Research ArticleExperimental Studies
Open Access

Antagonistic Effect of Erythropoietin Receptor Signaling in Macrophages and Type II Alveolar Epithelial Cells on Pulmonary Fibrosis in Mice

PENGFEI WU, JIALIN JIA, TIANRONG JIN, BANGWEI LUO, ZHIREN ZHANG and GUANSONG WANG
In Vivo May 2026, 40 (3) 1517-1529; DOI: https://doi.org/10.21873/invivo.14302

Abstract

Background/Aim: Although stimulation of erythropoietin receptor (EPOR) signaling demonstrates cytoprotective effects–including anti-apoptosis, pro-proliferation, and promotion of inflammation resolution–in various disease models, its alterations and specific role in the process of pulmonary fibrosis are still not well understood. The study aimed at investigating the changes of lung EPOR signal in pulmonary fibrosis and the effect of different cell EPOR signal on pulmonary fibrosis.

Materials and Methods: In a bleomycin-induced C57BL/6J mice model, fibrosis was assessed via Hematoxylin and eosin (H&E) staining, Masson’s trichrome staining, and hydroxyproline content. EPOR expression was measured in lung tissue and specific cells. Conditioned media (CM) from BLM/EPO-treated MLE-12, PMs, and C166 cells were applied to 3T3 fibroblasts, which were also treated with TGF-β1/EPO. Fibroblast activation was evaluated by α-SMA and Col-1 expression using RT-qPCR. Macrophage-specific (MΦ-EPORcko) and type II alveolar epithelial cell-specific (Sftpc-EPORcko) knockout mice were used to assess fibrosis severity.

Results: Lung overall EPOR expression decreased after fibrosis. Type II alveolar epithelial cells and macrophages showed the highest baseline EPOR expression, which declined post-fibrosis. CM from BLM+EPO-treated MLE-12 cells inhibited fibroblast activation, while media from PMs and C166 cells promoted it. Direct EPOR activation in fibroblasts enhanced their activation. MΦ-EPORcko mice exhibited attenuated fibrosis, whereas Sftpc-EPORcko mice displayed exacerbated fibrosis.

Conclusion: EPOR signaling in macrophages and type II alveolar epithelial cells exerts opposing effects on pulmonary fibrosis, with targeted activation EPOR signaling in alveolar epithelial cells representing a potential therapeutic strategy.

Keywords:

Introduction

The erythropoietin receptor (EPOR) was initially believed to operate solely as a homodimer on erythroid progenitors within the bone marrow, where it plays a crucial role in regulating red blood cell differentiation and maturation (1). However, subsequent research has identified its expression across a diverse range of tissues and cell types. In these non-hematopoietic contexts, EPOR frequently forms heterodimers with the common beta chain (βcR), facilitating cytoprotective functions such as anti-apoptotic activity, antioxidant defense, and the promotion of inflammation resolution (2-4). In a murine model of LPS-induced acute respiratory distress syndrome (ARDS), Fei Cao and colleagues demonstrated that erythropoietin (EPO), through activation of the EPOR/JAK2/STAT3 signaling pathway, inhibits the formation of the NLRP3 inflammasome and reduces the release of inflammatory cytokines, including IL-1β and IL-18. Conversely, administration of the EPOR antagonist EMP9 suppressed JAK2/STAT3 signaling, resulting in an increased production of IL-1β and IL-18. Collectively, these findings imply that maintaining pulmonary EPOR activity may alleviate the severity of ARDS (5). Priya Ravikumar and colleagues investigated the therapeutic potential of nanoparticles encapsulating EPOR cDNA in the treatment of hyperoxia-induced lung injury in SD rats. The study demonstrated that rats administered EPOR cDNA nanoparticles showed increased pulmonary levels of phosphorylated ERK1/2 and STAT5, a reduced lung wet-to-dry ratio, and decreased levels of Caspase-8 and 8-OHdG compared to control groups. These findings indicate that sustaining activated EPOR signaling in the lung mitigates oxidative damage and apoptosis induced by hyperoxia (6). However, a significant unresolved question from this research is the identification of the specific cell types expressing EPOR that are relevant to these effects. In support of cell-specific functions, Wen Zhang and colleagues provided direct evidence that deficiency of EPOR in macrophages exacerbates LPS-induced lung injury and weight loss compared to wild-type mice (7). Given the widespread expression of EPOR across various pulmonary cell types, it remains an open question whether EPOR signaling from distinct cellular compartments acts synergistically or antagonistically in the pathogenesis of lung diseases.

