5–7]. Clinically, BLM-induced ALI is primarily managed with corticosteroid therapy. Nevertheless, 1%-4% of patients exhibit no response to corticosteroid treatment, which may lead to lethal consequences [3]. This underscores the urgent need to further elucidate the pathogenesis of BLM-induced ALI and develop novel therapeutic strategies.
The pathogenesis of ALI centers on excessive inflammatory responses. Damage-associated molecular patterns (DAMPs) are currently recognized as critical mediators in ALI progression. Various injurious stimuli induce pulmonary epithelial cells damage [8, 9] and subsequent DAMP release, which interact with pattern recognition receptors (PRRs) such as nucleotide-binding oligomerization domain (NOD)-like receptors and Toll-like receptors (TLRs). These interactions activate the immune system, triggering severe inflammatory responses [10–12]. In recent years, DAMPs such as mitochondrial DNA (mtDNA) and high-mobility group box 1 (HMGB1) have been implicated in the pathogenesis of ALI [13]. However, effective clinical therapeutic strategies targeting these DAMPs remain elusive. Therefore, identifying novel DAMPs involved in BLM-induced ALI and uncovering their roles in pathogenesis may facilitate the discovery of potential therapeutic targets, underscoring significant clinical and translational implications, pending further validation.
Peroxiredoxin 1 (Prdx1), a member of the peroxiredoxin family, is predominantly expressed in the cytoplasm and participates in peroxide scavenging and cellular signaling regulation [14]. Emerging evidence identifies extracellular Prdx1 as a novel DAMP capable of binding PRRs including TLR2/4 and Mincle, thereby activating innate immunity and promoting inflammation [15, 16]. Prdx1 deficiency has been shown to reduce mortality in septic shock models [17], while circulating Prdx1 levels correlate with aggravated acute liver and kidney injury [16, 18]. However, the role of Prdx1 in BLM-induced ALI and its underlying mechanisms remain poorly understood.
In this study, we measured Prdx1 levels in bronchoalveolar lavage fluid (BALF) and serum from BLM-induced ALI murine model and generated Prdx1-knockout mice to investigate its functional significance in BLM-induced ALI pathogenesis. Our findings provide novel insights into the mechanisms underlying BLM-induced ALI and identify potential therapeutic targets for this condition.
Methods
Materials
BLM for in vivo applications was purchased from Nippon Kayaku Co., Ltd., while the in vitro grade BLM (#60257ES08) was acquired from Yeasen Biotechnology. BCA protein assay kit (#PA115) was obtained from ANGEN Biotech. Primary antibodies included: NOD1 (bs-7085R) and NOD2 (bs-7084R) from Bioss; IκB (#9242), phospho-IκB (Ser32/36, #2859), IKKα/β (#2682), phospho-IKKα/β (Ser176/180, #2697), HRP-conjugated anti-rabbit (#7074), and anti-mouse IgG (#7076) from Cell Signaling Technology; β-actin (#66009-1-Ig), GST (#10000-0-AP) and Alix(#12422-1-AP) from Proteintech; F4/80 (#ab300421), TSG101(#ab125011) and Calnexin (#ab22595) from Abcam; and Prdx1 (#PA5-27487) from Thermo Fisher Scientific. ELISA kits for mouse Prdx1 (#CSB-EL018653MO), MCP-1 (#BMS6005), and IL-6 (#BMS603-2) kits were purchased from CUSABIO and Thermo Fisher Scientific. The cell transfection reagent Lipofectamine 2000(#11668030) was purchased from Thermo Fisher Scientific. Prdx1-neutralizing monoclonal antibody and control IgG were obtained from ABclonal. Prdx1 recombinant murine protein (rPrdx1) (#P7974) was obtained from Abnova. NF-κB inhibitor (BAY 11-7082, #HY-13453) and NOD1 inhibitor (Nodinitib-1, #HY-18639) were obtained from Medchemexpress. HiPure Total RNA Plus Kit (#R411103) was obtained from Magen Biotech. cDNA Synthesis (#AU341-02-V2) and Green qPCR (#AQ601-02-V2) SuperMix kit were obtained from TransGen Biotech.
Animal models
All procedures were approved by the Animal Ethics Committee of Central South University (APU-2025-0221). Prdx1−/− mice (C57BL/6J background) were generated as described16. Wild-type male C57BL/6J mice (8–10 weeks, Silaike Co.) were housed under SPF conditions (12-hr light/dark cycle). Animals were randomly assigned to experimental or control groups before the initiation of the study to eliminate selection bias.
BLM, a clinically utilized antineoplastic agent, is a well-established inducer of ALI and pulmonary fibrosis in both clinical settings and experimental models [19]. This model recapitulates a biphasic pathological progression: (1) an acute inflammatory phase (days 1–7 post-instillation) characterized by inflammatory cells infiltration, (2) a transitional phase (days 7–10) featuring dynamic tissue remodeling, and (3) a fibrotic phase (day 10 onward) marked by collagen deposition and attenuated acute inflammation [6, 20, 21].
rPrdx1 was diluted in sterile saline and administered to the mice via tail vein injection. Prdx1-neutralizing monoclonal antibody, control IgG and Nodinitib-1 were diluted in sterile saline and administered to the mice via intraperitoneal injection. The control group mice received the same volume of sterile saline. The doses of rPrdx1, Prdx1-neutralizing monoclonal antibody and Nodinitib-1 were selected based on previous studies [16, 22]. At the end of the experiment, the animals were euthanized, and blood samples, BALF, as well as lung tissue were collected for subsequent experiments.
