Identification And Treatment Of Antigen-specific Neutrophils In Allergic Asthma

Objective:The pathogenesis of allergic asthma(AA)needs further study.Neutrophils(PMN)are one of the main inflammatory cells in AA.This study was designed to determine allergen(Ag)specific PMN(sPMN)and its role in the pathogenesis of AA.Study on the construction of tExo exosomes and its therapeutic mechanism of AA.Methods:First,AA mouse models were established by using OVA as specific Ag(sAg)and Freund’s adjuvant(CFA group)as immune enhancers.For comparison,AA mouse models were also established using OVA as sAg and aluminum hydroxide(Alum group)as immune enhancers.Then test airway hyperresponsiveness(AHR),OVA specific antibody(sIgE,sIgG),PMN and inflammatory mediators neutrophil elastase(NE)/tumor necrosis factor α(TNF-α),eosinophil(Eos)and inflammatory mediators alkaline protease(MBP)/peroxidase(EPX),macrophages(M(p)in bronchoalveolar lavage fluid(BALF),Serum sIgE,sIgG,Th1 inflammatory mediators(IFN-y),Th2 inflammatory mediators(interleukin 4 IL4,IL5,IL13)and airway epithelial barrier permeability,lung tissue inflammation score and the correlation analysis with inflammation score of lung tissue;The proportion and activation of Eos/PMN/Mφ were detected by flow cytometry(FCM).Meanwhile,sIgG and CD64(FcyRI)were detected by western blot(WB)and immunoprecipitation(IP)to investigate the characteristics of PMN in AA and its mechanism.Bone marrow derived dendritic cells(BMDC)were collected to construct tExo exosomes carrying CD64/OVA fusion protein.Then An OVA/CFA-sensitized AA mouse model was established and tExo exosomes carrying CD64/OVA fusion protein were used for nasal drip treatment.AHR,mouse lung inflammation,expression levels of NE/TNF-α/sIgE/sIgG/IL-4/IL-5/IL-13 and IFN-in BALF,and the proportion of PMNs/Eos/Mφ in BALF were detected.To investigate the efficacy and mechanism of tExo in treating AA.Results:The proportion of PMN and inflammatory mediators NE and TNF-α in OVA/CFA-sensitized AA mice were positively correlated with the inflammatory score of lung tissue.AA mice contained small amounts of sPMN,which were activated when exposed to specific Ag(sAg).sPMN expresses higher levels of FcyRI,binds to sIgG to form a complex,and PMN carrying sIgG/FcyRI complex is activated by sAg,and these PMNS can be specified as sPMN.In order to treat AA by depleting sPMN,tExo exosomes carrying an aCD64/OVA fusion protein were constructed.After tExo treatment,AHR of mice was significantly reduced,the expression of NE,TNF-,sIgE,sIgG,IL-4,IL-5,IL-13 and IFN-in BALF was also significantly reduced,and the proportion of PMNs and Eos in BALF was also significantly improved.The therapeutic mechanism may be specific recognition of sPMN,inducing apoptosis of sPMN to reduce the expression of inflammatory cytokines such as NE and TNF-α secreted by sPMN,and inhibiting inflammation in lung tissues of AA mice.Meanwhile,tExo treatment will not affect other PMN or human defense systems.Conclusions:PMN plays an important role in OVA/CFA sensitized asthma mice.sPMN was identified in the airway of mice.sPMN can be activated by sAg to induce or aggravate AA.TExo can induce apoptosis of sPMN and inhibit AA,which has a good therapeutic potential for AA.The results provide a new technique and a new method for the treatment of allergic diseases such as AA,which are mainly infiltrated by PMN.