Idiopathic pulmonary fibrosis (IPF) is a chronic and progressive interstitial lung disease with an unknown etiology, characterized by potential episodes of acute exacerbation. A widely accepted hypothesis suggests that IPF is primarily driven by epithelial processes, initiated by damage to alveolar epithelial cells and involving a complex interaction among various cell types, including endothelial cells, fibroblasts, and immune cells (8-10). During the fibrotic process, alveolar epithelial type II cells (AEC II) exhibit functional and proliferative abnormalities, which compromise their ability to effectively repair the damaged alveolar epithelium. Furthermore, these cells may assume a pro-fibrotic phenotype, secreting factors such as TGF-β1 and MMPs, which disrupt the function of adjacent cells (11). The role of vascular endothelial cells is complex and varied. They contribute to extracellular matrix remodeling by releasing MMPs and support epithelial progenitor cell proliferation (12). Additionally, they recruit VEGFR+ macrophages, which subsequently induce sustained upregulation of the Wnt/β-catenin-dependent Notch ligand Jagged1 in endothelial cells. This signaling cascade further activates Notch signaling in perivascular fibroblasts, thereby facilitating fibrogenesis (13). Previous research has established that EPO mitigates experimental pulmonary fibrosis by inhibiting apoptosis in alveolar epithelial and endothelial cells (14, 15). However, the role of endogenous pulmonary EPOR signaling in the pathogenesis of IPF remains insufficiently investigated. Given that EPO exerts its effects through the activation of EPOR and its downstream signaling pathways (4), and considering that EPO does not selectively target EPOR on specific cell types, it is unclear whether EPOR signaling has uniform or divergent effects across various pulmonary cell populations in the context of IPF. This study seeks to elucidate the spatiotemporal dynamics of pulmonary EPOR expression during fibrotic progression and to assess the functional impact of cell type-specific alterations in EPOR signaling on fibrosis severity. The overall experimental design is summarized in Figure 1.

Figure 1.
Figure 1.

The figure delineates the methodology employed to investigate alterations in pulmonary erythropoietin receptor (EPOR) signaling during the progression of lung fibrosis. Initially, in vivo assessments were conducted to evaluate changes in lung EPOR levels before and after the induction of fibrosis. This was succeeded by an analysis of EPOR expression modifications in various cell types, including macrophages, alveolar type II epithelial cells (AEC II), endothelial cells, and fibroblasts. Notably, the most significant alterations were detected in macrophages and alveolar type II epithelial cells. Subsequently, EPOR was selectively knocked out in these cell types to assess its impact on the severity of lung fibrosis. In vitro, conditioned medium was produced by activating EPOR signaling, and its influence on fibroblast activation was examined following EPOR stimulation across different cell types.

Materials and Methods

Materials. Primary mouse peritoneal macrophages (PMs) were isolated following established protocols (16). The mouse alveolar epithelial cell line (MLE-12), mouse vascular endothelial cell line (C166), and mouse embryonic fibroblast cell line (3T3) were procured from Zhongqiao Xinzhou Biotechnology Co. Ltd (Shanghai, PR China). Myeloid-specific EPOR knockout mice (MΦ-EPORcko) and EPOR-KOloxp mice were bred and maintained in-house. Sftpc-creER mice were acquired from Cyagen Biosciences (Santa Clara, CA, USA). Heterozygous offspring resulting from crosses between EPOR-KOloxp and Sftpc-creER mice were either intercrossed or backcrossed to parental strains over multiple generations to produce homozygous Sftpc-EPORcko mice.

