Vitamin D supplementation in chronic obstructive pulmonary disease patients with low serum vitamin D: a randomized controlled trial.

BACKGROUND: Vitamin D deficiency is frequently found in patients with chronic obstructive pulmonary disease (COPD). Vitamin D has antimicrobial, anti-inflammatory, and immunomodulatory effects. Therefore, supplementation may prevent COPD exacerbations, particularly in deficient patients. OBJECTIVES: We aimed to assess the effect of vitamin D supplementation on exacerbation rate in vitamin D-deficient patients with COPD. METHODS: We performed a multicenter, double-blind, randomized controlled trial. COPD patients with ≥1 exacerbations in the preceding year and a vitamin D deficiency (15-50 nmol/L) were randomly allocated in a 1:1 ratio to receive either 16,800 International Units (IU) vitamin D3 or placebo once a week during 1 y. Primary outcome of the study was exacerbation rate. Secondary outcomes included time to first and second exacerbations, time to first and second hospitalizations, use of antibiotics and corticosteroids, pulmonary function, maximal respiratory mouth pressure, physical performance, skeletal muscle strength, systemic inflammatory markers, nasal microbiota composition, and quality of life. RESULTS: The intention-to-treat population consisted of 155 participants. Mean ± SD serum 25-hydroxyvitamin D [25(OH)D] concentration after 1 y was 112 ± 34 nmol/L in the vitamin D group, compared with 42 ± 17 nmol/L in the placebo group. Vitamin D supplementation did not affect exacerbation rate [incidence rate ratio (IRR): 0.90; 95% CI: 0.67, 1.21]. In a prespecified subgroup analysis in participants with 25(OH)D concentrations of 15-25 nmol/L (n = 31), no effect of vitamin D supplementation was found (IRR: 0.91; 95% CI: 0.43, 1.93). No relevant differences were found between the intervention and placebo groups in terms of secondary outcomes. CONCLUSIONS: Vitamin D supplementation did not reduce exacerbation rate in COPD patients with a vitamin D deficiency.This trial was registered at clinicaltrials.gov as NCT02122627.

Impact of the Local Inflammatory Environment on Mucosal Vitamin D Metabolism and Signaling in Chronic Inflammatory Lung Diseases.

Vitamin D plays an active role in the modulation of innate and adaptive immune responses as well as in the protection against respiratory pathogens. Evidence for this immunomodulatory and protective role is derived from observational studies showing an association between vitamin D deficiency, chronic airway diseases and respiratory infections, and is supported by a range of experimental studies using cell culture and animal models. Furthermore, recent intervention studies have now shown that vitamin D supplementation reduces exacerbation rates in vitamin D-deficient patients with chronic obstructive pulmonary disease (COPD) or asthma and decreases the incidence of acute respiratory tract infections. The active vitamin D metabolite, 1,25-dihydroxy-vitamin D (1,25(OH)2D), is known to contribute to the integrity of the mucosal barrier, promote killing of pathogens (via the induction of antimicrobial peptides), and to modulate inflammation and immune responses. These mechanisms may partly explain its protective role against infections and exacerbations in COPD and asthma patients. The respiratory mucosa is an important site of local 1,25(OH)2D synthesis, degradation and signaling, a process that can be affected by exposure to inflammatory mediators. As a consequence, mucosal inflammation and other disease-associated factors, as observed in e.g., COPD and asthma, may modulate the protective actions of 1,25(OH)2D. Here, we discuss the potential consequences of various disease-associated processes such as inflammation and exposure to pathogens and inhaled toxicants on vitamin D metabolism and local responses to 1,25(OH)2D in both immune- and epithelial cells. We furthermore discuss potential consequences of disturbed local levels of 25(OH)D and 1,25(OH)2D for chronic lung diseases. Additional insight into the relationship between disease-associated mechanisms and local effects of 1,25(OH)2D is expected to contribute to the design of future strategies aimed at improving local levels of 1,25(OH)2D and signaling in chronic inflammatory lung diseases.

TGF-ß1 Impairs Vitamin D-Induced and Constitutive Airway Epithelial Host Defense Mechanisms.

