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Granulomatosis with polyangiitis (GPA) and microscopic polyangiitis (MPA) are the most frequent primary necrotizing small vessel vasculitides. In these formerly fatal diseases remission can be induced by stage- and activity-adapted immunosuppressive regimens in the majority of patients. This does not lead to drug-free long-term remission or even cure. Consequently, maintenance of remission medication is needed. Recent randomized controlled trials demonstrated that maintenance treatment with the anti-B-cell antibody Rituximab, administered 6-monthly as opposed to azathioprine leads to a significantly lower relapse rate but a similar profile of adverse events. These data enabled the extension of the approval of Rituximab for maintenance of remission treatment of GPA and MPA in Germany in 2018. Guidelines and expert recommendations concerning measures of infection prevention and vaccination of immunosuppressed patients as well as the management of hypogammaglobulinemia and cytopenia on Rituximab are presented in this review.Die Granulomatose mit Polyangiitis (GPA) und die mikroskopische Polyangiitis (MPA) stellen als primäre nekrotisierende Kleingefäßvaskulitiden die häufigsten ANCA-assoziierten Vaskulitiden dar. Bei diesen, früher oft tödlich verlaufenden Erkrankungen kann heute durch eine Stadien-adaptierte Immunsuppression in den meisten Fällen eine Remission erzielt werden. Diese mündet jedoch nicht in eine Heilung oder medikamentenfreie Langzeitremission. Es bedarf deshalb einer remissionserhaltenden Therapie. Aktuelle randomisierte kontrollierte Studien zeigen, dass unter einer remissionserhaltenden Behandlung mit halbjährlich verabreichtem anti-B-Zell-Antikörper Rituximab weniger Rezidive auftreten als unter der bisherigen Standardmedikation mit Azathioprin bei vergleichbarem Nebenwirkungsprofil. Diese Daten führten 2018 zur Zulassung von Rituximab als remissionserhaltende Therapie der GPA und MPA. Unter einer solchen Rituximab-Therapie muss mit einer Immunglobulindepletion sowie späten Zytopenien mit begleitenden Infekten gerechnet werden. Leitlinien und Expertenempfehlungen zu Infektprophylaxen, Impfungen und Immunglobulinsubstitution werden in diesem Beitrag vorgestellt.

Chronic hepatitis C virus (HCV) infection is the most common cause of cryoglobulinemic vasculitis. In contrast, non-cryoglobulinemic vasculitis is rare in chronic HCV infection. Cryoglobulinemic vasculitis is a systemic immune complex-mediated vasculitis predominantly affecting small vessels and associated with the presence of serum cryoglobulins, i.e. cold-precipitable immunoglobulins. HCV is a hepato- and lymphotropic virus. Notably, secondary transition from benign lymphoproliferative disease to malignant non-Hodgkin lymphoma (NHL) as well as primary co-manifestation of cryoglobulinemic vasculitis and NHL has been reported in chronic hepatitis C. While cryoglobulinemia is detected in 50% or more of the patients with chronic hepatitis C, less than 5% develop related vasculitis. In this disorder, HCV induces clonal proliferation of memory phenotype marginal zone-like B-lymphocytes with restricted Ig heavy chain variable (VH) 1-69 gene expression encoding for the IgM rheumatoid factor (RF) WA idiotype. Monoclonal IgM RF binding to the Fc region of polyclonal IgG with anti-HCV reactivity facilitate the formation of cryoprecipitable multi-molecular immune-complexes. Cryoglobulins are preferentially deposited in tissues with high blood flow per unit mass of tissues, e.g. skin, synovium, choroid plexus and glomerulus, where they bind to endothelial cells via the C1q receptor. Endothelial cryoglobulin deposition induces the activation and recruitment of circulating neutrophil granulocytes and other immune cells to the endothelial lesion eventually resulting in complement-consuming vasculitis. The histopathologic changes in HCV-related vasculitis range from cutaneous leukocytoclastic vasculitis to severe necrotizing arteritis. Type I membranoproliferative glomerulonephritis is frequently found in patients with renal involvement. Clinical features of HCV-associated vasculitis comprise purpura, Meltzer`s triad (purpura, arthralgia, asthenia), polyneuropathy, renal involvement and Raynaud`s phenomenon. Hemorrhagic alveolitis, interstitial lung disease, gastrointestinal vasculitis, cardiac involvement, osteosclerosis and hyperviscosity syndrome are less common manifestations. Diagnostic criteria have been developed for HCV-associated cryoglobulinemic vasculitis. International therapeutic guidelines recommend treatment of HCV-related vasculitis to be guided according to the severity of disease. In patients with mild to moderate disease, interferon-free therapy regimens with direct acting antivirals (DAA) are considered as first-line treatment. In patients with severe manifestations, rituximab is given for the control of vasculitic manifestations, followed by HCV eradication using DAA. High rates of clinical responses and sustained virologic responses (SVR) have been reported for DAA treatment in HCV-associated cryoglobulinemic vasculitis. However, relapse of cryoglobulinemic vasculitis may occur in patients despite earlier treatment-induced clinical response and SVR. Persistence of clonal B-lymphocyte proliferation and perseverance of perturbations of the immune homeostasis have been shown in HCV-cured patients with relapse of cryoglobulinemic vasculitis. Disclosure of Interests: None declared

Background Previous studies identified functional autoantibodies against the angiotensin receptor type-1 (AT1R) and the endothelin receptor type A (ETAR) in about 85% of the patients with systemic sclerosis (SSc, 1). The antibodies are cross-reactive, agonistic and functionally active by increasing the effects of the natural ligands as well as by specific activation of the receptors (2, 3). The levels of the antibodies are associated with clinical findings such as pulmonary arterial hypertension (PAH). Patients with highest antibody levels show worst prognosis and do not respond well to receptor blocker therapy (2-4). Several in vitro effects of the antibodies depend on the antibody levels and on the cell type bearing the receptors. Receptor expression of ETAR and AT1R was highest in early disease (3). Objectives To determine predictors of PAH, DU and clinical complications by measuring the antibody levels and the receptor expression on peripheral mononuclear cells in patients with systemic sclerosis. Methods The current study analyzed the serological levels of anti-AT1R and anti-ETAR antibodies and the extracellular and intracellular expression of AT1R, AT2R, ETAR and ETBR on circulating CD4pos T cells, CD8pos T cells, CD14pos Macrophages, CD15pos Granulocytes and CD19pos B cells in SSc (n=41) using sandwich ELISA and flow cytometry. Clinical data (PAH, history of digital ulcers, digital-ulcers score, mRSS, pulmonary fibrosis, therapy) and serological markers (ESR, CRP, NT-proBNP) were gathering at the time of serum sampling and every three-month up to 27month after baseline. Results Patients with PAH demonstrated a lower AT1R MFI and AT1R/AT2R MFI ratio on all PBMC. Levels of NT-proBNP correlated negatively with the AT1R MFI and AT1R/AT2R MFI ratio on all PBMC. The levels of anti-AT1R ab correlated with the NT-proBNP in SSc patients with levels of NT-proBNP Conclusion Expression of AT1R and AT2R on PBMC could be of diagnostic value identifying clinical progress and/or subgroups in SSc. Their role in the pathophysiology, e.g. their impact of endothelial damage, has to be further investigated in SSc. References [1] Riemekasten G. et al., Involvement of functional autoantibodies against vascular receptors in systemic sclerosis. Ann Rheum Dis. 2011Mar;70(3):530-6. [2] Kill A. et al., Autoantibodies to angiotensin and endothelin receptors in systemic sclerosis induce cellular and systemic events associated with disease pathogenesis. Arthritis Res Ther. 2014Jan28;16(1):R29. [3] Becker MO. Et al., Vascular receptor autoantibodies in pulmonary arterial hypertension associated with systemic sclerosis. Am J Respir Crit Care Med. 2014Oct1;190(7):808-17. [4] Gunther J. et al., Angiotensin receptor type 1 and endothelin receptor type A on immune cells mediate migration and the expression of IL-8 and CCL18 when stimulated by autoantibodies from systemic sclerosis patients. Arthritis Res Ther. 2014Mar11;16(2):R65. Acknowledgement We thank Actelion Pharmaceutical GmbH for their financial support. Disclosure of Interests Sebastian Klapa: None declared, Silke Pitann: None declared, Gabriela Marschner: None declared, Susanne Riepe: None declared, Andreas Koch: None declared, Antje Muller: None declared, Harald Heidecke: None declared, Peter Lamprecht: None declared, Gabriela Riemekasten Consultant for: Chugai, F. Hoffmann-La Roche, Speakers bureau: Chugai, F. Hoffmann-La Roche

