Immunomodulation Of The Allergic Airway Inflammation By Allergen-DNA-targeted Dendritic Cells In Mouse Models

Background:Asthma is a chronic inflammatory disorder of the airways, characterized by Th2-dominated immune responses. Allergen gene vaccines have been shown to be effective in modulationing the allergic immune response in mouse models,especially shifting the immune response fron a Th2- to a Th1-dominated immune response. To enhance the effectivemess of DNA vaccination and potentially treat patients with ongoing airway inflammation,we constructed a DNA vaccination plasmid containing cDNA for a prototypic allergen,OVA,fused to the cDNA of a potent immune modulating Fc.This approach is based on fact that Fc,enhancing DCs'absorption,and is very powerful in driving the production of Th1 cytokine synthesis in na?ve and memory T cells.Objective:We investigated the combined effect of OVA and Fc vaccination on asthma development in BALB/c mice.It would lay a foundation for developing a new DNA vaccine.The aim of the study was to analyze whether dendritic cells targeted with allergen-DNA conjugates are able to stimulate autologous CD₄⁺ T cells, CD₈⁺ T cells,or both from atopic individuals to produce TH1 cytokines instead of TH2 cytokines.Methods:1. Animals BALB/c mice,6–8 weeks old at the onset of experiments and sex matched within each experiment.Mice care and use were performed in accordance with the guidelines of Dutch Committee of Animal Experiments.2.DNA constructs and purification The PCR products of OVA and Fc were selected as target DNA fragments and cloned into pcDNA_3.1 (+) to construct the recombinant plasmids OVA-pcDNA_3.1and OVA-Fc- pcDNA_3.1 respectively. The recombinant plasmids were constructed and expressed successfully in CHO cell lines. All Plasmids were purified by the EndoFree Plasmid Giga Kit.The recombinant plasmids were identified by restriction enzyme analysis and PCR reaction and confirmed by DNA sequencing.3.Study groups BALB/c mice were divided into five groups. 4.Immunization protocols Except for the control group,BALAB/c mice from all groups were first sensitized with OVA (gradeâ…¡)before vaccination with the DNA plasmids.OVA (50μɡ) absorbed to alum was adminiatered i.p. once on day 0 . On days 8 and 9 the mice were challenged with aerosols of 1% OVA (gradeâ…¡) diluted in PBS for 30 min each day.On days 10 and 25 the different endoFree DNA constructs were injected i.m. in the quadriceps muscles (100μɡin 100μl 0.9%Nacl).On day 39 and 40 the mice were challenged with aerosols of 1% OVA (gradeâ…¤) diluted in PBS for 30 min each day. Mice from control group received a mock sensitization with intraperitonealn alum alone and a mock challenge with aerosol PBS at the corresponding time. Mice were sacrificed within 1 days of the last OVA challenge. 5. Collection of bronchoalveolar lavage fluid(BALF) Mice were anesthetized with urethane 24 h after the last OVA challenge, and the abdominal cavity was opened. Blood samples for serum were collected from the vena cava . The tracheas were cannulated ,and BAL was performed by two lavages with 0.5 ml ice-cold PBS. The total cell number in BALF was determined. The BALF was centrifuged and supernatant used to test for cytokine prouduction and the cell pellet used to prepare slides for differential cell counting.Cytospin slides were fixed and stained with Diffquik for leucocyte differential analysis.and the number of monocytes, lymphocytes,and eosinophils in a total of 200 cells were counted in each slide. 6. Concentrations of cytokines in BALF were measured with commercial ELISA kits according to the manufacture's instructions.Murine IFN-γ,IL-5 and IL-2,IL-10 ELISA kits.7.OVA-specific IgE assay Mice were bled et the time of sacrifice, and OVA-specific IgE was determined using a modified Ag-specific ELISA. 8. Generation of DCs from pulmonary and culture Pulmonary-derived DCs were enriched according to the published methods. The DCs were collected and immediately used in our assays. 9.Immunofluoresence and flow cytometry analysis Pulmonary-derived DCs were incubated with FITC-labeled CD_11c, PE -labeled anti-CD₈₀ (B7-1) MAb, PE-labeled anti-CD86 MAb on ice for 30 min and washed with PBS. Ten thousand cells were collected for each sample, and the data were analyzed with FACScan flow cytometer and CELLQUEST software. DCs of pulmonary express levels of the costimulatory molecules CD_11cCD₈₀ and CD_11cCD₈₆ surface marker.10.Flow cytometric analysis Autologous CD₄⁺ and CD₈⁺ T cells were stimulated with these targeted DCs,and proliferation and cytokine production were measured.Flow-cytometry assay was carried to detect the numbers of CD₄⁺ and CD₈⁺T cells in peripheral blood of mice at the 24 h after the last OVA challenge. FITC-labeled CD₄⁺ and CD₈⁺ Mab,hemolytic agentand IgG2a.11.Histopathology Formalin-fixed lungs were embedded in paraffin,sectioned in 6-μm thick slices,and stained with hematoxylin and eosin for routine hidtology. 