Airway epithelium is an important site for local vitamin D (VD) metabolism; this can be negatively affected by inflammatory mediators. VD is an important regulator of respiratory host defense, for example, by increasing the expression of hCAP18/LL-37. TGF-ß1 is increased in chronic obstructive pulmonary disease (COPD), and known to decrease the expression of constitutive host defense mediators such as secretory leukocyte protease inhibitor (SLPI) and polymeric immunoglobulin receptor (pIgR). VD has been shown to affect TGF-ß1-signaling by inhibiting TGF-ß1-induced epithelial-to-mesenchymal transition. However, interactions between VD and TGF-ß1, relevant for the understanding host defense in COPD, are incompletely understood. Therefore, the aim of the present study was to investigate the combined effects of VD and TGF-ß1 on airway epithelial cell host defense mechanisms. Exposure to TGF-ß1 reduced both baseline and VD-induced expression of hCAP18/LL-37, partly by increasing the expression of the VD-degrading enzyme CYP24A1. TGF-ß1 alone decreased the number of secretory club and goblet cells and reduced the expression of constitutive host defense mediators SLPI, s/lPLUNC and pIgR, effects that were not modulated by VD. These results suggest that TGF-ß1 may decrease the respiratory host defense both directly by reducing the expression of host defense mediators, and indirectly by affecting VD-mediated effects such as expression of hCAP18/LL-37.

Aberrant epithelial differentiation by cigarette smoke dysregulates respiratory host defence.

It is currently unknown how cigarette smoke-induced airway remodelling affects highly expressed respiratory epithelial defence proteins and thereby mucosal host defence.Localisation of a selected set of highly expressed respiratory epithelial host defence proteins was assessed in well-differentiated primary bronchial epithelial cell (PBEC) cultures. Next, PBEC were cultured at the air-liquid interface, and during differentiation for 2-3 weeks exposed daily to whole cigarette smoke. Gene expression, protein levels and epithelial cell markers were subsequently assessed. In addition, functional activities and persistence of the cigarette smoke-induced effects upon cessation were determined.Expression of the polymeric immunoglobulin receptor, secretory leukocyte protease inhibitor and long and short PLUNC (palate, lung and nasal epithelium clone protein) was restricted to luminal cells and exposure of differentiating PBECs to cigarette smoke resulted in a selective reduction of the expression of these luminal cell-restricted respiratory host defence proteins compared to controls. This reduced expression was a consequence of cigarette smoke-impaired end-stage differentiation of epithelial cells, and accompanied by a significant decreased transepithelial transport of IgA and bacterial killing.These findings shed new light on the importance of airway epithelial cell differentiation in respiratory host defence and could provide an additional explanation for the increased susceptibility of smokers and patients with chronic obstructive pulmonary disease to respiratory infections.

Proinflammatory Cytokines Impair Vitamin D-Induced Host Defense in Cultured Airway Epithelial Cells.

Vitamin D is a regulator of host defense against infections and induces expression of the antimicrobial peptide hCAP18/LL-37. Vitamin D deficiency is associated with chronic inflammatory lung diseases and respiratory infections. However, it is incompletely understood if and how (chronic) airway inflammation affects vitamin D metabolism and action. We hypothesized that long-term exposure of primary bronchial epithelial cells to proinflammatory cytokines alters their vitamin D metabolism, antibacterial activity, and expression of hCAP18/LL-37. To investigate this, primary bronchial epithelial cells were differentiated at the air-liquid interface for 14 days in the presence of the proinflammatory cytokines, TNF-α and IL-1ß (TNF-α/IL-1ß), and subsequently exposed to vitamin D (inactive 25(OH)D3 and active 1,25(OH)2D3). Expression of hCAP18/LL-37, vitamin D receptor, and enzymes involved in vitamin D metabolism (CYP24A1 and CYP27B1) was determined using quantitative PCR, Western blot, and immunofluorescence staining. Furthermore, vitamin D-mediated antibacterial activity was assessed using nontypeable Haemophilus influenzae. We found that TNF-α/IL-1ß treatment reduced vitamin D-induced expression of hCAP18/LL-37 and killing of nontypeable H. influenzae. In addition, CYP24A1 (a vitamin D-degrading enzyme) was increased by TNF-α/IL-1ß, whereas CYP27B1 (that converts 25(OH)D3 to its active form) and vitamin D receptor expression remained unaffected. Furthermore, we have demonstrated that the TNF-α/IL-1ß-mediated induction of CYP24A1 was, at least in part, mediated by the transcription factor specific protein 1, and the epidermal growth factor receptor-mitogen-activated protein kinase pathway. These findings indicate that TNF-α/IL-1ß decreases vitamin D-mediated antibacterial activity and hCAP18/LL-37 expression via induction of CYP24A1 and suggest that chronic inflammation impairs protective responses induced by vitamin D.