Background: Various alterations of the peripheral T-cell compartment have been reported in granulomatosis with polyangiitis (GPA) such as elevated populations of CD4+CD8+ double-positive and CD4+CD161+ and CD28- single-positive effector memory T-cells (TEM) within the total CD3+ T-cell population (1). Analysis of antigen-specific T-cell subpopulations shows, that PR3-specific T-cells display Th2-type, Th17 and Th22 cytokine profiles in GPA (2). Moreover, concomitant cellular CMV- and Epstein Barr virus (EBV)-infection has been found to be associated with the expansion of CD28- TEM in GPA (1,2). Notably, C-type lectin CD161+CD8+ T-cells displaying a polyfunctional memory profile directed against several common viruses have been reported. Furthermore, CD161+ T-cells are involved in the pathogenesis of early stage autoimmune hepatitis (3). CD161 expression on proteinase 3 (PR3)-specific T-cells in comparison to other antigen-specific T-cells has not been described in GPA as yet. Objectives: To determine the amount of C-type lectin CD161 on antigen-specific CD8+ single-positive and CD4+CD8+ double positive T-cells in patients with GPA Methods: In this study, we analyzed the expression of CD161 and CD28 on circulating antigen-specific CD8+ single-positive and CD4+CD8+ double positive T-cells in HLA-A2 positive patients with GPA (n=21) and healthy controls (n=21) using flow cytometry. Antigen-specific T cells were detected using peptide/MHC class 1 dextramers containing major peptide epitopes for PR3, Epstein Barr virus (EBV) BMLF1, and Cytomegalovirus (CMV) pp65 (aa 196-177, aa 280-288, and aa 495-504, respectively). Results: Patients with GPA showed a higher frequency of circulating CD8+ single-positive and CD4+CD8+double-positive PR3-specific T-cells with increased expressions of CD161 compared to HC. Compared to EBV- or CMV-specific T-cells, there was an increased expression of CD161 on PR3-specific T-cells in GPA. In contrast to HC and EBV- or CMV-specific T-cells, the percentage of CD28+ T-cells was expanded within the PR3-specific CD8+ T-cell subset in GPA. Conclusion: These findings suggest a potential role of CD161 as an additional TCR-independent co-stimulatory receptor on PR3-specific T-cells in GPA. The role of these cells in the pathophysiology and as a potential therapeutic target remains be further investigated. References [1] Kerstein A, et al., Environmental factors and inflammation-driven alteration of the total peripheral T-cell compartment in granulomatosis with polyangiitis; J Autoimmun. 2017Mar;78-79-91 [2] Lamprecht P, et al., Pathogenetic and clinical aspects of anti-neutrophil cytoplasmatic autoantibody associated vasculitis. Front. Immunol. 2018; 9:680 [3] Renand A, et al. Immune alterations in patinents with type 1 autoimmune hepatitis persist upon standard immunosuppressive treatment. Hepatol Commun. 2018 Aug 6;2(8):868-981 Disclosure of Interests: Sebastian Klapa: None declared, Anja Kerstein: None declared, Andreas Koch: None declared, Silke Pitann: None declared, Relana Nieberding: None declared, Gabriela Riemekasten Consultant for: Chugai, F. Hoffmann-La Roche, Speakers bureau: Chugai, F. Hoffmann-La Roche, Antje Muller: None declared, Peter Lamprecht: None declared

Background: Dysregulated miRNA expression profiles have been described in diverse chronic inflammatory diseases. We previously did a microarray screening of 847 miRNAs in nasal tissue from GPA patients and we found a disease associated alteration of miRNA expression compared to healthy controls (HC) and chronic rhinosinusitis (CRS). Objectives: In order to identify new miRNA targets of potential pathophysiological relevance in GPA, we validated dysregulated miRNAs by qPCR in GPA nasal tissue biopsies and sera. Moreover, we screened GPA associated miRNAs for their potential to regulate proteinase-3 (PRTN3). Methods: In an independent validation cohort (tissue and sera from 14 GPA-patients, 10 disease controls: CRS and Crohn’s/CD) 12 miRNAs were examined by qPCR. Validated and computational miRNA targets were identified by mirDIP algorithms. The inhibitory capacity of miRNAs on Proteinase-3 (PRTN3) was estimated by a dual-luciferase reporter system (Promega®). The 3’UTR-PRTN3 sequence was cloned and inserted into the pmirGlo vector and co-transfected with the hsa-mirna mimics (Dharmacon®) into HeLa cells. As a second method, the effect of miR-184 transfection on the endogenous PRTN3 expression in the human myeloid leukemia cell line HL-60 was estimated by western blot. Results: Microarray screening revealed alterations of 24 miRNAs in GPA nasal tissue compared to HC and CRS. qPCR confirmed dysregulation of 6 tissue related miRNAs also in GPA sera. Compared to CD 4 miRNAs (miR-10b, -99a/100, -125b, -532–3p) were down regulated in GPA tissue. The miRNA with the highest expression level in nasal tissue from GPA was miR-184. miR-184 along with miR-708 and mir-214-5p were also predicted to target PRTN3 by the mirDIP algorithm. The dual-luciferase reporter assay revealed a significant reduction of PRTN3 expression by miR-184, while these effects could not be observed for miR-708 or miR-214-5p. The transfection of miR-184 into HL-60 cells resulted in a dose-dependent knockdown of PRTN3 expression as detected by Western blot. Conclusion: Characteristic miRNA signatures in GPA, CRS and CD suggest distinct pathophysiological mechanisms. It indicates at a local miRNA dysregulation in inflamed GPA tissue with a corresponding serum signature that might serve as novel biomarkers. To our knowledge this is the first analysis that attempts to correlate GPA-associated miRNA expression patterns in tissue with serum. Moreover, this is the first description of a miRNA (miR-184) that potentially regulates the expression of the GPA autoantigen PRTN3. References: [1] O’Connell R et.al. Physiological and pathological roles for microRNAs in the immune system. Nat Rev Immunol. 2010;10(2):111-122. [2] Fasseu M et al. Identification of restricted subsets of mature microRNA abnormally expressed in inactive colonic mucosa of patients with inflammatory bowel disease. PLoS One. 2010 Oct 5;5(10). [3] Neudecker, V. MicroRNAs in mucosal inflammation. J Mol Med (Berl). 2017 September; 95(9): 935–949 [4] Coit P et al. An update on the role of epigenetics in systemic vasculitis. Curr Opin Rheumatol. 2018; 30(1): 4–15. Disclosure of Interests: Susanne Schinke Grant/research support from: travel and congress expenses from different companies pfizer, ucb, chemocentryx, Janssen-Cilag, msd, Nick Reichard: None declared, Barbara Russo: None declared, Antje Muller: None declared, Martin Laudien Paid instructor for: Olympus, Speakers bureau: Novartis, Robert Hasler: None declared, Gabriela Riemekasten Consultant for: Chugai, F. Hoffmann-La Roche, Speakers bureau: Chugai, F. Hoffmann-La Roche, Peter Lamprecht: None declared

To prevent serious complications such as permanent loss of vision and structural vascular damage, treatment must be initiated quickly in patients with giant-cell arteritis (GCA). So far, usually long-term corticosteroids in cumulative high dosages have been the standard therapy option. However, steroids are often insufficient to achieve adequate disease control and are associated with serious adverse events. Therefore, steroid-sparing therapy options are the focus of interest.

