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BACKGROUNDLittle is known about the autoreactive B cells in antineutrophil cytoplasmic antibody-associated (ANCA-associated) vasculitis (AAV). We aimed to investigate tolerance checkpoints of circulating antigen-specific proteinase 3-reactive (PR3+) B cells.METHODSMulticolor flow cytometry in combination with bioinformatics and functional in vitro studies were performed on baseline samples of PBMCs from 154 well-characterized participants of the RAVE trial (NCT00104299) with severely active PR3-AAV and myeloperoxidase-AAV (MPO-AAV) and 27 healthy controls (HCs). Clinical data and outcomes from the trial were correlated with PR3+ B cells (total and subsets).RESULTSThe frequency of PR3+ B cells among circulating B cells was higher in participants with PR3-AAV (4.77% median [IQR, 3.98%-6.01%]) than in participants with MPO-AAV (3.16% median [IQR, 2.51%-5.22%]) and participants with AAV compared with HCs (1.67% median [IQR, 1.27%-2.16%], P < 0.001 for all comparisons), implying a defective central tolerance checkpoint in patients with AAV. Only PBMCs from participants with PR3-AAV contained PR3+ B cells capable of secreting PR3-ANCA IgG in vitro, proving they were functionally distinct from those of participants with MPO-AAV and HCs. Unsupervised clustering identified subtle subsets of atypical autoreactive PR3+ memory B cells accumulating through the maturation process in patients with PR3-AAV. PR3+ B cells were enriched in the memory B cell compartment of participants with PR3-AAV and were associated with higher serum CXCL13 levels, suggesting an increased germinal center activity. PR3+ B cells correlated with systemic inflammation (C-reactive protein and erythrocyte sedimentation rate, P < 0.05) and complete remission (P < 0.001).CONCLUSIONThis study suggests the presence of defective central antigen-independent and peripheral antigen-dependent checkpoints in patients with PR3-AAV, elucidating the selection process of autoreactive B cells.Trial registrationClinicalTrials.gov NCT00104299.FundingThe Vasculitis Foundation, the National Institute of Allergy and Infectious Diseases of the NIH, and the Mayo Foundation for Education and Research.

ObjectivesTo characterise renal tissue metabolic pathway gene expression in different forms of glomerulonephritis.MethodsPatients with nephrotic syndrome (NS), antineutrophil cytoplasmic antibody-associated vasculitis (AAV), systemic lupus erythematosus (SLE) and healthy living donors (LD) were studied. Clinically indicated renal biopsies were obtained at time of diagnosis and microdissected into glomerular and tubulointerstitial compartments. Microarray-derived differential gene expression of 88 genes representing critical enzymes of metabolic pathways and 25 genes related to immune cell markers was compared between disease groups. Correlation analyses measured relationships between metabolic pathways, kidney function and cytokine production.ResultsReduced steady state levels of mRNA species were enriched in pathways of oxidative phosphorylation and increased in the pentose phosphate pathway (PPP) with maximal perturbation in AAV and SLE followed by NS, and least in LD. Transcript regulation was isozymes specific with robust regulation in hexokinases, enolases and glucose transporters. Intercorrelation networks were observed between enzymes of the PPP (eg, transketolase) and macrophage markers (eg, CD68) (r=0.49, p<0.01). Increased PPP transcript levels were associated with reduced glomerular filtration rate in the glomerular (r=−0.49, p<0.01) and tubulointerstitial (r=−0.41, p<0.01) compartments. PPP expression and tumour necrosis factor activation were tightly co-expressed (r=0.70, p<0.01).ConclusionThis study demonstrated concordant alterations of the renal transcriptome consistent with metabolic reprogramming across different forms of glomerulonephritis. Activation of the PPP was tightly linked with intrarenal macrophage marker expression, reduced kidney function and increased production of cytokines. Modulation of glucose metabolism may offer novel immune-modulatory therapeutic approaches in rare kidney diseases.

Antineutrophil cytoplasmic antibody (ANCA)-associated vasculitides (AAV) are rare autoimmune diseases with multisystemic organ involvement. Their pathogenesis remains incompletely understood, and reliable biomarkers for disease activity and treatment response are lacking. This review explores current and emerging biomarkers in AAVs to identify disease phenotypes and activity, aiming to optimize management and immunosuppressive treatment. Serological, cellular, and urinary biomarkers will be discussed, focusing on their current utility in clinical practice for assessing disease activity, damage related to the disease, and prognosis. Promising biomarkers and novel methodologies for detecting future biomarkers will also be briefly discussed.

