Pathogenesis Of ANCA-associated Vasculitis Research Articles

Novel Therapies for ANCA-associated Vasculitis: Apilimod Ameliorated Endothelial Cells Injury through TLR4/NF-κB Pathway and NLRP3 Inflammasome.

Antineutrophil cytoplasmic antibody-associated vasculitis (AAV) is a rapidly progressive form of glomerulonephritis for which effective therapeutic drugs are currently lacking, and its underlying mechanism remains unclear. This study aimed to investigate new treatment options for AAV through a combination of bioinformatics analysis and cell molecular experiments. The research utilized integrated bioinformatics analysis to identify genes with differential expression, conduct enrichment analysis, and pinpoint hub genes associated with AAV. Potential therapeutic compounds for AAV were identified using Connectivity Map and molecular docking techniques. In vitro experiments were then carried out to examine the impact and mechanism of apilimod on endothelial cell injury induced by MPO-ANCA-positive IgG. The findings revealed a set of 374 common genes from differentially expressed genes and key modules of WGCNA, which were notably enriched in immune and inflammatory response processes. A proteinprotein interaction network was established, leading to the identification of 10 hub genes, including TYROBP, PTPRC, ITGAM, KIF20A, CD86, CCL20, GAD1, LILRB2, CD8A, and COL5A2. Analysis from Connectivity Map and molecular docking suggested that apilimod could serve as a potential therapeutic cytokine inhibitor for ANCA-GN based on the hub genes. In vitro experiments demonstrated that apilimod could mitigate tight junction disruption, endothelial cell permeability, LDH release, and endothelial activation induced by MPO-ANCA-positive IgG. Additionally, apilimod treatment led to a significant reduction in the expression of proteins involved in the TLR4/NF-κB and NLRP3 inflammasome-mediated pyroptosis pathways. This study sheds light on the potential pathogenesis of AAV and highlights the protective role of apilimod in mitigating MPO-ANCA-IgG-induced vascular endothelial cell injury by modulating the TLR4/ NF-kB and NLRP3 inflammasome-mediated pyroptosis pathway. These findings suggest that apilimod may hold promise as a treatment for AAV and warrant further investigation.

FTY720 ameliorates experimental MPO-ANCA-associated vasculitis by regulating fatty acid oxidation via the neutrophil PPARα-CPT1a pathway.

Increasing studies demonstrated the importance of C5a and anti-neutrophil cytoplasmic antibody (ANCA)-induced neutrophil activation in the pathogenesis of ANCA-associated vasculitis (AAV). Sphingosine-1-phosphate (S1P) acts as a downstream effector molecule of C5a and enhances neutrophil activation induced by C5a and ANCA. The current study investigated the role of a S1P receptor modulator FTY720 in experimental autoimmune vasculitis (EAV) and explored the immunometabolism-related mechanisms of FTY720 in modulating ANCA-induced neutrophil activation. The effects of FTY720 in EAV were evaluated by quantifying hematuria, proteinuria, crescent formation, tubulointerstitial injury and pulmonary hemorrhage. RNA sequencing of renal cortex and gene enrichment analysis were performed. The proteins of key identified pathways were analyzed in neutrophils isolated from peripheral blood of patients with active AAV and normal controls. We assessed the effects of FTY720 on ANCA-induced neutrophil respiratory burst and neutrophil extracellular traps formation (NETosis). FTY720 treatment significantly attenuated renal injury and pulmonary hemorrhage in EAV. RNA sequencing analyses of renal cortex demonstrated enhanced fatty acid oxidation (FAO) and peroxisome proliferators-activated receptors (PPAR) signalling in FTY720-treated rats. Compared with normal controls, patients with active AAV showed decreased FAO in neutrophils. FTY720-treated differentiated HL-60 cells showed increased expression of carnitine palmitoyltransferase 1A (CPT1a) and PPARα. Blocking or knockdown of CPT1a or PPARα in isolated human neutrophils and HL-60 cells reversed the inhibitory effects of FTY720 on ANCA-induced neutrophil respiratory burst and NETosis. FTY720 attenuated renal injury in EAV through upregulating FAO via the PPARα-CPT1a pathway in neutrophils, offering potential immunometabolic targets in AAV treatment.