Antibacterial Defense of Human Airway Epithelial Cells from Chronic Obstructive Pulmonary Disease Patients Induced by Acute Exposure to Nontypeable Haemophilus influenzae: Modulation by Cigarette Smoke.

Antimicrobial proteins and peptides (AMPs) are a central component of the antibacterial activity of airway epithelial cells. It has been proposed that a decrease in antibacterial lung defense contributes to an increased susceptibility to microbial infection in smokers and patients with chronic obstructive pulmonary disease (COPD). However, whether reduced AMP expression in the epithelium contributes to this lower defense is largely unknown. We investigated the bacterial killing activity and expression of AMPs by air-liquid interface-cultured primary bronchial epithelial cells from COPD patients and non-COPD (ex-)smokers that were stimulated with nontypeable Haemophilus influenzae (NTHi). In addition, the effect of cigarette smoke on AMP expression and the activation of signaling pathways was determined. COPD cell cultures displayed reduced antibacterial activity, whereas smoke exposure suppressed the NTHi-induced expression of AMPs and further increased IL-8 expression in COPD and non-COPD cultures. Moreover, smoke exposure impaired NTHi-induced activation of NF-κB, but not MAP-kinase signaling. Our findings demonstrate that the antibacterial activity of cultured airway epithelial cells induced by acute bacterial exposure was reduced in COPD and suppressed by cigarette smoke, whereas inflammatory responses persisted. These findings help to explain the imbalance between protective antibacterial and destructive inflammatory innate immune responses in COPD.

Prevention of exacerbations in patients with COPD and vitamin D deficiency through vitamin D supplementation (PRECOVID): a study protocol.

BACKGROUND: Vitamin D is well known for its function in calcium homeostasis and bone mineralisation, but is increasingly studied for its potential immunomodulatory properties. Vitamin D deficiency is a common problem in patients with COPD. Previous studies have not demonstrated a beneficial effect of vitamin D on exacerbation rate in COPD patients. However, subgroup analyses suggested protective effects in vitamin D deficient patients. Our objective is to assess the effect of vitamin D supplementation on exacerbation rate specifically in vitamin D deficient COPD patients. METHODS/DESIGN: We will perform a randomised, multi-center, double-blind, placebo-controlled intervention study. The study population consists of 240 COPD patients aged 40 years and older with vitamin D deficiency (25-hydroxyvitamin D concentration < 50 nmol/L). Participants will be recruited after an exacerbation and will be randomly allocated in a 1:1 ratio to receive vitamin D3 16800 IU or placebo orally once a week during 1 year. Participants will receive a diary card to register the incidence of exacerbations and changes in medication during the study period. Visits will be performed at baseline, at 6 months and at 12 months after randomisation. Participants will undergo spirometry, measurement of total lung capacity and assessment of maximal respiratory mouth pressure. Several physical performance and hand grip strength tests will be performed, questionnaires on quality of life and physical activity will be filled in, a nasal secretion sample and swab will be obtained and blood samples will be taken. The primary outcome will be exacerbation rate. DISCUSSION: This study will be the first RCT aimed at the effects of vitamin D supplementation on exacerbation rate in vitamin D deficient COPD patients. Also, in contrast to earlier studies that used infrequent dosing regimens, our trial will study effects of a weekly dose of vitamin D supplementation. Secondly, the immunomodulatory effects of vitamin D on host immune response of COPD patients and underlying mechanisms will be studied. Finally, the effects on physical functioning will be examined. TRIAL REGISTRATION: This trial is registered in ClinicalTrials.gov, ID number NCT02122627 . Date of Registration April 2014.

IL-4 and IL-13 exposure during mucociliary differentiation of bronchial epithelial cells increases antimicrobial activity and expression of antimicrobial peptides.