Background:The immunomodulatory cytokine IL-16 is increased in several inflammatory and autoimmune diseases1. IL-16 recruits and activates CD4+ immune cells such as T cells, dendritic cells, or monocytes. IL-16 is produced by various immune and non-immune cells, but synthesis and storage of IL-16 is regulated differentially depending on the cell type and stimulation. For its biological activity, IL-16 cleavage by caspase-3 is required1. Necrotizing granulomatous inflammation is a hallmark of granulomatosis with polyangiitis (GPA) with neutrophil dysregulation as a central driver of chronic inflammation and autoimmunity2. Earlier studies showed a correlation between increased serum IL-16 and disease parameters in AAV, including GPA3, but functional evidence for a direct link between IL-16 and neutrophils in granulomatous inflammation is missing so far.Objectives:In this study we aim to identify a functional link between increased IL-16, neutrophils, and the autoantigen proteinase 3 (PR3) with regard to chronic inflammation and autoimmunity in GPA.Methods:IL-16 was measured in sera of GPA patients (n = 40) and healthy controls (HC, n = 50) by ELISA and correlated with clinical features, such as disease activity (BVAS), creatinine, GFR, VDI and PR3-ANCA status. IL-16 protein expression was analyzed in peripheral blood mononuclear cells (PBMC) and polymorphonuclear cells (PMN) from GPA patients and HC (n = 5, each) by SDS-PAGE and western blot. Binding affinity of recombinant pro-IL-16 to native human PR3 was assessed by microscale thermophoresis. Cleavage of pro-IL-16 by active human PR3 was performed at various time points at 37°C. Cleavage products were analyzed by SDS-PAGE and western blot.Results:Circulating IL-16 was significantly increased in GPA patients compared to HC. Elevated IL-16 positively correlated with BVAS, creatinine, VDI and PR3-ANCA status and negatively correlated with GFR. In PMBC and PMN from GPA and HC we identified different expression patters of precursor and active forms of IL-16. In healthy PBMC we found high amounts of precursor (80kD), pro-IL-16 (55kD) and active IL-16 (17kD). In contrast, PBMC from GPA patients had lower amounts of pro-IL-16 and no active IL-16, indicating activation and secretion of IL-16 due to inflammatory stimulation, as shown earlier5. In GPA PMN we detected no precursor IL-16, but pro-IL-16 and its active form, in contrast to very low amounts of all IL-16 forms in healthy PMN. Processing and release of IL-16 in neutrophils has been linked to apoptosis and secondary necrosis5. By interaction studies we demonstrated direct binding of pro-IL-16 to PR3 with a Kd of 10 nM. In a subsequent cleavage assay we confirmed IL-16 processing by PR3 in a time-dependent manner.Conclusion:Correlation of serum IL-16 with clinical features of GPA suggests that IL-16 is associated with markers of disease activity, tissue damage and autoreactivity. We showed that PBMC and PMN represent a source of IL-16 in GPA. By the identification of PR3 as an additional IL-16-activating enzyme we could demonstrate a potential link between excessive PR3 expression, cell death and IL-16-dependent mechanisms, contributing to chronic granulomatous inflammation and autoimmunity in GPA.References:[1]Glass, W. G. et al. Not-so-sweet sixteen: The role of IL-16 in infectious and immune-mediated inflammatory diseases. J. Interf. Cytokine Res. 26, 511–520 (2006).[2]Millet, A. et al. Proteinase 3 on apoptotic cells disrupts immune silencing in autoimmune vasculitis. J. Clin. Invest. 125, 4107–4121 (2015).[3]Yoon, T. et al. Serum interleukin-16 significantly correlates with the Vasculitis Damage Index in antineutrophil cytoplasmic antibody-associated vasculitis. Arthritis Res. Ther. 22, 1–6 (2020).[4]Elssner, A. et al. IL-16 Is Constitutively Present in Peripheral Blood Monocytes and Spontaneously Released During Apoptosis. J. Immunol. 172, 7721–7725 (2004).[5]Roth, S. et al. Secondary necrotic neutrophils release interleukin-16C and macrophage migration inhibitory factor from stores in the cytosol. Cell Death Discov. 1, 15056 (2015).Disclosure of Interests:None declared

Autoimmune diseases are initiated by a combination of predisposing genetic and environmental factors resulting in self-perpetuating chronic inflammation and tissue damage. Autoantibody production and an imbalance of effector and regulatory T-cells are hallmarks of autoimmune dysregulation. While expansion of circulating effector memory T-cells is linked to disease pathogenesis and progression, the causes driving alterations of the peripheral T-cell compartment have remained poorly understood so far. In granulomatosis with polyangiitis (GPA), a prototypical autoimmune disorder of unknown aetiology, we performed for the first time a combined approach using phenotyping, transcriptome and functional analyses of T-cell populations to evaluate triggers of memory T-cell expansion. In more detail, we found increased percentages of circulating CD4+CD28-, CD8+CD28- and CD4+CD161+ single-positive and CD4+CD8+ double-positive T-cells in GPA. Transcriptomic profiling of sorted T-cell populations showed major differences between GPA and healthy controls reflecting antigen- (bacteria, viruses, fungi) and cytokine-driven impact on T-cell populations in GPA. Concomitant cytomegalovirus (CMV) and Epstein-Barr virus (EBV) - positivity was associated with a significant increase in the percentage of CD28- T-cells in GPA-patients compared to sole CMV- or EBV-positivity or CMV- and EBV-negativity. T-cells specific for other viruses (influenza A virus, metapneumovirus, respiratory syncytial virus) and the autoantigen proteinase 3 (PR3) were infrequently detected in GPA. Antigen-specific T-cells were not specifically enriched in any of the T-cell subsets. Altogether, on a genetic and cellular basis, here we show that alterations of the peripheral T-cell compartment are driven by inflammation and various environmental factors including concomitant CMV and EBV infection. Our study provides novel insights into mechanisms driving autoimmune disease and on potential therapeutic targets.

Granulomatosis with polyangiitis (GPA) is a potentially life-threatening, rare disease. The etiology is unknown. GPA is histomorphologically characterized by extravascular necrotizing granulomatous inflammation and a systemic necrotizing vasculitis of small to medium-sized vessels. Clinically, a pulmonary-renal syndrome with pulmonary infiltrates, alveolar hemorrhage and a rapidly progressive glomerulonephritis is seen in about 80% of the cases with generalized disease. GPA is associated with proteinase 3-specific anti-neutrophil cytoplasmic autoantibodies (PR3-ANCA). Treatment is guided by severity of organ involvement and disease activity. Cytostatic immunosuppressants or the monoclonal anti-CD20 antibody rituximab are applied.

Die mit Anti-Neutrophilen zytoplasmatischen Autoantikorpern (ANCA)-assoziierten Vaskulitiden (AAV) erfordern abhangig vom Grad der Organdysfunktion und der Krankheitsaktivitat ein differenziertes therapeutisches Vorgehen. Bei Organdysfunktion und vitaler Bedrohung wird eine remissionsinduzierende Therapie mit Cyclophosphamid oder Rituximab empfohlen, bei fehlender Organdysfunktion und Fehlen einer vitalen Bedrohung mit Methotrexat oder Mycophenolat-Mofetil. In der remissionserhaltenden Therapie kommen Azathioprin oder Methotrexat zur Anwendung. Bei Kontraindikationen, Unvertraglichkeit oder fruherem Therapieversagen von Azathioprin und Methotrexat werden Rituximab, Leflunomid oder Mycophenolat-Mofetil als Alternativen verwendet. Die remissionserhaltende Therapie wird im Allgemeinen uber mindestens 2 Jahre durchgefuhrt. Die Therapiedeeskalation erfordert ein engmaschiges klinisches Monitoring und erfolgt durch eine schrittweise Dosisreduktion der Glukokortikoid- und ubrigen immunsuppressiven Therapie. Jede Therapiedeeskalation birgt jedoch die Gefahr eines Rezidivs in sich.

Rheumatologist should be familiar with the concept of IgG4-related disease (IgG4-RD). Due to the clinical spectrum IgG4-RD can fall directly within the scope of rheumatology and are often diagnosed primarily by rheumatologists. Furthermore, IgG4RD are relevant differential diagnoses for many other rheumatic conditions. Finally, there are an increasing amount of data suggesting an important role of immunological processes observed in IgG4-RD for other rheumatic diseases.

Endothelin 1 (ET-1) is a potent vasoactive peptide hormone produced by endothelial cells, macrophages and vascular smooth muscle cells (VSMCs). Elevated ET-1 plasma levels have been reported in patients with ischaemic complications in giant-cell arteritis (GCA), a systemic vasculitis predominantly affecting large vessels and their branches.1 In temporal artery biopsy specimens, increased expression of ET-1 and the G protein-coupled vasoconstrictive endothelin receptor A (ETAR) has been found.2 ETAR activation induces focal adhesion kinase-mediated morphological changes and increased motility of VSMC potentially contributing to intimal hyperplasia and vascular occlusion in GCA.3 VSMC migration can be blocked by specific ETAR antagonists.3 Notably, disease-specific anti-G protein-coupled receptor autoantibody (aab) signatures have been found in different autoimmune diseases.4 5 Anti-ETAR aabs are increased and associated with pulmonary arteriolar occlusion and hypertension in systemic sclerosis.5 6 Anti-ETAR aabss induce vascular adhesion molecules, interleukin (IL)-8 and chemokine ligand CCL-18 productions and exhibit chemotactic activity by mediating neutrophil and T cell migration.5 6 To address …

