RANKL promotes airway inflammation via pro-inflammatory alveolar macrophage activation in COPD

Abstract

Background

Chronic airway inflammation is a key mechanism involved in the pathogenesis and progression of chronic obstructive pulmonary disease (COPD). Altered activation states of alveolar macrophages (AMs) and the release of inflammatory cytokines represent critical phenotypes in this inflammatory process. Although RANKL is known to participate in immune regulation and cytokine secretion, its potential involvement in pro-inflammatory activation of AMs and the associated contribution to airway inflammation in COPD remains incompletely understood. The purpose of this study is to investigate the RANKL pathway in COPD and to elucidate its role in AM activation and airway inflammation.

Methods

First, we quantified and localized RANKL and its receptor RANK in lung tissues and assessed CD86-associated pro-inflammatory macrophage activation in bronchoalveolar lavage fluid from COPD patients, smokers, and non-smokers. Next, wild-type mice were exposed to either air or cigarette smoke (CS) for 24 weeks. CS-exposed mice received intraperitoneal injections of either an anti-mouse RANKL monoclonal antibody or a rat IgG2a kappa isotype control antibody; macrophage activation status and airway inflammation were subsequently evaluated. Finally, we investigated the in vitro biological function of RANKL in CS-induced pro-inflammatory macrophage activation and airway inflammation.

Results

We found that the expression of both RANKL and RANK, along with enhanced CD86-associated pro-inflammatory macrophage activation, was increased in the lung tissues of COPD patients. In these tissues, RANKL and RANK were localized to AMs. In CS-exposed mice, pro-inflammatory macrophage activation and airway inflammation were significantly increased; however, these effects were ameliorated in CS-exposed mice treated with the anti-RANKL monoclonal antibody. In vitro, cigarette smoke extract (CSE) up-regulated the expression of RANKL and RANK in AMs. AMs responded to CSE and RANKL stimulation by exhibiting pro-inflammatory activation and enhanced cytokine expression. Furthermore, CSE-induced pro-inflammatory macrophage activation and cytokine expression were partially inhibited by the addition of a neutralizing anti-RANKL monoclonal antibody.

Conclusion

RANKL contributes to airway inflammation through pro-inflammatory alveolar macrophage activation in COPD. These findings extend current understanding of the role of the RANKL pathway in airway inflammation and highlight it as a potential therapeutic target in COPD.

Introduction

Chronic obstructive pulmonary disease (COPD) is a complex chronic airway condition characterized by persistent airway inflammation and emphysema. The sharp decline in lung function observed in approximately half of all COPD patients is linked to chronic airway inflammation [1]. This evidence suggests that the development of airway inflammation plays a critical role in the onset and progression of COPD.

Alveolar macrophages (AMs) play a critical role in the regulation of inflammation and in maintaining lung homeostasis in COPD [2]. One study found that the number of macrophages increases by 5 to 10 times in the airways, pulmonary interstitium, bronchoalveolar lavage fluid (BALF), and sputum of COPD patients [3]. Additionally, AMs exhibit functional abnormalities, such as increased oxidative stress and apoptotic cells, as well as decreased phagocytosis [4]. Macrophages are traditionally classified into classically activated (M1) and alternatively activated (M2) phenotypes. M1 macrophages are induced by lipopolysaccharide (LPS) and interferon-γ (IFN-γ), and produce reactive oxygen species (ROS), inducible nitric oxide synthase (iNOS), and cytokines such as tumor necrosis factor-α (TNF-α), IFN-γ, interleukin-1β (IL-1β), and interleukin-6 (IL-6), thereby exerting pro-inflammatory effects and regulating Th1 immune responses [5, 6]. M2 macrophages are induced by interleukin-4 (IL-4), interleukin-10 (IL-10), and interleukin-13 (IL-13), and secrete IL-10 and transforming growth factor-β (TGF-β), which are involved in immunoregulatory and tissue remodeling processes [5, 7]. Although studies have indicated that both M1-polarized and M2-polarized AMs increase significantly in smokers and COPD patients [8], growing evidence indicates that COPD lung macrophages frequently exhibit non-classical, mixed, or incompletely polarized activation states. Such heterogeneous activation states, particularly those biased toward pro-inflammatory responses, are thought to contribute to chronic airway inflammation in COPD, although the precise regulatory mechanisms remain to be fully defined.

Receptor activator of nuclear factor-κB ligand (RANKL) was initially identified for its key roles in immune regulation and later in bone metabolism [9–12]. In our previous studies, we demonstrated that cigarette smoke (CS) exposure induced the expression of RANKL and its receptor RANK, alongside an increase in MMP-9 in lung tissues, which may contribute to the pathogenesis of emphysema [13]. We also found that the RANKL pathway plays a potentially important role in the lung, involved in CS-induced lymphoid neogenesis in COPD [14]. Studies on COPD complications have shown that RANKL is associated with bone loss and muscle atrophy [15, 16]. Additionally, RANKL has been implicated in cytokine production [17, 18] and macrophage activation and inflammatory responses [19]. However, whether RANKL participates in CS-induced pro-inflammatory activation of AMs and airway inflammation has not been investigated.

