Increasing S1P promotes M1 macrophage in chronic obstructive pulmonary disease and chronic obstructive pulmonary disease-obstructive sleep apnea overlap syndrome via S1PR1/HDAC1 signaling

Introduction

Chronic obstructive pulmonary disease (COPD), characterized by irreversible airflow limitation, chronic airway inflammation, and progressive bronchial obstruction, is one of the leading causes of death worldwide (1). Obstructive sleep apnea (OSA) is a common clinical condition with partial or complete pharyngeal collapses occurring repeatedly during sleep, causing apneas and hypopneas, and leading to intermittent hypoxia and sleep disruption (2). OSA is highly prevalent in patients with moderate to severe COPD (3). Patients with overlap syndrome (OS), namely, the coexistence of COPD and OSA, exhibit dyspnea, cough, and sputum caused by airway and/or alveolar organic abnormalities, daytime hypoxemia and sleepiness, as well as snoring and apnea due to upper airway collapse or stenosis. COPD-OSA patients experience lower quality of life (4). Chronic inflammation caused by smoking or harmful particles is an important pathogenesis and clinical manifestation in COPD patients, characterized by increasing inflammation cells and cytokines. Macrophages are key immune effector cells in solid tissues, including lung. Evidence has demonstrated that macrophage immune response is involved in COPD progression (5-7). Macrophages could polarize into pro-inflammatory M1 subtype and anti-inflammatory M2 subtype. The balance of M1/M2 macrophages is critical for lung homeostasis. Accumulating evidence has indicated that modulation of M1/M2 balance could be a potential therapeutic approach for COPD (8,9). Notably, the change of macrophage polarization in COPD-OSA remains unclear. Moreover, the mechanism involved in M1/M2 imbalance in COPD requires further elucidation.

Sphingosine-1-phosphate (S1P), a sphingolipid metabolite synthesized by sphingosine kinase 1 (SphK1) or sphingosine kinase 2 (SphK2), plays crucial roles in cellular proliferation, angiogenesis, maturation, immune response, and lymphocyte trafficking (10-12). S1P receptor 1 (S1PR1) is a receptor for S1P. De Cunto et al.’s study showed that S1P signaling up-regulation follows the disease progression in a COPD mice model and is involved in the development of airway hyperresponsiveness (13). Evidence has shown that S1P signaling block could regulate pulmonary vascular permeability by preserving the integrity of endothelial barrier (14). Additionally, Chen’s study demonstrated that inhibition of S1P signaling could alleviate pulmonary inflammation in a smoking mice model (15). However, the change of S1P in COPD-OSA and its potential function during macrophage polarization needs further clarification.

In this study, we postulated that the S1P signaling is implicated in the inflammatory regulation in COPD and COPD-OSA overlap patients. Our research found higher M1 macrophage polarization tendency in COPD and COPD-OSA patients, and more significantly in COPD-OSA. Plasma S1P levels were increased in both COPD and COPD-OSA patients [vs. healthy controls (HCs)], and more significantly in COPD-OSA. Moreover, this study found that increasing S1P signaling could exert an influence on macrophage polarization via S1PR1/histone deacetylase 1 (HDAC1) pathway. These results provided the evidence of the function and potential mechanism of S1P signaling in COPD and COPD-OSA OS. We present this article in accordance with the MDAR reporting checklist (available at https://jtd.amegroups.com/article/view/10.21037/jtd-24-1719/rc).

Methods

COPD and COPD-OSA subjects

20 patients with COPD and 20 patients with COPD-OSA OS were recruited in this study. The diagnosis of COPD was based on the 2023 GOLD guidelines (16), which encompassed a post-bronchodilator use forced expiratory volume in 1 second (FEV1)/forced vital capacity (FVC) ratio ≤70%, excluding patients with concomitant structural or functional lung disease, asthma, or other evident respiratory disorders. OSA diagnosis was according to polysomnography (PSG), which showed an apnea-hypopnea index (AHI) ≥5 events per hour. Patients with COPD-OSA OS were required to fulfill simultaneously both the diagnostic criteria for COPD and OSA. The exclusion criteria were as follows: (I) existence of other respiratory diseases, such as bronchial asthma, tuberculosis, and lung cancer; (II) combined tumors; and (III) currently experiencing any infections. Sex- and age-matched healthy individuals (n=20) in the same period were recruited as the control group. All the peripheral blood and lavage fluid specimens of this project were derived from the patients who received medical treatment at the Second People’s Hospital of Shenzhen. The study was approved by the ethics committee of the Second People’s Hospital of Shenzhen (No. 20201208001). All patients included in this study provided written informed consent. The study was conducted in accordance with the Declaration of Helsinki (as revised in 2013) (17).