12.Statistical analysis Results were shown as mean±SD.Statistical analysis was performed with the statistical software package SigmaStat.All assays were compared using ANOVA followed by least squares difference-t analysis.Results and methods:1.Histologic changes The inflammatory characteristics caused by OVA sensitization/challenge are shown in Figure 2-1, Figure 2-2, Figure 2-3, Figure 2-4, Figure 2-5. Histologic scores of peribronchiolitis,perivasculitis, alveolitis,and peribronchial eosinophilia in all groups with OVA sensitization/challeng (OVA, pcDNA_3.1,OVA-pcDNA_3.1,and OVA-Fc-pcDNA_3.1 groups) were significantly higher than that in the control group. Comparison among all groups with OVA sensitization/challenge showed that the OVA-Fc-pcDNA_3.1 group had significantly milder peribronchiolitis,perivasculitis,alveolitis, and peribronchial eosinophilia than other groups. 2.Cellular composition of the lung inflammatory responses In comparison with epithelial cells , which constituted the majorison of BAL cells in the control group,lymphocytes were the major inflammatory cells found in BALF from other groups that underwent OVA sensitization and challenge. The number of eosinophilsin the BALF of OVA, asthma, OVA-pcDNA_3.1,and OVA-Fc-pcDNA_3.1 groups were all significantly greater than in control group (p<0.01). The number of total cells and eosinophils in the OVA-Fc-pcDNA_3.1 group was significantly lower than in the OVA, asthma,and OVA-pcDNA_3.1 groups, with the lymphocyte number significantly lower than in the asthma, pcDNA_3.1,and OVA-pcDNA_3.1 groups. However, there was no difference in the number of monocytes and neutrophils between the four groups 3.Serum OVA-specific IgE OVA-sensitized/challenged mice (asthma, pcDNA_3.1 , OVA-pcDNA_3.1,and OVA-Fc-pcDNA_3.1 groups) showed significantly higher serum OVA-specific IgE titers than control mice. There were significant differences among all of the OVA-sensitized or–challenged groups.OVA-specific IgE was very high in OVA-immunized BALB/c mice.Vaccination with the different DNA vectors after immunization with OVA significantly reduced the level of OVA-specific IgE. The inhibitory effect on IgE production was strongest with the OVA-Fc fusion construct..4.Cytokine production in BALF The levels of IFN-γ,IL-2,IL-5,and IL-10 in BALF were assayed by ELISA.As showed in Figure 5, all OVA-sensitized/challenged groups (asthma, pcDNA_3.1, and pcDNA_3.1/OVA groups) had significantly lower IL-10,IL-2,IFN-γand higher IL-5 level than the control.IL-10 in OVA-Fc- pcDNA_3.1 group was significantly higher than in the asthma, pcDNA_3.1,and OVA-pcDNA_3.1 groups. 5. The numbers of CD₄⁺ and CD₈⁺ T cells in OVA-Fc- pcDNA_3.1 groups were higher significantly than in the asthma, pcDNA_3.1and OVA-pcDNA_3.1 groups.6.CD11cCD80 and CD11cCD86 were up-regulated on pulmonary-derived DCs from BALB/c mice sensitized and challenged with OVA. Vaccination with OVA-Fc- pcDNA_3.1 resulted in a dramatic decrease of CD11cCD80 and CD11cCD86. targeted DCs of pulmonary express lower levels of the costimulatory molecules CD11cCD80 and CD11cCD86 compared with that of asthma DCs.Conclusion:1.DNA sequencing and restriction endonuclease digestion analysis indicated that the eukarytotic expression vector OVA-Fc-pcDNA_3.1 had been concess of this plasmid constructed successfully.OVA-Fc expression could be detected in CHO cells by Western blotting,ELISA,and flow cytometry.2.EndoFree OVA-Fc-pcDNA_3.1 decreased asthmatic inflammation in OVA-ensitized/challended mouse model .Moreover,our studies,demonstrating that endoFree OVA-Fc fusion constructs have much greater immunogenicity than allergen-only cDNA constructs,suggest that EndoFree allergen-Fc DNA constructs may provide,effective,and potentially curative therapy for allergic disease and asthma.DCs can be targeted with OVA-DNA conjugates very efficiently by using Fc vector yielding DCs with high T-cell stimulatory capacities,directing the atopic-allergic immune response from Th2 dominance toward Th1 dominance.3.The data demonstrated that EndoFree OVA-Fc- pcDNA_3.1 recombinant plasmid inhibits lung inflammation and serum IgE in vivo. This effect may be due to reduced expression of CD11cCD80 and CD11cCD86 by DCs.4.EndoFree OVA-Fc DNA vaccine not only can enhance immunity effect of vaccine but also is safe and innocuous to mice.