Combined Treatment With Vitamin D And DNase Ⅰ In Neutrophilic Asthma Mice

Background:Asthma is considered to be an immune dysregulation disease,and it is a chronic inflammation process involving many immune cells,mainly mediated by cells such as T_H2,ILC2,and T_H17.In addition to promoting the absorption of calcium and phosphorus,VD is also an immunoregulatory factor.VD not only could induce macrophages and epithelial cells to produce Cathelicidins,enhance the innate immune response,but also could inhibit the differentiation of B cells into plasma cells and reduce the production of immunoglobulins,and inhibit the differentiation of T cells,and upregulate the number of Treg cells.DNase Ⅰ is abbreviation of deoxyribonuclease I,a DNA hydrolase that can hydrolyze large amounts of extracellular DNA released by neutrophils in allergic asthma,especially neutrophilic asthma,DNase Ⅰ can reduce the viscosity of airway secretions and alleviate airway obstruction.In this research,firstly,we induced macrophages or PBMCs with LPS in the presence of VD,to verify the anti-inflammatory ability of VD in vitro,and then established a co-culture system consisting of NHBE cells,BALF cells and neutrophils to observe the inhibition of inflammation by VD or VD combined with DNase Ⅰ,and finally build a mouse model of neutrophilic asthma induced by OVA/CFA to explore the effect of anti-inflammation of VD or VD combined with DNase Ⅰ.Through researching the anti-inflammatory effect in neutrophilic asthma of VD combined with DNase Ⅰ in vitro and in vivo,we aim to provide a feasible plan and basis for clinical treatment of allergic asthma,especially refractory asthma with hormone tolerance.Objective:1.To research the anti-inflammatory effect of VD and evaluate the therapeutic effect of VD combined with DNase Ⅰ on airway inflammation and lung function in neutrophilic asthma mice in vitro and in vivo.2.To explore the application of human primary cell 3D co-culture system in area of respiratory deseases,through constructing NHBE cell co-culture system.Methods:1.PMA-induced macrophages or PBMCs were stimulated with LPS,and then treated with different concentrations of 1,25（OH）₂D₃.Cell culture supernatants were analysed by ELISA to detect the production of IL-β,TNF-α,IL-6 and IL-8 to evaluate the anti-inflammatory effects of VD.2.Neutrophils NETosis was induced with PMA,the cf-DNA was detected by Pico Green DNA dye in the cell culture supernatant,to verify the ability of DNase Ⅰ to hydrolyze e DNA and VD whether inhibit neutrophil NETosis.3.16HBE cells were stimulated with IL-17A and TNF-αin the presence of 1,25（OH）₂D₃,the expression levels of CXCL1,CXCL2,CXCL3 and CCL8 were analysed by RT-PCR.4.A co-culture system consisting of NHBE cells,BALF cells and neutrophils were established in the presence of 1,25（OH）₂D₃ and DNase Ⅰ,cell culture supernatants were analysed with Multiple factor assay（Lumi Nex）.5.Mouse neutrophilic asthma model induced by OVA/CFA was constructed,1,25（OH）₂D₃ combined with DNase Ⅰ were administrated during the sensitization and challenge periods.The alveolar lavage fluid（BALF）was analysed by flow cytometry to detect BALF cells classification,and the expression of IL-4,IL-17A,IL-1β,IL-6and TNF-αin BALF were detected by ELISA.HE&PAS staining was used to evaluate the airway inflammation of neutrophilic asthma.Result:1.The results of cell culture supernatants analysed by ELISA showed that VD inhibited the production of cytokines such as IL-1β,IL-6,IL-8 and TNF-αinduced by LPS.2.The results of cf-DNA in the cell culture supernatants detected by Pico Green DNA dye indicated that DNase Ⅰ had a strong hydrolysis effect on e DNA,cf-DNA decreased significantly（P=0.0002）,but VD could not inhibit PMA-induced neutrophil NETosis.3.The RT-PCR results showed that low concentration of 1,25（OH）₂D₃down-regulated the expression levels of T_H17 signature gens such as CXCL1,CXCL2,CXCL3 and CCL8.4.The results of multiple factor assay showed that VD combined with DNase Ⅰ decreased the expression of IL-1β,IL-6,TNF-α,GM-CSF and other cytokines in the NHBE co-culture system.5.The results of animal experiments indicated that VD combined with DNase Ⅰ significantly alleviated mice hyperresponsiveness（P=0.0001）,reduced inflammatory cell infiltration and airway mucus secretion,and decreased the level of inflammatory cytokines such as IL-1β,IL-6 and TNF-α.Conclusion:Various experiments in vitro and in vivo showed that VD inhibited the inflammation produced by LPS-induced macrophages or PBMC,but VD could not inhibit PMA-induced neutrophil NETosis,and low concentrations of VD down-regulated the expression of T_H17 signature cytokines such as CXCL1,CXCL2,CXCL3,CCL8.VD combined with DNase Ⅰ reduced the inflammation produced by asthma patients’BALF cells and neutrophlis in vitro,and significantly reduced airway inflammation in neutrophilic asthma mice and had a certain blocking effect on the development of neutrophilic asthma.