Reagents and antibodies. The reagents used are as follows: Bleomycin (MCE, USA, #HY-17565), Fetal Bovine Serum (Hyclone, Logan, UT, USA, #SH30070), Dulbecco’s Modified Eagle Medium (Hyclone, #SH30022), Cell Dissociation Solution (Gibco, Waltham, MA, USA, #13151014), Hematoxylin-Eosin (H&E) Stain Kit (Solarbio, Beijing, PR China, #G1120), Masson’s Trichrome Stain Kit (Solarbio, #G1346), RNA Fast 200 Kit (Fastagen, Shanghai, PR China, #220011), RT Reagent Kit with gDNA Eraser (Takara, Shiga, Japan, #RR047A), SYBR Green qPCR Mix (MCE, USA, #HY-K0501), Citrate Antigen Retrieval Solution (Beyotime, Shanghai, PR China, #P0083), Bradford Protein Quantification Kit (Beyotime, #P0006C), DAPI (Biosharp, Beijing, PR China, #BL105A), TGF-β1 (Sino Biological, Beijing, PR China, #10804-HNAC).

The antibodies used are as follows: α-SMA (Selleck, Houston, TX, USA), Collagen-I (Selleck), CD31 (Abcam, Cambridge, UK), F4/80 (Proteintech, Wuhan, PR China), PDGFRa (CST, Danvers, MA, USA), Sftpc (Thermo Fisher, Waltham, MA, USA), EPOR (Mybiosource, San Diego, CA, USA).

Cell culture and in vitro modeling of injury. MLE-12, C166, and PMs cells were divided into three groups: Control, BLM, and BLM+EPO. The Control group received DMEM alone, the BLM group was treated with DMEM containing bleomycin (10 μg/ml), and the BLM+EPO group was exposed to DMEM supplemented with both bleomycin (10 μg/ml) and erythropoietin (EPO, 100 U/ml). All cells were then incubated at 37°C with 5% CO2 for 72 h. 3T3 cells were divided into three groups: Control, TGF-β1, and TGF-β1+EPO. The Control group received DMEM alone, the TGF-β1 group was treated with DMEM containing TGF-β1 (10 ng/ml), and the TGF-β1+EPO group was exposed to DMEM supplemented with both TGF-β1 (10 ng/ml) and erythropoietin (EPO, 100 U/ml). All cells were subsequently incubated at 37°C with 5% CO2 for 48 h.

Preparation of conditioned media and culture of 3T3 cells. Following a 72-h treatment with either BLM or BLM+EPO, MLE-12, C166, and PMs cells were washed twice with PBS and incubated in fresh DMEM for an additional 24 h. The supernatant was then collected, centrifuged, and mixed with an equal volume of fresh DMEM to generate CM. Subsequently, 3T3 cells were divided into three groups and treated accordingly: the Control group received fresh DMEM; the BLM group received CM from BLM-treated cells; and the BLM+EPO group received CM from BLM+EPO-treated cells. All cultures were maintained at 37°C under 5% CO2 for 48 h. After a 72-h exposure to either BLM or a combination of BLM and EPO (BLM+EPO), MLE-12, C166, and PMs cells underwent two washes with PBS and were subsequently incubated in fresh DMEM for an additional 24 h. The supernatant was collected, centrifuged, and combined with an equal volume of fresh DMEM to produce CM. Subsequently, 3T3 cells were divided into three groups: the Control group received fresh DMEM; the BLM group received CM derived from BLM-treated cells; and the BLM+EPO group received CM from BLM+EPO-treated cells. All cell cultures were maintained at 37°C in an atmosphere containing 5% CO2 for 48 h.

Model of pulmonary fibrosis and animal group design. Pulmonary fibrosis was induced in male mice aged 6–8 weeks through intratracheal instillation of bleomycin (2.5 mg/kg in 50 μl) under pentobarbital anesthesia. The mice were randomly assigned to either the Control or BLM groups and were euthanized on day 21 for subsequent analysis. All animal experiments were conducted in accordance with relevant ethical guidelines.

Lung histopathology. Lung sections (5 μm), embedded in paraffin, were prepared for histological analysis. Hematoxylin and eosin (H&E)-stained sections were evaluated for alveolar inflammation using the Szapiel method (17). Collagen deposition was visualized using Masson’s trichrome staining and analyzed semiquantitatively via the Ashcroft method (18).

