Can we attenuate ischaemia-reperfusion injury of allografts in a porcine left lung transplant models by adsorption of cytokines?

OBJECTIVES: Primary graft dysfunction resulting from ischaemia-reperfusion injury remains a major obstacle after lung transplantation (LTx) and is associated with morbidity and mortality. Continuous release of inflammatory cytokines, due to the process of ischaemia and reperfusion, triggers a complex cascade of apoptosis and necrosis resulting in graft dysfunction. Previous studies demonstrated successful graft improvement by cytokine filtration during ex vivo lung perfusion. We hypothesize that plasma cytokine filtration with CytoSorb® during in vivo graft perfusion immediately after implantation may attenuate ischaemia-reperfusion injury after left LTx in a porcine model. METHODS: Left porcine LTx was performed with allografts preserved for 24 h at 4°C. In the treatment group [T] (n = 7), a veno-venous shunt was created to insert the cytokine filter (CytoSorbents, Berlin, Germany). In the sham group [S] (n = 4), the shunt was created without the filter. Haemodynamic parameters, lung mechanics, blood gases and plasma cytokines were assessed during 6 h in vivo reperfusion. RESULTS: During 6 h of reperfusion, significant differences in plasma pro-inflammatory cytokine [interferon (IFN)-α, IFN-γ and interleukin (IL)-6] concentrations were observed between [T] and [S], but surprisingly with higher plasma levels in the [T] group. Plasma concentrations of other pro-inflammatory cytokines (IL-1ß, IL-12p40, IL-4, IL-6, IL-8, IFN-α, IFN-γ and tumour necrosis factor-α) and anti-inflammatory cytokines (IL-10) did not find any evidence for a difference. Furthermore, our study failed to show meaningful difference in haemodynamics and blood gases. Also, no statistically significant differences were found between [T] and [S] in biopsies and wet-to-dry ratio at the end of the experiment. CONCLUSIONS: In our porcine left LTx model cytokine filtration did not achieve the intended effect. This is in contrast to previous studies with CytoSorb use during ex vivo lung perfusion as a surrogate LTx model. Our findings might highlight the fact that the theoretical benefit of inserting an additional cytokine adsorber to improve graft function in clinical practice should be critically evaluated with further studies.

Effects of repeated infections with non-typeable Haemophilus influenzae on lung in vitamin D deficient and smoking mice.

BACKGROUND: In chronic obstructive pulmonary disease (COPD), exacerbations cause acute inflammatory flare-ups and increase the risk for hospitalization and mortality. Exacerbations are common in all disease stages and are often caused by bacterial infections e.g., non-typeable Heamophilus influenzae (NTHi). Accumulating evidence also associates vitamin D deficiency with the severity of COPD and exacerbation frequency. However, it is still unclear whether vitamin D deficiency when combined with cigarette smoking would worsen and prolong exacerbations caused by repeated infections with the same bacterial strain. METHODS: Vitamin D sufficient (VDS) and deficient (VDD) mice were exposed to nose-only cigarette smoke (CS) for 14 weeks and oropharyngeally instilled with NTHi at week 6, 10 and 14. Three days after the last instillation, mice were assessed for lung function, tissue remodeling, inflammation and immunity. The impact of VDD and CS on inflammatory cells and immunoglobulin (Ig) production was also assessed in non-infected animals while serum Ig production against NTHi and dsDNA was measured in COPD patients before and 1 year after supplementation with Vitamin D3. RESULTS: VDD enhanced NTHi eradication, independently of CS and complete eradication was reflected by decreased anti-NTHi Ig's within the lung. In addition, VDD led to an increase in total lung capacity (TLC), lung compliance (Cchord), MMP12/TIMP1 ratio with a rise in serum Ig titers and anti-dsDNA Ig's. Interestingly, in non-infected animals, VDD exacerbated the CS-induced anti-NTHi Ig's, anti-dsDNA Ig's and inflammatory cells within the lung. In COPD patients, serum Ig production was not affected by vitamin D status but anti-NTHi IgG increased after vitamin D3 supplementation in patients who were Vitamin D insufficient before treatment. CONCLUSION: During repeated infections, VDD facilitated NTHi eradication and resolution of local lung inflammation through production of anti-NTHi Ig, independently of CS whilst it also promoted autoantibodies. In COPD patients, vitamin D supplementation could be protective against NTHi infections in vitamin D insufficient patients. Future research is needed to decipher the determinants of dual effects of VDD on adaptive immunity. TRAIL REGISTRATION: ClinicalTrials, NCT00666367. Registered 23 April 2008, https://www.clinicaltrials.gov/ct2/show/study/NCT00666367 .