Bronchoalveolar lavage fluid collection
BALF was collected from each mouse using a standardized procedure. BALF was collected by injection and retraction of 0.5 mL precooled PBS with repeated three times. The BALF was centrifuged at 3000 g for 15 min at 4 °C, after which the supernatant was collected and stored at -20 °C for subsequent analysis. The total protein concentration in the collected BALF supernatant was measured using the BCA Protein Assay Kit.
Depletion of macrophages
Clodronate liposomes (CL; #40337ES08, Yeasen) and control liposomes (#40338ES05, Yeasen) were i.p. and i.t. injected into WT mice. The efficiency of macrophage depletion was assessed by immunohistochemical staining of macrophages in lung tissue.
Histopathology, immunohistochemistry and immunofluorescence
The mouse lung tissue was excised, with the right lung preserved in liquid nitrogen for future use, while the left lung was fixed in 4% paraformaldehyde. The right caudal lobe was used to determine wet/dry ratio. The fixed left lung was then embedded in paraffin and sectioned into 4 μm-thick slices, which were stained with Hematoxylin and Eosin (H&E). A semi-quantitative pathological score (ranging from 0 to 4) was independently assessed by three blinded pathologists based on established criteria evaluating inflammatory cell infiltration, alveolar structure disruption, and congestion.
Immunohistochemical staining was used to detect the expression level of Prdx1 or F4/80 in tissues. After inhibiting peroxidase activity and blocking antigens, the Prdx1 or F4/80 primary antibody was diluted with goat serum (1:500) and incubated with the slides overnight at 4 °C. Subsequently, the slides were incubated with secondary antibodies and the samples Were subjected to Staining reaction with DAB.
For tissue IF, sections were incubated overnight at 4 °C with anti-Prdx1 (1:100), followed by fluorophore-conjugated secondary antibodies (1:1000) for 1 h. For cellular IF, treated cells grown on confocal dishes were fixed, permeabilized, and blocked, then incubated overnight with anti-GST (1:500) followed by fluorescent secondary antibodies (1:1000). All samples were mounted with antifade mountant containing DAPI.
Isolation of primary peritoneal macrophages and cell culture
Primary peritoneal macrophages (PPMs) isolated from mice as previously described and iBMDM cells were cultured in RPMI-1640 medium [16]. Primary mouse bronchial epithelial cells (mBEpiC, #PC-006 m) were purchased from SAIOS Biotechnology Company and nurtured in Bronchial Epithelial Cell Medium (BEpiCM, #3262, sclencell). All cell culture media used were supplemented with 10% fetal bovine serum and 1% penicillin-streptomycin solution. All cells were maintained in an incubator set to an environment of 37 °C and 5% CO2.
The “Prdx1 + Prdx1 mAb” complex was prepared by mixing 50 µM rPrdx1 with 100–150 µM of a Prdx1 mAb for 30 min at room temperature.
Plasmids transfection and co-immunoprecipitation
The mouse-derived PRDX1-GST plasmid was constructed by Genechem Company. The pcDNA3.1(+)-PRDX1-GST plasmids were transfected into iBMDM cells using Lipofectamine 2000. After 48 h of transfection, the cells were lysed with 200 µl of IP lysis buffer and centrifuged at 12,000 rpm for 15 min at 4 °C to collect the supernatant.20 µl of supernatant was reserved as the Input control. To the remaining supernatant, 25 µl of protein G magnetic beads (#P3296, Sigma-Aldrich) and an appropriate amount of primary antibody were added and incubated overnight at 4 °C. Subsequently, the magnetic beads were washed five times with IP buffer, and then 2× SDS solution was added and boiled for western blot.
Small interfering RNA transfection
iBMDM cells were seeded in appropriate culture plates and transfected with either negative control siRNA or NOD1-targeting siRNA (siNOD1 antisense: CGUCAAUACUGACCCAGUA/dT//dT/) using Lipofectamine 2000 for 48 h.
Flow cytometry analysis
Lung tissues were digested with collagenase IV (#C5138, Sigma) to generate a cell suspension. Then, cells were costained with the corresponding antibodies: anti-Mo F4/80 (#123116, BioLegend), anti-Mo CD206 (#141706, BioLegend), anti-Mo CD80 (#104716, BioLegend) and anti-Mo CD11b (#101216, BioLegend). Flow cytometry was performed using a Cytek Dxp Athena™ flow cytometer. and staining data were analyzed using FlowJo CE software. The data were quantified by analyzing the percentage of these positive cells.