Peripheral blood, plasma, and bronchoalveolar lavage fluid (BALF) collection

Peripheral blood samples of COPD (male: n=2; female: n=1), COPD-OSA (male: n=2; female: n=1), and HC (male: n=1; female: n=2) were collected in the morning. The plasma was collected after centrifugation with 4,000 round per minute and stored at −80 ℃. BALF samples were collected according to the standard procedures (18).

Macrophage polarization

Peripheral blood mononuclear cells (PBMCs) were cultured with X-VIVO15 VIVO (Lonza, Walkersville, MD, USA; cat: 04-418Q) medium in a 5% CO₂ incubator at 37 ℃. After incubation for 3 hours, adherent cells were retained while non-adherent cells were gently removed. Then, the cells were used to prepare monocyte-derived macrophages (MDMs). Human monocyte cell line-THP-1 [human; American Type Culture Collection (ATCC), Manassas, VA, USA; BFN60700157] was cultured in Roswell Park Memorial Institute (RPMI)-1640 (Gibco, Waltham, MA, USA; cat: C11875500BT) supplemented with 10% fetal bovine serum (FBS; NEWZERUM, Christchurch, New Zealand; cat: FBS-CP500) in a 5% CO₂ incubator at 37 ℃.

To induce macrophage polarization into M1 phenotype, cells were treated with a combination of 50 ng/mL macrophage colony-stimulating factor (M-CSF; Sino Biological, Beijing, China, cat: 11792-HNAH), 100 ng/mL LPS (Sigma, St. Louis, MO, USA; cat: L2880) and 25 ng/mL interferon-γ (IFN-γ; Sino Biological; cat: 11725-HNAE) for 72 hours. For M2 polarization, cells were treated with 50 ng/mL interleukin-4 (IL-4; Sino Biological; cat: 11846-HNAE) for 72 hours to promote macrophage polarization towards M2 phenotype.

Extraction of PBMCs and induction of macrophage polarization

PBMCs were obtained using lymphocyte separation medium (TBD, Tianjin, China; cat: LTS1077) according to the operation manual. Cells in liquid layer were obtained and washed thrice with saline solution and resuspended in X-VIVO15 VIVO medium. After an incubation period of 3 hours, adherent cells were retained while non-adherent cells were gently removed.

To induce macrophage polarization towards M1 phenotype, we employed a combination of 50 ng/mL M-CSF, 100 ng/mL lipopolysaccharide (LPS), and 25 ng/mL IFN-γ for a duration of 72 hours. Additionally, we treated the cells with IL-4 at a concentration of 50 ng/mL for 72 hours to promote macrophage polarization towards M2 phenotype.

Cigarette smoke extract (CSE) preparation

The preparation method of CSE was as follows. Briefly, Yan’an cigarettes (10 mg tar, 0.8 mg flue-cured nicotine, 11 mg carbon monoxide) obtained from Yan’an Cigarette Factory of China Tobacco Shaanxi Industrial Co., Ltd. (Yan’an, China) were completely combusted after removing the filter tip. The smoke generated by burning six cigarettes was thoroughly reacted with 25 mL serum-free RPMI-1640, the optical density (OD) was refracted at 320 nm, and the absorbance was calibrated to 2.0 using a sterile medium in order to obtain 100% CSE. Subsequently, the resulting solution was filtered through a 0.22 µm filter.

COPD and COPD-OSA cell models

By using THP-1 cells, COPD and COPD-OSA cell models were constructed, as described previously (19). COPD cell models: THP-1 cells cultured in RPMI-1640 + 10% FBS, supplemented with 1% CSE, in a 5% CO₂ incubator at 37 ℃ for 24 hours. COPD-OSA cell models: THP-1 cells cultured in RPMI-1640 + 10% FBS, supplemented with 1% CSE, in an incubator with intermittent hypoxia conditions [cycles: 35 min normoxia and 25 min hypoxia (1% oxygen concentrations)].

