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• W2157428298 abstract "STAT6 plays a prominent role in adaptive immunity by transducing signals from extracellular cytokines. We now show that STAT6 is required for innate immune signaling in response to virus infection. Viruses or cytoplasmic nucleic acids trigger STING (also named MITA/ERIS) to recruit STAT6 to the endoplasmic reticulum, leading to STAT6 phosphorylation on Ser407 by TBK1 and Tyr641, independent of JAKs. Phosphorylated STAT6 then dimerizes and translocates to the nucleus to induce specific target genes responsible for immune cell homing. Virus-induced STAT6 activation is detected in all cell-types tested, in contrast to the cell-type specific role of STAT6 in cytokine signaling, and Stat6–/– mice are susceptible to virus infection. Thus, STAT6 mediates immune signaling in response to both cytokines at the plasma membrane, and virus infection at the endoplasmic reticulum. STAT6 plays a prominent role in adaptive immunity by transducing signals from extracellular cytokines. We now show that STAT6 is required for innate immune signaling in response to virus infection. Viruses or cytoplasmic nucleic acids trigger STING (also named MITA/ERIS) to recruit STAT6 to the endoplasmic reticulum, leading to STAT6 phosphorylation on Ser407 by TBK1 and Tyr641, independent of JAKs. Phosphorylated STAT6 then dimerizes and translocates to the nucleus to induce specific target genes responsible for immune cell homing. Virus-induced STAT6 activation is detected in all cell-types tested, in contrast to the cell-type specific role of STAT6 in cytokine signaling, and Stat6–/– mice are susceptible to virus infection. Thus, STAT6 mediates immune signaling in response to both cytokines at the plasma membrane, and virus infection at the endoplasmic reticulum. Virus infection activates STAT6 and regulates specific target genes Virus-induced STAT6 activation is JAK-independent but TBK1-dependent STING and MAVS are required for virus-induced STAT6 signaling STAT6 is required for antiviral innate immunity in vivo Innate immunity is the first line of defense against microbial infection. Recognition of pathogens is mainly mediated by pattern recognition receptors (PRRs), including Toll-like receptors (TLRs), RIG-I-like receptors (RLRs) and NOD-like receptors (NLRs) (Takeuchi and Akira, 2010Takeuchi O. Akira S. Pattern recognition receptors and inflammation.Cell. 2010; 140: 805-820Abstract Full Text Full Text PDF PubMed Scopus (5704) Google Scholar), that trigger signal cascades to upregulate the expression of various cytokines. In the case of viral infection, endosomal TLRs and cytoplasmic RLRs detect viral DNAs or RNAs and induce the production of type I IFN, which are potent inhibitors of viral replication (Gitlin et al., 2006Gitlin L. Barchet W. Gilfillan S. Cella M. Beutler B. Flavell R.A. Diamond M.S. Colonna M. Essential role of mda-5 in type I IFN responses to polyriboinosinic:polyribocytidylic acid and encephalomyocarditis picornavirus.Proc. Natl. Acad. Sci. USA. 2006; 103: 8459-8464Crossref PubMed Scopus (909) Google Scholar, Kato et al., 2005Kato H. Sato S. Yoneyama M. Yamamoto M. Uematsu S. Matsui K. Tsujimura T. Takeda K. Fujita T. Takeuchi O. et al.Cell type-specific involvement of RIG-I in antiviral response.Immunity. 2005; 23: 19-28Abstract Full Text Full Text PDF PubMed Scopus (1104) Google Scholar, Kato et al., 2006Kato H. Takeuchi O. Sato S. Yoneyama M. Yamamoto M. Matsui K. Uematsu S. Jung A. Kawai T. Ishii K.J. et al.Differential roles of MDA5 and RIG-I helicases in the recognition of RNA viruses.Nature. 2006; 441: 101-105Crossref PubMed Scopus (2887) Google Scholar). RLRs, including RIG-I and Mda5, are sensors of viral RNAs in the cytoplasm; in response to viral infection, RLRs associate with the adaptor protein MAVS/Cardif/IPS-1/VISA (Kawai et al., 2005Kawai T. Takahashi K. Sato S. Coban C. Kumar H. Kato H. Ishii K.J. Takeuchi O. Akira S. IPS-1, an adaptor triggering RIG-I- and Mda5-mediated type I interferon induction.Nat. Immunol. 