#933 Serum and kidney complement and severity of ANCA associated vasculitis in the kidney

Abstract Background and Aims Kidney involvement in ANCA associated vasculitis (AAV) is commonly described as pauci-immune based on histological characteristics. However, we now know that the complement system is activated and contributes to the pathogenesis of AAV – with Avacopan (C5a inhibitor) demonstrating superior efficacy in maintenance of remission over Prednisolone in the ADVOCATE trial. Our aim was to evaluate if there is an association between kidney disease severity and the presence of serum and histological complement activation markers. Method Retrospective cohort study of 59 patients with AAV diagnosed between November 2015 and May 2019. Complement activation markers were serum complements (C3 and C4) levels and presence of complement (C3, C1q and C4) in glomerular capillary walls in immunofluorescence with intensity of 2+ and 3+. Kidney disease severity markers were peak serum creatinine value at presentation, anti-MPO or anti-PR3 levels, and number and proportion of glomeruli with crescents on kidney biopsy. Associations were evaluated using Spearman correlation or Mann Whitney U test as appropriate Results Among 59 patients with biopsy-proven AAV in the kidney, the median (25th percentile, 75th percentile) age was 68.8 (60.7, 73.4) years, serum creatinine was 263 (156, 560) µmol/L, and anti-MPO and anti-PR3 levels were 100.0 (81.5, 199.2) and 128.8 (22.9, 200) in 41 and 7 patients, respectively. respectively. The number and proportion of glomeruli with crescents were 6 (3, 12) and 33.3% (12.0%, 42.6%), respectively. Serum C3 and C4 were performed in 41 patients and the median levels were 1.00 (0.83, 1.14) and 0.24 (0.18, 0.31), respectively. Serum C3 level did not correlate with serum creatinine, anti-MPO or anti-PR3 titers, and number and proportion of glomeruli with crescents. Serum C4 level was negatively correlated with anti-MPO titer (34 patients; r = −0.393, p = 0.02) but did not correlate with serum creatinine or number or proportion of crescents. Complement in the glomerular capillary wall was present in 8 patients (13.6%): C3, C1q and C4 were present in 8, 3 and 1 patients, respectively. Serum creatinine [median 299 (IQR 133, 998) µmol/L versus 261 (156, 359) µmol/L, p = 0.39], anti-MPO titer [100 (88.3, 200) IU/ml versus 100 (79.4, 198) IU/ml, p = 0.74], number of crescents [14 (4, 21) versus 5 (3, 12), p = 0.90] and proportion of crescents [38.9% (12.5%, 42.3%) versus 31.3% (11.1%, 44.3%), p = 0.59] were not different in those with and without complement in the kidney biopsy. Conclusion The presence of complement activation markers was not associated with kidney disease severity in AAV. However, the detection of clinically significant associations may have been limited by our cohort size.

Von Willebrand factor antigen as a marker of disease activity in childhood-onset antineutrophil cytoplasmic antibody–associated vasculitis

Abstract Objective Von Willebrand factor (VWF) antigen plays a role in vascular inflammation and thrombosis, both of which are important in the pathogenesis of ANCA-associated vasculitis (AAV). Previous work found that VWF correlates with disease activity in childhood-onset primary CNS vasculitis. We sought to determine the relationship between VWF and disease activity over time in children with AAV. Methods AAV patients with more than one VWF level measured were included in this retrospective study, and the relationships between active vasculitis, VWF and other disease measures were analysed. Generalized estimating equations analysis was used to account for repeated VWF measurements within a patient. Repeated measures correlation was used to determine associations of paired laboratory observations. Diagnostic performance was evaluated using receiver operating curve analysis. Results A total of 732 total VWF measurements were collected in 33 AAV patients. VWF antigen levels were higher during active disease [median 2.03 IU/ml, interquartile range (IQR) 1.35, 2.55] compared with inactive disease (median 1.18 IU/ml, IQR 0.94, 1.53). VWF antigen was the only variable that was significantly associated with active disease (odds ratio 3.01, P < 0.001, 95% CI 2.3, 3.93). The effect of VWF did not show a substantial difference between the disease subtypes. There was a moderate positive correlation between VWF antigen and disease activity, with an acceptable sensitivity and specificity rates. Conclusion Increased VWF antigen levels correlate with active vasculitis in this paediatric-onset AAV cohort and may be used as an additional biomarker in childhood AAV.

Association between the AKT1 single nucleotide polymorphism (rs2498786, rs2494752 and rs5811155) and microscopic polyangiitis risk in a Chinese population

Microscopic polyangiitis (MPA) is an autoimmune disease, characterized by ANCA in blood and necrotizing inflammation of small and medium-sized vessels, one of the three clinical phenotypes of ANCA-associated vasculitis (AAV). Autophagy has been confirmed to be involved in the pathogenesis of AAV. AKT1 is one of the autophagy-regulated proteins. Its single nucleotide polymorphisms (SNPs) are associated with multiple immune-related diseases, but there are rarely studies in AAV. The incidence rate of AAV has a notable geographic difference, and MPA is predominant in China. The aim of this study was to investigate the association between AKT1 SNP and MPA risk. Genotypes of 8 loci in AKT1 were evaluated by multiplex polymerase chain reaction (PCR) and high-throughput sequencing in 416 people, including 208 MPA patients and 208 healthy volunteers from Guangxi in China. Additionally, data of 387 healthy volunteers from China were obtained from the 1000Genomes Project on public database. Differences were observed between the loci (rs2498786, rs2494752, and rs5811155) genotypes in AKT1 and MPA risk (P = 7.0 × 10–4, P = 3.0 × 10–4, and P = 5.9 × 10–5, respectively). A negative association was detected in the Dominant model (P = 1.2 × 10–3, P = 2.0 × 10–4 and P = 3.6 × 10–5, respectively). A haplotype (G-G-T) was associated with MPA risk negatively (P = 7.0 × 10–4). This study suggests that alleles (rs2498786 G, rs2494752 G and rs5811155 insT) are protective factors for MPA and alleles (rs2494752 G and rs5811155 insT) for MPO-ANCA in patients with MPA. There is a haplotype (G-G-T), which is a protective factor for MPA. It suggests that the role of AKT1 in MPA/AAV needs further study to provide more intervention targets for MPA/AAV.

