1,25-dihydroxyvitamin D₃ Pretreatment Enhances The Effects Of Allergen Immunotherapy In A Mouse Model Of Allergic Asthma And Related Mechanisms

Objective:Bronchial asthma (asthma) is one of the common chronic respiratory diseases. In recent years, its prevalence around the world was growing year by year. Allergic asthma is a disease which was irritated by allergen. Airway allergic inflammation was involveed a variety of cells including airway inflammatory cells and structural cells (such as eosinophils, mast cells, T cells, neutrophils, smooth muscle cells and airway epithelial cells, etc.) and the cellular components, and its pathogenesis is not yet clearly[1-3]. The characteristics of allergic asthma includes infiltration of inflammatory cells in the airway submucosa and airway hyperresponsiveness[4, 5]. Among them, inflammatory response is an important mechanism for the pathogenesis of asthma, T cells were activated by antigen presenting cells, activated helper T cells (mainly Th2 cells) produce interleukin (IL) -4, IL-5, IL- 10 and IL-13, to activate B cells, mast cells, eosinophils,alveolar macrophages and other inflammatory cells, resulting in airway inflammation. Therefore, in allergic asthma, activation of T cells by antigen presenting cells is one of important steps of the initiating phase.Specific immunotherapy (SIT) is usually referred to as desensitization therapy. SIT for the treatment of allergic asthma provides a different way of treatment with conventional drugs (e.g. corticosteroids, aminophylline andβ2-receptor agonists, etc.). It changes the natural course of allergic by induction of immune tolerance[6, 7]. Looking back, by the previous treatment of infectious diseases, vaccination great success of the enlightenment, as early as 1911, Noon and Freeman started with specific allergen treatment of allergic diseases[8]. Since then, the clinical treatment of subcutaneous SIT gradually be developed and improved. Nearly a century, the subcutaneous SIT is considered to be allergic diseases (for example, insect venom, house dust mites, pollen or animal dander and other allergens) is an important reference for treatment measures[8, 9].The role of the mechanism of SIT is not entirely clear[10, 11]. Successful SIT can induce allergic patients with antigen-specific tolerance to the body. In the course, the induction of regulatory T cells (Tregs) plays an important role[10-14]. However, there may be induced adverse events (such as severe anaphylactic shock) and treatment length and the effect of uncertainty effector, SIT in clinical application has certain limitations.[15].1,25 dihydroxyvitamin D3 [1,25(OH)2D3, VitD3] is the active metabolite of vitamin D. VitD3 not only plays an important role in bone formation and calcium metabolism, but also plays an immunoregulatory role of specific receptors in response to allergen through the expression of antigen-presenting cells and activated T cells [16].In immune-related diseases, VitD3 not only prevents human typeⅠdiabetes mellitus [17] and reduces the incidence of animal models of inflammatory bowel disease [18], but also prolongs allograft survival, reduces the acute and chronic rejection[19]. VitD3 can induce monocyte-derived immature dendritic cells (DCs) surface antigen-presenting molecules MHC II and costimulatory molecules (CD40, CD80 and CD86) expression, affects the immune process[20].Different studies have shown that SIT and VitD3 contribute to immune tolerance, and the treatment of autoimmune diseases, this study observed the use of VitD3 pretreatment united SIT in sensitized mice asthma model with the ovalbumin (OVA) to Th1/Th2 cytokine secretion and spleen-derived DCs express of costimulatory molecules, and Tregs polarization role.Discusses the use of rmIL-4, rmGM-CSF combined VitD3 induced myeloid-derived DCs, DCs were observed in the OVA and lipopolysaccharide (LPS) plus stimulation on marrow-derived DCs in the expression of Jagged1 and Jagged2. We explored the role of VitD3 pretreatment in immunotherapy.Methods:1.In vivo: To study the VitD3 pretreatment united SIT and its mechanism①Prepare OVA sensitized (adjuvant is aluminum hydroxide gel) - desensitization - mice stimulated, and were identified by HE staining.②Use of VitD3 50 ng injected as a pretreatment, SIT with OVA 100μg subcutaneous injection, then 1% OVA aerosol inhalation 30min. After 24 h of the last challenge, Th1/Th2 cytokine levels in lavage were detected by ELISA.③Detect levels of spleen DCs surface molecules CD11c, CD80 and CD86 costimulatory molecules in negative control, asthma control,VitD3 pretreatment alone, SIT treatment alone, and VitD3 pretreatment combined SIT by Flow cytometry.