The Role And Mechanism Of LL37on PDCs Activation In ANCA Associated Vasculitis

BackgroudAnti-neutrophil cytoplasmic antibody (anti-neutrophil cytoplasmic antibodies, ANCA)associated vasculitis (ANCA associated vasculitis, AAV) represents a group of systemicautoimmune diseases. Approximately80%patients with AAV get kidney injury, and alsoAAV can lead to irreversible renal failure rapidly. In order to propose more effectivetreatment strategies, it is urgent for us to do further study on the pathogenesis of AAV.Immune response is based on the recognition of "self" or "non-self", which dedicatedthe immune tolerance or activation. Plasmacytoid dendritic cells (pDCs) play a key role inimmunity recognition. pDCs are a particular subset of DCs, which are responsible for thesubstantial production of type1interferon. So they are also known as interferon-producingcells. In innate immunity, pathogen-associated molecular patterns (PAMP) and patternrecognition receptors (PPRs) are recognized to distinguish "self" and "non-self”. In recentyears, pDCs have become central issue in autoimmune diseases, because they highlyexpress Toll-like receptor7(TLR7) and TLR9. Remarkable progress has been made in theoccurrence of psoriasis: pDCs highly express TLR7and TLR9, which activate the pathwayof signal interferon regulatory factor-7(IRF-7) by interacting with myeloid differentiationfactor88(MyD88), thus inducing a strong IFN-I response. In this case, NK cells andmyeloid dendritic cells are activated, which promotes Th1and Th17cells responses, andstart adaptive immune response.In steady conditions, pDCs promote immune responses through identification withpathogen DNA, but ignore of self-DNA released from dead cells. This kind of immunetolerance for self-DNA is essential for maintaining homeostasis. However, in autoimmunediseases, endogenous IFN-α inducer can replace pathogen DNA and RNA and breakimmune tolerance, which continuously stimulates pDC producing IFN-α and startingautoimmune response. Studies have found that patients with active AAV express high levelsof IFN-α, which suggests that the abnormal prodction of IFN-α released from activation of pDC play a role in the pathogenesis of autoimmune diseases. In accordance with psoriasis,peptide LL37plays a key role in abnormal activation of pDC. We, herein, speculate thatLL37may relate to the high level of IFN-αin AAV patients.Antimicrobial peptides participate in innate immune. LL-37is the only Cathelicidinpeptides found in the human race, and it has been verified that LL37can directly mediatepDCs to identify self-DNA, and take part in the occurrence of autoimmune inflammation.Neutrophil extracellular traps (NETs) are a newly recognized mode of neutrophilcell-death, which extrudes its structures of DNA and disturbs the release of self-DNA.LL37are components of NETs, and are also expressed in kidney tissue. As LL37is the keymediator in the pathogenesis of psoriasis, we speculate that ANCA-induced NETs wouldparticipate in ANCA-associated vasculitis (AVV) by identify self-DNA, anomalyidentification pathogenesis. LL37activated pDCs may play a key role in it.This project intends (1) to detectpeptide LL37and pDC producting IFN-α of serumand renal tissue of patient with AAV, and to analyze the relationship of expression of LL37,IFN-α and the extent of renal tissue injury with clinical cases, so as to elaborate the role ofLL37in the pathogenesis of AAV.(2) to observe the NETs formation and LL37expressionin neutrophils from patients with AAV in vitro, so as to confirm the LL37in patients withAAV is released from NETs.(3) pDCs were separated and purified by immunomagneticbeads, and then were incubated with NETs, or adding anti-LL37antibody, ODN-TTAGG.We observed the maturation marker expression on pDCs and the production of IFN-α,so asto elaborate whether NETs from AAV patients activate pDCs through LL37, and prove thepathway that LL37induced pDCs recognize self-DNA. With comprehensive analysis of theclinical casesin vitro experimental results, we intend to investigate the mechanism ofantimicrobial peptide LL37activated pDCs in ANCA-associated vasculitis, and the role ofpDCs in the process of disease development.MethodsPart1The expression and significance of LL37and IFN-α in serum and renaltissue of patients with AAV1. The peripheral blood serum samples of patients with AAV and healthy volunteerswere collected. The secretion of LL37and IFN-α was detected by ELISA, so as to knowwhether LL37and IFN-α increases or not. 2. Medical records of patients with AAV were retrospectively analyzed. Through thecollected data weevaluated the relationship of LL37/IFN-α and impaired renal function andrenal tissue crescent formation.3. Collecting renal tissue biopsy specimens of AAV patients, LL37, and