A CASE OF MICROSCOPIC POLYANGIITIS (MPA) FOLLOWING SARS-COV-2 INFECTION

A CASE OF MICROSCOPIC POLYANGIITIS (MPA) FOLLOWING SARS-COV-2 INFECTION

Down-regulated FcγRII expression on plasma cells is associated with the disease activity of ANCA-associated vasculitis.

Inhibitory FcγRIIB/CD32B on B cells are critical for immunity regulation to help maintain peripheral tolerance. Altered FcγRIIB expression on B cells has been observed in several autoimmune diseases, and animal studies have suggested that FcγRIIB on B cells participates in the pathogenesis of ANCA-associated vasculitis (AAV). Here, we investigated the expression of FcγRII (FcγRIIB) on various B cell subsets and the correlation of FcγRII/CD32 expression with disease activity in AAV patients. Blood samples of patients with AAV in active stage and in remission were collected. FcγRII/CD32 expressions on various B cell subsets of the whole blood were detected by flow cytometry, and their correlation with clinical and pathological data was analysed. The expression of FcγRII/CD32 on plasma cells was significantly lower in AAV patients in active stage than those in both AAV patients in remission and healthy donors. Furthermore, the expression of FcγRII/CD32 on plasma cells negatively correlated with BVAS and percentages of cellular crescents in renal biopsies. There is a down-regulation of FcγRIIB/CD32B expression on B cells in patients with AAV, which is associated with the disease activity of AAV.

CX3CL1-induced CD16+ monocytes extravasation in myeloperoxidase-ANCA-associated vasculitis correlates with renal damage.

BackgroundMonocytes are involved in the pathogenesis of ANCA-associated vasculitis (AAV). Monocyte/macrophages are the dominant infiltrating cells in the glomeruli of patients with myeloperoxidase-AAV (MPO-AAV). However, how human monocyte subsets extravasate to the kidney in MPO-AAV with renal damage is unclear.Methods30 MPO-AAV patients with renal damage and 22 healthy controls were enrolled in this study. Monocyte subsets and monocyte-related chemokines in the blood and kidneys of MPO-AAV patients were detected. The chemoattractant activity of the CX3CL1-CX3CR1 axis on CD16+ monocytes was observed. The effect of MPO-ANCA on the migration of CD16+ monocytes to human glomerular endothelial cells (HGECs) was detected by flow cytometry and transwell migration assay.ResultsCompared with controls, CD16+ monocytes were significantly decreased in the blood and increased in the glomeruli of MPO-AAV patients with renal damage. The level of CX3CL1, but not CCL2, was significantly increased in the plasma of MPO-AAV patients. CX3CL1 co-localized with glomerular endothelial cells in MPO-AAV patients with renal damage. Moreover, we initially found that MPO-ANCA promotes an increase of the chemokine CX3CL1 on HGECs, imposing recruitment on CD16+ monocytes. Finally, the percentage of CD16+ monocytes in the blood was found to be positively correlated with estimated glomerular filtration rate (eGFR) and negatively correlated with urinary protein creatinine ratio in MPO-AAV patients with renal damage. Furthermore, the urinary protein creatinine ratio was positively correlated with the infiltrating of CD14+ and CD16+ cells in the kidneys.ConclusionEnhanced extravasation of CD16+ monocytes to the kidney via the CX3CL1-CX3CR1 axis may be involved in renal damage in MPO-AAV.

Predicting bacterial infection risk in patients with ANCA-associated vasculitis in southwest China: development of a new nomogram.

