Study On Anti-asthma Effect Of Plasmid PSN In An OVA-induced Murine Model Of Asthma

BackgroundAllergic asthma is preceived as a complicated Th2immune responsepredominant disease. Th2cytokines, such as IL-4, IL-5and IL-13are demonstrated toplay crucial roles in promoting and maintenance of asthma symptoms which serves astarget in asthma therapy. However, biologicals for allergic asthma therapy bydown-regulating expression or blocking its binding to the receptor of a single Th2cytokine, such as IL-4, failed to achieve significant outcomes in asthma clinical trialsdue to the complex compensatory mechanism mediated by distinct Th2cytokines inallergic asthma pathogenesis. Previous researches have shown that the production ofpathogen microbes can stimulate innate immune and modulate Th1/Th2balance,which were suggested with great potential to be used in asthma treatment.In this study, we hypothesized that combined using the Helicobacter Pylorineutrophil-activating protein (HP-NAP) which has ability to elevate Th1response andthe IL-4blocking soluble IL-4receptor (sIL-4R), could modulate a variety of Th1/Th2cytokines involved in asthma pathogenesis, which might achieve betterprotective outcomes in asthma treatment. To this end, plasmid expressing fuesdsIL-4R and HP-NAP was constructed, and its anti-asthma effect was evaluated in anOVA-induced mouse model of asthma.Methods1. Recombinant plasmid pcDNA3.1-sIL-4R-NAP, named PSN, was modified toexpress fused HP-NAP and sIL-4R protein. HP-NAP and sIL-4R genes were linkedby flexible peptide (GSGSGS) coding sequence. Plasmid PSN was transientlytransfected to COS-7cells and the expression of fusion protein sIL-4R-NAP wasdetected by RT-PCR and Western Bolt.2. The OVA-induced BALB/c mouse model of asthma was established toevaluate the anti-asthma efficacy of plasmid PSN and investigate the possiblemechanisms underlain. 3. Mice were sacrificed24hours after the last OVA aerosol exposure, lungswere bronchoalveolar lavaged then number of total cells, eosinophils and neutrophilsin bronchoalveolar lavage fluid (BALF) was counted. Airway inflammation wasanalysed by lung tissue H&E staining.4. IL-4and IFN-γ levels in BALF and plasma OVA specific IgE productionwere detected by ELISA.5. The mRNA levels of cytokines (IL-5, IL-12and IL-13) or chemokines(eotaxin-1, eotaxin-2, CXCL2, CXCL5) and inflammatory factors (IL-6, TNF-α,IL-17A) that involved in asthma pathology process were analysed by qRT-PCR.6. The mRNA levels of IL-10and Foxp3, which involved inimmunosuppression, were analysed by qRT-PCR.Results1. RT-PCR and Western Blot analysis confirmed the fused HP-NAP and sIL-4R(sIL-4R-NAP) protein was successfully expressed in COS-7cells and was secreted tothe outside of cells.2. Plasmid PSN significantly improved asthma symptoms and attenuated lunginflammation with reduced number of eosinophils and total cells in BALF, whilepercentage of neutrophils in BALF remained unchanged.3. Significantly decreased mRNA level of Th2cytokines (IL-5and IL-13) andeosinophil-attracting chemokine eotaxin-2in lung were observed under PSNimmunization, while the mRNA levels of neutrophil chemoattractant (CXCL2andCXCL5), inflammatory factors (IL-6, TNF-α, IL-17A) and immunosuppressionrelative gene (IL-10and Foxp3) in lung tissue were not influenced.4. Plasmid PSN significantly increased IFN-γ expression in BALF and reducedplasma IgE level and IL-4expression in BALF.ConclusionsPlasmid PSN immunization effectively reversed Th2predominant immuneresponse, inhibited airway inflammation and decreased total cells number of BALF.Number of eosinophils in BALF, eosinophil-attracting chemokine eotaxin-2mRNA level in lung and plasma IgE production was also significantly decreased underplasmid PSN treatment. While percentage of neutrophils in BALF, inflammatoryfactors mRNA level in lung were not influenced. In addition, the anti-asthma efficacyof PSN was independent of immunosuppression functions derived from Treg cells.