Vitamin D deficiency is associated with increased risks of chronic obstructive pulmonary disease (COPD). Nevertheless, the mechanisms remain unknown. This study analyzed the correlations between vitamin D levels and inflammation in COPD patients. One hundred and one patients with COPD and 202 control subjects were enrolled. Serum 25(OH)D level and inflammatory cytokines were detected. Serum 25(OH)D was decreased and inflammatory cytokines were increased in COPD patients. According to forced expiratory volume in 1 s, COPD patients were divided into three grades. Furthermore, serum 25(OH)D was gradually decreased in COPD patients ranging from grade 1–2 to 4. Serum 25(OH)D was inversely associated with inflammatory cytokines in COPD patients. Further analysis found that NF-κB and AP-1 signaling were activated in COPD patients. Besides, inflammatory signaling was gradually increased in parallel with the severity of COPD. By contrast, pulmonary nuclear vitamin D receptor was decreased in COPD patients. In vitro experiments showed that 1,25(OH)2D3 inhibited LPS-activated inflammatory signaling in A549 cells (human lung adenocarcinoma cell). Mechanically, 1,25(OH)2D3 reinforced physical interactions between vitamin D receptor with NF-κB p65 and c-Jun. Our results indicate that vitamin D is inversely correlated with inflammatory signaling in COPD patients. Inflammation may be a vital mediator of COPD progress in patients with low vitamin D levels.

Chronic obstructive pulmonary disease (COPD) is a progressive disease with persistent airflow obstruction and chronic systemic inflammation caused by noxious particles and gases, hence inducing irreversible airflow limitation and dyspnea (1). It is widely accepted that COPD results in pulmonary chronic inflammatory injury, primarily coupled with intrathoracic airways disease and emphysematous destruction (2). Epidemiologic studies have suggested that COPD will become the fourth main cause of morbidity and mortality all over the world by 2030 (3, 4). However, the accurate cause of COPD is not completely illuminated. Accumulating data have demonstrated that activated inflammatory cells and inflammatory cytokines lead to systemic inflammation, which is associated with extrarespiratory manifestations of COPD (5). C-reactive protein (CRP) and systemic inflammatory markers were increased in COPD patients (6, 7). However, the potential molecular mechanism of inflammation-induced COPD is unclear.

Vitamin D, a secosteroid hormone, is famous for its classical functions in calcium uptake and bone metabolism (4, 8). Currently, vitamin D is discovered in some nonclassical actions, such as immunomodulation, antioxidant effect, antiapoptosis effect, regulating epithelial–mesenchymal transition, and anti-inflammatory activity (914). Recently, numerous researchers indicated that 1,25(OH)2D3, the active form of vitamin D, recovered elastane-induced reduction of forced expiratory volume in 1 s (FEV1) to the forced vital capacity (FVC) and alveolar destruction in a mouse model of COPD (15). A population-based study found that vitamin D supplementation obviously alleviated acute exacerbation in COPD patients (16). Otherwise, experiment in vitro also found that vitamin D receptor (VDR) knock-out mice showed increased lung inflammation and accelerated lung function decline in a COPD mouse model (17). Several epidemiological reports found that low concentrations of 25(OH)D were correlated with lung function impairment and increased the risk of COPD (18). Nevertheless, the underlying mechanisms of vitamin D states linking to the pathophysiology of COPD is often complex and not fully understood.

Vitamin D has an anti-inflammatory role in the lung (13, 19). Several reports showed that vitamin D could repress inflammation via reinforcing the interaction between VDR and NF-κB p65 subunit (20). Hence, it is especially interesting to study whether vitamin D deficiency increased the incidence of COPD and the level of inflammation in COPD patients. Our results showed that there was a correlation among COPD, vitamin D levels, and inflammation in a population-based case-control study. The inflammatory cytokines were elevated in COPD patients with low vitamin D levels. Besides, VDR expression was reduced in COPD patients. In contrast, NF-κB signaling and AP-1 signaling were activated in COPD patients with low vitamin D concentrations.

This was a hospital-based case-control study. All participators were recruited from the Second Affiliated Hospital of Anhui Medical University in China between February 2017 and October 2018. Altogether, 303 individuals were recruited. This current research comprised 101 patients with newly diagnosed COPD. All participators performed pulmonary function tests, and those who showed a ratio of FEV1 to FVC of <0.7 and an FEV1 <80% were included. The patients were enrolled during stable state; active autoimmune diseases or cancer within the last 5 y were causes for exclusion. A total of 202 control subjects were selected from the physical examination center in the First Affiliated Hospital in the Anhui Medical University. Every case was matched with two control subjects regarding age, sex, and the season in which the blood sample was collected. The demographic features between patients and control subjects were demonstrated in Table I. Serum samples of all cases and controls were collected at the same season and stored at −80°C in a refrigerator. To measure NF-κB signaling, AP-1 signaling, and VDR expression in the lung, lung tissues were obtained from surgical operation on COPD patients. Paracancerous tissues were collected from lung cancer patients without COPD in the Second Affiliated Hospital. Every case was matched with one control subject in view of age and gender. A total of 25 controls and 25 cases were recruited at last. This study was approved by the Ethics Committee of Anhui Medical University. All control subjects and COPD patients received written and oral information and provided informed signed consent.

