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Nardilysin Is Involved in Autoimmune Arthritis Via the Regulation of Tumour Necrosis Factor Alpha Secretion

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Nardilysin Is Involved in Autoimmune Arthritis Via the Regulation of Tumour Necrosis Factor Alpha Secretion Animal models RMD Open: first published as 10.1136/rmdopen-2017-000436 on 13 July 2017. Downloaded from ORIGINAL articLE Nardilysin is involved in autoimmune arthritis via the regulation of tumour necrosis factor alpha secretion Takayuki Fujii,1 Eiichiro Nishi,2,3 Hiromu Ito,1 Hiroyuki Yoshitomi,4 Moritoshi Furu,5 Namiko Okabe,6 Mikiko Ohno,2 Kiyoto Nishi,2 Yusuke Morita,2 Yugo Morita,1 Masayuki Azukizawa,1 Akinori Okahata,1 Takuya Tomizawa,1 Takeshi Kimura,2 Shuichi Matsuda1 To cite: Fujii T, Nishi E, ABSTRACT Key messages Ito H, et al. Nardilysin is Objective Tumour necrosis factor alpha (TNF-α) plays involved in autoimmune an important role in rheumatoid arthritis (RA). TNF-α is arthritis via the regulation synthesised as a membrane-anchored precursor and is What is already known about this subject? of tumour necrosis factor fully activated by a disintegrin and metalloproteinase 17 ► Tumour necrosis factor alpha (TNF-α), which alpha secretion. RMD Open (ADAM17)-mediated ectodomain shedding. Nardilysin is activated by ectodomain shedding, plays an 2017;3:e000436. doi:10.1136/ important role in rheumatoid arthritis (RA). rmdopen-2017-000436 (NRDC) facilitates ectodomain shedding via activation of ADAM17. This study was undertaken to elucidate the role ► Nardilysin facilitates TNF-α shedding via enhancing of NRDC in RA. a disintegrin and metalloproteinase 17 activity. Additional material is –/– ► Methods NRDC-deficient Nrdc( ) mice and macrophage- published online only. To view What does this study add? specific NRDC-deficientNrdc ( delM) mice were examined please visit the journal online ► Deletion or inhibition of nardilysin prevents (http:// dx. doi. org/ 10. 1136/ in murine RA models, collagen antibody-induced arthritis autoantibody-induced arthritis, murine models of rmdopen- 2017- 000436). (CAIA) and K/BxN serum transfer arthritis (K/BxN STA). RA. We evaluated the effect of gene deletion or silencing of ► Concentration of nardilysin in synovial fluid is on ectodomain shedding of TNF- in macrophages Nrdc α markedly increased in patients with RA compared or monocytes. NRDC concentration in synovial fluid from http://rmdopen.bmj.com/ Received 16 January 2017 with that in patients with osteoarthritis, suggesting Revised 23 March 2017 patients with RA and osteoarthritis (OA) were measured. a crucial role of nardilysin in the pathogenesis of RA. Accepted 19 April 2017 We also examined whether local gene silencing of Nrdc ameliorated CAIA. How might this impact on clinical practice? Results CAIA and K/BxN STA were significantly attenuated ► Nardilysin could be a novel biomarker and –/– delM in Nrdc mice and Nrdc mice. Gene deletion or therapeutic target for RA. silencing of Nrdc in macrophages or THP-1 cells resulted in the reduction of TNF-α shedding. The level of NRDC is higher in synovial fluid from RA patients compared with 2 that from OA patients. Intra-articular injection of anti- mice develop arthritis, TNF-α is produced on September 26, 2021 by guest. Protected copyright. Nrdcsmall interfering RNA ameliorated CAIA. in synovial tissues of murine experimental Conclusion These data indicate that NRDC plays crucial models of RA,3 murine experimental arthritis roles in the pathogenesis of autoimmune arthritis and is attenuated in TNF-α-deficient mice4 could be a new therapeutic target for RA treatment. and neutralisation of TNF-α ameliorates murine collagen-induced arthritis.5 TNF-α is produced mostly by macrophages in RA INTRODUCTION synovial tissues6 and, alone or in combination Rheumatoid arthritis (RA) is a chronic with other cytokines or chemokines, recruits inflammatory disease that causes joint inflam- inflammatory cells such as B cells, T cells and mation and destruction affecting 0.5%–1.0% neutrophils, and induces production of cyto- of the population.1 Anti-tumour necrosis kines, chemokines, matrix metalloproteinases factor alpha (TNF-α) biologic agents have or receptor activator of nuclear factor κB For numbered affiliations see led to a revolutionary improvement in the ligand, resulting in joint inflammation and end of article. management of RA patients, indicating that bone resorption.7–11 this inflammatory cytokine plays a key role in One of the earliest subclinical features of RA Correspondence to Dr Eiichiro Nishi; nishi@ kuhp. RA pathogenesis. The importance of TNF-α is a breakdown of self-tolerance. Autoantibodies kyoto-u. ac. jp and Dr Hiromu Ito; has been also demonstrated in murine models such as rheumatoid factor (RF) and anti-citrul- hiromu@ kuhp. kyoto- u. ac. jp of RA; for example, human TNF-α transgenic linated peptide antibody (ACPA) are found in Fujii T, et al. RMD Open 2017;3:e000436. doi:10.1136/rmdopen-2017-000436 1 RMD Open RMD Open: first published as 10.1136/rmdopen-2017-000436 on 13 July 2017. Downloaded from the patients’ serum long before arthritis occurs.12 While the been admitted to