ELISA. Lung tissue samples (100 mg) were homogenized on ice in 900 μl PBS, centrifuged at 12,000 × g for 10 min at 4°C, and the supernatant was analyzed for EPOR concentration using a commercial ELISA kit according to the manufacturer’s instructions to compare levels among groups.

Immunofluorescence. Following standard deparaffinization and rehydration procedures, paraffin-embedded lung tissue sections were immersed in deionized water for a duration of 5 min. Antigen retrieval was conducted via microwave heating in a citrate buffer for 15 min, followed by allowing the sections to naturally cool to room temperature. Subsequently, the sections were washed three times with PBS, blocked with a blocking buffer for 1 h at room temperature, and incubated with diluted primary antibodies overnight at 4°C. After the primary antibodies were removed and the sections were washed three times with PBS, they were incubated with the appropriate secondary antibodies for 1 h at room temperature in the dark. The sections were then washed three times with PBS, counterstained with DAPI to visualize nuclei, washed twice more with PBS, mounted, and examined under a fluorescence microscope.

RT-qPCR. The lung tissue was placed on a 70 μm cell strainer. Following the addition of 100 μl of sterile, nuclease-free water, the tissue was gently homogenized using the plunger of a 1 ml syringe. An additional 100 μl of water was employed to rinse the strainer, and the resulting homogenate was collected. Total RNA was subsequently extracted using a commercial RNA extraction kit in accordance with the manufacturer’s instructions. RNA concentration and purity were determined using a Nanodrop 1000 spectrophotometer. cDNA was synthesized from 1 μg of total RNA using a reverse transcription kit. Quantitative real-time PCR (qPCR) was performed in a Bio-Rad CFX96 system with SYBR Green Master Mix. β-Actin served as the endogenous control for normalization. The relative mRNA expression levels of target genes were calculated using the 2−ΔΔCt method. Primer sequences are listed in Table I.

View this table:
Table I.

Primer sequence used for RT-qPCR

Hydroxyproline assay. The hydroxyproline content in lung tissue, an indicator of collagen deposition, was measured using a commercial assay kit based on the alkaline hydrolysis method. Briefly, 50 mg of tissue was homogenized and processed following the manufacturer’s protocol.

Western blot. In accordance with the previously established method (7), proteins were extracted from lung tissues utilizing RIPA lysis buffer, which was supplemented with protease and phosphatase inhibitors. The protein concentration was quantified using a Bradford assay kit (Beyotime). Equivalent protein quantities (20–60 μg) were separated via SDS-PAGE on 10% Tris-glycine gels and subsequently transferred to PVDF membranes (Millipore, Burlington, MA, USA). The membranes were blocked with a blocking buffer for 1 h at room temperature and then incubated overnight at 4°C with primary antibodies specific to α-SMA (1:1,000) and Collagen-1 (1:1,000). Following a series of washes, HRP-conjugated secondary antibodies were applied for 1 h at room temperature. Protein bands were visualized using ECL substrate (Epizyme, Shanghai, PR China) with the FUSION SOLO S imaging system (Vilber Lourmat, Collégien, France), and band intensities were quantified using ImageJ software.

Ethical statement. Animal experiments complied with institutional guidelines and were approved by the Army Medical University Animal Care and Use Committee (Chongqing, China).

Statistical analysis. Data are presented as mean±standard deviation (SD) and were analyzed using GraphPad Prism 9.0 software. Multiple group comparisons were conducted using one-way analysis of variance (ANOVA) with Tukey’s post hoc test, while comparisons between two groups employed Student’s t-test, assuming a normal distribution. p<0.05 was considered statistically significant.

Results

Pulmonary EPOR expression is downregulated post fibrosis. EPOR is acknowledged as a cytoprotective receptor (19), however, the alterations in pulmonary EPOR levels during the progression of pulmonary fibrosis remain inadequately understood. To investigate this, a pulmonary fibrosis model was developed through the administration of bleomycin via intratracheal. The hematoxylin and eosin (H&E) staining revealed that, in comparison to the Control group, mice treated with bleomycin exhibited pronounced alveolar septal thickening, increased infiltration of inflammatory cells, and disruption of normal alveolar architecture, accompanied by a significant elevation in the alveolar inflammation score (Figure 2A). Masson’s trichrome staining indicated substantial collagen deposition within lung tissue, along with a markedly elevated fibrosis score relative to the Control group (Figure 2B). Additionally, the lung hydroxyproline content was significantly greater in the bleomycin-treated group than in the Control group (Figure 2C), thereby confirming the successful establishment of the fibrosis model. Subsequent evaluation of pulmonary EPOR expression, conducted using RT-qPCR, ELISA, and immunofluorescence staining, revealed a significant reduction in total EPOR levels in fibrotic lungs, discernible at both transcriptional and protein levels (Figure 2D-E).