The following information was collected from the electronic medical records of each participant: age, gender, history of smoking, comorbidities, pulmonary function during the stable stage without cough, expectoration and asthmatic syndrome, WBC count, neutrophil, and FEV1/FVC. COPD was graded into three stages on the basis of FEV1 (1): grade 1–2: FEV1 >50%, mild and moderate COPD; grade 3: FEV1 30–49%, severe COPD; and grade 4: FEV1 <30% or FEV1 30–50%, very severe COPD.

Serum 25(OH)D level was detected by radioimmunoassay (RIA) following the manufacturer’s instructions (21). The RIA kits, with 125I labeled 25(OH)D as a tracer, were purchased from DiaSorin (DiaSorin, Stillwater, MN). Serum 25(OH)D unit was in nanograms per milliliter. A 25(OH)D status under 15 ng/ml was considered vitamin D deficiency (22).

Commercial ELISA kits (CRP, TNF-α, and IL-1β) were purchased from Cusabio, Wuhan, China (https://www.cusabio.com/). IL-6 and MCP-1 kits were from Wuhan ColorfulGene Biological Technology (http://www.jymbio.com/). All inflammatory cytokines were measured by microplate assay according to the manufacturer’s protocol (23).

A549 cells (pulmonary adenocarcinoma–derived cell line) were purchased from the Chinese Academy of Sciences Shanghai Branch (Shanghai, China). A549 cells were grown in RPMI 1640 medium (HyClone, Logan, UT) supplemented with 100 U/ml penicillin, 100 μg/ml streptomycin, and 7.5% (v/v) heat-inactivated FBS in a humidified chamber with 5% CO2/95% air at 37°C. A549 cells were seeded into 75-cm2 flasks at a density of 1 × 106 cells per well and incubated for 12 h to allow them to adhere to flasks. Approximately 50% confluent, the cells were pretreated twice with 1,25(OH)2D3 (100 nM) at 24 and 12 h before LPS (2 μg/ml) incubation. After 6 h of exposure of LPS, the cells were washed with chilled PBS three times and then harvested for Western blotting and coimmunoprecipitation (Co-IP).

Western blotting was performed using standard protocols. Total lysate was prepared by homogenizing 50 mg of human lung tissue in 500 μl of RIPA lysis buffer containing phosphatase inhibitor mixture. For nuclear protein extraction, total lysate from human lung was suspended in hypotonic buffer (24). The suspension was then centrifuged for 30 s at 14,000 × g. The nuclear pellet was lysed in complete lysis buffer. Protein level was measured with BCA protein assay reagents. Equivalent amount of proteins among different groups was electrophoresed in 10% SDS-PAGE gels and then transferred to a polyvinylidene fluoride membrane. Membranes were then washed and blocked in 5% skim milk and incubated subsequently with mouse monoclonal p-IκBα Ab (1:3000) and β-actin (1:10,000) from Abcam, rabbit mAbs for IκBα (1:3000), p-p65 (1:2000), p–c-Fos (1:2000), p–c-Jun (1:2000), VDR (1:2000) from Abcam, and goat Polyclonal Lamin A/C Ab (1:2000) from Millipore. HRP-conjugated monoclonal goat anti-rabbit, anti-mouse, or anti-donkey IgG Abs were used as the secondary Ab. The membranes were visualized followed by film exposure using an ECL reagent detection kit.

Lungs were fixed in 4% paraformaldehyde and embedded in paraffin according to the standard procedure (25). Paraffin-embedded pulmonary tissue was cut in 4-μm-thick sections. All sections were deparaffinized and then rehydrated. For Ag retrieval, we put the slides in the microwave using sodium citrate solution with pH 6, and then they were blocked with serum reagent. Sections were incubated with p65 or p50 Abs and diluted at 1:200 in Ab diluent overnight at 4°C. Sections were washed. The color reaction was detected with the HRP-linked polymer detection system and counterstained with hematoxylin. Immunostaining of NF-κB in the lung was graded on a semiquantitative scale: 0, absent staining/no color; 1, weak staining/pale brown color; 2, distinct staining/dark brown color; and 3, strong staining/brownish-black color. The immunohistochemistry was evaluated by two pathologists separately (26).

Total lysate from A549 cells was prepared in a lysis buffer (0.6% Nonidet P-40, 0.5% sodium deoxycholate, 150 mM NaCl, and 50 mM Tris-HCl, pH 7.5) containing protease inhibitors (0.5 mg/ml aprotinin, 0.5 mg/ml trans-epoxy succinyl-l-leucylamido-[4-guanidino] butane [E-64], 0.5 mg/ml pepstatin, 0.5 mg/ml bestatin, 10 mg/ml chymostatin, and 0.1 ng/ml leupeptin). Cells lysates (400 μg) were precleared with protein A/G-agarose (Santa Cruz, CA) and then incubated with agarose-conjugated VDR Ab at 4°C overnight. The precipitates were washed with cold nondenature lysis buffer before Western blotting. Interaction efficiency was measured using Western blotting (27).

