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PAD4 is not essential for disease in the K/BxN murine autoantibody-mediated model of arthritis

PAD4 is not essential for disease in the K/BxN murine autoantibody-mediated model of arthritis Introduction: Both murine and human genome-wide association studies have implicated peptidyl arginine deiminase (PAD4) as a susceptibility gene in rheumatoid arthritis (RA). In addition, patients with RA commonly have autoantibodies which recognize PAD4 or and/or citrullinated peptides. This study aims to evaluate the role of PAD4 in the effector phase of arthritis. Methods: PAD4 knock out (KO) and wild type (WT) C57BL/6J mice were injected with K/BxN sera to induce disease. Progression of disease was monitored by measuring paw and ankle swelling and clinical indexes of disease, and pathogenesis was assessed by indexing of clinical progression on paws collected from WT and PAD4 KO mice injected with K/BxN serum. PAD4 activity was determined by visualization of neutrophil extracellular traps (NETs) and immunohistological analysis of histone citrullination. Results: PAD4 activity is readily detectable in the inflamed synovium of WT but not PAD4 deficient animals, as demonstrated by histone citrullination and NET formation. However, PAD4 WT and KO animals develop K/BxN serum transfer disease with comparable severity and kinetics, with no statistically significant differences noted in clinical scores, swelling, joint erosion or joint invasion. Conclusions: PAD4 WT and KO mice develop disease in the K/BxN serum transfer model of arthritis with similar severity and kinetics, indicating that PAD4 is dispensable in this effector phase model of disease. Introduction anti-citrullinated peptide antibodies (ACPAs) exhibit Citrulline-containing proteins are generated through high specificity and sensitivity as diagnostic markers of posttranslational modification of arginine residues in a the disease [7]. Anti-citrulline peptide antibodies can 2+ reaction catalyzed by the Ca -dependent peptidyl argi- appear before disease onset and correlate with the most nine deiminases (PADs). There are five PAD family erosive form of RA [8]. members, but only PAD2 and PAD4 expression are clo- PAD4 shows elevated expression in RA [1,9], and RA sely linked with inflammation in RA synovial tissue patients generate high affinity anti-PAD4 autoantibodies [1,2]. While PAD2 is broadly expressed across tissue which correlate with more severe disease [10-12]. types, including by immune cells, PAD4 exhibits an Further, variants of PAD4 are linked to RA in several expression pattern restricted to immune cell types, in Japanese and Korean cohorts, although this association particular macrophages and granulocytes [1,3]. has not held up in most North American and European Rheumatoid arthritis (RA) is a chronic autoimmune study groups, despite the prevalence of ACPA in all eth- nic groups [13]. Thus, the development of autoantibo- disease characterized by systemic inflammation, chronic synovitis, joint destruction and bone loss, affecting dies to citrullinated epitopes and PAD4 and elevated approximately 2% of the world population [4]. Plasma PAD4 expression in RA, suggests that aberrant PAD and synovial biopsy specimens from patients with RA activity may contribute to disease pathogenesis. contain high levels of citrullinated proteins [5,6], and The offspring of an intercross between the KRN TCR transgenic mouse specific for a bovine RNase (42-56) in k g7 the context of I-A and the I-A -expressing non-obese * Correspondence: [email protected] Department of Chemical Physiology, The Scripps Research Institute, 10550 diabetic (NOD) background, spontaneously develop a North Torrey Pines Road, La Jolla, California, 92037, USA progressive, inflammatory joint disease with features Full list of author information is available at the end of the article © 2012 Rohrbach et al.; licensee BioMed Central Ltd. This is an open access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/2.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. Rohrbach et al. Arthritis Research & Therapy 2012, 14:R104 Page 2 of 10 http://arthritis-research.com/content/14/3/R104 similar to human RA (K/BxN mice) [14]. The autoanti- Recently, using a genome-wide screen of mice, John- gen in this model is glucose-6-phosphate isomerase sen et al. identified the region containing Padi4 as being (GPI), a ubiquitous cytoplasmic enzyme [15]. Treatment putatively involved in the development of K/BxN arthri- with the sera of K/BxN mice or purified anti-GPI auto- tis and demonstrated that increases in the transcripts of antibodies is sufficient to transfer disease to healthy ani- both PAD2 and PAD4 correlates with increased severity mals, even in animals devoid of B and T cells [14,15]. of disease [35]. Because the K/BxN model is neutrophil dependent, PAD4 activity is required for NET formation Because autoantibodies are passively transferred, this and PAD4 overexpression correlates with rheumatoid model focuses on immune recruitment and joint arthritis in patients, we sought to determine the extent destruction (effector phase), rather than the breaking of immune tolerance (priming phase). Innate immune sig- to which PAD4 would contribute to the effector phase nals are critical for this model because mice deficient in of arthritis using the K/BxN-serum transfer model the alternative complement pathway, the C5a receptor, [20,25,27,36]. the CXCR2 chemokine receptor, interleukin-1 receptor (IL-1R), and myeloid differentiation primary response Materials and methods protein (MyD88) are resistant to disease [14,16,17]. Mice Further, arthritis was not sustained in toll-like receptor PAD4 knockout (KO) mice were generated as previously 4 (TLR4) mutant mice [16]. Passively transferred arthri- described [25]. KRN T cell receptor (TCR) transgenic tis also requires the presence of mast cells and neutro- mice were a gift from Drs D Mathis and C Benoist phils [18-21]. (Harvard Medical School, Boston, MA) and the Institut Neutrophils are among the first immune cell types to de GénétiqueetdeBiologieMoléculaire et Cellulaire accumulate during an inflammatory response [22]. In (Strasbourg, France) [14]. Wildtype C57BL/6J mice were response to inflammatory stimuli, neutrophils decon- obtained from the Scripps Research Institute rodent dense their chromatin and actively expel their DNA- breeding colony. Experiments were reviewed and producing neutrophil extracellular traps (NETs) that are approved by the Scripps Research Institute Institutional decorated with granular and nuclear proteins, including Animal Care and Use Committee. citrullinated histones [23,24]. Incubation of neutrophils with phorbol 12-myristate 13-acetate (PMA), hydrogen Induction of serum-transfer arthritis peroxide, lipopolysaccharide (LPS), bacteria, and yeast K/BxN serum was collected from 6- to 10-week-old K/ induces NET formation [24-27]. Our lab and others BxN mice and pooled, and 5- to 6-week-old male PAD4 have shown that PAD4 is essential for the production of KO or C57BL/6J WT control mice were injected intra- NETs and NET-associated histone citrullination peritoneally (i.p.) with 150 μL pooled K/BxN sera on [24,25,27]. PAD4-mediated histone citrullination is days 0 and 2. Caliper measurements of paw and ankle thoughttoplayamechanicalroleinNET formation, swelling were taken daily. Clinical indexes of swelling where the conversion of positively charged arginine resi- were assigned according to previously described criteria dues into the neutral citrulline amino acid by PAD4 [37]. Animals were sacrificed at day 5 or day 10 and the promotes chromatin decondensation [24]. PAD4- hind paws were prepared for fluorescence microscopy mediated NET formation is critical for controlling at or histology. least a subset of bacterial infections because PAD4-defi- cient mice are more susceptible to infectious disease in Fluorescence Microscopy of Histone Citrullination and a necrotizing fasciitis model [27]. NETs NET formation, although critical for the full activation Hind paws were removed from euthanized animals at of the innate immune response [27], has also been day 5, post K/BxN serum injection. Ankle joints were implicated in inflammatory disease pathogenesis, includ- exposed and flushed with Roswell Park Memorial Insti- ing the autoimmune disorder lupus [28], cystic fibrosis tute media (RPMI) with 0.5% BSA. Peripheral blood was ([29-31], sepsis [32], and thrombosis [33]. Interestingly, collected by eye bleed. Erythrocytes were lysed in 10 it has been suggested that NETs offer a possible mM KHCO , 150 mM NH Cl NS 1 mM EDTA and 3 4 mechanism by which PAD4 may be liberated from the remaining cells were washed with PBS. Ankle or periph- cell to generate citrullinated antigens and exacerbate eral blood cells