To investigate whether CS-induced pro-inflammatory activation of AMs and airway inflammation are related to RANKL, we first examined the correlation between the expression of RANKL, its receptor RANK, and macrophage activation status in COPD patients. We found that both RANKL and RANK were up-regulated, accompanied by enhanced CD86-associated pro-inflammatory macrophage activation. Consequently, we employed a long-term CS exposure mouse model with RANKL neutralization. To our knowledge, this study provides further evidence that both pro-inflammatory macrophage activation and airway inflammation were reduced in CS-exposed mice following RANKL neutralization. In an in vitro culture of AMs, cigarette smoke extract (CSE) up-regulated the expression of RANKL and RANK. The cells responded to CSE or RANKL stimulation with enhanced cytokine production and increased pro-inflammatory macrophage activation, both of which were inhibited by a neutralizing monoclonal anti-RANKL antibody. Together, these findings suggest that the RANKL pathway participates in CS exposure-induced pro-inflammatory macrophage activation and airway inflammation, identifying it as a potential therapeutic target for managing airway inflammation in COPD.

Methods

Human study subjects

Patients undergoing surgery for solitary lung tumors at The First Affiliated Hospital of Zhengzhou University were enrolled in this study. Prior to surgery, all patients received bronchoscopy. Lung resection samples were obtained from three groups: smokers with COPD, smokers with normal lung function, and never-smokers with normal lung function. A pathologist collected lung tissues from sites at the maximum distance from the pulmonary lesions, obtained at least 5 cm away from the tumor margin, ensuring no signs of retro-obstructive pneumonia or tumor invasion [20–22]. The subjects’ characteristics are presented in Table 1. The study was approved by the Ethics Committee of the First Affiliated Hospital of Zhengzhou University, and written informed consent was obtained from all participants.

Table 1.

Characteristics of the study population

	Never-smokers	Smoker	COPD					P Value (ANOVA)
			GOLD I	GOLDII	GOLDIII		GOLDIV
Subjects (n)	6	12	4	8	1	0
Male / Female (n)	1/5	11/1	12/1					< 0.001*
Age (years)	50.67 ± 2.56	58.00 ± 2.20	57.38 ± 2.23					NS
Body mass index	24.63 ± 2.31	25.59 ± 1.04	23.66 ± 0.84					NS
Smoker/ex-smoker	N/A	10/2	6/5
Pack-years	N/A	43.46 ± 9.25	44.62 ± 8.94					NS#
FEV1, % predicted	94.58 ± 6.86	87.78 ± 4.24	71.52 ± 4.50					0.020
FEV1/FVC	82.43 ± 1.91	78.17 ± 1.64	61.59 ± 1.76					< 0.001

ANOVA analysis of variance, COPD chronic obstructive pulmonary disease, GOLD Global Initiative for Chronic Obstructive Lung Disease, N/A not applicable, NS not significant

Values are mean ± SEM

*Categorical variables were analyzed with Chi-Square tests

#is used Student’s t test

Animals and experimental design

Six- to eight-week-old female C57BL/6 mice were supplied by Beijing Vital River Laboratory and bred in-house. Food and water were provided ad libitum. All mice were housed under a 12-hour light-dark cycle in specific pathogen-free conditions. All in vivo procedures were approved by the Ethics Committee of the First Affiliated Hospital of Zhengzhou University. After one week of acclimation, the mice were randomly divided into two main cohorts: a room air group (n = 12) and a CS exposure group (n = 36). The CS group was subsequently further divided into three sub-groups: a CS-only group (n = 12), a CS group with intraperitoneal injection of a monoclonal anti-RANKL antibody (n = 12), and a CS group with intraperitoneal injection of a rat IgG2a kappa isotype control antibody (n = 12). For RANKL neutralization, the dosage and administration regimen were based on previous studies [16, 23], CS-exposed mice received an intraperitoneal injection of either an anti-mouse RANKL monoclonal antibody (100 µg/mouse, IK22-5, BioXcell) or a rat IgG2a kappa isotype control antibody (100 µg/mouse, 2A3, BioXCell) twice per week from week 1 to week 24 during the CS exposure period. Mice in the CS group were placed in an in-house designed glass chamber (100 × 100 × 100 cm) for 24 weeks to introduce cigarette smoke exposure (10 cigarettes/hour, 1 h/session, 2 sessions/day, 6 days/week). The cigarette type used was Hongqiqu with filter, produced by Henan Cigarette Industry (tar: 11 mg, nicotine: 0.9 mg, CO: 11 mg). At the endpoint, mice were anesthetized with an intraperitoneal injection of 1% pentobarbital sodium (70 mg/kg; Sigma-Aldrich). Bronchoalveolar lavage (BAL) was then performed: 1 ml of cold PBS was injected via a tracheal cannula and the lavage sample was collected. The mice were subsequently euthanized by exsanguination. The left lung was collected for histopathological analysis, and the right lung was stored at -80 °C for real-time quantitative PCR (RT-qPCR) analysis.