All statistical analyses were performed using SPSS 21.0. All data were expressed as mean ± SEM or median plus range. The difference in continuous variables between two independent groups was analyzed via two independent sampling t test or the Mann-Whitney U test. Comparative analyses of categorical variables were implemented by the χ2 test. Multivariable logistic regression was carried out to distinguish the association between 25(OH)D and COPD. The relationships between 25(OH)D states and inflammatory cytokines were analyzed using scatter plots and linear correlation. A p value < 0.05 was considered statistically significant.

Demographic characteristics are summarized in Table I. A total of 101 patients (73.3% males) aged between 54 and 75 y (mean 68.1 y) were included in this study. A total of 202 control subjects were recruited. No differences in age, gender, and eosinophils were found between control subjects and COPD patients. COPD patients had higher levels of leukocyte and neutrophil than these in control subjects. As expected, COPD patients contained a higher proportion of current smokers. In addition, FEV1%, FEV1/FVC%, FVC, and FEV1 were reduced in COPD patients (Table I).

Table I.
The demographic and biochemical characteristics between COPD patients and control subjects
Controls (n = 202)Cases (n = 101)p
Male, n (%) 145 (71.8) 74 (73.3) 0.126 
Age (y) 65.12 ± 0.89 68.10 ± 0.50 0.2103 
Smoker (%) 32.46 78.56 <0.01 
Leukocyte (109/l) 6.34 (6.52, 8.74) 7.69 (5.12, 9.74) <0.05 
Neutrophil (109/l) 4.22 (2.78, 4.92) 6.51 (3.35, 8.14) <0.01 
Eosinophils (109/l) 0.19 (0.08, 0.34) 0.12 (0.01, 0.21) 0.051 
FVC (l) NS 2.17 (1.60, 2.89) NS 
FEV1 (%) NS 29.0 (20.0, 57.0) NS 
FEV1 (l) NS 1.17 (0.79, 1.79) NS 
FEV1/FVC (%) NS 57.69 (48.75, 65.71) NS 
Controls (n = 202)Cases (n = 101)p
Male, n (%) 145 (71.8) 74 (73.3) 0.126 
Age (y) 65.12 ± 0.89 68.10 ± 0.50 0.2103 
Smoker (%) 32.46 78.56 <0.01 
Leukocyte (109/l) 6.34 (6.52, 8.74) 7.69 (5.12, 9.74) <0.05 
Neutrophil (109/l) 4.22 (2.78, 4.92) 6.51 (3.35, 8.14) <0.01 
Eosinophils (109/l) 0.19 (0.08, 0.34) 0.12 (0.01, 0.21) 0.051 
FVC (l) NS 2.17 (1.60, 2.89) NS 
FEV1 (%) NS 29.0 (20.0, 57.0) NS 
FEV1 (l) NS 1.17 (0.79, 1.79) NS 
FEV1/FVC (%) NS 57.69 (48.75, 65.71) NS 

To explore the association between serum 25(OH)D and pulmonary function, serum 25(OH)D states were detected in all subjects. As shown in Fig. 1A, serum 25(OH)D concentration in COPD patients was lower than these in control subjects. COPD patients were further classified into three groups based on 2017 Global Initiative for Chronic Obstructive Lung Disease Standard. Grade 1–2 is mild and moderate COPD; grade 3 is severe COPD; and grade 4 is very severe COPD. We further analyzed 25(OH)D concentration in different grades of COPD patients. As expected, 25(OH)D concentration gradually decreased from grade 1–2 to grade 4 in COPD patients (Fig. 1B). The correlation between pulmonary function and serum 25(OH)D status was analyzed using multivariate logistic regression. As shown in Table II, FEV1% was negatively correlated with serum 25(OH)D concentration.

FIGURE 1.

Serum 25(OH)D levels in patients with COPD and control subjects. Serum 25(OH)D was measured by RIA. (A) Serum 25(OH)D level was compared between COPD patients and control subjects (n = 101 for COPD patients; n = 202 for control subjects). (B) Serum 25(OH)D level was compared among patients with different grades of COPD (n = 28 for grade 1–2 of COPD patients; n = 29 for grade 3 of COPD patients; n = 44 for grade 4 of COPD patients). All data were expressed as means ± SEM. *p < 0.05, **p < 0.01.

FIGURE 1.

Serum 25(OH)D levels in patients with COPD and control subjects. Serum 25(OH)D was measured by RIA. (A) Serum 25(OH)D level was compared between COPD patients and control subjects (n = 101 for COPD patients; n = 202 for control subjects). (B) Serum 25(OH)D level was compared among patients with different grades of COPD (n = 28 for grade 1–2 of COPD patients; n = 29 for grade 3 of COPD patients; n = 44 for grade 4 of COPD patients). All data were expressed as means ± SEM. *p < 0.05, **p < 0.01.

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Table II.
Correlation between 25(OH)D and COPD using multivariable logistic regression
VariableβWaldpOR (95% CI)
Unadjusted −0.169 6.976 0.008 0.845 (0.745, 0.957) 
Adjusted −0.156 4.262 0.039 0.856 (0.738, 0.992) 
VariableβWaldpOR (95% CI)
Unadjusted −0.169 6.976 0.008 0.845 (0.745, 0.957) 
Adjusted −0.156 4.262 0.039 0.856 (0.738, 0.992) 

Adjusted for smoker (percentage), leukocyte, and neutrophil.

