Fibrocytes with different phenotypes are recruited in chronic obstructive pulmonary disease

Abstract

Previous studies showed that fibrocytes are increased in the circulation and in the airway walls of chronic obstructive pulmonary disease (COPD) patients. However, it is unclear whether fibrocytes express different phenotypes. The aim of the study was to further evaluate the recruitment and phenotypic characteristics of fibrocytes in patients and murine models with COPD. By using flow cytometry analysis, blood fibrocytes were enumerated and characterized in the fresh peripheral blood samples from 10 control subjects and 25 COPD patients. Tissue fibrocytes were detected by using multiplex staining and multispectral imaging analysis in COPD patients and murine models. Our analysis revealed no significant difference in the counts of total (CD45⁺Collagen I⁺) circulating fibrocytes, or those positive for the thymic stromal lymphopoietin receptor (TSLPR) or the chemokine CXCL12 receptor (CXCR4), between COPD patients and control subjects. In contrast, a significantly higher proportion of circulating fibrocytes expressed the IL-25 receptor (IL-17RA/IL-17RB) and the IL-33 receptor (sT2L) in COPD patients compared to controls. Furthermore, we demonstrated an increase in total fibrocytes, as well as IL-17RA/IL-17RB⁺, sT2L⁺, TSLPR⁺, CXCR4⁺, and smooth muscle actin α (α-SMA)⁺ fibrocytes, within the airway walls, alveolar walls, and alveolar spaces of both COPD patients and corresponding murine models. These results indicate that fibrocytes are elevated and exhibit an activated phenotype (α-SMA-positive) in the lung tissues of patients with COPD. These cells also express receptors for IL-25, IL-33, TSLP, and CXCL12. Taken together, these findings suggest that fibrocytes may participate in the development of peribronchial and pulmonary fibrosis in COPD, potentially mediated by epithelial-derived cytokines and chemokines.

Supplementary Information

The online version contains supplementary material available at https://doi.org/10.1038/s41598-026-56766-1.

Introduction

Chronic obstructive pulmonary disease (COPD) is a very common airway disease that affects more than 200 million and 99.9 million persons worldwide and in China, respectively^1,2. COPD is characterized by persistent bronchial and pulmonary inflammation and structural remodeling³. These pathological processes result in epithelial mucous metaplasia, alveolar destruction, collagen deposition in small airway walls (known as peribronchiolar fibrosis), persistent airflow obstruction, and accelerated decline in lung function⁴. Current pharmacological interventions are largely ineffective at inhibiting or reversing these changes, which may periodically present as acute exacerbations⁵. This clinical progression underscores that the underlying pathogenic mechanisms of COPD remain poorly understood.

Fibrocytes are bone marrow-derived progenitor cells with both hematopoietic and mesenchymal characteristics. These cells are a distinct population that coexpress Collagen I and CD45 and/or CD34^6–8. They reside in the circulation and can be recruited into injured tissues, contributing to tissue inflammation and remodeling. We and others have shown that fibrocytes are increased in the circulation and airway and contribute to airway inflammation and remodeling in asthma patients^9–13. Dupin et al. previously showed that during COPD exacerbations, circulating fibrocytes (defined as Collagen I⁺ and CD45⁺ nonadherent non-T [NANT] cells) increase via the CXCR4/CXCL12 axis¹⁴. Moreover, they found that fibrocytes (defined as CD45⁺ and fibroblast-specific protein 1 [FSP1]⁺ cells) also accumulate in the airway walls of patients with COPD¹⁵. Although fibrocyte infiltration occurs in COPD lungs, it has not been established whether these cells undergo a phenotypic transition involving activation and differentiation, such as the acquisition of smooth muscle actin α (α-SMA) expression.

