Reactive Oxygen Species and Neutrophil Function.

Neutrophils are essential for killing bacteria and other microorganisms, and they also have a significant role in regulating the inflammatory response. Stimulated neutrophils activate their NADPH oxidase (NOX2) to generate large amounts of superoxide, which acts as a precursor of hydrogen peroxide and other reactive oxygen species that are generated by their heme enzyme myeloperoxidase. When neutrophils engulf bacteria they enclose them in small vesicles (phagosomes) into which superoxide is released by activated NOX2 on the internalized neutrophil membrane. The superoxide dismutates to hydrogen peroxide, which is used by myeloperoxidase to generate other oxidants, including the highly microbicidal species hypochlorous acid. NOX activation occurs at other sites in the cell, where it is considered to have a regulatory function. Neutrophils also release oxidants, which can modify extracellular targets and affect the function of neighboring cells. We discuss the identity and chemical properties of the specific oxidants produced by neutrophils in different situations, and what is known about oxidative mechanisms of microbial killing, inflammatory tissue damage, and signaling.

Causal relationship between immune cells and the risk of myeloperoxidase antineutrophil cytoplasmic antibody-associated vasculitis: A Mendelian randomization study.

Eosinophilic granulomatosis with polyangiitis (EGPA) is a rare inflammatory disease categorized as antineutrophil cytoplasmic antibody (ANCA)-associated vasculitis. The majority of patients are ANCA-positive, predominantly against myeloperoxidase (MPO). Previous studies have predominantly concentrated on the association between EGPA and neutrophils, but recent research has emphasized the role of lymphocytes in the development of EGPA. The objective of our research was to examine the causal association between immune cells and MPO + ANCA EGPA. A two-sample bidirectional Mendelian randomization (MR) analysis was performed, which included 159 MPO + ANCA EGPA cases and 6688 controls and utilized Genome-Wind Associaton Studies (GWAS) summary statistics of immune traits from approximately 3757 individuals, encompassing around 22 million single nucleotide polymorphisms (SNPs). Our findings revealed that 23 immunophenotypes were associated with MPO + ANCA EGPA. Furthermore, the reverse MR analysis showed that MPO + ANCA EGPA had significant causal effects on three immunophenotypes within the Treg panel. By integrating existing research, our study unveiled the contributions of Tregs, B cells, and monocytes to the development of EGPA. Subgroup analysis specifically examined the roles of lymphocyte subtypes, cytokines, and their surface molecules in the pathogenic mechanisms of the disease. This comprehensive approach provides a novel perspective on the biological mechanisms and early intervention strategies for MPO + ANCA EGPA by focusing on immune cells.

Myeloperoxidase-ANCA IgG induces different forms of small vessel vasculitis based on type of synergistic immune stimuli.

Anti-neutrophil cytoplasmic autoantibody (ANCA) vasculitis has diverse patterns of injury including microscopic polyangiitis (MPA), granulomatosis with polyangiitis (GPA), and eosinophilic granulomatosis with polyangiitis (EGPA). Necrotizing and crescentic glomerulonephritis (NCGN) occurs in all syndromes and as renal limited vasculitis (RLV). Single-dose intravenous ANCA IgG-specific for mouse myeloperoxidase (MPO) causes RLV in mice. Although multiple mouse models have elucidated ANCA-IgG induced necrotizing and crescentic glomerulonephritis (NCGN), pathogenesis of ANCA-induced granulomatosis and vasculitis outside the kidney has not been clarified. To investigate this, we used intravenous MPO-ANCA IgG in the same strain of mice to induce different patterns of lung disease mirroring patients with granulomatosis with polyangiitis (GPA), microscopic polyangiitis (MPA), and eosinophilic granulomatosis with polyangiitis (EGPA). Repeated intravenous MPO-ANCA IgG induced GPA with NCGN, lung capillaritis, arteritis and granulomatosis. Lung leukocyte phenotypes were evaluated by immunohistochemical image analysis and by flow cytometry. ANCA lung capillaritis and microabscesses began within one day and evolved into granulomas in under seven days. Influenza plus single-dose MPO-ANCA IgG induced MPA with NCGN, lung capillaritis and arteritis, but no granulomatosis. Allergic airway disease caused by house dust mites or ovalbumin plus single-dose intravenous MPO-ANCA IgG induced EGPA with eosinophilic bronchiolitis, NCGN, capillaritis, arteritis, and granulomatosis. Thus, our study shows that the occurrence and pattern of lung lesions are determined by the same ANCA IgG accompanied by different synergistic immune factors.

