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. 2015 Sep 16;16(1):110.
doi: 10.1186/s12931-015-0271-x.

Vitamin D deficiency exacerbates COPD-like characteristics in the lungs of cigarette smoke-exposed mice

Nele Heulens  1 , Hannelie Korf  2 , Nele Cielen  3 , Elien De Smidt  4 , Karen Maes  5 , Conny Gysemans  6 , Erik Verbeken  7 , Ghislaine Gayan-Ramirez  8 , Chantal Mathieu  9 , Wim Janssens  10
Affiliations

Vitamin D deficiency exacerbates COPD-like characteristics in the lungs of cigarette smoke-exposed mice

Nele Heulens et al. Respir Res. .

Abstract

Background: Chronic obstructive pulmonary disease (COPD) is characterized by excessive inflammation and disturbed bacterial clearance in the airways. Although cigarette smoke (CS) exposure poses a major risk, vitamin D deficiency could potentially contribute to COPD progression. Many in vitro studies demonstrate important anti-inflammatory and antibacterial effects of vitamin D, but a direct contribution of vitamin D deficiency to COPD onset and disease progression has not been explored.

Methods: In the current study, we used a murine experimental model to investigate the combined effect of vitamin D deficiency and CS exposure on the development of COPD-like characteristics. Therefore, vitamin D deficient or control mice were exposed to CS or ambient air for a period of 6 (subacute) or 12 weeks (chronic). Besides lung function and structure measurements, we performed an in depth analysis of the size and composition of the cellular infiltrate in the airways and lung parenchyma and tested the ex vivo phagocytic and oxidative burst capacity of alveolar macrophages.

Results: Vitamin D deficient mice exhibited an accelerated lung function decline following CS exposure compared to control mice. Furthermore, early signs of emphysema were only observed in CS-exposed vitamin D deficient mice, which was accompanied by elevated levels of MMP-12 in the lung. Vitamin D deficient mice showed exacerbated infiltration of inflammatory cells in the airways and lung parenchyma after both subacute and chronic CS exposure compared to control mice. Furthermore, elevated levels of typical proinflammatory cytokines and chemokines could be detected in the bronchoalveolar lavage fluid (KC and TNF-α) and lung tissue (IP-10, MCP-1, IL-12) of CS-exposed vitamin D deficient mice compared to control mice. Finally, although CS greatly impaired the ex vivo phagocytic and oxidative burst function of alveolar macrophages, vitamin D deficient mice did not feature an additional defect.

Conclusions: Our data demonstrate that vitamin D deficiency both accelerates and aggravates the development of characteristic disease features of COPD. As vitamin D deficiency is highly prevalent, large randomized trials exploring effects of vitamin D supplementation on lung function decline and COPD onset are needed.

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Figures

Fig. 1
Fig. 1
Effect of vitamin D deficiency on lung function parameters in air- and CS-exposed mice after 6 and 12 weeks. At the age of 8 weeks, C57Bl/6J vitamin D deficient and control mice were exposed to CS or ambient air for a period of 6 or 12 weeks. Lung function was measured with whole-body plethysmography after 6 and 12 weeks of smoking. a The total lung capacity (TLC) and b lung compliance (Cchord). n = 10–12 per group per timepoint; mean ± SEM; *p < 0.05, **p < 0.01
Fig. 2
Fig. 2
Effect of vitamin D deficiency on lung structure in air- and CS-exposed mice after 6 and 12 weeks. At the age of 8 weeks, C57Bl/6J vitamin D deficient and control mice were exposed to CS or ambient air for a period of 6 or 12 weeks. a Representative figures of H&E-stained lung sections of paraffinized lungs of each experimental group (100X magnification) after 6 weeks of smoking. b The irregularity of airspaces on histological lung sections was given a semi-quantitative score ranging from 0 to 3 by a pathologist in a blinded manner (0 = absent, 0.5 = minimal, 1 = mild, 2 = moderate and 3 = severe). c The mean linear intercept (Lm), as a measure of the interalveolar wall distance, was determined on histological lung sections (at 200X magnification). The Lm was measured in 15 randomly selected fields per lung slide and calculated as the total length of the grid lines x random fields divided by the sum of the alveolar intercepts. d Relative expression levels of MMP-12 in lung homogenates, analyzed with RT-PCR. Data were normalized using RPL-27 as housekeeping gene and analyzed with the comparative cycle threshold (Ct) method. n = 10–12 per group per timepoint; mean ± SEM; *p < 0.05, **p < 0.01, ****p < 0.0001
Fig. 3
Fig. 3
Effect of vitamin D deficiency on airway inflammation in air- and CS-exposed mice after 6 and 12 weeks. At the age of 8 weeks, C57Bl/6J vitamin D deficient and control mice were exposed to CS or ambient air for a period of 6 or 12 weeks. The number of a neutrophils (CD11c−CD11b+Ly6G+), d CD4+ T cells and e CD8+ T cells was determined in the BAL fluid using flow cytometry. Data are expressed as number of cells (×104) per milliliter of recovered BAL fluid. Levels of b KC and c TNF-α were measured in the supernatant of the BAL fluid. n = 10–12 per group per time point; mean ± SEM; **p < 0.01, ***p < 0.001, ****p < 0.0001
Fig. 4
Fig. 4
Effect of vitamin D deficiency on parenchymal inflammation in air- and CS-exposed mice after 6 and 12 weeks. At the age of 8 weeks, C57Bl/6J vitamin D deficient and control mice were exposed to CS or ambient air for a period of 6 or 12 weeks. a Representative figures of H&E-stained lung sections of each experimental group (200X magnification) after 6 weeks of smoking. Full arrows show the presence of peribronchial lymphocytic inflammation, while dashed arrows show the presence of pigmented macrophages in CS-exposed vitamin D deficient mice. b The diffuse presence of macrophages and the presence of c alveolar and d peribronchial lymphocytic inflammation on histological lung sections was given a semiquantitative score ranging from 0 to 3 by a pathologist in a blinded manner (0 = absent, 0.5 = minimal, 1 = mild, 2 = moderate and 3 = severe). n = 10–12 per group per time point; mean ± SEM; *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001
Fig. 5
Fig. 5
Effect of vitamin D deficiency on expression levels of proinflammatory cytokines and chemokines in lung tissue of air- and CS-exposed mice after 6 and 12 weeks. At the age of 8 weeks, C57Bl/6J vitamin D deficient and control mice were exposed to CS or ambient air for a period of 6 or 12 weeks. Relative expression levels of a IL-12, b MCP-1 and c IP-10 in lung homogenates were analyzed with RT-PCR. Data were normalized using RPL-27 as housekeeping gene and analyzed with the comparative cycle threshold (Ct) method. n = 10–12 per group per time point; mean ± SEM; *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001
Fig. 6
Fig. 6
Effect of vitamin D deficiency on the antibacterial function of alveolar macrophages in air- and CS-exposed mice after 6 and 12 weeks. At the age of 8 weeks, C57Bl/6J vitamin D deficient and control mice were exposed to CS or ambient air for a period of 6 or 12 weeks. The ex vivo phagocytic and oxidative burst capacity of alveolar macrophages was assessed following interaction of BAL cells with E. coli bacteria. a Percentage of alveolar macrophages (CD11c+autofluorescenthigh) that have internalized E. coli bacteria. b Percentage of alveolar macrophages (CD11c+autofluorescenthigh) that have produced reactive oxygen radicals. n = 10–12 per group per time point; mean ± SEM; *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001
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