Local nebulization of 1α,25(OH)2D3 attenuates LPS-induced acute lung inflammation.

BACKGROUND: Evidence supports a critical role of vitamin D status on exacerbation in chronic obstructive pulmonary disease, indicating the need to avoid vitamin D deficiency in these patients. However, oral vitamin D supplementation is limited by the potential risk for hypercalcemia. In this study, we investigated if local delivery of vitamin D to the lungs improves vitamin D-mediated anti-inflammatory action in response to acute inflammation without inducing hypercalcemia. METHODS: We studied vitamin D sufficient (VDS) or deficient (VDD) mice in whom 1α,25(OH)2D3 (0.2 µg/kg) or a vehicle followed by lipopolysaccharide (LPS 25 µg) were delivered to the lung as a micro-spray. RESULTS: Local 1α,25(OH)2D3 reduced LPS-induced inflammatory cells in bronchoalveolar lavage (BAL) in VDS (absolute number of cells: - 57% and neutrophils - 51% p < 0.01) and tended to diminish LPS-increased CXCL5 BAL levels in VDS (- 40%, p = 0.05) while it had no effect on CXCL1 and CXCL2 in BAL and mRNA in lung of VDS and VDD. It also significantly attenuated the increased IL-13 in BAL and lung, especially in VDD mice (- 41 and - 75%, respectively). mRNA expression of Claudin-18 in lung was significantly lower in VDS mice with local 1α,25(OH)2D3 while Claudin-3, -5 and -8 mRNA levels remained unchanged. Finally, in VDD mice only, LPS reduced lung mRNA expression of adhesion junction Zona-occludens-1, in addition to increasing uric acid and total protein in BAL, which both were prevented by local 1α,25(OH)2D3. CONCLUSION: Under normal levels of vitamin D, local 1α,25(OH)2D3 nebulization into the lung efficiently reduced LPS induction of inflammatory cells in BAL and slightly attenuated LPS-increase in CXCL5. In case of severe vitamin D deficiency, although local 1α,25(OH)2D3 nebulization failed to significantly minimize cellular inflammation in BAL at this dose, it prevented epithelial barrier leakage and damage in lung. Additional research is needed to determine the potential long-term beneficial effects of local 1α,25(OH)2D3 nebulization on lung inflammation.

Enhanced lung inflammatory response in whole-body compared to nose-only cigarette smoke-exposed mice.

BACKGROUND: Chronic obstructive pulmonary disease (COPD) is characterized by a progressive and abnormal inflammatory response in the lungs, mainly caused by cigarette smoking. Animal models exposed to cigarette smoke (CS) are used to mimic human COPD but the use of different CS protocols makes it difficult to compare the immunological and structural consequences of using a nose-only or whole-body CS exposure system. We hypothesized that when using a standardized CS exposure protocol based on particle density and CO (carbon monoxide) levels, the whole-body CS exposure system would generate a more severe inflammatory response than the nose-only system, due to possible sensitization by uptake of CS-components through the skin or via grooming. METHODS: In this study focusing on early COPD, mice were exposed twice daily 5 days a week to CS either with a nose-only or whole-body exposure system for 14 weeks to assess lung function, remodeling and inflammation. RESULTS: At sacrifice, serum cotinine levels were significantly higher in the whole-body (5.3 (2.3-6.9) ng/ml) compared to the nose-only ((2.0 (1.8-2.5) ng/ml) exposure system and controls (1.0 (0.9-1.0) ng/ml). Both CS exposure systems induced a similar degree of lung function impairment, while inflammation was more severe in whole body exposure system. Slightly more bronchial epithelial damage, mucus and airspace enlargement were observed with the nose-only exposure system. More lymphocytes were present in the bronchoalveolar lavage (BAL) and lymph nodes of the whole-body exposure system while enhanced IgA and IgG production was found in BAL and to a lesser extent in serum with the nose-only exposure system. CONCLUSION: The current standardized CS-exposure protocol resulted in a higher internal load of serum cotinine in the whole-body exposure system, which was associated with more inflammation. However, both exposure systems resulted in a similar lung function impairment. Data also highlighted differences between the two models in terms of lung inflammation and remodelling, and potential sensitization to CS. Researchers should be aware of these differences when designing their future studies for an early intervention in COPD.