Immunohistochemistry and Immunofluorescence

Lung tissues obtained from humans and mice were fixed by immersion in formalin and embedded in paraffin. The tissues were sectioned into 5 μm thick slices and mounted on glass slides. After dewaxing and hydration, the sections were incubated in 0.3% hydrogen peroxide for 15 min and then subjected to antigen retrieval by heating in 5mM citrate buffer (pH 6.0) using a microwave oven. Subsequently, the sections were blocked with goat serum (ZSGB-Bio, Beijing, China) and incubated overnight with primary antibodies against RANKL (Abcam, Cambridge, UK) and RANK (Abcam). Following this, the sections were incubated for 30 min with a horseradish peroxidase (HRP)-conjugated goat anti-mouse IgG secondary antibody (ZSGB-Bio). Immunoreactivity was visualized using a DAB detection kit (ZSGB-Bio), and the sections were counterstained with Mayer’s hematoxylin. For quantitative analysis, the number of RANKL- or RANK-positive cells was counted and expressed as positive cells per mm². Quantification was performed in a blinded manner; 20 randomly selected non-overlapping fields per sample were analyzed at 400× magnification, and positive cells were counted to calculate cell density (cells/mm²). Negative controls, used to assess nonspecific binding, were processed by omitting the primary antibody.

Confocal microscopy was used to evaluate the co-expression of RANKL with the AMs markers CD68 (human). A similar protocol was applied to assess the expression of RANK (Abcam) alongside the same macrophage markers. Briefly, after dewaxing and hydration, tissue sections were subjected to antigen retrieval by heating in 5 mM citrate buffer (pH 6.0) using a microwave oven for 15 min. Subsequently, the sections were blocked with goat serum (ZSGB-Bio) and incubated overnight with primary antibodies. Following this, the sections were incubated for 30 min at 37 °C with secondary antibodies, including goat anti-rabbit IgG conjugated to Alexa Fluor 488 and goat anti-mouse IgG conjugated to Alexa Fluor 594 (Jackson ImmunoResearch, West Grove, PA, USA). All stained slides were stored at 4 °C and analyzed within 24 h. Negative controls for nonspecific binding were processed by omitting the primary antibody. Immunofluorescence was evaluated using a confocal microscope (TCS SP8; Leica Microsystems, Wetzlar, Germany).

Cell culture

The mouse alveolar macrophage cell line MH-S was purchased from Wuhan Servicebio Technology (Wuhan, Hubei, China). MH-S cells were cultured in RPMI-1640 medium (Thermo Fisher Scientific, Waltham, MA, USA) supplemented with 10% fetal bovine serum (FBS) and 1% penicillin/streptomycin (both from Thermo Fisher Scientific). The cells were maintained at 37 °C in a humidified incubator with 5% CO₂.

CSE Preparation

CSE was prepared by bubbling smoke from five commercial cigarettes (Hongqiqu with filter; tar: 11 mg, nicotine: 0.9 mg, CO: 11 mg) through 10 ml of serum-free cell culture medium at a constant flow rate. Each cigarette was smoked for 5 min. The resulting solution was then sterile-filtered through a 0.22 μm membrane, which was designated as the 100% CSE stock solution [24]. Cell proliferation was assessed under treatment with various CSE concentrations (0.5%, 1%, 2%, 4%) using a Cell Counting Kit-8 (CCK-8).

Cell stimulation

AMs were stimulated with CSE at 37 °C under 5% CO₂ for 48 h to assess the expression of RANKL and RANK. To evaluate cytokine expression and pro-inflammatory macrophage activation, cells were stimulated for 48 h under the same conditions with one of the following: CSE, recombinant RANKL (100 ng/mL; R&D Systems, Minneapolis, MN, USA), anti-RANKL monoclonal antibody (10 µg/mL; BioLegend, San Diego, CA, USA), or a rat IgG2a kappa isotype control antibody (10 µg/mL; BioLegend).

Flow cytometry

Human BAL samples were obtained using a standardized protocol for fiberoptic bronchoscopy and filtered through nylon gauze [25]. The protocol for obtaining BAL samples from mice is described in the “Animals and Experimental Design” section. After centrifugation at 400×g for 10 min at 4 °C, the cells were resuspended in fluorescence activated cell sorting (FACS) buffer (PBS supplemented with 0.5% bovine serum albumin and 2 mM EDTA). Cells were stained with fluorochrome-conjugated monoclonal antibodies to detect surface markers. The following antibodies were used for human BAL samples: anti-CD68-FITC and anti-CD86-PE(both from eBiosciences, San Diego, CA, USA). The following antibodies were used for mouse BAL samples: anti-F4/80-APC and anti-CD86-FITC (both from eBiosciences). At the end of the cell culture period, cells in 12-well plates were harvested and incubated with the following fluorescence-conjugated antibodies: anti-mouse RANKL-PE, anti-mouse RANK-APC, rat IgG2a κ-PE and rat IgG2a κ-APC (all from BioLegend, CA, USA). Data acquisition was performed on a BD FACS Calibur flow cytometer, with at least 10,000 events counted per sample.

