Deletion of vitamin D receptor leads to premature emphysema/COPD by increased matrix metalloproteinases and lymphoid aggregates formation

Abstract

Deficiency of vitamin D is associated with accelerated decline in lung function. Vitamin D is a ligand for nuclear hormone vitamin D receptor (VDR), and upon binding it modulates various cellular functions. The level of VDR is reduced in lungs of patients with chronic obstructive pulmonary disease (COPD) which led us to hypothesize that deficiency of VDR leads to significant alterations in lung phenotype that are characteristics of COPD/emphysema associated with increased inflammatory response. We found that VDR knock-out (VDR(-/-)) mice had increased influx of inflammatory cells, phospho-acetylation of nuclear factor-kappaB (NF-κB) associated with increased proinflammatory mediators, and up-regulation of matrix metalloproteinases (MMPs) MMP-2, MMP-9, and MMP-12 in the lung. This was associated with emphysema and decline in lung function associated with lymphoid aggregates formation compared to WT mice. These findings suggest that deficiency of VDR in mouse lung can lead to an early onset of emphysema/COPD because of chronic inflammation, immune dysregulation, and lung destruction.

Fig. 1. Neutrophil and macrophage influx into BAL fluid of VDR^−/− and WT mice

The number of neutrophils (A), macrophages (B) and total cells (C) on cytospin slides was determined by Diff-Quik staining. Lung sections of VDR^−/− and WT mice were stained with anti-mouse Mac-3 antibody (D). Representative figure of Mac-3 positive cells identified by immunohistochemical staining (dark brown stained macrophages were indicated by arrows), Original magnification: x200. Histogram (E) Values are mean ± SEM (n= 3–5 mice per group). ** P < 0.01, ***P < 0.001, significant compared to WT mice.

Fig. 2. Phospho/acetylation of NF-κB subunit RelA/p65 and NF-κB responsive proinflammatory cytokines in the lung of VDR^−/− and WT mice

Phosphorylation (S276) and acetylation (K310) of NF-κB RelA/p65 was assessed in the whole lung homogenates of VDR^−/− and WT mice by immunoblot analysis (A) and bands were measured by densitometry (relative band intensity versus total RelA/p65 or β-actin) (B). β-actin was used as a loading control. After densitometric analysis, values were normalized against loading controls. The levels of proinflammatory cytokines, MCP-1 (C) and KC (D) were measured by ELISA using the lung homogenates from VDR ^−/− and WT mice. Values are mean ± SEM (n=3–4 mice per group). *P < 0.05, **P < 0.01, significant compared to WT mice.

Fig. 3. Expression levels of matrix metalloproteinases (MMPs) and tissue inhibitors of metalloproteinases (TIMPs) in the lungs of VDR^−/− and WT mice

The protein abundance of MMP-2 (A), MMP-9 (B), MMP-12 (C) MMP-2 and MMP-9 enzyme activity (D), levels of TIMP-1 (E), TIMP-2 (F), TIMP-3 (G) and TIMP-4 (H) were assessed in the whole lung homogenates of VDR^−/− and WT mice by immunoblot analysis and zymography assay. β-actin was used as a loading control. β-actin was used as a loading control. After densitometric analysis, values were normalized against loading controls. Values are mean ± SEM (n=3–4 mice per group). *P < 0.05, **P < 0.01, ***P < 0.001 significant compared to WT mice.

Fig. 4. Morphometric analysis, trichrome staining and respiratory mechanics in lungs of VDR^−/− and WT mice

Representative figure of hematoxylin and eosin (H&E) stained lung sections from VDR^−/− and WT mice. Original magnification: x200. Mean linear intercept (Lm) was calculated using H&E stained slides (A). Representative figure of trichrome stained lung sections from WT and VDR^−/− mice showing peribronchial collagen deposition stained dark blue and lymphoid aggregates indicated by the dark arrow. Original magnification: x100 (B). Significant increase in Lm between the VDR^−/− and WT mice was observed. Dark arrow indicates alveolar airspace enlargement. Dynamic lung resistance (C) lung elastance (D) and lung compliance (E) as a measure of lung mechanics were determined in WT and VDR^−/− mice using Flexivent. Values are mean ± SEM (n=3–5 mice per group). *P < 0.05, ***P < 0.001, significant compared to WT mice.