Immune Tolerance Induced By Ovalbumin-loaded Nanovaccine Targeting Dendritic Cells And Characteristics Of Skin,Oral Cavity And Gut Microbiota In Patients With Atopic Dermatitis

Part I:Immune tolerance induced by ovalbumin-loaded nanovaccine targeting dendritic cellsAtopic dermatitis is a chronic recurrent inflammatory disease.The Th2-type immune response to allergens plays an important role in the pathogenesis.Traditional treatments have good effects on controlling symptoms,but none of them can prevent recurrence.Allergen specific immunotherapy(AIT)is the only effective way for etiological treatment.PLGA nanoparticle is a research focus of vaccine drug delivery systems and it is a comprehensive solution to the slow and continuous release,improvement of targeting and bioavailability.In this study,PLGA nanoparticles loaded with allergen ovalbumin(OVA)were prepared,and nanoparticles were modified by mannan to target dendritic cells and increase their uptake efficiency.Furthermore,the polarization direction of T cells is affected to achieve the target of allergen specific immunotherapy.In this study,PLGA nanoparticles loaded with OVA were prepared by the double emulsion-solution volatilization method.Mannan was modified on the surface of nanoparticles by adsorption method.Scanning electron microscopy showed that the morphology of the nanoparticles was smooth and spherical in appearance.Zeta potential and laser particle size analyzers detected the diameter of the nanoparticles was between 250 nm and 300nm.The surface of the nanoparticles loaded with allergen had an absolute Zeta potential between 20-30 mv.The distribution of diameters was uniform.BCA protein assay determined the entrapment efficiency of allergen OVA was 69.5%±4.1%,and it could be released slowly and continuously in vitro.The CCK-8 method was used to detect the cytotoxicity of PLGA nanoparticles and it proved the safety of materials.In this study,OVA-loaded PLGA-OVA nanoparticles and PLGA-OVA-Mannan nanoparticles were co-incubated with peripheral blood-derived dendritic cells(MoDCs)for 24 hours.Flow cytometry results showed different degrees of reduction of the expression of CD80 and CD86 which were costimulatory molecules and mature markers CD83 and HLA-DR on the DC surface.The levels of pro-inflammatory cytokines IL-6 and TNF-a secreted by DCs were detected by CBA cytokines kit and qPCR method.The results showed their secretion levels were higher than those of negative controls and lower than the free OVA solution stimulation group significantly.The secretion level of IL-10 was significantly higher than the negative control and OVA solution group.The experimental results suggest that PLGA nanoparticles loaded with OVA can suppress the activation of DCs and induce DCs to semi-mature or immature status.In this study,DCs stimulated with nanoparticles were co-cultured with autologous T cells in vitro for 6 days.Flow cytometry was used to detect the polarization direction of T cells.The results showed that compared with the OVA solution-stimulated group,the proportion of Th2 cell subsets secreting IL-4 was significantly reduced,and the proportion of Th22,Thl7,and Thl cell subsets secreting IL-22,IL-17,and IFN-y showed a downward trend.The proportion of Treg expressing FoxP3 was significantly increased,suggesting that the OVA loaded nanoparticles can inhibit the polarization of T cells towards Th2 direction and induce T cells to polarize in the direction of Treg,and then participate in inducing immune tolerance.The above experimental results show that the nanoparticles loaded with allergens can inhibit dendritic cells from developing toward maturation in vitro,thereby inducing differentiation of T cells into Tregs and inducing immune tolerance.It is expected to be a novel allergen vaccine targeting DCs,and it is expected to provide new ideas for allergen-specific immunotherapy.Part ?:Characteristics of skin,oral cavity and gut microbiota in patients with atopic dermatitis and the association between themPrevious studies independently delineated the microbiota from the skin and gut of atopic dennatitis(AD)patients;however,the microbiota in another important body habitat,the oral cavity,has not been characterized,and the relationship among the microbiota from different habitats in AD patients is unclear.Therefore we did this research to comparatively analyze the microbial community structure and function across the skin,oral cavity and gut of AD patients.The microbiota of the three major body habitats from 172 AD patients and 120 healthy controls were analyzed by using 16S rRNA gene amplicon sequencing.Clinical information was also obtained,and total IgE was analyzed by ImmunoCap.The results showed that the skin and oral cavity,but not gut.of AD patients demonstrated differential reduction in the microbial diversity,which was distinctly correlated with disease severity.There were hierarchical shifts in the community structure among different habitats,and the lineage distance between the skin and oral microbiota of AD patients was closer than that of controls.Different habitats of AD patients had site-specific alterations in microbes,and the direction of enrichment was opposite between the skin and oral cavity for many oral-specific microbes of AD.Most interestingly,there was inverse association in the functional pathways between the skin and oral microbiota in AD patients.In conclusion,the skin,oral cavity and gut had site-specific alterations in the microbial composition and function in AD patients.The oral microbiota was closely associated with that of skin in AD patients,and might play a regulatory role against skin inflammation.