Isolation and identification of exosomes
mBEpiCs were cultured in medium supplemented with exosome-depleted serum and stimulated with BLM (400 ng/mL) for 24 h, after which the supernatant was collected for exosome isolation. The supernatant was first centrifuged at 500 g for 10 min at room temperature to remove dead cells, followed by centrifugation at 3,000 g for 20 min at 4 °C to eliminate apoptotic bodies. Exosomes were then pelleted by ultracentrifugation at 120,000 g for 70 min at 4 °C, and the resulting pellet was resuspended in PBS to obtain the final exosome preparation. The protein concentration of the exosomes was quantified using a BCA assay kit, and aliquots were stored at -80 °C. Exosome morphology and size distribution were analyzed by transmission electron microscopy and nanoparticle tracking analysis (Servicebio Biotechnology Co., Ltd.), and the presence of exosomal markers (TSG101, Alix) and the absence of the negative marker Calnexin were confirmed by Western blot.
RNA extraction and quantitative real-time PCR
Total RNA from cells was extracted using the HiPure Total RNA Plus Kit. The Biometra TOne real-time PCR system (Analytik Jena) was used to perform reverse transcription on the obtained RNA samples. CFX96 Real-Time System (Bio-Rad) was Conducted to Quantitative Real-time PCR analysis. β-actin was used as an internal reference for mRNA expression. The primer sequences for the target genes used in this study were listed in Table S1.
Western blot
Proteins were extracted from mouse lung tissue or cells using SDS and boiled for 10 min at 100 °C. Subsequently, the protein concentration was determined using a BCA protein assay kit. For western blot analysis, 20-60ug of protein was separated on SDS-PAGE and transferred to PVDF membranes according to previous study, followed by blocking with fast blocking solution for 15 min at room temperature. membranes were incubated with primary antibodies overnight at 4℃, including NOD1 (1:1000), NOD2 (1:1000), P-IKK (1:1000), IKK (1:1000), P-IκB (1:1000), IκB (1:1000), Prdx1 (1:1000), Alix (1:1000), TSG101 (1:1000), Calnexin (1:1000), β-actin (1:10000), and then incubated with corresponding secondary antibodies for 1 h at room temperature. β-actin was used as an internal reference for protein expression. The target protein bands were visualized by the ECL substrate and analysed by ImageJ software.
Enzyme-linked immunosorbent assay
The concentrations of Prdx1, IL-6, and MCP-1 in mouse BALF and cell supernatants, were measured using the corresponding ELISA kits according to the manufacturer’s instructions. Prdx1, IL-6 and MCP-1 levels were normalized to total volume.
LDH assay
Cell death was analyzed by measuring LDH activity in cell culture supernatants using a commercial LDH Cytotoxicity Assay Kit (#C0017, Beyotime, Shanghai, China).
Single-cell RNA sequencing
At 7 days post-BLM exposure, lung tissues were harvested from both Prdx1−/− and WT mice. The collected lung tissues were stored in the sCelLiveTM Tissue Preservation Solution (Singleron Biotechnologies, Nanjing, China) and then transported to the Singleron lab on ice as soon as possible. Sequencing was performed on the Illumina NovaSeq 6000 platform. Subsequent data analysis was conducted with technical support provided by Singleron Biotechnologies.
RNA sequencing and analysis
Total RNA was extracted using trizol reagent (15596026, Invitrogen, USA). RNA-seq was performed on DNBSEQ-T7 platform (Geneplus-Shenzhen, Shenzhen, China). Subsequent data analysis was conducted with technical support provided by Geneplus Biotechnologies.
Statistical analysis
All data were expressed in mean ± standard deviation of at least three independent experiments. Each “n” represents an independent biological replicate. For comparisons between two groups, Student’s t-test was applied. For comparisons among multiple groups, one-way analysis of variance (ANOVA) was used, followed by Tukey’s post hoc test for specific group comparisons. For high-throughput sequencing data, rigorous multiple testing corrections were applied to control the false discovery rate (FDR): differential expression analysis for bulk RNA-seq data was performed using DESeq2, with significance defined by an adjusted p-value (FDR) < 0.05; similarly, for single-cell RNA-seq data, differentially expressed genes between clusters were identified using the Wilcoxon rank-sum test with Bonferroni correction. All statistical analyses were performed using SPSS (version 22.0, IBM, USA) and relevant bioinformatics packages. Statistical significance was defined as p < 0.05 or FDR < 0.05.
Result
Both BALF and serum Prdx1 levels dynamically elevate in BLM-induced murine ALI model
ALI model was established in mice via intratracheal instillation of BLM. H&E staining revealed that BLM exposure in WT mice induced substantial inflammatory cell infiltration, alveolar septal thickening, and architectural disruption in a time-dependent manner. The pulmonary histopathological changes progressively worsened with prolonged BLM exposure, reaching peak severity on day 7 post-BLM administration (Fig. 1A,B). Excessive inflammatory response and alveolar space exudation/edema are hallmark features of ALI. Consistent with histopathological alterations, quantitative analysis of BALF components showed progressive increases in IL-6, MCP-1, and total protein levels, peaking at day 7 post-BLM exposure (Fig. 1E-G). Notably, while histopathological features at day 10 remained comparable to those at day 7 post-BLM exposure, BALF cytokine levels significantly declined (Fig. 2E,F), suggesting a transition from acute injury to fibrotic remodeling. This pathological progression informed our selection of the 7-day timepoint for subsequent investigations. In parallel, we quantified Prdx1 levels in BALF and serum using ELISA. We observed that Prdx1 levels progressively increased with prolonged BLM exposure, peaking on day 7 and declining by day 10, which paralleled the severity of pulmonary histopathological changes and inflammatory responses (Fig. 1C,D). Notably, Prdx1 levels in BALF exhibited a significant increase starting from day 3 post-modeling, providing a rationale for subsequent experimental designs. Collectively, these findings suggest that Prdx1 may serve as a potential biomarker and contribute to the pathogenesis of BLM-induced ALI.