Quantitative real-time polymerase chain reaction (qRT-PCR)

The total RNA from cells was extracted using TRIzol reagent (Invitrogen, Carlsbad, CA, USA; cat: 10296028) following the manufacturer’s protocol. Subsequently, purified RNA samples were reverse-transcribed using the PrimerScript regent kit (Takara, Shiga, Japan; cat: RR047A). qRT-PCR was conducted employing the Blastaq^TM 2× qPCR MasterMix (Applied Biological Materials, Vancouver, Canada; cat: G891). The relative expression levels of messenger RNA (mRNA) were normalized to glyceraldehyde 3-phosphate dehydrogenase (GAPDH) expression and calculated utilizing the 2^−ΔΔCt method. All the primer sequences were listed in Table 1.

Western blot

A total protein extraction kit (Solarbio, Beijing, China; cat: BC3790) was used to obtain total cellular proteins, following the manufacturer’s protocol. Then, cell protein was subjected to 10% sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE), followed by transfer of the bands to a polyvinylidene fluoride (PVDF) membrane using electrophoresis. The membrane was immersed in blocking buffer for approximately 1 hour, followed by overnight incubation at 4 ℃ with primary antibody against Actin (1:40,000; MedChemExpress, Shanghai, China; cat: HY-P80438), HDAC1 (1:1,000; Abcam, Cambridge, MA, USA; cat: ab109411) or S1PR1 (1:800; Affinity Biologicals, Ontario, Canada; cat: DF2785). Then, membranes were incubated with secondary antibody for 1 hour and visualized with an electrochemiluminescence (ECL) kit (4A Biotech Co., Ltd., Beijing, China; cat: 4AW011-500).

Flow cytometry (FCM) analysis

FCM was used to detect the ratio of M1 (CD14⁺CD86⁺) and M2 (CD14⁺CD206⁺) cells. The collected cells were incubated with fluorochrome-conjugated antibodies for 30 minutes at room temperature. Antibodies: anti-human CD14-phycoerythrin (PE) antibody (BioLegend, San Diego, CA, USA; cat: 301806); anti-human CD86-fluorescein isothiocyanate (FITC) (BioLegend; cat: 374204); anti-human CD206-FITC (BioLegend; cat: 321104). Then, cells were washed twice and resuspended with phosphate-buffered saline (PBS). Fluorescent signals were quantitated by flow cytometer (Aglient Novocyte, Hangzhou, China).

Cytokines detection

Cytokine levels were detected by multiple microsphere flow immunofluorescence assay, by using a multiple cytokine detection kit (RAISECARE, Qingdao, China; cat: 20200077). In brief, plasma and BALF were centrifuged at 3,000 rpm for 5 minutes. Then, 50 µL samples were incubated with microsphere labeled with multiple antibodies and biotin-labeled detected antibody for 2 hours, washed twice with buffer, PE-streptavidin was added, and incubated for 30 minutes. Fluorescent signals were quantitated by flow cytometer (Aglient Novocyte). The concentration of cytokines was calculated according to the standard curves.

Enzyme-linked immunosorbent assay (ELISA)

Plasma S1P levels were detected by ELISA, according to the manufacturer’s instructions (Yuannuo Technology Co., Ltd., Qingdao, China, cat: YMS9693-A). In brief, 50 µL plasma was added into the plate and incubated for 1 hour at 37 ℃. After being washed five times, horseradish peroxidase (HRP)-labeled detection antibody was added into plate and incubated for 1 hour at 37 ℃. Finally, the 3,3',5,5'-tetramethylbenzidine (TMB) substrate was added, and the OD (450 nm) was measured. S1P concentration was calculated according to the standard curve.

Statistical analysis

All data were obtained from three independent biological samples or replicates. The statistical analysis was performed using the R software (version 4.3.0; R Foundation for Statistical Computing, Vienna, Austria). Normally distributed continuous variables were presented as mean ± standard deviation (SD) and analyzed by Student’s t-test. Student’s t-test was employed to compare differences between two groups, whereas the one-way analysis of variance (ANOVA) was used for comparative analysis among multiple groups. Statistical significance was defined as P<0.05.

Results

Clinical characteristics in patients with COPD and COPD-OSA

In total, 20 patients with COPD and 20 patients with COPD-OSA OS were included in this study, and 20 age-matched HC were selected as the control group (Table 2). Compared with the control group, there were no significant differences in body mass index (BMI) in COPD and COPD-OSA patients. AHI and oxygen desaturation index (ODI) were significantly higher in COPD-OSA patients than in COPD and HC. The FVC, FEV1, FEV1%, and FEV1/FVC% were significantly lower in COPD and COPD-OSA patients than that in HC.