Regulatory T cells effectively downregulate the autoimmune anti-MPO response and ameliorate anti-MPO induced glomerulonephritis in mice.

Regulation of autoreactive cells is key for both prevention and amelioration of autoimmune disease. A better understanding of the key cell population(s) responsible for downregulation of autoreactive cells would provide necessary foundational insight for cellular-based therapies in autoimmune disease. Utilizing a mouse model of anti-myeloperoxidase (MPO) glomerulonephritis, we sought to understand which immune cells contribute to downregulation of the anti-MPO autoimmune response. MPO-/- mice were immunized with whole MPO to induce an anti-MPO response. Anti-MPO splenocytes were then transferred into recipient mice (Rag2-/- mice or WT mice). Anti-MPO titers were followed over time. After anti-MPO splenocyte transfer, WT mice are able to downregulate the anti-MPO response while anti-MPO titers persist in Rag2-/- recipients. Reconstitution with WT splenocytes into Rag2-/- recipients prior to anti-MPO splenocyte transfer enabled mice to downregulate the anti-MPO immune response. Therefore, wildtype splenocytes contain a cellular population that is capable of downregulating the autoimmune response. Through splenocyte transfer, antibody depletion experiments, and purified cell population transfers, we confirmed that the regulatory T cell (Treg) population is responsible for the downregulation of the anti-MPO autoimmune response. Further investigation revealed that functional Tregs from WT mice are capable of downregulating anti-MPO antibody production and ameliorate anti-MPO induced glomerulonephritis. These data underscore the importance of functional Tregs for control of autoimmune responses and prevention of end-organ damage due to autoimmunity.

Blockade of neutrophil extracellular trap components ameliorates cholestatic liver disease in Mdr2 (Abcb4) knockout mice.

Primary sclerosing cholangitis (PSC) is an (auto)immune-mediated cholestatic liver disease with a yet unclear etiology. Increasing evidence points to an involvement of neutrophils in chronic liver inflammation and cirrhosis but also liver repair. Here, we investigate the role of the neutrophil extracellular trap (NET) component myeloperoxidase (MPO) and the therapeutic potential of DNase I and of neutrophil elastase (NE) inhibitor GW311616A on disease outcome in the multidrug resistance 2 knockout (Mdr2-/-) mouse, a PSC animal model. Initially, we observed the recruitment of MPO expressing cells and the formation of NETs in liver biopsies of PSC patients and in Mdr2-/- livers. Furthermore, sera of Mdr2-/- mice contained perinuclear anti-neutrophil cytoplasmic antibody (p-ANCA)-like reactivity similar to PSC patient sera. Also, hepatic NE activity was significantly higher in Mdr2-/- mice than in wild type littermates. Flow cytometry analyses revealed that during disease development a highly active neutrophil subpopulation established specifically in the liver of Mdr2-/- mice. However, absence of their MPO activity, as in MPO-deficient Mdr2-/- mice, showed no effect on hepatobiliary disease severity. In contrast, clearance of extracellular DNA by DNase I reduced the frequency of liver-resident neutrophils, plasmacytoid dendritic cells (pDCs) and CD103+ conventional DCs and decreased cholangiocyte injury. Combination of DNase I with a pDC-depleting antibody was additionally hepatocyte-protective. Most importantly, GW311616A, an orally bioavailable inhibitor of human NE, attenuated hepatobiliary injury in a TNFα-dependent manner and damped hyperproliferation of biliary epithelial cells. Further, hepatic immigration and activity of CD11b+ DCs as well as the secretion of IFNγ by hepatic CD4 and CD8 T cells were reduced. Our findings delineate neutrophils as important participants in the immune cell crosstalk that drives cholestatic liver disease and identify NET components as potential therapeutic targets.

Dual MPO/PR3 ANCA positivity and vasculitis: insights from a 7-cases study and an AI-powered literature review.