Fig. 1.
Temporal dynamics of Prdx1 in BLM-induced ALI model. BLM (5 mg/kg) was administered via intratracheal instillation. BLM groups sampled at days 1, 3, 5, 7 and 10 (n = 5 in each group). A Representative H&E-stained lung sections (10x: scale bar = 1000 μm; 200x: scale bar = 50 μm). B Semi-quantitative lung injury scores across groups. C BALF Prdx1 levels in each group. D Serum Prdx1 levels in each group. E BALF IL-6 concentrations in each group. F BALF MCP-1 concentrations in each group. G Total BALF protein content in each group
Fig. 2.
Knockout of Prdx1 attenuates BLM-induced ALI. BLM groups sampled at days 3 and 7 (n = 5 in each group). A H&E-stained lung sections across groups (10x: 1000 μm; 200x: 50 μm). B Lung injury scores across groups. C BALF MCP-1 concentrations in each group. D BALF IL-6 concentrations in each group. E Total BALF protein content in each group. F Lung wet/dry ratios in each group
Prdx1 deficiency mitigates BLM-induced ALI
In BLM-induced ALI model, the parallel elevation of Prdx1 levels in BALF and serum with the severity of pulmonary histopathological damage suggested Prdx1’s potential involvement in ALI pathogenesis. To investigate the pathologic contributions of Prdx1 to ALI, we generated Prdx1−/− mice and subjected them to BLM-induced injury. H&E staining revealed that Prdx1 deficiency significantly attenuated pulmonary histopathological alterations (Fig. 2A,B) and BALF analysis revealed decreased IL-6, MCP-1, and total protein levels (Fig. 2C-E) compared to WT model mice, with the most pronounced differences observed on day 7 post-BLM administration. Additionally, the lung wet/dry ratio (a common indicator of pulmonary edema) also demonstrated that Prdx1 deficiency mitigated the severity of bleomycin-induced lung injury (Fig. 2F). These results indicate that Prdx1 deficiency effectively attenuates BLM-induced ALI, highlighting the critical role of Prdx1 in the pathogenesis and progression of BLM-induced ALI.
Exogenous Prdx1 exacerbates BLM-induced ALI
Previous studies have demonstrated that circulating Prdx1 functions as a DAMP in sterile encephalitis, IRI-induced acute kidney injury, and APAP-induced acute liver injury [15, 16, 18]. To establish causal relationship between circulating Prdx1 elevation and disease progression, we delivered rPrdx1 via tail vein to Prdx1−/− model mice. H&E staining demonstrated significant aggravation of pulmonary histopathological alterations (Fig. 3A-C) and BALF analysis revealed pronounced increases in IL-6, MCP-1, and total protein levels (Fig. 3D-F) in rPrdx1-treated group compared to BLM group.
Fig. 3.
Exogenous administration of rPrdx1 exacerbates BLM-induced ALI, whereas Prdx1-neutralizing antibody treatment significantly attenuates BLM-triggered pulmonary damage. Prdx1−/− mice received rPrdx1 (10ug/kg, i.v.) on day 3 post-BLM. WT mice administered Prdx1 mAb (150 µg/dose, i.p.) on days 3 and 5 post-BLM (n = 5 in each group). A Experimental timeline. B, G H&E-stained sections across groups (10x: 1000 μm; 200x: 50 μm). C, H Lung injury scores across groups. D, I BALF MCP-1 concentrations in each group. E, J BALF IL-6 concentrations in each group. F, K Total BALF protein content in each group
The timing of tail vein injection with rPrdx1 was determined based on our observation that Prdx1 levels in BALF were significantly elevated starting on day 3 post-BLM exposure, indicating the onset of detectable lung tissue injury at this timepoint. Similarly, the dosage of rPrdx1 was established according to the kinetic profile of serum Prdx1 concentrations during BLM-induced ALI progression and our previously research [16, 18] (Fig. 1C,D).