Higher M1 macrophage polarization in COPD and COPD-OSA patients

Previous research showed that macrophage polarization was involved in COPD progression. We investigated the M1 and M2 macrophage profile in BALF from COPD and COPD-OSA patients. Compared with HC, the ratio of CD14⁺CD86⁺ M1 macrophage was increased in COPD and COPD-OSA patients, whereas the ratio of CD14⁺CD206⁺ M2 macrophage was decreased (Figure 1A,1B). Notably, there were higher M1 macrophage ratio and lower M2 ratio in COPD-OSA than that in COPD. Additionally, we observed higher interleukin-6 (IL-6) and interleukin-8 (IL-8) levels in both COPD and COPD-OSA than those in HC (Figure 1C-1F).

Figure 1 Higher M1 polarization tendency and inflammation in COPD and OS. (A,B) The ratio of CD14⁺CD86⁺ M1 and CD14⁺CD206⁺ M2 macrophage in BALF from COPD, OS, and healthy subjects. (C,D) The levels of IL-6 and IL-8 in BALF from COPD, OS, and healthy subjects. (E,F) The plasma IL-6 and IL-8 levels in COPD, OS, and healthy subjects. (G,H) Detection of the M1 and M2 polarization efficiency of human MDM from COPD, OS, and healthy subjects in response to LPS/IFN-γ or IL-4 stimulation. (I,J) Detection of the M1 and M2 polarization efficiency of THP-1, COPD cell model (1% CSE treated THP-1 cells) and OS cell model (1% CSE and intermittent hypoxia-treated THP-1 cells) in response to LPS/IFN-γ or IL-4 stimulation. The Student’s t-test was employed to compare difference between two groups. *, P<0.05; **, P<0.01; ***, P<0.001. BALF, bronchoalveolar lavage fluid; COPD, chronic obstructive pulmonary disease; CSE, cigarette smoke extract; FITC, fluorescein isothiocyanate; IFN-γ, interferon-γ; IL-4, interleukin-4; IL-6, interleukin-6; IL-8, interleukin-8; LPS, lipopolysaccharide; MDM, monocyte-derived macrophage; OS, COPD-OSA overlap syndrome; OSA, obstructive sleep apnea; PE, phycoerythrin.

Furthermore, human MDM from HC, COPD, and COPD-OSA patients were prepared. Then, MDM were induced to polarized to M1 by LPS/IFN-γ and to M2 by IL-4. We observed a significant difference in MDM differentiation in their responses to the same polarization stimuli. Compared with MDM from HC, the MDM from COPD and COPD-OSA showed a higher tendency to M1 polarization, and a lower tendency to M2 polarization (Figure 1G,1H). Notably, flowing the same stimuli, MDM from COPD-OSA displayed more M1 and less M2 polarization than MDM from COPD (Figure 1G,1H). Moreover, analyses of differentiation of THP-1 under COPD (CSE treatment) and COPD-OSA (CSE + hypoxia treatment) conditions in response to polarization stimuli were also performed. Consistent with MDM, THP-1 under COPD and COPD-OSA conditions also showed a higher tendency to M1 polarization (vs. normal culture condition; Figure 1I,1J).

S1P was increased in plasma of COPD and COPD-OSA and promoted M1 polarization

Plasma S1P levels were detected by ELISA in HC, COPD and COPD-OSA patients. Compared with HC, plasma S1P levels were significantly increased in COPD and COPD-OSA patients. Notably, plasma S1P levels were higher in COPD-OSA patients than in COPD patients (Figure 2A). In HC (Figure S1A-S1G) and COPD patients (Figure S1H-S1N), there was no significant correlation between plasma S1P levels and AIH, ODI, FVC, FEV1, FEV1/FVC. In COPD-OSA patients, plasma S1P levels showed a significant positive correlation with AIH, FVC, FEV1, and FEV1% (Figure S1O-S1U). Notably, there was a positive correlation between plasma S1P levels and M1 cell rate (MDM responses to M1 polarization stimuli) (r=0.679; P=0.044) (Figure S2, left), yet a negative correlation between S1P levels and M2 rate (MDM responses to M2 polarization stimuli) (r=−0.700; P=0.043) (Figure S2, right).

