ObjectiveAnti-neutrophil cytoplasmic antibody (ANCA)-associated vasculitides (AAV) are chronic, relapsing inflammatory diseases, yet reliable biomarkers for detecting relapse and organ involvement remain limited. This study aimed to identify plasma protein biomarkers that distinguish active disease from remission and to explore markers associated with lung and kidney involvement.MethodsPlasma samples from 113 patients with granulomatosis with polyangiitis or microscopic polyangiitis (68 active disease and 45 remission) were profiled using a proximity extension assay targeting 181 inflammation- and cardiovascular-related proteins. Clinical data, including CRP and Birmingham vasculitis activity score (BVAS), were collected at sampling. Differential protein expression was assessed using ANOVA, with top candidates validated in independent sample cohorts (plasma samples, n = 74; serum samples, n = 34). Correlations with BVAS and CRP and discriminatory performance (AUC) were evaluated. Associations with chest- and kidney-specific BVAS were also examined.ResultsA total of 57 proteins were differentially expressed between active disease and remission. Seven proteins (ST2, OPN, IL-2RA, CCL23, IL-6, Flt3L, and SCF) were validated in independent cohorts and showed strong associations with disease activity. Several demonstrated high discriminatory ability (AUC ≥ 0.80) between active disease and remission. Multiple proteins correlated with organ-specific BVAS scores: seven for chest involvement and 16 for kidney involvement, after adjustment for kidney function-related protein variation.ConclusionThis study identifies a robust panel of plasma proteins that differentiate active AAV from remission and correlate with global and organ-specific disease activity. These biomarkers may enhance non-invasive disease monitoring and support earlier recognition of relapse and organ involvement in AAV.

OBJECTIVE:The interferon (IFN) system is activated in systemic sclerosis (SSc), and various stimuli may induce IFN in SSc patients. We investigated whether type I and III IFN responses to Toll-like receptor (TLR)-7/8/9 stimulation differ between patients with SSc and healthy individuals, and whether IFN production is associated with clinical features. METHOD:Peripheral blood mononuclear cells (PBMCs), monocyte-depleted PBMCs, and monocytes were prepared from 45 SSc patients and 47 healthy controls. Cells were stimulated with RNA-containing immune complexes (RNA-IC), an RNA oligonucleotide (ORN8L), or inactivated herpes simplex virus (HSV) targeting TLR7, TLR8, and TLR9, respectively. IFN-α, -β, -λ1, and -λ2 levels were measured by immunoassays, and intracellular IFN-α by flow cytometry. RESULTS:SSc PBMCs produced type I and III IFNs in response to all three stimuli, with the strongest response following TLR9 activation. Compared with controls, SSc PBMCs produced less IFN-α (p < 0.02), whereas IFN-β levels were higher in SSc monocytes (342 vs 59.9 pg/mL, p = 0.041) when activated via TLR9. IFN-λ1/2 levels were consistently lower than type I IFNs. IFN-α priming increased TLR7/8-induced IFN production in PBMCs. Strong TLR9-mediated IFN-α production was associated with diffuse cutaneous SSc, anti-RNA-polymerase III autoantibodies, and interstitial lung disease (ILD). CONCLUSION:Leucocytes from SSc patients produce type I and III IFNs in response to a broad range of TLR stimuli. IFN-α priming enhanced TLR7/8-induced IFN production, suggesting that strong IFN inducers, e.g. certain viruses, may amplify IFN synthesis in response to endogenous TLR ligands and contribute to SSc pathogenesis, including the development of ILD.