The airway epithelium forms a barrier against infection but also produces antimicrobial peptides (AMPs) and other inflammatory mediators to activate the immune system. It has been shown that in allergic disorders, Th2 cytokines may hamper the antimicrobial activity of the epithelium. However, the presence of Th2 cytokines also affects the composition of the epithelial layer which may alter its function. Therefore, we investigated whether exposure of human primary bronchial epithelial cells (PBEC) to Th2 cytokines during mucociliary differentiation affects expression of the human cathelicidin antimicrobial protein (hCAP18)/LL-37 and human beta defensins (hBD), and antimicrobial activity.PBEC were cultured at an air-liquid interface (ALI) for two weeks in the presence of various concentrations of IL-4 or IL-13. Changes in differentiation and in expression of various AMPs and the antimicrobial proteinase inhibitors secretory leukocyte protease inhibitor (SLPI) and elafin were investigated as well as antimicrobial activity.IL-4 and IL-13 increased mRNA expression of hCAP18/LL-37 and hBD-2. Dot blot analysis also showed an increase in hCAP18/LL-37 protein in apical washes of IL-4-treated ALI cultures, whereas Western Blot analysis showed expression of a protein of approximately 4.5 kDa in basal medium of IL-4-treated cultures. Using sandwich ELISA we found that also hBD-2 in apical washes was increased by both IL-4 and IL-13. SLPI and elafin levels were not affected by IL-4 or IL-13 at the mRNA or protein level. Apical wash obtained from IL-4- and IL-13-treated cultures displayed increased antimicrobial activity against Pseudomonas aeruginosa compared to medium-treated cultures. In addition, differentiation in the presence of Th2 cytokines resulted in increased MUC5AC production as has been shown previously.These data suggest that prolonged exposure to Th2 cytokines during mucociliary differentiation contributes to antimicrobial defence by increasing the expression and release of selected antimicrobial peptides and mucus.

Smoking status and anti-inflammatory macrophages in bronchoalveolar lavage and induced sputum in COPD.

BACKGROUND: Macrophages have been implicated in the pathogenesis of COPD. M1 and M2 macrophages constitute subpopulations displaying pro- and anti-inflammatory properties. We hypothesized that smoking cessation affects macrophage heterogeneity in the lung of patients with COPD. Our aim was to study macrophage heterogeneity using the M2-marker CD163 and selected pro- and anti-inflammatory mediators in bronchoalveolar lavage (BAL) fluid and induced sputum from current smokers and ex-smokers with COPD. METHODS: 114 COPD patients (72 current smokers; 42 ex-smokers, median smoking cessation 3.5 years) were studied cross-sectionally and underwent sputum induction (M/F 99/15, age 62 ± 8 [mean ± SD] years, 42 (31-55) [median (range)] packyears, post-bronchodilator FEV1 63 ± 9% predicted, no steroids past 6 months). BAL was collected from 71 patients. CD163+ macrophages were quantified in BAL and sputum cytospins. Pro- and anti-inflammatory mediators were measured in BAL and sputum supernatants. RESULTS: Ex-smokers with COPD had a higher percentage, but lower number of CD163+ macrophages in BAL than current smokers (83.5% and 68.0%, p = 0.04; 5.6 and 20.1 × 10(4)/ml, p = 0.001 respectively). The percentage CD163+ M2 macrophages was higher in BAL compared to sputum (74.0% and 30.3%, p < 0.001). BAL M-CSF levels were higher in smokers than ex-smokers (571 pg/ml and 150 pg/ml, p = 0.001) and correlated with the number of CD163+ BAL macrophages (Rs = 0.38, p = 0.003). No significant differences were found between smokers and ex-smokers in the levels of pro-inflammatory (IL-6 and IL-8), and anti-inflammatory (elafin, and Secretory Leukocyte Protease Inhibitor [SLPI]) mediators in BAL and sputum. CONCLUSIONS: Our data suggest that smoking cessation partially changes the macrophage polarization in vivo in the periphery of the lung towards an anti-inflammatory phenotype, which is not accompanied by a decrease in inflammatory parameters.

Airway proteoglycans are differentially altered in fatal asthma.