Figure 2 S1P levels were increased in COPD and OS patients and promoted M1 polarization. (A) Plasma S1P levels in COPD, OS, and healthy subjects. (B) qRT-PCR showed the relative mRNA expression of S1P receptors in PBMC from COPD, OS, and healthy subjects. (C,D) qRT-PCR and western-blot showed the S1PR1 mRNA and protein expression in MDM from COPD, OS, and healthy subjects. (E,F) The effects of S1P and FTY720 treatment on S1PR1 mRNA and protein expression in THP-1, COPD, and OS cell models. (G) The effect of S1P and FTY720 treatment on M1 polarization of THP-1 in response to LPS/IFN-γ stimulation. (H) The effect of S1P and FTY720 treatment on IL-6 production of THP-1 cells. The Student’s t-test was employed to compare difference between two groups. *, P<0.05; **, P<0.01; ***, P<0.001; ns, not significant (P>0.05). COPD, chronic obstructive pulmonary disease; DMSO, dimethyl sulfoxide; IFN-γ, interferon-γ; IL-6, interleukin-6; LPS, lipopolysaccharide; MDM, monocyte-derived macrophage; mRNA, messenger RNA; OS, COPD-OSA overlap syndrome; OSA, obstructive sleep apnea; PBMC, peripheral blood mononuclear cell; qRT-PCR, quantitative real-time polymerase chain reaction; S1P, sphingosine-1-phosphate; S1PR1, S1P receptor 1; WT, wild type.

Furthermore, the expression levels of S1P receptors were investigated. The results showed that S1PR1 expression was significantly increased in COPD and COPD-OSA patients (vs. HCs; Figure 2B). Moreover, MDM from COPD and COPD-OSA showed higher S1PR1 expression levels than did MDM from HC (Figure 2C,2D). In vitro, we observed that S1P treatment could enhance the S1PR1 expression levels in THP-1 cells, CSE-treated THP-1 (COPD cell model), and CSE + hypoxia-treated THP-1 cells (COPD-OSA cell model), which could be reversed by FTY720 (an S1P antagonist) (Figure 2E,2F).

Furthermore, the effects of S1P on macrophage polarization were investigated. The results showed that S1P treatment could promote M1 polarization efficiency (Figure 2G). Correspondingly, the promoted effect of S1P treatment could be reversed by FTY720 (Figure 2G). The levels of IL-6 production by THP-1 were also enhanced by S1P treatment and inhibited by FTY720 (Figure 2H). Taken together, these data demonstrated that the increased levels of plasma S1P in COPD and COPD-OSA might enhance the M1 polarization tendency via S1PR1.

S1PR1/HDAC1 signaling mediates the influence of S1P in COPD and COPD-OSA

Firstly, to confirm the role of S1PR1 in macrophage polarization progress, S1PR1 overexpression cell models were constructed (Figure 3A,3B). Under M1 polarization stimuli, THP-1 cells with S1PR1 overexpression displayed higher M1 polarization efficiency (Figure 3C). Additionally, S1PR1-overexpressed THP-1 cells with either COPD (1% CSE treatment) or COPD-OSA (1% CSE + hypoxia treatment) conditioning showed the more higher M1 polarization efficiency (Figure 3C). Correspondingly, inducible nitric oxide synthase (iNOS) and IL-6 expression levels were increased in S1PR1-overexpressed cells in response to M1 polarization stimuli (Figure 3D,3E).

Figure 3 S1PR1 overexpression in THP-1 promote M1 polarization. (A,B) Identification of S1PR1 over-expressed THP-1 cell models. (C) The effect of S1PR1 overexpression on M1 polarization of THP-1, CSE treated, and CSE + hypoxia-treated THP-1 in response to LPS/IFN-γ stimulation. (D,E) The effects of S1PR1 overexpression on IL-6 and iNOS expression of THP-1, CSE treated and CSE + hypoxia-treated THP-1. The Student’s t-test was employed to compare difference between two groups. *, P<0.05; **, P<0.01; ***, P<0.001. CSE, cigarette smoke extract; IFN-γ, interferon-γ; IL-6, interleukin-6; iNOS, inducible nitric oxide synthase; LPS, lipopolysaccharide; mRNA, messenger RNA; S1P, sphingosine-1-phosphate; S1PR1, S1P receptor 1.