OBJECTIVES: Anti-neutrophil cytoplasm antibodies (ANCA)-associated vasculitides (AAV) are rare conditions characterized by inflammatory cell infiltration in small blood vessels, leading to tissue necrosis. While most patients with AAV present antibodies against either myeloperoxidase (MPO) or proteinase 3 (PR3), rare cases of dual positivity for both antibodies (DP-ANCA) have been reported, and their impact on the clinical picture remains unclear. The goal of this study was to investigate the clinical implications, phenotypic profiles and outcomes of patients with DP-ANCA. METHODS: A retrospective screening for DP-ANCA cases was conducted at Brest University Hospital's immunology laboratory (France), analysing ANCA results from March 2013 to March 2022. Clinical, biological, imaging, and histological data were collected for each DP-ANCA case. Additionally, a comprehensive literature review on DP-ANCA was performed, combining an artificial intelligence (AI)-based search using BIBOT software with a manual PUBMED database search. RESULTS: The report of our cases over the last 9 years and those from the literature yielded 103 described cases of patients with DP-ANCA. We identified four distinct phenotypic profiles: (i) idiopathic AAV (∼30%); (ii) drug-induced AAV (∼25%); (iii) autoimmune disease associated with a low risk of developing vasculitis (∼20%); and (iv) immune-disrupting comorbidities (infections, cancers, etc) not associated with AAV (∼25%). CONCLUSION: This analysis of over a hundred DP-ANCA cases suggests substantial diversity in clinical and immunopathological presentations. Approximatively 50% of DP-ANCA patients develop AAV, either as drug-induced or idiopathic forms, while the remaining 50%, characterized by pre-existing dysimmune conditions, demonstrates a remarkably low vasculitis risk. These findings underscore the complex nature of DP-ANCA, its variable impact on patient health, and the necessity for personalized diagnostic and management approaches in these cases.

Neutrophil Extracellular Trap Formation and Deoxyribonuclease I Activity in Patients with Otitis Media with Antineutrophil Cytoplasmic Antibody-Associated Vasculitis.

INTRODUCTION: No previous studies have evaluated the levels of neutrophil extracellular trap (NET) remnants or the importance of deoxyribonuclease (DNase) I activity based on the disease activity of otitis media with antineutrophil cytoplasmic antibody-associated vasculitis (OMAAV). The aim of this study was to explore the formation of NETs in the middle ear of patients with OMAAV during the onset and remission phases of the disease, with a particular focus on the relationships between the quantifiable levels of NET remnants and DNase I activity. METHODS: OMAAV patients were eligible for inclusion. Patients with otitis media with effusion (OME) were examined as controls. The levels of cell-free deoxyribonucleic acid (DNA), citrullinated-histone H3 (cit-H3)-DNA complex, and myeloperoxidase (MPO)-DNA complex were quantified using an enzyme-linked immunosorbent assay. DNase I activity was measured using a fluorometric method. RESULTS: The quantifiable levels of cell-free DNA, cit-H3-DNA complex, and MPO-DNA complex in the middle ear lavage of patients with OMAAV at onset were significantly higher than those in patients with OMAAV at remission and in patients with OME. DNase I activity in the patients with OMAAV at onset was significantly lower than those in patients with OMAAV at remission and OME and was negatively correlated with the level of MPO-DNA complex. CONCLUSIONS: This study suggests that NET remnants and DNase I activity may be potentially useful biomarkers for the diagnosis and disease activity of OMAAV.

First-year cumulative myeloperoxidase-ANCA titres are associated with all-cause mortality in patients with microscopic polyangiitis.