Post hoc analysis of a randomised controlled trial: effect of vitamin D supplementation on circulating levels of desmosine in COPD.

BACKGROUND: Vitamin D supplementation lowers exacerbation frequency in severe vitamin D-deficient patients with COPD. Data regarding the effect of vitamin D on elastin degradation are lacking. Based on the vitamin's anti-inflammatory properties, we hypothesised that vitamin D supplementation reduces elastin degradation, particularly in vitamin D-deficient COPD patients. We assessed the effect of vitamin D status and supplementation on elastin degradation by measuring plasma desmosine, a biomarker of elastin degradation. METHODS: Desmosine was measured every 4 months in plasma of 142 vitamin D-naïve COPD patients from the Leuven vitamin D intervention trial (100 000 IU vitamin D3 supplementation every 4 weeks for 1 year). RESULTS: No significant association was found between baseline 25-hydroxyvitamin D (25(OH)D) and desmosine levels. No significant difference in desmosine change over time was found between the placebo and intervention group during the course of the trial. In the intervention arm, an unexpected inverse association was found between desmosine change and baseline 25(OH)D levels (p=0.005). CONCLUSIONS: Vitamin D supplementation did not have a significant overall effect on elastin degradation compared to placebo. Contrary to our hypothesis, the intervention decelerated elastin degradation in vitamin D-sufficient COPD patients and not in vitamin D-deficient subjects.

Local expression profiles of vitamin D-related genes in airways of COPD patients.

Treatment of Chronic Obstructive Pulmonary Disease (COPD) is based on bronchodilation, with inhaled corticosteroids or azithromycin associated when frequent exacerbations occur. Despite the proven benefits of current treatment regimens, the need for new interventions in delineated subgroups remains. There is convincing evidence for oral vitamin D supplementation in reducing exacerbations in COPD patients severely deficient for circulating vitamin D. However, little is known about local vitamin D metabolism in the airways and studies examining expression of the vitamin D receptor (VDR), the activating enzyme (CYP27B1) and inactivating enzyme (CYP24A1) of vitamin D in lung tissue of COPD patients are lacking. Therefore, the expression and localization of key enzymes and the receptor of the vitamin D pathway were examined in tissue of 10 unused donor lungs and 10 COPD explant lungs. No differences in the expression of CYP27B1 and CYP24A1 were found. Although protein expression of VDR was significantly lower in COPD explant tissue, there was no difference in downstream expression of the antimicrobial peptide cathelicidin. Whereas CYP27B1 and CYP24A1 were present in all layers of the bronchial epithelium, VDR was only expressed at the apical layer of a fully differentiated bronchial epithelium with no expression in vascular endothelial cells. By contrast, CYP24A1 expression was highly present in lung endothelial cells suggesting that systemic vitamin D can be inactivated before reaching the epithelial compartment and the tissue immune cells. These data support the idea of exploring the role of vitamin D inhalation in patients with COPD.

Targeting Vitamin D Deficiency to Limit Exacerbations in Respiratory Diseases: Utopia or Strategy With Potential?

Patients with respiratory diseases such as cystic fibrosis, chronic obstructive pulmonary disease, or asthma often experience an acute worsening of respiratory symptoms, termed exacerbations. Although the course of exacerbations is disease specific, they are mostly triggered by a respiratory infection. Exacerbations often require hospitalization and are an important cause of mortality. Treatments of exacerbations aim to minimize the negative impact and to prevent subsequent events. Despite many existing therapy options, many patients do not benefit from therapy and suffer from recurrent events. Vitamin D deficiency is a worldwide problem and is extremely prevalent in these patients. Vitamin D, known for its calcemic effects, also has immunomodulatory and anti-infectious actions and can therefore be a possible agent to treat or prevent exacerbations. This review will focus on vitamin D as a potential candidate to treat or prevent exacerbations in CF, COPD, and asthma.

Distinct autophagy-apoptosis related pathways activated by Multi-walled (NM 400) and Single-walled carbon nanotubes (NIST-SRM2483) in human bronchial epithelial (16HBE14o-) cells.