Figure 2.
Figure 2.

The lung EPOR expression declines after fibrosis. (A) H&E staining was used to assess the degree of pulmonary pathological changes, bar=625 μm, n=4. (B) Masson staining was used to evaluate the degree of collagen deposition in the lungs, bar=625 μm, n=4. (C) Quantification of lung hydroxyproline levels, n=4. (D) The mRNA and protein level of EPOR in the lung was measured by qPCR and ELISA, n=4. (E) Representative images of immunofluorescence showing EPOR level in murine lung sections, bar=50μm, n=4. **p<0.01, ***p<0.001, ****p<0.0001. EPOR: Erythropoietin receptor. H&E staining: Hematoxylin and eosin staining.

Alterations in EPOR levels across distinct cell types following fibrosis. Pulmonary fibrosis is currently conceptualized as an epithelial pathology, initiated by recurrent damage to alveolar epithelial cells and involving various cell types, including endothelial cells, fibroblasts, and immune cells (20). Considering the numerical predominance of AEC II within the epithelium and the prevalence of macrophages among immune cells, immunofluorescence analysis was conducted to assess alterations in EPOR expression following fibrosis in these critical cell populations: macrophages, vascular endothelial cells, AEC II cells, and fibroblasts. The analysis confirmed EPOR expression in macrophages (Figure 3A), endothelial cells (Figure 3B), AEC II cells (Figure 3C), and fibroblasts (Figure 3D), with AEC II cells exhibiting the highest basal expression. Following fibrosis, all cell types demonstrated reduced EPOR levels compared to controls, with the most pronounced decrease observed in AEC II cells (Figure 3C). This suggests a potentially significant role for EPOR in macrophages and, particularly, AEC II cells in the pathogenesis of fibrosis.

Figure 3.
Figure 3.

Alterations in EPOR levels in different cell types in response to fibrosis. Representative images of immunofluorescence showing EPOR (green) level in different type of cells after fibrosis (A) Macrophage: F4/80, red, n=4. (B) Endothelium: CD31, red, n=4. (C) Type 2 alveolar epithelial cells: SPC, red, n=4. (D) Fibroblasts: PDGFRa, red, n=4. Upper: Bar=50 μm, lower: enlarged view of the boxed region. *p<0.05, **p<0.01, ****p<0.0001.

The impact of activating EPOR across distinct cell types on 3T3 fibroblasts. To evaluate the influence of cell type-specific EPOR signaling activation on the differentiation of 3T3 fibroblasts into myofibroblasts, primary macrophages (PMs), vascular endothelial cells (C166), and type II alveolar epithelial cells (MLE-12) were exposed to either BLM or a combination of BLM and EPO for a duration of 72 h. Subsequently, the media were replaced with fresh DMEM, and the cells were incubated for an additional 24 h. Conditioned media were then collected and applied to 3T3 cells for 48 h. In the MLE-12 cells, 3T3 cells treated with conditioned media from the BLM group demonstrated elevated expression levels of α-SMA and col-1 compared to the control group. Conversely, conditioned media from the BLM+EPO group resulted in a reduction of these markers relative to the BLM group (Figure 4A). In the case of C166 cells and PMs, conditioned media from the BLM+EPO group further enhanced 3T3 cell activation compared to the BLM group (Figure 4B, C). However, direct activation of EPOR in 3T3 cells intensified TGF-β1-induced fibroblast activation (Figure 4D). These in vitro results suggest that the effects of EPOR activation on fibroblast activation are dependent on the specific cell type, exhibiting diverse outcomes.

Figure 4.
Figure 4.

