CD109 Regulates the Inflammatory Response and Is Required for The
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Rheumatoid arthritis Ann Rheum Dis: first published as 10.1136/annrheumdis-2019-215473 on 27 August 2019. Downloaded from TRANSLATIONAL SCIENCE CD109 regulates the inflammatory response and is required for the pathogenesis of rheumatoid arthritis Guanhua Song,1 Tingting Feng,2 Ru Zhao,2 Qiqi Lu,3 Yutao Diao,1 Qingwei Guo,4 Zhaoxia Wang,1 Yuang Zhang,3 Luna Ge,5 Jihong Pan,5 Lin Wang, 5 Jinxiang Han5 Handling editor Josef S ABStract Key message Smolen Objective The aim of this study was to investigate the role of CD109 in rheumatoid arthritis (RA) fibroblast-like ► Additional material is What is already known about this subject? published online only. To synoviocytes (FLSs) and to evaluate its potential as a ► CD109 regulates diverse pathogenic processes, view please visit the journal therapeutic target. including fibrosis, osteoporosis and tumour online (http:// dx. doi. org/ 10. Methods CD109 expression was examined in metastasis. 1136annrheumdis- 2019- synovial tissues and FLSs from RA patients and 215473). CD109 inhibits transforming growth factor collagen-induced arthritis (CIA) model mice. CD109- ► (TGF)- signalling by enhancing SMAD7/ deficient mice were developed to evaluate the severity β For numbered affiliations see Smurf2-dependent degradation of the TGF- end of article. of CIA. Small interfering RNAs and a neutralising β receptor. antibody against CD109 (anti-CD109) were designed Correspondence to for functional or treatment studies in RA FLSs and CIA. Dr Lin Wang, Key Laboratory for What does this study add? Results CD109 was found to be abundantly Rare and Uncommon Diseases CD109 is required to sustain rheumatoid expressed in the synovial tissues from RA patients ► of Shandong Province, Research arthritis (RA) fibroblast-like synoviocyte (FLS)- and CIA mice. CD109 expression in RA FLSs Center for Medicinal induced inflammation. Biotechnology, Shandong First was upregulated by inflammatory stimuli, such CD109 interacts with and stabilises ENO1 to Medical University & Shandong as interleukin-1β and tumour necrosis factor-α. ► Academy of Medical Sciences, regulate the RA FLS-mediated inflammatory Silencing of CD109 or anti-CD109 treatment reduced Jinan 250062, China; response. wanglin. 83@ 163. com and proinflammatory factor production, cell migration, ► An anti-CD109 antibody shows both Professor Jinxiang Han; invasion, chemoattractive potential and osteoclast prophylactic and therapeutic effects in collagen- sams- h2016@ 163. com differentiation, thereby reducing the deleterious induced arthritis mice. inflammatory response ofRA FLSs in vitro. Mice Received 2 April 2019 lacking CD109 were protected against arthritis in the Revised 31 July 2019 How might this impact on clinical practice or CIA model. Anti-CD109 treatment prevented the onset Accepted 6 August 2019 future developments? and ameliorated the severity of CIA lesions. CD109 could be a promising treatment target Conclusion Our study uncovers an antiarthritic role ► for RA. for CD109 and suggests that CD109 inhibition might serve as a promising novel therapeutic strategy for RA. http://ard.bmj.com/ reticulum (ER) chaperone, to inhibit transforming growth factor (TGF-β) signalling in response to INTRODUCTION 8 Rheumatoid arthritis (RA) is a systemic autoim- ER stress. Interestingly, GRP78 acts as a patho- mune disease characterised by chronic inflamma- logical factor that contributes to the severity of RA,9 suggesting that CD109 is likely involved in tion and hyperplasia of fibroblast-like synoviocytes on September 29, 2021 by guest. Protected copyright. (FLSs).1 RA FLSs display an aggressive phenotype the pathological progression of RA. and produce excessive amounts of proinflammatory A previous study demonstrated that CD109 cytokines and matrix-degrading enzymes, resulting inhibits TGF-β signalling by enhancing SMAD7/ in joint dysfunction and destruction.2 Hence, identi- Smurf2-dependent degradation of the TGF-β 10 11 fying key factors that specifically target RA FLS-me- receptor TGFβRI. More interestingly, diated inflammation may provide novel therapeutic although CD109 inactivates TGF-β signalling and targets for RA. subsequent fibrosis, fibroblasts with increased CD109 is a cell-surface antigen that belongs CD109 protein levels still produce excessive 12 © Author(s) (or their to the α2-macroglobulin/C3, C4, C5 family of amounts of extracellular matrix (ECM). It is thus employer(s)) 2019. Re-use thioester-containing proteins.3 As a glycosylphos- believed that CD109 is insufficient to completely permitted under CC BY-NC. No phatidylinositol-anchored protein,4 increased counteract the effect of TGF-β activation on commercial re-use. See rights fibrosis, and the exact role of elevated CD109 in and permissions. Published expression of CD109 in various tumours is indic- by BMJ. ative of a more aggressive phenotype