Antineutrophil cytoplasmic antibody (ANCA)-associated vasculitis (AAV) constitutes life-threatening autoimmune diseases affecting every organ, including the kidneys, where they cause necrotizing crescentic glomerulonephritis. ANCA activates neutrophils and activated neutrophils damage the endothelium, leading to vascular inflammation and necrosis. Better understanding of neutrophil-mediated AAV disease mechanisms may reveal novel treatment strategies. Here we report that ANCA induces neutrophil extracellular traps (NETs) via receptor-interacting protein kinase (RIPK) 1/3- and mixed-lineage kinase domain-like (MLKL)-dependent necroptosis. NETs from ANCA-stimulated neutrophils caused endothelial cell (EC) damage in vitro. This effect was prevented by (i) pharmacologic inhibition of RIPK1 or (ii) enzymatic NET degradation. The alternative complement pathway (AP) was recently implicated in AAV, and C5a inhibition is currently being tested in clinical studies. We observed that NETs provided a scaffold for AP activation that in turn contributed to EC damage. We further established the in vivo relevance of NETs and the requirement of RIPK1/3/MLKL-dependent necroptosis, specifically in the bone marrow-derived compartment, for disease induction using murine AAV models and in human kidney biopsies. In summary, we identified a mechanistic link between ANCA-induced neutrophil activation, necroptosis, NETs, the AP, and endothelial damage. RIPK1 inhibitors are currently being evaluated in clinical trials and exhibit a novel therapeutic strategy in AAV.

Complement activation is a relevant driver in the pathomechanisms of vasculitis. The involved proteins in the interaction between endothelia, complement, and platelets in these conditions are only partially understood. Thrombospondin-1 (TSP-1), found in platelet α-granules and released from activated endothelial cells, interacts with factor H (FH) and vWF. However, to our knowledge, direct regulatory interaction with the complement cascade has not yet been described. Our study shows that TSP-1 is a potent, FH-independent inhibitor of the alternative complement pathway. TSP-1 binds to complement proteins and inhibits cleavage of C3 and C5 and the formation of the membrane attack complex. We validated complement-regulatory function in blood samples from patients with primary complement defects. The physiological relevance of TSP-1 was demonstrated in patients with antineutrophil cytoplasmic antibody-associated vasculitis (AAV) by significantly enhanced TSP-1 staining in glomerular lesions and increased complement activity and NETosis after TSP-1 deficiency in an in vitro and in vivo model of AAV. The complement-inhibiting function of TSP-1 represents an important mechanism in the interaction of endothelia and complement. In particular, the interplay between released TSP-1 and the complement system locally, especially on surfaces, influences the balance between complement activation and inhibition and may be relevant in various vascular diseases.

The kidney is a primary target organ in systemic lupus erythematosus (SLE) and ANCA-associated vasculitis (AAV), which frequently manifest as lupus nephritis (LN) and renal AAV, respectively. A severe acute presentation of both diseases is rapidly progressive glomerulonephritis (RPGN), leading to rapid loss of renal function. However, the shared molecular mechanisms underlying this aggressive phenotype remain poorly defined. This study aimed to identify key biomarkers and pathological processes in acute autoimmune-related glomerulonephritis, with a focus on LN and AAV presenting as RPGN. Through comparative transcriptomic analysis of glomerular samples from patients with LN, AAV, and RPGN against healthy controls, we identified a total of 180 common differentially expressed genes, comprising 33 upregulated and 147 downregulated genes. Pathway analysis revealed enhanced immune and inflammatory responses, including neutrophil extracellular trap (NET) formation, accompanied by significant impairments in amino acid and fatty acid metabolism. A core set of 12 hub genes was identified, and network analysis suggested specific transcriptional and post-transcriptional regulatory axes. Immune infiltration analysis demonstrated a common pattern characterized by decreased resting immune cells and increased activated cells-including memory CD4 ⁺ T cells, NK cells, and mast cells-along with elevated monocyte levels, particularly in LN. Notably, epidermal growth factor (EGF) was significantly downregulated and strongly correlated with impaired renal function, demonstrating good diagnostic performance. These findings suggest that EGF may serve as a potential biomarker for LN and RPGN. This study provides the first systematic transcriptomic analysis of LN and AAV presenting as RPGN, highlighting enhanced inflammation (including NETosis) and dysregulated amino acid/fatty acid metabolism as key glomerular pathological features. Targeting EGF signaling and monocyte differentiation may offer novel therapeutic strategy for LN and AAV presenting as RPGN.