Effects of EPOR activation in various cell types upon 3T3 fibroblasts. The effect of conditioned medium of BLM, BLM+EPO incubating MLE-12 (A), C166 (B), PMS (C) on the expression of α-SMA, Col-1 in 3T3 cells. (D) Activation of 3T3 cells EPOR promotes TGF-β1-induced expression of α-SMA and Col-1. n=4. *p<0.05, **p<0.01, ***p<0.001, ****p<0.0001. PMs: Peritoneal macrophages; α-SMA: α-smooth muscle actin; Col-1: collagen I.

Targeted deletion of EPOR in macrophages ameliorates experimental pulmonary fibrosis in mice. To elucidate the function of macrophage EPOR in the context of pulmonary fibrosis, wild-type C57BL/6J mice were utilized as the control group. EPOR-KOloxp (EPOR-C) mice served as a positive control, while mice with a macrophage-specific EPOR knockout (MΦ-EPORcko) were designated as the experimental group. Both EPOR-C and MΦ-EPORcko mice underwent intratracheal administration of BLM to induce pulmonary fibrosis. Hematoxylin and eosin (H&E) staining revealed that MΦ-EPORcko mice exhibited enhanced alveolar architecture and a reduced alveolar inflammation score compared to the EPOR-C group (Figure 5A). Masson’s trichrome staining indicated a diminished deposition of blue collagen and a lower fibrosis score in the lungs of MΦ-EPORcko mice relative to EPOR-C group (Figure 5B). The hydroxyproline content assay demonstrated a significant decrease in lung hydroxyproline levels in the MΦ-EPORcko group compared to the EPOR-C group (Figure 5C). Western blot analysis showed reduced protein levels of α-SMA and Col-1 in the lungs of MΦ-EPORcko mice compared to the EPOR-C group (Figure 5D). Collectively, these findings suggest that macrophage-specific EPOR knockout mitigates BLM-induced pulmonary fibrosis in mice.

Figure 5
Figure 5

Knockout of macrophage EPOR ameliorates pulmonary fibrosis in mice. (A) H&E staining was used to assess the degree of pulmonary pathological changes bar=625μm, n=4. (B) Masson staining was used to evaluate the degree of collagen deposition in the lungs. bar=625μm, n=4. (C) Quantification of lung hydroxyproline levels.n=4. (D) The protein level of EPOR in the lung was measured by western blot. n=3. *p<0.05, **p<0.01, ***p<0.001, ****p<0.0001. MΦ-EPORcko: Bleomycin-treated macrophage-specific EPOR knockout mice; EPOR-C: bleomycin-treated EPOR-KOloxp homozygous mice; α-SMA: α-smooth muscle actin; Col-1: collagen I.

AEC II-specific EPOR deficiency worsens lung fibrosis. To elucidate the role of EPOR in type II alveolar epithelial cells in the context of pulmonary fibrosis, we employed Sftpc-EPORcko mice and EPOR-KOloxp (EPOR-C) mice in a BLM-induced fibrosis model. Hematoxylin and eosin (H&E) staining revealed that Sftpc-EPORcko mice exhibited more severe disruption and collapse of alveolar architecture, as well as an increased alveolar inflammation score, compared to the EPOR-C group (Figure 6A). Masson’s trichrome staining further demonstrated an exacerbated deposition of blue collagen fibers and a higher fibrosis score in the lungs of Sftpc-EPORcko mice relative to EPOR-C controls (Figure 6B). The hydroxyproline content assay indicated a significant elevation in lung hydroxyproline levels in the Sftpc-EPORcko group compared to the EPOR-C group (Figure 6C). Additionally, Western blot analysis revealed upregulated protein expression ofα-SMA and Col-1 in the lungs of Sftpc-EPORcko mice compared to the EPOR-C group (Figure 6D). Collectively, these findings indicate that the knockout of EPOR specifically in AEC II exacerbates BLM-induced pulmonary fibrosis in mice.

Figure 6.
Figure 6.