were attached to poly-lysine-coated glass inflammation [9,34]. Recently, Dwievedi et al. described coverslips and fixed in 4% paraformaldehyde (PFA) with hypercitrullination in neutrophils from arthritic patients, 1% Nonidet P-40 (NP-40) and 0.5% Triton X-100 in as well as the specific reactivity of arthritic serum to PBS. Slides were blocked with 2.5% BSA and 5% goat activated neutrophils and citrullinated histones [34]. It is serum in PBS with 0.5% Tween-20. Citrullinated his- unclear whether PAD4-induced NET formation plays a tones were detected with anti-H4Cit3 (Millipore, Biller- role in the RA inflammatory process. ica, MA, USA) and goat anti-rabbit Alexa488 (Life Rohrbach et al. Arthritis Research & Therapy 2012, 14:R104 Page 3 of 10 http://arthritis-research.com/content/14/3/R104 Technologies, Grand Island, NY, USA). DNA was within synovium throughout the serum-transfer during labeled with DAPI (Sigma, St. Louis MO, USA). Cover- the course of arthritis disease [17,20]. Importantly, slips were mounted with Prolong Gold Antifade reagent PAD4 is highly expressed in the granulocyte lineage, (Life Technologies, Grand Island, NY, USA). including neutrophils [3] and is known to be required for the formation of Neutrophil Extracellular Traps Histology (NETs) [25,27]. NETs have been shown to occur in sev- Hindpaws were removedfromeuthanizedanimals, eral inflammatory disease states, including lupus [28], fixed in 4% paraformaldehyde in PBS overnight and dec- sepsis [32,33] and cystic fibrosis [29-31] and are thought alcified in 0.375 M EDTA pH 7.5 for 3 weeks. Paws to serve as a means for PAD4 to enter into the extracel- were embedded in paraffin and affixed to slides for lular space, where it may citrullinate substrates and immunohistochemistry or pathological analysis by The exacerbate inflammation [9]. NETs are common to sev- Scripps Research Institute Histology Core. For immuno- eral other inflammatory disease states, but to our knowl- histochemistry, paraffin-embedded slices on slides were edge, NET structures, per se, have not been reported in deparaffinized with xylene and rehydrated. Antigens patients with RA or in murine models of disease. How- were unmasked by boiling in PBS pH 7.4. Slides were ever, recently, Dwivedi et al. reported that serum IgGs blocked with 10% normal goat serum and endogenous from patients with Felty’s syndrome react to NETs and peroxidases were quenched with 3% peroxide in metha- preferentially to citrullinated histones over unmodified nol. Sections were then incubated with anti-H4cit3 or histones, indicating that NET markers, and thus NETs, control rabbit immunoglobulin (IgG) antibodies (Milli- maybepresent in Felty’ssyndrome[34]. SinceACPA pore, Billerica, MA, USA). Labeling was detected with a and anti-PAD4 antibodies are frequently found in biotinylated anti-rabbit secondary antibody (Life Tech- patients with RA [9], it is presumed that aberrant PAD nologies, Grand Island, NY, USA), Vectastain Elite ABC activity influences the priming phase of arthritis. How- reagent (Vector Labs, Burlingame, CA, USA) and DAB ever, it is unknown where PAD4 may also regulate the (Life Technologies, Grand Island, NY, USA). Slides were effector phase of arthritis. We therefore considered counterstained with Shandon Harris hematoxylin whether PAD4-mediated NET formation could contri- (Thermo Scientific, Waltham, MA, USA). Histopatholo- bute to K/BxN-induced inflammation. gical scoring was performed blinded as described pre- To determine whether NET formation occurs in the viously [16]. Briefly, ankles of arthritic mice were given K/BxN serum transfer model, fluorescence microscopy scores of 0 to 5 for inflammation, according to the fol- of flushed synovial cells was performed. PAD4 WT and lowing criteria: 0 = normal; 1 = minimal infiltration of KO mice were injected with K/BxN serum on days 0 inflammatory cells in the periarticular area; 2 = mild and 2. Mice were sacrificed at day 5 to collect synovial infiltration; 3 = moderate infiltration; 4 = marked infil- and peripheral blood cells. Cells flushed from the ankles tration; and 5 = severe infiltration. Knees of arthritic of arthritic WT mice displayed citrullinated histones, a mice were given scores of 0 to 5 for bone resorption, hallmark of PAD4 activity, and NET formation, while according to the following criteria: 0 = normal; 1 = those from PAD4 KO mice exhibited no detectable his- minimal (small areas of resorption, not readily apparent tone citrullination or NET formation, as detected by on low magnification); 2 = mild (more numerous areas anti-H4Cit3 and 4’,6-diamidino-2-phenylindole (DAPI) of resorption, not readily apparent on low magnification, (Figure 1A). As expected, few cells could be flushed in trabecular or cortical bone); 3 = moderate (obvious from the ankles of WT control mice, and none were resorption of trabecular and cortical bone, without full- observed to have citrullinated histones or to form NETs thickness defects in the cortex; loss of some trabeculae; (Figure 1A). Histone citrullination, but not NET forma- lesions apparent on low magnification); 4 = marked tion, was detected in the peripheral blood of WT K/ (full-thickness defects in cortical bone and marked tra- BxN mice, but not WT control mice (Figure 1B). becular bone loss; without distortion of the profile of Neither histone citrullination nor NET formation was the remaining cortical surface) and 5 = severe (full- found in control mice of either genotype (Figure 1B). thickness defects in the cortical bone and marked trabe- Thus, NET formation does occur in the K/BxN model cular bone loss with distortion of the profile of the of the effector phase of RA and is specific for the remaining cortical surface). arthritic ankle. Results There is abundant PAD4 activity during K/BxN serum- NETs are found in the joints of K/BxN serum-transfer transferred arthritis animals PAD4 is found in the synovial fluid and infiltrating syno- Neutrophils are the predominant infiltrating cell type in vial cells of patients with RA [9,38,39]. In the K/BxN the K/BxN serum transfer arthritis model and are found serum transfer model, elevated expression of PAD4 in the Rohrbach et al. Arthritis Research & Therapy 2012, 14:R104 Page 4 of 10 http://arthritis-research.com/content/14/3/R104 DAPI H4Cit3-Alexa488 Overlay WT Ankle K/BxN PAD4KO Ankle K/BxN WT Ankle Control WT Peripheral Blood K/BxN PAD4 KO Peripheral Blood K/BxN WT Peripheral Blood Control 0.1 mm Figure 1 Wild type (WT) K/BxN serum transfer mice generate synovial neutrophil extracellular traps (NETs). Mice were injected with K/ BxN serum on days 0 and 2 and sacrificed on day 5. Synovial cells were flushed from the ankle joint (A) and peripheral blood was collected from the venous plexus of arthritic or healthy mice (B) and stained with antibodies to citrullinated histone H4 and 4’,6-diamidino-2-phenylindole (DAPI). Histone citrullination and NETs were readily detectable in the arthritic joints of WT mice, but were not observed in peptidyl arginine deiminase 4 knockout (PAD4 KO) K/BxN mice or WT healthy controls (A). Histone citrullination was present in the peripheral blood of WT K/BxN mice, however, no NET formation was observed. Images were taken at 80× magnification and are representative of at least three independent experiments with two mice per experimental group. Rohrbach et al. Arthritis Research & Therapy 2012, 14:R104 Page 5 of 10 http://arthritis-research.com/content/14/3/R104 spleen correlates with the severity of arthritis [35]. To disease and the overall kinetics of disease were similar determine whether PAD4 enzymaticactivitywas ahall- between WT and PAD4 KO mice (Figure 3). Although mark of the effector phase of arthritis, immunohistochem- there was a trend toward reduced swelling and lower istry of the PAD4-dependent citrullinated histone H4 was disease scores in PAD4 KO mice, the differences were performed [25,27]. Mice were injected with K/BxN serum small and did not reach statistical significance (Figure on days 0 and 2. On day 10, mice were sacrificed, hind 3). Disease is transient in the serum transfer model. We limbs were harvested and prepared for histology, and tis- foundthatPAD4KO micerecoveredwithsimilar sue sections were immunostained for citrullinated histone kinetics to WT mice (data not shown). Thus, PAD4 is H4. WT mice displayed robust histone H4 citrullination, not required for the development or maintenance of K/ with the most prominent staining in infiltrating cells BxN-serum transferred arthritis. within the synovial sublining, which were most likely neu- trophils. This modification was dependent upon PAD4 Comparable histological features between arthritic WT because PAD4 KO mice exhibited no staining for citrulli- and PAD4-deficient mice nated histone H4 (Figure 2). These data demonstrate that