OBJECTIVES: We investigated whether first-year cumulative myeloperoxidase (MPO)-antineutrophil cytoplasmic antibody (ANCA) and proteinase 3 (PR3)-ANCA titres were associated with all-cause mortality and relapse during follow-up in patients with microscopic polyangiitis (MPA) and granMETHODS: Altogether, 74 patients with MPA and 40 with GPA were included in this study. Their clinical data at diagnosis were collected. First-year cumulative ANCA titres were defined as the area under the curve (AUC) of ANCA titres during the first year after MPA or GPA diagnosis, which was obtained using the trapezoidal rule. All-cause mortality and relapse were considered poor outcomes of MPA and GPA. RESULTS: The median ages of patients with MPA and GPA were 65.5 and 60.5 years, respectively. No significant correlation was observed between ANCA titres at diagnosis and concurrent MPA and GPA activity or the inflammatory burden. First-year cumulative MPO-ANCA titres exhibited a significant AUC for all-cause mortality during follow-up in patients with MPA. The optimal cut-off of first-year cumulative MPO-ANCA titres for all-cause mortality was determined as 720.8 IU/mL using receiver operating characteristic curve analysis. MPA patients with first-year cumulative MPO-ANCA titres ≥720.8 IU/mL exhibited a significantly higher risk for all-cause mortality than those without (relative risk 13.250). Additionally, MPA patients with first-year cumulative MPO-ANCA titres ≥720.8 IU/mL exhibited a significantly lower cumulative patients' survival rate than those without. CONCLUSIONS: This is the first study to demonstrate the association between first-year cumulative MPO-ANCA titres and all-cause mortality during follow-up in patients with MPA.

Neutrophils impaired by anti-galectin-3 antibodies elicit inflammation of endothelial cells to aggregate the development of lupus cutaneous vasculitis.

OBJECTIVES: To investigate whether the interplay of anti-galectin-3 antibodies (anti-Gal3 Abs) with neutrophils contributes to the development of lupus cutaneous vasculitis. METHODS: Enzyme-linked immunosorbent assay was used to determine the serum level of anti-Gal3 Abs in lupus patients. Flow cytometry, quantitative PCR and western blot were performed to investigate the expression of cell surface receptors, proinflammatory cytokines and signalling molecules in neutrophils stimulated by serum from lupus patients or healthy controls (HCs) or anti-Gal3 Ab, respectively. Immunofluorescence was performed to visualise the formation of neutrophil extracellular traps (NETs). Human umbilical vein endothelial cells were co-cultured with the supernatants from neutrophils stimulated by anti-Gal3 Ab, and cytokine production was measured at mRNA and protein levels. Immunohistochemistry was adopted to reveal the distribution of Gal3, cytokines and myeloperoxidase within lupus skin lesions. RESULTS: Serum levels of anti-Gal3 Abs were negatively correlated with peripheral counts of neutrophils. Anti-Gal3 Abs positive sera from SLE patients accelerated neutrophil death, altered cell phenotype and promoted formation of NETs with the involvement of p38 MAPK pathway. Supernatants collected from neutrophils co-cultured with anti-Gal3 Ab provoked endothelial cells to produce cytokines such as IL-1, ICAM-1, SELE and particularly IL-6. Consistently, IL-6 was higher in SLE patients with anti-Gal3 Ab positive sera and enriched in the area of vascular inflammation together with enhanced expression of Gal3 protein and infiltration of neutrophils. CONCLUSIONS: Overall, these findings suggested that neutrophils were crucial mediators in anti-Gal3 Ab induced lupus cutaneous vasculitis.

Two cases of MPO-ANCA-positive hypertrophic pachymeningitis mimicking as intracranial infection.

Hypertrophic pachymeningitis (HP) is a rare disorder marked by thickening of the dura mater due to diverse etiologies. MPO-ANCA-positive HP represents a variant of AAV confined to the central nervous system, distinguished by the presence of serum MPO antibodies. Distinguishing HP triggered by MPO-ANCA from other causes can be challenging.In this study, we present two cases of MPO-ANCA-positive HP initially misdiagnosed as intracranial infections. Case 1 underwent surgery for chronic suppurative otitis media, with histopathological findings revealing inflammatory changes without definitive suppuration. He was presumed to have a secondary intracranial infection resulting from the surgery. However, his condition deteriorated despite two weeks of antibiotic and antiviral treatment. Case 2 presented with headache and was initially suspected of having intracranial Brucellosis given his serum Brucella positivity. Despite treatment for brucellosis, his symptoms persisted, and he developed visual and hearing impairments. Both patients were ultimately diagnosed with MPO-ANCA-positive HP, exhibiting serum MPO antibody positivity. Their symptoms showed improvement with glucocorticoid and immunosuppressive therapy.Based on these observations, we propose that MPO-ANCA-positive HP may initially present as intracranial infection. For HP patients presenting with headache, mastoiditis, otitis media, and visual loss, it is imperative to conduct ANCA antibody-related tests to enhance diagnostic precision.