Real-Time quantitative PCR

Total RNA was extracted from the lung tissues of humans and mice, as well as from cultured cells, using TRIZOL reagent (Thermo Fisher Scientific) according to the manufacturer’s instructions. Subsequently, 1 µg of isolated mRNA was reverse-transcribed into complementary DNA (cDNA) using a reverse transcription system with oligo(dT) primers (Promega, Madison, WI, USA).

RT-qPCR was used to determine the expression levels of specific genes. In human and mouse lung tissues, we analyzed RANKL and RANK. In mouse lung tissues and MH-S cells, we measured the expression of pro-inflammatory cytokines, including IL-1β, IL-6, IFN-γ, and TNF-α. RT-qPCR was performed using a Bio-Rad CFX system with a 20µL reaction mixture and the SYBR Green One-Step qRT-PCR Kit (Tiangen, Beijing, China).

The primer sequences used were as Table 2. The thermal cycling conditions were as follows: an initial step at 95 °C for 15 min, followed by 40 cycles of 95 °C for 10 s and 60 °C for 32 s. The results were analyzed using the comparative Ct (2^(-ΔΔCt)) method and are presented as fold changes normalized to the GAPDH reference gene.

Table 2.

The primer sequences that was used in the RT-qPCR analysis

Gene Name	Forward	Reverse
GAPDH(human)	5′-GCACCGTCAAGGCTGAGAAC-3′	5′-TGGTGAAGACGCCAGTGGA-3′
RANK(human)	5′-AGGATCCGTTGGTGGGAATTTA-3′	5′-CAGTGAGATGACAGTCACAGCAGAA-3′
RANKL(human)	5′-TGGATGCCTTGAATAATAAGCAGGA-3′	5′-AATTTGCGGCACTTGTGGAA-3′
GAPDH(mouse)	5′-GCAAATTCAACGGCACAGTCAAG-3′	5′-TCGCTCCTGGAAGATGGTGATG-3′
RANK(mouse)	5’-TGAGCCTCCGAGCAGAACTGAC-3′	5′-CTGCCTGTGTAGCCATCTGTTGAG-3′
RANKL(mouse)	5’-ATGGAAGGCTCATGGTTGGATGTG-3′	5′-TGGCAGCATTGATGGTGAGGTG-3′
TNF-α(mouse)	5’-GCCTCTTCTCATTCCTGCTTGTGG-3′	5′-GTGGTTTGTGAGTGTGAGGGTCTG-3′
IFN-γ(mouse)	5’-CTGGAGGAACTGGCAAAAGGATGG-3′	5′-GACGCTTATGTTGTTGCTGATGGC-3′
IL-6(mouse)	5’-CTTCTTGGGACTGATGCTGGTGAC-3′	5′-AGGTCTGTTGGGAGTGGTATCCTC-3′
IL-1β(mouse)	5’-TCGCAGCAGCACATCAACAAGAG-3′	5′- AGGTCCACGGGAAAGACACAGG-3′

Cytokine level quantification

Cytokine levels (IL-1β, IL-6, IFN-γ, TNF-α) were measured in samples of mouse BAL fluid, serum, and cell culture supernatant. Mouse BAL fluid and serum were analyzed via a bead-based flow cytometry assay (ABplex Mouse 6-Plex Custom Panel, ABclonal), while the cell culture supernatant was assessed using ELISA (Neobioscience, Shenzhen, China).

Statistical analysis

Statistical analyses were performed using SPSS 20.0 (IBM, Chicago, IL, USA). Data are expressed as the mean ± standard error of the mean (SEM). Comparisons between two groups were conducted using Student’s t-test. For comparisons among three or more groups, one-way analysis of variance (ANOVA) was employed, followed by Bonferroni post hoc tests (when equal variances were assumed) or Dunnett’s T3 tests (when equal variances were not assumed). Categorical variables were analyzed using the Chi-squared test. A P value of less than 0.05 was considered statistically significant.

