Kinin B1 Receptor Is Important in the Pathogenesis of Myeloperoxidase-Specific ANCA GN.

BACKGROUND: Myeloperoxidase-specific ANCA (MPO-ANCA) are implicated in the pathogenesis of vasculitis and GN. Kinins play a major role during acute inflammation by regulating vasodilatation and vascular permeability and by modulating adhesion and migration of leukocytes. Kinin system activation occurs in patients with ANCA vasculitis. Previous studies in animal models of GN and sclerosing kidney diseases have demonstrated protective effects of bradykinin receptor 1 (B1R) blockade via interference with myeloid cell trafficking. METHODS: To investigate the role of B1R in a murine model of MPO-ANCA GN, we evaluated effects of B1R genetic ablation and pharmacologic blockade. We used bone marrow chimeric mice to determine the role of B1R in bone marrow-derived cells (leukocytes) versus nonbone marrow-derived cells. We elucidated mechanisms of B1R effects using in vitro assays for MPO-ANCA-induced neutrophil activation, endothelial adherence, endothelial transmigration, and neutrophil adhesion molecule surface display. RESULTS: B1R deficiency or blockade prevented or markedly reduced ANCA-induced glomerular crescents, necrosis, and leukocyte influx in mice. B1R was not required for in vitro MPO-ANCA-induced neutrophil activation. Leukocyte B1R deficiency, but not endothelial B1R deficiency, decreased glomerular neutrophil infiltration induced by MPO-ANCA in vivo. B1R enhanced ANCA-induced neutrophil endothelial adhesion and transmigration in vitro. ANCA-activated neutrophils exhibited changes in Mac-1 and LFA-1, important regulators of neutrophil endothelial adhesion and transmigration: ANCA-activated neutrophils increased surface expression of Mac-1 and increased shedding of LFA-1, whereas B1R blockade reduced these effects. CONCLUSIONS: The leukocyte B1R plays a critical role in the pathogenesis of MPO-ANCA-induced GN in a mouse model by modulating neutrophil-endothelial interaction. B1R blockade may have potential as a therapy for ANCA GN and vasculitis.

ANCA autoantigen gene expression highlights neutrophil heterogeneity where expression in normal-density neutrophils correlates with ANCA-induced activation.

ANCA vasculitis is an autoimmune disease with increased expression of the autoantigen genes, myeloperoxidase (MPO) and proteinase 3 (PRTN3), but the origin and significance of expression is less distinct. To clarify this, we measured MPO and PRTN3 messenger RNA in monocytes, normal-density neutrophils, and in enriched leukocytes from peripheral blood mononuclear cells. Increased autoantigen gene expression was detected in normal-density neutrophils and enriched leukocytes from patients during active disease compared to healthy individuals, with the largest difference in enriched leukocytes. RNA-seq of enriched leukocytes comparing active-remission pairs identified a gene signature for low-density neutrophils. Cell sorting revealed low-density neutrophils contained mature and immature neutrophils depending on the presence or absence of CD10. Both populations contributed to autoantigen expression but the frequency of immature cells in low-density neutrophils did not correlate with low-density neutrophil MPO or PRTN3 expression. Low-density neutrophils were refractory to MPO-ANCA induced oxidative burst, suggesting an alternative role for low-density neutrophils in ANCA vasculitis pathogenesis. In contrast, normal-density neutrophils were activated by MPO-ANCA and monoclonal anti-PR3 antibody. Normal-density neutrophil activation correlated with MPO and PRTN3 mRNA. Increased autoantigen gene expression originating from the mature low-density and normal-density neutrophils suggests transcriptional dysregulation is a hallmark of ANCA vasculitis. Thus, the correlation between autoantigen gene expression and antibody-mediated normal-density neutrophil activation connects autoantigen gene expression with disease pathogenesis.

Common E-Cigarette Flavoring Chemicals Impair Neutrophil Phagocytosis and Oxidative Burst.