OBJECTIVE:The antineutrophil cytoplasmic antibody (ANCA)-associated vasculitides are heterogeneous disorders. The aim of this study was to identify and characterize subgroups of patients based on sex, ANCA, age at diagnosis, and organ involvement. METHODS:In total, 1,167 patients with granulomatosis with polyangiitis (GPA) or microscopic polyangiitis (MPA) were retrospectively recruited to the study. Data including cumulative involvement of 10 different organ systems, end-stage kidney disease (ESKD), sex, proteinase (PR) 3-ANCA, myeloperoxidase (MPO)-ANCA, age at diagnosis, disease duration, and relapse were obtained from medical records. Clinical variables were analyzed for associations with sex, age at diagnosis, and relapse using logistic regression analysis. Thirteen clinical variables were included in hierarchical cluster analyses using the Ward method. RESULTS:In patients with GPA, PR3-ANCA, renal and pulmonary involvement, and ESKD were significantly associated with male sex, whereas MPO-ANCA was associated with female sex. Patients with GPA who were younger than 32 years of age at diagnosis were significantly more often females and had more ear-nose-throat involvement than patients older than 32 years. In patients with MPA, female patients were significantly younger at diagnosis than male patients. Relapse was significantly associated with young age at diagnosis and pulmonary involvement in GPA and with musculoskeletal involvement in MPA. Hierarchical cluster analyses identified five and seven patient clusters among individuals with GPA and MPA, respectively. PR3-/MPO-ANCA defined the largest clusters, whereas heart, gastrointestinal, and central nervous system involvement were hallmarks for three clusters for both patients with GPA and MPA. CONCLUSION:Sex, age at diagnosis, and specific organ involvements define clinically relevant subgroups among patients with ANCA-associated vasculitides.

ObjectiveDespite recent advances, the pathophysiological mechanisms underlying anti-neutrophil cytoplasmic antibody (ANCA)-associated vasculitides (AAV) remain incompletely understood, and comparative proteomic analyses of AAV subtypes are lacking. This study aimed to identify key molecular signaling pathways activated in AAV and to elucidate molecular distinctions between AAV with proteinase 3 ANCA (PR3-AAV) and AAV with myeloperoxidase ANCA (MPO-AAV).MethodsPlasma samples from 41 cases with active PR3-AAV, 24 with active MPO-AAV and 138 population controls were analyzed for 185 proteins using proximity extension assay and Luminex. Differential expression was assessed between PR3-AAV, MPO-AAV and controls using univariate and partial least squares discriminant analyses. Protein-protein interactions and pathway enrichment were explored using STRING and Cytoscape databases.ResultsCompared with controls, 31 proteins were significantly upregulated in PR3-AAV and 29 in MPO-AAV; 18 were shared, whereas 13 and 11 were specific to PR3-AAV and MPO-AAV, respectively. Shared proteins were enriched in general immune pathways, including IL-6 signaling. AAV subgroup-specific proteins were combined with proteins differentiating between PR3-AAV and MPO-AAV in a direct comparison. MMP-1, MMP-9, HGF, and OSM were uniquely upregulated in PR3-AAV, while TNF, TNF-R1/R2, TNFRSF14, and TNFRSF9 were prominent in MPO-AAV. Functional enrichment analyses underscored STAT3 signaling in PR3-AAV and TNF signaling in MPO-AAV.ConclusionsThis study identifies distinct and shared signaling pathways in PR3-AAV and MPO-AAV, highlighting STAT3 and TNF pathways as potential subtype-specific mechanisms. These findings offer insight into AAV pathogenesis and may guide the development of more targeted, less toxic treatments tailored to AAV subtypes.