Results

Increased expression of RANKL and RANK in the lung tissues of COPD patients

In our previous studies, RANKL and RANK were found increased expression and involved in lymphoid follicles. To examine expressions of RANKL and RANK in COPD, we performed immunostaining and RT-qPCR of lung tissues from COPD patients, smokers and non-smokers with normal lung function. First, lung tissues from COPD patients exhibited more severe airway inflammation than those from smokers or never-smokers with normal lung function (Fig. 1A-C). Furthermore, immunohistochemical analysis revealed the presence of RANKL in the cytoplasm of cells within the small airways, bronchiolar walls, and lung parenchyma in the COPD group compared to the never-smoker and smoker groups (Fig. 1D-F). A similar expression pattern was observed for RANK (Fig. 1G-I). To support these qualitative observations, we quantified RANKL⁺ and RANK⁺ cells in lung sections and found that both RANKL⁺ cell density (Fig. 1J) and RANK⁺ cell density (Fig. 1L) were significantly increased in COPD patients compared to never-smokers and smokers without airway obstruction (P < 0.05).

Fig. 1.

Increased expression of RANKL and RANK in COPD. Representative lung histology from (A) a smoker with COPD, (B) a smoker without COPD, and (C) a never-smoker, shown by H&E staining. Scale bar = 250 μm. Immunohistochemical staining for RANKL in lung tissues from (D) a smoker with COPD, (E) a smoker without COPD, and (F) a never-smoker. Scale bar = 100 μm. Immunohistochemical staining for RANK in lung tissues from (G) smoker with COPD, (H) smoker without COPD, and (I) never-smoker. Scale bar = 25 μm. Positive immunoreactivity is indicated by brown DAB chromogen; nuclei are counterstained with Mayer’s hematoxylin (blue). Quantification of RANKL⁺ (J) and RANK⁺ (K) cells in human lung tissues, expressed as positive cells per mm². L RANKL and (M) RANK mRNA levels in total human lung tissues (n = 6–13). Data are presented as mean ± SEM. *P < 0.05, **P < 0.01

We next analyzed and quantified the mRNA levels of RANKL and RANK in total lung tissues from human subjects. The mRNA levels of both RANKL (Fig. 1L) and RANK (Fig. 1M) were significantly elevated in COPD patients compared to never-smokers or smokers without airway obstruction. These results indicate that COPD is associated with increased pulmonary RANKL and RANK expression.

While RANKL cellular localization has been documented in various tissues and cells, its expression pattern in COPD remained to be clarified. To address this, we performed immunofluorescence co-staining for RANKL and specific cellular markers, with a focus on AMs. Interestingly, we found that RANKL-positive cells were identified as AMs in COPD subjects (Fig. 2A). We further examined the cellular localization of its receptor, RANK, in the lungs of COPD patients and confirmed its expression by AMs in human lung tissues (Fig. 2B).

Fig. 2.

Cellular localization of RANKL and RANK in the lungs of smokers with COPD. A Co-immunofluorescence staining for RANKL (red; Alexa Fluor 594) and the macrophage marker CD68 (green; Alexa Fluor 488). B Co-immunofluorescence staining for RANK (red; Alexa Fluor 594) and CD68 (green; Alexa Fluor 488). Nuclei were counterstained with DAPI (blue). Arrows indicate double-positive cells. Scale bars: 10 μm (A) and 50 μm (B)

Pro-inflammatory alveolar macrophage activation is increased in COPD patients

Previous studies have established a correlation between altered macrophage activation states and the development of COPD. To further investigate this, we analyzed pro-inflammatory macrophage activation in BAL fluid by performing surface staining for CD68 and CD86. Our results demonstrated that the proportion of CD68⁺CD86⁺ pro-inflammatory macrophages was significantly higher in COPD subjects than in never-smokers or smokers without airway obstruction (Fig. 3A-D).

Fig. 3.

Increased M1 macrophage polarization in smokers with COPD. Representative flow cytometry plots showing M1 macrophages from bronchoalveolar lavage (BAL) fluid in (A) a never-smoker, B a smoker without COPD, and C a smoker with COPD. D Quantitative analysis of the frequencies of M1 macrophages in the three study groups. Data are presented as mean ± SEM. ***P < 0.001

Cigarette smoke induces RANKL and RANK expression in mice lungs

We investigated the effect of CS on the expression of RANKL and RANK using C57BL/6 wild-type mice. After 24 weeks of CS exposure, increased expression of RANKL was evident in the CS-exposed mice compared to the air-exposed controls. Immunohistochemical staining confirmed that the expression of both RANKL (Fig. 4A-B) and RANK (Fig. 4C-D) was elevated in the lung tissues of CS-exposed mice. Consistent with this, RT-qPCR analysis revealed a significant increase in RANKL mRNA levels in the lungs of CS-exposed mice (Fig. 4E). Interestingly, a similar upregulation was also observed for RANK (Fig. 4F).

Fig. 4.