Although WT and PAD4 KO mice exhibited a similar robust PAD4 activity is present within the ankle joint dur- level of inflammation, the degree of joint destruction ing the effector phase of arthritis. and inflammatory infiltrates could be affected by PAD4 deficiency. Thus, hind limbs were harvested from WT PAD4 deficiency does not protect against disease in and PAD4 KO mice on day 10 following serum transfer. serum-transferred arthritis Histological evaluation by an investigator blinded to the To test the importance of PAD4 in the effector phase of animal genotype for degree of inflammation and erosion arthritis, we injected K/BxN serum into age-matched of bone was performed. Inflammation and bone erosions WT and PAD4 KO C57BL6/J mice on days 0 and 2 and were quantified by scoring joint sections on a 0 to 5 monitored disease progression. Measurements of paw scale. Hematoxylin and eosin stain (H&E) sections of swelling and clinical disease index were taken daily for the ankle joints revealed a comparable, moderate degree ten days. Paw swelling was calculated as the percentage of inflammatory cell infiltration in WT and PAD4 KO increase in paw size compared to day 0. The onset of mice (Figure 4 and Table 1). In healthy mice, the Figure 2 Peptidyl arginine deiminase 4 (PAD4) activity is detected in K/BxN arthritic legs in wild type (WT) but not PAD4 knockout (KO) mice. PAD4 activity was measured by immunohistochemical detection of citrullinated histone H4 (H4Cit3). WT and PAD4 KO mice were immunized with pooled K/BxN serum as described. Mice were sacrificed 10 days post injection (p.i.) and their legs prepared for histology. Sections were blocked in goat serum and probed with either anti-H4Cit3 antibody or isotype control. Images were taken at 80× magnification. Specific staining of citrullinated histone H4 was apparent on WT but not PAD4 KO slides and is representative of at least 4 independent experiments. Rohrbach et al. Arthritis Research & Therapy 2012, 14:R104 Page 6 of 10 http://arthritis-research.com/content/14/3/R104 Figure 3 Progression of K/BxN serum transfer arthritis in peptidyl arginine deiminase 4 (PAD4) wild type (WT) (n = 29) and knockout (KO) (n = 27) mice. WT and PAD4 KO mice were immunized with pooled K/BxN serum as described and monitored for signs of disease severity for 10 days. Paw swelling (A) is expressed as the average percent size increase of four paws and two ankles compared to day 0 measured by caliper. Clinical indexes (B) of 0 to 3 were assigned for each paw as previously described [37]. Data are combined from three independent experiments and represented as the average ± standard error of the mean (SEM). No statistically significant difference in the progression of K/BxN disease was apparent between WT and PAD4 KO mice. Rohrbach et al. Arthritis Research & Therapy 2012, 14:R104 Page 7 of 10 http://arthritis-research.com/content/14/3/R104 Figure 4 Pathological evaluation of immune invasion and joint destruction in wild type (WT) and peptidyl arginine deiminase 4 knockout (PAD4 KO) K/BxN mice.WT(n = 27) and PAD4 KO (n = 29) animals were immunized with pooled K/BxN serum as described, sacrificed at day 10 post injection (p.i.) and their legs prepared for histology. Hematoxylin- and eosin-stained stained slides were scored blinded by a trained observer for immune cell invasion (0 to 5) and joint erosion (0 to 5). Pictures were taken at 40× magnification and are representative of the average scores for invasion (4) and erosion (2), shown in Table 1. Areas of immune cell invasion into cartilage of joint (closed arrows) and bone reabsorption (open arrows) are highlighted. synovium consists of a single layer. Both WT and PAD4 benefit in the collagen-induced arthritis model but has KO mice developed a proliferative synovial response fol- no benefit when arthritic disease is induced by the lowing K/BxN serum treatment. Furthermore, WT and administration of anti-collagen antibodies. Collagen PAD4 KO mice exhibited a similar level of bone erosion. induced arthritis and injection of anti-collagen antibo- Therefore, the histological features of the K/BxN serum- dies represent the priming and effector phases of dis- mediated model of arthritis are not altered in the ease, respectively. We have now shown that PAD4 is absence of PAD4. active but not required for disease, in another model of the effector phase, the K/BxN serum transfer model, Discussion which is consistent with the finding that PAD inhibitors In this study, we find that the PAD4 enzyme is active have no effect on disease when induced by administra- tion by anti-collagen antibodies [40]. Interestingly, Cl- within the joint tissue of arthritic mice and leads to NET formation. However, the disease course and histo- amidine preferentially inhibits PADs 1 and 4 over logical features of the arthritic joints following K/BxN PAD3, and its effects have not been reported for PADs serum injection were comparable between WT and 2 or 6 [41]. PAD4-deficient mice, demonstrating that PAD4 is not Indeed, Johnsen et al. identified the Padi locus, which required for the effector phase of arthritis. Our data are contains the PAD4 gene along with the genes for PADs consistent with the findings by Willis et al., showing 1, 2, 3, and 6, as being linked with disease in the K/BxN that the PAD inhibitor Cl-amidine provides therapeutic 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 Table 1 Clinical scores of inflammation and joint erosion types, whereas the expression of PADs 1, 3, and 6 are in wild-type and peptidyl arginine deiminase 4 knockout restricted to the epidermis, hair follicle, and oocyte, K/BxN mice respectively. Indeed, increased splenic expression of Group Inflammation (0 to 5) Joint Erosion (0 to 5) both PAD2 and PAD4 correlated with disease severity (mean ± STDEV) (mean ± STDEV) in the K/BxN model [35]. Further, within the PAD WT 3.4 ± 1.3 2.5 ± 1.4 region, a SNP found within the Padi2 locus showed the PAD4 KO 3.6 ± 1.0 2.2 ± 1.7 most significant association with disease [35]. We specu- Pathological scoring of immune invasion and joint destruction in wild type late that the loss of PAD2 and PAD4 together may pro- (WT) and peptidyl arginine deiminase 4 knockout (PAD4 KO) K/BxN mice. WT (n = 27) and PAD4 KO (n = 29) animals were immunized with pooled K/BxN duce a more apparent phenotype in the K/BxN model serum as described, sacrificed at day 10 post injection (p.i.), and their legs [40]. PAD2 KO mice have been reported [42], however prepared for histology. Hematoxylin- and eosin-stained slides were scored blinded by a trained observer for immune cell invasion (0 to 5) and joint the PAD2 and PAD4 loci are approximately 1 centimor- erosion (0 to 5). Results are represented as the average of 27 and 29 mice per gan apart, and therefore, the recombination frequency genotype ± standard deviation. Average scores for joint invasion and erosion between the targeted PAD2 and PAD4 alleles would be were similar in WT and PAD4 KO mice. Rohrbach et al. Arthritis Research & Therapy 2012, 14:R104 Page 8 of 10 http://arthritis-research.com/content/14/3/R104 quite small, making the generation of Padi2/Padi4 DKO vessels [51]. Systemic lupus erythematous (SLE) is a sys- mice unlikely. While our data demonstrate that PAD4 is temic autoimmune disease characterized by the forma- not required for the development of the K/BxN serum- tion of pathogenic immune complexes. When activated transfer model, it is possible that there might be redun- by autoantibodies, neutrophils isolated from patients dant activity of other PADs or they may independently with SLE produce NETs, exposing immunostimulatory contribute to the pathogenesis of antibody-mediated proteins and potential autoantigens and leading to the induction of Type I interferons by plasmacytoid dendri- arthritis. tic cells [28,52,53]. Collectively, these results support the The blood of RA patients contains autoantibodies notion that NET production can contribute to disease directed against a number of self-antigens. Many auto- antibodies in RA are directed against citrullinated pro- pathogenesis in inflammatory conditions. While hyperci- teins. In fact, the presence of anti-citrulline antibodies is trullination of neutrophil histones has been reported in a better predictor of RA than rheumatoid factor [43]. patients with RA [34], it is unclear whether NETs have Variants of PAD4 are linked to RA in several Japanese a role in RA inflammation. Our results suggest that and Korean cohorts, and the mRNA of a disease-asso- PAD4 activity and subsequent NET formation is present ciated allele is more stable than a non-disease associated in the K/BxN serum transfer model, but is not required allele [36,44]. It has been proposed that PAD4 is linked for this model of effector phase of disease. to RA because PAD4 