Background The pathogenesis of ANCA-associated vasculitis (AAV) remains largely unknown. Proteinase 3 (PR3)- and myeloperoxidase (MPO)-AAV are two cateogories of AAV with distinct genetic background, but mechanistic differences between the two are poorly characterized. We hypothesized that in-depth studies of the plasma proteome in patients with active AAV would provide clues to the molecular and cellular mechanisms behind these disorders. Objectives To improve our understanding of the disease mechanisms behind AAV and pathophysiological differences between PR3- and MPO-AAV. Methods Plasma samples were collected at six Swedish rheumatological and/or nephrological centers from 42 PR3-AAV- and 25 MPO-AAV patients with active disease prior to commencement of therapy and from 138 healthy matched controls. All patients were classified into granulomatosis with polyangiitis or microscopic polyangiitis according to the European Medicines Agency algorithm. Samples were analysed for the relative levels of 181 proteins associated with inflammation or cardiovascular disease, using proximity extension assay (OLINK Proteomics). Differentially expressed proteins (DEPs) between groups were analyzed using ANOVA, where proteins with a fold change ≥ 1.5 and adjusted P value < 0.05 were considered as significant DEPs. Partial least square discriminant analysis (PLS-DA) was used to identify proteins contributing most to PR3-AAV/MPO-AAV separation from healthy controls. The STRING database was used to analyse protein–protein interaction networks. Gene ontology, KEGG and Reactome databases were used for pathway enrichment analyses using ClueGO. Results In comparison with healthy controls, 63 DEPs were identified for PR3-AAV and 62 for MPO-AAV; of these, 49 DEPs were common to both AAV groups. Pathway enrichment analysis of the 49 common DEPs identified IL-17-, IL-10-, TNF-α- and NF-kappa B signaling and neutrophil chemotaxis among the significantly enriched processes. The 14 DEPs unique for PR3-AAV formed a functional and physical protein-protein interaction network in STRING analysis, with significant enrichment for regulation of B cell proliferation, activation of matrix metalloproteinases, collagen degradation and IL-17- and TNF-α signaling pathways. The 13 DEPs unique for MPO-AAV did not show any significant functional enrichment. Of the top 15 proteins contributing most to group separation in the PLS-DA analysis, 11 proteins where common to both PR3- and MPO-AAV and 4 proteins were unique for PR3-AAV and MPO-AAV, respectively (Table 1). Conclusion Combining quantitative proteomics and bioinformatics analyses, we have identified a large group of DEPs characterizing both active PR3- and MPO-AAV and have determined their associated biological mechanisms. DEPs unique for PR3-AAV formed an interconnected protein network associated with biological processes of high relevance for AAV-pathogenesis. In conclusion, these findings may provide new insights into similarities and differences in the pathogenesis of MPO- and PR3-AAV. REFERENCES: NIL. Acknowledgements: NIL. Disclosure of Interests None Declared.Table 1PLS-DA results showing the top 15 proteins contributing most to separation of PR3-AAV and MPO-AAV patients, respectively, from healthy controls.Common for MPO-AAV and PR3-AAV vs healthy controlsUnique for MPO-AAV vs healthy controlsUnique for PR3-AAV vs healthy controlsCCL23EPHB4EN-RAGECD40LTBRMCP-3IL2-RAPLCPRTN3OPNRETNST2TGF-alphaTIMP-1TNF-R1TNF-R2TNFRSF14U-PARVEGFA

Objective To identify and characterize genetic loci associated with the risk of developing ANCA-associated vasculitides (AAV). Methods Genetic association analyses were performed after Illumina sequencing of 1853 genes and subsequent replication with genotyping of selected single nucleotide polymorphisms in a total cohort of 1110 Scandinavian cases with granulomatosis with polyangiitis or microscopic polyangiitis, and 1589 controls. A novel AAV-associated single nucleotide polymorphism was analysed for allele-specific effects on gene expression using luciferase reporter assay. Results PR3-ANCA(+) AAV was significantly associated with two independent loci in the HLA-DPB1/HLA-DPA1 region [rs1042335, P = 6.3 x 10(-61), odds ratio (OR) 0.10; rs9277341, P = 1.5 x 10(-44), OR 0.22] and with rs28929474 in the SERPINA1 gene (P = 2.7 x 10(-10), OR 2.9). MPO-ANCA(+) AAV was significantly associated with the HLA-DQB1/HLA-DQA2 locus (rs9274619, P = 5.4 x 10(-25), OR 3.7) and with a rare variant in the BACH2 gene (rs78275221, P = 7.9 x 10(-7), OR 3.0), the latter a novel susceptibility locus for MPO-ANCA(+) granulomatosis with polyangiitis/microscopic polyangiitis. The rs78275221-A risk allele reduced luciferase gene expression in endothelial cells, specifically, as compared with the non-risk allele. Conclusion We identified a novel susceptibility locus for MPO-ANCA(+) AAV and propose that the associated variant is of mechanistic importance, exerting a regulatory function on gene expression in specific cell types.