Upregulation of RANKL and RANK in CS-exposed mice. Immunohistochemical staining for RANKL in lung tissues from (A) air-exposed and B CS-exposed mice. Immunohistochemical staining for RANK in lung tissues from (C) air-exposed and (D) CS-exposed mice. Nuclei were counterstained with Mayer’s hematoxylin (blue). Scale bars: 50 μm (A-D). E RANKL and F RANK mRNA levels in total lung tissues from mice. Data are presented as mean ± SEM. *P < 0.05, ***P < 0.001

CS-Induced Pro-inflammatory macrophage activation in the lungs depends on the RANKL pathway

Given the established roles of RANKL and RANK in cytokine secretion and COPD pathogenesis, and their observed upregulation in COPD, we first sought to determine whether CS-induced pro-inflammatory macrophage activation and cytokines expression depend on RANKL signaling. To this end, we employed a RANKL-neutralization approach in which CS-exposed mice received intraperitoneal injections of either an anti-RANKL monoclonal antibody or a rat IgG2a kappa isotype control antibody. After 24 weeks of CS exposure, mice developed significant airway inflammation compared to the air-exposed controls (Fig. 5A-B). This inflammatory response was markedly attenuated in CS-exposed mice treated with the anti-RANKL antibody, relative to those receiving the isotype control (Fig. 5C-D). Furthermore, while CD86-associated pro-inflammatory macrophage activation was significantly enhanced in CS-exposed mice compared to controls (Fig. 5E-F), it was significantly reduced in the anti-RANKL treatment group compared to the isotype control group (Fig. 5G-I).

Fig. 5.

RANKL neutralization attenuates CS-induced M1 macrophage polarization. Representative lung histology from (A) the air-exposed group, B the CS-exposed group, C the CS-exposed group treated with anti-RANKL antibody, and D the CS-exposed group treated with an isotype control antibody, shown by H&E staining. Scale bar = 100 μm (A-D). E–H Flow cytometry plots of M1 macrophages from bronchoalveolar lavage (BAL) fluid in the corresponding groups. I Quantitative analysis of the frequencies of M1 macrophages across all groups. Data are presented as mean ± SEM. *P < 0.05, **P < 0.01, ***P < 0.001

CS exposure upregulates cytokine Expression, which is attenuated by Anti-RANKL treatment in mice

Furthermore, levels of the cytokines TNF-α, IL-6, IL-1β, and IFN-γ were quantified in BAL fluid and serum samples from mice using a flow cytometry-based bead immunoassay. The results demonstrated that the concentrations of these cytokines in BAL fluid were highest in the CS-exposed group. Notably, cytokine levels were significantly lower in the CS-exposed group treated with the anti-RANKL antibody than in both the CS-exposed alone group and the CS-exposed group treated with the isotype control antibody (Fig. 6A-D). A similar expression pattern was observed in serum samples (Fig. 6E-H). Consistent with the protein-level findings, mRNA expression of these cytokines in lung tissues, as detected by RT-qPCR, followed a comparable pattern (Fig. 6I-L).

Fig. 6.

Cytokine levels in bronchoalveolar lavage fluid, serum, and lung tissues of mice. Protein levels of (A) TNF-α, (B) IL-6, (C) IL-1β, and (D) IFN-γ were measured in bronchoalveolar lavage (BAL) fluid from the four experimental groups. Protein levels of (E) TNF-α, (F) IL-6, (G) IL-1β, and (H) IFN-γ were quantified in serum. mRNA levels of (I) TNF-α, (J) IL-6, (K) IL-1β, and (L) IFN-γ were determined in lung tissues. Data are presented as mean ± SEM. *P < 0.05, **P < 0.01, ***P < 0.001

CSE Induces Cytokine Expression and Pro-inflammatory Macrophage Activation in a RANKL-Dependent Manner In Vitro

The optimal stimulating concentration of CSE for MH-S cells, as determined by a CCK-8 assay, was identified to be 0.5%(Fig. 7A), and this concentration was used for all subsequent treatments. Our in vivo study demonstrated that CS stimulates AMs and upregulates the expression of RANKL and RANK. To confirm this in vitro, the MH-S alveolar macrophage cell line was treated with CSE. Flow cytometric analysis revealed a significant increase in the mean fluorescence intensity of RANKL(Fig. 7B-C) and RANK(Fig. 7D-E) compared to the control.

Fig. 7.

RANKL regulates M1 polarization and cytokine expression in vitro. A Viability of MH-S cells following 48-hour treatment with CSE, as assessed by CCK-8 assay. B, C Flow cytometric analysis of RANKL mean fluorescence intensity (MFI) in MH-S cells cultured in medium alone or stimulated with 0.5% CSE. D, E Flow cytometric analysis of RANK MFI under the same conditions. Histogram overlays: isotype control (black), medium control (orange), and CSE (blue). F Representative flow cytometry plots showing M1 polarization in MH-S cells treated with CSE, recombinant RANKL, or CSE combined with a neutralizing anti-RANKL antibody. Levels of (G) TNF-α, H IL-6, I IL-1β, and J IFN-γ in the culture supernatant of MH-S cells under the indicated treatments. mRNA levels of (K) TNF-α, L IL-6, and M IL-1β in MH-S cells under the same treatments. Data are presented as mean ± SEM. *P < 0.05, **P < 0.01, ***P < 0.001

Given that AMs are a major source of cytokines and that pro-inflammatory macrophage activation plays a critical role in COPD related airway inflammation, we first assessed pro-inflammatory macrophage activation by flow cytometry. We found that pro-inflammatory macrophage activation was enhanced following CSE stimulation (Fig. 7F). Concurrently, we measured cytokine levels (TNF-α, IL-6, IL-1β, and IFN-γ) in the cell supernatant and their mRNA expression in MH-S cells. Both protein (Fig. 7G-J) and mRNA levels (Fig. 7K-M) of the cytokines were significantly elevated after CSE treatment compared to the control.