Background B cell depletion with rituximab (RTX) is approved for treatment of rheumatoid arthritis (RA) and ANCA-associated vasculitides (AAV). Recently, RTX has been shown to be effective in AAV maintenance therapy, but an optimal RTX treatment schedule is unknown and the time to B cell repopulation after RTX has not been studied. Methods Retrospective single-center analysis of B cell repopulation in patients with AAV, RA or connective tissue disease (CTD) treated with RTX. Results Beginning B cell repopulation within the first year after RTX treatment was observed in 93% of RA and 88% of CTD patients. Only 10% of patients with granulomatosis with polyangiitis (GPA) and microscopic polyangiitis (MPA) and no patient with eosinophilic granulomatosis with polyangiitis (EGPA) showed B cell repopulation within this time. Median time of B cell depletion was 26 months in GPA/MPA, and 21 months in EGPA compared to 9 months in RA, and 8 months in CTD ( p  < 0.0001). In 25 AAV-patients B cell depletion lasted for at least 44 months. There was a significant decline in serum immunoglobulin concentrations in GPA/MPA patients, but not in patients with RA or CTD. Significantly more GPA/MPA patients developed hygogammaglobulinemia (IgG <7 g/L) compared to patients with RA or CTD. Conclusions In contrast to RA and CTD, in AAV RTX induces long-lasting depletion of B cells that is associated with decreased antibody production. This observation points toward potential defects in the B cell compartment in AAV that are unmasked by immunosuppressive treatment and has important implications for the design of maintenance treatment schedules using RTX.

The complement system is crucial in antineutrophil cytoplasmic antibody-associated vasculitis (AAV) pathogenesis. Extracellular vesicles (EVs) serve as carriers of bioactive substances, influencing the immune system. We aimed to investigate myeloperoxidase-positive EVs (MPO EVs) exposing complement components (C3a, C4d), terminal complement complex (TCC) and complement factor B (CFB) in relation to disease activity and kidney involvement. Plasma MPO EVs carrying complement products, C3a, C4d, TCC or CFB, were analysed by flow cytometry. Clinical data, including kidney biopsy findings, were retrieved. Disease activity was assessed using the Birmingham Vasculitis Activity Score (BVAS). Eighty-one patients with AAV with granulomatosis with polyangiitis (n=59) or microscopic polyangiitis (n=22) were included. Active disease was noted in 73 (90.1%) patients and 50 (68.5%) had kidney involvement. Patients with kidney involvement had higher concentrations of MPO , MPO C3a , MPO C4d and MPO TCC EVs compared to patients without, and concentrations correlated positively with BVAS (p<0.05), respectively. Levels of EVs showed a negative correlation with estimated glomerular filtration rate, and a positive correlation with the proportions of necrosis and crescents in kidney biopsies. ROC curve analysis indicated that MPO EVs could distinguish patients with kidney involvement from non-renal AAV. A binary multivariable logistic regression confirmed an independent association between levels of EV subsets and kidney involvement. Elevated levels of MPO EVs exposing complement components in patients with kidney involvement indicate activation of the classical or lectin and common complement pathways in renal AAV. Furthermore, the association between levels of MPO EVs with the presence of crescents and necrotic lesions in kidney tissue supports a role in renal inflammatory activity.

Evidence is lacking for direct pathogenicity of human anti-proteinase-3 (PR3) antibodies in development of systemic vasculitis and granulomatosis with polyangiitis (GPA, Wegener's granulomatosis). Progress in study of these antibodies in rodents has been hampered by lack of PR3 expression on murine neutrophils, and by different Fc-receptor affinities for IgG across species. Therefore, we tested whether human anti-PR3 antibodies can induce acute vasculitis in mice with a human immune system. Chimeric mice were generated by injecting human haematopoietic stem cells into irradiated NOD-scid-IL2Rγ⁻/⁻ mice. Matched chimera mice were treated with human IgG from patients with: anti-PR3 positive renal and lung vasculitis; patients with non-vasculitic renal disease; or healthy controls. Six-days later, 39% of anti-PR3 treated mice had haematuria, compared with none of controls. There was punctate bleeding on the surface of lungs of anti-PR3 treated animals, with histological evidence of vasculitis and haemorrhage. Anti-PR3 treated mice had mild pauci-immune proliferative glomerulonephritis, with infiltration of human and mouse leukocytes. In 3 mice (17%) more severe glomerular injury was present. There were no glomerular changes in controls. Human IgG from patients with anti-PR3 autoantibodies is therefore pathogenic. This model of anti-PR3 antibody-mediated vasculitis may be useful in dissecting mechanisms of microvascular injury.