The aim of this study was to develop and assess a risk nomogram of bacterial infection in patients with ANCA-associated vasculitis (AAV) in southwest China. We established a prediction model based on a training dataset of 249 AAV patients. The least absolute shrinkage and selection operator (Lasso) was used to screen feature variables. Multivariate logistic regression analysis was used to build a prediction model for feature variables. Nomogram was used to predict the risk of bacterial infection in AAV patients. Receiver operating characteristic (ROC) curve was used to evaluate and verify the prediction accuracy of the model. Calibration and clinical useful range was assessed using calibration curve and decision curve analysis, respectively. Bactericidal permeability enhancement protein of ANCAs (BPI-ANCAs), procalcitonin (PCT), and white blood cell (WBC) were the characteristic variables in this study. Nomogram showed that positive BPI-ANCAs and PCT had higher positive predictive value for bacterial infection in AAV patients. The area under curve (AUC) of the model was 0.703 (95% confidence interval: 0.640-0.766). In the validation model, the AUC was 0.745 (95% confidence interval: 0.617-0.872). Decision curve analysis showed that the nonadherence nomogram was clinically useful within the threshold probability range of 0.31-0.85. Nomogram combined with BPI-ANCAs and PCT has the guiding significance for predicting bacterial infection risk in AAV. As an ANCA-specific autoantibody, BPI-ANCAs is helpful for clinicians to understand the role of specific autoantibodies in the pathogenesis of AAV. Key Points • BPI-ANCAs, PCT, and WBC could predict bacterial infection in AAV patients. • Nomogram showed that positive BPI-ANCAs had a high positive predictive value for bacterial infection in AAV patients.

MO241: Nets and Terminal Complement Pathway as Potential Biomarkers for Complement Overactivation Assessment in Anca-Associated Vasculitis

Abstract BACKGROUND AND AIMS Clinical, in vitro, and animal model-derived evidence has demonstrated a critical involvement of the alternative complement pathway (aCP) in the pathogenesis of ANCA-associated vasculitis (AAV). In this regard, neutrophil extracellular traps (NETs) have been suggested to be a key element between ANCA-induced neutrophil activation and aCP [1]. However, the role of the terminal complement pathway (tCP) is less well studied [2]. METHOD A prospective, observational, multicenter study analyzing first episodes and relapses of patients with AAV, with a minimum follow-up of 6 months, was performed. Blood samples were collected at diagnosis (AAV-t1) and at remission (AAV-t2). Control population consisted of age and sex-matched individuals. Complement activation was assessed by analyzing the complement membrane attack complex (C5b-9) deposition on cultured endothelial cells (HMEC-1), by immunofluorescence, after exposing them to activated plasma (a-plasma: obtained by mixing patient's citrated plasma with healthy subjects’ sera pool, 1:1). C5b-9 deposits induced by patients’ a-plasma were calculated as percentage of labeled area with respect to the total area analyzed. Results from patient and control samples were expressed as fold increase (mean ± SEM) versus those obtained with the pool of a-plasma from healthy subjects. Plasma levels of tCP and aCP soluble factors, such as sC5b-9 and sFBb (respectively), were also measured (mean ± SEM). Circulating NETs were indirectly measured by quantifying circulant dsDNA plasmatic concentration (mean ± SEM) as a NET surrogate. RESULTS The present results were obtained with samples from 13 AAV-MPO patients who achieved complete remission (38% men, age 63 ± 14 years) and 10 controls (45% men, age 66 ± 6 years). At AAV-t1, there was a statistically significant increase (P < .05) of C5b-9 deposition on HMEC-1 in response to patients’ a-plasma (fold increase of 5.3 ± 1.3) compared with control samples (fold increase of 1.2 ± 0.2). Samples obtained at AAV-t2 induced less C5b-9 deposition than at AAV-t1 (fold increase of 0.9 ± 0.2; P < .05), with values similar to controls. Regarding soluble factors, levels of both sC5b-9 and sFBb were significantly increased in AAV-t1 (1882 ± 418 ng/mL and 3.2 ± 0.4 µg/mL, respectively; P < .05) versus AAV-t2 (852 ± 104 ng/mL and 1.9 ± 0.2 µg/mL, respectively; P < .05). At AAV-t2, levels were similar to controls (708 ± 42 ng/mL for sC5b-9 and 2.4 ± 0.2 µg/mL for sFBb). Circulating NETs were also increased in AAV-t1 (22.2 ± 3.5 µg/mL) compared with both AAV-t2 and controls (13.6 ± 1.1 µg/mL and 13.7 ± 0.3 µg/mL, respectively; P < .05). Moreover, NETs in AAV-t1 presented a significant correlation with sFBb levels, both in AAV-t1 (r = 0.709; P < .05) and in AAV-t2 (r = 0.585; P < .05). CONCLUSION There is a relationship between NETs, AAV activity and sFBb, which supports the role of NETs and cAP in AAV pathogenesis. Moreover, differences in C5b-9 deposition between the two stages of the disease suggest that tCP may be dysregulated in AAV. Further characterization of this dysregulation may lead to new diagnostic or disease activity biomarkers, as well as new therapeutic options for the management of patients with AAV.

Serum ANCA and Overall Mortality: A 10-Year Retrospective Cohort Study on 1,024 Italian Subjects.