CI, confidence interval; OR, odds ratio.

To further explore the associations between inflammatory cytokines and pulmonary function, the levels of serum proinflammatory cytokines and chemokines were measured in COPD patients and control subjects. As shown in Table III, serum CRP, TNF-α, MCP-1, IL-6, and IL-1β in COPD patients were higher than these in control subjects. Serum proinflammatory cytokine and chemokine levels in patients with different grades of COPD were further analyzed. As shown in Table III, serum CRP, TNF-α, and MCP-1 levels were increased in a grade-dependent manner in COPD patients. The more severe the COPD is, the greater the inflammation is. Moreover, patients with grade 4 COPD had higher levels of serum IL-6 and IL-1β than in patients with grade 1–2 COPD. However, there was no difference in the level of serum IL-6 and IL-1β between grade 1–2 and grade 3 in COPD patients (Table III). Moreover, the levels of inflammatory cytokines were compared between control subjects and different grades of COPD patients. As shown in Table III, the levels of CRP, TNF-α, MCP-1, and IL-1β were higher in the different grades of COPD patients than these in control subjects. The level of IL-6 was elevated in grade 1–2 COPD patients compared with control subjects. Meanwhile, the associations between the levels of serum proinflammatory cytokines and chemokines with 25(OH)D status were analyzed in patients with COPD. The results indicated that serum CRP, TNF-α, and MCP-1 levels were inversely correlated with serum 25(OH)D (Fig. 2A, r = −0.252, p < 0.01; Fig. 2B, r = −0.208, p < 0.05; Fig. 2C, r = −0.268, p < 0.01).

Table III.
Serum inflammatory cytokines in COPD patients and control subjects
nCRP (μg/ml)TNF-α (ng/ml)MCP-1 (pg/ml)IL-6 (pg/ml)IL-1β (pg/ml)
All subjects       
 Control 202 54.3 ± 8.4 45.3 ± 1.8 78.4 ± 12.3 28.3 ± 4.3 38.3 ± 4.3 
 COPD 101 104.2 ± 12.3b 81.8 ± 2.9b 196.0 ± 15.7b 45.3 ± 5.4b 77.1 ± 6.2b 
Grade according to FEV1%       
 Grade 1–2 28 72.3 ± 4.3a 60.1 ± 3.5b 155.7 ± 5.8b 29.6 ± 1.80 65.2 ± 2.0b 
 Grade 3 29 94.3 ± 5.6b,c 80.3 ± 5.6b,d 182.5 ± 10.6b,c 41.14 ± 7.92 71.0 ± 2.7b 
 Grade 4 44 134.1 ± 8.9b,d,f 109.3 ± 6.6b,d,e 224.6 ± 13.4b,d,f 55.6 ± 9.5b,d 87.6 ± 7.0b,c,e 
nCRP (μg/ml)TNF-α (ng/ml)MCP-1 (pg/ml)IL-6 (pg/ml)IL-1β (pg/ml)
All subjects       
 Control 202 54.3 ± 8.4 45.3 ± 1.8 78.4 ± 12.3 28.3 ± 4.3 38.3 ± 4.3 
 COPD 101 104.2 ± 12.3b 81.8 ± 2.9b 196.0 ± 15.7b 45.3 ± 5.4b 77.1 ± 6.2b 
Grade according to FEV1%       
 Grade 1–2 28 72.3 ± 4.3a 60.1 ± 3.5b 155.7 ± 5.8b 29.6 ± 1.80 65.2 ± 2.0b 
 Grade 3 29 94.3 ± 5.6b,c 80.3 ± 5.6b,d 182.5 ± 10.6b,c 41.14 ± 7.92 71.0 ± 2.7b 
 Grade 4 44 134.1 ± 8.9b,d,f 109.3 ± 6.6b,d,e 224.6 ± 13.4b,d,f 55.6 ± 9.5b,d 87.6 ± 7.0b,c,e 

All data were expressed as means ± SEM.

a

p < 0.05, bp < 0.01 as compared with control subjects.

c

p < 0.05, dp < 0.01 as compared with grade 1–2.

e

p < 0.05, fp < 0.01 as compared with grade 3.

FIGURE 2.

Correlation analysis between serum 25(OH)D and inflammation in patients with COPD. (A) Correlation analysis between serum 25(OH)D and CRP in patients with COPD. (B) Correlation analysis between serum 25(OH)D and TNF-α in patients with COPD. (C) Correlation analysis between serum 25(OH)D and MCP-1 in patients with COPD.

FIGURE 2.

Correlation analysis between serum 25(OH)D and inflammation in patients with COPD. (A) Correlation analysis between serum 25(OH)D and CRP in patients with COPD. (B) Correlation analysis between serum 25(OH)D and TNF-α in patients with COPD. (C) Correlation analysis between serum 25(OH)D and MCP-1 in patients with COPD.