Previous research has shown that S1P could bind to HDAC1 and inhibit HDAC1 activity (20). In this study, we observed that HDAC1 expression levels were significantly decreased in PBMC from COPD and COPD-OSA patients (vs. HCs; Figure 4A). Moreover, MDM from COPD and COPD-OSA showed lower HDAC1 expression levels than MDM from HC (Figure 4B,4C). Notably, there might be a negative correlation between S1PR1 and HDAC1 expression (Figure 4C). S1P treatment significantly inhibited the HDAC1 expression in THP-1 cells, yet could be reversed by S1P antagonist-FTY720 (Figure 4D,4E). Furthermore, we investigated the effect of HDAC1 on macrophage polarization. By using HDAC1 overexpressed THP-1 cell model (Figure 4F,4G), we observed that high HDAC1 expression could inhibit the M1 polarization and the expression iNOS and IL-6 (Figure 4H-4J). Taken together, these data demonstrated that S1P could affect the macrophage polarization via activating SIPR1 or inhibiting HDAC1 signaling.

Figure 4 HDAC1 was decreased in COPD and OS patients. S1P treatment could inhibit HDAC1 expression in THP-1. (A) The mRNA expression levels of HDAC1 in PBMC from COPD, OS, and healthy subjects. (B,C) The mRNA and protein expression levels in MDM from COPD, OS, and healthy subjects. (D,E) The effect of S1P and FTY720 on HDAC1 expression in THP-1 cells. (F,G) Identification of HDAC1 over-expressed THP-1 cell models. (H) The effect of HDAC1 overexpression on M1 polarization of THP-1 in response to LPS/IFN-γ stimulation. (I,J) The effects of HDAC1 overexpression on IL-6 and iNOS expression of THP-1. The Student’s t-test was employed to compare difference between two groups. *, P<0.05; **, P<0.01; ***, P<0.001; ns, not significant (P>0.05). COPD, chronic obstructive pulmonary disease; DMSO, dimethyl sulfoxide; HDAC1, histone deacetylase 1; IFN-γ, interferon-γ; IL-6, interleukin-6; iNOS, inducible nitric oxide synthase; LPS, lipopolysaccharide; MDM, monocyte-derived macrophage; mRNA, messenger RNA; NC, negative control; OS, COPD-OSA overlap syndrome; OSA, obstructive sleep apnea; PBMC, peripheral blood mononuclear cell; S1P, sphingosine-1-phosphate; S1PR1, S1P receptor 1.

Discussion

COPD is a chronic respiratory disease characterized by persistent airway obstruction. The pathogenesis of COPD is complex and has remained unclear. Airway inflammation is a hallmark of COPD. Growing evidence has suggested that the imbalance of macrophage differentiation and subsequent excessive inflammatory response contribute to COPD development (21-24). Patients with COPD-OSA, a co-occurrence OS of COPD and OSA, experience more prolonged and severe events than those with either OSA or COPD alone, including more serious clinical symptoms and higher mortality rate (25).

In this study, we firstly investigated the status of macrophage polarization and inflammation in COPD and COPD-OSA patients. The results showed that the ratio of pro-inflammatory M1 macrophage was increased in BALF from COPD and COPD-OSA patients (vs. HCs), whereas anti-inflammatory M2 macrophage was decreased. Notably, compared with COPD alone, COPD-OSA showed more M1 macrophages and less M2 macrophages. Consistently, in response to the same macrophage polarization stimulus, human MDM from COPD and COPD-OSA tended to polarize into more M1 cells and less M2 cells than the MDM from HC, and this trend was more significant in MDM from COPD-OSA patients. Our results were also verified by using COPD and COPD-OSA cells models in vitro. Notably, IL-6 and IL-8 levels were increased in plasma and BALF from COPD and COPD-OSA patients (level: COPD-OSA > COPD > HC). Taken together, these data demonstrated the increased pro-inflammatory mediators in COPD patients, and more in COPD-OSA patients. Consistent with our study, Eapen et al.’s study reported increased M1 polarization in COPD patients (26). Moreover, macrophages could release large amounts of pro-inflammatory cytokines including IL-6 and IL-8 in COPD patients, thereby exacerbating inflammation and tissue damage (27). Inhibition of M1 polarization and pro-inflammatory cytokines may help to preserve airway epithelial homeostasis and improve clinical symptoms in COPD (7,28). Thus, because of the more significant increasing trend of M1 polarization and inflammation in COPD-OSA, potential useful therapies in COPD might also be helpful in COPD-OSA.