IL-25, IL-33 and thymic stromal lymphopoietin (TSLP) are a group of epithelial-derived cytokines which have been shown to exert an important role on airway inflammation and remodeling in asthma patients¹⁶. However, the pathogenic role of these cytokines in COPD remains elusive. Our previous work demonstrated that IL-25 drives a pro-fibrotic phenotypic transformation in fibrocytes, marked by increased proliferation, migration, and differentiation, as well as elevated expression of extracellular matrix (ECM) components (collagen I/III, fibronectin) and pro-fibrotic factors like connective tissue growth factor (CTGF)⁹. IL-33 has also been shown to promote the migration and proliferation of circulating fibrocytes in patients with allergen-exacerbated asthma, and to enhance proliferation along with α-SMA, IL-13, and IL-5 expression in fibrocytes from healthy volunteers^17,18. In keloid tissue of BALB/c mice, intradermal TSLP injection increased the infiltration of CXCR4⁺ fibrocytes¹⁹. Furthermore, in an allergic asthma model, intraperitoneal administration of an anti-TSLP antibody reduced fibrocyte numbers and thereby prevented changes in both airway and vascular architecture²⁰. These findings indicate that fibrocytes exhibit considerable plasticity in response to specific cytokine milieus.

Herein, we hypothesize that in the context of COPD, fibrocytes may express receptors for CXCL12, IL-25, IL-33, and TSLP. Within this cytokine and chemokine milieu, fibrocytes can migrate to the airways and lung tissues, where they may become activated and subsequently express α-SMA in response to these mediators. Therefore, the objective of this study was to quantify the counts of peripheral and tissue fibrocytes exhibiting distinct phenotypes in patients with COPD and to compare them with those in control subjects. These differences were further evaluated in a murine model of COPD.

Methods

Study populations

To quantify circulating fibrocytes (CFs), a total of 25 patients with acute exacerbations of COPD (AECOPD; 5 women and 20 men) and 10 control subjects (2 women and 8 men) were prospectively enrolled from Beijing Tongren Hospital, Capital Medical University. AECOPD was defined according to the Global Initiative for Chronic Obstructive Lung Disease (GOLD) criteria²¹. Peripheral blood samples were obtained immediately upon hospital admission (during acute exacerbation), prior to the initiation of corticosteroid or other therapeutic interventions. All healthy volunteers demonstrated normal pulmonary function test results and had no history of pulmonary disease. In this study, we did not include a control group of smokers without COPD. Therefore, a direct comparison to isolate the specific effect of smoking independent of COPD was not performed. To quantify fibrocytes in lung tissue, specimens were obtained from 4 patients with COPD who underwent lung volume reduction surgery. Lung tissue samples adjacent to the tumor were collected from 3 patients undergoing lung cancer surgery and served as the control group. Written informed consent was obtained from all participants after a comprehensive explanation of the study procedures. All study protocols adhere to the Declaration of Helsinki and was approved by the Ethics Committee of Beijing Tongren Hospital, Capital Medical University. The characteristics of the study groups are shown in Tables 1 and 2.

Table 1.

Clinical characteristics of study populations for detecting circulating fibrocytes.

Parameters	Normal subjects (N = 10)	COPD patients (N = 25)	P value
Sex (male/female)	8/2	20/5	1.000
Age (years)	66.30 ± 6.11	72.40 ± 8.88	0.056
Smoking time (years)	-	26.50 ± 19.94	-
smoking index (pack-year)		24.78 ± 26.10	-
Eosinophil Count‌ (×109/L)	-	0.096 ± 0.097	-
Acute exacerbations in the previous year		0.88 ± 0.85
FEV1/FVC (%)	86.14 ± 4.91	56.02 ± 14.20	<0.0001
FEV1%pred.	111.60 ± 10.32	55.84 ± 27.63	<0.0001

Data are presented as means ± SD. P values indicating differences between the two groups were calculated using the Mann–Whitney U test. Comparisons of proportions were performed using Fisher’s exact test.

Abbreviations: FEV₁, forced expiratory volume in 1 s; FVC, forced vital capacity.

Table 2.

Clinical characteristics of study populations for detecting tissue fibrocytes.

