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. 2017 Jun:80:39-47.
doi: 10.1016/j.jaut.2017.01.006. Epub 2017 Feb 7.

Peptidylarginine deiminase 2 is required for tumor necrosis factor alpha-induced citrullination and arthritis, but not neutrophil extracellular trap formation (V体育官网)

Mandar Bawadekar  1 , Daeun Shim  1 , Chad J Johnson  1 , Thomas F Warner  2 , Ryan Rebernick  1 , Dres Damgaard  3 , Claus H Nielsen  3 , Ger J M Pruijn  4 , Jeniel E Nett  5 , Miriam A Shelef  6
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"VSports手机版" Peptidylarginine deiminase 2 is required for tumor necrosis factor alpha-induced citrullination and arthritis, but not neutrophil extracellular trap formation

Mandar Bawadekar et al. J Autoimmun. 2017 Jun.

Abstract

Citrullination, the post-translational conversion of arginines to citrullines, may contribute to rheumatoid arthritis development given the generation of anti-citrullinated protein antibodies (ACPAs). However, it is not known which peptidylarginine deiminase (PAD) catalyzes the citrullination seen in inflammation. PAD4 exacerbates inflammatory arthritis and is critical for neutrophil extracellular traps (NETs). NETs display citrullinated antigens targeted by ACPAs and thus may be a source of citrullinated protein. However, PAD4 is not required for citrullination in inflamed lungs VSports手机版. PAD2 is important for citrullination in healthy tissues and is present in NETs, but its role in citrullination in the inflamed joint, NETosis and inflammatory arthritis is unknown. Here we use mice with TNFα-induced inflammatory arthritis, a model of rheumatoid arthritis, to identify the roles of PAD2 and PAD4 in citrullination, NETosis, and arthritis. In mice with TNFα-induced arthritis, citrullination in the inflamed ankle was increased as determined by western blot. This increase was unchanged in the ankles of mice that lack PAD4. In contrast, citrullination was nearly absent in the ankles of PAD2-deficient mice. Interestingly, PAD2 was not required for NET formation as assessed by immunofluorescence or for killing of Candida albicans as determined by viability assay. Finally, plasma cell numbers as assessed by flow cytometry, IgG levels quantified by ELISA, and inflammatory arthritis as determined by clinical and pathological scoring were all reduced in the absence of PAD2. Thus, PAD2 contributes to TNFα-induced citrullination and arthritis, but is not required for NETosis. In contrast, PAD4, which is critical for NETosis, is dispensable for generalized citrullination supporting the possibility that NETs may not be a major source of citrullinated protein in arthritis. .

Keywords: Citrullination; Murine model; Neutrophil extracellular trap; Peptidylarginine deiminase; Rheumatoid arthritis; TNFα V体育安卓版. .

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Figures

Figure 1
Figure 1. Joint citrullination is increased in TNFα overexpressing mice without a requirement for PAD4
Ankle protein lysates from 4–5 month old TNF+ and littermate control (WT) mice were subjected to western blot to detect citrullinated proteins and gel electrophoresis to detect total protein. A. Representative blot (left) and gel (right). B. The density of the entire lanes was determined and citrullinated protein signal was normalized to total protein signal. Average and SEM are graphed (n=9, *p<0.05). Ankle lysates from 4–5 month old TNF+PAD4+/+ and TNF+PAD4−/− littermates were similarly assessed. C. Representative blot (left) and gel (right). D. Graph with average and SEM (n=8).
Figure 2
Figure 2. PAD2 is required for joint citrullination
Protein lysates from the ankles of 4–5 month old TNF+PAD2+/+ and TNF+PAD2−/− mice were subjected to western blot to detect citrullinated proteins and gel electrophoresis to detect total protein. A. Representative blot (left) and gel (right). B. The density of the entire lanes was determined and citrullinated protein signal was normalized to total protein signal. Average and SEM are graphed for the entire lane (TNF+PAD2+/+ n=9, TNF+PAD2−/− n=8, *p<0.05). Ankle lysates from PAD2+/+ and PAD2−/− littermates were similarly assessed. C. Representative blot (left) and gel (right). D. Graph with average and SEM (PAD2+/+ n=4, PAD2−/− n=5, **p<0.01).
Figure 3
Figure 3. PAD2 is not increased in inflamed joints in the absence of PAD4, but is upregulated in PAD4-deficient neutrophils
A. RNA from PAD4+/+ and PAD4−/− neutrophils was subjected to qPCR to assess PAD2 gene expression levels with average and SEM graphed for the fold change (n=6). B. Protein lysates from the ankles of TNF+PAD2+/+ and TNF+PAD2−/− mice were assessed for PAD2 protein by western blot. Representative blots (upper panel) and average with SEM for quantification of PAD2 normalized to actin in the lower panel (TNF+PAD2+/+ n=9, TNF+PAD2−/− n=8). C. Protein lysates from the ankles of TNF+PAD4+/+ and TNF+PAD4−/− mice were assessed for PAD2 protein by western blot. Representative blots (upper panel) and average with SEM for quantification of PAD2 normalized to actin in the lower panel (n=7). For all panels, *p<0.05.
Figure 4
Figure 4. PAD2 is not required for NETosis
Neutrophils from PAD4+/+ and PAD4−/− mice were untreated or treated with TNFα and LPS followed by staining for citrullinated histone H4 (red) and DNA (blue). A. Representative images at 400x. B. Graph with average and SEM for the percent of cells that formed NETs (n=3, *p=0.05). C. Neutrophils from PAD2+/+ and PAD2−/− mice were treated identically to the PAD4+/+ and PAD4−/− neutrophils. C. Representative images at 400x. D. Graph with average and SEM for the percent of cells that formed NETs (n=5). E. PAD2+/+ and PAD2−/− neutrophils were incubated with Candida in the presence or absence of Cytochalasin D. The percent of Candida killed was detected by XTT. Average and SEM are graphed (n = 3).
Figure 5
Figure 5. PAD2 contributes to plasma cells and IgG levels in TNFα-induced arthritis
A. Total serum IgG was quantified for 4–5 month old TNF+PAD2+/+ and TNF+PAD2−/− mice by ELISA with average and SEM graphed (n=8, **p<0.01). B. Total serum IgG was quantified for 2 month old PAD2+/+ and PAD2−/− mice by ELISA with average and SEM graphed (n=7, *p<0.05). Bone marrow from TNF+PAD2+/+ and TNF+PAD2−/− mice was stained for B220 and CD138 and subjected to flow cytometry. C. Representative zebra plots of live cells. D. Average and SEM for the percentage of B220LOCD138HI plasma cells is graphed (n=9, *p<0.05).
Figure 6
Figure 6. PAD2 is required for TNFα-induced inflammatory arthritis
A. Severity of clinical arthritis was scored on a scale of 0–3 in 2, 3, and 4 month old TNF+PAD2+/+ and TNF+PAD2−/− mice including (left) or not including (right) grip strength with average and SEM graphed (2 and 3 months: n=25 TNF+PAD2+/+ and n=24 TNF+PAD2−/− mice; 4 months: n=19 pairs; *p<0.05). The ankles of 4–5 month old TNF+PAD2+/+ and TNF+PAD2−/− mice were fixed, sectioned, and stained with H&E. B. Representative images with arrows indicating inflamed synovium/pannus invading the tibio-talar joint (Magnification 100x, bar 500 μm). The percentage of surface erosion (C) and extent of synovitis on a scale of 0–4 (D) at the tibio-talar joint was determined in a blinded manner with average and SEM graphed (TNF+PAD2+/+ n=11, TNF+PAD2−/− n=10, *p<0.05).
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