To investigate the specific role of RANKL in this process, we stimulated alveolar macrophages with recombinant RANKL in vitro. This treatment similarly increased pro-inflammatory macrophage activation (Fig. 7F). Likewise, both cytokine protein secretion (Fig. 7G-J) and mRNA expression (Fig. 7K-M) were upregulated in RANKL stimulated MH-S cells compared to controls.

Since both CSE and RANKL promoted pro-inflammatory macrophage activation and cytokine expression, and given that CSE upregulates RANKL expression, we hypothesized that CSE exerts its effects, at least partially, through the RANKL pathway. To test this, we added an anti-RANKL monoclonal antibody to the culture medium of CSE stimulated AMs. Notably, the CSE induced cytokine expression (Fig. 7G-M) and pro-inflammatory macrophage activation (Fig. 7F) were partially inhibited. These results suggest that CS exposure induces airway inflammation partly by upregulating RANKL, supporting a role for the RANKL pathway in regulating pro-inflammatory activation of AMs.

Discussion

In this study, we demonstrate that pro-inflammatory macrophage activation in COPD patients is associated with increased expression of RANKL and its receptor RANK. We also demonstrated that RANKL blockade alleviated airway inflammation induced by long-term CS exposure in a well-established mouse model of COPD. Specifically, RANKL neutralization attenuated airway inflammation, reduced cytokine production in lung tissue and serum, and decreased CD86-associated pro-inflammatory macrophage activation in BAL fluid. In in vitro experiments using MH-S cells, CSE induced the expression of RANKL and RANK. Both CSE and RANKL stimulation enhanced pro-inflammatory macrophage activation and cytokine production in AMs. Importantly, these CSE-induced effects were partially blocked by an anti-RANKL antibody. Thus, our data support an important role for RANKL in regulating CS-induced airway inflammation, by promoting pro-inflammatory activation of alveolar macrophages, extending previous observations in COPD-related inflammatory processes.

The RANKL/RANK pathway was initially recognized for its key roles in immune regulation [9, 10] and was later identified in bone tissues as a critical mediator of bone homeostasis and remodeling [11, 12]. Subsequent studies found RANKL to be essential for lymph node development and lymphoid neogenesis in the small intestine [23, 26]. A very recent study revealed the involvement of RANKL and RANK in mammary/breast cancer metastasis [27]. Research on inflammatory mechanisms has shown that the RANKL/RANK axis plays a critical role in controlling inflammation in ischemic brains [28] and that RANKL induces the production of TNF-α, IL-1α, and IL-1β in myocardial inflammation [17]. A key finding demonstrated that RANKL activates monocytes/macrophages and regulates their function by inducing cytokine secretion [18]. The involvement of RANKL in COPD has recently garnered increasing attention. Previous studies reported elevated circulatory levels of RANKL in COPD patients, which were associated with bone loss [15, 29]. We were the first to demonstrate that long-term CS exposure induces the expression of RANKL and its receptor RANK in AMs within lung tissues, and that they play a critical role in the pathogenesis of emphysema in COPD [13]. We also found that the RANKL pathway is involved in lymphoid follicle formation [14] as well as in muscle atrophy and dysfunction in COPD [16]. Consistent with our previous studies, we confirmed that the expression of RANKL and RANK is increased in both COPD patients and a mouse model of COPD induced by long-term CS exposure. To elucidate the biological function of RANKL, we localized RANKL and its receptor RANK to AMs. Collectively, these findings support a contributory role for the RANKL–RANK pathway in COPD-related inflammatory processes by promoting pro-inflammatory activation of AMs.