Parameters	Normal subjects (N = 3)	COPD patients (N = 4)	P value
Sex (male/female)	2/1	3/1	1.000
Age (years)	62.33 ± 11.09	68.20 ± 7.77	0.2855
Smoking time (years)	-	28.00 ± 4.55	-
smoking index (pack-year)		25.88 ± 12.77	-
FEV1/FVC (%)	94.01 ± 7.57	63.62 ± 7.37	0.0571
FEV1%pred.	105.20 ± 4.46	55.63 ± 4.92	0.0571

Data are presented as means ± SD. P values indicating differences between the two groups were calculated using the Mann–Whitney U test. Comparisons of proportions were performed using Fisher’s exact test.

Abbreviations: FEV₁, forced expiratory volume in 1 s; FVC, forced vital capacity.

Identification and characterization of circulating fibrocytes

CFs in blood samples obtained from patients with COPD and healthy volunteers were identified by flow cytometry as previously described^9,10. CFs were defined as CD45⁺Collagen I⁺ cells among peripheral blood mononuclear cells (PBMCs). Expression of chemokine and cytokine receptors was also evaluated using flow cytometry with specific fluorescent antibodies targeting CXCR4, IL-17RA/RB, IL-33R (sT2L), and TSLPR (BD Biosciences, San Jose, CA). The total and phenotypically distinct subpopulations of CFs were expressed as a percentage of the total PBMC population. The gating strategy is shown in Fig. S1-S2. The detailed methods are provided in the supporting materials.

Identification and characterization of tissue fibrocytes

Fibrocytes in airway and lung tissues were detected by multiplex immunohistochemistry and multispectral image analysis. Lung tissue samples were inflated with 10% buffered formalin, embedded in paraffin, and sectioned at a thickness of 4 μm. Multiplex immunofluorescence staining and multispectral imaging were employed to identify cell subsets co-expressing CD45, collagen I, IL-25R (IL-17RA/RB), IL-33R (sT2L), TSLPR, CXCR4, or α-SMA in lung sections, as previously described⁹. Detailed methodologies are provided in the supporting materials.

Establishment of a murine model of COPD

Six- to eight-week-old male C57BL/6J mice were randomly assigned to two groups (n = 10 per group): a control group and a cigarette smoke-induced COPD group. A custom-designed smoking chamber was utilized for cigarette smoke exposure. Mice in the smoke-exposed group were placed inside the chamber, which featured two 1 cm diameter vents on the top. A cigarette was attached to a cigarette holder, and smoke was continuously drawn using a 60 mL syringe and delivered into the chamber via an infusion tube connected to a three-way connector. Commercially available cigarettes (Marlboro, Philip Morris, USA; containing 1.0 mg nicotine and 11 mg tar per cigarette) were used to generate the smoke. Smoke exposure was performed twice daily, each session lasting 2 h, with 9 cigarettes burned per hour. The exposure was conducted 6 days per week, and the total duration of the model establishment period was 24 weeks. During smoke exposure, the animals had free access to food and water inside the chamber. Control group mice were exposed to normal air under identical conditions. All mice completed the entire modeling protocol, with no unintended losses. For endpoint analysis, 3 mice from the control group and 4 mice from the COPD group were randomly selected. These animals were euthanized by intraperitoneal injection of 200 µL of 1% sodium pentobarbital, and lung tissues were collected. Subsequent processing included histopathological examination (hematoxylin‑eosin and Masson staining) and fibrocyte identification via multiplex immunohistochemistry combined with multispectral image analysis. All animal experimental procedures were performed according to the NIH animal care guidelines and the ARRIVE guidelines and were approved by the Institutional Animal Care and Use Committee of Beijing Tongren Hospital, Capital Medical University.

Statistical analysis

Data are presented as mean ± standard deviation (SD) or median (interquartile range). Due to the small sample size and non-normal distribution of data, the two-tailed, Mann-Whitney U test was employed to compare differences between the Normal and COPD groups. A P value of less than 0.05 was considered statistically significant. All statistical analyses were conducted using GraphPad Prism version 8 (GraphPad Software, San Diego, CA, USA).

Results

Patients characteristics

A total of 25 COPD patients were enrolled for the detection of circulating fibrocytes. Their baseline characteristics are presented in Table 1. The mean age was 72.40 ± 8.88 years, and the majority of participants were male (80.0%). The mean FEV₁% predicted and FEV₁/FVC ratio of COPD patients were 55.84 ± 27.63 and 56.02 ± 14.20, respectively, which was significantly reduced in comparison with normal subjects. The study included 10 healthy control subjects, aged 66.30 ± 6.11 years, comprising 8 males and 2 females.