and poor fibroblasts remains unclear. Recently, CD109 has prognosis.5–7 Since RA FLSs exhibit tumour- been shown to activate Jak-Stat3 signalling, which To cite: Song G, like proliferation and invasion behaviours,2 it consequently leads to a metastatic phenotype in Feng T, Zhao R, et al. 13 Ann Rheum Dis Epub ahead is possible that CD109 may play a role in the lung cancer cells. This evidence indicates that of print: [please include Day aggressive phenotype of RA FLSs. It has been CD109 functions in a cell-specific manner, and it Month Year]. doi:10.1136/ reported that CD109 interacts with glucose-regu- would be interesting to characterise the exact role annrheumdis-2019-215473 lated protein 78 (GRP78), a classical endoplasmic of CD109 in RA FLSs. Song G, et al. Ann Rheum Dis 2019;0:1–10. doi:10.1136/annrheumdis-2019-215473 1 Rheumatoid arthritis Ann Rheum Dis: first published as 10.1136/annrheumdis-2019-215473 on 27 August 2019. Downloaded from factor (M-CSF; 30 ng/mL, R&D) and RANKL (50 ng/mL, R&D). The dilution buffer, phosphate buffer saline, was applied as vehicle control. RESULTS CD109 is abundantly expressed in the synovial tissues of patients with RA CD109 was first found to be abundantly expressed in all RA patient synovial tissues but dramatically reduced in osteoar- thritis (OA) samples (figure 1A). In vitro, CD109 mRNA expres- sion in RA FLSs was relatively higher than that in OA FLSs (figure 1B). Immunohistochemical staining showed that CD109 was expressed at higher levels in RA synovial tissues than in OA synovial tissues and mainly distributed in the lining layer of the RA synovial tissues (figure 1C). A similar increase in CD109 expression was also observed in FLSs (figure 1D,E) and synovial tissues (figure 1F) from CIA mice when compared with those from wild-type (WT) mice. In addition, CD109 expression was elevated at the mRNA (figure 1G,I) and protein (figure 1H,J) levels in FLSs from RA patients and CIA mice following TNF-α and IL-1β stimulation, but not following IL-1α, IL-6 and IL-17 14 Figure 1 Expression of CD109 in synovial tissues extracted from stimulation (online supplementary figure 1). RA patients and CIA model mice. (A) Western blot analysis of CD109 protein in synovial tissues from RA (n=7) and OA (n=7) patients. Effects of CD109 on the inflammatory phenotype of RA FLSs β-Actin was used as a loading control. (B) Quantitative real-time (qRT) RA FLSs can secrete IL-6, IL-8, matrix metalloproteinase PCR analysis of CD109 mRNA levels in FLSs from RA (n=7) and OA (MMP)-1, MMP-3 and MMP-13 in response to IL-1β and (n=7) patients. ***p<0.001. (C) Immunohistochemical staining of RA TNF-α stimulation, and these factors are implicated in the RA 15–22 (n=16) and OA (n=7) synovial tissues using antibodies against CD109. FLS-mediated inflammatory response. We next measured Original magnification is 200×. FLSs were collected from DBA/1J the levels of these proteins in the supernatant of siCD109-trans- mice immunised with collagen (CIA mice) or bovine serum albumin fected or siCtrl-transfected RA FLS. The silencing efficiency of (BSA, referred to as Ctrl mice). qRT-PCR and Western blot assays were small interfering RNAs (siRNAs) against CD109 was confirmed performed to assess the levels of CD109 mRNA (D) and protein (E). by Western blot (figure 2A). The data showed that silencing of n=3 per group, **p<0.01. (F) Immunohistochemical staining of CD109 CD109 significantly reduced the levels of IL-6, IL-8, MMP-1 from the hind paw in CIA and Ctrl mice. FLSs from RA patients (G and and MMP-3 (figure 2B–E), but not MMP-13 (data not shown), H) or CIA model mice (I and J) were stimulated with TNF-α or IL-1β in the supernatants of RA FLSs with or without TNF-α and IL-1β for 24 hours. Total RNA and protein levels were subjected to qRT-PCR stimulation. Additionally, CD109 knockdown resulted in an 23 24 25–27 and Western blotting for the analysis of CD109 mRNA (G and I) and obvious reduction in TNF-α-induced or IL-1β-induced protein (H and J) levels. qRT-PCR data in G and I are expressed as phosphorylation of AKT serine/threonine kinase (Akt), nuclear http://ard.bmj.com/ the mean of six samples (three male and three female) or three mice factor-kappa B (NF-κB), signal transducer and activator of tran- from two independent experiments. Western blot data in H and J scription 3 (Stat3) and p38 MAPK signalling proteins (figure 2F). represent two independent experiments from six samples (three males CXCL9/10 modulates RA immune responses by activating and 28 29 and three females) or three mice with similar results. *p<0.05 and recruiting leucocytes. CD109 knockdown significantly **p<0.01 compared with vehicle. CIA, collagen-induced arthritis; FLSs, reduced the production of CXCL9 and CXCL10 (figure 2G,H) fibroblast-like synoviocytes; IL-1β, interleukin-1β; OA, osteoarthritis; RA, in RA FLSs. The number of migrating leucocytes cultured with rheumatoid arthritis; TNF-α, tumour necrosis factor-α.