6346 MOLECULAR MEDICINE REPORTS 16: 6346-6353, 2017 Tumor necrosis factor‑α‑induced protein‑8 like 2 regulates lipopolysaccharide‑induced rat rheumatoid arthritis immune responses and is associated with Rac activation and interferon regulatory factor 3 phosphorylation CHUNYAN SHI1,2*, YUE WANG1*, GUOHONG ZHUANG1*, ZHONGQUAN QI1, YANPING LI1 and PING YIN3 1Organ Transplantation Institute, Anti‑Cancer Research Center, Medical College, Xiamen University, Xiamen, Fujian 361100; 2The Department of Oncology, Jiujiang People's Hospital, Jiujiang, Jiangxi 332000; 3Department of Pathology, Zhongshan Hospital Affiliated to Xiamen University, Xiamen, Fujian 361100, P.R. China Received October 28, 2016; Accepted June 19, 2017 DOI: 10.3892/mmr.2017.7311 Abstract. The endogenously activated rheumatoid arthritis Introduction (R A) synovial fibroblasts (RSFs) a re li kely to be the key to cur ing the disease. RSFs express Toll‑like receptors (TLRs) rendering Rheumatoid arthritis (RA) is a chronic inflammatory disease them prone to activation by exogenous and endogenous TLR characterized by articular inflammation and leads to joint ligands, resulting in the production of chemokines and cyto- destruction (1,2). RA pathogenesis involves a complex kines Germline deletion of tumor necrosis factor‑α-induced humoral and cellular immune response, including the infiltra- protein‑8 like 2 (TIPE2, also known as TNFAIP8L2) results tion of lymphocytes and monocytes into the synovium. These in fatal inflammation and hypersensitivity to TLR and T cell infiltrating cells and synoviocytes release numerous proin- receptor stimulation. The present study demonstrates an flammatory cytokine and chemokines, including interleukin inverse association between TIPE2 and cytokine gene expres- IL‑6, IL‑1 and tumor necrosis factor‑α (TNF‑α), which may sion in RSFs following lipopolysaccharide (LPS) stimulation. serve a significant role in RA pathogenesis (3,4), and may Enhanced TIPE2 expression decreased Ras‑related C3 botu- determine the activation and proliferation of the synovial linum toxin substrate (Rac) activation and interferon regulatory lining and the recruitment of inflammatory cells that induce factor 3 phosphorylation, and phosphoinositide 3‑kinase and inflammation and promote destruction. Elevated levels of Rac inhibition significantly diminished LPS‑induced cytokine proinflammatory cytokines have been observed in the sera gene expression in RSFs. In conclusion, the findings of the and synovial fluids of patients with RA, such as IL‑6, IL‑8, present study demonstrate that TIPE2 serves a negative role in TNF‑α, IL-20, IL-17 and IL-33 (5-9). Neutralizing these activating the Rac signaling pathway and in the initiation of the cytokines with monoclonal antibodies or soluble receptors has immune response by decreasing the activity of proinflamma- previously been developed as a novel treatment for RA (10). tory cytokines. These results may be useful in designing novel The TNF‑α‑inducible protein 8 (TNFAIP8) family consists strategies for the prevention and treatment of RA. of TNFAIP8, TNF‑α‑induced protein 8‑like 1 (TIPE1, also termed TNFAIP8L1), TIPE2 (also termed TNFAIP8L2) and TIPE3 (also termed TNFAIP8L3) (11). These are recently identified proteins that share considerable sequence homology for the regulation of cellular and immune homeostasis (12). Correspondence to: Dr Guohong Zhuang, Organ Transplantation TIPE2 is preferentially expressed in lymphoid tissues and Institute, Anti‑Cancer Research Center, Medical College, Xiamen hematopoietic cells, and negatively regulates immunity (12,13). University, Xiangan South Road, Xiamen, Fujian 361100, P.R. China E‑mail: [email protected] TIPE2 deletion in mice leads to multi‑organ inflammation, splenomegaly and premature death (14-16). TIPE2‑deficient Dr Ping Yin, Department of Pathology, Zhongshan Hospital cells hyperactivate toll‑like receptors (TLRs) and T cell recep- Affiliated to Xiamen University, Hubin South Road, Xiamen, tors (11,12). TIPE2 is significantly downregulated in during Fujian 361100, P.R. China infection or autoimmune disease (17,18). TIPE2‑deficient cells E‑mail: [email protected] lead to the production of various kinds of proinflammatory *Contributed equally cytokines and activation of the phosphoinositide 3‑kinase (PI3K)‑Ras‑related C3 botulinum toxin substrate (Rac) Key words: rheumatoid arthritis, toll‑like receptors, tumor necrosis signaling pathway, which enhances protein kinase B (AKT), factor‑α‑induced protein‑8 like 2, Ras‑related C3 botulinum toxin Rac and interferon (IFN) regulatory factor 3 activities (19-21). substrate 1 In the present study, TIPE2 regulated lipopolysaccharide (LPS)‑induced RA immune responses by targeting Rac SHI et al: TIPE2 REGULATES RHEUMATOID ARTHRITIS IMMUNE RESPONSES IN RATS 6347 GTPases in a PI3K‑dependent manner. Tipe2 protein has the were as follows: 5'‑GTT GAC AGC CAC TGC CTT C C ‑ 3 ' potential to