The number of differentiated circulating fibrocytes is elevated in patients with COPD

No significant differences were observed in the total number of circulating fibrocytes (CD45⁺Collagen I⁺ PBMCs) between COPD patients and control subjects (Fig. 1A). However, the numbers of CD45⁺Collagen I⁺ fibrocytes expressing IL-25R (IL-17RA/IL-17RB, Fig. 1B), IL-33R (sT2L, Fig. 1C), or co-expressing IL-25R/IL-33R/TSLPR (Fig. 1E) were all significantly increased (P < .05) in COPD patients compared with controls. In contrast, the numbers of fibrocytes expressing TSLPR (Fig. 1D) or CXCR4 (Fig. 1F) were not elevated in COPD patients.

Fig. 1.

Increased subgroups of circulating fibrocytes in COPD patients. Fibrocytes were quantified by flow cytometry. Total fibrocytes (A), IL-17RA⁺/IL-17RB⁺ fibrocytes (B), sT2L⁺ fibrocytes (C), TSLPR⁺ fibrocytes (D), IL-17RA⁺/IL-17RB⁺ sT2L⁺ TSLPR⁺ fibrocytes (E), CXCR4⁺ fibrocytes (F) were expressed as a percentage of NANT PBMCs. Horizontal lines represent median. **p < .01, ***p < .001, nonparametric Mann-Whitney test, n = 25 of COPD patients and n = 10 of normal control subjects.

Tissue fibrocyte numbers are increased in COPD patients

The baseline characteristics of COPD patients used for detecting tissue fibrocytes are presented in Table 2. We demonstrated that fibrocytes were located, occasionally in clusters, in the peribronchial region (including within the airway lumen, the epithelial layer, or the subepithelial area, Fig. 2A), as well as in alveolar walls (Fig. 2B) and alveolar spaces (Fig. 2C) in COPD patients. All values in the COPD group were higher than those in the Normal group, with complete separation of the datasets. Direct observation of histological sections revealed a markedly increased total number of fibrocytes in the lungs of COPD patients compared to control subjects. A Mann-Whitney U test also revealed a marginally significant difference between groups in the proportion of total tissue fibrocytes (CD45⁺Collagen I⁺ cells) (U = 0, p = .057, two-sided; Fig. 2E).

Fig. 2.

Total and IL-25R-positive fibrocytes are elevated in lung tissues of patients with COPD. Tissue fibrocytes were quantified by multiplex immunohistochemistry and multispectral image analysis. Total and IL-17RA⁺/IL-17RB⁺ (IL-25R⁺) fibrocytes were observed in the peribronchial regions (A), alveolar walls (B) and alveolar spaces (C) in COPD patients compared with normal control (D). E and F: Statistical analysis for the percentages of total fibroctyes (CD45⁺Collagen I⁺) and IL-25R expressing fibrocytes (IL-17RA⁺IL-17RB⁺ CD45 + Collagen I⁺). Bars represent the mean ± SEM. Data were analyzed with the Mann-Whitney U test. A marginally significant difference was observed (U = 0, p = .057, two-sided; n = 4 for COPD patients, n = 3 for control subjects).

Differentiated tissue fibrocyte numbers are increased in COPD patients

We next examined whether tissue fibrocytes express receptors for epithelial-derived cytokines (IL-25, IL-33, and TSLP) and CXCL12 (CXCR4). Our results showed that the majority of tissue fibrocytes expressed IL-17RA/IL-17RB (Fig. 2A-C), as well as sT2L, TSLPR (either individually or in combination as sT2L/TSLPR; Fig. 3A-C), and CXCR4 (Fig. 4A). The Mann-Whitney U test indicated a marginally significant difference between groups in the proportions of fibrocytes positive for IL-17RA/IL-17RB, ST2L, TSLPR, the ST2L/TSLPR combination, and CXCR4 (U = 0, p = .057, two-sided; Figs. 2F, 3E-G and 4D). We further investigated whether tissue fibrocytes exhibit an activated phenotype through expression of α-SMA. Analysis revealed a marginally significant increase in α-SMA–positive fibrocytes in COPD lung tissues compared to controls (U = 0, p = .057, two-sided; Fig. 4A-C).