Knockout of type II alveolar epithelial cells EPOR exacerbates pulmonary fibrosis in mice. (A) H&E staining was used to assess the degree of pulmonary pathological changes. Bar=625 μm, n=4. (B) Masson staining was used to evaluate the degree of collagen deposition in the lungs. Bar=625 μm, n=4. (C) Quantification of lung hydroxyproline levels, n=4. (D) The protein level of EPOR in the lung was measured by western blot, n=3. *p<0.05, **p<0.01, ****p<0.0001. Sftpc-EPORcko: Bleomycin-treated type II alveolar epithelial cells-specific EPOR knockout mice; EPOR-C: bleomycin-treated EPOR-KOloxp homozygous mice; α-SMA: α-smooth muscle actin; Col-1: collagen I.

Discussion

EPOR is acknowledged as a cytoprotective receptor, whose activation enhances cellular functions such as anti-apoptotic activity, antioxidant defense, and the resolution of inflammation (19). Nevertheless, the changes in pulmonary EPOR levels during the progression of pulmonary fibrosis remain poorly understood. Our findings demonstrated a significant downregulation of total pulmonary EPOR during fibrosis, highlighting the potential therapeutic significance of maintaining or restoring EPOR signaling to mitigate fibrotic progression. Previous research has shown that EPOR is broadly expressed across various lung cell types, with the exception of type I alveolar epithelial cells (21). However, the specific roles of EPOR signaling in different cell types concerning the pathogenesis of pulmonary fibrosis have yet to be elucidated. Consequently, we evaluated EPOR levels in four cell types critically involved in pulmonary fibrosis using immunofluorescence staining. Under physiological conditions, EPOR was predominantly expressed in macrophages and AEC II, with lower expression levels observed in endothelial cells and fibroblasts. Following the induction of fibrosis, a pronounced reduction in EPOR expression was specifically noted in macrophages and AEC II. These findings indicate that EPOR signaling, which contributes to pulmonary fibrosis, may primarily originate from two specific cell populations.

The dysregulated activation of fibroblasts, serving as the main agents of fibrosis, is influenced by intricate interactions with parenchymal and immune cells. Damage to macrophages, endothelial cells, or alveolar epithelial cells can disrupt fibroblast homeostasis through paracrine signaling (22). In vitro experiments demonstrated that 3T3 fibroblasts treated with conditioned media from cells with activated EPOR signaling exhibited varied responses. CM from MLE-12 cells inhibited the expression of α-SMA and Col-1 in 3T3 cells, whereas CM from PMs and C166 cells enhanced their expression. Additionally, direct EPOR activation in 3T3 cells intensified TGF-β1-induced fibroblast activation. These in vitro findings tentatively suggest that EPOR activation has diverse, cell type-specific effects on fibrotic processes. Macrophages, as the most prevalent immune cells in the lung, are implicated in the promotion of pulmonary fibrosis. Patients with IPF show a significant increase in alveolar macrophage numbers compared to healthy individuals, with these macrophages expressing higher levels of pro-fibrotic genes (23, 24). M2-polarized macrophages are recognized as pivotal contributors to IPF, promoting fibroblast activation and fibrogenesis through the paracrine secretion of mediators such as TGF-β1 and PDGF (25). Interestingly, research utilizing ARDS mouse models has demonstrated that EPOR activation in macrophages enhances the expression of TGF-β1 and IL-10, thereby facilitating tissue repair (7). Our experimental findings revealed that mice with macrophage-specific EPOR knockout exhibited significantly reduced pulmonary fibrosis, indicating that the deletion of EPOR in macrophages mitigates bleomycin-induced lung fibrosis. Additionally, we observed a decrease in EPOR expression within macrophages following fibrosis. We propose two non-mutually exclusive hypotheses to explain this apparent discrepancy. First, pulmonary macrophages are composed of alveolar macrophages (AMs) and interstitial macrophages (IMs), and the specific subtypes predominantly expressing EPOR have yet to be identified. Although the total number of macrophages increases during fibrosis, the expanding population may include subtypes with inherently lower EPOR expression, resulting in an overall reduction in average EPOR levels. Secondly, the dynamics of macrophage EPOR expression may exhibit a complex, non-linear temporal pattern. Given that our analysis was limited to a single time point, it may not have fully captured the trajectory of EPOR changes or its precise relationship with fibrotic progression.

