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. 2015 Dec;74(12):2199-206.
doi: 10.1136/annrheumdis-2014-205365. Epub 2014 Aug 7.

Peptidylarginine deiminase inhibition disrupts NET formation and protects against kidney, skin and vascular disease in lupus-prone MRL/lpr mice

Jason S Knight  1 , Venkataraman Subramanian  2 , Alexander A O'Dell  1 , Srilakshmi Yalavarthi  1 , Wenpu Zhao  3 , Carolyne K Smith  3 , Jeffrey B Hodgin  4 , Paul R Thompson  2 , Mariana J Kaplan  3
Affiliations

Peptidylarginine deiminase inhibition disrupts NET formation and protects against kidney, skin and vascular disease in lupus-prone MRL/lpr mice

Jason S Knight et al. Ann Rheum Dis. 2015 Dec.

Abstract

Objectives: An imbalance between neutrophil extracellular trap (NET) formation and degradation has been described in systemic lupus erythematosus (SLE), potentially contributing to autoantigen externalisation, type I interferon synthesis and endothelial damage. We have demonstrated that peptidylarginine deiminase (PAD) inhibition reduces NET formation and protects against lupus-related vascular damage in the New Zealand Mixed model of lupus. However, another strategy for inhibiting NETs--knockout of NOX2--accelerates lupus in a different murine model, MRL/lpr. Here, we test the effects of PAD inhibition on MRL/lpr mice in order to clarify whether some NET inhibitory pathways may be consistently therapeutic across models of SLE.

Methods: NET formation and autoantibodies to NETs were characterised in lupus-prone MRL/lpr mice. MRL/lpr mice were also treated with two different PAD inhibitors, Cl-amidine and the newly described BB-Cl-amidine. NET formation, endothelial function, interferon signature, nephritis and skin disease were examined in treated mice.

Results: Neutrophils from MRL/lpr mice demonstrate accelerated NET formation compared with controls. MRL/lpr mice also form autoantibodies to NETs and have evidence of endothelial dysfunction. PAD inhibition markedly improves endothelial function, while downregulating the expression of type I interferon-regulated genes. PAD inhibition also reduces proteinuria and immune complex deposition in the kidneys, while protecting against skin disease.

Conclusions: PAD inhibition reduces NET formation, while protecting against lupus-related damage to the vasculature, kidneys and skin in various lupus models. The strategy by which NETs are inhibited will have to be carefully considered if human studies are to be undertaken.

Keywords: Autoimmune Diseases; Autoimmunity; Systemic Lupus Erythematosus.

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Conflict of interest statement

Competing interests None.