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For the sake of analyzing the mechanism of the elevation of inflammation, NF-κB and AP-1 signaling were measured in human lung tissues. As shown in Fig. 3, the level of pulmonary phosphorylated IκBα was elevated in COPD patients. On the contrary, pulmonary IκBα was decreased in COPD patients (Fig. 3A–C). As shown in Fig. 3A and 3D, the level of pulmonary p-p65 was increased in COPD patients than that in control subjects. In addition, NF-κB p65 and p50 subunits were then detected in the lungs by immunohistochemistry. As expected, NF-κB p65 and p50 positive nuclei in COPD patients were increased than these in controls (Fig. 4). Moreover, immunostaining of nuclear NF-κB signaling was further analyzed in the gradation system. The staining intensity of p65 and p50 in COPD patients was greater than these in controls (Supplemental Table I). Pulmonary p-IκBα, IκBα, and p-p65 were further measured in different grades of COPD patients. As shown in Fig. 3E–H, the expression of p-IκBα and p-p65 was elevated in parallel with the severity of COPD patients. Moreover, the level of IκBα was lower in grade 4 than these in grade 1–2 and grade 3 COPD patients (Fig. 3G).

FIGURE 3.

NF-κB signaling in patients with COPD and control subjects. Phosphorylated IκBα, IκBα, and phosphorylated p65 were measured between patients with COPD and control subjects in the lungs. (A) Pulmonary phosphorylated IκBα, IκBα, and phosphorylated p65 were detected in COPD patients and control subjects using Western blotting. (BD) Quantitative analysis of scanning densitometry was performed. (B) p-IκBα/IκBα. (C) IκBα. (D) p-p65. (E) Pulmonary phosphorylated IκBα, IκBα, and phosphorylated p65 were detected in the different grades of COPD patients through Western blotting. (FH) Quantitative analysis of scanning densitometry was performed. (F) p-IκBα/IκBα. (G) IκBα. (H) p-p65. All data were expressed as means ± SEM of six different lung samples (n = 6). All experiments were repeated three times. *p < 0.05, **p < 0.01.

FIGURE 3.

NF-κB signaling in patients with COPD and control subjects. Phosphorylated IκBα, IκBα, and phosphorylated p65 were measured between patients with COPD and control subjects in the lungs. (A) Pulmonary phosphorylated IκBα, IκBα, and phosphorylated p65 were detected in COPD patients and control subjects using Western blotting. (BD) Quantitative analysis of scanning densitometry was performed. (B) p-IκBα/IκBα. (C) IκBα. (D) p-p65. (E) Pulmonary phosphorylated IκBα, IκBα, and phosphorylated p65 were detected in the different grades of COPD patients through Western blotting. (FH) Quantitative analysis of scanning densitometry was performed. (F) p-IκBα/IκBα. (G) IκBα. (H) p-p65. All data were expressed as means ± SEM of six different lung samples (n = 6). All experiments were repeated three times. *p < 0.05, **p < 0.01.

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FIGURE 4.

The levels of nuclear translocation of NF-κB p65 and p50 in COPD patients and control subjects. Nuclear translocation of NF-κB p65 and NF-κB p50 was detected in human lung tissues. (A) Pulmonary NF-κB p65 was measured by immunohistochemistry. (B) NF-κB p65 positive nuclei were analyzed. (C) Pulmonary NF-κB p50 was determined using immunohistochemistry (blue arrows). (D) NF-κB p50 positive nuclei were calculated (blue arrows). Scale bar, 50 μm. Original magnification ×400. All data were expressed as means ± SEM of 25 different lung samples (n = 25). All experiments were repeated three times. **p < 0.01.

FIGURE 4.

The levels of nuclear translocation of NF-κB p65 and p50 in COPD patients and control subjects. Nuclear translocation of NF-κB p65 and NF-κB p50 was detected in human lung tissues. (A) Pulmonary NF-κB p65 was measured by immunohistochemistry. (B) NF-κB p65 positive nuclei were analyzed. (C) Pulmonary NF-κB p50 was determined using immunohistochemistry (blue arrows). (D) NF-κB p50 positive nuclei were calculated (blue arrows). Scale bar, 50 μm. Original magnification ×400. All data were expressed as means ± SEM of 25 different lung samples (n = 25). All experiments were repeated three times. **p < 0.01.

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AP-1 signaling was evaluated in human lung tissues between COPD patients and control subjects. As shown in Fig. 5A–C, phosphorylated c-Fos and c-Jun were elevated in COPD patients. Pulmonary p–c-Fos and p–c-Jun protein were further measured in patients with different grades of COPD. As shown in Fig. 5D–F, phosphorylated c-Fos in grade 1–2 COPD patients was lower than in grade 3 and grade 4 COPD patients. Phosphorylated c-Jun was increased in parallel with the severity of COPD in patients (Fig. 5D–F).

FIGURE 5.

AP-1 signaling in patients with COPD and control subjects. Pulmonary phosphorylated c-Fos and c-Jun were determined in human lung tissues between COPD patients and control subjects. (A) Pulmonary phosphorylated c-Fos and c-Jun were determined using Western blotting in two groups. (B and C) Quantitative analysis of scanning densitometry was performed. (B) p–c-Fos. (C) p–c-Jun. (D) Pulmonary phosphorylated c-Fos and c-Jun were determined using Western blotting in different grades of COPD patients. (E and F) Quantitative analysis of scanning densitometry was performed. (E) p–c-Fos. (F) p–c-Jun. All data were expressed as means ± SEM of six different lung samples from six cases (n = 6). All experiments were repeated three times. *p < 0.05, **p < 0.01.

