Peptidylarginine Deiminase Inhibitor Suppresses Neutrophil Extracellular Trap Formation and MPO-ANCA Production

Abstract

Myeloperoxidase-antineutrophil cytoplasmic antibody (MPO-ANCA)-associated vasculitis is a systemic small-vessel vasculitis, wherein, MPO-ANCA plays a critical role in the pathogenesis. Neutrophil extracellular traps (NETs) released from activated neutrophils are composed of extracellular web-like DNA and antimicrobial proteins, including MPO. Diverse stimuli, such as phorbol myristate acetate (PMA) and ligands of toll-like receptors (TLR), induce NETs. Although TLR-mediated NET formation can occur with preservation of living neutrophilic functions (called vital NETosis), PMA-stimulated neutrophils undergo cell death with NET formation (called suicidal NETosis). In the process of suicidal NETosis, histones are citrullinated by peptidylarginine deiminase 4 (PAD4). Since this step is necessary for decondensation of DNA, PAD4 plays a pivotal role in suicidal NETosis. Although NETs are essential for elimination of microorganisms, excessive formation of NETs has been suggested to be implicated in MPO-ANCA production. This study aimed to determine if pan-PAD inhibitors could suppress MPO-ANCA production in vivo. At first, NETs were induced in peripheral blood neutrophils derived from healthy donors (1 × 10(6)/ml) by stimulation with 20 nM PMA with or without 20 μM propylthiouracil (PTU), an anti-thyroid drug. We then determined that the in vitro NET formation was inhibited completely by 200 μM Cl-amidine, a pan-PAD inhibitor. Next, we established mouse models with MPO-ANCA production. BALB/c mice were given intraperitoneal (i.p.) injection of PMA (50 ng at days 0 and 7) and oral PTU (2.5 mg/day) for 2 weeks. These mice were divided into two groups; the first group was given daily i.p. injection of PBS (200 μl/day) (n = 13) and the other group with daily i.p. injection of Cl-amidine (0.3 mg/200 μl PBS/day) (n = 7). Two weeks later, citrullination as an indicator of NET formation in the peritoneum and serum MPO-ANCA titer was compared between the two groups. Results demonstrated that citrullination in the peritoneum was significantly reduced in the Cl-amidine-treated mice compared with the vehicle-injected control mice (38% reduction). Additionally, the serum MPO-ANCA titer of the Cl-amidine-treated mice (32.3 ± 31.0 ng/ml) was significantly lower than that in the vehicle-injected mice (132.1 ± 41.6 ng/ml). The collective findings indicate that excessive formation of NETs may be implicated in MPO-ANCA production in vivo.

Keywords: MPO-ANCA; MPO-ANCA-associated vasculitis; neutrophil extracellular trap; peptidylarginine deiminase 4; peptidylarginine deiminase inhibitor.

Figure 1

Effect of Cl-amidine on NET induction in vitro. Human peripheral blood neutrophils were seeded in wells of 4-well chamber slides (1 × 10⁶/ml). After incubation for 30 min at 37°C, the cells were exposed to 0 or 20 nM PMA with or without 20 μM PTU. Fifteen minutes prior to PMA/PTU administration, 200 μM Cl-amidine was added alternately into the wells. After 2 h of incubation at 37°C, the medium containing the reagents was removed, and the remaining cells were washed with PBS followed by fixation with 4% paraformaldehyde for 15 min. Thereafter, immunofluorescent staining for citrullinated histone 3 was carried out followed by mounting with the DAPI-containing solution. Representative photos (A). Red, citrullinated histone 3; Blue: DNA (original magnification: ×400). Quantification of NET formation (B). NET formation was quantified by counting the citrullinated histone 3-positive cells per × 100 power field of view. Data from five random fields of view (×100) were subjected to quantitative analysis. **p < 0.01 in Mann–Whitney U-test.

Figure 2

Establishment of mouse models with MPO-ANCA production. Experimental protocol (A). BALB/c, NZW, B6/N, B6/J, and DBA mice (14-week-old female) were given i.p. injection of PMA (50 ng at days 0 and 7) and oral PTU (2.5 mg/day) for 4 weeks (n = 5/strain, p.o.: Per Os). Mouse urine was collected during the last 24 h using metabolic cages, and then hematuria was assessed by dipsticks. Blood samples were obtained at days 14 and 28, and then the concentrations of BUN and Cr were determined. The lungs, kidneys, and peritoneal tissues were obtained at day 28. Serum titers of MPO-ANCA determined by ELISA were shown with the results of the urinalysis and concentrations of Cr in the serum (B). The normal limit of serum Cr level is 0.4 mg/dl.

Figure 3

Effect of Cl-amidine on citrullination and MPO-ANCA production in vivo. Experimental protocol (A). BALB/c mice (14-week-old female) were given i.p. injection of PMA (50 ng at days 0 and 7) and oral PTU (2.5 mg/day) for 2 weeks (p.o.: Per Os). These mice were divided into two groups. The first group of mice was given daily i.p. injection of PBS (200 μl/day) (n = 13). The other group was given daily i.p. injection of Cl-amidine (0.3 mg/200 μl PBS/day) (n = 7). Mouse urine was collected during the last 24 h using metabolic cages. Blood and tissue samples were obtained at day 14. NET formation in peritoneal tissues (B). The formalin-fixed paraffin-embedded sections of peritoneal tissues were subjected to immunohistochemistry for citrullinated histone 3. Representative photos among five random fields of view (×100) were shown. NET induction was quantified by calculating the rate of citrullinated histone 3-positive cells in polymorphonuclear cells in the five random fields of view. *p < 0.05 in Mann–Whitney U-test. Serum titers of MPO-ANCA determined by ELISA (C). **p < 0.01 in Mann–Whitney U-test.