The Role And Mechanism Of Pentraxin3in Anti-neutrophil Cytoplasmic Antibodies Associated Vasculitis

Objectives:Anti-neutrophil cytoplasmic antibody (anti-neutrophil cytoplasmic antibodies, ANCA)associated vasculitis (ANCA associated vasculitis, AAV) was a group of systemicautoimmune inflammation diseases. There is not rare especially in the aged population. Theincidence rate is increasing after the ANCA detection was universal in China in recentyears.The clinical manifestations were comlex and associated with multiple organs wereinvolved frequently. The kidneys are frequently affected in AAV patients, and also can beled to irreversible renal failure rapidly. It is urgent for us to further study the pathogenesisof AAV to provide more effective treatment strategies.The pentraxin family contains mainly the two short serumamyloid P (SAP) andC-reactive protein (CRP)(also referred to as acute-phase proteins), and the prototypic longpentraxin is PTX3. As a soluble pattern recognition receptor (PRR) in the innate immuneresponse, which had verified participated in various autoimmunity disease and correlationswith activity and local inflammation of tissues. Neutrophil extracellular traps (NETs) are anewly recognized mode of cell-death of neutrophil, which extrudes its structures of DNAand disturbs the release of self-DNA. PTX3are components of NETs, and may also expressin kidney tissue. Recent studies suggested that PTX3could counteract the pathogens and inthe form of a kind of natural antibodies to protect the body.ANCA play a core role in the pathogenesis of AAV and also correlated with diseaseactivity as a biomarker. ANCA itself also had the pathogenic and had been involved in theprogress of the AAV directly, and also participate in the formation of a vicious cycle of theautoimmune inflammation. Our previous study had shown that ANCA could induce NETsformation and exposured to endogenous danger signals through released the DNA andautoantigens. As PTX3is the key mediator in the pathogenesis of inflammation, we speculate that ANCA-induced NETs would release the PTX3, which may reflect the localinflammation more rapidly than CRP levels or ESR and could be a new acute inflammationbiomarker of disease activity.The purpose of our project is to explore the the role and mechanism of PTX3in thepathogenesis of AAV through by (1) detcting the sera levels of PTX3and the expression ofPTX3in the renal tissue of patient with AAV, and to further analyze the correlation betweenthe sera levels of PTX3and urine proteins and BVAS score;(2) investigating the sources ofPTX3whether were released from neutrophils through extracelluar traps formation inducedby ANCA in vitro,. These results raise the possibility that PTX3may play a role and asbiomarker in the AAV.Methods:Part1The expression and significance of PTX3in serum and renal tissue ofpatients with AAV1. The expressions of PTX3were detected in active AAV renal section byimmunofluorescent staining using confocal microscope to investigate whether or PTX3were existed at the sites of renal inflammation.2. The differences serum levels of PTX3and CRP between the AAV patients andhealthy controls were evaluated by ELISA.3. The active and remission stage of AAV patients were evaluated by BVAS scorebased on their medical records, retrospectively. To evaluate the relationship between theactive and remission in patients with sera levels of PTX3and CRP and renal functiondamage, independent non paired t test, Mann Whitney U test of two independent samplesnonparametric and pearson correlation analysis were performed. The accuracy wereanalysised by ROC curves of the PTX3, CRP and ESR, respectively.Part2PTX3released from neutrophils induced by ANCA through NETsformation1. Neutrophils were isolated by density gradient centrifugation method usingPolymorphprepTMfrom fresh peripheral vein blood samples of AAV patients and healthyvolunteers. The purity of neutrophils was identified labeled with FITC-antihuman-CD16antibody by the flow cytometry analysis. The average cell survival rate of neutrophils was calculated by trypan blue staining method.2. The supernatant levels of PTX3were assayed by ELISA.3. The expression of PTX3were detected by immunofluorescence staining in eachgroup of the resting neutrophils, incubated with LAMP-2IgG (H4B4), TNF-α,the serafrom AAV patients and healthy volunteers for180min, respectively.Results:Part11. The expression of PTX3in the renal tissue section of active AAV weresignificantly higher than those of mesangial proliferative glomerulonephritis and IgAnephtitis.2. The sera levels of PTX3in active AAV patients were significantly higher thanthose in inactive AAV patients, and also were higher than in healthy patients or those withSLE. However, the sera levels of PTX3in SLE patients were significantly lower than thehealthy controls (HC).3. The levels of CRP and ESR were also elevated in patients with active comparedwith inactive AAV. However, there were higher levels of CRP and ESR in patients withSLE which had lower levels of PTX3.4. There were significant positive correlation between serum PTX3concentration and24-h urinary protein quantity. There were no correlations between the levels of PTX3andWBC, NEUT, absolute value of lymphocytes, complemente3, serum creatinine and eGFR.On the contrary, there were no correlations between CRP,ESR and24-h urinary proteinquantity. There were significant positive correlation between the levels of PTX3, CRP andBVAS score. There were no correlations between ESR levels and BVAS score.5. ROC curve showed that serum PTX3level between the active and remission stageAAV accuracy is higher than that of serum CRP and ESR levels. The area of ROC curveunder the PTX3curve was0.9567(P<0.0001). The area of ROC curve under the CRPcurve was0.7792(P=0.0105) and the that of ESR was0.7424(P=0.0263). To evaluate theactive and remission stage AAV patients, the serum level of PTX3was0.721ng/mL as thedistinction between the cutoff value, the sensitivity and specificity were90.48%and100% Part26. Immunofluorescence staining showed that PTX3was stored in quiescent state ofneutrophils ex vivo. The expression levels of PTX3in neutrophils from AAV patients (n=3)were significantly higher than those of healthy controls (n=9)(P=0.0402).7. PTX3was released from neutrophils was responsed to anti-LAMP-2antibody andthe sera of AAV patients. The levels of PTX3were higher in anti-LAMP-2antibody groupcompared with normal controls (P=0.0020). The immunofluorescence analysis had shownthat the mean flourscence indensity (MFI) of PTX3in neutrophils incubated with AAVserum was higher than the healthy controls (P=0.0029).8. PTX3was released along with MPO through the neutrophil extracelluar traps(NETs) formation detected by laser scanning confocal microscope.Conclusions:1. The serum levels of PTX3were significantly higher in active AAV patients than HC.and expressed at local inflammation of renal tissue in active AAV patients. There was apositive correlation between the serum levels of PTX3and disease activity,24hour urineprotein excretion.The PTX3may be a novel acute-phase protein to evaluate the diseaseactivity of AAV.2. Amounts of PTX3were released from neutrophils of AAV patients and could bereleased with MPO and DNA induced by ANCA from neutrophils of HC through NETsformation. These process may be involved the pathophysiological state in AAV.