In this study, we find that the PAD4 enzyme is active within the joint tissue of arthritic mice and leads to NET formation. However, the disease course and histological features of the arthritic joints following K/BxN serum injection were comparable between WT and PAD4-deficient mice, demonstrating that PAD4 is not required for the effector phase of arthritis. Our data are consistent with the findings by Willis et al
., showing that the PAD inhibitor Cl-amidine provides therapeutic benefit in the collagen-induced arthritis model but has no benefit when arthritic disease is induced by the administration of anti-collagen antibodies. Collagen induced arthritis and injection of anti-collagen antibodies represent the priming and effector phases of disease, respectively. We have now shown that PAD4 is active but not required for disease, in another model of the effector phase, the K/BxN serum transfer model, which is consistent with the finding that PAD inhibitors have no effect on disease when induced by administration by anti-collagen antibodies [40
]. Interestingly, Cl-amidine preferentially inhibits PADs 1 and 4 over PAD3, and its effects have not been reported for PADs 2 or 6 [41
Indeed, Johnsen et al.
identified the Padi
locus, which contains the PAD4 gene along with the genes for PADs 1, 2, 3, and 6, as being linked with disease in the K/BxN serum transfer model [35
]. PAD2 and PAD4 are the most likely candidates to regulate the effector stage of arthritis as they are both expressed in immune cell types, whereas the expression of PADs 1, 3, and 6 are restricted to the epidermis, hair follicle, and oocyte, respectively. Indeed, increased splenic expression of both PAD2 and PAD4 correlated with disease severity in the K/BxN model [35
]. Further, within the PAD
region, a SNP found within the Padi2
locus showed the most significant association with disease [35
]. We speculate that the loss of PAD2 and PAD4 together may produce a more apparent phenotype in the K/BxN model [40
]. PAD2 KO mice have been reported [42
], however the PAD2 and PAD4 loci are approximately 1 centimorgan apart, and therefore, the recombination frequency between the targeted PAD2 and PAD4 alleles would be quite small, making the generation of Padi2/Padi4
DKO mice unlikely. While our data demonstrate that PAD4 is not required for the development of the K/BxN serum-transfer model, it is possible that there might be redundant activity of other PADs or they may independently contribute to the pathogenesis of antibody-mediated arthritis.
The blood of RA patients contains autoantibodies directed against a number of self-antigens. Many autoantibodies in RA are directed against citrullinated proteins. In fact, the presence of anti-citrulline antibodies is a better predictor of RA than rheumatoid factor [43
]. Variants of PAD4 are linked to RA in several Japanese and Korean cohorts, and the mRNA of a disease-associated allele is more stable than a non-disease associated allele [36
]. It has been proposed that PAD4 is linked to RA because PAD4 citrullination of peptides leads to a breakdown in tolerance to self-antigens [43
]. In support of this, treatment of mice with the PAD inhibitor Cl-amidine in the collagen-induced arthritis (CIA) model reduces the levels of citrulline found in the serum and synovial tissue, diminishes the formation of autoantibodies, and ameliorates disease [40
]. Thus, it will be interesting to determine whether the effects of Cl-amidine are attributable to PAD4 activity by examining the susceptibility of PAD4 KO mice to arthritic disease using the CIA model.
Incubation of human neutrophils with lipopolysaccharides (LPS), TNFα, N-formyl-methionine- leucine-phenylalanine (fMLP), or lipoteichoic acid and murine neutrophils with LPS or bacteria, has been shown to induce histone deimination and NET formation, marking PAD4 activity [24
]. Further, we detected deiminated histone H4 in lung leukocytes isolated from influenza-infected mice [25
]. In this report, we find that PAD4 activity is readily detected within the affected arthritic joint. In WT mice receiving K/BxN serum, the presence of deiminated histones corresponded primarily to the infiltrating cells of the joint sublining, which is consistent with the expression pattern of PAD4 found in patients with RA [46
]. The stimulus that induces PAD activity during autoimmune-mediated inflammation is undefined. However, the LPS receptor TLR4, has been linked to animal models of arthritis, perhaps because of the activation of TLR4 by endogenous ligands, such as Tenascin-C [16
NETs possess potent microbicidal capabilities but have also been implicated in chronic inflammatory diseases [23
]. NET formation is linked to cystic fibrosis [29
]. Similarly, the formation of NETs contributes to endothelial and tissue injury during sepsis [32
]. In autoimmune small-vessel vasculitis, anti-neutrophil cytoplasm antibodies (ANCA) trigger the formation of NETs, promoting necrotic inflammation of the blood vessels [51
]. Systemic lupus erythematous (SLE) is a systemic autoimmune disease characterized by the formation of pathogenic immune complexes. When activated by autoantibodies, neutrophils isolated from patients with SLE produce NETs, exposing immunostimulatory proteins and potential autoantigens and leading to the induction of Type I interferons by plasmacytoid dendritic cells [28
]. Collectively, these results support the notion that NET production can contribute to disease pathogenesis in inflammatory conditions. While hypercitrullination of neutrophil histones has been reported in patients with RA [34
], it is unclear whether NETs have a role in RA inflammation. Our results suggest that PAD4 activity and subsequent NET formation is present in the K/BxN serum transfer model, but is not required for this model of effector phase of disease.
Our data demonstrate that PAD4 is not necessary for the antibody-dependent, effector stage of arthritis. It is also possible that compensation by other PAD family members, PAD2 in particular, may mask the function of PAD4 in arthritis, although we note that PAD2 expression is not upregulated in PAD4-deficient neutrophils [25
]. Finally, since the Padi
locus is linked to disease severity in the K/BxN serum transfer model, it may be necessary to eliminate several PAD family members, either by targeting multiple locations within the PAD locus or by combining treatment with specific PAD inhibitors with targeted PAD alleles. Further studies will be necessary to dissect the role of PAD4 in the priming phase of arthritis.