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Rac Pathway − Targeting the Phosphatidylinositol 3-Kinase

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Rac Pathway − Targeting the Phosphatidylinositol 3-Kinase TIPE2 Controls Innate Immunity to RNA by Targeting the Phosphatidylinositol 3-Kinase −Rac Pathway This information is current as Honghong Sun, Guohong Zhuang, Lihui Chai, Zhaojun of October 1, 2021. Wang, Derek Johnson, Yuanfang Ma and Youhai H. Chen J Immunol published online 17 August 2012 http://www.jimmunol.org/content/early/2012/08/17/jimmun ol.1103477 Downloaded from Why The JI? Submit online. • Rapid Reviews! 30 days* from submission to initial decision http://www.jimmunol.org/ • No Triage! Every submission reviewed by practicing scientists • Fast Publication! 4 weeks from acceptance to publication *average Subscription Information about subscribing to The Journal of Immunology is online at: by guest on October 1, 2021 http://jimmunol.org/subscription Permissions Submit copyright permission requests at: http://www.aai.org/About/Publications/JI/copyright.html Email Alerts Receive free email-alerts when new articles cite this article. Sign up at: http://jimmunol.org/alerts The Journal of Immunology is published twice each month by The American Association of Immunologists, Inc., 1451 Rockville Pike, Suite 650, Rockville, MD 20852 Copyright © 2012 by The American Association of Immunologists, Inc. All rights reserved. Print ISSN: 0022-1767 Online ISSN: 1550-6606. Published August 17, 2012, doi:10.4049/jimmunol.1103477 The Journal of Immunology TIPE2 Controls Innate Immunity to RNA by Targeting the Phosphatidylinositol 3-Kinase–Rac Pathway Honghong Sun,*,1 Guohong Zhuang,*,†,1 Lihui Chai,*,‡ Zhaojun Wang,* Derek Johnson,* Yuanfang Ma,‡ and Youhai H. Chen* RNA receptors such as TLR3 and retinoid acid-inducible gene I/melanoma differentiation-associated gene 5 play essential roles in innate immunity to RNAviruses. However, how innate immunity to RNAs is controlled at the molecular level is not well understood. We describe in this study a new regulatory pathway of anti-RNA immunity that is composed of PI3K, its target GTPase Rac, and the newly described immune regulator TNF-a–induced protein 8 like-2 (TIPE2, or TNFAIP8L2). Polyinosinic-polycytidylic acid [Poly (I:C)], a dsRNA receptor ligand, activates Rac via its guanine nucleotide exchange factor Tiam; this leads to the activation of cytokine genes and, paradoxically, downregulation of the Tipe2 gene. TIPE2 is a negative regulator of immunity; its deficiency leads to hyperactivation of the PI3K–Rac pathway as exemplified by enhanced AKT, Rac, P21-activated kinase, and IFN regu- Downloaded from latory factor 3 activities. As a consequence, TIPE2 knockout myeloid cells are hyperreactive to Poly (I:C) stimulation, and TIPE2 knockout mice are hypersensitive to Poly (I:C)-induced lethality. These results indicate that TIPE2 controls innate immunity to RNA by targeting the PI3K–Rac pathway. Therefore, manipulating TIPE2 or Rac functions can be effective for controlling RNA viral infections. The Journal of Immunology, 2012, 189: 000–000. oll-like receptor 3 and retinoid acid-inducible gene I gene transcription, and death (13, 14). However, whether and how (RIG-I)/melanoma differentiation-associated gene 5 the small GTPases are involved in innate immunity to dsRNAs is http://www.jimmunol.org/ T (MDA5) have been identified as receptors for dsRNAs (1–3). not clear. RNA ligation of its receptors induces type I IFN and proinflamma- TIPE2, or TNF-a–induced protein 8 (TNFAIP8)-like 2 tory cytokines via a MyD88-independent pathway, which involves (TNFAIP8L2), is a newly described immune regulator of the Toll/IL-1R domain-containing adapter inducing IFN-b or IFN-b TNFAIP8 family (15, 16). It is preferentially expressed in hema- promoter stimulator (2, 4). Toll/IL-1R domain-containing adapter topoietic cells and significantly downregulated in patients with in- inducing IFN-b relays signals to the kinases TANK-binding ki- fectious or autoimmune disorders (17). The mammalian TNFAIP8 nase 1 (TBK1) and IkB kinase, which in turn activate the down- family consists of four members: TNFAIP8 (TIPE), TIPE1, stream transcription factors IFN regulatory factor 3 (IRF3), NF- TIPE2, and TIPE3, the functions of which are largely unknown. by guest on October 1, 2021 kB, and AP-1, leading to the production of type I IFN and We report in this study that TIPE2 regulates Poly (I:C)-induced proinflammatory cytokines (4–7). Recent studies indicate that the innate immune responses by targeting Rac GTPases in a PI3K- PI3K–AKT pathway and small GTPases may also be involved dependent manner. in TLR signaling (8–12). Small GTPases are enzymes that hy- drolyze GTP. They are active when bound to GTP and inactive Materials and Methods when bound to GDP and therefore serve as molecular on-and- Mice off switches of signaling pathways that control a wide variety of Wild-type (WT) C57BL/6 (B6) mice were purchased from The Jackson cellular processes including growth, motility, vesicle trafficking, 2 2 Laboratory. The Tipe2 / B6 mice were generated by backcrossing Tipe22/2 129 mice to B6 mice for 12 generations. Age- and sex-matched WT and Tipe2-deficient mice were used in all experiments. Mice were *Department of Pathology and Laboratory Medicine, University of Pennsylvania † housed in the University of Pennsylvania Animal Care Facilities under School of Medicine, Philadelphia, PA 19104; Cancer Center, University of Xiamen specific pathogen-free conditions. All animal procedures were preapproved School of Medicine, Xiamen, Fujian 361005, People’s Republic of China; and by the Institutional Animal Care and Use Committee of the University of ‡Laboratory of Cellular and Molecular Immunology, Henan University School of Medicine, Kaifeng 475001, People’s Republic of China Pennsylvania. 