FIGURE 5.

AP-1 signaling in patients with COPD and control subjects. Pulmonary phosphorylated c-Fos and c-Jun were determined in human lung tissues between COPD patients and control subjects. (A) Pulmonary phosphorylated c-Fos and c-Jun were determined using Western blotting in two groups. (B and C) Quantitative analysis of scanning densitometry was performed. (B) p–c-Fos. (C) p–c-Jun. (D) Pulmonary phosphorylated c-Fos and c-Jun were determined using Western blotting in different grades of COPD patients. (E and F) Quantitative analysis of scanning densitometry was performed. (E) p–c-Fos. (F) p–c-Jun. All data were expressed as means ± SEM of six different lung samples from six cases (n = 6). All experiments were repeated three times. *p < 0.05, **p < 0.01.

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Activated VDR could repress NF-κB signaling. So, pulmonary VDR was then detected in human lung tissues between COPD patients and control subjects. As shown in Fig. 6A and 6B, pulmonary nuclear VDR was reduced in patients with COPD. Simultaneously, nuclear VDR level was also measured in the lungs from different grades of COPD patients. As shown in Fig. 6C and 6D, nuclear VDR level in patients with grade 1–2 COPD was higher than these in patients with grade 3 and grade 4 COPD.

FIGURE 6.

Pulmonary VDR in patients with COPD and control subjects. The protein level of VDR was detected in human lung tissues between COPD patients and control subjects. (A) Pulmonary VDR was measured using Western blotting between COPD patients and control subjects. (B) Quantitative analysis of scanning densitometry was performed. (C) Pulmonary VDR was measured using Western blotting in different grades of COPD patients. (D) Quantitative analysis of scanning densitometry was performed. All data were expressed as means ± SEM (n = 6). All experiments were repeated three times. **p < 0.01.

FIGURE 6.

Pulmonary VDR in patients with COPD and control subjects. The protein level of VDR was detected in human lung tissues between COPD patients and control subjects. (A) Pulmonary VDR was measured using Western blotting between COPD patients and control subjects. (B) Quantitative analysis of scanning densitometry was performed. (C) Pulmonary VDR was measured using Western blotting in different grades of COPD patients. (D) Quantitative analysis of scanning densitometry was performed. All data were expressed as means ± SEM (n = 6). All experiments were repeated three times. **p < 0.01.

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To further elucidate the mechanism that vitamin D deficiency activated pulmonary NF-κB and AP-1 signaling in COPD patients, A549 cells were pretreated with 1,25(OH)2D3 (the active form of vitamin D3) and then exposed to LPS to observe whether 1,25(OH)2D3 pretreatment inhibited NF-κB and AP-1 signaling in A549 cells. The present study investigated the effect of pretreatment with 1,25(OH)2D3 on LPS-activated NF-κB and AP-1 signaling in vitro experiment. As shown in Fig. 7A and 7B, the level of p-IκBα was increased in the LPS group. In contrast, pretreatment with 1,25(OH)2D3 repressed LPS-induced IκBα phosphorylation. Moreover, pretreatment with 1,25(OH)2D3 also inhibited p65 phosphorylation in A549 cells (Fig. 7A, 7C). Further analysis found that LPS exposure activated AP-1 signaling in A549 cells, and c-Jun and c-Fos phosphorylation were elevated in the LPS group (Fig. 7D–F). Interestingly, pretreatment with 1,25(OH)2D3 repressed LPS-evoked elevation of c-Jun phosphorylation (Fig. 7D–F).

FIGURE 7.

Active vitamin D3 downregulates LPS-activated NF-κB and AP-1 signaling pathways in A549 cells. A549 cells were cocultured with 1,25(OH)2D3 (100 nM) before LPS (2 μg/ml) exposure. After 6 h of exposure, the cells were collected for Western blotting. (A) p-p65, p-IκBα, and IκBα were detected using Western blotting. (B and C) Quantitative analysis of scanning densitometry was performed. (B) p-IκBα/IκBα. (C) p-p65. (D) p–c-Jun and p–c-Fos were detected using Western blotting. (E and F) Quantitative analysis of scanning densitometry was performed. (E) p–c-Jun. (F) p–c-Fos. All data were expressed as means ± SEM of six different lung samples (n = 6). All experiments were repeated three times. *p < 0.05, **p < 0.01.

FIGURE 7.

Active vitamin D3 downregulates LPS-activated NF-κB and AP-1 signaling pathways in A549 cells. A549 cells were cocultured with 1,25(OH)2D3 (100 nM) before LPS (2 μg/ml) exposure. After 6 h of exposure, the cells were collected for Western blotting. (A) p-p65, p-IκBα, and IκBα were detected using Western blotting. (B and C) Quantitative analysis of scanning densitometry was performed. (B) p-IκBα/IκBα. (C) p-p65. (D) p–c-Jun and p–c-Fos were detected using Western blotting. (E and F) Quantitative analysis of scanning densitometry was performed. (E) p–c-Jun. (F) p–c-Fos. All data were expressed as means ± SEM of six different lung samples (n = 6). All experiments were repeated three times. *p < 0.05, **p < 0.01.