Given the recent development in the field of particle and fibre toxicology, parallels have been drawn between Carbon nanotubes (CNTs) and asbestos. It is now established that both multi-walled (MWCNTs) and single-walled (SWCNTs) carbon nanotubes might contribute to pulmonary disease. Although multiple mechanisms might be involved in CNT induced pathogenesis, systematic understanding of the relationship between different CNT exposure (MWCNT vs SWCNT) and autophagy/ apoptosis/ necrosis, in human lung epithelial cells remains limited. In this study, we demonstrate that exposure to MWCNT (NM-400), but not SWCNT (NIST-SRM2483), leads to an autophagic response after acute exposure (24 h). MWCNT exposure was characterized by an increase in anti-apoptotic BCL2, downregulation of executor Caspase-3/7 and increase in expression of genes from the autophagy machinery. For SWCNT exposure however, we observed an overexpression of executor Caspase-3/7 and upregulation of pro-apoptotic BAX; enrichment for processes like cornification, apoptotic process, cell differentiation from proteomic analysis. These results clearly indicate a major difference in the pathways initiated by the CNTs, in vitro. While the present study design provides mechanistic understanding after an acute exposure for the tested CNTs, we believe that the information obtained here would have relevance in better understanding of CNT toxicity and pathogenesis in general.

Vitamin D Modulates the Response of Bronchial Epithelial Cells Exposed to Cigarette Smoke Extract.

In chronic obstructive pulmonary disease (COPD), the bronchial epithelium is the first immune barrier that is triggered by cigarette smoke. Although vitamin D (vitD) has proven anti-inflammatory and antimicrobial effects in alveolar macrophages, little is known about the direct role of vitD on cigarette smoke-exposed bronchial epithelial cells. We examined the effects of vitD on a human bronchial epithelial cell line (16HBE) and on air-liquid culture of primary bronchial epithelial cells (PBEC) of COPD patients and controls exposed for 24 h to cigarette smoke extract (CSE). VitD decreased CSE-induced IL-8 secretion by 16HBE cells, but not by PBEC. VitD significantly increased the expression of the antimicrobial peptide cathelicidin in 16HBE and PBEC of both COPD subjects and controls. VitD did not affect epithelial to mesenchymal transition or epithelial MMP-9 expression and was not able to restore impaired wound healing by CSE in 16HBE cells. VitD increased the expression of its own catabolic enzyme CYP24A1 thereby maintaining its negative feedback. In conclusion, vitD supplementation may potentially reduce infectious exacerbations in COPD by the upregulation of cathelicidin in the bronchial epithelium.

Airway morphometry in COPD with bronchiectasis: a view on all airway generations.

The pathophysiological processes underlying bronchiectasis in chronic obstructive pulmonary disease (COPD) are not understood. In COPD, both small and large airways are progressively lost. It is currently not known to what extent the different airway generations of patients with COPD and bronchiectasis are involved.COPD explant lungs with bronchiectasis were compared to COPD explant lungs without bronchiectasis and unused donor lungs as controls. In order to investigate all airway generations, a multimodal imaging approach using different resolutions was conducted. Per group, five lungs were frozen (n=15) and underwent computed tomography (CT) imaging for large airway evaluation, with four tissue cores per lung imaged for measurements of the terminal bronchioles. Two additional lungs per group (n=6) were air-dried for lobar microCT images that allow airway segmentation and three-dimensional quantification of the complete airway tree.COPD lungs with bronchiectasis had significantly more airways compared to COPD lungs without bronchiectasis (p<0.001), with large airway numbers similar to control lungs. This difference was present in both upper and lower lobes. Lack of tapering was present (p=0.010) and larger diameters were demonstrated in lower lobes with bronchiectasis (p=0.010). MicroCT analysis of tissue cores showed similar reductions of tissue percentage, surface density and number of terminal bronchioles in both COPD groups compared to control lungs.Although terminal bronchioles were equally reduced in COPD lungs with and without bronchiectasis, significantly more large and small airways were found in COPD lungs with bronchiectasis.

Data on inflammatory cytokines and pathways involved in clearance of Nontypeable Haemophilus influenzae from the lungs during cigarette smoking and vitamin D deficiency.