Neutralizing circulating Prdx1 attenuates BLM-induced ALI
To functionally validate the central role of circulating Prdx1 in BLM-induced ALI, we further generated a Prdx1-mAb and its isotype control IgG. We first evaluated the neutralizing efficacy of Prdx1-mAb both in vivo and in vitro. In vivo, we observed a significant reduction in circulating Prdx1 levels in model mice following treatment (Figue S1A). In vitro, the antibody was also found to effectively block the pro-inflammatory effects induced by rPrdx1 (Figure S1B, C). H&E staining revealed that, compared to the model group and IgG-treated group, Prdx1-mAb treatment markedly alleviated pulmonary damage (Figs. 3G,H) and BALF analysis revealed decreased IL-6, MCP-1, and total protein levels (Figs. 3I-K). These results suggest that Prdx1-mAb treatment attenuates BLM-induced ALI, further confirming the critical role of circulating Prdx1 in the pathogenesis of ALI. Our findings highlight Prdx1-mAb as a novel therapeutic agent and provide a promising strategy for ALI management.
Bronchial epithelial cells constitute the primary source of Prdx1
To identify the source of elevated Prdx1 in BLM-induced ALI, we conducted IHC and IF analyses. In WT mice, Prdx1 was primarily localized to bronchial epithelial cells. BLM challenge markedly reduced its intracellular levels there (Fig. 4A, B). However, Prdx1 mRNA levels in lung tissues between WT and BLM groups were unchanged (Fig. 4C), ruling out transcriptional downregulation. Vitro experiment revealed that BLM stimulation did not influence Prdx1 mRNA levels in mBEpiCs, which remained stable or slightly increased (Fig. 4D), yet triggered a time-dependent rise in Prdx1 protein within the cell supernatant (Figs. 4E,F). This demonstrates that extracellular Prdx1 accumulation results from cellular damage, not transcriptional upregulation, aligning with prior reports [16, 18]. Furthermore, we also observed that alveolar epithelial cells release Prdx1 upon BLM stimulation (Figure S4B). However, both their basal Prdx1 expression levels and its release following injury were markedly lower than those observed in bronchial epithelial cells (Fig. 4A, E and S4B). Thus, we conclude that bronchial epithelial cells serve as the primary source of extracellular Prdx1.
Fig. 4.
Bronchial epithelial cells constitute the primary source of Prdx1. A Immunofluorescence staining for Prdx1 in lung tissues (200x: 50 μm) from control and BLM-challenged groups (n=5 in each group). B Immunohistochemical staining for Prdx1 in lung tissues (200x: 50 μm) from control and BLM-challenged groups (n=5 in each group). C Prdx1 mRNA levels in lung tissues from control and BLM-challenged groups (n=5 in each group). D-F Primary mouse bronchial epithelial cells were exposed to BLM (400 μg/mL) for 6,12,24 hours (n=3 in each group): (D) Prdx1 mRNA levels, (E) Prdx1 levels in the supernatant measured by ELISA, (F) Prdx1 protein levels in the supernatant measured by western blot. G LDH release of Primary mouse bronchial epithelial cells exposed to BLM (400 μg/mL) for 6,12,24 hours (n=3 in each group). H-J Primary mouse bronchial epithelial cells were exposed to BLM (400 μg/mL) for 24 hours, followed by the collection of the supernatant for exosome isolation: (H) Exosome electron microscopy images of control and BLM-exposed groups. (I) Particle size distribution histogram of exosomes in the control and BLM-exposed groups. (J) Western blot analysis of Calnexin, TSG101, Alix and Prdx1 in exosomes from control and BLM-exposed groups (mBEpiC lysis was used as cell group).
Based on reports that LPS or TNF-α induce peroxidation and exosomal release of Prdx1 [23], we investigated its release mechanism in BLM-stimulated mBEpiCs. First, we detected LDH release and found that BLM exposure induced mBEpiCs cell death, evidenced by a time-dependent increase in LDH release (Fig. 4G). Subsequently, we collected supernatant after 24 h of BLM exposure and isolated exosomes (Fig. 4H, I). Western blot analysis showed that the level of Prdx1 secreted via exosomes increased after BLM stimulation (Fig. 4J). These results indicate that Prdx1 is released into the extracellular space via both passive (cell death) and active (exosomal secretion) mechanisms under BLM stimulation, contributing to its role in the pathogenesis of acute lung injury.
Prdx1 exacerbates BLM-induced ALI via enhanced macrophage infiltration and M1 polarization
To investigate the molecular mechanism of Prdx1 promoting ALI, single-cell RNA sequencing technology was used. The single-cell sequencing, based on UMAP clustering, was mapped to eight major cell types: Epithelial cells, Endothelial cells, Fibroblasts, Plasma cells, B cells, T and NK cells, Neutrophils, and Mononuclear phagocytes (Fig. 5A). Characteristic marker genes for each cell type are displayed (Figure S3C). Heatmap analysis between the WT model and the Prdx1−/− model groups revealed elevated expression of monocyte chemokine genes such as Ccl2 and Ccl7 in the WT model group (Figure S3A). GO enrichment analysis revealed terms primarily related to leukocyte migration and cytokine production (Figure S3B). Quantitative assessment of cell subsets showed a significant increase in the monocyte-macrophage population in BLM-treated WT mice compared to the control group. In contrast, Prdx1 deficiency significantly reduced BLM-induced pulmonary infiltration of monocyte-macrophages (Fig. 5B). Notably, baseline macrophage numbers were comparable between unchallenged WT and Prdx1−/− mice (Figure S1D, E). These findings suggest that Prdx1 may function by modulating macrophages.