Fig. 3.

IL-33R and TSLPR-positive fibrocytes are elevated in lung tissues of patients with COPD. Tissue fibrocytes were quantified by multiplex immunohistochemistry and multispectral image analysis. sT2L⁺ (IL-33R⁺) and TSLPR⁺ fibrocytes were observed in the peribronchial regions (A), alveolar walls (B) and alveolar spaces (C) in COPD patients compared with normal control (D). E-G: Statistical analysis for the percentages of IL-33R (sT2L⁺CD45⁺Collagen I⁺), TSLPR (TSLPR⁺CD45⁺Collagen I⁺) and IL-33R/TSLPR expressing fibrocytes (sT2L⁺TSLPR⁺CD45⁺Collagen I⁺). Bars represent the mean ± SEM. Data were analyzed with the Mann-Whitney U test. A marginally significant difference was observed (U = 0, p = .057, two-sided; n = 4 for COPD patients, n = 3 for control subjects).

Fig. 4.

CXCR4 and α-SMA -positive fibrocytes are elevated in lung tissues of patients with COPD. Tissue fibrocytes were quantified by multiplex immunohistochemistry and multispectral image analysis. A and B: CXCR4⁺ and α-SMA⁺ fibrocytes were elevated in COPD patients compared with normal control. C and D: Statistical analysis for the percentages of α-SMA (α-SMA ⁺CD45⁺Collagen I⁺) and CXCR4 expressing fibrocytes (CXCR4⁺CD45⁺Collagen I⁺). Bars represent the mean ± SEM. Data were analyzed with the Mann-Whitney U test. A marginally significant difference was observed (U = 0, p = .057, two-sided; n = 4 for COPD patients, n = 3 for control subjects).

Total and differentiated tissue fibrocyte numbers are increased in COPD murine model

To further investigate the infiltration of fibrocytes in lung tissue, a cigarette smoke-induced COPD murine model was established, and lung samples were collected to assess the number and distribution of fibrocytes. The H&E and Masson staining of lung tissues from smoke-exposed mice reveal characteristic pathological changes, including alveolar structural destruction, emphysematous enlargement, and inflammatory/fibrotic remodeling around the airways (Fig. S3 and Fig.S4). Analysis revealed fibrocytes expressing IL-17RA/IL-17RB (Fig. 5A–C), IL-33R, TSLPR, IL-33R/TSLPR (Fig. 6A–C), or CXCR4 (Fig. 7A–C) located in peribronchial regions (top panels), alveolar walls (middle panels), and alveolar spaces (bottom panels). A Mann-Whitney U test was performed to compare the proportions of total fibrocytes and fibrocytes of varying phenotypes between control and COPD mice (Figs. 5E-F, 6E-G and 7E). The analysis indicated a marginally significant difference between groups (U = 0, p = .057, two-sided). Notably, the values exhibited no overlap, with all measurements in the control group being lower than those in the COPD group.

Fig. 5.

Total and IL-25R-positive fibrocytes are elevated in lung tissues of COPD mice model. Tissue fibrocytes were quantified by multiplex immunohistochemistry and multispectral image analysis. Total and IL-17RA⁺/IL-17RB⁺ (IL-25R⁺) fibrocytes were observed in the peribronchial regions (A), alveolar walls (B) and alveolar spaces (C) in COPD mice compared with normal control (D). E and F: Statistical analysis for the percentages of total fibroctyes (CD45⁺Collagen I⁺) and IL-25R expressing fibrocytes (IL-17RA⁺IL-17RB⁺ CD45⁺Collagen I⁺). Bars represent the mean ± SEM. Data were analyzed with the Mann-Whitney U test. A marginally significant difference was observed (U = 0, p = .057, two-sided; n = 4 for COPD mice, n = 3 for control mice).

Fig. 6.