It has been suggested that airway remodelling is responsible for the persistent airway obstruction and decline in lung function observed in some asthmatic patients. The small airways are thought to contribute significantly to this functional impairment. Proteoglycans (PGs) are important components of the extracellular matrix (ECM) in the lungs. Besides controlling biophysical properties of the ECM, they play important roles in the regulation of some cytokines. Increased subepithelial PG deposition in the airways of mild asthmatics has been reported. However, there are no data on the PG content in small airways in asthma. This study has compared the content and distribution of PGs in large and small airways of patients who died of asthma with those in control lungs. Immunohistochemistry and image analysis were used to determine the content of lumican, decorin, biglycan, and versican in large (internal perimeter >6 mm) and small (internal perimeter < or =6 mm) airways of 18 patients who had died of asthma (A) and ten controls (C). The results were expressed as PG area (microm2)/epithelial basement membrane length (microm). The main differences between asthmatics and controls were observed in the small airways. There was a significant decrease in decorin and lumican contents in the external area of small airways in asthmatics (decorin: A = 1.05 +/- 0.27 microm, C = 3.97 +/- 1.17 microm, p = 0.042; lumican: A = 1.97 +/- 0.37 microm, C = 5.66 +/- 0.99 microm, p = 0.002). A significant increase in versican content in the internal area of small and large airways in asthmatics was also observed (small: A = 7.48 +/- 0.84 microm, C = 5.16 +/- 0.61 microm, p = 0.045; large: A = 18.38 +/- 1.94 microm, C = 11.90 +/- 2.86 microm, p = 0.028). The results show that PGs are differentially expressed in the airways of fatal asthma and may contribute to airway remodelling. These data reinforce the importance of the small airways in airway remodelling in asthma.

Are rhinovirus-induced airway responses in asthma aggravated by chronic allergen exposure?

Airway inflammation in asthma may represent a favorable environment for respiratory viral infections, augmenting virus-induced exacerbations in asthma. We postulated that repeated low-dose allergen exposure preceding experimental rhinovirus 16 (RV16) infection increases the severity of RV-induced airway obstruction and inflammation. Thirty-six house dust mite-allergic patients with mild to moderate asthma participated in a three-arm, parallel, placebo-controlled, double-blind study. Patients inhaled a low dose of house dust mite allergen for 10 subsequent working days (Days 1-5 and 8-12) and/or were subsequently infected with RV16 (Days 15 and 16). Allergen exposure resulted in a significant fall in FEV1 (p < 0.001) and provocative concentration of histamine causing a 20% fall in FEV1 (p < 0.001) and an increase in exhaled nitric oxide (p < 0.001) and percentage of sputum eosinophils (p < 0.001). RV16 infection led to a fall in FEV1 (p = 0.02) and increases in the percentage of sputum neutrophils (p = 0.01), sputum interleukin-8 (p = 0.04), and neutrophil elastase (p = 0.04). Successive allergen exposure and RV16 infection had no synergistic or additive effect on any of the clinical or inflammatory outcomes. In conclusion, repeated low-dose allergen exposure and RV16 infection induce distinct inflammatory profiles within the airways in asthma without apparent interaction between these two environmental triggers. This suggests that preceding allergen exposure, at the used dose and duration, is not a determinant of the severity of RV-induced exacerbations in patients with mild to moderate asthma.

Asymptomatic worsening of airway inflammation during low-dose allergen exposure in asthma: protection by inhaled steroids.

Asthma is a chronic inflammatory disease that persists even during adequate therapy and asymptomatic episodes. We questioned whether "silent" chronic allergen exposure can induce and maintain airway inflammation and whether this still occurs during regular treatment with inhaled steroids. Twenty-six patients with house dust mite allergy and mild asthma (dual responders) participated in a parallel, double-blind study. All patients inhaled a low-dose of allergen on 10 subsequent working days (Days 1-5, 8-12). They were treated with 400 micro g budesonide once daily (n = 13) or placebo (n = 13) from Days -3 to 19. At baseline (Day -6) and on Days 5, 12, and 19 we measured the provocative concentration of methacholine causing a 20% fall in FEV(1) (PC(20)), and percent eosinophils, interleukin (IL)-5/interferon-gamma messenger RNA ratio (in sputum cells by real-time reverse transcription-polymerase chain reaction [RT-PCR]), and eosinophilic cationic protein (ECP) in induced sputum. Symptoms, peak expiratory flow (PEF), FEV(1), and exhaled nitric oxide (NO) were recorded repeatedly during the study. In the placebo group, repeated low-dose allergen exposure resulted in a significant increase in sputum eosinophils (p = 0.043), ECP (p = 0.011), IL-5/IFN-gamma messenger RNA ratio (p = 0.04), and in exhaled NO (p = 0.001), without worsening of symptoms, PEF, or baseline FEV(1) (p > 0.07). In the budesonide group, the changes in PC(20), sputum ECP, and exhaled NO were significantly different as compared with the placebo group (p < 0.03). We conclude that repeated low-dose allergen exposure in asthma can lead to airway inflammation without worsening of symptoms, which can be prevented by inhaled steroid treatment. This suggests that antiinflammatory therapy is beneficial during allergen exposure, even during asymptomatic episodes.