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To further explore the mechanism through which low vitamin D statues activates NF-κB and AP-1 signaling in COPD patients. The interactions between VDR and NF-κB p65 as well as VDR and c-Jun were analyzed using Co-IP. As shown in Fig. 8A and 8B, 1,25(OH)2D3 pretreatment did not affect the level of NF-κB p65 in the immunocomplexes precipitated by anti-VDR Ab, and vitamin D3 plus LPS elevated the level of NF-κB p65 and VDR in the immunocomplexes precipitated by anti-VDR Ab (Fig. 8A, 8B). Moreover, the interaction between VDR and c-Jun in A549 cells was evaluated. As expected, vitamin D3 pretreatment had no effect on the level of c-Jun in the total protein precipitated by anti-VDR Ab, and vitamin D3 plus LPS elevated c-Jun in the immunocomplexes precipitated by anti-VDR Ab (Fig. 8C, 8D).

FIGURE 8.

The interaction between VDR and p65 as well as between VDR and c-Jun in A549 cells. A549 cells were cocultured with 1,25(OH)2D3 (100 nM) before LPS (2 μg/ml) exposure. After 6 h of exposure, the cells were collected for Western blotting. Total lysate was isolated. (A) Total lysate fractions were incubated with agarose-conjugated Ab against VDR or NF-κB p65. Co-IP (IP): anti-VDR; immunoblot: p65. (B) Quantitative analysis of scanning densitometry was performed. (C) Total lysate fractions were incubated with agarose-conjugated Ab against VDR or c-Jun. IP: anti-VDR; immunoblot: c-Jun. (D) Quantitative analysis of scanning densitometry was performed. All experiments were duplicated three times of three different samples (n = 3). *p < 0.05, **p < 0.01.

FIGURE 8.

The interaction between VDR and p65 as well as between VDR and c-Jun in A549 cells. A549 cells were cocultured with 1,25(OH)2D3 (100 nM) before LPS (2 μg/ml) exposure. After 6 h of exposure, the cells were collected for Western blotting. Total lysate was isolated. (A) Total lysate fractions were incubated with agarose-conjugated Ab against VDR or NF-κB p65. Co-IP (IP): anti-VDR; immunoblot: p65. (B) Quantitative analysis of scanning densitometry was performed. (C) Total lysate fractions were incubated with agarose-conjugated Ab against VDR or c-Jun. IP: anti-VDR; immunoblot: c-Jun. (D) Quantitative analysis of scanning densitometry was performed. All experiments were duplicated three times of three different samples (n = 3). *p < 0.05, **p < 0.01.

Close modal

This research mainly analyzed the correlations among COPD, vitamin D concentration, and inflammation in COPD patients. Our discoveries indicated that serum 25(OH)D level was decreased in COPD patients. On the contrary, serum CRP, TNF-α, IL-6, IL-1β, and MCP-1 were elevated in patients with COPD. Vitamin D concentration was negatively associated with serum inflammatory cytokines in COPD patients. Therefore, these results have demonstrated first (to our knowledge) that low vitamin D concentration is inversely associated with inflammation in COPD patients.

The accumulation of inflammatory cytokines may induce respiratory histopathologic changes. Increasing evidence supports that the presence of systemic inflammatory cytokines increased the risks of COPD and the comorbidities occurrence (7). Circulatory proinflammatory cytokine CRP is an important marker of systemic chronic inflammation. It reflects the total systemic burden of an individual’s inflammation (28). The severity of COPD and CRP levels is associated with each other (29, 30). Numerous studies have reported that IL-6 level in serum was negatively associated with decreased lung function, dyspnea, and muscle weakness in COPD patients (31, 32). IL-1β was increased in the COPD patients’ lungs tissue and bronchoalveolar lavage fluid (33). Additionally, a higher level of TNF-α was observed in BALF from chronic smokers and sputum from COPD patients (34, 35). Several studies have revealed that MCP-1 was increased in COPD patients (3638). We found that 25(OH)D concentration was gradually decreased in COPD patients ranging from grade 1–2 to grade 4 in this study. Even more impressively, the levels of serum inflammatory cytokines were gradually increased in parallel with the severity of COPD. These results demonstrate that low vitamin D concentration is correlated with inflammation and the severity of COPD.

As we all know, vitamin D can exert an anti-inflammation action in the lung. Recently, one report from our laboratory found that pretreatment with calcitriol attenuated LPS-induced elevation of inflammation (19). An earlier in vitro study has demonstrated that 1,25(OH)2D3 alleviated LPS-induced elevation of inflammatory cytokines in BEAS-2B cells (39). This research also explored the correlations between vitamin D status and serum inflammatory cytokines in COPD patients with different grades. It was interesting that 25(OH)D concentration in serum was inversely correlated with inflammatory cytokines in COPD patients. These results provide a mechanistic explanation for the association of low vitamin D status with high levels of inflammatory cytokines in patients with COPD.

