Expression profiling of TRIM protein family in THP1-derived macrophages following TLR stimulation

www.nature.com/scientificreports OPEN received: 16 April 2016 accepted: 16 January 2017 Published: 17 February 2017 Expression profiling of TRIM protein family in THP1-derived macrophages following TLR stimulation Mei-Xiu Jiang1, Xuan Hong1, Bin-Bin Liao1, Shui-Zhen Shi1, Xiao-Fang Lai1, Huai-Yu Zheng1, Lin Xie1, Yuan Wang1, Xiao-Lei Wang1, Hong-Bo Xin1, Mingui Fu2 & Ke-Yu Deng1 Activated macrophages play an important role in many inflammatory diseases including septic shock and atherosclerosis. However, the molecular mechanisms limiting macrophage activation are not completely understood. Members of the tripartite motif (TRIM) family have recently emerged as important players in innate immunity and antivirus. Here, we systematically analyzed mRNA expressions of representative TRIM molecules in human THP1-derived macrophages activated by different toll-like receptor (TLR) ligands. Twenty-nine TRIM members were highly induced (>3 fold) by one or more TLR ligands, among which 19 of them belong to TRIM C-IV subgroup. Besides TRIM21, TRIM22 and TRIM38 were shown to be upregulated by TLR3 and TLR4 ligands as previous reported, we identified a novel group of TRIM genes (TRIM14, 15, 31, 34, 43, 48, 49, 51 and 61) that were significantly up-regulated by TLR3 and TLR4 ligands. In contrast, the expression of TRIM59 was down-regulated by TLR3 and TLR4 ligands in both human and mouse macrophages. The alternations of the TRIM proteins were confirmed by Western blot. Finally, overexpression of TRIM59 significantly suppressed LPS-induced macrophage activation, whereas siRNA-mediated knockdown of TRIM59 enhanced LPSinduced macrophage activation. Taken together, the study provided an insight into the TLR ligandsinduced expressions of TRIM family in macrophages. Macrophages are the major components of innate immunity that enable the body to combat bacteria and other pathogens. However, over-activation of macrophages plays a central role in a variety of inflammatory diseases, such as septic shock, atherosclerosis, arthritis and inflammatory bowel diseases. In these disease settings, activated macrophages elaborate a large array of cytokines, growth factors and proteolytic enzymes that are critical for tissue damage and repair1,2. Macrophages are activated in response to the pathogen-associated molecular patterns by various pattern-recognition receptors (PRRs), such as the Toll-like receptors (TLRs) and the RIG-I-like receptors (RLR)3,4. There are 13 TLRs that sense various pathogen components and trigger intracellular signaling pathways that eventually mediate the induction of inflammatory cytokines, chemokines and type I interferons, which are critical for antimicrobial activity4,5. The molecular mechanisms of regulation of macrophage activation in response to TLR ligands have been largely unknown. Tripartite motif (TRIM) proteins contain a RING finger, one or two B-box motifs and a coiled-coil motif, and are involved in many biological processes including innate immunity, viral infection, carcinogenesis and development6. There are over 70 members of TRIM protein family described in humans7. Recently, several systematic analyses suggest that many TRIM proteins are implicated in the regulation of innate immune pathways and anti-viral activities8–11. For example, Carthagena et al. identified 27 of the 72 human TRIM genes are sensitive to interferon (IFN) by performing a systematic analysis of TRIM gene expressions in human primary lymphocytes and monocyte-derived macrophages in response to IFNs10. In addition, Rajsbaum et al. found that the genes encoding a subset of TRIM proteins located on chromosome 7 were up-regulated by type I IFN in macrophages/ DC, suggesting that they may have anti-viral functions11. TRIM8 negatively regulates PIAS3-mediated repression 1 Institute of Translational Medicine, Nanchang University, Nanchang, Jiangxi, 330031, China. 2Department of Basic Medical Science, Shock/Trauma Research Center, School of Medicine, University of Missouri Kansas City, Kansas City, MO, 64108, USA. Correspondence and requests for materials should be addressed to H.-B.X. (email: hongboxin@ yahoo.com) or M.F. (email: ) or K.-Y.D. (email: ) Scientific Reports | 7:42781 | DOI: 10.1038/srep42781 1 www.nature.com/scientificreports/ of NF-κB by inducing translocation of PIAS3 from nucleus to cytoplasm as well as its turnover12–14, whereas TRIM16 (also known as EBBP) was reported to promote IL-1βsecretion. TRIM22 is involved in anti-viral pathways by activating NF-κB signaling15–18. TRIM30 induces the lysosomal degradation of TAB2 and TAB3, thereby negatively regulating NF-κB induction in the LPS-triggered TLR4 signaling pathway19. TRIM21 negatively regulates TLR3, −4, −7, and −9 and RLR signaling pathways by modulating the activities of IKKs and interferon regulatory factors (IRFs)20,21. TRIM27 targets all IKKs and negatively regulates the PRR pathways21,22. CARD domain ubiquitination by TRIM25 is essential for RIG-I-mediated type I interferon induction21,23. TRIM56 facilitates double-strand DNA-stimulated interferon induction by ubiquitination of STING (stimulator of interferon genes)21,24. However, the functions of most of TRIM family members remain to be characterized. In the present study, we systematically profiled the expressions of TRIM gene family in human THP1-derived macrophages activated by different TLR ligands. The up-regulated or down-regulated TRIM genes were further confirmed by quantitative real-time polymerase chain reaction (qRT-PCR) and Western blot analysis. The function of TRIM59 in macrophage activation was further studied. Results Expression profiling of TRIM gene family in TLR ligand-activated THP1-derived macrophages. Macrophages are equipped with almost all TLRs, which sense different pathogens and initiate inflammatory responses. To understand the regulatory mechanisms that control macrophage activation in response to TLR ligands, we employed qRT-PCR to profile the expression changes of 72 TRIM genes in THP1-derived macrophages upon stimulation by different TLR ligands. TNFαand oxLDL were included to serve as positive controls. The primer set for 72 human TRIM gene members was tested by melting curve and product sequencing before the experiments. The entire data set were log-transformed and cluster analyzed and presented as a heat map (Fig. 1a). As shown in Fig. 1a and S1, 29 TRIM genes were highly induced (>3 fold) by one or more TLR ligands, among which 15 of them were increased by more than 10 folds compared with controls and 19 of them belong to TRIM C-IV subgroup. Moreover, the expressions of 46 TRIM members were inhibited by at least one of TLR ligands, among which 19 of them were showed an inhibition of more than 50% of control and the inhibitory ranges for other members of the TRIM family were from <75~<67% of control. Interestingly, 9 TRIM genes (TRIM10, 15, 43, 48, 49, 50, 51, 60 and 64) in TRIM C-IV subgroup, 4 genes (TRIM29, 31, 40 and 61) i (...truncated)