Kyoto University Hospital. All of the RA autoantigens have not been fully identified, autoantibodies patients met 2010 American College of Rheumatology may contribute to the pathogenesis of RA via immunocom- and European League against Rheumatism (ACR/ plex (IC) formation and deposition in synovial tissues.13 EULAR) classification criteria for RA. Peripheral blood The importance of autoantibodies has been demonstrated samples were obtained and the level of C-reactive protein in mouse autoantibody-induced arthritis (AAIA) models of (CRP), erythrocyte sedimentation rate (ESR), RF, and RA, such as collagen antibody-induced arthritis (CAIA) and ACPA was examined before surgery. Disease activity score K/BxN serum transfer arthritis (K/BxN STA). Antibodies 28 (DAS28) was calculated according to the results of against intra-articular antigens, type II collagen in CAIA14 15 physical examinations and blood tests.32 The full methods and glucose-6-phophoisomerase in K/BxN STA16 17 are arthro- are provided in the online supplementary material. genic in mice. TNF-α has been demonstrated to be important in both models: anti-TNF-α antibody attenuates CAIA,18 while RESULTS TNF-α-deficient mice are resistant to K/BxN STA.4 Nrdc-deficient mice are resistant to CAIA Importantly, TNF-α is synthesised as a membrane-an- To explore the role of NRDC in RA, we compared wild- chored precursor and is fully activated by a disintegrin and type (WT) and Nrdc–/– mice with a BALB/c background metalloproteinase 17 (ADAM17)-mediated ectodomain using the CAIA model. After the intraperitoneal admin- shedding,19 indicating that ADAM17 is an attractive alter- istration of a five-clone cocktail of monoclonal anti-type native target for treatment of RA. However, the multiple II collagen antibodies, we assessed the severity of arthritis disease phenotypes of ADAM17-deficient patients and mice daily for 2 weeks. The onset of arthritis was delayed (on have raised concerns about the potential adverse effects of day 3 in WT mice and on day 6 in Nrdc–/– mice, respec- anti-ADAM17 therapy.20 21 We have previously shown that tively) and the average of arthritis score was significantly ADAM17 activity and TNF-α shedding are enhanced by and markedly lower in Nrdc–/– mice (figure 1A,B). Ankle nardilysin, N-arginine dibasic convertase (NRDC), via its thickness was also significantly less in Nrdc–/– mice from direct binding to ADAM17.22–24 days 4 to 9 compared with that in WT mice. Histological NRDC is a zinc peptidase of the M16 family that selec- examination of the ankle joints demonstrated that syno- tively cleaves dibasic sites.25 26 We identified NRDC on vial hypertrophy with infiltration of inflammatory cells the cell surface as a specific binding partner of hepa- and bone and cartilage erosion were reduced or absent rin-binding epidermal growth factor (EGF)-like growth in Nrdc–/– mice (figure 1C). These results suggest that factor (HB-EGF) and demonstrated that NRDC enhances NRDC is crucial for AAIA development and progression. ectodomain shedding of HB-EGF.27 Our subsequent studies revealed that NRDC enhances ectodomain shed- Macrophage-specificNrdc -deficient mice are resistant to ding of not only HB-EGF but also a wide range of other CAIA http://rmdopen.bmj.com/ 23 28 29 membrane proteins including TNF-α, neuregulin 1 Because macrophages play an important role in the 22 and amyloid precursor protein. We have also demon- pathogenesis of RA, we next generated NrdcdelM mice by strated that NRDC-induced ectodomain shedding plays crossing Nrdcflox/flox mice with Lyz2-Cre mice, in which Cre critical roles in myelination, gastric cancer and Alzhei- recombinase is expressed under the control of the lyso- 22–24 28–31 mer’s disease. zyme 2 gene (Lyz2) promoter. Because the Nrdcflox/flox and –/– We demonstrate here that NRDC-deficient (Nrdc ) NrdcdelM mice have a C57BL/6 genetic background, which delM mice and macrophage-specific NRDC-deficient (Nrdc ) is more resistant to CAIA than BALB/c, we administered flox/flox delM mice show attenuation of AAIA. We also demonstrate 2.5 times more antibody to Nrdc and Nrdc mice on September 26, 2021 by guest. Protected copyright. that gene deletion or silencing of Nrdc in mouse macro- for the CAIA experiments compared with the amount phages or human monocytic cells results in reduction that was used for the Nrdc–/– mice. The results showed of TNF-α shedding. Importantly, the level of NRDC is that the onset of arthritis was delayed and the severity increased in synovial fluid from RA patients compared of arthritis was markedly reduced in NrdcdelM mice with that from osteoarthritis (OA) patients. Moreover, compared with controls (figure 2A, B). As was the case intra-articular injection of anti-Nrdc small interfering for Nrdc–/– mice, synovial hypertrophy and bone erosion RNA (siRNA) ameliorates CAIA. Together, these data were attenuated in NrdcdelM mice (figure 2C). These find- suggest that NRDC controls AAIA via the regulation of ings demonstrated that macrophage-specific deletion of TNF-α shedding and could be a new therapeutic target NRDC almost completely recapitulated the resistance of for RA treatment.
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