IL-33R and TSLPR-positive fibrocytes are elevated in lung tissues of COPD mice model. Tissue fibrocytes were quantified by multiplex immunohistochemistry and multispectral image analysis. sT2L⁺ (IL-33R⁺) and TSLPR⁺ fibrocytes were observed in the peribronchial regions (A), alveolar walls (B) and alveolar spaces (C) in COPD mice compared with normal control (D). E-G: Statistical analysis for the percentages of IL-33R (sT2L⁺CD45⁺Collagen I⁺), TSLPR (TSLPR⁺CD45⁺Collagen I⁺) and IL-33R/TSLPR expressing fibrocytes (sT2L⁺TSLPR⁺CD45⁺Collagen I⁺). Bars represent the mean ± SEM. Data were analyzed with the Mann-Whitney U test. A marginally significant difference was observed (U = 0, p = .057, two-sided; n = 4 for COPD mice, n = 3 for control mice).

Fig. 7.

CXCR4 positive fibrocytes are elevated in lung tissues of COPD mice model. Tissue fibrocytes were quantified by multiplex immunohistochemistry and multispectral image analysis. CXCR4⁺ fibrocytes were observed in the peribronchial regions (A), alveolar walls (B) and alveolar spaces (C) in COPD mice compared with normal control (D). E: Statistical analysis for the percentages of CXCR4 expressing fibrocytes (CXCR4⁺CD45⁺Collagen I⁺). Bars represent the mean ± SEM. Data were analyzed with the Mann-Whitney U test. A marginally significant difference was observed (U = 0, p = .057, two-sided; n = 4 for COPD mice, n = 3 for control mice).

Discussion

These results demonstrate a significant increase in the proportion of fibrocytes in COPD. Furthermore, these elevated fibrocyte populations express receptors for IL-25, IL-33, TSLP, and CXCL12, and can adopt an activated phenotype characterized by α-SMA expression. This suggests that fibrocytes may contribute to peribronchial and pulmonary fibrosis in COPD in response to epithelial-derived cytokines and chemokines.

One of the typical pathological features of COPD is extracellular matrix (ECM) remodeling. The major ECM compartments in the lung include the basement membrane, the lamina propria of the airways, and the alveolar interstitium. Disruption of the ECM can have significant consequences, leading to remodeling of lung tissue that may affect all pulmonary compartments—such as airway wall fibrosis and emphysema—in COPD²². Peri-airway remodeling and fibrosis primarily result from enhanced collagen deposition within the airway walls and structural alterations in collagen fibrils, which exhibit a more disorganized arrangement. The principal sources of extracellular matrix (ECM) proteins in the airway are fibroblasts and their activated counterparts, myofibroblasts, with additional contributions from airway smooth muscle cells and airway epithelial cells.

Fibrocytes, a population of hematopoietic-derived cells characterized by the expression of CD45 and Collagen I, play a direct role in tissue fibrosis through collagen production in response to injury, disease progression, and aging⁶. Studies, including our own, have demonstrated elevated fibrocyte levels in asthma patients, particularly in cases of severe and chronic obstructive asthma^9,11,12. Elevated levels of circulating fibrocytes exhibit increased myofibroblastic differentiation and reduced responsiveness to corticosteroids. These elevated levels are associated with an annual decline in FEV₁, greater asthma severity, and a higher frequency of acute exacerbations, and may contribute to airway obstruction and fibrosis in patients with asthma. Dupin et al. observed a significant elevation in circulating fibrocyte levels among COPD patients during acute exacerbations compared to control subjects¹⁴. This increase was associated with a higher mortality risk and exhibited negative correlations with several pulmonary function parameters, including forced expiratory volume in 1 s (FEV₁), forced vital capacity (FVC), the FEV₁/FVC ratio, diffusing capacity of the lung for carbon monoxide (DLCO), and arterial oxygen tension (PaO₂)¹⁴. Furthermore, they also demonstrated that fibrocyte numbers were elevated in both distal and proximal lung tissue specimens from COPD patients¹⁵. Fibrocyte density was inversely correlated with lung function measures and positively correlated with bronchial wall thickness as quantified by computed tomography (CT)¹⁵. In the present study, although the total number of circulating fibrocytes was not significantly elevated in COPD patients compared with normal controls (Fig. 1), we demonstrated that tissue fibrocytes are present in multiple regions of the lungs in both COPD patients and murine models, including airway walls, alveolar walls, and airway/alveolar spaces (Figs. 2 and 5). These cells were sometimes observed to aggregate into clusters, and in their active form (α-SMA positive). Their numbers in lung tissues were significantly increased in COPD patients and mice relative to normal subjects (Figs. 2 and 5). Taken together, these studies indicate that fibrocytes are elevated in the context of COPD and, as one of the cellular sources of collagen, may contribute to airway remodeling and fibrosis.