This article contains data related to the inflammatory cytokine and investigated pathways involved in bacterial clearance reported in "Airway infection with Nontypeable Haemophilus influenzae is more rapidly eradicated in vitamin D deficient mice" (Serré et al., 2018) [1]. Vitamin D deficient or sufficient mice were oropharyngeally instilled with 106 NTHi and sacrificed at 4, 8, 24 and 72 h post-infection. We measured proinflammatory cytokines (KC, TNF-α, IL-1ß, IL6 and MCP-1) markers of bacterial clearance pathways (myeloid peroxidase, nitric oxide, complement C5a and immunoglobulin A) in bronchoalveolar fluid (BALF) during infection and mRNA expression levels of innate immune defense mechanism markers (mucin glycoproteins, pathogen recognitions receptor TLR2 and TLR4, antimicrobial peptides SLPI, REG3γ, lysozyme, BD-1, BD-2, BD-3 and surfactant proteins SP-A and SP-D) in lung homogenate. Finally, genomic DNA of NTHi (protein D) measured in lung homogenate was used as an indicator of NTHi invasion of alveolar macrophages or epithelial cells.

Vitamin D to prevent exacerbations of COPD: systematic review and meta-analysis of individual participant data from randomised controlled trials.

BACKGROUND: Randomised controlled trials (RCTs) of vitamin D to prevent COPD exacerbations have yielded conflicting results.Individual participant data meta-analysis could identify factors that explain this variation. METHODS: PubMed, Embase, the Cochrane Central Register of Controlled Trials and Web of Science were searched from inception up to and including 5 October 2017 to identify RCTs of vitamin D supplementation in patients with COPD that reported incidence of acute exacerbations. Individual participant data meta-analysis was performed using fixed effects models adjusting for age, sex, Global Initiative for Chronic Obstructive Lung Disease spirometric grade and trial. RESULTS: Four eligible RCTs (total 560 participants) were identified; individual participant data were obtained for 469/472 (99.4%) participants in three RCTs. Supplementation did not influence overall rate of moderate/severe COPD exacerbations (adjusted incidence rate ratio (aIRR) 0.94, 95% CI 0.78 to 1.13). Prespecified subgroup analysis revealed that protective effects were seen in participants with baseline 25-hydroxyvitamin D levels <25 nmol/L (aIRR 0.55, 95% CI 0.36 to 0.84) but not in those with baseline 25-hydroxyvitamin D levels ≥25 nmol/L (aIRR 1.04, 95% CI 0.85 to 1.27; p for interaction=0.015). Vitamin D did not influence the proportion of participants experiencing at least one serious adverse event (adjusted OR 1.16, 95% CI 0.76 to 1.75). CONCLUSIONS: Vitamin D supplementation safely and substantially reduced the rate of moderate/severe COPD exacerbations in patients with baseline 25-hydroxyvitamin D levels <25 nmol/L but not in those with higher levels. TRIAL REGISTRATION NUMBER: CRD42014013953.

Airway infection with Nontypeable Haemophilus influenzae is more rapidly eradicated in vitamin D deficient mice.

Chronic obstructive pulmonary disease (COPD), which is characterized by an excessive inflammatory response of the airways, is often complicated by exacerbations. Vitamin D deficiency has been associated with an increased risk for COPD and may predispose COPD patients to a higher exacerbation rate, particularly during smoking. In the current study, we investigated the effect of vitamin D deficiency and cigarette smoke (CS)-exposure on lung inflammation and bacterial clearance after an acute infection with Nontypeable Haemophilus influenzae (NTHi). Vitamin D deficient or sufficient mice were exposed to nose-only CS or ambient air for 6 weeks and oropharyngeally instilled with 106 NTHi. Residual viable NTHi were measured at different time points post-infection. Mechanisms of bacterial clearance (e.g. phagocytosis, pattern recognition receptors, antimicrobial peptides, surfactant proteins and mucin) and lung remodeling (e.g. metalloproteinases, MMP's) were assessed. Although smoking resulted in reduced phagocytosis capacity of macrophages and neutrophils, bacterial clearance was similar to control mice. By contrast and independent of smoking, bacterial clearance was significantly accelerated in vitamin D deficient mice already from 24 h post-infection (p = 0.0087). This faster and complete eradication was associated with a more rapid resolution of cytokines and neutrophils 72 h post-infection and dominated by an upregulation of cathelicidin-related antimicrobial peptide (CRAMP) mRNA during infection (p = 0.026). However, vitamin D deficiency also resulted in more MMP12 protein in broncho-alveolar lavage and a shift in mRNA expression of MMP12/TIMP1 (p = 0.038) and MMP9/TIMP1 (p = 0.024) ratio towards more protease activity. Overall, vitamin D deficient mice resolved NTHi infection faster with a faster resolution of local lung inflammation, possibly through upregulation of CRAMP. This was associated with a disruption of the protease/anti-protease balance, which may potentially scale towards a higher extracellular matrix breakdown.