BackgroundAntineutrophil cytoplasmic antibodies (ANCA) are primarily involved in the pathogenesis of ANCA-associated vasculitides (AAV). However, ANCA may also be present in healthy subjects and in patients with autoimmune disorders different from AAV. We hypothesized that serum ANCA are associated with a worse prognosis in disorders other than AAV.ObjectiveWe investigated the association between the overall survival and the presence of serum ANCA in 1,024 Italian subjects with various testing indications in a 10-year interval.MethodsIn this retrospective cohort study, a population of 6,285 patients (many of whom were subsequently excluded due to our criteria) who tested for ANCA at a single center in 10 years was considered, and life status and comorbidities of subjects were collected. We compared the overall survival of ANCA-positive and ANCA-negative patients by means of Kaplan-Meier curves, while a multivariable adjusted Cox regression was used to evaluate the association between the ANCA status and the outcome (death) in terms of hazard ratios (HR) with 95% confidence intervals (CI).ResultsThe positivity of perinuclear ANCA (pANCA) increased significantly mortality (HR, 1.60; 95% CI, 1.10–2.32), while cytoplasmic ANCA (cANCA) positivity failed to show a significant association (HR, 1.43; 95% CI, 0.77–2.68). The increased mortality rate was observed for both pANCA and cANCA in patients suffering from rheumatic disorders. No association was found between mortality and anti-MPO (HR, 0.63; 95% CI, 0.20–2.00) or anti-PR3 (HR, 0.98; 95% CI, 0.24–3.96) after adjusting for confounders.ConclusionsSerum pANCA and cANCA are independent negative prognostic factors in patients with concurrent autoimmune diseases.

Altered circulating CCR6+and CXCR3+ T cell subsets are associated with poor renal prognosis in MPO-ANCA-associated vasculitis

BackgroundEffector memory T cells are pivotal effectors of adaptive immunity with enhanced migration characteristics and are involved in the pathogenesis of ANCA-associated vasculitis (AAV). The diversity of effector memory T cells in chemokine receptor expression has been well studied in proteinase 3 (PR3)-AAV. However, few studies have been conducted in myeloperoxidase (MPO)-AAV. Here, we characterized chemokine receptor expression on effector memory T cells from patients with active MPO-AAV.MethodsClinical data from newly diagnosed MPO-AAV patients and healthy subjects were collected and analyzed. Human peripheral blood mononuclear cells (PBMCs) isolated from patients with active MPO-AAV were analyzed by flow cytometry. The production of effector memory T cell-related chemokines in serum was assessed by ELISA.ResultsWe observed decreased percentages of CD4+ and CD8+ T cells in the peripheral blood, accompanied by a significant decrease in CCR6-expressing T cells but an increase in CXCR3+ T cells, in active MPO-AAV. Furthermore, the decrease in CCR6 and increase in CXCR3 expression were mainly limited to effector memory T cells. Consistent with this finding, the serum level of CCL20 was increased. In addition, a decreasing trend in the TEM17 cell frequency, with concomitant increases in the frequencies of CD4+ TEM1 and CD4+ TEM17.1 cells, was observed when T cell functional subsets were defined by chemokine receptor expression. Moreover, the proportions of peripheral CD8+ T cells and CD4+ TEM subsets were correlated with renal prognosis and inflammatory markers.ConclusionsOur data indicate that dysregulated chemokine receptor expression on CD4+ and CD8+ effector memory T cells and aberrant distribution of functional CD4+ T cell subsets in patients with active MPO-AAV have critical roles related to kidney survival.

Relationship between Renal Damage and Serum Complement C3 in Children with Anti-Neutrophil Cytoplasmic Antibody-Associated Vasculitis

Background: Anti-neutrophil cytoplasmic antibody-associated vasculitis (AAV) disease is a well-known antibody-induced autoimmune disease. The pathogenesis of AAV has not yet been completely clarified, but may be related to heredity, infection, environmental factors, cellular immunity, etc. In recent years, complement in AAV pathogenesis has become the latest research hotspot, and the decrease of serum complement C3 is associated with poor prognosis of anti-neutrophil cytoplasmic antibody-associated glomerulonephritis. In the current study, we investigated the associations between serum complement C3 and kidney injury in AAV children. Methods: Twenty-four children with AAV admitted to our hospital from June 2014 to June 2019 were divided into the low C3 group and the normal C3 group. All the children have undergone renal biopsy. The clinical manifestations, laboratory tests, renal pathology, treatment, and prognosis of the 2 groups were observed. The primary end point was end-stage renal disease (ESRD). Results: It was shown that kidney injury was more obvious in patients with low C3 than in patients with normal C3 serum. The values of ESR, Scr, and UA before treatment in the low C3 group were higher than those in the normal C3 group (p < 0.01); the values of RBC, Hb, PLT, ALB, LDH, and eGFR in the normal C3 group were higher than those in the low C3 group (p < 0.01). The values of urinary protein and NAG enzyme in the low C3 group were higher than those in the normal C3 group (p < 0.01). The area of glomerular abandonment, sclerosis, segmental sclerosis, crescent, cellular crescent, cellular fibrous crescent, fibrous crescent, segmental loop necrosis, and the number of cases with acute renal tubulointerstitial lesions in the low C3 group were bigger than those in the normal C3 group (p < 0.05 and < 0.01). The number of cases with C3 deposition in the low C3 group was higher than that in the normal C3 group (p < 0.05). The number of patients receiving CRRT and PE in the low C3 group was higher than that in the normal C3 group (p < 0.05 and < 0.01). In this study, 3 children entered the stage of ESRD and 1 died in the low C3 group. Conclusion: The kidney injury of AAV children with low complement C3 is serious, and the prognosis is poor. We should pay attention to the influence of decreased complement C3 on the condition and prognosis of AAV children.

