Intraarticular overexpression of Smad7 ameliorates experimental arthritis

Rheumatoid arthritis (RA) and Crohn’s disease (CD) are autoimmune disorders with a crosstalk between their pathogenesis such as increased expression of TNF in the target organs. Despite a successful clinical trial with an oral Smad7 antisense oligonucleotide in CD, intraarticular (i.a.) modulation of Smad7 expression has not been performed in rheumatoid joint yet. In this study, contradictory to the findings in CD mucosa, higher levels of pSmad2/3 were found in RA synovium. In vitro experiments with synovial fibroblasts revealed that higher acetylated Smad7 expression was associated with lower activation status. Abundant expression of synovial pSmad2/3 with increased levels during the progression of arthritis was detected in collagen-induced arthritis (CIA) mice. To prove the concept that overexpressing Smad7 as a therapeutic strategy in rheumatoid joint, the i.a. injection of lentiviral vectors carrying Smad7 (LVSmad7) was carried out in CIA mice. In LVSmad7-injected joints, there were lower arthritis and histological scores with less synovitis, synovial hyperplasia and erosion on cartilage and bone as well as reduced IL-17 and TNF expression levels in comparison with other control groups. In conclusion, we demonstrate that lentiviral vector-mediated i.a. overexpression of Smad7 can ameliorate rheumatoid joint, implicating a pharmacological development of Smad7-based molecular strategy in RA.

Scientific RepoRts | 6:35163 | DOI: 10.1038/srep35163 Increased expression levels of pSmad2/3 in CIA synovial tissues. Next, we examined an experimental arthritis model for synovial expression of pSmad2/3 and Smad7. There were abundant expression of synovial pSmad2/3 with increased levels from day 10 onward during the progression of CIA, and lower Smad7 levels with a similar kinetic pattern except a decline on day 21 (Fig. 2), suggesting an upregulated TGF-β signaling activity in rheumatoid joint.

Higher expression levels of acetylated Smad7 associated with lower activation status in SFs.
Furthermore, in vitro experiments were carried out by culturing SFs. With the presence of HDAC1 inhibitor trichostatin A (TSA) in the SFs culture for 24 hr, there was a dose-dependent increase in acetylation levels of Smad7 (Fig. 3a). Notably, higher acetylated Smad7 levels were associated with down-regulated expression levels of Snail, a TGF-β -inducible transcription factor capable of activating SFs to perpetuate the RA activity 9 . In addition, the phosphorylated nuclear factor (NF)-κ B p65 signaling intensities of cultured SFs were reduced in the presence of TSA (data not shown). Collectively, together with the finding of higher synovial expression levels of HDAC1, these results suggest that overexpressing Smad7 to down-regulate the TGF-β signaling can be a beneficent approach in the RA therapy.
Amelioration of CIA by i.a. overexpression of Smad7 with reduced synovial IL-17 and TNF expression. The efficacy of overexpressing Smad7 in CIA joints was verified by analyzing the expression levels of Smad7 and Flag in LVSmad7, LVnull and medium alone-treated synovial tissues with the immunoblot assay. In Fig. 4a, there were significantly increased levels of Smad7 with the presence of Flag in LVSmad7-injected synovial tissues. In order to prove the concept that overexpressing Smad7 as a therapeutic strategy in rheumatoid joint, we performed the i.a. injection of lentiviral vectors in CIA mice during the progression of arthritis (all groups with 100% incidence in this study). There were lower arthritis and histological scores with less synovitis, synovial hyperplasia and erosion on cartilage and bone in LVSmad7-injected joints in comparison with other control groups (Fig. 4b,c). Furthermore, LVSmad7-treated synovial tissues had reduced IL-17 and TNF expression levels as compared with LVnull or medium alone-treated synovium (Fig. 4d,e); however, there were no differences in the IL-6 expression levels among three treatment groups (data not shown), suggesting that the activation of non-canonical TGF-β -NF-κ B cross-talk is in favor of the TNF-induced NF-κ B signaling pathway in rheumatoid joint 10 . Discussion RA and CD are organ-specific autoimmune disorders with a crosstalk between their pathogenesis such as increased expression of TNF in their target organs 6,11 . In particular, together with IL-6 through the activation of   STAT3, TGF-β can induce RORγ t to orchestrate the IL-17 expression in naïve CD4-positive T cells, resulting in the differentiation into Th17 cells, a therapeutic target under active pharmacological development in both diseases 12,13 . Indeed, in this study, interfering with the TGF-β signaling could reduce synovial IL-17 and TNF expression levels. Notably, there are conflicting results regarding the expression levels of pSmad2/3 with up-regulation in RA synovium and down-regulation in CD mucosa, suggesting that different regulatory mechanisms exist in the TGF-β signaling pathway of two distinct autoimmunity status 7,14 . Consequently, contradictory approach has been used by overexpressing Smad7 and silencing this molecule in rheumatoid joint and inflamed bowel, respectively, with therapeutic responses in both diseases.
Interestingly, Smad7 participates in the crosstalk between TGF-β and diverse signaling pathways 3 . Inhibition of NF-κ B activation by Smad7 can be mediated through the binding to IRAK1 and TAB2/TAB3 to block IL-1R and TNFR signaling, respectively, resulting in lower levels of pro-inflammatory cytokines and less anti-apoptotic signaling activity 3,15,16 . Notably, in tumor cells, a cross-talk has been identified between TGF-β and NF-κ B signaling pathways mediated through TAK1 and Smad7 10 . Indeed, in this study, there was a decrease in the signaling intensities of phosphorylated NF-κ B p65 in SFs, raising a possibility that such a cross-talk is mediated through Smad7 in the SFs from rheumatoid joint. Moreover, Smad7 can reduce the Wnt signaling, a pathogenic pathway up-regulated by TNF in RA, through the complex formation with β -catenin and Smurf2 with degradation of the former molecule via the action of proteasome 3,9,17 . Thus, in addition to the TGF-β -dependent signaling, Smad7 can modulate other pathogenesis-related signaling pathways to ameliorate rheumatoid joint.
In conclusion, we demonstrate that lentiviral vector-mediated i.a. overexpression of Smad7 can ameliorate rheumatoid joint, implicating a pharmacological development of Smad7-based molecular strategy in RA.