The alveolar epithelium consists of AEC I and AEC II. AEC I primarily facilitate gas exchange and act as a mechanical barrier. Upon injury to AEC I, AEC II cells proliferate and differentiate to restore the epithelium, thereby preserving normal alveolar architecture and function. Repeated injury and aberrant repair of the alveolar epithelium, triggered by various insults, are recognized as critical initiating events in IPF (20, 26). Consistent with this, lung tissues from IPF patients exhibit prominent pathological features in AEC II, including increased levels of apoptosis, senescence, abnormal differentiation, and impaired proliferative capacity (20). Single-cell RNA sequencing data have revealed a significant reduction in the number and compromised reparative capacity of isolated AEC II from the lungs of patients with IPF compared to healthy controls (27). To support a causal relationship, a transgenic mouse model with alveolar epithelial cell type II-specific injury induced by diphtheria toxin exhibited a twofold increase in lung hydroxyproline content by days 21 and 28 post-injury, with histopathological analysis confirming the development of fibrotic lesions. This suggests that targeted injury to AEC II is sufficient to initiate pulmonary fibrosis (9). Conversely, the inhibition of AEC II apoptosis or ferroptosis has been demonstrated to mitigate fibrotic progression. Yoshimi Michihiro et al. reported that EPO attenuates bleomycin-induced pneumonitis by inhibiting apoptosis in alveolar epithelial cells. In a separate study employing an ARDS mouse model (15), Fei Cao et al. observed that EPO suppresses the activation of the pulmonary NLRP3 inflammasome and promotes the resolution of edema. however, these protective effects were negated when the EPOR was pharmacologically blocked with EMP9, resulting in persistent edema (5). Consistent with these observations, our results demonstrated that Sftpc-EPORcko mice developed significantly exacerbated pulmonary fibrosis, confirming that the deletion of EPOR specifically in type II alveolar epithelial cells aggravates bleomycin-induced lung fibrosis.

Conclusion

This study is the first comprehensive analysis of EPOR expression dynamics across various pulmonary cell populations during fibrosis development. We identified contrasting roles for EPOR signaling in different cellular contexts: its activation in macrophages seems to facilitate fibrogenesis, whereas in AEC II, it likely exerts a protective, anti-fibrotic effect. Nonetheless, our findings also highlight unresolved questions. The mechanisms by which the observed downregulation of EPOR in macrophages paradoxically contributes to exacerbated fibrosis, and the nature of the potential crosstalk between macrophage and AEC II EPOR signaling pathways, remain to be elucidated. Further research into these interactive mechanisms is necessary to provide deeper theoretical insights into the spatiotemporal regulation of EPOR signaling in pulmonary fibrosis.

Footnotes

  • Authors’ Contributions

    Zhiren Zhang provided the idea and designed the experiments. Pengfei Wu, Jialin Jia and Tianrong Jin performed the experiments. Bangwei Luo provided technical support. Pengfei Wu analyzed and interpreted the data, wrote the draft of the manuscript. Guansong Wang, Bangwei Luo and Zhiren Zhang revised the manuscript, supervised the research, and provided funding.

  • Conflicts of Interest

    The Authors have declared that no conflicts of interest exist.

  • Funding

    This research was supported by a grant (CSTB2025NSCQ-GPX0629) from the Chongqing Natural Science Foundation (to ZZ), and supported by Scientific and Technological Research Program of Chongqing Municipal Education Commission (to BL, Grant No. KJQN202512831).

  • Artificial Intelligence (AI) Disclosure

    No artificial intelligence (AI) tools, including large language models or machine learning software, were used in the preparation, analysis, or presentation of this manuscript.

  • Received December 19, 2025.
  • Revision received January 25, 2026.
  • Accepted February 3, 2026.

This is an open access article under the terms of the Creative Commons Attribution License, which permits use, distribution and reproduction in any medium, provided the original work is properly cited.

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Antagonistic Effect of Erythropoietin Receptor Signaling in Macrophages and Type II Alveolar Epithelial Cells on Pulmonary Fibrosis in Mice
PENGFEI WU, JIALIN JIA, TIANRONG JIN, BANGWEI LUO, ZHIREN ZHANG, GUANSONG WANG
In Vivo May 2026, 40 (3) 1517-1529; DOI: 10.21873/invivo.14302
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