citrullination of peptides leads to a Our data demonstrate that PAD4 is not necessary for breakdown in tolerance to self-antigens [43,45]. In sup- the antibody-dependent, effector stage of arthritis. It is port of this, treatment of mice with the PAD inhibitor also possible that compensation by other PAD family Cl-amidine in the collagen-induced arthritis (CIA) members, PAD2 in particular, may mask the function of model reduces the levels of citrulline found in the PAD4 in arthritis, although we note that PAD2 expres- serum and synovial tissue, diminishes the formation of sion is not upregulated in PAD4-deficient neutrophils autoantibodies, and ameliorates disease [40]. Thus, it [25]. Finally, since the Padi locus is linked to disease will be interesting to determine whether the effects of severity in the K/BxN serum transfer model, it may be Cl-amidine are attributable to PAD4 activity by examin- necessary to eliminate several PAD family members, ing the susceptibility of PAD4 KO mice to arthritic dis- either by targeting multiple locations within the PAD ease using the CIA model. locus or by combining treatment with specific PAD Incubation of human neutrophils with lipopolysac- inhibitors with targeted PAD alleles. Further studies will be necessary to dissect the role of PAD4 in the priming charides (LPS), TNFa, N-formyl-methionine- leucine- phase of arthritis. phenylalanine (fMLP), or lipoteichoic acid and murine neutrophils with LPS or bacteria, has been shown to induce histone deimination and NET formation, mark- Conclusions ing PAD4 activity [24-27]. Further, we detected deimi- NET formation is known to correlate with inflammatory nated histone H4 in lung leukocytes isolated from disease [28,32,33,52,53], however NET formation has influenza-infected mice [25]. In this report, we find that not been reported in RA. NET formation is dependent PAD4 activity is readily detected within the affected on PAD4 and the association between PAD4 and RA is arthritic joint. In WT mice receiving K/BxN serum, the well-established [9]. In this report, we show that in the presence of deiminated histones corresponded primarily K/BxN murine model of arthritis, which emulates the to the infiltrating cells of the joint sublining, which is effector phase of disease, PAD4 activity and NET forma- consistent with the expression pattern of PAD4 found tion are detected. PAD4 activity is not, however, in patients with RA [46,47]. The stimulus that induces required for disease, as PAD4 WT and KO mice develop PAD activity during autoimmune-mediated inflamma- K/BxN-induced arthritis with similar severity and tion is undefined. However, the LPS receptor TLR4, has kinetics. Overall our data indicate that PAD4 is dispen- been linked to animal models of arthritis, perhaps sable in this model. However this does not eliminate a because of the activation of TLR4 by endogenous role for PAD4 in the priming phase of disease. Future ligands, such as Tenascin-C [16,48,49]. studies will consider the role of PAD4 in priming phase NETs possess potent microbicidal capabilities but have models of RA, including the CIA model. also been implicated in chronic inflammatory diseases [23,50]. NET formation is linked to cystic fibrosis Abbreviations [29-31]. Similarly, the formation of NETs contributes to ANCA: anti-neutrophil cytoplasmic antibodies; CIA: Collagen-induced arthritis; endothelial and tissue injury during sepsis [32]. In auto- DAPI: 4’,6-diamidino-2-phenylindole; fMLP: formyl-methionine-leucine- phenylalanine; H&E: hematoxylin and eosin stain; IgG: immunoglobulin; IL-1R: immune small-vessel vasculitis, anti-neutrophil cyto- interleukin-1 receptor; KO: knockout; LPS: lipopolysaccharide; MyD88: myeloid plasm antibodies (ANCA) trigger the formation of differentiation primary response protein; PAD: Peptidyl arginine deiminase; NETs, promoting necrotic inflammation of the blood PMA: phorbol 12-myristate 13-acetate; NET: neutrophil extracellular trap; Rohrbach et al. Arthritis Research & Therapy 2012, 14:R104 Page 9 of 10 http://arthritis-research.com/content/14/3/R104 NOD: non-obese diabetic;RA: rheumatoid arthritis; SLE: systemic lupus 12. Pollmann S, Stensland M, Halvorsen EH, Sollid LM, Kvien TK, Fleckenstein B, erythematous; SNP: single nucleotide polymorphism; TCR: T cell receptor; Molberg O: Anti-PAD4 autoantibodies in rheumatoid arthritis: levels in TLR: toll-like receptor; WT: wild type. serum over time and impact on PAD4 activity as measured with a small synthetic substrate. Rheumatol Int 2012, 32:1271-1276. Acknowledgements 13. van der Helm-van Mil AH, Huizinga TW: Advances in the genetics of Dr Arandjelovic was supported by an Arthritis Foundation Fellowship award. rheumatoid arthritis point to subclassification into distinct disease Ms. Rohrbach, and Drs Hemmers, Arandjelovic, and Mowen were supported subsets. Arthritis Res Ther 2008, 10:205. by the NIH (National Institute of Allergy and Infectious Diseases [NIAID] 14. Kouskoff V, Korganow AS, Duchatelle V, Degott C, Benoist C, Mathis D: grant AI067460 and National Institute of General Medicine [NIGMS] grant Organ-specific disease provoked by systemic autoimmunity. Cell 1996, GM085117-01). Dr Corr’s work was supported by the Arthritis Foundation. 87:811-822. This is manuscript #21621 from The Scripps Research Institute. 15. Matsumoto I, Staub A, Benoist C, Mathis D: Arthritis provoked by linked T and B cell recognition of a glycolytic enzyme. Science 1999, Author details 286:1732-1735. Department of Chemical Physiology, The Scripps Research Institute, 10550 16. Choe JY, Crain B, Wu SR, Corr M: Interleukin 1 receptor dependence of North Torrey Pines Road, La Jolla, California, 92037, USA. Department of serum transferred arthritis can be circumvented by toll-like receptor 4 Medicine, University of California, San Diego, 9500 Gilman Drive, La Jolla, signaling. J Exp Med 2003, 197:537-542. California, 92093, USA. 17. Jacobs JP, Ortiz-Lopez A, Campbell JJ, Gerard CJ, Mathis D, Benoist C: Deficiency of CXCR2, but not other chemokine receptors, attenuates Authors’ contributions autoantibody-mediated arthritis in a murine model. Arthritis Rheum 2010, KM conceived the experiments. AR and KM designed the experiments, 62:1921-1932. analyzed data and prepared the manuscript, with input from SA, SH and MC. 18. Mancardi DA, Jonsson F, Iannascoli B, Khun H, Van Rooijen N, Huerre M, AR, SA and SH prepared reagents and executed experiments. MC scored the Daeron M, Bruhns P: The murine high-affinity IgG receptor Fc(gamma)RIV histological samples. The manuscript was read and approved by all authors. is sufficient for autoantibody-induced arthritis. J Immunol 2011, 186:1899-1903. Competing interests 19. 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Suzuki A, Yamada R, Chang X, Tokuhiro S, Sawada T, Suzuki M, Nagasaki M, Nakayama-Hamada M, Kawaida R, Ono M, Ohtsuki M, Furukawa H, Yoshino S, Yukioka M, Tohma S, Matsubara T, Wakitani S, Teshima R, Nishioka Y, Sekine A, Iida A, Takahashi A, Tsunoda T, Nakamura Y, Yamamoto K: Functional haplotypes of PADI4, encoding citrullinating enzyme peptidylarginine deiminase 4, are associated with rheumatoid arthritis. Nat Genet 2003, 34:395-402. 45. Yamada R, Suzuki A, Chang X, Yamamoto K: Peptidylarginine deiminase type 4: identification of a rheumatoid arthritis-susceptible gene. Trends Mol Med 2003, 9:503-508. 46. Nakayama-Hamada M, Suzuki A, Kubota K, Takazawa T, Ohsaka M, Kawaida R, Ono M, Kasuya A, Furukawa H, Yamada R, Yamamoto K: Comparison of enzymatic properties between hPADI2 and hPADI4. Biochem Biophys Res Commun 2005, 327:192-200. Submit your next manuscript to BioMed Central 47. Stahl EA, Raychaudhuri S, Remmers EF, Xie G, Eyre S, Thomson BP, Li Y, and take full advantage of: Kurreeman FA, Zhernakova A, Hinks A, Guiducci C, Chen R, Alfredsson L, Amos CI, Ardlie KG, Consortium B, Barton A, Bowes J, Brouwer E, Burtt NP, • Convenient online submission Catanese JJ, Coblyn J, Coenen MJ, Costenbader KH, Criswell LA, Crusius JB, Cui J, de Bakker PI, De Jager PL, Ding B, et al: Genome-wide association • Thorough peer review study meta-analysis identifies seven new rheumatoid arthritis risk loci. • No space constraints or color figure charges Nat Genet 2010, 42:508-514. • Immediate publication on acceptance 48. Lee EK, Kang SM, Paik DJ, Kim JM, Youn J: Essential roles of Toll-like receptor-4 signaling in arthritis induced by type II collagen antibody • Inclusion in PubMed, CAS, Scopus and Google Scholar and LPS. Int Immunol 2005, 17:325-333. • Research which is freely available for redistribution 49. 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PAD4 is not essential for disease in the K/BxN murine autoantibody-mediated model of arthritis

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Copyright © 2012 by Rohrbach et al.; licensee BioMed Central Ltd.
