Therapeutic Myeloperoxidase Inhibition Attenuates Neutrophil Activation, ANCA-Mediated Endothelial Damage, and Crescentic GN

Abstract

Background: Myeloperoxidase 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.

Methods: We 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.

Results: All 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.

Conclusions: Myeloperoxidase 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.

Keywords: ANCA; glomerular endothelial cells; glomerulonephritis; myeloperoxidase; neutrophils; vasculitis.

Graphical abstract

Figure 1.

Serum MPO levels are elevated in patients with active AAV and are reduced when disease is in remission. Median (interquartile range) plasma concentration of circulating MPO in (A) MPO (n=13) and PR3 (n=14) AAV ANCA subtypes, renovascular disease (RVD) controls (n=5), and healthy controls (HC; n=10); and (B) active PR3-ANCA AAV and PR3-ANCA AAV in remission (n=58). Nonparametric Kruskal–Wallis test and Dunn multiple comparison post-test, Wilcoxon test *P<0.05; **P<0.01; ***P<0.001.

Figure 2.

Extracellular (non-leukocyte associated) MPO deposition is seen in the renal biopsies of patients with diverse forms of crescentic GN. Immunohistochemistry for MPO (brown) and CD15 (red) in inflamed glomeruli from patients with CGN (all ×60 magnification) due to (A) PR3-ANCA and IgG control (bottom left), (B) MPO-ANCA, (C) ANCA-negative disease, and (D) crescentic IgA nephropathy. Extracellular (extraleukocyte) deposition along capillary walls can be seen in various areas (highlighted by yellow arrows). Negative control staining with isotype IgG for (E) MPO and (F) CD15. Scale bars are shown in the right corner of each image.

Figure 3.

Total (neutrophil-associated and extracellular) whole kidney and glomerular as well as extraleukocyte glomerular MPO deposition alone correlates with histological and clinical disease severity in patients with diverse forms of CGN (MPO-ANCA n=5, crescentic IgA n=4, SLE n=4, PR3-ANCA n=3, ANCA-negative n=2). Percentage of total whole kidney (n=18) and glomerular intra- and extraleukocyte MPO deposition (n=11) (percentage of MPO-stained area per whole kidney section or glomerulus) with eGFR (ml/min per 1.73 m²) (A, C, and E, respectively) and percentage of active cellular crescents (B, D, and F, respectively). Nonparametric Spearman rank correlation analysis, *P<0.05; **P<0.01; ***P<0.001.

Figure 4.

MPO Inhibition with AZM198 reduces ROS production, neutrophil degranulation and NET formation in vitro. (A) Rhodamine 123 (FITC) MFI expression in TNFα-primed/PR3-ANCA–stimulated neutrophils analyzed by flow cytometry (n=7; four healthy controls, three patients with PR3-ANCA). (B) HNP 1–3 (pg/ml) release in the supernatants from TNFα-primed/PR3-ANCA–stimulated neutrophils (n=8; five healthy controls, three patients with PR3-ANCA). (C) Quantification of NET formation in TNFα-primed/PR3-ANCA–stimulated neutrophils in patients with active AAV (n=3) and healthy controls (n=5); Friedman test, *P<0.05; **P<0.01; ***P<0.001. (D–F) NET formation in unstimulated neutrophils (D) or in the absence (E) or presence (F) of AZM198 (10 μM) after 1 hour neutrophil stimulation with TNFα/PR3-ANCA from a healthy control. Extracellular DNA and permeable cells (Sytox) are shown in green. Nuclei are stained blue with DAPI. NET index was defined as the ratio between the cumulative Sytox Green–stained area, corrected for the number of imaged neutrophils.

Figure 5.

PR3-ANCA stimulated PMNs induce EC damage that is reduced by pharmacological MPO inhibition with AZM198. (A) Median (interquartile range) BrdU release and (B) vWf release in the supernatants of EC/PMN coculture. BrdU-labeled ECs were primed with TNFα for 18 hours. After rinsing to remove all traces of TNFα ECs were cultured without or with inclusion of PMN in the absence or presence of TNFα/PR3-ANCA, and with AZM198 or DNAse for a further 18 hours, after which vWf release and BrdU-labeled DNA fragments from ECs was analyzed; Friedman test **P<0.01; ***P<0.001. Merged microscopic images of TNFα-primed ECs in monoculture (C) or coculture (D–F) with unstimulated PMN (D), TNFα/PR3-ANCA–stimulated PMNs in the absence (E) or presence (F) of AZM198 (10 μM). ECs are stained with vascular endothelial (VE) cadherin (red), PMNs are stained with MPO (green), and nuclei are stained with DAPI (blue).

Figure 6.

Delayed MPO inhibition with AZM198 reduces glomerular inflammation in neprotoxic nephritis (NTN). Median (interquartile range) (A) proteinuria, (B) plasma creatinine, and (C) glomerular thrombosis score in mice with CGN secondary to NTN treated with vehicle (n=14) or AZM198 dosed at two different doses, 400 μmol/kg (n=7) and 133 μmol/kg (n=8, but only six were analyzed for urine protein). Kruskal–Wallis, *P<0.05; ***P<0.001. Periodic acid–Schiff stain showing glomerular thrombosis in kidney sections of mice with NTN treated with (D) vehicle or (E) MPOi at 133 μmol/kg (both ×20 magnification).

Figure 7.

MPO inhibition with AZM198 reduces glomerular MPO deposition as well as macrophage and T cell infiltration in nephrotoxic nephritis with no significant effect on neutrophil recruitment. Median (interquartile range) glomerular CTCF and immunofluorescence staining for (A, C, and D) F4/80-expressing macrophages, (B, E, and F) MPO, and (G) glomerular CD4-positive cells in the glomeruli of mice with NTN treated with (C and E) vehicle or (D and F) MPOi at 133 μmol/kg. Mann–Whitney test, *P<0.05; **P<0.01. (H) Effect of MPOi on glomerular neutrophil accumulation: mice were dosed with either vehicle (n=4) or MPOi (n=4) twice a day at 133 μmol/kg for 48 hours after the injection of NTS (12 hours after the last dosing).There is no significant effect on neutrophil accumulation at 2 hours after NTS injection; yellow lines outline glomeruli.

Figure 8.

MPO inhibition with AZM198 did not increase cellular and humoral responses in nephrotoxic nephritis (A–D): (A) median (interquartile range) frequency of CD44^high CD62L^low CD4⁺ splenocytes from mice with NTN (n=8 per group). (B–D) Effect of MPO inhibition on serum humoral immune responses to sheep globulin: median (IQR) IgG subclass concentrations IgG1, IgG2b, and IgG3, respectively (n=8 per group). (E and F) Antigen-specific T cell responses using adoptive transfer of DO11.10 lymphocytes into OVA-immunized mice: median (interquartile range) frequency of CD44^highCD4⁺ cells (E) and CD44^high KJ⁺ CD4⁺ cells (F). Mann–Whitney test, P=NS.