Background:Granulomatosis with polyangiitis (GPA) is characterized by extravascular necrotizing granulomatous inflammation and systemic ANCA – associated (AAV) vasculitis with neutrophils as a key player in the pathogenesis (1). We and others have shown that neutrophil-related cell death mechanisms contribute to chronic inflammatory processes in AAV (2, 3). Recently, another form of inflammatory cell death primarily described in monocytes called pyroptosis was also discovered in neutrophils (4). A cardinal feature of pyroptosis is the activation of the NLRP3 inflammasome, a sensor of different pathogen- and damage-associated molecular patterns (PAMP, DAMP), following caspase-1-mediated processing and secretion of IL-1beta (5).Objectives:The aim of this study was to investigate, if neutrophils from GPA patients express pyroptosis-related components NLRP3, active caspase 1 and cleaved IL-1beta.Methods:Polymorphonuclear leukocytes (PMN) were isolated from peripheral blood of GPA patients and healthy controls (HC) (n = 10 each). Expression of NLRP3, inactive/active caspase 1 and active IL-1beta was determined by western blot. In addition, peripheral blood mononuclear cells (PBMC) were isolated from GPA and HC. mRNA expression ofnlrp3andil1bwas determined by qPCR. To exclude false-positive results by contamination with monocytes we performed flow cytometry analysis of whole blood samples with markers CD3, CD14, CD15, CD66b and NLRP3.Results:PMN from GPA patients showed markedly increased expression of NLRP3, active caspase 1 and active IL-1beta compared to HC. In contrast, there was no difference between GPA and HC on the mRNA level of neithernlrp3noril1bin PBMC. In addition, we confirmed by flow cytometry increased expression of NLRP3 in PMN from GPA, but not in monocytes.Conclusion:Here we provide evidence, that neutrophils from GPA undergo pyroptosis, demonstrated by increased NLRP3, active caspase 1 expression as well as IL-1beta processing. Neutrophils are present in high numbers at the site of granulomatous lesions of inflamed tissue in GPA and IL-1beta is increased in GPA sera (2). Therefore, neutrophils represent a potential source of IL-1beta in GPA. Given the fact that GPA-associated features such as massive release of necrosis-related DAMP or microbial agents such asStaphylococcus aureus(6) can activate the NLRP3-inflammasome, we identified here a potential relevant mechanism of neutrophils contributing to chronic inflammation of GPA.References:[1]Jennette, J.C., and Falk, R.J. (2014). Pathogenesis of antineutrophil cytoplasmic autoantibody-mediated disease. Nat. Rev. Rheumatol.10, 463–473.[2]Millet, A., Martin, K.R., Bonnefoy, F., Saas, P., Mocek, J., Alkan, M., Terrier, B.,Kerstein,A., Tamassia, N., Satyanarayanan, S.K., et al. (2015). Proteinase 3 on apoptotic cells disrupts immune silencing in autoimmune vasculitis. J. Clin. Invest. 125, 4107–4121.[3]Schreiber, A., Rousselle, A., Becker, J.U., von Mässenhausen, A., Linkermann, A., and Kettritz, R. (2017). Necroptosis controls NET generation and mediates complement activation, endothelial damage, and autoimmune vasculitis. Proc. Natl. Acad. Sci. 201708247.[4]Tourneur, L., and Witko-Sarsat, V. (2019). Inflammasome activation: Neutrophils go their own way. J. Leukoc. Biol.105, 433–436.[5]Bergsbaken, T., Fink, S.L., and Cookson, B.T. (2009). Pyroptosis: Host cell death and inflammation. Nat. Rev. Microbiol.7, 99–109.[6]Lamprecht, P.,Kerstein, A., Klapa, S., Schinke, S., Karsten, C.M., Yu, X., Ehlers, M., Epplen, J.T., Holl-Ulrich, K., Wiech, T., et al. (2018). Pathogenetic and Clinical Aspects of Anti-Neutrophil Cytoplasmic Autoantibody-Associated Vasculitides. Front. Immunol.9, 1–10.Disclosure of Interests:Anja Kerstein-Staehle: None declared, Nadja Leinung: None declared, Jannik Meyer: None declared, Silke Pitann: None declared, Antje Müller: None declared, Gabriela Riemekasten Consultant of: Cell Trend GmbH, Janssen, Actelion, Boehringer Ingelheim, Speakers bureau: Actelion, Novartis, Janssen, Roche, GlaxoSmithKline, Boehringer Ingelheim, Pfizer, Peter Lamprecht: None declared

Background:Activation of the alternative and final common pathways have been shown in ANCA-associated vasculitis (AAV) (1). Circulating titers of C5a are elevated and correlate with disease activity in AAV. Binding to the corresponding G protein-coupled receptor (GPCR) C5aR1 enhances the influx of neutrophils, leading to ROS generation and severe necrotizing of vascular walls (2). Moreover, subsequent interaction of C5a with C5aR1 may represent a proinflammatory amplification loop (3). Blocking of the receptor is protective in a murine model in AAV (4). In humans, avacopan, a C5aR1-inhibitor showed promising results as glucocorticoid-sparing agent in two randomized phase II and one ongoing phase III clinicals trials in AAV (NCT02994927). Notably, disease-specific anti-GPCR autoantibody (aab) signatures have been found in different autoimmune diseases (5).Objectives:The aim of the present study was to examine whether (patho)physiological anti-C3aR and anti-C5aR1 aabs correlate with clinical findings in AAV, and whether this is linked to the clinical outcome.Methods:Sera and plasma of AAV patients [granulomatosis with polyangiitis (GPA), n=64; microscopic polyangiitis (MPA), n=26; eosinophilic granulomatosis with polyangiitis (EGPA), n=11] were measured by Elisa for circulating autoantibodies against complement receptors C3a (anti-C3aR aab) and C5a (anti-C5aR1 aab) and plasma levels of C3a and C5a. Expression of C3aR and C5aR1 on T-cells was determined using flow cytometry. Clinical data were assessed at the time of serum sampling and during follow-up for 48 monthsResults:GPA displayed low titers of anti-C3aR aab (GPA:5.33±2.54vs. HD:6.47±2.61, P=0.0031). Anti-C5aR1 aab were decreased in AAV, especially in GPA (GPA:1.02±1.07vs. HD:6.63±2.91, P=vs. HD:3.44±0.68%, P=0.0021; CD8+C5aR1+T-cells GPA:9.74±2.10%vs.HD:4.11±0.92%, P=0.0198). Reduced titers of anti-C5aR1 aab Conclusion:As potential diagnostic marker, anti-C5aR1 aab titer may additionally be useful to monitor disease activity in AAV.References:[1]Chen M et al.Complement deposition in renal histopathology of patients with ANCA-associated pauci-immune glomerulonephritis.Nephrol Dial Transpl. 2009;24:1247-1252[2]Schreiber A et al.C5a receptor mediates neutrophil activation an ANCA-induced glomerulonephritis.J Am Soc Nephrol. 2009; 20:289-298[3]Lamprecht P et al.: Pathogenetic and clinical aspects of Anti-Neutrophil Cytoplasmic Autoantibody-associated vasculitides.Front Immunol.2018 Apr 9;9-680[4]Xiao H et al.C5a receptor (CD88) blockade protects against MPO-ANCA GN.J Am Soc Nephrol. 2014;25(2):225-31[5]Klapa S et al. Decreased endothelin receptor A autoantibody levels are associated with early ischaemic events in patients with giant-cell arteritis.Ann Rheum Dis2019 Oct;78(19):1443-1444Disclosure of Interests:Sebastian Klapa Grant/research support from: Actelion, Consultant of: Pfizer, Abbvie, Antje Müller: None declared, Andreas Koch: None declared, Anja Kerstein-Staehle: None declared, Wataru Kaehler: None declared, Harald Heidecke Shareholder of: Cell Trend GmbH, Employee of: Cell Trend GmbH, Speakers bureau: Cell Trend GmbH, Susanne Schinke Speakers bureau: Pfizer, Markus Huber-Lang: None declared, Martin Nitschke: None declared, Silke Pitann: None declared, Christian Karsten: None declared, Gabriela Riemekasten Consultant of: Cell Trend GmbH, Janssen, Actelion, Boehringer Ingelheim, Speakers bureau: Actelion, Novartis, Janssen, Roche, GlaxoSmithKline, Boehringer Ingelheim, Pfizer, Peter Lamprecht: None declared