The mechanism by which circulating fibrocytes migrate to lung tissues in COPD remains incompletely understood. Phillips et al. demonstrated that CD45⁺Collagen I⁺ circulating fibrocytes express CXCR4 and are capable of migrating toward CXCL12, trafficking to the lungs in a murine model of bleomycin-induced pulmonary fibrosis²³. Treatment of bleomycin-exposed animals with specific neutralizing anti-CXCL12 antibodies inhibited the intrapulmonary recruitment of CD45⁺Collagen I⁺CXCR4⁺ circulating fibrocytes and attenuated lung fibrosis²³. Shipe et al. reported that an increase in CD45⁺Collagen I⁺CXCR4⁺ fibrocytes was primarily observed in patients with severe asthma compared to those with milder asthma¹¹. Dupin et al. further indicated that fibrocytes highly express CXCR4 and CCR3, the chemokine receptors for CXCL12 and CCL11, respectively¹⁴. Fibrocytes isolated from patients with COPD exhibited enhanced chemotactic migration in response to CXCL12 but not to CCL11, relative to those from control subjects. Plerixafor, a CXCR4 antagonist, reduced fibrocyte migration toward plasma from patients experiencing COPD exacerbation¹⁴. In the present study, we also confirmed that a subset of CD45⁺Collagen I⁺ fibrocytes are CXCR4-positive, and that CD45⁺Collagen I⁺CXCR4⁺ fibrocytes are significantly increased in lung tissues from COPD patients and in a murine model (Figs. 4 and 7). Collectively, these findings support the involvement of the CXCL12/CXCR4 axis in the recruitment and accumulation of fibrocytes in lung tissues.

Type 2 inflammation, typically linked to asthma, has also been observed in COPD patients with no prior history of asthma, and is considered to represent a distinct endotype of COPD²⁴. Recent studies commonly propose that epithelium-derived cytokines—specifically IL-25, IL-33, and TSLP—act as key regulatory factors connecting immune-pathogenic mechanisms in allergic rhinitis (AR), chronic rhinosinusitis (CRS), and asthma. These cytokines are recognized as important regulators of type 2 immune responses and serve as critical links between innate and adaptive immunity¹⁶. Previous studies have demonstrated that airway levels of IL-25, IL-33, and TSLP are elevated in patients with asthma compared to healthy individuals and correlate with disease severity^25,26. Moreover, our prior investigations indicate that chronic airway exposure to either IL-25 or IL-33 alone is sufficient to elicit allergen- and IgE-independent asthma-like airway inflammation, remodeling, and hyperresponsiveness in mice^27–29. The cellular targets of IL-25, IL-33, and TSLP are diverse, encompassing dendritic cells, T-helper type 2 (Th2) cells, group 2 innate lymphoid cells (ILC2s), eosinophils, macrophages, and fibroblasts³⁰. In our recently published study⁹, we demonstrated that bronchial epithelial cells (BECs) and fibrocytes represent significant cellular targets of IL-25. Upon IL-25 stimulation, BECs displayed phenotypic alterations consistent with epithelial-mesenchymal transition (EMT), while fibrocytes exposed to IL-25 secreted elevated levels of extracellular matrix (ECM) proteins and connective tissue growth factor (CTGF). Previous studies have also demonstrated that IL-33 and TSLP can promote the recruitment, migration, proliferation, activation, and cytokine expression of fibrocytes in both in vitro and in vivo models^17–20. In the present study, we identified distinct subpopulations of circulating and tissue fibrocytes that express receptors for IL-25, IL-33, and TSLP. Fibrocytes expressing the receptors for IL-25, IL-33, and TSLP are significantly increased in the lung tissue of COPD patients and murine models compared with their respective controls. These findings suggest that, in the context of COPD, IL-25, IL-33, and TSLP may contribute to airway inflammation, fibrosis, and remodeling by inducing a pro-fibrotic phenotypic shift in fibrocytes. In other words, fibrocytes may become activated, produce excessive ECM proteins, and thereby contribute to airway fibrosis and remodeling within the epithelial-derived cytokine (IL-25, IL-33, and TSLP) milieu.