Fig. 5.
Prdx1 exacerbates BLM-induced ALI via enhanced macrophage infiltration and M1 polarization. CL: clodronate liposomes; rPrdx1 (10 µg/kg, i.v.) administered on day 3 post-BLM. A Single-cell clustering of lung tissues from experimental groups. B The specific proportions of various cell types among each group. C Macrophage depletion protocol schematic. D H&E-stained sections across groups (10x: 1000 μm; 200x: 50 μm, n = 5 in each group). E Lung injury scores across groups. F BALF IL-6 concentrations in each group. G BALF MCP-1 concentrations in each group. H Total BALF protein content in each group. I, J Quantitative analysis of M1 (F4/80+CD11b+CD80+) and M2 (F4/80+CD11b+CD206+) macrophages in lung tissues (n = 5 in each group). Four-color flow cytometry was performed on samples from WT and Prdx1−/− mice with BLM-induced ALI
A total of 6111 macrophages were identified, unsupervised clustering dividing them into 6 heterogeneous subgroups (Figure S3D). GO enrichment analysis revealed that functional differences between the WT and Prdx1−/− model groups primarily involved cytokine-mediated signaling pathways and innate immune regulation (Figure S3E). KEGG pathway analysis indicated that the differentially enriched pathways between the two groups included the NOD-like receptor signaling pathway and the Chemokine signaling pathway (Figure S3F).
To functionally validate the critical role of macrophages in BLM-induced ALI, we administered CL, a widely used macrophage-depleting agent, to selectively eliminate macrophages in both the circulation and lung tissues (Fig. 5C). Efficiency of macrophage depletion was confirmed by F4/80 immunohistochemistry in lung and liver tissues (Figure S2A-C). CL treatment significantly ameliorated BLM-induced pulmonary histopathological damage, reduced lung injury scores (Fig. 5D, E), and decreased levels of IL-6, MCP-1, and total protein in BALF (Fig. 5F-H). Macrophage M1 and M2 polarization plays a significant role in BLM-induced lung injury, with M1 polarization promoting early inflammation. We observed that following BLM challenge, the F4/80⁺CD11b⁺ macrophage population in the lungs of WT mice showed a greater enrichment of CD80⁺ M1 macrophages compared to Prdx1−/− mice (Fig. 5I, J). In contrast, the proportion of CD206⁺ M2 macrophages did not differ significantly between the two groups. Consistent with this, prior in vitro experiments confirmed that rPrdx1 stimulation induces M1, but not M2, polarization in PPMs [16]. Together, these results support the conclusion that Prdx1 exacerbates ALI by promoting macrophage infiltration and M1 polarization.
Prdx1 activates macrophage NOD1/NF-κB signaling to exacerbate BLM-induced ALI
To validate these findings, we performed transcriptomic sequencing on rPrdx1-stimulated PPMs. GO and KEGG enrichment analysis confirmed significant enrichment of the “NOD-like receptor signaling pathway” and “cytokine-cytokine receptor interaction” (Fig. 6A, B). Since Prdx1 may act as a DAMP, we hypothesized it interacts with PRRs in the NLR pathway such as NOD1 or NOD2 [24, 25]. Western blot analysis showed that NOD1, but not NOD2, was significantly upregulated in lung tissues from BLM-induced ALI mice, an effect reversed by Prdx1 deficiency (Fig. 6C and S4A). In vitro, rPrdx1 stimulation increased both mRNA and protein levels of NOD1 in PPMs (Fig. 6D,E). Consistent with NOD1’s known role in activating NF-κB, rPrdx1 induced phosphorylation of IKK and IκB, and increased secretion of IL-6 and MCP-1 in PPMs (Fig. 6F,G). These effects were attenuated by a NOD1 inhibitor (Nodinitib-1), an NF-κB inhibitor, or NOD1 silencing in iBMDMs (Fig. 6F,G; Fig. S4C, D). In vivo, Nodinitib-1 treatment alleviated BLM-induced lung injury and inflammation (Fig. 7A-E) and abolished the aggravating effect of rPrdx1 in Prdx1−/− mice (Fig. 7F-I).
Fig. 6.
Prdx1 activates macrophage NOD1/NF-κB signaling to exacerbate BLM-induced ALI. A GO enrichment analysis of the transcriptome in rPrdx1-treated PPMs (n = 3 in each group). B Transcriptomic profiling of KEGG pathway activation in rPrdx1-treated PPMs (n = 3 in each group). C NOD1 protein levels in WT and Prdx1−/− mice with BLM-challenged (n = 5 in each group). D Western blot analysis of NOD1 protein expression in PPMs treated with 50 nM rPrdx1 for 6, 12, or 24 h (n = 3 in each group). E qPCR analysis of NOD1 mRNA expression in PPMs treated with 50 nM rPrdx1 for 6, 12, or 24 h (n = 3 in each group). F PPMs were pretreated for 3 h with 30 μm Nodinitid-1 before rPrdx1 (50 nM). Cells were harvested 24 h after rPrdx1 stimulation, followed by Western blot analysis for P-IKK and P-IKB. IL-6 and MCP-1 levels in the supernatants were measured by ELISA (n = 3 in each group). G PPMs were pretreated for 1 h with 10 μm BAY 11-7082 before rPrdx1 (50 nM). Cells were harvested 24 h after rPrdx1 stimulation, followed by Western blot analysis for P-IKK and P-IKB. IL-6 and MCP-1 levels in the supernatants were measured by ELISA (n = 3 in each group)
Fig. 7.