Background Patients with rheumatoid arthritis (RA) are at increased risk of developing diffuse large B-cell lymphoma (DLBCL). Immunotherapy blocking programmed cell death protein 1 (PD-1) and its ligand PD-L1 has substantially improved outcome in a number of malignant diseases but has so far been disappointing in DLBCL. However, current research seeks to identify DLBCL subgroups responsive to PD-1 blocking agents. Whether patients with pre-existing rheumatoid arthritis (RA) might constitute such a subgroup is unknown. Expression of PD-1 and PD-L1 has not previously been studied in a cohort of RA-DLBCL patients. Objectives To determine intratumoural expression of PD-1 and PD-L1 in DLBCL patients with pre-existing RA and relate the expression to clinical characteristics and survival outcome. Methods We included 103 RA-DLBCL patients and 74 DLBCL controls without rheumatic disease. PD-1 and PD-L1 immunohistochemical markers were applied on tumour tissue microarrays. The number of PD-1+ tumour infiltrating leukocytes (TILs) and proportions of PD-L1+ tumour cells and TILs were calculated and correlated to clinical data. For RA patients, disease activity had been individually scored repeatedly over time based on swollen and tender joint counts, erythrocyte sedimentation rate (ESR), and the treating physician’s global assessment of disease activity from onset of RA until lymphoma diagnosis(1). Values of cumulative disease activity for each patient were calculated as area under the curve (AUC). To investigate a possible influence of RA disease severity on the expression of PD-1 and PD-L1, RA patients with DLBCL were divided in two groups using the upper quartile of the AUC values as cut off for the highest disease activity group. Results Expression of PD-1 in TILs and PD-L1 in tumour cells was similar in RA-DLBCL and DLBCL controls. In RA-DLBCL, high expression of PD-L1 in tumour cells was significantly more common in patients with the most severe RA disease (45% vs 20%, p=0.04) (Table 1) and was associated with inferior overall survival in multivariable analysis (HR 4.62, 95% CI, 1.55-13.71). Table 1. Clinical and lymphoma related parameters and their distribution in the RA-DLBCL patients according to high and low expression of PD-L1 in tumour cells Entire cohort High PD-L1 in tumour cells 1 Low PD-L1 in tumour cells P-value* All patients, n (% ) 103 (100 ) 11 (100 ) 89 (100 ) Women, n (% ) 61 (59 ) 6 (55 ) 54 (61 ) 0.70 Age ≥60, n (% ) 85 (83 ) 10 (91 ) 72(81 ) 0.68 DLBCL subtype, n (% ) 0.35 GCB 30 (29 ) 2 (18 ) 27 (30 ) Non-GCB 69 (67 ) 9 (82 ) 57 (64 ) Missing 4 (4 ) 0 (0 ) 5 (6 ) Ann Arbor stage III-IV, n (% ) 65 (63 ) 5 (45 ) 57 (64 ) 0.31 Missing 4 (4 ) 1 (10 ) 3 (3 ) EBV status, n (% ) <0.001 Positive 9 (9 ) 4 (36 ) 5 (6 ) Missing 2 (2 ) 0 (0 ) 2 (2 ) RA highest disease severity group, n (% ) 0.04 Yes 25 (25 ) 5 (45 ) 18 (20 ) Missing 4 (4 ) 1 (10 ) 3 (3 ) Active RA treatment, n (% ) † 0.96 Yes 39 (38 ) 4 (36 ) 34 (38 ) Missing 3 (3 ) 1 (10 ) 2 (2 ) RA, rheumatoid arthritis; DLBCL, diffuse large B-cell lymphoma; GCB, germinal centre B-cell like; EBV, Epstein-Barr virus; DMARD, disease modifying anti-rheumatic drug; TIL, tumour-infiltrating leukocytes; 1 High expression ≥17% PD-L1+ tumour cells of all tumour cells; *According to chi-square or Fischer’s exact test; † Active RA treatment= DMARD and/or corticosteroids; Conclusion Our results indicate an association between a more severe RA disease and increased expression of PD-L1 in DLBCL tumour cells. This might have implications for the prognosis of the lymphoma as well as the chance of response to immunotherapy and warrants further studies also in other chronic inflammatory condidions. References [1]Baecklund E, Iliadou A, Askling J, Ekbom A, Backlin C, Granath F, et al. Association of chronic inflammation, not its treatment, with increased lymphoma risk in rheumatoid arthritis. Arthritis Rheum. 2006;54(3):692-701. Disclosure of Interests None declared