Prognosis of microscopic polyangiitis is well predictable in the first 2 weeks of treatment.

BACKGROUND: Kidney and life outcomes remain unsatisfactory in patients with microscopic polyangiitis (MPA). Appropriate treatment intensity must be provided to the appropriate patients. To identify severe cases early, we investigated the factors related to kidney and life outcomes. METHODS: We included patients diagnosed with MPA based on myeloperoxidase-antineutrophil cytoplasmic antibody (MPO-ANCA) positivity and kidney histopathology results after kidney biopsies between January 1, 2021, and May 11, 2023, at 10 affiliated centers, including our hospital. Death, maintenance dialysis, and estimated glomerular filtration rate (eGFR) < 15 after 6 months of treatment were defined as poor prognosis groups, and factors associated with these conditions were investigated. RESULTS: We included 84 (36 men and 48 women) patients in this study. Median age was 73.8 (interquartile range: 71-81) years. After 6 months of treatment, the proportion of patients in the poor prognosis group was 16.7 %, with a mortality of 7.1 % and a poor kidney prognosis rate of 9.5 %. Area under the receiver operating characteristic curve showed that eGFR at 2 weeks had a comparable prognostic performance equal as eGFR at 4 weeks (area under the curve: 0.875 and 0.896, respectively). After adjustment by various factors, eGFR at 2 weeks was related with prognosis significantly (p = 0.031). CONCLUSION: Kidney function 2 weeks after the start of treatment for MPA can predict prognosis.

Diagnostic accuracy of ANCA serology in ANCA-associated vasculitis with renal involvement.

BACKGROUND: Pauci-immune glomerulonephritis (GN) due to antineutrophil cytoplasmic antibody (ANCA)-associated vasculitis (AAV) is a common cause of crescentic GN. Despite advances in treatment, rates of mortality and progression to end-stage kidney disease remain high. Renal involvement is diagnosed by histological examination of kidney tissue. Serum ANCAs play a significant role in AAV; however, the value of serum ANCA quantification to predict renal involvement is not well-established. AIM: We aimed to evaluate the diagnostic accuracy of serum ANCA titres in diagnosing AAV with renal involvement. METHODS: We conducted a retrospective study of consecutive native kidney biopsies reported at our centre from 2016 to 2021. We included all adults who had both a kidney biopsy and ANCA serology. ANCA serology was tested using indirect immunofluorescence with reporting of titres. Antibodies to proteinase 3 and myeloperoxidase were measured using a chemiluminescent immunoassay. RESULTS: Eight hundred and forty-eight native kidney biopsies were reported during the study period. Five hundred and seven cases were included. The biopsy prevalence of pauci-immune GN in paired samples was 41/507 (8.1%). Most of the cohort had haematuria (66.6%), proteinuria (93.4%) and/or acute kidney injury (65.0%). A positive ANCA at any titre demonstrated a sensitivity of 97.6% and a specificity of 71.2% for a diagnosis of pauci-immune GN. The area under the curve for the receiver operator characteristic was 0.93 (95% confidence interval [CI]: 0.89-0.97). A cutoff ANCA titre of 1:160 provided the optimum balance between a sensitivity of 75.6% (95% CI: 59.7%-87.6%) and a specificity of 94.0% (95% CI: 91.6%-96.0%). CONCLUSIONS: ANCA titres are highly predictive of pauci-immune GN in the appropriate context. While serum ANCA quantitation may not replace renal biopsy, reporting will assist in the decision to start treatment early for patients with organ or life-threatening disease.

Stratified genetic analysis reveals sex differences in MPO-ANCA-associated vasculitis.