According to two recent reports from our laboratory, vitamin D inhibited LPS-induced proinflammatory cytokines by reinforcing the physical interaction between VDR and NF-κB p65 (20, 23). The activated NF-κB subunits then translocate into the nucleus where it binds to specific sequences of DNA response elements and activates the gene transcription. Many proinflammatory cytokines are the downstream target of NF-κB signaling. In consequence, vitamin D exerts an anti-inflammatory role through inhibiting NF-κB transcriptional activity. This study analyzed pulmonary VDR and NF-κB signaling in COPD patients. As expected, pulmonary nuclear VDR was decreased and NF-κB signaling was elevated in COPD patients. Moreover, we found that nuclear VDR was gradually decreased ranging from grade 1–2 to grade 4 in COPD patients. However, the expression of NF-κB signaling was opposite to nuclear VDR. NF-κB signaling was gradually elevated in parallel with the severity of COPD. These results suggest that NF-κB signaling is reversely associated with vitamin D deficiency in COPD patients.

AP-1 (transcription activator-1), one of the important transcription factors (40), regulates a wide range of cellular processes, including cell proliferation, survival, and differentiation (21). AP-1 is consisted of a variety of combinations of dimerized proteins that belong to the Jun, Fos, Maf, and ATF subfamilies. C-Jun and c-Fos are two most significant components of AP-1 transcription factor (41). Nuclear translocation of AP-1 initiates the transcription of genes encoding proinflammatory mediators and cytokines (42). The previous studies found that proinflammatory cytokines were increased and AP-1 pathway was activated after LPS exposure in human airway epithelial cells (43, 44). Interestingly, two studies have revealed that vitamin D can interact with AP-1 (c-Jun/c-Fos) and form a c-Fos/c-Jun-VDR complex (4547). In the current study, we found c-Jun and c-Fos phosphorylation were elevated in COPD patients. Pulmonary c-Jun phosphorylation was gradually increased in COPD patients ranging from grade 1–2 to grade 4. These results demonstrate that low vitamin D is linked with pulmonary AP-1 activation in COPD patients.

To further elucidate the mechanism that vitamin D deficiency activated pulmonary NF-κB and AP-1 signaling in COPD patients, this study investigated the effect of 1,25(OH)2D3 pretreatment on LPS-activated NF-κB and AP-1 signaling in vitro experiment. As expected, 1,25(OH)2D3 inhibited LPS-activated NF-κB and AP-1 signaling in A549 cells. Further analysis found that 1,25(OH)2D3 pretreatment promoted physical interaction between VDR and NF-κB p65 as well as between VDR and c-Jun. Activated VDR inhibits NF-κB and AP-1 from cytoplasm translocating into the nucleus where it binds to specific sequences of DNA response elements and activates the gene transcription, at last repressing the inflammation. These results provide a mechanistic explanation for the association among low vitamin D deficiency and pulmonary NF-κB and AP-1 signaling activation in COPD patients.

There are some flaws in this study. First, this was single-center research, and the sample size was small; large-sample multicenter research is needed in future work. Second, this was only a hospital-based case-control study; the causal link between vitamin D deficiency and inflammation in COPD patients was not clear. The cohort study and animal experiments are needed in future research. Third, reduction of serum 25(OH)D inhibited VDR nuclear translocation. However, elevating inflammation also repressed VDR nuclear translocation. Therefore, the reason that led to VDR reduction remained unclear. More in vivo experiments are demanded next. Fourth, this study did not analyze whether vitamin D deficiency promoted the occurrence and development of COPD in vivo, and the causality of vitamin D deficiency and COPD remained unknown. Animal experiments could resolve this confusion. Fifth, because of limited experimental condition, we only explored the effect of 1,25(OH)2D3 pretreatment on LPS-activated inflammatory signaling. We cannot observe whether replacement of activated VDR alleviated LPS-activated inflammatory signaling in vitro.

To summarize, the present research explored the correlations among COPD, vitamin D concentration, and inflammation. Our data found that serum 25(OH)D was reduced and proinflammatory cytokines were increased in COPD patients. Our results proved that vitamin D concentration was inversely correlated with proinflammatory cytokines in COPD patients. We also found that pulmonary VDR was decreased in COPD patients. On the contrary, pulmonary NF-κB and AP-1 signaling were elevated in COPD patients. Mechanistically, vitamin D alleviated inflammation through the interaction between VDR and NF-κB p65 as well as VDR and c-Jun in pulmonary epithelial cells. Our results provide evidence that there is a negative correlation between vitamin D deficiency and inflammatory signaling in COPD patients.

We thank all colleagues who generously shared reagents. We thank all members of the Department of Toxicology of Anhui Medical University for experimental input and constructive criticisms.

This work was supported by the National Natural Science Foundation of China (81630084) and National Natural Science Foundation Incubation Program of the Second Affiliated Hospital of Anhui Medical University (2019GQFY06).

The online version of this article contains supplemental material.

Abbreviations used in this article:

Co-IP

coimmunoprecipitation

COPD

chronic obstructive pulmonary disease

CRP

C-reactive protein

FEV1

forced expiratory volume in 1 s

FVC

forced vital capacity

RIA

radioimmunoassay

VDR

vitamin D receptor.

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The authors have no financial conflicts of interest.

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