Vitamin D3 deficiency has been recognized as a pandemic with serious health consequences including chronic respiratory diseases. Unfortunately, improvement in this situation by using vitamin D supplementation has failed. The direct delivery of 1,25(OH)₂-vitamin D3 and its precursor into the respiratory tract, by nebulization, seemed to be a better option, as verified in the presented study. To induce vitamin D deficiency, mice received a diet with 0.05 IU/g cholecalciferol, while control animals were given feed with 0.5 IU/g cholecalciferol. Vitamin-D-deficient mice were exposed to different doses of calcidiol or calcitriol via nebulization for at least 7 days. At the end of the experiment, whole-body plethysmography was conducted. Pulmonary and serum levels of calcitriol were examined using ELISA. The calcitriol concentrations in mice on standard vs. deficient diet were 30.31/18.20 pg/mg (lungs) and 132.24/98.61 pg/mL (serum), respectively. Restoration of the physiological level of calcitriol in vitamin-D-deficient mice required 1-week exposure to 100 pg/g of calcidiol or 5 pg/g of calcitriol. The inhalations did not cause any side changes in murine respiratory function. The presented study revealed the usefulness and safety of chronic inhalation with a bioactive form of vitamin D3 or its precursor for the restoration of physiological calcitriol levels in animals with vitamin D deficiencies.

Despite numerous scientific reports on the negative impact of vitamin D3 deficiency on many respiratory diseases, little is known about the influence of this phenomenon on the development and progression of hypersensitivity pneumonitis (HP). The presented study is an attempt to shed light on this occurrence. The research was performed on mouse strain C57BL/6J exposed to the antigen of Pantoea agglomerans (etiological factor of HP). To induce vitamin D3 deficiency, mice received a diet with a 10 times lower amount of cholecalciferol than the main control group. VD3-deficient mice inhaled 25(OH)-VD3 or 1,25(OH)2-VD3 used separately or with SE-PA. At the beginning of the experiment and after 14 and 28 days of inhalation, respiratory function was examined using whole-body plethysmography. Moreover, at indicated time points, mice were sacrificed and samples collected for histological examination, flow cytometry, and ELISA. The performed study revealed that inhalations with 25(OH)-VD3 and 1,25(OH)2-VD3 effectively eliminated most of the negative changes in the respiratory system caused by vitamin D3 deficiency by restoring the physiological concentration of 1,25(OH)₂-VD3 in the body. VD3-deficient mice which inhaled P. agglomerans antigen and vitamin D3 metabolites also demonstrated the ability of the tested compounds to eliminate, or at least weaken, the negative effects of the HP causative factor and desired effect, including improvement of respiratory functions and attenuation of inflammation and signs of fibrosis. The obtained results suggested that the beneficial influence of inhaled vitamin D3 metabolites on HP development was associated with the restoration of the physiological concentration of 1,25(OH)2-VD3 in the pulmonary compartments in VD3-deficient mice.

Although vitamin D3 (VD3) deficiency has been recognized as a harmful agent in several respiratory diseases, the present study is the first one to investigate its influence on the development of hypersensitivity pneumonitis (HP). This research was conducted in a murine model of HP, wherein pulmonary fibrosis was induced by antigen of Pantoea agglomerans. VD3 deficiency was provoked by diet with 10-times less cholecalciferol than feed given to VD3-sufficient mice. Before and after 14 and 28 days of nebulization, lung function was evaluated. Moreover, at indicated time points, lungs were collected and subjected to histological assessment, flow cytometry, gene expression assays, and ELISA. The performed research showed a higher sensitivity of VD3-deficient mice to fibrosis response to P. agglomerans antigen, which was strongly associated with enhanced epithelial-to-mesenchymal transition, the signs of which were over-expression of EMT-transcription factors (Snail2, Zeb1, Zeb2) and mesenchymal cell markers (Cdh2/N-cadherin, Acta2/SMA, Fn1/Fibronectin, Vim/Vimentin). Indicated negative changes in VD3-deficient mice with developed HP were supported by deepening calcitriol deficiency and worsening respiratory functions, including the frequency of breathing, minute volume, total cycle times, expiratory and inspiratory time. Moreover, typical for VD3-deficient mice with HP, there was also an increased influx of immune cells into the lungs (especially neutrophils, macrophages, dendritic cells and lymphocytes Tc), a disturbed cytokine profile with over-production of growth factors favoring fibrosis (FGF2 and TGFβ), and lowered synthesis of several cytokines (IL1β, IL6, IL12, IL4 IL10, IL13). The present study reveals that VD3 deficiency promotes the development of pulmonary fibrosis in the murine model of HP.

Our earlier studies have revealed the beneficial impact of vitamin D3 (VD3) metabolites on the development of pulmonary fibrosis in a murine model of hypersensitivity pneumonitis (HP). Despite the discovery of the great antifibrotic potential of both the bioactive form of VD3, 1,25(OH)₂-VD3 and its direct precursor, 25(OH)-VD3, study designs based on the co-administration of VD3-metabolites and fibrosis-inducers do not reflect the clinical situation. Thus, the presented study aims to examine the effectiveness of therapeutic interventions based on the mentioned inhalations, initiated during the acute phase of HP at the first signs of pulmonary fibrosis. The research was performed using VD3-deficient mice with HP, while VD3-sufficient mice were used as the main control. The data were collected after 28 days of daily exposure of the mice to a fibrosis-inducer (an antigen of Pantoea agglomerans, a well-known etiological factor in HP), with 25(OH)-VD3 or 1,25(OH)₂-VD3 administered daily during the last 14 days of the experiment. The effect of the intervention was monitored by whole-body plethysmography, histological assessment, flow cytometry, RealTime-PCR, and ELISA. Treatment with VD3 metabolites initiated during the acute phase of HP alleviated the disease courses and restored respiratory function, thanks to inhibition of pathological extracellular matrix deposition, immune-cell infiltration of lung parenchyma, profibrotic cytokine production, and EMT progression. Despite the discovery of the beneficial features of the investigated VD3-based therapies, none of them caused full recovery in mice with HP. Nevertheless, after verification in additional animal models and a deeper understanding of the underlying molecular mechanisms, the identified beneficial features of the investigated metabolites have the potential to serve as a foundation for future therapies for HP fibrosis.