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Medicine & Public Health; Rheumatology; Orthopedics
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Abstract

Introduction: Both murine and human genome-wide association studies have implicated peptidyl arginine deiminase (PAD4) as a susceptibility gene in rheumatoid arthritis (RA). In addition, patients with RA commonly have autoantibodies which recognize PAD4 or and/or citrullinated peptides. This study aims to evaluate the role of PAD4 in the effector phase of arthritis. Methods: PAD4 knock out (KO) and wild type (WT) C57BL/6J mice were injected with K/BxN sera to induce disease. Progression of disease was monitored by measuring paw and ankle swelling and clinical indexes of disease, and pathogenesis was assessed by indexing of clinical progression on paws collected from WT and PAD4 KO mice injected with K/BxN serum. PAD4 activity was determined by visualization of neutrophil extracellular traps (NETs) and immunohistological analysis of histone citrullination. Results: PAD4 activity is readily detectable in the inflamed synovium of WT but not PAD4 deficient animals, as demonstrated by histone citrullination and NET formation. However, PAD4 WT and KO animals develop K/BxN serum transfer disease with comparable severity and kinetics, with no statistically significant differences noted in clinical scores, swelling, joint erosion or joint invasion. Conclusions: PAD4 WT and KO mice develop disease in the K/BxN serum transfer model of arthritis with similar severity and kinetics, indicating that PAD4 is dispensable in this effector phase model of disease. Introduction anti-citrullinated peptide antibodies (ACPAs) exhibit Citrulline-containing proteins are generated through high specificity and sensitivity as diagnostic markers of posttranslational modification of arginine residues in a the disease [7]. Anti-citrulline peptide antibodies can 2+ reaction catalyzed by the Ca -dependent peptidyl argi- appear before disease onset and correlate with the most nine deiminases (PADs). There are five PAD family erosive form of RA [8]. members, but only PAD2 and PAD4 expression are clo- PAD4 shows elevated expression in RA [1,9], and RA sely linked with inflammation in RA synovial tissue patients generate high affinity anti-PAD4 autoantibodies [1,2]. While PAD2 is broadly expressed across tissue which correlate with more severe disease [10-12]. types, including by immune cells, PAD4 exhibits an Further, variants of PAD4 are linked to RA in several expression pattern restricted to immune cell types, in Japanese and Korean cohorts, although this association particular macrophages and granulocytes [1,3]. has not held up in most North American and European Rheumatoid arthritis (RA) is a chronic autoimmune study groups, despite the prevalence of ACPA in all eth- nic groups [13]. Thus, the development of autoantibo- disease characterized by systemic inflammation, chronic synovitis, joint destruction and bone loss, affecting dies to citrullinated epitopes and PAD4 and elevated approximately 2% of the world population [4]. Plasma PAD4 expression in RA, suggests that aberrant PAD and synovial biopsy specimens from patients with RA activity may contribute to disease pathogenesis. contain high levels of citrullinated proteins [5,6], and The offspring of an intercross between the KRN TCR transgenic mouse specific for a bovine RNase (42-56) in k g7 the context of I-A and the I-A -expressing non-obese * Correspondence: [email protected] Department of Chemical Physiology, The Scripps Research Institute, 10550 diabetic (NOD) background, spontaneously develop a North Torrey Pines Road, La Jolla, California, 92037, USA progressive, inflammatory joint disease with features Full list of author information is available at the end of the article © 2012 Rohrbach et al.; licensee BioMed Central Ltd. This is an open access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/2.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. Rohrbach et al. Arthritis Research & Therapy 2012, 14:R104 Page 2 of 10 http://arthritis-research.com/content/14/3/R104 similar to human RA (K/BxN mice) [14]. The autoanti- Recently, using a genome-wide screen of mice, John- gen in this model is glucose-6-phosphate isomerase sen et al. identified the region containing Padi4 as being (GPI), a ubiquitous cytoplasmic enzyme [15]. Treatment putatively involved in the development of K/BxN arthri- with the sera of K/BxN mice or purified anti-GPI auto- tis and demonstrated that increases in the transcripts of antibodies is sufficient to transfer disease to healthy ani- both PAD2 and PAD4 correlates with increased severity mals, even in animals devoid of B and T cells [14,15]. of disease [35]. Because the K/BxN model is neutrophil dependent, PAD4 activity is required for NET formation Because autoantibodies are passively transferred, this and PAD4 overexpression correlates with rheumatoid model focuses on immune recruitment and joint arthritis in patients, we sought to determine the extent destruction (effector phase), rather than the breaking of immune tolerance (priming phase). Innate immune sig- to which PAD4 would contribute to the effector phase nals are critical for this model because mice deficient in of arthritis using the K/BxN-serum transfer model the alternative complement pathway, the C5a receptor, [20,25,27,36]. the CXCR2 chemokine receptor, interleukin-1 receptor (IL-1R), and myeloid differentiation primary response Materials and methods protein (MyD88) are resistant to disease [14,16,17]. Mice Further, arthritis was not sustained in toll-like receptor PAD4 knockout (KO) mice were generated as previously 4 (TLR4) mutant mice [16]. Passively transferred arthri- described [25]. KRN T cell receptor (TCR) transgenic tis also requires the presence of mast cells and neutro- mice were a gift from Drs D Mathis and C Benoist phils [18-21]. (Harvard Medical School, Boston, MA) and the Institut Neutrophils are among the first immune cell types to de GénétiqueetdeBiologieMoléculaire et Cellulaire accumulate during an inflammatory response [22]. In (Strasbourg, France) [14]. Wildtype C57BL/6J mice were response to inflammatory stimuli, neutrophils decon- obtained from the Scripps Research Institute rodent dense their chromatin and actively expel their DNA- breeding colony. Experiments were reviewed and producing neutrophil extracellular traps (NETs) that are approved by the Scripps Research Institute Institutional decorated with granular and nuclear proteins, including Animal Care and Use Committee. citrullinated histones [23,24]. Incubation of neutrophils with phorbol 12-myristate 13-acetate (PMA), hydrogen Induction of serum-transfer arthritis peroxide, lipopolysaccharide (LPS), bacteria, and yeast K/BxN serum was collected from 6- to 10-week-old K/ induces NET formation [24-27]. Our lab and others BxN mice and pooled, and 5- to 6-week-old male PAD4 have shown that PAD4 is essential for the production of KO or C57BL/6J WT control mice were injected intra- NETs and NET-associated histone citrullination peritoneally (i.p.) with 150 μL pooled K/BxN sera on [24,25,27]. PAD4-mediated histone citrullination is days 0 and 2. Caliper measurements of paw and ankle thoughttoplayamechanicalroleinNET formation, swelling were taken daily. Clinical indexes of swelling where the conversion of positively charged arginine resi- were assigned according to previously described criteria dues into the neutral citrulline amino acid by PAD4 [37]. Animals were sacrificed at day 5 or day 10 and the promotes chromatin decondensation [24]. PAD4- hind paws were prepared for fluorescence microscopy mediated NET formation is critical for controlling at or histology. least a subset of bacterial infections because PAD4-defi- cient mice are more susceptible to infectious disease in Fluorescence Microscopy of Histone Citrullination and a necrotizing fasciitis model [27]. NETs NET formation, although critical for the full activation Hind paws were removed from euthanized animals at of the innate immune response [27], has also been day 5, post K/BxN serum injection. Ankle joints were implicated in inflammatory disease pathogenesis, includ- exposed and flushed with Roswell Park Memorial Insti- ing the autoimmune disorder lupus [28], cystic fibrosis tute media (RPMI) with 0.5% BSA. Peripheral blood was ([29-31], sepsis [32], and thrombosis [33]. Interestingly, collected by eye bleed. Erythrocytes were lysed in 10 it has been suggested that NETs offer a possible mM KHCO , 150 mM NH Cl NS 1 mM EDTA and 3 4 mechanism by which PAD4 may be liberated from the remaining cells were washed with PBS. Ankle or periph- cell to generate citrullinated antigens and exacerbate eral blood cells were attached to poly-lysine-coated glass inflammation [9,34]. Recently, Dwievedi et al. described coverslips and fixed in 4% paraformaldehyde (PFA) with hypercitrullination in neutrophils from arthritic patients, 1% Nonidet P-40 (NP-40) and 0.5% Triton X-100 in as well as the specific reactivity of arthritic serum to PBS. Slides were blocked with 2.5% BSA and 5% goat activated neutrophils and citrullinated histones [34]. It is serum in PBS with 0.5% Tween-20. Citrullinated his- unclear whether PAD4-induced NET formation plays a tones were detected with anti-H4Cit3 (Millipore, Biller- role in the RA inflammatory process. ica, MA, USA) and goat anti-rabbit Alexa488 (Life Rohrbach et al. Arthritis Research & Therapy 2012, 14:R104 Page 3 of 10 http://arthritis-research.com/content/14/3/R104 Technologies, Grand Island, NY, USA). DNA was within synovium throughout the serum-transfer during labeled with DAPI (Sigma, St. Louis MO, USA). Cover- the course of arthritis disease [17,20]. Importantly, slips were mounted with Prolong Gold Antifade reagent PAD4 is highly expressed in the granulocyte lineage, (Life Technologies, Grand Island, NY, USA). including neutrophils [3] and is known to be required for the formation of Neutrophil Extracellular Traps Histology (NETs) [25,27]. NETs have been shown to occur in sev- Hindpaws were removedfromeuthanizedanimals, eral inflammatory disease states, including lupus [28], fixed in 4% paraformaldehyde in PBS overnight and dec- sepsis [32,33] and cystic fibrosis [29-31] and are thought alcified in 0.375 M EDTA pH 7.5 for 3 weeks. Paws to serve as a means for PAD4 to enter into the extracel- were embedded in paraffin and affixed to slides for lular space, where it may citrullinate substrates and immunohistochemistry or pathological analysis by The exacerbate inflammation [9]. NETs are common to sev- Scripps Research Institute Histology Core. For immuno- eral other inflammatory disease states, but to our knowl- histochemistry, paraffin-embedded slices on slides were edge, NET structures, per se, have not been reported in deparaffinized with xylene and rehydrated. Antigens patients with RA or in murine models of disease. How- were unmasked by boiling in PBS pH 7.4. Slides were ever, recently, Dwivedi et al. reported that serum IgGs blocked with 10% normal goat serum and endogenous from patients with Felty’s syndrome react to NETs and peroxidases were quenched with 3% peroxide in metha- preferentially to citrullinated histones over unmodified nol. Sections were then incubated with anti-H4cit3 or histones, indicating that NET markers, and thus NETs, control rabbit immunoglobulin (IgG) antibodies (Milli- maybepresent in Felty’ssyndrome[34]. SinceACPA pore, Billerica, MA, USA). Labeling was detected with a and anti-PAD4 antibodies are frequently found in biotinylated anti-rabbit secondary antibody (Life Tech- patients with RA [9], it is presumed that aberrant PAD nologies, Grand Island, NY, USA), Vectastain Elite ABC activity influences the priming phase of arthritis. How- reagent (Vector Labs, Burlingame, CA, USA) and DAB ever, it is unknown where PAD4 may also regulate the (Life Technologies, Grand Island, NY, USA). Slides were effector phase of arthritis. We therefore considered counterstained with Shandon Harris hematoxylin whether PAD4-mediated NET formation could contri- (Thermo Scientific, Waltham, MA, USA). Histopatholo- bute to K/BxN-induced inflammation. gical scoring was performed blinded as described pre- To determine whether NET formation occurs in the viously [16]. Briefly, ankles of arthritic mice were given K/BxN serum transfer model, fluorescence microscopy scores of 0 to 5 for inflammation, according to the fol- of flushed synovial cells was performed. PAD4 WT and lowing criteria: 0 = normal; 1 = minimal infiltration of KO mice were injected with K/BxN serum on days 0 inflammatory cells in the periarticular area; 2 = mild and 2. Mice were sacrificed at day 5 to collect synovial infiltration; 3 = moderate infiltration; 4 = marked infil- and peripheral blood cells. Cells flushed from the ankles tration; and 5 = severe infiltration. Knees of arthritic of arthritic WT mice displayed citrullinated histones, a mice were given scores of 0 to 5 for bone resorption, hallmark of PAD4 activity, and NET formation, while according to the following criteria: 0 = normal; 1 = those from PAD4 KO mice exhibited no detectable his- minimal (small areas of resorption, not readily apparent tone citrullination or NET formation, as detected by on low magnification); 2 = mild (more numerous areas anti-H4Cit3 and 4’,6-diamidino-2-phenylindole (DAPI) of resorption, not readily apparent on low magnification, (Figure 1A). As expected, few cells could be flushed in trabecular or cortical bone); 3 = moderate (obvious from the ankles of WT control mice, and none were resorption of trabecular and cortical bone, without full- observed to have citrullinated histones or to form NETs thickness defects in the cortex; loss of some trabeculae; (Figure 1A). Histone citrullination, but not NET forma- lesions apparent on low magnification); 4 = marked tion, was detected in the peripheral blood of WT K/ (full-thickness defects in cortical bone and marked tra- BxN mice, but not WT control mice (Figure 1B). becular bone loss; without distortion of the profile of Neither histone citrullination nor NET formation was the remaining cortical surface) and 5 = severe (full- found in control mice of either genotype (Figure 1B). thickness defects in the cortical bone and marked trabe- Thus, NET formation does occur in the K/BxN model cular bone loss with distortion of the profile of the of the effector phase of RA and is specific for the remaining cortical surface). arthritic ankle. Results There is abundant PAD4 activity during K/BxN serum- NETs are found in the joints of K/BxN serum-transfer transferred arthritis animals PAD4 is found in the synovial fluid and infiltrating syno- Neutrophils are the predominant infiltrating cell type in vial cells of patients with RA [9,38,39]. In the K/BxN the K/BxN serum transfer arthritis model and are found serum transfer model, elevated expression of PAD4 in the Rohrbach et al. Arthritis Research & Therapy 2012, 14:R104 Page 4 of 10 http://arthritis-research.com/content/14/3/R104 DAPI H4Cit3-Alexa488 Overlay WT Ankle K/BxN PAD4KO Ankle K/BxN WT Ankle Control WT Peripheral Blood K/BxN PAD4 KO Peripheral Blood K/BxN WT Peripheral Blood Control 0.1 mm Figure 1 Wild type (WT) K/BxN serum transfer mice generate synovial neutrophil extracellular traps (NETs). Mice were injected with K/ BxN serum on days 0 and 2 and sacrificed on day 5. Synovial cells were flushed from the ankle joint (A) and peripheral blood was collected from the venous plexus of arthritic or healthy mice (B) and stained with antibodies to citrullinated histone H4 and 4’,6-diamidino-2-phenylindole (DAPI). Histone citrullination and NETs were readily detectable in the arthritic joints of WT mice, but were not observed in peptidyl arginine deiminase 4 knockout (PAD4 KO) K/BxN mice or WT healthy controls (A). Histone citrullination was present in the peripheral blood of WT K/BxN mice, however, no NET formation was observed. Images were taken at 80× magnification and are representative of at least three independent experiments with two mice per experimental group. Rohrbach et al. Arthritis Research & Therapy 2012, 14:R104 Page 5 of 10 http://arthritis-research.com/content/14/3/R104 spleen correlates with the severity of arthritis [35]. To disease and the overall kinetics of disease were similar determine whether PAD4 enzymaticactivitywas ahall- between WT and PAD4 KO mice (Figure 3). Although mark of the effector phase of arthritis, immunohistochem- there was a trend toward reduced swelling and lower istry of the PAD4-dependent citrullinated histone H4 was disease scores in PAD4 KO mice, the differences were performed [25,27]. Mice were injected with K/BxN serum small and did not reach statistical significance (Figure on days 0 and 2. On day 10, mice were sacrificed, hind 