IFN-α andBDCA2were detected by immunofluorescence., in order to assay whether LL37and IFN-αexpression and pDC cell recruitment occurs in renal injured tissue.Part2The expression of LL37and NETs formation in patients with AAV1. Collecting fresh peripheral blood samples of AAV patients and healthy volunteers,neutrophils were isolated by density gradient centrifugation method.2. Neutrophil extracellular traps (NETs) formation was detected byimmunofluorescence and scanning electron microscopy.3. The expression of LL37released from neutrophil extracellular traps (NETs)formation was detected by immunofluorescence, and the production of LL37was assayedby ELISA.4. The components of NETs, including histones, lysosomal membrane protein-2(LAMP2), myeloperoxidase (MPO) and protease3(PR3), were detected byimmunofluorescence, so as to know whether neutrophils localized in renal inflammatoryinjuried tissue produce NETs or not.Part3The mechanism of LL37activate pDCs1. Collecting fresh peripheral blood samples of AAV patients and healthy volunteers,neutrophils were isolated by density gradient centrifugation method, and then supernatantswere collected.2. pDCs were separated by immunomagnetic beads and purified by flow cytometry,then CD4+CD11c-Lineage-pDCs were harvested.3. pDCs were incubated in the supernatants of NETs from AAV patients, and theinfluences on MFI of pDCs activation markers (CD80, CD83) were detected by flowcytometry.4. After adding anti-LL37antibody or ODN-TTAGG, the influences on MFI of pDCsactivation markers (CD80, CD83) were detected by flow cytometry, to verify whether NETsfrom AAV patients activate pDCs via LL37, and LL37activate pDCs through TLR9.5. Collecting all supernatants of pDCs and NETs, the levels of IFN-α were assayed by ELISA.ResultsPart11. The levels LL37and IFN-αsignificantly increased in the serum of AAV patients.The serum levels of LL37and IFN-αin patients with crescentic glomerulonephritis(CGN)were significantly higher than those in patients without CGN.2. Patients with high LL37and INF-α expression had a greater risk of havingcrescentic GN as compared to patients with low LL37and INF-αexpression.3. We found a positive correlation between the serum LL37and IFN-α levels as wellas significant positive correlations between the serum levels of LL37and Cr and betweenthe serum levels of IFN-α and Cr, however, neither LL37nor IFN-α showed anycorrelation with complement C3.4. We found that a large amount of LL37and IFN-α in renal biopsies from AAVpatients with crescentic GN, while a little of LL37and IFN-α was found in renal biopsiesfrom AAV patients without CGN.Part21. The neutrophils isolated from patients with AAV had NETs formation.2. NETs from patients with AAV exposed histones, LAMP2, MPO and PR3.3. Antimicrobial peptide LL37, NETs, LAMP-2, MPO and PR3were observed at renalinjured location in AAV patients with CGN.4. Antimicrobial peptide LL37, NETs and MPO were all located at renal injured part inAAV patients with CGN.Part31. NETs supernatant of AAV patients significantly increased the markers indicative ofmaturation (CD80, CD83) on pDCs..2. NETs supernatant of AAV patients decreased the markers indicative of maturation(CD80, CD83) on pDCs, after adding anti-LL37antibody.3. NETs supernatant of AAV patients decreased the markers indicative of maturation(CD80, CD83) on pDCs, after adding ODN-TTAGG.4. NETs supernatant of AAV patients significantly increased the secretion of INF-α.5. NETs supernatant of AAV patients decreased the secretion of INF-α, after adding anti-LL37antibody or ODN-TTAGG.Conclusions:1. The levels of LL37and INF-αsignificantly increased in AAV patients, especiallywith crescentic GN, and LL37and IFN-α express at local inflammation renal tissue in AAVpatients with CGN2. There is a positive correlation between the serum LL37and IFN-α levels in theserum of AAV patients, and LL37and IFN-α levels and renal function in patients withAAV crescent formation related. The level of LL37and IFN-α are related to renal functionand crescent formation.3. The neutrophils isolated from patients with AAV had amounts of NETs formation.4. NETs formation observed from AAV patients neutrophils, releases antimicrobialpeptide LL37, NETs, LAMP-2, MPO and PR3.5. NETs supernatant of AAV patients activate pDCs, and promote the secretion ofIFN-α.NETs supernatant of AAV patients activate pDCs, and promote the secretion of IFN-α,however, the increase of these effects on pDCs were inhibited by blocking LL37or TLR9function.To sum up, NETs formation increases in AAV patient peripheral blood and renalinjured tissue. The formation of NETs releases a large amount of LL37and self-DNA, andactivates pDCs to secrect INF-α through TLR9pathway, which participates in AAVinflammation and immune response.