OBJECTIVE: To identify and genetically characterize subgroups of patients with ANCA-associated vasculitides (AAV) based on sex and ANCA subtype. METHODS: A previously established SNP dataset derived from DNA sequencing of 1853 genes and genotyping of 1088 Scandinavian cases with AAV and 1589 controls was stratified for sex and ANCA subtype and analysed for association with five top AAV SNPs. rs9274619, a lead variant at the HLA-DQB1/HLA-DQA2 locus previously associated with AAV positive for myeloperoxidase (MPO)-ANCA, was analysed for association with the cumulative disease involvement of ten different organ systems. RESULTS: rs9274619 showed a significantly stronger association to MPO-ANCA-positive females than males [P = 2.0 × 10-4, OR = 2.3 (95% CI 1.5, 3.5)], whereas proteinase 3 (PR3)-ANCA-associated variants rs1042335, rs9277341 (HLA-DPB1/A1) and rs28929474 (SERPINA1) were equally associated with females and males with PR3-ANCA. In MPO-ANCA-positive cases, carriers of the rs9274619 risk allele were more prone to disease engagement of eyes [P = 0.021, OR = 11 (95% CI 2.2, 205)] but less prone to pulmonary involvement [P = 0.026, OR = 0.52 (95% CI 0.30, 0.92)]. Moreover, AAV with both MPO-ANCA and PR3-ANCA was associated with the PR3-ANCA lead SNP rs1042335 [P = 0.0015, OR = 0.091 (95% CI 0.0022, 0.55)] but not with rs9274619. CONCLUSIONS: Females and males with MPO-ANCA-positive AAV differ in genetic predisposition to disease, suggesting at least partially distinct disease mechanisms between the sexes. Double ANCA-positive AAV cases are genetically similar to PR3-ANCA-positive cases, providing clues to the clinical follow-up and treatment of these patients.

Altered follicular regulatory T (Tfr)- and helper T (Tfh)-cell subsets are associated with autoantibody levels in microscopic polyangiitis patients.

Antineutrophil cytoplasmic antibodies (ANCA)-associated vasculitis (AAV) is an autoimmune disease characterized by B cells-derived ANCAs, and ANCA was proved to be a key factor in its pathogenesis. Follicular regulatory T (Tfr) and follicular helper T (Tfh) cells were T-cell subsets that play important roles in B-cell maturation and antibody production. However, their significances in microscopic polyangiitis (MPA) patients, one type of AAV, has not been thoroughly studied. In this study, comprehensive pattern analyses of circulating Tfr and Tfh were performed in MPA patients and healthy controls (HCs), and we found Tfr levels and Tfr/Tfh ratios were significantly decreased in MPA patients. Compared with HCs, Helios+, CD45RA-FoxP3hi, and Ki-67+ Tfr were lower in MPA patients, while CD226+ Tfr cells were higher. These phenotypes suggest that function and proliferation ability of Tfr cells were relatively impaired. Tfh subsets, including ICOS+PD-1+ and Ki-67+ Tfh, were significantly increased, suggesting that the function of Tfh was enhanced in MPA although the total Tfh levels did not change significantly. Circulating memory B cells and plasmablasts were significantly elevated and negatively correlated with Tfr levels and Tfr/Tfh ratios in MPA patients. In addition, Tfr levels and Tfr/Tfh ratios were negatively while Tfh was positively correlated with serum myeloperoxidase (MPO)-ANCA levels. Furthermore, Tfr and Tfr/Tfh ratio were also reversely associated with SCr, BUN, IL-4, and IL-21 levels. Our results suggest that the imbalance of Tfr and Tfh functional subsets is related to increased level of autoantibodies in MPA patients, and we propose a new mechanism for the pathogenesis of MPA.

2022 American College of Rheumatology/European Alliance of Associations for Rheumatology classification criteria for microscopic polyangiitis.

OBJECTIVE: To develop and validate classification criteria for microscopic polyangiitis (MPA). METHODS: Patients with vasculitis or comparator diseases were recruited into an international cohort. The study proceeded in five phases: (1) identification of candidate items using consensus methodology, (2) prospective collection of candidate items present at the time of diagnosis, (3) data-driven reduction of the number of candidate items, (4) expert panel review of cases to define the reference diagnosis and (5) derivation of a points-based risk score for disease classification in a development set using least absolute shrinkage and selection operator logistic regression, with subsequent validation of performance characteristics in an independent set of cases and comparators. RESULTS: The development set for MPA consisted of 149 cases of MPA and 408 comparators. The validation set consisted of an additional 142 cases of MPA and 414 comparators. From 91 candidate items, regression analysis identified 10 items for MPA, 6 of which were retained. The final criteria and their weights were as follows: perinuclear antineutrophil cytoplasmic antibody (ANCA) or anti-myeloperoxidase-ANCA positivity (+6), pauci-immune glomerulonephritis (+3), lung fibrosis or interstitial lung disease (+3), sino-nasal symptoms or signs (-3), cytoplasmic ANCA or anti-proteinase 3 ANCA positivity (-1) and eosinophil count ≥1×109/L (-4). After excluding mimics of vasculitis, a patient with a diagnosis of small- or medium-vessel vasculitis could be classified as having MPA with a cumulative score of ≥5 points. When these criteria were tested in the validation data set, the sensitivity was 91% (95% CI 85% to 95%) and the specificity was 94% (95% CI 92% to 96%). CONCLUSION: The 2022 American College of Rheumatology/European Alliance of Associations for Rheumatology classification criteria for MPA are now validated for use in clinical research.