NOD1 inhibitor (Nodinitid-1) attenuates BLM-induced ALI. Nodinitid-1 (20 mg/kg, i.p.) administered on days 3 and 5 post-BLM (n = 5 in each group). A Experimental timeline. B, F H&E-stained sections across groups (10x:1000 μm; 200x:50 μm). C, G Lung injury scores across groups. D, H BALF MCP-1 concentrations in each group. E, I BALF IL-6 concentrations in each group
NOD1 is primarily localized in the cytoplasm, and the mechanism by which extracellular Prdx1 influences NOD1 remains unclear. To investigate this, we employed heat stress—a potent inducer of programmed necroptosis—to generate Prdx1-enriched cell supernatants [16, 26]. Specifically, MLE-12 cells overexpressing Prdx1-GST plasmids were subjected to heat stress, and the resulting Prdx1-GST-rich supernatants were used to culture iBMDMs (Fig. 8A). Immunofluorescence analysis revealed that extracellular Prdx1-GST translocated across the cell membrane into the cytoplasm of iBMDMs (Fig. 8B). Subsequent co-immunoprecipitation assays confirmed a direct interaction between Prdx1 and NOD1 (Fig. 8C). These data demonstrate that extracellular Prdx1 enters the cytoplasm and activates macrophages via its interaction with NOD1.
Fig. 8.
Interaction of Prdx1 and macrophage-inducible NOD1. The Prdx1-GST plasmid were transfected into the MLE12 cells, and after 48 h, the MLE12 cells were heated at 50 °C for 1 h, followed by incubation at 37 °C for 5 h before collecting the supernatant, which was then used to culture IBMDMs for 24 h. A Schematic illustration of the study design. B GST immunofluorescence staining. The original magnification was 1000x. C Prdx1-GST/NOD1 interaction by co-IP
Discussion
While BLM remains an effective chemotherapeutic agent, its dose-limiting pulmonary toxicity poses a significant clinical challenge in the absence of effective targeted therapies. Our study elucidates a novel pathological axis in BLM-induced ALI: damaged epithelial cells release Prdx1 into the extracellular space via both active and passive mechanisms, where it functions as a DAMP to activate the NOD1/NF-κB signaling pathway in macrophages, thereby driving dysregulated inflammation and tissue injury (Fig. 9). This Prdx1/NOD1/NF-κB axis not only provides a mechanistic explanation for the inflammatory cascade in ALI but also unveils new opportunities for biomarker and therapeutic development.
Fig. 9.
Schematic illustration of the proposed mechanism by which Prdx1 exacerbates BLM-induced ALI
Excessive inflammation is a hallmark of ALI. During its progression, injured pulmonary epithelial cells release DAMPs. As critical mediators bridging innate and adaptive immunity, DAMPs activate PRRs on immune cells, triggering downstream signaling cascades that drive inflammation and tissue damage [11, 27–29]. Well-studied DAMPs such as HMGB1, heat shock proteins, and mtDNA have been confirmed to play significant pro-inflammatory roles in ALI [30]. For instance, Hsp70 can prevent BLM-induced inflammation and injury, with its overexpression reducing pulmonary leukocyte recruitment and parenchymal cell apoptosis [31]. HMGB1 may contribute to the state of vascular hyperpermeability in BLM-induced ALI via the RAGE pathway [32]. Our study confirms that extracellular Prdx1 acts as a DAMP, playing an important role in BLM-induced ALI. Targeting Prdx1 represents a promising therapeutic strategy, as the Prdx1-mAb significantly reduces circulating Prdx1 levels and alleviates lung injury. However, this protective effect is incomplete, and the potential synergistic or compensatory roles of other DAMP molecules (e.g., HMGB1) in ALI require further assessment.
Under normal physiological conditions, Prdx1 primarily functions as an intracellular antioxidant protein. However, once released into the extracellular space, extracellular Prdx1 assumes the role of a DAMP [15, 33]. Consequently, intracellular and extracellular Prdx1 exhibit distinct functional roles. Previous studies have reported that in bleomycin-induced pulmonary fibrosis models, intracellular Prdx1 mitigates fibrotic injury through its antioxidant activity [34]. This indicates that Prdx1 plays divergent roles at different stages of bleomycin-induced lung injury, which aligns with our observation that circulating Prdx1 declines as the disease progresses to the fibrotic phase in BLM-induced lung injury. This dual functionality may expand its potential clinical utility in managing bleomycin-associated lung injury. For instance, our findings suggest that therapeutic strategies could involve neutralizing extracellular Prdx1 with antibodies during the acute lung injury phase, while supplementing recombinant Prdx1 may confer benefits during the fibrotic phase.