be used to treated RA (19,21-25). (forward) and 5'‑CTG ACA GTG C A T C ATC GCT G‑3' (reverse) for IL‑6; 5 ' ‑ C C T CTC CAT CGA CTA CAA GC‑3' (forward) and Materials and methods 5 ' ‑ C T C TTC TCC ATC TGT GAC GG‑3' (reverse) for IFNG; 5 ' ‑ C C A GGA GAA AGT CAG CCT CC‑3' (forward) and 5'‑GTT Cell lines and plasmids. The induction of adjuvant arthritis GAC CTC AGC GCT GAG C‑3' (reverse) for TNF; 5'‑CTG TGA in rats, the preparation of rheumatoid arthritis (RA) syno- TAG GCC TCT CAA GG‑3' (forward) and 5'‑CAC CAT GGC vial fibroblasts (RSF) and identification techniques were CAA TGT AGG TG‑3' (reverse) for TLR2; 5 ' ‑ C C A GCT GTT performed according to previous publications (10,26). AGC AAC CAG C‑3' (forward) and 5'‑CGA GGG ACA GAT Normal synovial fibroblasts (NSFs) and RSFs were grown ACT TCA GG‑3' (reverse) for TLR3; 5 ' ‑ G T A GCC GCT CTG in Dulbecco's modified Eagle's medium (Gibco; Thermo GCA TCA TC‑3' (forward) and 5'‑CTC CCC AGA GCA TTG Fisher Scientific, Inc., Waltham, MA, USA) supplemented TCC TC‑3' (reverse) for TLR4; 5 ' ‑ T G C GTG ACA TCA AAG with 10% fetal bovine serum (Hangzhou Sijiqing Biological AGA AG‑3' (forward) and 5'‑TCC ATA CCC AAG AAG GAA Engineering Materials Co., Ltd., Hangzhou, China), penicillin GG‑3' (reverse) for β‑actin 5'‑TGA TTC TAC CCA CGG CAA and streptomycin. To generate stable cell lines, 5x105 adju- GTT‑3' (forward) and 5'‑TGA TGG GTT TCC CAT TGA T G A ‑ 3 ' vant arthritis fibroblast‑like synoviocytes were transduced (reverse) for GAPDH; 5'‑CCT TGT GCA AGT GTC TGA with MIGR1/TIPE2‑overexpression recombinant lentiviral AGC‑3' (forward) and 5'‑CCC AAG TCA AGG GCT TGG A A ‑ 3 ' vectors and a MIGR1 lentiviral vector was used as the (reverse) for IL‑1; and, 5'‑GGG AAC ATC CAA GGC A A G ‑ 3 ' negative control (gift from Dr Chen Youhai, University of (forward) and 5'‑AGC TCA TCT AGC ACC TCA CT‑3' (reverse) Pennsylvania School of Medicine, Philadelphia, PA, USA). for TNFAIP8L2. The lentiviral vectors contained human and rat inserts for the gene encoding TIPE2 and the sequences are at: http://www. LPS stimulation assay. Control RFSs and TIPE2‑ addgene.org/27490/sequences/#addgene‑partial. The cells over exp r ess e d RSFs wer e t r e at e d by L PS (10 0 ng /ml; 9001‑62‑1; were cultured at 37˚C and the culture medium was replaced Sigma‑Aldrich; Merck KGaA, Darmstadt, Germany) at 24 h after transduction. After an additional 48 h culture at 3 time‑points (0 min, 2, 8 h). After LPS treatment the cells 37˚C, transduced cells were observed visually under a fluo- were collected and Total RNA was isolated using TRIzol rescent microscope, 12 fields were counted and infected cells reagent (Invitrogen; Thermo Fisher Scientific, Inc.). Analysis constituted >90% of the total cell count. The infected cells of relative gene expression data was performed using the 2-ΔΔCq were selected by flow cytometry and identified by reverse tran- method (27). scription‑quantitative polymerase chain reaction (RT‑qPCR) and western blot analysis (11,26). PI3K and Rac inhibition assay. Control RSFs were treated with or without the indicated concentrations of LY294002 RT‑qPCR. Total RNA was isolated using TRIzol reagent or NSC23766 inhibitors for 20 min prior to stimulation with (Invitrogen; Thermo Fisher Scientific, Inc.) from NSFs LPS (100 ng/ml) for 2 h. Cytokine expression was determined and RSFs and purified with RNeasy Mini kits (Qiagen, using RT‑qPCR (19,26). Inc., Valencia, CA, USA) according to the manufacturer's protocol (11,26). Western blot analysis. The protein was isolated from RSFs After processing with RNase‑free DNase I (Invitrogen; and NSFs using the ProteoPrep Total Extraction Sample Thermo Fisher Scientific, Inc.), RNA samples were reverse kit (PROTTOT‑1KT; Sigma‑Aldrich; Merck KGaA), and transcribed with oligo (dT) and SuperScript II transcrip- the protein concentration was determined by BCA assay tase (Invitrogen; Thermo Fisher Scientific, Inc.). PCR was (p0012 BCA; Beyotime Institute of Biotechnology; Shanghai, performed using an Applied Biosystems 7500 system and China). Then, 0.6 µg aliquots of synovial cell lysates were Power SYBR‑Green PCR Master mix (Applied Biosystems, loaded and separated by 12% SDS‑PAGE, transferred onto Thermo Fisher Scientific, Inc.). Relative gene expression levels a polyvinylidene difluoride membrane, blocked by 5% were determined using GAPDH as the control. The PCR FBS (Hangzhou Sijiqing Biological Engineering Materials products were run in an agarose gel and were in all cases Co., Ltd.) for 1 h at room temperature and probed with the confined to a single band of the expected size. A melting‑curve following primary antibodies for 75 min at room tempera- analysis was also performed to ensure specificity of the prod- ture: rabbit anti‑β‑actin (m1210‑5; 1:1,000; Hangzhou ucts (11,24). Analysis of relative gene expression data was Huaan Biotechnology Co., Ltd., Hangzhou, China), rabbit performed using the 2-ΔΔCq method (27).