Neutrophil extracellular traps contribute to immunothrombosis in COVID-19 acute respiratory distress syndrome.

COVID-19 affects millions of patients worldwide, with clinical presentation ranging from isolated thrombosis to acute respiratory distress syndrome (ARDS) requiring ventilator support. Neutrophil extracellular traps (NETs) originate from decondensed chromatin released to immobilize pathogens, and they can trigger immunothrombosis. We studied the connection between NETs and COVID-19 severity and progression. We conducted a prospective cohort study of COVID-19 patients (n = 33) and age- and sex-matched controls (n = 17). We measured plasma myeloperoxidase (MPO)-DNA complexes (NETs), platelet factor 4, RANTES, and selected cytokines. Three COVID-19 lung autopsies were examined for NETs and platelet involvement. We assessed NET formation ex vivo in COVID-19 neutrophils and in healthy neutrophils incubated with COVID-19 plasma. We also tested the ability of neonatal NET-inhibitory factor (nNIF) to block NET formation induced by COVID-19 plasma. Plasma MPO-DNA complexes increased in COVID-19, with intubation (P < .0001) and death (P < .0005) as outcome. Illness severity correlated directly with plasma MPO-DNA complexes (P = .0360), whereas Pao2/fraction of inspired oxygen correlated inversely (P = .0340). Soluble and cellular factors triggering NETs were significantly increased in COVID-19, and pulmonary autopsies confirmed NET-containing microthrombi with neutrophil-platelet infiltration. Finally, COVID-19 neutrophils ex vivo displayed excessive NETs at baseline, and COVID-19 plasma triggered NET formation, which was blocked by nNIF. Thus, NETs triggering immunothrombosis may, in part, explain the prothrombotic clinical presentations in COVID-19, and NETs may represent targets for therapeutic intervention.

Characterisation of an enhanced preclinical model of experimental MPO-ANCA autoimmune vasculitis.

Experimental autoimmune vasculitis (EAV) is a model of antineutrophil cytoplasm antibody (ANCA)-associated vasculitis (AAV) induced by immunisation of susceptible rat strains with myeloperoxidase (MPO). Animals develop circulating MPO-ANCA, pulmonary haemorrhage, and glomerulonephritis, although renal injury is mild and recovers spontaneously without treatment. In this study we aimed to augment the severity of glomerulonephritis. Following induction of EAV on day 0, a sub-nephritogenic dose of nephrotoxic serum (NTS) containing heterologous antibodies to glomerular basement membrane was administered on day 14. This resulted in a significant increase in disease severity at day 28 compared to MPO immunisation alone - with more urinary abnormalities, infiltrating glomerular leucocytes, and crescent formation that progressed to glomerular and tubulointerstitial scarring by day 56, recapitulating important features of human disease. Importantly, the glomerulonephritis remained pauci-immune, and was strictly dependent on the presence of autoimmunity to MPO, as there was no evidence of renal disease following administration of sub-nephritogenic NTS alone or after immunisation with a control protein in place of MPO. Detailed phenotyping of glomerular leucocytes identified an early infiltrate of non-classical monocytes following NTS administration that, in the presence of autoimmunity to MPO, may initiate the subsequent influx of classical monocytes which augment glomerular injury. We also showed that this model can be used to test novel therapeutics by using a small molecule kinase inhibitor (fostamatinib) that rapidly attenuated both glomerular and pulmonary injury over a 4-day treatment period. We believe that this enhanced model of MPO-AAV will prove useful for the study of glomerular leucocyte behaviour and novel therapeutics in AAV in the future. © 2021 The Authors. The Journal of Pathology published by John Wiley & Sons, Ltd. on behalf of The Pathological Society of Great Britain and Ireland.