Background:Micro-RNAs (miRNA) are short non-coding RNAs that regulate inflammation mostly by translational repression. Previously, we screened 847 miRNAs in nasal tissue from GPA patients and found a disease associated alteration of miRNA expression compared to healthy controls and chronic rhinosinusitis. MiR-184 was most over expressed in nasal tissue from GPA (13.4x). The dual-luciferase reporter assay confirmed a significant reduction of Proteinase-3 (PRTN3) expression by miR-184 (1).PRTN3 transcripts with an alternative 3’ untranslated region (UTR) have been described in GPA (2). The pathophysiological relevance of this alternative transcript remains unclarified.Objectives:To identify new miRNA targets of potential pathophysiological relevance in GPA, we validated the effect of the 21 most dysregulated miRNAs on the mRNA of PRTN3. Further, we included the alternative PRTN3 mRNA in our screen to look for new regulatory differences.Methods:The inhibitory capacity of miRNAs on Proteinase-3 mRNA was estimated by a dual-luciferase reporter system. The sequences of the alternative (132bp longer) and the regular 3’UTR-PRTN3 were cloned and inserted into the pmirGLO vector and co-transfected with 21 miRNA mimics into HeLa cells. Co-transfection withCaenorhabditis elegansmiRNA 67 mimic (cel-miR-67) was used as negative control. Statistical significance was evaluated by students t-test adjusted for multiple comparisons (Holm-Sidak).Results:For 18 of 21 investigated miRNAs no effects could be observed on the alternative and the regular 3’UTR-PRTN3. But there were remarkable differential effects of let-7f, miR-184 and miR-708. Let-7f (-29,2%) and miR-708 (-23,6%) both showed a suppression of the alternative 3’UTR-PRTN3 but no effect on the regular 3’UTR-PRTN3 while miR-184 only suppressed the regular 3’UTR (-17,5 %) and not the alternative variant (fig. 1-2).Fig. 1.Dual-luciferase reporter assay with the regular 3’UTR of PRTN3 cloned into the pmirGLO vector compared to empty vector (NTC). Significant effect for miR-184 (17,5 %), miR-708 no effect and let-7f small but not significant reduction in luciferase activity (12,3 %). Data represent 3 independent experiments with triplicate measurements. miR-184 was tested 6 times. *PFig. 2.Dual-luciferase reporter assay with the alternative 3’UTR of PRTN3. Significant effects of let-7f (29,2 %) and miR-708 mimic (23,6 %) but no significant effects of miR-184 of luciferase activity. 3 independent experiments with triplicate measurements. *PConclusion:Disease specific miRNA signatures together with an increased PRTN3 level and in alternative PRTN3 mRNA in GPA suggest a dysregulation of PRTN3 expression in GPA. To our knowledge this is the first analysis in GPA showing that miRNAs can differentially regulate the expected and the alternative 3’UTR variants of PRTN3-mRNA. As miR-184 is markedly upregulated in GPA, a repression of PRTN3 is to be anticipated, possibly as a reaction to previous neutrophil activation with PRTN3 overexpression. Our findings also strengthen the potential pathophysiological role of the alternative PRTN3 mRNA.References:[1]Schinke S et alPROTEINASE-3 REGULATING MICRO-RNA IN GRANULOMATOSIS WITH POLYANGIITIS. Ann Rheum Dis 2019 (78 Suppl 2):437[2]McInnes E et alDysregulation of Autoantigen Genes in ANCA-Associated Vasculitis Involves Alternative Transcripts and New Protein Synthesis J Am Soc Nephrol. 2015 26(2): 390–399Acknowledgments:Vasculitis foundation for fundingDisclosure of Interests:Nick Reichard: None declared, Anja Kerstein-Staehle: None declared, Antje Müller: None declared, Gabriela Riemekasten Consultant of: Cell Trend GmbH, Janssen, Actelion, Boehringer Ingelheim, Speakers bureau: Actelion, Novartis, Janssen, Roche, GlaxoSmithKline, Boehringer Ingelheim, Pfizer, Peter Lamprecht: None declared, Susanne Schinke Speakers bureau: Pfizer

Eosinophilic granulomatosis with polyangiitis (EGPA: formerly Churg-Strauss syndrome) is a rare inflammatory disease of unknown cause. 30% of patients have anti-neutrophil cytoplasm antibodies (ANCA) specific for myeloperoxidase (MPO). We performed a genome-wide association study (GWAS) of EGPA, testing 7.5 million genetic variants in 684 cases and 6,838 controls. Case-control analyses were performed for EGPA as a whole, and stratified by ANCA. To increase power, we used a conditional false discovery rate method to leverage findings from GWASs of related phenotypes. In total, 11 variants were associated with EGPA, two specifically with ANCA-negative EGPA, and one (HLA-DQ) with MPO+ANCA EGPA. Many variants were associated with asthma, eosinophilic and immune-mediated diseases and, strikingly, nine were associated with eosinophil count in the general population. Through Mendelian randomisation, we show that a primary tendency to eosinophilia underlies EGPA susceptibility. We demonstrate that EGPA comprises two genetically and clinically distinct syndromes, with ANCA-negative EGPA genetically more similar to asthma. MPO+ ANCA EGPA is an eosinophilic autoimmune disease sharing certain clinical features and an MHC association with MPO+ ANCA-associated vasculitis, while ANCA-negative EGPA may instead have a mucosal/barrier dysfunction origin. Five identified candidate genes are targets of therapies in development, supporting their exploration in EGPA.

Dysbiosis¸ i.e. changes in microbial composition at a mucosal interface, is implicated in the pathogenesis of many chronic inflammatory and autoimmune diseases. To assess the composition of the microbial upper respiratory tract (URT) community in patients with granulomatosis with polyangiitis (GPA), we used culture-independent high-throughput methods. In this prospective clinical study, nasal swabs were collected from patients with GPA, patients with rheumatoid arthritis (RA, disease control), and healthy controls. Nasal bacterial taxa were assessed using V3-V4 region 16S rRNA amplicon sequencing. Staphylococcus aureus, Haemophilus influenza, and entero- and rhinoviruses were detected using qPCR. Unbiased metagenomic RNA sequencing (UMERS) was performed in a subset of samples to determine the relative abundance of bacterial, fungal, and viral species. A trend toward reduced microbiome diversity was detected in GPA samples compared with healthy controls. The abundance of bacterial taxa and microbial richness were significantly decreased in GPA samples compared with RA samples. The relative abundance of bacterial families shifted, with increased Planococcaceae and decreased Moraxellaceae, Tissierellaceae, Staphylococcaceae, and Propionibacteriaceae in GPA and RA. Further, decreased abundance of Corynebacteriaceae, and Aerococcaceae was observed in GPA samples. Significantly more colonization of S. aureus was seen in the nasal microbiome of GPA compared with RA and healthy control samples. H. influenzae colonization was also observed in GPA samples. UMERS detected the presence of rhinoviral sequences in some GPA samples. Thus, our study uncovered changes in the URT microbial composition in patients with GPA and RA, suggesting that both immunosuppression and disease background affect the URT microbiome. Complex alterations of host-microbiome interactions in the URT could influence chronic endonasal inflammation in GPA.

PURPOSE: We aimed to assess the impact of (18)F-fluorodeoxyglucose (FDG) positron emission tomography (PET) on the management of patients with suspected large vessel vasculitis. METHODS: An international expert panel determined diagnoses and clinical management in patients with suspected large vessel vasculitis, with and without the results of (18)F-FDG PET, respectively. The accuracy of the clinical diagnosis and the resulting clinical management with and without the (18)F-FDG PET results were compared using logistic regression models. RESULTS: The analysis included 30 patients referred to a tertiary care centre with large vessel vasculitis and 31 controls. (18)F-FDG PET had an overall sensitivity of 73.3% [95% confidence interval (CI) 54.1-87.7%], a specificity of 83.9% (95% CI 66.3-94.5%), a positive predictive value of 81.5% (95% CI 61.9-93.7%) and a negative predictive value of 76.5% (95% CI 58.8-89.3%). The diagnostic accuracy of (18)F-FDG PET was higher in patients not receiving immunosuppressive drugs (93.3 vs 64.5%, p = 0.006). Taken in context with other available diagnostic modalities, the addition of (18)F-FDG PET increased the clinical diagnostic accuracy from 54.1 to 70.5% (p = 0.04). The addition of (18)F-FDG PET increased the number of indicated biopsies from 22 of 61 patients (36.1%) to 25 of 61 patients (41.0%) and changed the treatment recommendation in 8 of 30 patients (26.7%) not receiving immunosuppressive medication and in 7 of 31 patients (22.6%) receiving immunosuppressive medication. CONCLUSION: (18)F-FDG PET is a sensitive and specific imaging tool for large vessel vasculitis, especially when performed in patients not receiving immunosuppressive drugs. It increases the overall diagnostic accuracy and has an impact on the clinical management in a significant proportion of patients.