3). Disease is transient in the serum transfer model. We limbs were harvested and prepared for histology, and tis- foundthatPAD4KO micerecoveredwithsimilar sue sections were immunostained for citrullinated histone kinetics to WT mice (data not shown). Thus, PAD4 is H4. WT mice displayed robust histone H4 citrullination, not required for the development or maintenance of K/ with the most prominent staining in infiltrating cells BxN-serum transferred arthritis. within the synovial sublining, which were most likely neu- trophils. This modification was dependent upon PAD4 Comparable histological features between arthritic WT because PAD4 KO mice exhibited no staining for citrulli- and PAD4-deficient mice nated histone H4 (Figure 2). These data demonstrate that Although WT and PAD4 KO mice exhibited a similar robust PAD4 activity is present within the ankle joint dur- level of inflammation, the degree of joint destruction ing the effector phase of arthritis. and inflammatory infiltrates could be affected by PAD4 deficiency. Thus, hind limbs were harvested from WT PAD4 deficiency does not protect against disease in and PAD4 KO mice on day 10 following serum transfer. serum-transferred arthritis Histological evaluation by an investigator blinded to the To test the importance of PAD4 in the effector phase of animal genotype for degree of inflammation and erosion arthritis, we injected K/BxN serum into age-matched of bone was performed. Inflammation and bone erosions WT and PAD4 KO C57BL6/J mice on days 0 and 2 and were quantified by scoring joint sections on a 0 to 5 monitored disease progression. Measurements of paw scale. Hematoxylin and eosin stain (H&E) sections of swelling and clinical disease index were taken daily for the ankle joints revealed a comparable, moderate degree ten days. Paw swelling was calculated as the percentage of inflammatory cell infiltration in WT and PAD4 KO increase in paw size compared to day 0. The onset of mice (Figure 4 and Table 1). In healthy mice, the Figure 2 Peptidyl arginine deiminase 4 (PAD4) activity is detected in K/BxN arthritic legs in wild type (WT) but not PAD4 knockout (KO) mice. PAD4 activity was measured by immunohistochemical detection of citrullinated histone H4 (H4Cit3). WT and PAD4 KO mice were immunized with pooled K/BxN serum as described. Mice were sacrificed 10 days post injection (p.i.) and their legs prepared for histology. Sections were blocked in goat serum and probed with either anti-H4Cit3 antibody or isotype control. Images were taken at 80× magnification. Specific staining of citrullinated histone H4 was apparent on WT but not PAD4 KO slides and is representative of at least 4 independent experiments. Rohrbach et al. Arthritis Research & Therapy 2012, 14:R104 Page 6 of 10 http://arthritis-research.com/content/14/3/R104 Figure 3 Progression of K/BxN serum transfer arthritis in peptidyl arginine deiminase 4 (PAD4) wild type (WT) (n = 29) and knockout (KO) (n = 27) mice. WT and PAD4 KO mice were immunized with pooled K/BxN serum as described and monitored for signs of disease severity for 10 days. Paw swelling (A) is expressed as the average percent size increase of four paws and two ankles compared to day 0 measured by caliper. Clinical indexes (B) of 0 to 3 were assigned for each paw as previously described [37]. Data are combined from three independent experiments and represented as the average ± standard error of the mean (SEM). No statistically significant difference in the progression of K/BxN disease was apparent between WT and PAD4 KO mice. Rohrbach et al. Arthritis Research & Therapy 2012, 14:R104 Page 7 of 10 http://arthritis-research.com/content/14/3/R104 Figure 4 Pathological evaluation of immune invasion and joint destruction in wild type (WT) and peptidyl arginine deiminase 4 knockout (PAD4 KO) K/BxN mice.WT(n = 27) and PAD4 KO (n = 29) animals were immunized with pooled K/BxN serum as described, sacrificed at day 10 post injection (p.i.) and their legs prepared for histology. Hematoxylin- and eosin-stained stained slides were scored blinded by a trained observer for immune cell invasion (0 to 5) and joint erosion (0 to 5). Pictures were taken at 40× magnification and are representative of the average scores for invasion (4) and erosion (2), shown in Table 1. Areas of immune cell invasion into cartilage of joint (closed arrows) and bone reabsorption (open arrows) are highlighted. synovium consists of a single layer. Both WT and PAD4 benefit in the collagen-induced arthritis model but has KO mice developed a proliferative synovial response fol- no benefit when arthritic disease is induced by the lowing K/BxN serum treatment. Furthermore, WT and administration of anti-collagen antibodies. Collagen PAD4 KO mice exhibited a similar level of bone erosion. induced arthritis and injection of anti-collagen antibo- Therefore, the histological features of the K/BxN serum- dies represent the priming and effector phases of dis- mediated model of arthritis are not altered in the ease, respectively. We have now shown that PAD4 is absence of PAD4. active but not required for disease, in another model of the effector phase, the K/BxN serum transfer model, Discussion which is consistent with the finding that PAD inhibitors In this study, we find that the PAD4 enzyme is active have no effect on disease when induced by administra- tion by anti-collagen antibodies [40]. Interestingly, Cl- within the joint tissue of arthritic mice and leads to NET formation. However, the disease course and histo- amidine preferentially inhibits PADs 1 and 4 over logical features of the arthritic joints following K/BxN PAD3, and its effects have not been reported for PADs serum injection were comparable between WT and 2 or 6 [41]. PAD4-deficient mice, demonstrating that PAD4 is not Indeed, Johnsen et al. identified the Padi locus, which required for the effector phase of arthritis. Our data are contains the PAD4 gene along with the genes for PADs consistent with the findings by Willis et al., showing 1, 2, 3, and 6, as being linked with disease in the K/BxN that the PAD inhibitor Cl-amidine provides therapeutic 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 Table 1 Clinical scores of inflammation and joint erosion types, whereas the expression of PADs 1, 3, and 6 are in wild-type and peptidyl arginine deiminase 4 knockout restricted to the epidermis, hair follicle, and oocyte, K/BxN mice respectively. Indeed, increased splenic expression of Group Inflammation (0 to 5) Joint Erosion (0 to 5) both PAD2 and PAD4 correlated with disease severity (mean ± STDEV) (mean ± STDEV) in the K/BxN model [35]. Further, within the PAD WT 3.4 ± 1.3 2.5 ± 1.4 region, a SNP found within the Padi2 locus showed the PAD4 KO 3.6 ± 1.0 2.2 ± 1.7 most significant association with disease [35]. We specu- Pathological scoring of immune invasion and joint destruction in wild type late that the loss of PAD2 and PAD4 together may pro- (WT) and peptidyl arginine deiminase 4 knockout (PAD4 KO) K/BxN mice. WT (n = 27) and PAD4 KO (n = 29) animals were immunized with pooled K/BxN duce a more apparent phenotype in the K/BxN model serum as described, sacrificed at day 10 post injection (p.i.), and their legs [40]. PAD2 KO mice have been reported [42], however prepared for histology. Hematoxylin- and eosin-stained slides were scored blinded by a trained observer for immune cell invasion (0 to 5) and joint the PAD2 and PAD4 loci are approximately 1 centimor- erosion (0 to 5). Results are represented as the average of 27 and 29 mice per gan apart, and therefore, the recombination frequency genotype ± standard deviation. Average scores for joint invasion and erosion between the targeted PAD2 and PAD4 alleles would be were similar in WT and PAD4 KO mice. Rohrbach et al. Arthritis Research & Therapy 2012, 14:R104 Page 8 of 10 http://arthritis-research.com/content/14/3/R104 quite small, making the generation of Padi2/Padi4 DKO vessels [51]. Systemic lupus erythematous (SLE) is a sys- mice unlikely. While our data demonstrate that PAD4 is temic autoimmune disease characterized by the forma- not required for the development of the K/BxN serum- tion of pathogenic immune complexes. When activated transfer model, it is possible that there might be redun- by autoantibodies, neutrophils isolated from patients dant activity of other PADs or they may independently with SLE produce NETs, exposing