Platelet CXCL4 mediates neutrophil extracellular traps formation in ANCA-associated vasculitis

Neutrophils form neutrophil extracellular traps (NETs), which are involved in the pathogenesis of ANCA-associated vasculitis (AAV). Recent reports suggest that platelets stimulated via toll-like receptor (TLR) pathways can induce NETs formation. However, the mechanism underlying the involvement of platelets in NETs formation in AAV is unknown. We investigated the role of platelets in the pathogenesis of AAV. Platelets from AAV patients and healthy controls (HCs) were co-cultured with peripheral neutrophils, and NETs formation was visualized and quantified. The expression levels of TLRs on platelets were examined by flow cytometry. Platelets were treated with a TLR agonist, platelet-derived humoral factor, CXCL4 (platelet factor 4: PF4), and/or anti-CXCL4 antibody to investigate the effects of TLR–CXCL4 signaling on NETs formation. Platelets from AAV significantly upregulated NETs formation in vitro. Flow cytometric analysis revealed that the proportion of TLR9 positive platelets was significantly higher in AAV than HCs. CXCL4 released from TLR9 agonist-stimulated platelets was significantly enhanced in AAV, which subsequently increased NETs formation. Further, neutralizing anti-CXCL4 antibody significantly inhibited NETs formation enhanced by platelets from AAV. TLR9 signaling and CXCL4 release underlie the key role that platelets play in NETs formation in the pathogenesis of AAV.

Mass spectrometry-based proteomic exploration of the small urinary extracellular vesicles in ANCA-associated vasculitis in comparison with total urine

ANCA-associated vasculitis (AAV) is a rare, but potentially severe autoimmune disease, even nowadays displaying increased mortality and morbidity. Finding early biomarkers of activity and prognosis is thus very important. Small extracellular vesicles (EVs) isolated from urine can be considered as a non-invasive source of biomarkers. We evaluated several protocols for urinary EV isolation. To eliminate contaminating non-vesicular proteins due to AAV associated proteinuria we used proteinase K treatment. We investigated the differences in proteomes of small EVs of patients with AAV compared to healthy controls by label-free LC–MS/MS. In parallel, we performed an analogous proteomic analysis of urine samples from identical patients. The study results showed significant differences and similarities in both EV and urine proteome, the latter one being highly affected by proteinuria. Using bioinformatics tools we explored differentially changed proteins and their related pathways with a focus on the pathophysiology of AAV. Our findings indicate significant regulation of Golgi enzymes, such as MAN1A1, which can be involved in T cell activation by N-glycans glycosylation and may thus play a key role in pathogenesis and diagnosis of AAV. SignificanceThe present study explores for the first time the changes in proteomes of small extracellular vesicles and urine of patients with renal ANCA-associated vasculitis compared to healthy controls by label-free LC–MS/MS. Isolation of vesicles from proteinuric urine samples has been modified to minimize contamination by plasma proteins and to reduce co-isolation of extraluminal proteins. Differentially changed proteins and their related pathways with a role in the pathophysiology of AAV were described and discussed. The results could be helpful for the research of potential biomarkers in renal vasculitis associated with ANCA.

The alternative complement pathway in ANCA-associated vasculitis: further evidence and a meta-analysis.

We compared the common pathway components C3a, C5a and membrane attack complex (MAC), also known as C5b-9, and the alternative pathway components factor B and properdin in patients with ANCA-associated vasculitis (AAV) and healthy controls, and conducted a meta-analysis of the available clinical evidence for the role of complement activation in the pathogenesis of AAV. Complement components were evaluated in 59patients with newly diagnosed or relapsing granulomatosis with polyangiitis or microscopic polyangiitis and 36 healthy volunteers. In 28 patients, testing was repeated in remission. Next, we performed a meta-analysis by searching databases to identify studies comparing complement levels in AAV patients and controls. A random-effects model was used for statistical analyses. The median concentrations of MAC, C5a, C3a and factor B were higher in active AAV patients (P<0·001). Achievement of remission was associated with reductions in C3a (P=0·005), C5a (P=0·035) and factor B levels (P=0·045), whereas MAC and properdin levels did not change. In active AAV, there were no effects of ANCA specificity, disease phenotype, previous immunosuppression or disease severity on complement levels. A total of 1122articles were screened, and five studies, including this report, were entered into the meta-analysis. Plasma MAC, C5a and factor B in patients with active AAV were increased compared to patients in remission (excluding factor B) and controls. Changes in C3a were of borderline significance. Our findings and the results of the meta-analysis support activation of the complement system predominantly via the alternative pathway in AAV patients.