Methods
Ethics statement. The Institutional Review Board of National Cheng Kung University Hospital approved the permission to obtain human synovial specimens, and informed consent was obtained from all subjects. The Institutional Animal Care and Use Committee of National Cheng Kung University approved the animal experiments. All methods relating to humans were performed in accordance with the relevant guidelines and regulations, and were approved by the Institutional Review Board of National Cheng Kung University Hospital. All animal experiments were conducted in accordance with the approved institutional guidelines.
Immunohistochemical analysis. Paraffin-embedded synovial sections were processed and stained with anti-Smad7 (R&D systems), anti-phosphorylated Smad2/3 (Santa Cruz), anti-histone deacetylase 1 (HDAC1, Santa Cruz), or isotype control IgG (Santa Cruz), followed by secondary antibody and substrate chromogen, and their averaged expression intensities in 5 blindly chosen random fields were quantified with HistoQuest analysis software (TissueGnostics), as previously described 9,18 . Construction and production of lentiviral plasmids. The pCMV-Tag2B plasmid (Addgene) was digested with NdeI and EcoRI to release the CMV immearly promoter with N-terminal Flag, subcloned into pCMV5-Smad7-HA (Addgene), resulting in pCMV-Flag-Smad7-HA. It was further excised and subcloned into the lentiviral plasmid pLKO.1-shLuc (National RNAi Core Facility, Academia Sinica, Taiwan) by digestion with NdeI and XbaI to generate pLKO.1-Flag-Smad7-HA. The control plasmid pWPXL-null encoding no transgene was constructed from the pWPXL by digestion with PmeI and EcoRI to delete the GFP cDNA. Recombinant lentiviral vectors were produced by transfecting 293T cells with pLKO.1-Flag-Smad7-HA or pWPXL-null, along with packaging plasmids psPAX2 and envelope plasmids pMD2.G by using the calcium phosphate precipitation method, and LVSmad7 and LVnull vectors were concentrated with their titers expressed as viral particles (VPs), as previously described 9,18 . Induction of CIA and isolation of SFs. Male 8-week old DBA/1(J) mice housed under the specific pathogen-free condition, were immunized intradermally with bovine type II collagen 100 μ g in 50 μ l 0.1 M acetic acid (Elastin Products) emulsified with 50 μ l Freund's complete adjuvant (4 mg/ml, Chondrex) at the tail base on day 0, and received the intraperitoneal booster of bovine type II collagen 100 μ g in 50 μ l 0.1 M acetic acid without adjuvant on day 21, resulting in a more than 90% incidence of arthritis, as previously reported 18,19 . SFs isolated from CIA synovial tissues and cultured continuously until confluence, were used for further experiments with lines between the 4 th and 7 th passage.

Delivery of lentiviral vectors and evaluation of arthritis.
On day 36 during the progression of arthritis, CIA mice received i.a. injections of 1 × 10 9 VPs of LVSmad7 and LVnull into right and left ankle joints, respectively, with medium injection alone as another control group. Arthritis severity was scored on a 0 to 4 scale in each posterior paw with 0: no evidence of erythema and swelling, 1: erythema and mild swelling confined to the tarsals or ankle joint, 2: erythema and mild swelling extending from the ankle to the tarsals, 3: erythema and moderate swelling extending from the ankle to metatarsal joints, and 4: erythema and severe swelling encompassing the ankle, foot and digits, or ankylosis of the limb, as previously described 18,20 . Hematoxylin and eosin (H&E)-stained paraffin-embedded ankle joint sections were evaluated for synovial hyperplasia, cartilage erosion, and inflammatory cell infiltration, and a histologic score of 0-2 scale was assigned for each of these features (0: absent, 1: mild, 2: severe) with maximum of 6, as previously described 18,19 . Synovial immunohistochemical staining was performed with anti-Smad7 (R&D systems), anti-phosphorylated Smad2/3 (Santa Cruz), anti-IL-17 (eBioscience), anti-TNF (Santa Cruz) or isotype control IgG (Santa Cruz) with the expression intensities quantified by HistoQuest software (TissueGnostics).