Changes in the composition of the upper respiratory tract microbial community in granulomatosis with polyangiitis.

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.

Circulating CD4+CD8+ double-positive T-cells display features of innate and adaptive immune function in granulomatosis with polyangiitis.

OBJECTIVES: To examine functional features of CD4+CD8+ double-positive T-cells in patients with granulomatosis with polyangiitis (GPA) using phenotypic and transcriptomic analysis. METHODS: 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. RESULTS: 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. CONCLUSIONS: 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.

Environmental factor and inflammation-driven alteration of the total peripheral T-cell compartment in granulomatosis with polyangiitis.

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.

Pathogenetic and Clinical Aspects of Anti-Neutrophil Cytoplasmic Autoantibody-Associated Vasculitides.

Anti-neutrophil cytoplasmic autoantibodies (ANCA) targeting proteinase 3 (PR3) and myeloperoxidase expressed by innate immune cells (neutrophils and monocytes) are salient diagnostic and pathogenic features of small vessel vasculitis, comprising granulomatosis with polyangiitis (GPA), microscopic polyangiitis, and eosinophilic GPA. Genetic studies suggest that ANCA-associated vasculitides (AAV) constitute separate diseases, which share common immunological and pathological features, but are otherwise heterogeneous. The successful therapeutic use of anti-CD20 antibodies emphasizes the prominent role of ANCA and possibly other autoantibodies in the pathogenesis of AAV. However, to elucidate causal effects in AAV, a better understanding of the complex interplay leading to the emergence of B lymphocytes that produce pathogenic ANCA remains a challenge. Different scenarios seem possible; e.g., the break of tolerance induced by a shift from non-pathogenic toward pathogenic autoantigen epitopes in inflamed tissue. This review gives a brief overview on current knowledge about genetic and epigenetic factors, barrier dysfunction and chronic non-resolving inflammation, necro-inflammatory auto-amplification of cellular death and inflammation, altered autoantigen presentation, alternative complement pathway activation, alterations within peripheral and inflamed tissue-residing T- and B-cell populations, ectopic lymphoid tissue neoformation, the characterization of PR3-specific T-cells, properties of ANCA, links between autoimmune disease and infection-triggered pathology, and animal models in AAV.

[Granulomatosis with polyangiitis]. / Granulomatose mit Polyangiitis.

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.

Proteinase 3 on apoptotic cells disrupts immune silencing in autoimmune vasculitis.

Granulomatosis with polyangiitis (GPA) is a systemic necrotizing vasculitis that is associated with granulomatous inflammation and the presence of anti-neutrophil cytoplasmic antibodies (ANCAs) directed against proteinase 3 (PR3). We previously determined that PR3 on the surface of apoptotic neutrophils interferes with induction of antiinflammatory mechanisms following phagocytosis of these cells by macrophages. Here, we demonstrate that enzymatically active membrane-associated PR3 on apoptotic cells triggered secretion of inflammatory cytokines, including granulocyte CSF (G-CSF) and chemokines. This response required the IL-1R1/MyD88 signaling pathway and was dependent on the synthesis of NO, as macrophages from animals lacking these pathways did not exhibit a PR3-associated proinflammatory response. The PR3-induced microenvironment facilitated recruitment of inflammatory cells, such as macrophages, plasmacytoid DCs (pDCs), and neutrophils, which were observed in close proximity within granulomatous lesions in the lungs of GPA patients. In different murine models of apoptotic cell injection, the PR3-induced microenvironment instructed pDC-driven Th9/Th2 cell generation. Concomitant injection of anti-PR3 ANCAs with PR3-expressing apoptotic cells induced a Th17 response, revealing a GPA-specific mechanism of immune polarization. Accordingly, circulating CD4+ T cells from GPA patients had a skewed distribution of Th9/Th2/Th17. These results reveal that PR3 disrupts immune silencing associated with clearance of apoptotic neutrophils and provide insight into how PR3 and PR3-targeting ANCAs promote GPA pathophysiology.