THU0321 URINARY T CELLS IDENTIFY ACTIVE RENAL ANCA ASSOCIATED VASCULITIS

Background:ANCA-associated vasculitis (AAV) causes necrotizing crescentic glomerulonephritis (NCGN) which is a major contributor to morbidity and mortality in AAV. Since therapy relies on cytotoxic agents with potentially severe adverse effects, a reliable non-invasive biomarker of disease activity is needed to determine the right balance between over- and undertreatment. Using the urine space as a window into the local inflammatory milieu of the kidney, quantification of urinary leukocytes using flow cytometry has become an upcoming marker of various other inflammatory kidney diseases. As recent studies have illuminated the emerging role of T regulatory (Treg) and Th17 cells in the pathogenesis of AAV, these could represent a viable non-invasive biomarker more closely displaying the underlying pathogenic processes than metabolites or epiphenomena of inflammation.Objectives:To quantify urinary T cells in active renal AAV and asses their biomarker characteristics.Methods:Using flow cytometry, T-lymphocytes and their subsets were quantified in peripheral blood and urine samples from patients with active AAV with or without NCGN, in stable remission with previous NCGN and healthy controls. Concentrations of urinary soluble metabolites and cytokines (Monocyte-attracting protein 1 (MCP-1), sCD163, sCD25 and C5a) were measured using Multiplex analysis. Results were verified in a separate validation cohort.Results:Patients with renal active AAV (n = 30) showed significantly higher urinary cell counts of total T cells, CD4+, CD8+, Treg and Th17 subsets than disease (n = 21) and healthy controls (n = 8). Patients with active renal AAV also showed a significantly higher percentage of Tregs in urine than in blood. While Tregs allowed a robust discrimination between active renal AAV and disease controls (receiver operator characteristics (ROC): area under the curve (AUC) 0.93, sensitivity 79%, specificity 95%) quantification of all T cells proved to be slightly more accurate (ROC: AUC 0.95, sensitivity 92%, specificity 95%). Soluble markers showed a slightly inferior discrimination (MCP-1 ROC: AUC 0.90, sensitivity 60%, specificity 100%, sCD163 ROC: AUC 0.92, sensitivity 96%, specificity 85%) while sCD25 and C5a were far less accurate.Conclusion:Urinary T cells are significantly elevated in active renal AAV and the increased frequency of Tregs in urine suggests active migration into inflamed glomeruli and thereby the urine rather than mere bleeding of ruptured capillaries. These cells show great potential for a non-invasive biomarker close to the local inflammatory milieu. Particularly the total count of urinary T cells showed slightly superior biomarker characteristics than previously established soluble markers. Further studies are needed to confirm these results and show potential prognostic value of these cellular markers.Disclosure of Interests:None declared

P0456LONG TERM PATIENT SURVIVAL AND RELAPSE RATE IN ANCA ASSOCIATED PATIENTS WITH RENAL INVOLVMENET - DATA FROM CROATIAN REFERRAL CENTER

Abstract Background and Aims The aim of the research was to evaluate patient and renal as well as relapse free survival in ANCA associated vasculitis (AAV) patients in our center. Despite the advances in understanding pathogenesis of AAVs and advances in treatment, the outcomes of AAV patient differ in various centers. Method This study included 106 consecutive AAV patients with renal involvement in the period from 2007-2017. We performed renal biopsy on patients using automatic 16 Gauge needle. Light, immunofluorescent and electronic microscopy were performed. All the patients were treated with cyclophosphamide and steroids in induction treatment with adjuvant PLEX and dialysis depending on renal function and lung manifestations. Primary outcomes were combined outcome progression to end-stage renal disease, defined as persistent (more than three months) need for renal replacement therapy or permanent reduction of EGFR to &amp;lt;15ml/minute (according to CKD EPI formula) and/or death (ESRDD), death (D) and ESRD alone, and disease relapse. Kaplan Meyer survival analysis and multivariate Cox proportional hazard regression analysis were used to explore difference between phenotypes and finding significant predictors regarding outcomes. Out of 106 patients (55,6% female, median age 61; IQR 51-70) there were 66 (61,1%) microscopic poliangitiis (MPA), 20 (18,5%) granulomatosis with angitiis and 20 (18,5%) with renal limited vasculitis (RLV),There were 14 (13%) PR3-ANCA positive patients, 57 (52,8%) MPO ANCA positive, 5 (4,6%) PR3-ANCA+MPO-ANCA positive and 32 (29,6%) ANCA negative patients. Histologically (Berden classification) 43 (39,8%) patients had crescentic, 19 (17,6%) focal, 34 (31,5%) mixed and 12 (11,1%) sclerotic class. Follow up time ranged from 1 to 127 months. Median follow up time was 21 months (IQR = 7-44). Median time to diagnosis was 3 months (IQR 2,0-6,0). Results During follow up 21 (19,8%) patients died, 26 (24,5%) patients reached ESRD and 10 (9,4%) patients relapsed. There was no significant difference in outcomes between clinical, serological or histological phenotypes. In multivariant analysis independent predictors for death were age (HR = 1,059, 95% CI =1,001-1,120; p = 0,046), anemia (HR = 0,952, 95% CI =0,908-0,998; p = 0,040) and BVAS (HR = 1,093, 95% CI =1,030-1,159; p = 0,003), for ESRD. the need for acute dialysis (HR = 4,674, 95% CI =1,996-10,946; p = &amp;lt; 0,001), and interstitial fibrosis and tubular atrophy (IFTA) percentage over 50% (HR = 2,652, 95% CI =1,157-6,081; p = 0,021). and for relapse rate younger age (HR = 0,924, 95% CI = 0,870-0,981; p =0,010), lower serum creatinine levels (HR = 0,996, 95% CI = 0,992-1,000; p = 0,033), and the need for acute dialysis (HR = 59,545, 95% CI =3,467-1022,665; p = 0,005). Event free survival after 12, 24, 36 and 60 months was for death 83,9, 81,2, 79 and 74,7%, for ESRD 80,6, 77,9, 76,1 and 71% and for relapse 95,3, 88,4, 88,4 and 85%. Conclusion Timely diagnosis and treatment can ensure better outcomes in AAV patients. Though there is an overlap in predictive factors between different cohorts, there are still distinctive differences especially between cohorts from clinical trials and those from observational studies. Our study is among few to show significance of anemia as clinical predictor and IFTA percentage as pathohistological predictor.