During the exudative phase of ALI/ARDS, excessive inflammation and immune cell infiltration are primarily driven by the overactivation of M1 macrophages [35, 36]. Our findings corroborate this, demonstrating massive recruitment and M1 polarization of monocyte-derived macrophages in BLM-induced ALI mice, a process significantly suppressed in Prdx1−/− mice, leading to attenuated lung injury. Macrophage depletion also markedly improved pulmonary inflammation and pathological damage. Previous studies have shown that intravenous injection of CL depletes 70–80% of peripheral blood monocytes within 24–48 h. CL exhibits tissue-specific distribution in vivo, with minimal accumulation in the lungs and predominant retention in the the liver, spleen, and bone marrow [37]. We employed a dual administration strategy—intratracheal instillation combined with intravenous injection of CL—to effectively deplete pulmonary macrophages, compensating for its limited lung accumulation.
Macrophage polarization is a highly regulated process involving complex signaling pathways, transcriptional/epigenetic regulation, and post-transcriptional control [38]. NF-κB, a family of transcription factors, plays a crucial role in cell differentiation, proliferation, and inflammation [39]. Activation of the NF-κB signaling pathway promotes M1 macrophage polarization and inflammatory responses, mediating the expression of various inflammatory mediators and cytokines, including IL-1β, IL-6, and MCP-1 [40]. Previous studies have shown that agents like GC-1 and curcumin can alleviate BLM-induced ALI by inhibiting the NF-κB pathway [41, 42]. Our study found that Prdx1 promotes macrophage M1 polarization and inflammatory cytokine secretion by activating the NF-κB signaling pathway, thereby exacerbating ALI in mice. These findings suggest that targeting macrophages and the NF-κB pathway may represent a focused and effective therapeutic approach for ALI.
DAMPs primarily function by interacting with PRRs such as TLRs and NLRs. For example, HMGB1 mediates its effects in macrophages through direct interaction with TLR2 and TLR4 [43]. Through single-cell sequencing and transcriptomics, our study revealed that Prdx1 acts via NLRs. Subsequent in vivo and in vitro experiments confirmed that Prdx1 exacerbates ALI by activating the NOD1/NF-κB pathway. NOD1 is closely associated with cancer and inflammatory diseases [44, 45]. Studies have shown that NOD1 activation exacerbates house dust mite-induced allergic airway inflammation [46]. Notably, the mechanism of extracellular Prdx1 internalization remains unclear. This knowledge gap parallels observations with other DAMPs: HMGB1 enters cells via receptor-mediated transport (e.g., RAGE engagement) [47], while the NOD2 ligand MDP is actively imported through the peptide transporter hPepT1 [48]. We propose two plausible hypotheses for Prdx1 uptake: 1) receptor-mediated endocytosis via unidentified membrane receptors, or 2) passive diffusion facilitated by its relatively small molecular size. This warrants further experimental validation in future studies.
Our study has several limitations. First, the lung wet-to-dry weight ratio is an important indicator of pulmonary inflammation and edema, yet this measurement was not performed in all animal experiments in the present study. Second, the interaction between Prdx1 and NOD1 was validated only by co-immunoprecipitation (co-IP), and the precise mechanism by which Prdx1 activates NOD1 warrants further investigation. In addition, the clinical relevance of our findings requires further clarification. Bleomycin is currently widely used in combination therapies for various malignancies. Before attempting to translate our findings—for instance, by exploring neutralizing antibodies against Prdx1 as a therapeutic strategy—it is necessary to verify whether BLM-induced lung injury follows the same mechanism in relevant tumor models (e.g., lymphoma models). This step is crucial for assessing the translational value of Prdx1 and for guiding the design of corresponding clinical trials.
Conclusion
Our current findings demonstrate that Prdx1, functioning as a DAMP, critically contributes to BLM-induced ALI. Notably, the development of neutralizing antibodies targeting Prdx1 significantly ameliorates BLM-triggered ALI, highlighting its translational potential. These results position Prdx1 as a promising therapeutic target for managing BLM-induced ALI, particularly in chemotherapy patients.
Supplementary Information
Acknowledgements
We thank all the participants of this study.
Authors’ contributions
GLJ designed and conducted the experiment, analyzed the data, and wrote the manuscript; YZ, LZL, XYZ and PY participated in revising the manuscript; TTY, XYC, LJZ and QXL assisted in data analysis; YJH, MJ supervised the experiment and reviewed the manuscript; JM conceived and designed the experiment, provided technical guidance and financial support, and reviewed and revised the manuscript.
Funding
This research was financially supported by the National Natural Science Foundation of China (Grant No.82270079 and 82300096).
Data availability
Data are available from the corresponding authors upon reasonable request.
Declarations
Ethics approval and consent to participate
All procedures were approved by the Animal Ethics Committee of Central South University (APU-2025-0221).
Consent for publication
Not applicable.
Competing interests
The authors declare no competing interests.
Footnotes
Publisher’s Note
Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.
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Data Availability Statement
Data are available from the corresponding authors upon reasonable request.