Background Since the time when Paul Ehrlich conceived the term “horror autotoxicus”, autoantibodies have been associated with the development of autoimmune diseases. However, several works have recently shown the presence of autoantibodies in sera from healthy subjects (n=489), who do not develop autoimmune diseases. Objectives Here, we report a network of immunoglobulin G (IgG) autoantibodies targeting G protein-coupled receptors (GPCRs), growth factors and growth factor-related molecules in sera from healthy subjects. Methods Autoantibody levels in sera were assessed using ELISA. Autoantibody network was analysed by exploratory factor analysis (EFA), dandelion plot method, hierarchical clustering, and multi-study factor analysis (MFA). We also reverse engineered autoantibody functions through in silico evaluation of autoantibody target interactions using STRING and gene ontology (GO). To test the autoantibodies functionality we assessed the in vitro production of IL-8 by PBMCs and neutrophil migration in response to IgG from healthy subjects as well as ETAR-immunised and control mice. Leukocyte cellularity to secondary immune organs was analysed comparing ETAR-immunised with control mice. Results Gender, age and the presence of pathological conditions (systemic sclerosis n=84, Alzheimer’s disease n=91 and ovarian cancer n=207) changed correlations between the autoantibodies and their hierarchical clustering distribution. Notably, subjects at age below and above 65 years or with pathological conditions exhibited particular autoantibody hierarchical clustering signatures. In addition, females at age above 65 years, representing the group of subjects with higher risk to develop SSc, displayed the closest link to SSc in terms of autoantibody hierarchical clustering. Finally, autoantibody directed against the endothelin receptor type A (ETAR) showed an essential role in the autoantibody network by orchestrating neutrophil trafficking in vitro and in ETAR-immunised mice. Conclusions Our data provide a framework for the existence of a physiological network of autoantibodies and reveal a new paradigmatic view on these physiological molecules. Disclosure of Interest None declared

Circulating anti-neutrophilic cytoplasmic autoantibodies targeting proteinase 3 (PR3-ANCA) are a diagnostic and pathogenic hallmark of granulomatosis with polyangiitis (GPA). It is, however, incompletely understood if inflamed tissue supports presence or emergence of PR3-ANCA

Autoantibodies have been associated with autoimmune diseases. However, studies have identified autoantibodies in healthy donors (HD) who do not develop autoimmune disorders. Here we provide evidence of a network of immunoglobulin G (IgG) autoantibodies targeting G protein-coupled receptors (GPCR) in HD compared to patients with systemic sclerosis, Alzheimer’s disease, and ovarian cancer. Sex, age and pathological conditions affect autoantibody correlation and hierarchical clustering signatures, yet many of the correlations are shared across all groups, indicating alterations to homeostasis. Furthermore, we identify relationships between autoantibodies targeting structurally and functionally related molecules, such as vascular, neuronal or chemokine receptors. Finally, autoantibodies targeting the endothelin receptor type A (EDNRA) exhibit chemotactic activity, as demonstrated by neutrophil migration toward HD-IgG in an EDNRA-dependent manner and in the direction of IgG from EDNRA-immunized mice. Our data characterizing the in vivo signatures of anti-GPCR autoantibodies thus suggest that they are a physiological part of the immune system., Autoantibodies are implicated in autoimmunity, but may also be present in healthy individuals. Here the authors find that the autoantibody specificity signatures against various G protein-coupled receptors are associated with multiple parameters, including disease states, to imply a physiological function in maintaining immune homeostasis.

Patients with antineutrophil cytoplasmic autoantibody (ANCA)-associated vasculitides (AAV) have an expansion of effector memory T‑cells in peripheral blood. The enlarged effector memory cell population contains distinct cell subsets, including T‑helper type 1 (Th1) CD4+ T‑cells lacking co-stimulatory CD28 expression and Th17 cells in granulomatosis with polyangiitis (GPA) and microscopic polyangiitis (MPA) and Th2 type and Th17 cells in eosinophilic granulomatosis with polyangiitis (EGPA). The cytokine response of autoreactive proteinase 3 (PR3)-specific effector memory T‑cells is skewed towards an increase of Th2 type, Th17 and Th22 cell fractions in GPA. Anomalous effector memory CD4+ T‑cell co-stimulation is suggested by the aberrant expression of P‑selectin glycoprotein ligand‑1, beta‑2 integrin, chemokine receptors, natural-killer group 2 member D (NKG2D) and other activating receptors. The increased expression of these receptors is accompanied by T‑cell activation and migration to inflamed tissues. The T‑cells are abundant and secrete proinflammatory cytokines in inflammatory lesions in AAV. The T‑cell mediated tissue damage correlates with renal outcome, whereas B-cell infiltration does not. Activation of lesional CD4+NKG2D+ effector memory T‑cells is independent of the antigen; moreover, CD4+NKG2D+ effector memory T‑cells display NK-cell-like cytotoxicity towards microvascular endothelial cells in vitro. Thus, effector memory T‑cells play an important role in tissue damage and disease progression in AAV. Sequentially administered or combined with B-cell depleting therapy, T‑cell-directed therapies, especially those directed against effector memory CD4+ T‑cells, may further improve the outcome and help to achieve long-term remission in AAV.

Patients with antineutrophil cytoplasmic autoantibody (ANCA)-associated vasculitides (AAV) have an expansion of effector memory T‑cells in peripheral blood. The enlarged effector memory cell population contains distinct cell subsets, including T‑helper type 1 (Th1) CD4+ T‑cells lacking co-stimulatory CD28 expression and Th17 cells in granulomatosis with polyangiitis (GPA) and microscopic polyangiitis (MPA) and Th2 type and Th17 cells in eosinophilic granulomatosis with polyangiitis (EGPA). The cytokine response of autoreactive proteinase 3 (PR3)-specific effector memory T‑cells is skewed towards an increase of Th2 type, Th17, and Th22 cell fractions in GPA. Anomalous effector memory CD4+ T‑cell co-stimulation is suggested by the aberrant expression of P‑selectin glycoprotein ligand-1, β‑2 integrin, chemokine receptors, natural-killer group 2 member D (NKG2D) and other activating receptors. The increased expression of these receptors is accompanied by T‑cell activation and migration to inflamed tissues. T‑cells are abundant and secrete proinflammatory cytokines in inflammatory lesions in AAV. The T‑cell mediated tissue damage correlates with renal outcome, whereas B‑cell infiltration does not. Activation of lesional CD4+NKG2D+ effector memory T‑cells is independent of the antigen; moreover, CD4+NKG2D+ effector memory T‑cells display NK-cell-like cytotoxicity towards microvascular endothelial cells in vitro. Thus, effector memory T‑cells play an important role in tissue damage and disease progression in AAV. Sequentially administered or combined with B‑cell depleting therapy, T‑cell-directed therapies, especially those directed against effector memory CD4+ T‑cells, may further improve the outcome and help to achieve long-term remissions in AAV.

To examine functional features of CD4+CD8+ double-positive T-cells in patients with granulomatosis with polyangiitis (GPA) using phenotypic and transcriptomic analysis.Staining of cellular surface marker was performed using freshly collected whole blood. For intracellular cytokine staining freshly collected whole blood was stimulated with phorbol myristate acetate and ionomycin. Multicolor flow cytometric analysis was performed on a FACSCanto II cytometer using FACSDiva software. Lymphocytes were gated on CD3, CD4, and CD8 staining. FACS-sorted CD4+CD8+ double-positive T-cells of GPA-patients and HC (n=3 each) were subjected to transcriptional profiling using an Affymetrix Human Genome 2.0 microarray. Differently expressed genes were analysed using biological databases.Frequency of CD4+CD8+ double-positive T-cells was increased within the total CD3+ T-cell population in GPA, but no difference was detected between patients with active disease and remission. Percentages of interferon γ (Th1-type), interleukin 17 and interleukin 22 (Th17-type) producing CD4+CD8+ double-positive T-cells exceeded the percentage of interleukin 4 (Th2-type) producing cells. There were no significant differences in the percentages of the respective cytokine-positive CD4+CD8+ double-positive T-cells between GPA and HC. Up-regulated genes of CD4+CD8+ double-positive T-cells in GPA were enriched within Kyoto Encyclopedia of Genes and Genomes (KEGG) pathways related to nuclear factor kappa-lightchain-enhancer of activated B cells signalling, toll-like receptor signalling, nucleotide-binding oligomerisation domain-like receptor signalling as well as major histocompatibility complex class-II antigen presentation.Employing a combined phenotypic and transcriptomic approach we disclosed a Th1/Th17 phenotype as well as innate and adaptive functions of CD4+CD8+ double-positive T-cells in GPA.

Objective To prepare a dermatologic addendum to the 2012 revised International Chapel Hill Consensus Conference Nomenclature of Vasculitides (CHCC2012) to address vasculitides affecting the skin (D-CHCC). The goal was to standardize the names and definitions for cutaneous vasculitis. Methods A nominal group technique with a facilitator was used to reach consensus on the D-CHCC nomenclature, using multiple face-to-face meetings, e-mail discussions, and teleconferences. Results Standardized names, definitions, and descriptions were adopted for cutaneous components of systemic vasculitides (e.g., cutaneous IgA vasculitis as a component of systemic IgA vasculitis), skin-limited variants of systemic vasculitides (e.g., skin-limited IgA vasculitis, drug-induced skin-limited antineutrophil cytoplasmic antibody–associated vasculitis), and cutaneous single-organ vasculitides that have no systemic counterparts (e.g., nodular vasculitis). Cutaneous vasculitides that were not included in the CHCC2012 nomenclature were introduced. Conclusion Standardized names and definitions are a prerequisite for developing validated classification and diagnostic criteria for cutaneous vasculitis. Accurate identification of specifically defined variants of systemic and skin-limited vasculitides requires knowledgeable integration of data from clinical, laboratory, and pathologic studies. This proposed nomenclature of vasculitides affecting the skin, the D-CHCC, provides a standard framework both for clinicians and for investigators.