Pathogenesis of ANCA-associated vasculitis: an emerging role for immunometabolism.

ANCA-associated vasculitis (AAV) is a severe systemic autoimmune disease. A key feature of AAV is the presence of Anti-Neutrophil Cytoplasmic Antibodies (ANCA) directed against myeloperoxidase (MPO) or proteinase-3 (PR3). ANCA are key to the pathogenesis of AAV, where they activate innate immune cells to drive inflammation. Pre-activation or 'priming' of immune cells appears to be important for complete cellular activation in AAV. The burgeoning field of immunometabolism has illuminated the governance of immune cell function by distinct metabolic pathways. There is ample evidence that the priming events synonymous with AAV alter immune cell metabolism. In this review we discuss the pathogenesis of AAV and its intersection with recent insights into immune cell metabolism.

Evaluation of the Safety and Efficacy of Avacopan, a C5a Receptor Inhibitor, in Patients With Antineutrophil Cytoplasmic Antibody-Associated Vasculitis Treated Concomitantly With Rituximab or Cyclophosphamide/Azathioprine: Protocol for a Randomized, Double-Blind, Active-Controlled, Phase 3 Trial.

BackgroundAntineutrophil cytoplasmic antibody (ANCA)–associated vasculitis is a serious, often life-threatening disease. In new-onset disease or a relapse, the standard treatment is immunosuppressive therapy with glucocorticoids; these therapies are associated with substantial short- and long-term toxicity. Complement component 5a (C5a) binding to C5a receptor (C5aR) may play a central role in the pathogenesis of ANCA-associated vasculitis. Avacopan is a novel, orally bioavailable, and highly selective antagonist of human C5aR. Avacopan does not interfere with the production of C5b or the membrane attack complex (ie, terminal complement complex) and does not block C5a binding to a second receptor, C5L2 (also called C5aR2), shown to be protective in antimyeloperoxidase glomerulonephritis. This trial will evaluate if avacopan replaces the need for chronic glucocorticoids in the treatment of ANCA-associated vasculitis.ObjectiveThe aim of this study is to determine the proportions of patients in remission at week 26 and with sustained remission at week 52, defined as Birmingham Vasculitis Activity Score=0, and not taking glucocorticoids within the 4 weeks before week 26 and week 52, respectively.MethodsThe Avacopan Development in Vasculitis to Obtain Corticosteroid elimination and Therapeutic Efficacy study is a randomized, double-blind, active-comparator (prednisone), 2-arm study evaluating the safety and efficacy of avacopan versus prednisone, administered in combination with other immunosuppressive therapy. Eligible subjects will have active disease requiring induction of remission. Subjects are stratified based on the type of immunosuppressive therapy, ANCA subtype, and new or relapsing disease. Target sample size is 300 patients, enrolled at over 200 sites globally. All authors and local ethics committees approved the study design. All patients will provide informed consent.ResultsEnrollment of patients was completed in Q4 2018. Topline results are anticipated to be published by Q3 2020.ConclusionsResults will be released irrespective of whether the findings are positive or negative.Trial RegistrationClinicalTrials.gov NCT02994927; https://clinicaltrials.gov/ct2/show/NCT02994927International Registered Report Identifier (IRRID)DERR1-10.2196/16664