S1P signaling is emerging as a critical regulator of cellular behaviors and immune response. S1P signaling has been found to be involved in the progression of several diseases, including digestive system disease (29), lung fibrosis (30), and cardiovascular diseases (31). Previous research has shown that the S1P levels were increased in COPD mice models and COPD patients, and indicated the potential function of S1P signaling during COPD progression (32). In this present study, we observed significantly increased plasma S1P levels in COPD and COPD-OSA (level: COPD-OSA > COPD > HC). Furthermore, we assessed the effect of S1P on macrophage polarization. The results showed that S1P treatment could enhance the efficiency of M1 polarization of THP-1 in response to LPS/IFN-γ stimulation, which could be reversed by S1P antagonist-FTY720.

We explored the potential mechanism of S1P in macrophage polarization. The data showed that S1P treatment could enhance expression of S1PR1 in THP-1 cells, which could be reversed by FTY720. The overexpression of S1PR1, which is a S1P receptor, could facilitate the M1 polarization of THP-1 in response to LPS/IFN-γ. Correspondingly, in PBMC and MDM from COPD and COPD-OSA, the S1PR1 expression levels were higher than that from HC.

Class I HDACs are enzymes that remove acetyl groups from histones and other nuclear proteins, thereby inducing chromatin condensation and transcriptional repression (33). Hait et al.’s study showed that S1P could bind to HDAC1 and inhibit HDAC1 activity (20). Ebenezer et al. further showed that S1P lyase-generated ∆2-hexadecenal could inhibit HDAC1 activity in lung epithelial cells (34). In this study, we observed that HDAC1 expression was decreased in THP-1 by S1P treatment, which could be reversed by FTY720. Moreover, our data showed that high HDAC1 expression could inhibit M1 the polarization efficiency of THP-1 in response to LPS/IFN-γ. Notably, the expression of HDAC1 was lower in PBMC and MDM from COPD and COPD-OSA patients (vs. HCs), and even lower in COPD-OSA (vs. COPD). Thus, taken together, these data suggested that increasing S1P levels could facilitate macrophage polarization toward to M1 subtype in COPD and COPD-OSA, via enhancing S1PR1 signaling or inhibiting HDAC1 signaling.

There are certain limitations in this study. Firstly, there was no patient group including those with OSA alone. Secondly, an in vivo mice model experiment was not conducted. The function of S1P during COPD and COPD-OSA progression needs further verification in mice models.

Conclusions

Our results demonstrated that patients with COPD and COPD-OSA exhibit significantly elevated plasma S1P levels. The increasing S1P signaling may facilitate the polarization of macrophages towards to M1 phenotype via activating S1PR1 and inhibiting the HDAC1 signaling pathway. Thus, S1P might exert an aggravating influence on COPD and COPD-OSA progression, especially in COPD-OSA. Inhibition of S1P signaling might be a potential therapeutic avenue for COPD and COPD-OSA.

Acknowledgments

None.

Footnote

Reporting Checklist: The authors have completed the MDAR reporting checklist. Available at https://jtd.amegroups.com/article/view/10.21037/jtd-24-1719/rc

Data Sharing Statement: Available at https://jtd.amegroups.com/article/view/10.21037/jtd-24-1719/dss

Peer Review File: Available at https://jtd.amegroups.com/article/view/10.21037/jtd-24-1719/prf

Funding: This study was supported in part by grants from the National Natural Science Foundation of China (No. 82070096) and the Healthy Science and technology project of Nanshan District, China (No. NS2021005).

Conflicts of Interest: All authors have completed the ICMJE uniform disclosure form (available at https://jtd.amegroups.com/article/view/10.21037/jtd-24-1719/coif). The authors have no conflicts of interest to declare.

Ethical Statement: The authors are accountable for all aspects of the work in ensuring that questions related to the accuracy or integrity of any part of the work are appropriately investigated and resolved. The study was approved by the ethics committee of the Second People’s Hospital of Shenzhen (No. 20201208001). All patients included in this study provided written informed consent. The study was conducted in accordance with the Declaration of Helsinki (as revised in 2013).

Open Access Statement: This is an Open Access article distributed in accordance with the Creative Commons Attribution-NonCommercial-NoDerivs 4.0 International License (CC BY-NC-ND 4.0), which permits the non-commercial replication and distribution of the article with the strict proviso that no changes or edits are made and the original work is properly cited (including links to both the formal publication through the relevant DOI and the license). See: https://creativecommons.org/licenses/by-nc-nd/4.0/.

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