Vitamin D supplementation in respiratory diseases: evidence from randomized controlled trials.

Pulmonary diseases are one of the most important causes of morbidity and mortality. Although vitamin D is best known for its role in calcium, phosphorus, and bone homeostasis, it has gained attention in the recent years because of a wide range of extraskeletal effects, including its immunomodulatory and antibacterial potential. Vitamin D deficiency is highly prevalent in chronic pulmonary diseases such as chronic obstructive pulmonary disease (COPD), cystic fibrosis, tuberculosis, and asthma, and several clinical studies have been conducted investigating the effect of vitamin D supplementation on disease outcomes. In this review, we searched for positive evidence on vitamin D supplementation from randomized controlled trials and elaborated on the optimal serum vitamin D levels and dosing regimens for an effective intervention. While vitamin D supplementation seems to be beneficial as an add­on treatment for adult patients with asthma and a potent intervention to reduce exacerbations in patients with COPD, there is little evidence for its therapeutic use in cystic fibrosis, pneumonia, and tuberculosis.

Interleukin-1α induced release of interleukin-8 by human bronchial epithelial cells in vitro: assessing mechanisms and possible treatment options.

Survival after lung transplantation is hampered by chronic lung allograft dysfunction (CLAD). Persistently elevated BAL-neutrophilia is observed in some patients despite treatment with azithromycin, which may be induced by IL-1α. Our aim is to establish an in vitro model, assess mechanistic pathways and test different therapeutic strategies of IL-1α-induced release of IL-8 by human bronchial epithelial cells. Bronchial epithelial cells (16HBE) were stimulated with IL-1α with or without azithromycin or dexamethasone. IL-8 protein was analyzed in cell supernatant. Different MAP kinases (p38, JNK, ERK1/2 , Iκß) and targets known to be involved in tumor formation (PI3K, Akt) were investigated. Finally, different treatment options were tested for their potential inhibitory effect. IL-1α induced IL-8 in bronchial epithelial cells, which was dose-dependently inhibited by dexamethasone but not by azithromycin. IL-1α induced p38 and Akt phosphorylation, but activation of these MAPK was not inhibited by dexamethasone. JNK, ERK1/2 , Iκß and PI3K were not activated. None of the tested drugs reduced the IL-1α induced IL-8 production. We established an in vitro model wherein steroids inhibit the IL-1α-induced IL-8 production, while azithromycin was ineffective. Despite using this simple in vitro model, we could not identify a new treatment option for azithromycin-resistant airway neutrophilia.

1,25-Dihydroxyvitamin D Modulates Antibacterial and Inflammatory Response in Human Cigarette Smoke-Exposed Macrophages.

Cigarette smoking is associated with increased inflammation and defective antibacterial responses in the airways. Interestingly, vitamin D has been shown to suppress inflammation and to improve antibacterial defense. However, it is currently unknown whether vitamin D may modulate inflammation and antibacterial defects in human cigarette smoke (CS)-exposed airways. To explore these unresolved issues, alveolar macrophages obtained from non-smoking and smoking subjects as well as human cigarette smoke extract (CSE)-treated THP-1 macrophages were stimulated with 1,25-dihydroxyvitamin D (1,25(OH)2D) to address inflammatory and antibacterial responses. Although basal levels of inflammatory cytokines and chemokines did not differ between non-smoking and smoking subjects, 1,25(OH)2D did reduce levels of IL-6, TNF-α and MCP-1 in alveolar macrophages in response to LPS/IFN-γ, although not statistically significant for TNF-α and IL-6 in smokers. CSE did not significantly alter vitamin D metabolism (expression levels of CYP24A1 or CYP27B1) in THP-1 macrophages. Furthermore, stimulation with 1,25(OH)2D reduced mRNA expression levels and/or protein levels of IL-8, TNF-α and MCP-1 in CSE-treated THP-1 macrophages. 1,25(OH)2D did not improve defects in phagocytosis of E. coli bacteria or the oxidative burst response in CSE-treated THP-1 macrophages or alveolar macrophages from smokers. However, 1,25(OH)2D significantly enhanced mRNA expression and/or protein levels of the antimicrobial peptide cathelicidin in alveolar macrophages and THP-1 macrophages, independently of CS exposure. In conclusion, our results provide the first evidence that vitamin D could be a new strategy for attenuating airway inflammation and improving antibacterial defense in CS-exposed airways.