immunostimulatory contribute to the pathogenesis of antibody-mediated proteins and potential autoantigens and leading to the induction of Type I interferons by plasmacytoid dendri- arthritis. tic cells [28,52,53]. Collectively, these results support the The blood of RA patients contains autoantibodies notion that NET production can contribute to disease directed against a number of self-antigens. Many auto- antibodies in RA are directed against citrullinated pro- pathogenesis in inflammatory conditions. While hyperci- teins. In fact, the presence of anti-citrulline antibodies is trullination of neutrophil histones has been reported in a better predictor of RA than rheumatoid factor [43]. patients with RA [34], it is unclear whether NETs have Variants of PAD4 are linked to RA in several Japanese a role in RA inflammation. Our results suggest that and Korean cohorts, and the mRNA of a disease-asso- PAD4 activity and subsequent NET formation is present ciated allele is more stable than a non-disease associated in the K/BxN serum transfer model, but is not required allele [36,44]. It has been proposed that PAD4 is linked for this model of effector phase of disease. to RA because PAD4 citrullination of peptides leads to a Our data demonstrate that PAD4 is not necessary for breakdown in tolerance to self-antigens [43,45]. In sup- the antibody-dependent, effector stage of arthritis. It is port of this, treatment of mice with the PAD inhibitor also possible that compensation by other PAD family Cl-amidine in the collagen-induced arthritis (CIA) members, PAD2 in particular, may mask the function of model reduces the levels of citrulline found in the PAD4 in arthritis, although we note that PAD2 expres- serum and synovial tissue, diminishes the formation of sion is not upregulated in PAD4-deficient neutrophils autoantibodies, and ameliorates disease [40]. Thus, it [25]. Finally, since the Padi locus is linked to disease will be interesting to determine whether the effects of severity in the K/BxN serum transfer model, it may be Cl-amidine are attributable to PAD4 activity by examin- necessary to eliminate several PAD family members, ing the susceptibility of PAD4 KO mice to arthritic dis- either by targeting multiple locations within the PAD ease using the CIA model. locus or by combining treatment with specific PAD Incubation of human neutrophils with lipopolysac- inhibitors with targeted PAD alleles. Further studies will be necessary to dissect the role of PAD4 in the priming charides (LPS), TNFa, N-formyl-methionine- leucine- phase of arthritis. phenylalanine (fMLP), or lipoteichoic acid and murine neutrophils with LPS or bacteria, has been shown to induce histone deimination and NET formation, mark- Conclusions ing PAD4 activity [24-27]. Further, we detected deimi- NET formation is known to correlate with inflammatory nated histone H4 in lung leukocytes isolated from disease [28,32,33,52,53], however NET formation has influenza-infected mice [25]. In this report, we find that not been reported in RA. NET formation is dependent PAD4 activity is readily detected within the affected on PAD4 and the association between PAD4 and RA is arthritic joint. In WT mice receiving K/BxN serum, the well-established [9]. In this report, we show that in the presence of deiminated histones corresponded primarily K/BxN murine model of arthritis, which emulates the to the infiltrating cells of the joint sublining, which is effector phase of disease, PAD4 activity and NET forma- consistent with the expression pattern of PAD4 found tion are detected. PAD4 activity is not, however, in patients with RA [46,47]. The stimulus that induces required for disease, as PAD4 WT and KO mice develop PAD activity during autoimmune-mediated inflamma- K/BxN-induced arthritis with similar severity and tion is undefined. However, the LPS receptor TLR4, has kinetics. Overall our data indicate that PAD4 is dispen- been linked to animal models of arthritis, perhaps sable in this model. However this does not eliminate a because of the activation of TLR4 by endogenous role for PAD4 in the priming phase of disease. Future ligands, such as Tenascin-C [16,48,49]. studies will consider the role of PAD4 in priming phase NETs possess potent microbicidal capabilities but have models of RA, including the CIA model. also been implicated in chronic inflammatory diseases [23,50]. NET formation is linked to cystic fibrosis Abbreviations [29-31]. Similarly, the formation of NETs contributes to ANCA: anti-neutrophil cytoplasmic antibodies; CIA: Collagen-induced arthritis; endothelial and tissue injury during sepsis [32]. In auto- DAPI: 4’,6-diamidino-2-phenylindole; fMLP: formyl-methionine-leucine- phenylalanine; H&E: hematoxylin and eosin stain; IgG: immunoglobulin; IL-1R: immune small-vessel vasculitis, anti-neutrophil cyto- interleukin-1 receptor; KO: knockout; LPS: lipopolysaccharide; MyD88: myeloid plasm antibodies (ANCA) trigger the formation of differentiation primary response protein; PAD: Peptidyl arginine deiminase; NETs, promoting necrotic inflammation of the blood PMA: phorbol 12-myristate 13-acetate; NET: neutrophil extracellular trap; Rohrbach et al. Arthritis Research & Therapy 2012, 14:R104 Page 9 of 10 http://arthritis-research.com/content/14/3/R104 NOD: non-obese diabetic;RA: rheumatoid arthritis; SLE: systemic lupus 12. Pollmann S, Stensland M, Halvorsen EH, Sollid LM, Kvien TK, Fleckenstein B, erythematous; SNP: single nucleotide polymorphism; TCR: T cell receptor; Molberg O: Anti-PAD4 autoantibodies in rheumatoid arthritis: levels in TLR: toll-like receptor; WT: wild type. serum over time and impact on PAD4 activity as measured with a small synthetic substrate. Rheumatol Int 2012, 32:1271-1276. Acknowledgements 13. van der Helm-van Mil AH, Huizinga TW: Advances in the genetics of Dr Arandjelovic was supported by an Arthritis Foundation Fellowship award. rheumatoid arthritis point to subclassification into distinct disease Ms. Rohrbach, and Drs Hemmers, Arandjelovic, and Mowen were supported subsets. Arthritis Res Ther 2008, 10:205. by the NIH (National Institute of Allergy and Infectious Diseases [NIAID] 14. Kouskoff V, Korganow AS, Duchatelle V, Degott C, Benoist C, Mathis D: grant AI067460 and National Institute of General Medicine [NIGMS] grant Organ-specific disease provoked by systemic autoimmunity. Cell 1996, GM085117-01). Dr Corr’s work was supported by the Arthritis Foundation. 87:811-822. This is manuscript #21621 from The Scripps Research Institute. 15. Matsumoto I, Staub A, Benoist C, Mathis D: Arthritis provoked by linked T and B cell recognition of a glycolytic enzyme. Science 1999, Author details 286:1732-1735. Department of Chemical Physiology, The Scripps Research Institute, 10550 16. Choe JY, Crain B, Wu SR, Corr M: Interleukin 1 receptor dependence of North Torrey Pines Road, La Jolla, California, 92037, USA. Department of serum transferred arthritis can be circumvented by toll-like receptor 4 Medicine, University of California, San Diego, 9500 Gilman Drive, La Jolla, signaling. J Exp Med 2003, 197:537-542. California, 92093, USA. 17. 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Stahl EA, Raychaudhuri S, Remmers EF, Xie G, Eyre S, Thomson BP, Li Y, and take full advantage of: Kurreeman FA, Zhernakova A, Hinks A, Guiducci C, Chen R, Alfredsson L, Amos CI, Ardlie KG, Consortium B, Barton A, Bowes J, Brouwer E, Burtt NP, • Convenient online submission Catanese JJ, Coblyn J, Coenen MJ, Costenbader KH, Criswell LA, Crusius JB, Cui J, de Bakker PI, De Jager PL, Ding B, et al: Genome-wide association • Thorough peer review study meta-analysis identifies seven new rheumatoid arthritis risk loci. • No space constraints or color figure charges Nat Genet 2010, 42:508-514. • Immediate publication on acceptance 48. Lee EK, Kang SM, Paik DJ, Kim JM, Youn J: Essential roles of Toll-like receptor-4 signaling in arthritis induced by type II collagen antibody • Inclusion in PubMed, CAS, Scopus and Google Scholar and LPS. Int Immunol 2005, 17:325-333. • Research which is freely available for redistribution 49. Abdollahi-Roodsaz S, Joosten LA, Roelofs MF, Radstake TR, Matera G, Popa C, van der Meer JW, Netea MG, van den Berg WB: Inhibition of toll- Submit your manuscript at www.biomedcentral.com/submit

Journal

Arthritis Research & Therapy – Springer Journals

Published: May 2, 2012

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