Neutrophil Extracellular Traps Increase Airway Mucus Viscoelasticity and Slow Mucus Particle Transit.

Mucus obstruction is a key feature of many inflammatory airway diseases. Neutrophil extracellular traps (NETs) are released upon neutrophil stimulation and consist of extracellular chromatin networks studded with cytotoxic proteins. When released in the airways, these NETs can become part of the airway mucus. We hypothesized that the extracellular DNA and/or oxidative stress (e.g., by the release of reactive oxygen species and myeloperoxidase during NETs formation in the airways) would increase mucus viscoelasticity. We collected human airway mucus from endotracheal tubes of healthy patients admitted for elective surgery and coincubated these samples with NETs from phorbol 12-myristate 13-acetate-stimulated neutrophils. Unstimulated neutrophils served as controls, and blocking experiments were performed with dornase alfa for extracellular DNA and the free radical scavenger dimethylthiourea for oxidation. Compared with controls, the coincubation of mucus with NETs resulted in 1) significantly increased mucus viscoelasticity (macrorheology) and 2) significantly decreased mesh pore size of the mucus and decreased movement of muco-inert nanoparticles through the mucus (microrheology), but 3) NETs did not cause visible changes in the microstructure of the mucus by scanning EM. Incubation with either dornase alfa or dimethylthiourea attenuated the observed changes in macrorheology and microrheology. This suggests that the release of NETs may contribute to airway mucus obstruction by increasing mucus viscoelasticity and that this effect is not solely due to the release of DNA but may in part be due to oxidative stress.

Standardised immunophenotypic analysis of myeloperoxidase in acute leukaemia.

Given its myeloid-restricted expression, myeloperoxidase (MPO) is typically used for lineage assignment (myeloid vs. lymphoid) during acute leukaemia (AL) diagnostics. In the present study, a robust flow cytometric definition for MPO positivity was established based on the standardised EuroFlow protocols, the standardised Acute Leukaemia Orientation Tube and 1734 multicentre AL cases (with confirmed assay stability). The best diagnostic performance was achieved by defining MPO positivity as ≥20% of the AL cells exceeding a lymphocyte-based threshold. The methodology employed should be applicable to any form of standardised flow cytometry.

Phenotypical and Functional Characterization of Neutrophils in Two Pyrin-Associated Auto-inflammatory Diseases.

PURPOSE: Familial Mediterranean Fever (FMF) and Pyrin-Associated Autoinflammation with Neutrophilic Dermatosis (PAAND) are clinically distinct autoinflammatory disorders caused by mutations in the pyrin-encoding gene MEFV. We investigated the transcriptional, phenotypical, and functional characteristics of patient neutrophils to explore their potential role in FMF and PAAND pathophysiology. METHODS: RNA sequencing was performed to discover transcriptional aberrancies. The phenotypical features, degranulation properties, and phagocytic capacity of neutrophils were assessed by flow cytometry. Production of reactive oxygen species (ROS), myeloperoxidase (MPO) release, and chemotactic responses were investigated via chemiluminescence, ELISA, and Boyden chamber assays, respectively. RESULTS: Neutrophils from PAAND and FMF patients showed a partially overlapping, activated gene expression profile with increased expression of S100A8, S100A9, S100A12, IL-4R, CD48, F5, MMP9, and NFKB. Increased MMP9 and S100A8/A9 expression levels were accompanied by high plasma concentrations of the encoded proteins. Phenotypical analysis revealed that neutrophils from FMF patients exhibited an immature character with downregulation of chemoattractant receptors CXCR2, C5aR, and BLTR1 and increased expression of Toll-like receptor 4 (TLR4) and TLR9. PAAND neutrophils displayed an increased random, but reduced CXCL8-induced migration. A tendency for enhanced random migration was observed for FMF neutrophils. PAAND neutrophils showed a moderately but significantly enhanced phagocytic activity as opposed to neutrophils from FMF patients. Neutrophils from both patient groups showed increased MPO release and ROS production. CONCLUSIONS: Neutrophils from patients with FMF and PAAND, carrying different mutations in the MEFV gene, share a pro-inflammatory phenotype yet demonstrate diverse features, underscoring the distinction between both diseases.