The first step for understanding the molecular mechanism of the antifibrotic effect of inhalations with 25(OH)-Vitamin D3 and 1,25(OH)2-Vitamin D3 in the murine model of hypersensitivity pneumonitis

Marta Kinga Lemieszek^*Michał ChojnackiIwona PaśnikAlicja WilczyńskaWiktoria GawryśJakub AnisiewiczIlona Leśniowska

• Department of Medical Biology, Institute of Rural Health in Lublin, Lublin, Poland

Introduction: Pulmonary fibrosis occurs in several respiratory diseases, among which hypersensitivity pneumonitis (HP) is often ignored in designing therapeutic strategies. We strive to fill these knowledge gaps. Our earlier studies revealed antifibrotic potential of inhalations with 1,25(OH)2-VD3 and 25(OH)-VD3 based on modulation of immune response and deposition of extracellular matrix components, both of which are important components of epithelial-mesenchymal transition (EMT), which we focused on in this research. The study aimed to describe direct impact of VD3-metabolites on EMT in the course of pulmonary fibrosis in HP to understand their therapeutic effect. Methods: The research was performed in the HP model, wherein pulmonary fibrosis is induced by mice chronic exposure to antigen of Pantoea agglomerans (SE-PA). The study was conducted in VD3-deficient mice, while VD3-sufficient mice were used as the main control. VD3-deficient mice were inhaled with 100 pg/g of 25(OH)-VD3 or 5 pg/g of 1,25(OH)2-VD3 used separately or with SE-PA for 14-days and 28-days. The range of pulmonary fibrosis was determined after Masson Trichrome staining. Expression of EMT-molecules was examined using RealTime PCR and Western Blot. Results and discussion: Performed studies revealed that VD3-deficiency triggers EMT, the sign of which was increased expression of EMT-transcription factors (Snail1, Snail2, Zeb1, Zeb2), inhibited expression of epithelial cell markers (E-cadherin, Occludin), altered expression of mesenchymal cell markers including upregulated N-cadherin and Vimentin. Pathological changes caused by VD3-deficiencies accelerated in response to SE-PA the signs of which were: 1) upregulated expression of Snail1, Snail2, Zeb1, Zeb2, Acta2, Cdh2, Fn1, Vim; 2) downregulated expression of Cdh1, Ocln; 3) increased level of α-SMA, Fibronectin, Vimentin, Occludin; 4) decreased amount of N-cadherin; 5) increased deposition of fibers in lung tissue. All negative changes recorded on transcriptome level in VD3-deficient mice with HP were effectively reduced by inhalations with 25(OH)-VD3 and 1,25(OH)2-VD3, suggesting that their antifibrotic effects are associated with EMT inhibition. Nevertheless, beneficial impact of VD3-metabolites on proteome level was associated with restoration of the balance in the expression of EMT-molecules disturbed by cholecalciferol deficiency and SE-PA exposure; metabolites lowered overexpressed amount of Fibronectin, Vimentin and Occludin; simultaneously increased downregulated N-cadherin and enhanced expression of E-cadherin.