Mechanism Of Immune System Activation To Produce Autoantibody ANCA In WKY Rats Under The Background Of Chronic Bronchitis

Objective Anti-neutrophil cytoplasmic antibody-associated vasculitis(AAV)is a group of autoimmune disease affecting small vessels of multiple organs.A number of clinical and basic studies have confirmed that anti-neutrophil cytoplasmic antibodies(ANCA)are pathogenic and participate in the disease process of AAV through a variety of channels.Etiological studies also suggest that the incidence of ANCA-associated vasculitis is related to chronic inflammatory diseases such as chronic bronchitis.In this study,we proposes the hypothesis that the inflammatory microenvironment of chronic bronchitis with repeatedly NETs formation can activate the immune system and affect anti-neutrophil cytoplasmic antibodies’ production.This study repeatedly stimulate WKY rats with LPS to induce the production of NETs in WKY rats,and the cross-immunization of human recombinant myeloperoxidase protein to produce ANCA in WKY rats for chronic bronchitis produces two angles to verify the hypothesis.Methods 1.Chronic bronchitis WKY rats was repeatedly stimulated by LPS to mimic the process of repeated infection 40 WKY rats were randomly divided into four groups: the group of CB+LPS,the group of sham+LPS,The group of CB+PBS,The group of sham +PBS)2.Circulating MPO-ANCA and Cit H3 were detected and quantified by standardized ELISA kit The 4 groups of rats were stimulated with LPS/PBS for 24 hours each time,and the serum was collected and stored for later detection.The serum MPO-ANCA titer and Cit H3 level were detected by enzyme-linked immunosorbent assay.3.Double-labeled immunofluorescence histochemistry was performed for NETs in lungs The NETS related protein MPO CITH3 in the lungs of the four groups of rats was labeled by fluorescence double labeling and the nuclei were localized by DAPI.The formation of NETS was determined according to the co-localization of the three kinds of fluorescence and tissue staining morphology 4.Semi-quantitative assessment of renal chronic inflammatory cell infiltration by immunohistochemistry After 4 stimulations,All rats were sacrificed.Lung tissue were harvest and paraffinembedded.Anti-CD3 antibody and anti-CD20 antibody were respectively used to label T and B lymphocytes.H-SCORE was used to semi-quantitate the staining of each section.5.Chronic bronchitis rats cross-reacted immunized with recombination human MPO polypeptide 15 WKY rats randomly divided into 3 groups: the group of CB+h MPO,the group of Sham+h MPO,the group of sham+vehicle.In D1 and D14 all rats in the group of CB+h MPO and the group of Sham +h MPO were immunised by 400μg/kg h MPO mixed with an equal volume of Freund’s adjuvant(CFA)subcutaneously.Results 1.A stable WKY rat model of chronic bronchitis was established by endotracheal infusion of lipopolysaccharide combined with cigarette smoke exposure 2.The titer of MPO-ANCA in CB+LPS rats increased with the increase of the number of stimulations.After 4 stimulations,the titers of MPO-ANCA in the group of CB+LPS were significantly higher than those in the group of Sham+LPS,CB+PBS,and Sham+PBS.3.According to the immunofluorescence staining of NETs-related proteins MPO,Cit H3 and DAPI,it can be seen that a large number of NETs were produced in the lungs of rats in the group of CB+LPS group,a small amount of NETs were produced in the lungs of rats in the group of Sham+LPS group,and a small amount of NETs were produced in the lungs of the group of Sham+PBS.there was no obvious NETs in the group of CB+PBS.4.The renal interstitium of rats in the CB+LPS group was significantly infiltrated by T and B lymphocytes,but there was no T and B lymphocyte infiltration in the control group.5.In the context of chronic inflammation,WKY rats produced stronger antigenic and antibody responses to recombinant human MPO proteinConclusion Long-term repeated LPS stimulated simulated infections can cause chronic bronchitis rats to produce MPO-ANCA through NETs.The chronic bronchitis lung chronic disease environment can strengthen the immune response of the rat immune system to human recombinant MPO protein.This indicates that chronic bronchitis might be the pathogenic background for ANCA production in ANCA associated vasculitis.