In respiratory inflammation associated with COPD, various inflammatory cells—such as AMs, neutrophils, and T lymphocytes—are increased in lung tissue and secrete inflammatory mediators [4, 30, 31]. A prominent feature of COPD is macrophage activation [32]. Tissue-resident macrophages are derived from circulating monocytes, which originate from bone marrow hematopoietic stem cells [33, 34]. Macrophages play a critical role in regulating innate and acquired immunity, where they can potentiate inflammation and trigger immune responses. In patients with COPD, the number of macrophages is increased by 5 to 10 fold in the airways, lung parenchyma, and BAL fluid compared to healthy individuals, and this increase correlates with disease severity [3, 4]. Macrophage polarization is traditionally classified into two major types: M1 and M2 [35, 36]. M1 macrophages, induced by LPS and IFN-γ, produce ROS, iNOS, and pro-inflammatory cytokines such as TNF-α, IFN-γ, IL-1β, and IL-6, thereby representing a prototypical pro-inflammatory macrophage activation state and contributing to Th1 immune responses [5, 6]. In contrast, M2 macrophages are induced by IL-4, IL-10, and IL-13, and secrete IL-10 and TGF-β, which are involved in immunoregulation and tissue remodeling [5, 7]. Previous studies have shown that the proportions of both M1 and M2 associated macrophage activation states are significantly increased in COPD patients, with macrophage activation biased toward pro-inflammatory features showing a strong association with disease severity [3, 37]. However, the specific role of RANKL in regulating CS-induced pro-inflammatory macrophage activation had not been thoroughly investigated. In our study, we confirmed that our results are consistent with these earlier findings and demonstrated that CS-induced pro-inflammatory macrophage activation is regulated, at least in part, by the RANKL pathway. This conclusion was supported by our key finding that CSE-induced CD86-associated pro-inflammatory activation in AMs was partially blocked by an anti-RANKL antibody, thereby confirming our hypothesis.

Airway and systemic inflammation are principal features of COPD and are closely related to disease progression and mortality [38, 39]. The inflammatory response in COPD involves the production of pro-inflammatory mediators and the activation of inflammatory cells [40]. Previous studies have demonstrated that cytokine levels, including those of IL-1β, IL-6, IL-8, and TNF-α, are elevated in the serum of COPD patients [41], indicating the involvement of systemic inflammation in triggering COPD-related inflammatory responses. Similarly, the levels of pro-inflammatory mediators such as IL-1β, IL-6, and TNF-α are increased in the BAL fluid of both COPD patients and CS-exposed mice [42]. Our in vivo experiments showed that CS-induced production of pro-inflammatory mediators and associated airway inflammation were reduced following RANKL neutralization. Furthermore, our in vitro study supports an important role for RANKL in contributing to this inflammatory response.

Several limitations of the present study should be noted. First, macrophage activation in COPD is heterogeneous, and although CD86-associated pro-inflammatory activation was used here to characterize inflammatory macrophage responses, additional markers, functional assays, or transcriptomic analyses would further refine macrophage phenotyping. In this context, the potential involvement of RANKL in M2-associated or alternative macrophage activation states was not addressed and warrants future investigation. Second, downstream signaling pathways of the RANKL/RANK axis were not directly examined, and mechanistic interpretations are therefore based on functional outcomes rather than defined intracellular signaling events. Third, although human lung tissues were obtained from regions distant from tumor margins, tumor-related field effects and structural lung abnormalities were not systematically quantified, and relevant clinical variables were not comprehensively captured, representing potential sources of residual confounding. Fourth, mechanistic in vitro experiments relied on the murine MH-S alveolar macrophage cell line, which does not fully recapitulate primary human alveolar macrophage biology. Finally, while inflammatory mediators were measured in BAL fluid, lung tissue, and serum, the present study focused primarily on airway and lung inflammation, and inter-compartmental regulatory relationships were not examined. Despite these limitations, the integrated human, in vivo, and in vitro data consistently support a contributory role for RANKL signaling in cigarette smoke–induced airway inflammation through pro-inflammatory activation of alveolar macrophages. Future studies will aim to employ lung- or macrophage-specific RANKL deletion strategies to further delineate the cell-specific contribution of RANKL signaling to airway inflammation and macrophage activation in COPD.

Conclusion

In conclusion, the present study demonstrates an increase in the expression of RANKL and its receptor RANK in the AMs of COPD patients, which is associated with enhanced pro-inflammatory macrophage activation. Furthermore, we show that CS induces cytokine expression and pro-inflammatory macrophage activation in a RANKL-dependent manner both in vivo and in vitro. Collectively, these data further support the involvement of the RANKL pathway in airway inflammation in COPD and highlight its potential as a therapeutic target.

Authors’ contributions

L.Z., B.Z., H.W., J.L. and L.M. conceived and designed the study; L.Z., B.Z., H.W. and J.L carried out the studies; L.Z.,B.Z., H.W. and L.M. acquired and analyzed the data; L.Z., B.Z., H.W., J.L. and L.M. drafted the manuscript; L.Z. and B.Z. edited the manuscript. All authors read and approved the final manuscript.

Funding

This work was supported by the National Natural Science Foundation of China Youth Fund Project (82100048) and the Medical Science and Technology Research Project of Henan Province (LHGJ20200363).

Data availability

Data are available from the corresponding author on reasonable request.

Declarations

Ethics approval and consent to participate

The study was approved by the Ethics Committee of the First Affiliated Hospital of Zhengzhou University. Written informed consent was obtained from all participants.

Consent for publication

Not applicable. All patient-level data were de-identified.

Competing interests

The authors declare no competing interests.