Figures

Figure 1
Figure 1
MRL/lpr mice have enhanced neutrophil extracellular trap (NET) formation and vascular damage compared with controls. (A) In the left panel, bone marrow neutrophils were prepared from control MRL/MpJ mice (MRL) and cultured with 10% serum from the indicated 14-week-old mice for 4 h. To the right, bone marrow neutrophils were prepared from the indicated 14-week-old mice and cultured under serum-free conditions for 4 h. NET formation was quantified by fluorescence microscopy. (B) Representative image demonstrating that MRL/lpr NETs contain citrullinated histone H3. DNA is stained blue and citrullinated histone H3 green. The scale bar represents 10 μ. (C) Autoantibodies to cathelicidin-related antimicrobial peptide (CRAMP) were quantified in the serum of the indicated 14-week-old mice. OD index normalises absorbance to the mean value for MRL controls. (D) Aortic rings were isolated from the indicated 14-week-old mice, and acetylcholine-dependent relaxation was determined as described in ‘Methods’. N=5 mice for all experiments. Mean±SEM are plotted; *p<0.05; **p<0.01; ***p<0.001.
Figure 2
Figure 2
Synthesis and characterisation of BB-Cl-amidine. (A) Peptidylarginine deiminase (PAD) reaction mechanism. PADs catalyse the hydrolysis of peptidyl-arginine to form peptidyl-citrulline. (B) BB-Cl-amidine is a C-terminal bioisostere of Cl-amidine. The Cl-amidine structure is based on the structure of benzoyl arginine amide, a small molecule PAD substrate. In BB-Cl-amidine, the C-terminus is replaced by a benzimidazole to prevent proteolysis of the C-terminal amide, and the N-terminal benzoyl group is replaced by a biphenyl moiety to increase hydrophobicity and enhance cellular uptake. (C) Potency and selectivity profiles. The potency (kinact/KI) of Cl-amidine and BB-Cl-amidine for PADs 1 to 4 is depicted graphically. kinact/KI is used because it is the best measure of the potency of an irreversible inhibitor. (D) EC50 of Cl-amidine and BB-Cl-amidine in U2OS cells was assessed. Human osteosarcoma (U2OS) cells were treated with various concentrations of inhibitor for 72 h. Cell viability was measured using the XTT assay. (E) Cl-amidine (10 mg/kg) and BB-Cl-amidine (1 mg/kg) were injected into C57BL/6 mice. Plasma levels were determined by LC-MS/MS.
Figure 3
Figure 3
Peptidylarginine deiminase inhibition prevents neutrophil extracellular trap (NET) formation when studied with in vitro and ex vivo models. A, Bone marrow neutrophils were prepared from 8-week-old MRL/lpr mice and stimulated with Phorbol 12-myristate 13-acetate (PMA) for 4 h (all black bars received PMA stimulation). NET formation was quantified by fluorescence microscopy. For some samples, neutrophils were pretreated with either Cl-amidine (Cl-am) or BB-Cl-amidine (BB) at the indicated concentrations. To the right, MRL/lpr neutrophils were stimulated for 1 h with PMA in the presence of inhibitors as indicated. The no-inhibitor condition was arbitrarily set at 100% H2O2 production, after subtracting for background H2O2 production from unstimulated cells. DPI, diphenyleneiodonium. NAC, n-acetyl cysteine. The data represent three independent experiments; mean±SEM are plotted. (B) MRL/lpr mice were treated with either Cl-amidine (Cl-am; 10 mg/kg/day) or BB-Cl-amidine (BB-Cl-am; 1 mg/kg/day) by daily subcutaneous injection from 8 to 14 weeks of age. Bone marrow neutrophils were prepared from 14-week-old mice and cultured for 4–6 h, assaying both serum-free spontaneous NET formation (left panel) and PMA-stimulated NET formation (right panel). (C) Circulating neutrophils were determined in the same 14-week-old mice and are plotted as a percentage of total nucleated cells. (D) Spleen weight was measured in the same 14-week-old mice. Boxes represent the median, 25th centile and 75th centile, while whiskers delineate the minimum and maximum values. *p<0.05; **p<0.01; ***p<0.001; ****p<0.0001.
Figure 4
Figure 4
Peptidylarginine deiminase (PAD) inhibition improves vascular function, and decreases interferon (IFN) signature in bone marrow and kidney. MRL/lpr mice were treated with either Cl-amidine (Cl-am; 10 mg/kg/day) or BB-Cl-amidine (BB-Cl-am; 1 mg/kg/day) by daily subcutaneous injection from 8 to 14 weeks of age. (A) At 14 weeks of age, aortic rings were isolated from the indicated groups and acetylcholine-dependent relaxation was determined as described in ‘Methods’. (B) At 14 weeks of age, RNA was prepared from total bone marrow and gene expression was determined by quantitative PCR for several type I IFN-regulated genes. The data are expressed as fold change for PAD inhibitor-treated mice, relative to vehicle-treated mice. P values are indicated. (C) At 14 weeks of age, kidneys were harvested, fixed in formalin and stained for Mx1 protein expression (brown). Glomeruli were scored as positive or negative for Mx1, with a minimum of 10 glomeruli counted per mouse. Boxes represent the median, 25th centile and 75th centile, while whiskers delineate the minimum and maximum values. Representative negative (left) and positive (right) staining is shown. Positive staining especially highlights capillary loops. For all experiments, n=10 per group; *p<0.05; **p<0.01; ***p<0.001.
Figure 5
Figure 5
Peptidylarginine deiminase (PAD) inhibition decreases IC deposition in kidneys and reduces proteinuria. MRL/lpr mice were treated with either Cl-amidine (Cl-am; 10 mg/kg/day) or BB-Cl-amidine (BB-Cl-am; 1 mg/kg/day) by daily subcutaneous injection from 8 to 14 weeks of age. (A) Representative anti-IgG staining is shown with a vehicle-treated glomerulus on the left and PAD inhibitor-treated on the right. Quantification used a scoring system ranging from 0 to 3+ as described in ‘Methods’. (B) Representative anti-C3 staining is shown with a vehicle-treated glomerulus on the left and PAD inhibitor-treated on the right. Quantification used a scoring system ranging from 0 to 3+ as described in ‘Methods’. (C) Periodic acid-Schiff (PAS)-stained sections were scored for activity (activ.) and chronicity (chron.) as described in ‘Methods’; there were no statistical differences in the left graph. Interstitial inflammation was also quantified, and pooled data for Cl-am and BB-Cl-am is denoted as ‘PAD inh.’ (D) Proteinuria was determined at 14 weeks of age by calculating the ratio of albumin to creatinine in the urine. Pooled data for Cl-am and BB-Cl-am are denoted as ‘PAD inh.’ For all experiments, n=10 per group; *p<0.05; **p<0.01; ***p<0.001. Boxes represent the median, 25th centile and 75th centile, while whiskers delineate the minimum and maximum values.
Figure 6
Figure 6
Peptidylarginine deiminase (PAD) inhibition improves skin involvement in MRL/lpr mice. MRL/lpr mice were treated with either Cl-amidine (Cl-am; 10 mg/kg/day) or BB-Cl-amidine (BB-Cl-am; 1 mg/kg/day) by daily subcutaneous injection from 8 to 14 weeks of age. (A) Representative area of alopecia in a 14-week-old mouse treated with vehicle. To the right is quantification of lesion circumference in the presence or absence of PAD inhibition. (B) The area of alopecia (or the corresponding area in mice without visible lesions) was sampled and frozen sections were prepared. Dermal neutrophil extracellular trap were determined as areas of MPO-DNA overlap. At least five 400× fields were counted per mouse. Representative staining is shown to the right with DNA in blue and MPO in green; for orientation, the epidermis is marked with an ‘E.’ Scale bar=25 μ. (C) Formalin-fixed, paraffin-embedded sections were prepared, and Mx1-positive cells within the dermis were quantified. Representative vehicle-treated (left) and PAD inhibitor-treated (right) sections are shown. Mx1 protein is stained brown. The secondary antibody reacted non-specifically with the epidermis (E) as well as hair follicles (HF). Scale bar=50 μ. For all experiments, n=10 per group. Boxes represent the median, 25th centile and 75th centile, while whiskers delineate the minimum and maximum values; *p<0.05; **p<0.01; ***p<0.001.
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