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Endothelial NF-κB Blockade Abrogates ANCA-Induced GN.

ABSTRACT: ANCA-associated vasculitis (AAV) is a highly inflammatory condition in which ANCA-activated neutrophils interact with the endothelium, resulting in necrotizing vasculitis. We tested the hypothesis that endothelial NF-κB mediates necrotizing crescentic GN (NCGN) and provides a specific treatment target. Reanalysis of kidneys from previously examined murine NCGN disease models revealed NF-κB activation in affected kidneys, mostly as a p50/p65 heterodimer, and increased renal expression of NF-κB-dependent tumor necrosis factor α (TNF-α). NF-κB activation positively correlated with crescent formation, and nuclear phospho-p65 staining showed NF-κB activation within CD31-expressing endothelial cells (ECs) in affected glomeruli. Therefore, we studied the effect of ANCA on NF-κB activation in neutrophil/EC cocultures in vitro ANCA did not activate NF-κB in primed human neutrophils, but ANCA-stimulated primed neutrophils activated NF-κB in ECs, at least in part via TNF-α release. This effect increased endothelial gene transcription and protein production of NF-κB-regulated interleukin-8. Moreover, upregulation of endothelial NF-κB promoted neutrophil adhesion to EC monolayers, an effect that was inhibited by a specific IKKβ inhibitor. In a murine NCGN model, prophylactic application of E-selectin-targeted immunoliposomes packed with p65 siRNA to downregulate endothelial NF-κB significantly reduced urine abnormalities, renal myeloid cell influx, and NCGN. Increased glomerular endothelial phospho-p65 staining in patients with AAV indicated that NF-κB is activated in human NCGN also. We suggest that ANCA-stimulated neutrophils activate endothelial NF-κB, which contributes to NCGN and provides a potential therapeutic target in AAV.

SUBMITTER: Choi M

PROVIDER: S-EPMC5661273 | biostudies-literature | 2017 Nov

REPOSITORIES: biostudies-literature

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Endothelial NF-κB Blockade Abrogates ANCA-Induced GN.

Choi Mira M Schreiber Adrian A Eulenberg-Gustavus Claudia C Scheidereit Claus C Kamps Jan J Kettritz Ralph R

Journal of the American Society of Nephrology : JASN 20170707 11

ANCA-associated vasculitis (AAV) is a highly inflammatory condition in which ANCA-activated neutrophils interact with the endothelium, resulting in necrotizing vasculitis. We tested the hypothesis that endothelial NF-κB mediates necrotizing crescentic GN (NCGN) and provides a specific treatment target. Reanalysis of kidneys from previously examined murine NCGN disease models revealed NF-κB activation in affected kidneys, mostly as a p50/p65 heterodimer, and increased renal expressi ...[more]

PMID: 28687535

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Project description:BackgroundMyeloperoxidase released after neutrophil and monocyte activation can generate reactive oxygen species, leading to host tissue damage. Extracellular glomerular myeloperoxidase deposition, seen in ANCA-associated vasculitis, may enhance crescentic GN through antigen-specific T and B cell activation. Myeloperoxidase-deficient animals have attenuated GN early on, but augmented T cell responses. We investigated the effect of myeloperoxidase inhibition, using the myeloperoxidase inhibitor AZM198, to understand its potential role in treating crescentic GN.MethodsWe evaluated renal biopsy samples from patients with various forms of crescentic GN for myeloperoxidase and neutrophils, measured serum myeloperoxidase concentration in patients with ANCA-associated vasculitis and controls, and assessed neutrophil extracellular trap formation, reactive oxygen species production, and neutrophil degranulation in ANCA-stimulated neutrophils in the absence and presence of AZM198. We also tested the effect of AZM198 on ANCA-stimulated neutrophil-mediated endothelial cell damage in vitro, as well as on crescentic GN severity and antigen-specific T cell reactivity in the murine model of nephrotoxic nephritis.ResultsAll biopsy specimens with crescentic GN had extracellular glomerular myeloperoxidase deposition that correlated significantly with eGFR and crescent formation. In vitro, AZM198 led to a significant reduction in neutrophil extracellular trap formation, reactive oxygen species production, and released human neutrophil peptide levels, and attenuated neutrophil-mediated endothelial cell damage. In vivo, delayed AZM198 treatment significantly reduced proteinuria, glomerular thrombosis, serum creatinine, and glomerular macrophage infiltration, without increasing adaptive T cell responses.ConclusionsMyeloperoxidase inhibition reduced neutrophil degranulation and neutrophil-mediated endothelial cell damage in patients with ANCA-associated vasculitis. In preclinical crescentic GN, delayed myeloperoxidase inhibition suppressed kidney damage without augmenting adaptive immune responses, suggesting it might offer a novel adjunctive therapeutic approach in crescentic GN.

Project description:BackgroundNeutrophil gelatinase-associated lipocalin (NGAL) is a diagnostic marker of intrinsic kidney injury produced by damaged renal cells and by neutrophils. ANCA-associated vasculitis features necrotizing crescentic GN (NCGN), and ANCA-activated neutrophils contribute to NCGN. Whether NGAL plays a mechanistic role in ANCA-associated vasculitis is unknown.MethodsWe measured NGAL in patients with ANCA-associated vasculitis and mice with anti-myeloperoxidase (anti-MPO) antibody-induced NCGN. We compared kidney histology, neutrophil functions, T cell proliferation and polarization, renal infiltrating cells, and cytokines in wild-type and NGAL-deficient chimeric mice with anti-MPO antibody-induced NCGN. To assess the role of TH17 immunity, we transplanted irradiated MPO-immunized MPO-deficient mice with bone marrow from either wild-type or NGAL-deficient mice; we also transplanted irradiated MPO-immunized MPO/IL-17A double-deficient mice with bone marrow from either IL-17A-deficient or NGAL/IL-17A double-deficient mice.ResultsMice and patients with active ANCA-associated vasculitis demonstrated strongly increased serum and urinary NGAL levels. ANCA-stimulated neutrophils released NGAL. Mice with NGAL-deficient bone marrow developed worsened MPO-ANCA-induced NCGN. Intrinsic neutrophil functions were similar in NGAL-deficient and wild-type neutrophils, whereas T cell immunity was increased in chimeric mice with NGAL-deficient neutrophils with more renal infiltrating TH17 cells. NGAL-expressing neutrophils and CD3+ T cells were in close proximity in kidney and spleen. CD4+ T cells showed no intrinsic difference in proliferation and polarization in vitro, whereas iron siderophore-loaded NGAL suppressed TH17 polarization. We found significantly attenuated NCGN in IL-17A-deficient chimeras compared with MPO-deficient mice receiving wild-type bone marrow, as well as in NGAL/IL-17A-deficient chimeras compared with NGAL-deficient chimeras.ConclusionsOur findings support that bone marrow-derived, presumably neutrophil, NGAL protects from ANCA-induced NCGN by downregulating TH17 immunity.

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Endothelial NF-κB Blockade Abrogates ANCA-Induced GN.

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Endothelial NF-<i>κ</i>B Blockade Abrogates ANCA-Induced GN.

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