Our study has several limitations. First, the sample size for detecting circulating and tissue fibrocytes was modest. Thus, whether fibrocytes are increased in patients with COPD requires further validation in larger cohorts. Additionally, the heterogeneity of COPD should be considered, as the disease encompasses distinct phenotypes based on clinical, radiographic, and physiologic characteristics, as well as molecular and immune features^31,32. Secondly, experiments in this COPD mouse model revealed the accumulation of receptor‑positive fibrocyte subsets in the lung. However, these findings do not distinguish whether cigarette smoke directly induces receptor expression on fibrocytes or preferentially recruits pre‑existing receptor‑positive populations into the lung tissue. Thirdly, the present study did not establish whether elevated levels of total fibrocytes or those expressing IL-25, IL-33, and TSLP contribute to the pathogenesis of COPD. Further investigation using cell-based or animal models targeting the IL-25/IL-33/TSLP–fibrocyte axis is warranted. Despite these limitations, our findings demonstrate that fibrocytes—as key cellular sources of extracellular matrix proteins—can migrate into the lung and may promote airway fibrosis and remodeling in COPD patients within a pro-fibrotic epithelial-derived cytokine milieu.

Conclusions

In conclusion, this study reveals elevated fibrocyte levels in both COPD patients and murine models. The frequencies of fibrocytes expressing IL-17RA/IL-17RB, ST2L, TSLPR, CXCR4, and α-SMA were also markedly increased in COPD. CXCL12, together with IL-25, IL-33, and TSLP, may promote fibrocyte recruitment and activation. Further studies are needed to clarify the role of the IL-25/IL-33/TSLP–fibrocyte axis in COPD pathogenesis.

Supplementary Information

Below is the link to the electronic supplementary material.

Supplementary Material 1 ^{(970.6KB, docx)}

Abbreviations

AR

Allergic rhinitis

COPD

Chronic obstructive pulmonary disease

CRS

Chronic rhinosinusitis

CFs

Circulating fibrocytes

CTGF

Connective tissue growth factor

EMT

Epithelial-mesenchymal transition

ECM

Extracellular matrix

FEV₁

Forced expiratory volume in 1 s

FVC

Forced vital capacity

ILC2s

Innate lymphoid cells

PBMCs

Peripheral blood mononuclear cells

α-SMA

Smooth muscle actin α

TSLP

Thymic stromal lymphopoietin

Author contributions

Conception: LX, ZL, YX. Design: LX, ZL, YX. Data collection and analysis: YX, CQ. Drafting the manuscript: YX, CQ. interpretation: LX, ZL. All authors read and approved the final manuscript.

Funding

This study was financially supported by the following programs and organizations: the Natural Science Foundation of Beijing (Grant No. 7212018); the Beijing Municipal Hospital Management Center ‘Green Seedlings Program’ (Grant No. QML20230208); the Young Elite Talents Cultivation Program of Beijing Tongren Hospital, Capital Medical University (Grant NO. 2020-YXJ-ZZL-007).

Data availability

The datasets generated and/or analysed during the current study are available from the corresponding author on reasonable request.

Declarations

Competing interests

The authors declare no competing interests.

Ethics approval and consent to participate

Written informed consent was obtained from all participants after a comprehensive explanation of the study procedures. The research protocol was approved by the Ethics Committee of Beijing Tongren Hospital, Capital Medical University.