E-cigarette flavorings have not been thoroughly evaluated for inhalational toxicity. We have shown that the flavoring chemical cinnamaldehyde impairs human neutrophils, macrophages, and natural killer cells. Here we investigated the effects of other common e-liquid flavoring chemicals on phagocytosis and oxidative burst in neutrophils. We demonstrate that cinnamaldehyde and ethyl vanillin dose-dependently decrease oxidative burst and that benzaldehyde and benzaldehyde propylene glycol acetal dose-dependently impair phagocytosis. Isoamyl acetate did not affect either measure of neutrophil function. These data suggest that inhaling aromatic aldehydic flavoring chemicals, such as cinnamaldehyde, benzaldehyde, benzaldehyde propylene glycol acetal, or ethyl vanillin, could impair neutrophil function.

Mechanisms of vascular damage in ANCA vasculitis.

The discovery of anti-neutrophil cytoplasmic antibodies (ANCA) and their antigenic targets, myeloperoxidase (MPO) and proteinase 3 (PR3), has led to further understanding as to the pathophysiologic processes that underlie vascular and tissue damage in ANCA vasculitis. ANCA trigger neutrophil activation leading to vascular damage in ANCA vasculitis. However, decades of study have determined that neutrophil activation alone is not sufficient to cause disease. Inflammatory stimuli are drivers of ANCA autoantigen expression and ANCA production. Certain infections or bacterial peptides may be crucial players in the initial steps of ANCA immunopathogenesis. Genetic and epigenetic alterations of gene encoding for MPO and PR3 provide additional disturbances to the immune homeostasis which provide a substrate for pathogenic ANCA formation from an adaptive immune system predisposed to autoreactivity. Promoted by inflammatory cytokines, ANCA binding leads to neutrophil activation, a process characterized by conformational changes, production and release of cytotoxic substances, and alternative complement pathway activation, thus creating an intense inflammatory milieu. This cascade of events perpetuates a vicious cycle of further inflammatory cell recruitment and activation, culminating in tissue necrosis. Our understanding of the pathogenic process in ANCA vasculitis paves the way for the development of therapies targeting crucial steps in this process. The greater appreciation of the role for complement, monocytes, and the adaptive immune system has already led to novel complement blockers and is poised to lead to further innovations which will allow for tailored antigen- or cell-specific immunotherapy targeting the autoimmune process without exposure to undue risks or toxicities.

The frequency of Treg subsets distinguishes disease activity in ANCA vasculitis.

Objectives: T regulatory cells (Tregs) are a heterogeneous group of immunoregulatory cells that dampen self-harming immune responses and prevent the development of autoimmune diseases. In anti-neutrophil cytoplasmic autoantibody (ANCA) vasculitis, Tregs possess diminished suppressive capacity, which has been attributed to the expression of a FOXP3 splice-variant lacking exon 2 in T cells (FOXP3Δ2 CD4+ T cells). However, the suppressive capacity of Tregs varies between subsets. We evaluated the frequency of Treg subsets in ANCA vasculitis as a potential explanation for diminished suppressive capacity. Methods: We developed a custom mass cytometry panel and performed deep immune profiling of Tregs in healthy controls, patients with active disease and in remission. Using these data, we performed multidimensional reduction and discriminant analysis to identify associations between Treg subsets and disease activity. Results: Total Tregs were expanded in ANCA vasculitis, which was associated with remission and the administration of rituximab and/or prednisone. The frequency of FOXP3Δ2 CD4+ T cells did not distinguish disease activity and this population had high expression levels of CD127 and lacked both CD25 and Helios, suggesting that they are not conventional Tregs. The frequency of CXCR3+, CD103+ and CCR7+ Tregs distinguished disease activity, and the combination of the frequency of these three Treg subsets segregated active patients from patients in remission and healthy controls. From these three subsets, the frequency of CXCR3+ Tregs distinguished patients with active disease with renal involvement. Conclusion: Treg heterogeneity can discriminate disease activity and should be explored as a biomarker of disease activity in ANCA vasculitis.