Einleitung: Eine deutschsprachige Leitlinie zur Diagnostik und Therapie der ANCA-assoziierten Vaskulitiden sollte erstellt werden. Methoden: Eine Expertenkommission aus 13 Mitgliedern wurde von der DGRh mit der Erstellung der Leitlinie beauftragt. Für eine systematische Literaturrecherche wurden[zum vollständigen Text gelangen Sie über die oben angegebene URL], 45. Kongress der Deutschen Gesellschaft für Rheumatologie (DGRh), 31. Jahrestagung der Deutschen Gesellschaft für Orthopädische Rheumatologie (DGORh), 27. Jahrestagung der Gesellschaft für Kinder- und Jugendrheumatologie (GKJR)

Background There is growing evidence that nervous and immune system communicate with each other through soluble mediators.1 Immune cells such as neutrophils express muscarinic acetylcholine receptors (mAChR), which are neuroimmune receptors and highly prevalent in the nervous system.2 Aberrant neutrophil functioning plays an important role in various autoimmune diseases. Dysregulation of neutrophil immune responses such as oxidative burst and migration is one of the key mechanisms leading to tissue damage in autoimmune diseases.3 However, the impact of mAChR activation on neutrophils remains contradictory. Objectives We aimed to determine effects of muscarinic receptor activation on development and functions of neutrophils. Methods Neutrophils were isolated from peripheral blood of healthy donors by dextran sedimentation. After one hour in the absence or presence of the natural ligand acetylcholine (Ach) (10nM-100μM) or the muscarinic agonist oxotremorine-m (oxo-m) (10nM-100μM), neutrophil respiratory burst was analyzed by dihydrorhodamine (DHR) flow cytometry assay and migration assessed by transwell assay in response to N-formylmethionyl-leucyl-phenylalanine (fMLP). Cells that migrated were quantified by flow cytometry. To analyze the effects mAChR activation on the development of neutrophils, HL-60 cells were incubated in the presence of DMSO (1%), oxo-m (100μM) or DMSO plus oxo-m. After 6 days, cells were harvested and expression of maturation markers (CD15, CD63 and CD16) as well as mAChR (M1-M5) were measured by flow cytometry. Results We observed no effects of mAChR activation on the respiratory burst of neutrophils. However, both ACh and oxo-m inhibited neutrophil migration in a dose-dependent manner, but with peculiar differences. By increasing acetylcholine concentrations, we observed a reduction of neutrophil migration in a directly proportional manner. On the other hand, while the lowest dose (10nM) of oxo-m inhibited migration most effectively, the increase of oxo-m showed inversely proportional effects on neutrophil migration. Thus, we aimed to investigate, if the highest dose of oxo-m has a different effect on neutrophils ontogeny. In agreement with the results obtained with neutrophils, the incubation of HL-60 cells with the highest dose of oxo-m showed no effect on oxidative burst and migration and induced no changes in the expression of mAChRs (M1-M5), CD16 and CD63 in HL-60 cells. However, we observed that it resulted in significantly increased surface levels of the neutrophilic lineage marker CD15. Conclusions Our data indicate a differential activation of mAChR affecting different steps of neutrophil ontogeny. Considering this finding, abnormalities in the activation of muscarinic receptors as have been observed in autoimmune diseases might contribute to neutrophil dysfunction and need further investigation. References Blalock, J. E. The syntax of immune-neuroendocrine communication. Immunol Today 15, 504–511 (1994). Milara et al. Non-neuronal cholinergic system contributes to corticosteroid resistance in chronic obstructive pulmonary disease patients. Respiratory Research (2016) 17:145. Wright, H. L. et al. The multifactorial role of neutrophils in rheumatoid arthritis Nat Rev Rheumatol 10 593–601 (2014). Disclosure of Interest None declared

Break of tolerance driving autoimmune disease is initiated by a combination of predisposing genetic and environmental factors resulting in self-perpetuating chronic inflammation and tissue damage. Effector molecules and cells targeting tissues housing the inciting autoantigen(s) maintain tissue damage and the autoimmune response. Granulomatosis with polyangiitis (GPA, formerly Wegener9s granulomatosis) is a prototypical autoimmune disease characterized by extravascular necrotizing granulomatous inflammation and a systemic autoimmune vasculitis associated with anti-neutrophil cytoplasmic autoantibodies specific for the neutrophil- and monocyte-derived serine-protease proteinase 3 (PR3-ANCA). GPA is strongly associated with HLA-DPB1*0401 polymorphisms. Its association with the R620W variant of the PTPN22 gene has been linked to reduced immune-regulatory interleukin (IL)-10 transcription. Epidemiological studies and molecular analysis of immune cells suggests a multifactorial pathogenesis of GPA, in which a potentially metachronous and individually varying sequence of infectious agents contributes to the break of tolerance and chronic inflammation sustaining autoimmunity in GPA. Disclosure of Interest None declared

Background Endothelin-1 type A receptor (ETAR) antagonists (e.g. ambrisentan) are currently approved by the U.S Food and Drug administration, representing a well-tolerated treatment of pulmonary arterial hypertension (PAH)[1] for patients with connective tissue diseases such as systemic sclerosis (SSc). Noteworthy, increased numbers of infiltrating neutrophils have been associated with worse clinical outcome in PAH patients. In another context, several studies have reported that endothelin-1 and its receptor ETAR also play a central role in the development of tumour cell invasion and metastasis. However, the effects of ETAR antagonists on migration of neutrophils and tumour cells remain to be determined. Objectives The objective was to analyse the effects of two ETAR antagonists on migration of neutrophils and tumour cell lines. Methods The migratory ability of peripheral neutrophils from healthy donors (HD) and different tumour cell lines (myeloid leukaemia HL60 cells and human pancreatic adenocarcinoma COLO357 cells) was analysed in response to N-Formylmethionyl-leucyl-phenylalanine (FMLP) or Protease-activated receptor 2 (Par-2) agonist. Because it has been shown before [2], IgGs from HD and SSc patients were used as additional stimulus for migration. Neutrophils and HL60 cells were preincubated (1h) with sitaxentan or ambrisentan, respectively, before being tested for migration (1h) using the Transwell assay. COLO357 cells were incubated (48h) in the presence of sitaxentan and migration was tested in the Oris Pro Cell assay. Migration was analysed by automatic cell counting or digital photo analysis and a migration index was calculated. Results Sitaxentan and/or ambrisentan significantly blocked the migration of neutrophils and tumour cell lines. In more detail, neutrophil migration in response to FMLP, being set to 100%, was completely inhibited by sitaxentan (0,46%). Further, neutrophil migration in response to IgGs from HD and SSc patients was induced equally, again being set to 100%. In the presence of sitaxentan migration was reduced to 60%, respectively. In DMSO-differentiated HL60 cells the migratory capacity in response to FMLP (100%) was reduced to 66% by ambrisentan and to 14% by sitaxentan. Moreover, in the presence of sitaxentan and a Par-2 agonist the migratory ability of COLO357 cells was significantly decreased to 89% compared to Par-2 agonist only, being set to 100%. Conclusions Our results suggest a pivotal and non-redundant role of ETAR in cell migration, which needs further clarification in order to repurpose the use of ETAR inhibitors. Therapeutic switching of ETAR antagonists from PAH to cancer therapies is a promising adjuvant therapy. References Waxman AB. A review of sitaxsentan sodium in patients with pulmonary arterial hypertension. Vasc Health Risk Manag 2007;3:151–7. Kill A, Tabelin C, Undeutsch R, et al. Autoantibodies to angiotensin and endothelin receptors in systemic sclerosis induce cellular and systemic events associated with disease pathogenesis. Arthritis R&T, 2014, 16:R29. Disclosure of Interest None declared

La Presse Medicale - In Press.Proof corrected by the author Available online since samedi 2 mars 2013

Granulomatosis with polyangiitis (GPA) is a potentially life-threatening, rare disease. The etiology is unknown. GPA is histomorphologically characterized by extravascular necrotizing granulomatous inflammation and a systemic necrotizing vasculitis of small to medium-sized vessels. Clinically, a pulmonary-renal syndrome with pulmonary infiltrates, alveolar hemorrhage and a rapidly progressive glomerulonephritis is seen in about 80% of the cases with generalized disease. GPA is associated with proteinase 3-specific anti-neutrophil cytoplasmic autoantibodies (PR3-ANCA). Treatment is guided by severity of organ involvement and disease activity. Cytostatic immunosuppressants or the monoclonal anti-CD20 antibody rituximab are applied.