Current research seeks to identify subgroups of non-Hodgkin lymphoma (NHL) patients responsive to PD-1 blocking agents. Whether patients with pre-existing rheumatic diseases might constitute such a subgroup is unknown. We determined intratumoral expression of PD-1 and its ligands in lymphoma patients with pre-existing rheumatic diseases. We included 215 patients with rheumatoid arthritis (RA), systemic lupus erythematosus (SLE) or Sjögren's syndrome with subsequent lymphoma and 74 diffuse large B-cell lymphoma (DLBCL) controls without rheumatic disease. PD-1 and PD-ligand immunohistochemical markers were applied on tumor tissue microarrays. The number of PD-1+ tumor infiltrating leukocytes (TILs) and proportions of PD-L1+ and PD-L2+ tumor cells and TILs were calculated and correlated with clinical data. Expression of PD-L1 in tumor cells and TILs was highest in classical Hodgkin lymphoma and DLBCL. In DLBCLs, expression of PD-1 in TILs and PD-L1 in tumor cells was similar in RA, SLE and controls. In RA-DLBCL, high expression of PD-L1 in tumor cells was significantly more common in patients with the most severe RA disease and was associated with inferior overall survival in multivariable analysis.

Immunoglobulin G4-related disease (IgG4-RD) is an immune-mediated fibroinflammatory condition that can affect multiple organs. IgG4-RD may show a variety of initial symptoms. In the oral mucosa, lesions present as inflammatory fibrosis with a large number of IgG4-positive plasma cells. Evaluating treatment is a well-known problem in IgG4-RD due to the absence of an established assessment system. There are difficulties in defining the severity of the disease, which is why treatment is primarily based on its clinical manifestations. We present a case report of localized IgG4-RD with ulcerative and proliferative manifestations on the tongue, which clinically mimicked oral squamous cell carcinoma. A tumor-like lesion on the tongue can indicate something else other than the malignant or reactive changes commonly found in the oral mucosa. Multiple differential diagnoses of these atypical oral lesions, including localized IgG4-RD, should be considered.

Malignant lymphoma in granulomatosis with polyangiitis: subtypes, clinical characteristics and prognosis

ObjectiveSystemic lupus erythematosus (SLE) and systemic sclerosis (SSc) are autoimmune diseases that predominantly affect female patients, and therefore fewer investigations have been conducted in men. The aim of this study was to analyze sex hormone levels in male patients with SLE and those with SSc, compared to matched controls, in relation to the use of corticosteroids and cyclophosphamide (CYC).MethodsSex hormone levels were measured in fasting blood samples from male patients with SLE (n = 71) and those with SSc (n = 29) and compared to population‐based, age‐matched male controls. Relevant hormone profiles were identified using cluster analysis.ResultsMale SLE patients had higher levels of luteinizing hormone (LH) (P < 0.0001) and more frequent bioactive testosterone deficiency (P = 0.02) than their matched controls. The current dosage of prednisolone correlated inversely with the levels of bioactive testosterone (r = −0.36, P = 0.03). Cluster analysis identified a subset of SLE patients with increased levels of follicle‐stimulating hormone, LH, and prolactin as well as lower levels of bioactive testosterone (P < 0.0001) in relation to higher daily doses of prednisolone. In male SSc patients, levels of testosterone (P = 0.03) and bioactive testosterone (P = 0.02) were significantly lower than those in matched controls. Use of CYC during the previous year was associated with lower bioactive testosterone levels in both SLE patients (P = 0.02) and SSc patients (P = 0.01), after adjustment for age.ConclusionThe results of this study highlight the negative impact of corticosteroids on gonadal function in men with SLE. Furthermore, use of CYC during the year prior to study inclusion impaired bioactive testosterone levels in male patients with either SLE or SSc. Physicians should be more aware of the possibility of hypogonadism in male patients with autoimmune diseases. The need for hormonal supplementation remains to be formally evaluated in these patients.