1 H.S. and G.Z. contributed equally to this work. Reagents Received for publication December 5, 2011. Accepted for publication July 12, 2012. Poly (I:C), CpG, imiquimod, and rhodamine-labeled Poly (I: C) were This work was supported by grants from the National Institutes of Health (AI- purchased from InvivoGen. LPS and peptidoglycan (PGN) were from 077533, AI-050059, and GM-085112). Sigma-Aldrich. PI3K inhibitor LY294002 was obtained from Cell Signal- Address correspondence and reprint requests to Dr. Youhai H. Chen, Department of ing Technology. The p38 inhibitor SB203580, ERK kinase I inhibitor Pathology and Laboratory Medicine, University of Pennsylvania School of Medicine, PD98059, JNK inhibitor SP600123, and NF-kB inhibitor Bay were pur- 713 Stellar-Chance Labs, 422 Curie Boulevard, Philadelphia, PA 19104-6160. E-mail chased from Promega. Rac inhibitor NSC23766 was obtained from Tocris. address: [email protected] GM-CSF was from R&D Systems. ELISA reagents were purchased from Abbreviations used in this article: B6, C57BL/6; BMDC, bone marrow-derived den- BD Biosciences, which include purified and biotinylated rat anti-mouse dritic cell; BMM, bone marrow-derived macrophage; GAP, GTPase-activating pro- IL-6 and TNF-a. Abs used were as follows: rabbit anti-Rac1/2/3, rabbit tein; GEF, guanine nucleotide exchange factor; IRF3, IFN regulatory factor 3; MDA5, anti-Na/K ATPase, rabbit anti–phospho-P21–activated kinase (PAK) 1 melanoma differentiation-associated gene 5; NP-40, Nonidet P-40; PAK, P21-activated 144 141 kinase; PGN, peptidoglycan; PH, pleckstrin homology; Poly (I:C), polyinosinic- (Ser )/PAK2 (Ser ), rabbit anti-PAK1/2/3, rabbit anti–p-AKT, rabbit polycytidylic acid; RIG-I, retinoid acid-inducible gene I; TBK1, TANK-binding kinase anti-total AKT, rabbit anti-IRF3 (Cell Signaling Technology), rabbit anti- 1; TIPE2, TNF-a–induced protein 8 like-2; WT, wild-type. Tiam (Santa Cruz Biotechnology), and HRP-conjugated anti-mouse or anti-rabbit Ig (GE Healthcare). Quantitative real-time PCR primers for Copyright Ó 2012 by The American Association of Immunologists, Inc. 0022-1767/12/$16.00 IL-6, TNF-a, IL-1b, IFN-b1, and IFN-a4 were purchased from Qiagen. www.jimmunol.org/cgi/doi/10.4049/jimmunol.1103477 2 TIPE2 AND INNATE IMMUNITY TO RNA Preparation of bone marrow-derived dendritic cells and Retrovirus preparation and retrovirus-mediated gene transfer macrophages The murine RAW264.7 macrophages (American Type Culture Collection) Bone marrow-derived dendritic cells (BMDCs) were generated as described and HEK293T cells (American Type Culture Collection) were cultured in (18). Briefly, 2 3 106 bone marrow precursors from B6 and Tipe2-deficent DMEM containing 10% heat-inactivated FBS, 2 mM L-glutamine, and 100 mice were seeded in complete IMDM supplemented with 3.3 ng/ml GM- U/ml penicillin/streptomycin. To prepare retroviruses, packaging cells CSF in six-well plates. Two milliliters medium was added to the culture on (293T) were cultured in 10-mm culture dishes and transfected with nerve day 3, and half of the medium was replaced with new medium on days 5, growth factor receptor murine stem cell virus-based retroviral vector 6, and 7. To generate bone marrow-derived macrophages (BMMs), bone containing WT Rac, Rac1 T17N, and Rac1 Q61L along with pVSVG and marrow cells were cultured for 7 d in DMEM supplemented with 10% FCS, pCGP sequences using the CalPhos mammalian transfection kit (Clon- 1% penicillin/streptomycin, 1% glutamine, and 30% L-929 cell culture tech). The transfection medium was replaced with fresh medium 6 h supernatant. At the end of culturing, the cells were washed twice with posttransfection. At 24 and 48 h posttransfection, the culture medium cold PBS and rested overnight in complete DMEM before assays. BMDCs containing recombinant retroviruses was harvested, filtered (with a 0.20- were 80% CD11c+, and BMMs were .95% CD11b+ and F4/80+ as deter- mm filter) and used to infect Raw cells. The infection efficiency was ∼70% mined by flow cytometry. as determined by flow cytometry. TLR ligand and inhibitor treatment Poly (I:C) injection 2 2 TLR2 ligand PGN (10 mg/ml), TLR3 ligand Poly (I:C) (20 mg/ml), TLR9 Six- to 8-wk-old WT and Tipe2 / mice were i.p. injected with Poly (I:C) ligand CpG (5 mM), TLR4 ligand LPS (100 ng/ml), and TLR7 ligand (40 mg/kg body weight).
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