Vaskulitiden sind eine heterogene Gruppe von entzundlichen Erkrankungen der Blutgefase, die gleichermasen durch ihre Diversitat als auch durch eine Uberlappung klinischer und pathologischer Befunde und Manifestationen gekennzeichnet sind. Die Atiologie der meisten Vaskulitiden ist noch nicht geklart. Ihre Nomenklatur und Klassifikation stellt daher eine Herausforderung fur Internisten, Immunologen und Pathologen dar. Die revidierte Chapel Hill Consensus Conference (CHCC) 2012 Nomenklatur beinhaltet eine Aktualisierung der Namen und Definitionen der in der CHCC 1994 Nomenklatur genannten Vaskulitiden. Auserdem wurden 4 neue Kategorien eingefuhrt, um das Spektrum der Vaskulitiden umfassender abzubilden. Aufgrund des Nebenwirkungsspektrums von zytotoxisch wirksamen Immunsuppressiva besteht ein Bedarf an therapeutischen Alternativen, die eine effektivere und gezieltere Therapie bei gunstigerem Nebenwirkungsprofil ermoglichen. Erste Studienergebnisse zur Therapie von Vaskulitiden mit Biologicals lassen eine zukunftige Erweiterung der therapeutischen Optionen erwarten.

The nomenclature and classification of vasculitis has been difficult and controversial for many decades. This is problematic for both research on vasculitis as well as clinical care of patients with vasculitis. The first (1994) International Chapel Hill Consensus Conference on the Nomenclature of Systemic Vasculitides (CHCC) proposed names and definitions for the most common forms of vasculitis. Since then, there have been substantial advances in our understanding of vasculitis and changes in medical terminology. In addition, CHCC 1994 did not propose a nomenclature for some relatively common forms of vasculitis, such as vasculitis secondary to other diseases. To address these issues, a second International Chapel Hill Consensus Conference was held in 2012. The goals were to change names and definitions as appropriate, and add important categories of vasculitis not included in CHCC 1994. This overview summarizes the 2012 CHCC and points out the changes compared to the 1994 CHCC. Notable changes include the introduction of new terms such as granulomatosis with polyangiitis, eosinophilic granulomatosis with polyangiitis and IgA vasculitis, and the inclusion of categories for variable vessel vasculitis and secondary forms of vasculitis.

Expansion of circulating CD28- T-cells reminiscent of effector memory T-cells (T(EM)) has been reported in Wegener's granulomatosis (WG) recently. To investigate the role of T(EM) in WG, we analyzed the expression of the activating NK-receptor NKG2D and its ligand MIC on circulating T(EM) and in granulomatous lesions, respectively. NKG2D was anomalously expressed and preferentially detected on circulating CD4+CD28- T(EM) in WG. Compared to healthy controls, T(EM) display a more activated phenotype potentially favoring unbalanced proinflammatory responses in WG. Cluster-like formations of "Wegener's autoantigen" PR3 were surrounded by NKG2D+ and NKG2D-ligand MIC+ cells in WG-granulomata, but not in disease controls. Further, IL-15 - known to drive T(EM) differentiation and proliferation--was also expressed in WG-granulomata. Thus, through acquisition of NK-like "innate" properties, IL-15 stimulated NKG2D+ T(EM) could interact with MIC+ cells within WG-granulomata, thereby sustaining inflammation and autoimmunity and promoting self-perpetuating pathology in WG.

Defects in regulatory T (Treg) cells have been implicated in the pathogenesis of chronic inflammatory and autoimmune diseases, such as Wegener's granulomatosis (WG). This study aimed at evaluating numbers, phenotype and suppressive capacity of Treg cells in WG. Peripheral blood (PB) mononuclear cells from 22 WG-patients (17 active, 5 remission) and 22 sex- and age-matched healthy controls (HC) were examined for Treg cells by flow cytometry measuring CD4, CD25, transcription factor forkhead box P3 (FoxP3), chemokine receptor CCR4 and interferon receptor I (IFNRI). Suppressive function of CD4+CD25high Treg cells from 3 WG-patients and 3 HC was analysed using a carboxyfluoresceindiacetate-succinimidylester-based in vitro proliferation assay. Endonasal biopsies of 10 WG- and 5 sinusitis-patients were investigated for CD3+FoxP3+ cells, employing double immunohistochemistry. WG-patients displayed elevated numbers of CD4+CD25med T cells and of CD4+CD25high Treg cells. CD4+ T cells of WG-patients contained higher numbers of CCR4+ cells. However, CD4+CD25high Treg cells of WG-patients exhibited decreased numbers of cells co-expressing FoxP3 and CCR4. A low but significant increase of CD4+CD25highIFNRI+ Treg cells was detected in WG-patients. 9 days following stimulation with interferon (IFN)alpha + proteinase 3 (PR3), a reduced suppression of proliferation of responder T cells was observed for WG and proliferated CD4+CD25high Treg cells still showed downregulated co-expressions of FoxP3 and CCR4. Wegener's granuloma exhibited increased numbers of CD3+FoxP3+ cells. The results indicate upregulated numbers of Treg cells in PB and nasal mucosa as well as phenotypical and functional alterations of PB Treg cells in WG, some presumably mediated through PR3 and IFN-alpha.

Background: C-reactive protein (CRP) is a key molecule in inflammation and tissue homeostasis and is produced locally by renal tubular epithelial cells. Its significance of expression in contrast to expression of cytotoxic T cell (CTL) markers remains to be elucidated. Methods: By means of real-time PCR, we determined mRNA levels of CRP in 66 renal allograft biopsies with acute allograft failure and in 34 biopsies with chronic dysfunction. Results were compared to expression of CTL components (perforin, granzyme B) and were correlated with histologic diagnoses and outcome. Results: CTL markers were found in most biopsies, and thus were not specific for particular histologies, although expression levels increased significantly with Banff rejection grades. Expression of CRP was highly specific for rejection episodes in acute failure (p < 0.0001) as well as for transplant glomerulopathy or de novo/recurrent glomerulonephritis in chronic failure (p < 0.005). Finally, the presence of CRP expression in renal allografts was associated with a deteriorating 1-year graft function in acute (p < 0.01) as well as chronic allograft dysfunction (p = 0.077). Conclusions: Our data suggest local CRP expression of kidney transplants as an indicator for pathologic entities associated with unfavorable outcomes in the early and late course of kidney transplants.

Drugs and infections may induce antineutrophil cytoplasmic antibodies (ANCA) and vasculitic manifestations mimicking ANCA-associated vasculitides (AAV) and mechanisms relevant in their pathogenesis. This review summarizes the most recent findings in this field.Drug-induced and infection-induced proteinase 3 (PR3)-ANCA and myeloperoxidase (MPO)-ANCA may be associated with a vasculitis clinically resembling AAV. Mechanisms relevant for the break of tolerance and induction of ANCA (e.g. danger signals, superantigens, neutrophil extracellular traps, protease-activated receptor-2, IL-17 cells) may be shared to some extent between drug-induced and infection-induced ANCA-positive vasculitis and AAV, especially with regard to the potential role of infection in Wegener's granulomatosis. Differences in immunopathology, clinical presentation, and functional aspects of ANCA help to distinguish drug-induced and infection-induced ANCA-positive vasculitis from AAV, and present new avenues for future research in this field.Medications and infections, which induce PR3-ANCA and MPO-ANCA, have to be considered in the differential diagnosis of primary AAV. Moreover, there is clinical and experimental evidence for an association between certain drugs and infections with ANCA-production. Analysis of ANCA-induction in such conditions also sheds new light on our understanding of immune mechanisms relevant in the break of tolerance and ANCA-production in AAV.

Adult-onset Still's disease (AoSD), Schnitzler syndrome, and cases of adult-onset autoinflammatory syndromes [10-15% of cases of familial Mediterranean fever (FMF) and tumor necrosis factor (TNF) receptor-associated periodic syndrome (TRAPS)] are characterized by a genetic predisposition, with increased interleukin (IL)-1beta and IL-18 production and TNF-alpha signaling, respectively. As a result, periodic fever and inflammation at barrier tissues (synovial tissues, serous membranes, and the skin) are encountered in such patients. Pathophysiological insights into these diseases have renewed interest in research on IL-1beta in rheumatic diseases and have opened new therapeutic avenues. Recently published studies have shown that patients with Schnitzler syndrome, methotrexate-refractory AoSD, and colchicine-refractory FMF or contraindications to colchicines in FMF respond well to treatment with the soluble IL-1 receptor antagonist anakinra. For TRAPS patients, the p75 TNF-alpha receptor/Fc-IgG1 fusion protein etanercept is the treatment of first choice.