| Background: Non-eosinophilic asthma(NEA)constitutes nearly half of all asthma patients,and is associated with airway inflammation consisting predominatly of polymorphonuclear neutrophil(PMN)infiltration.NEA is often less sensitive to glucocorticoids.IL-17 A has been found to play an important role in PMN infiltration of asthma.IL-17 stimulates the expression of a variety of chemokines by its effector cells.Thus,blocking IL-17 A reduces PMN infiltration in non-eosinophilic asthma.Previously,our laboratory has successfully constructed a recombinant Mycobacterium smegmatis r MS-Ag85a-IL-17 A vaccine to induce autoantibodies against IL-17 A,which achieved a certain effect in a murine severe asthma model.This study aims to further elucidate the function and mechanism of recombinant Mycobacterium smegmatis r MS-Ag85a-IL-17 a vaccine in airway inflammation of NEA by using a DO11.10 murine model.Objective: To induce and extract the IL-17 A autoantibodies in serum by immunizing DO11.10 mice with r MS-Ag85a-IL-17 a vaccine,and to investigate inhibition of neutrophil function and airway inflammation of NEA.Methods: This study is divided into four parts:(1)Establish the NEA model and observe the function of PMNs in NEA: Establish the NEA model by using DO11.10 mouse,observations are made of the neutrophils expression and function by means of: the cell and differential count in broncheoalveolar lavage fluid(BALF);ELISA assay of BALF to measure the levels of IL-4,IFN-γ,IL-17 A,IL-6,CXCL-1 and CXCL-2;RT-PCR of lung tissue for detecting the m RNA expression levels of ENA-78,NAP-2,NE;determination of BALF and lung tissue MPO activity.(2)Establishing r MS-Ag85a-IL-17 a immunization programe together with extraction and purification of IL-17 A autoantibodies: Mice are nasally immunized with r MS-Ag85a-IL-17 a,monitoring IL-17 A autoantibody levels using ELISA.During peak expression,massive amounts of serums are collected to extract and purify IL-17 A autoantibody by protein A chromatography.The concentration of purified autoantibody are determined by spectrophotometry,integrity and stability of the autoantibodies are determined by SDS-Page,it’s activity determined by ELISA.(3)Studying the inhibition of PMN chemotaxis caused by polyclonal IL-17 A autoantibodies: Transwell lower chamber were inoculated with RAW264.7 cells,which then were stimulated with the commercial recombinant IL-17 A,while being blocked using IL-17 A autoantibodies.The upper chamber is added with PMNs extracted from the mice,whose passage into the lower chamber is indirectly measured by means of MTT.Using ELISA to determine the secretion levels of CXCL-1 and CXCL-2 in the cell culture supernatant,the results are then compared to the PBS group,IL-17 A group and IL-17 A autoantibody groups in parallel to observe the inhibition of PNM chemotaxis.Western blot to determine how the chemotaxis transcription pathway is affected by IL-17 A autoantibodies.(4)Studying r MS-Ag85a-IL-17 a anti-inflammatory mechanisms in NEA: DO11.10 mice were divided into a control group,asthma group,ICS group,r MS-Ag85a-IL-17a-group and MS group.H&E stained differential count were performed in BALF,ELISA assay in BALF to measure IL-17 A,IL-6,IL-23,CXCL-1 and CXCL-2 expression levels;RT-PCR to detect lung tissue’s ENA-78,NAP-2,NE,T-bet,GATA-3 and ROR-γt m RNA expression;Determine BALF and lung tissue MPO activity;performing lung histopathology H&E and PAS staining.Results:(1)DO11.10 asthmatic mice BALF shows PMN dominance and elevated levels of IL-17 A,IL-6,CXCL-1 and CXCL-2;whereas in lung tissue the expression of ENA-78,NAP-2 and NE is elevated and MPO activity increased.(2)r MS-Ag85a-IL-17 a induced serum IL-17 A autoantibody can maintain a high level of long-term titer,purified IL-17 A antibodies have good integrity,stability and high activity.(3)IL-17 A autoantibodies can inhibit PMN aggregation by reducing IL-17A-promoted secretion of chemokines.(4)r MS-Ag85a-IL-17 a treatment of NEA mice can significantly reduce the total leukocyte and PNM count in BALF;decreasing expression levels of IL-17 A,IL-6,IL-23,CXCL-1,CXCL-2 in BALF and ENA-78,NAP-2,NE in lung tissue,respectively;inhibiting MPO activity,in which lung tissue NE expression was significantly lower than in the ICS group;lung tissue T-bet,GATA-3,and ROR-γt expression showed no significant difference;lung tissue histopathology demonstrated significantly reduced airway inflammation in the r MS-Ag85a-IL-17 a intervention group,together with significantly reduced exudation of inflammatory cells and airway mucus secretion.Conclusion:(1)PMN recruitment and activation is the main cause of NEA asthma airway inflammation.(2)r MS-Ag85a-IL-17 a immunization resulted in sustained high levels of IL-17 A autoantibodies with good integrity,stability and high activity.(3)IL-17 A autoantibodies inhibits PMN chemotaxis.(4)r MS-Ag85a-IL-17 a immunization can effectively reduce the NEA asthma airway inflammation in mice. |
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