④Purified spleen CD4+ lymphocyte cells, to detect levels of CD4+CD25+Foxp3+ T cells in negative control, asthma control, VitD3 pretreatment alone, SIT treatment alone, and VitD3 pretreatment of spleen united SIT with Flow cytometry. 2.In vitro: To explore levels of Jagged1 and Jagged2 in VitD3 induced myeloid- derived DCs after stimulated with OVA/LPS①Isolated cells from mouse bone marrow cells, using rmIL-4, rmGM-CSF and combined VitD3 induced myeloid derived dendritic cells. Levels surface molecules CD11c, CD80, CD86 were detected with Flow cytometry.②Combined VitD3 to induce bone marrow, to detect levels of Jageed1 and Jagged2 on dendritic cells by RT-PCR and Western blotting.Results:1. Inflammatory cells in bronchoalveolar lavage fluid (BALF): Total cells and the number of eosinophils in asthma group (Asthma) and SIT alone group (VitD3) mice were significantly higher than the negative control group (Control); In VitD3 treatment combined OVA group (VitD3 + SIT), total cells and the number of eosinophils were significantly lower than Asthma and SIT alone Group (SIT) (P<0.05).2. Pulmonary pathology by HE staining: Lung tissue biopsies by HE staining from the asthma group (Asthma) compared with control mice, the volume of the lungs increased swelling, bronchi and blood vessels can be seen around the large number of inflammatory cells, endothelial part of the trachea off, irregular thickening of the basement membrane. In SIT alone group (VitD3) mice, tracheal basement membrane were thickening and massive infiltration of inflammatory cells, but less mitigation; In VitD3 treatment combined OVA group (VitD3 + SIT), tracheal basement membrane were significantly reduced pathological damages.3. OVA-specific IgE in serum levels: The OVA specific serum IgE levels in other groups were increased, compared with the control group. VitD3 pretreatment combined SIT group (VitD3 + SIT) compared with SIT alone Group (SIT), serum OVA-specific IgE level was significantly lower, statistically significant difference (P<0.05).4. Compared with the control group, Levels of IL-4 and IL-5 in BALF of asthmatic control (Asthma) group increased significantly; And IL-5 Levels of VitD3 pretreatment combined with SIT group (VitD3 + SIT) significantly reduced compared with VitD3 pretreatment SIT alone group (SIT). However, level of IL-10 in VitD3 pretreatment combined SIT group (VitD3 + SIT) increased significantly, compared with SIT alone group (SIT).5. Compare asthmatic control group (Asthma) with the control group, CD80 on DCs of spleen derived significant increase (85.37±4.06% vs 72.263.24% , P<0.05). VitD3 pretreatment combined SIT group (VitD3 + SIT) compared SIT alone group (SIT) was significantly lower (64.46±2.75% vs 75.27±5.13%, P<0.05). Another, CD86 on DCs in the combination therapy group (VitD3 + SIT) compared to SIT alone group (SIT) (50.14±2.75% vs 64.75±3.36%, P<0.05) also significantly reduced (P<0.05).6. Compare asthmatic control group (Asthma) with control group, CD4+CD25+Foxp3+ T cells in the spleen was significantly lower. Compare VitD3 pretreatment combined SIT group (VitD3 + SIT) with VitD3 pretreatment alone group (VitD3) or SIT alone group (SIT), CD4+CD25+Foxp3+ T cells increased significantly.7. Without the joint use of VitD3 induced marrow-derived dendritic cells, stimulated with LPS or OVA in 24h, the mRNA and protein expression of Jagged1 and Jagged2 in increased significantly by RT-PCR and Western Blotting detection; With VitD3 induced marrow-derived dendritic cells, stimulated with LPS or OVA in 24h, the mRNA and protein expression of Jagged1 and Jagged2 was not significantly change by RT-PCR and Western Blotting detection.Conclusion:1. Mice allergic asthma with OVA model, with VitD3 pretreatment can enhance the therapeutic effect of SIT, showed reducing the airway inflammation, correct the Th1/Th2 shift, costimulatory molecules CD80, CD86 expression on spleen-derived DCs were decreased and the induction of Tregs increases.2. Marrow-derived DCs in mice by VitD3, rmIL-4 and rmGM-CSF, after the plus OVA, LPS stimulation, can reduce significantly the mRNA and protein expression of Jagged1 and Jagged2 on DCs. Because Notch signaling pathway affects peripheral T cell differentiation, Jaagged1 and Jagged2 expression decreased may be one of the mechanisms of VitD3 involved in immune regulation.This study proved that VitD3 pretreatment can enhance effects of SIT in a mouse model of allergic asthma, contribute to the formation of immune tolerance;By VitD3 induced marrow-derived dendritic cells and stimulated with OVA/ LPS proved that VitD3 may reduce the level of Jagged1 and Jagged2 on marrow-derived DCs, Notch ligands may play immunomodulatory effects.