2005; 6: 981-988Crossref PubMed Scopus (2008) Google Scholar, Meylan et al., 2005Meylan E. Curran J. Hofmann K. Moradpour D. Binder M. Bartenschlager R. Tschopp J. Cardif is an adaptor protein in the RIG-I antiviral pathway and is targeted by hepatitis C virus.Nature. 2005; 437: 1167-1172Crossref PubMed Scopus (1951) Google Scholar, Seth et al., 2005Seth R.B. Sun L. Ea C.K. Chen Z.J. Identification and characterization of MAVS, a mitochondrial antiviral signaling protein that activates NF-kappaB and IRF 3.Cell. 2005; 122: 669-682Abstract Full Text Full Text PDF PubMed Scopus (2470) Google Scholar, Xu et al., 2005Xu L.G. Wang Y.Y. Han K.J. Li L.Y. Zhai Z. Shu H.B. VISA is an adapter protein required for virus-triggered IFN-beta signaling.Mol. Cell. 2005; 19: 727-740Abstract Full Text Full Text PDF PubMed Scopus (1503) Google Scholar), an integral membrane protein that functions on both mitochondria and peroxisomes through distinct mechanisms (Dixit et al., 2010Dixit E. Boulant S. Zhang Y. Lee A.S. Odendall C. Shum B. Hacohen N. Chen Z.J. Whelan S.P. Fransen M. et al.Peroxisomes are signaling platforms for antiviral innate immunity.Cell. 2010; 141: 668-681Abstract Full Text Full Text PDF PubMed Scopus (588) Google Scholar); the RLR/MAVS complex facilitates TBK1/IKKε-mediated activation of IRF3/7 and NF-κB, which lead to the induction of type I IFNs. Besides viral RNA, cytoplasmic double-stranded DNA (dsDNA) also induces type I IFNs, but the exact identity of the receptor in this situation is currently not fully established (Ishii et al., 2006Ishii K.J. Coban C. Kato H. Takahashi K. Torii Y. Takeshita F. Ludwig H. Sutter G. Suzuki K. Hemmi H. et al.A Toll-like receptor-independent antiviral response induced by double-stranded B-form DNA.Nat. Immunol. 2006; 7: 40-48Crossref PubMed Scopus (643) Google Scholar, Stetson and Medzhitov, 2006Stetson D.B. Medzhitov R. Recognition of cytosolic DNA activates an IRF3-dependent innate immune response.Immunity. 2006; 24: 93-103Abstract Full Text Full Text PDF PubMed Scopus (803) Google Scholar). A recently identified adaptor protein, endoplasmic reticulum IFN stimulator (STING, also named MITA/ERIS) (Ishikawa and Barber, 2008Ishikawa H. Barber G.N. STING is an endoplasmic reticulum adaptor that facilitates innate immune signalling.Nature. 2008; 455: 674-678Crossref PubMed Scopus (1945) Google Scholar, Sun et al., 2009Sun W. Li Y. Chen L. Chen H. You F. Zhou X. Zhou Y. Zhai Z. Chen D. Jiang Z. STING, an endoplasmic reticulum IFN stimulator, activates innate immune signaling through dimerization.Proc. Natl. Acad. Sci. USA. 2009; 106: 8653-8658Crossref PubMed Scopus (556) Google Scholar, Zhong et al., 2008Zhong B. Yang Y. Li S. Wang Y.Y. Li Y. Diao F. Lei C. He X. Zhang L. Tien P. et al.The adaptor protein MITA links virus-sensing receptors to IRF3 transcription factor activation.Immunity. 2008; 29: 538-550Abstract Full Text Full Text PDF PubMed Scopus (1010) Google Scholar) exhibits a vital role in dsDNA signaling (Ishikawa et al., 2009Ishikawa H. Ma Z. Barber G.N. STING regulates intracellular DNA-mediated, type I interferon-dependent innate immunity.Nature. 2009; 461: 788-792Crossref PubMed Scopus (1664) Google Scholar). The DNA sensors induce type I IFN production either through STING (IFI16 [Unterholzner et al., 2010Unterholzner L. Keating S.E. Baran M. Horan K.A. Jensen S.B. Sharma S. Sirois C.M. Jin T. Latz E. Xiao T.S. et al.IFI16 is an innate immune sensor for intracellular DNA.Nat. Immunol. 2010; 11: 997-1004Crossref PubMed Scopus (1184) Google Scholar]) or via the RIG-I–MAVS axis (involving RNA polymerase III mediated transcription of cytoplasmic DNA [Ablasser et al., 2009Ablasser A. Bauernfeind F. Hartmann G. Latz E. Fitzgerald K.A. Hornung V. RIG-I-dependent sensing of poly(dA:dT) through the induction of an RNA polymerase III-transcribed RNA intermediate.Nat. Immunol. 2009; 10: 1065-1072Crossref PubMed Scopus (673) Google Scholar, Chiu et al., 2009Chiu Y.H. Macmillan J.B. Chen Z.J. RNA polymerase III detects cytosolic DNA and induces type I interferons through the RIG-I pathway.Cell. 2009; 138: 576-591Abstract Full Text Full Text PDF PubMed Scopus (877) Google Scholar]), and both pathways ultimately result in the recruitment and activation of TBK1, which in turn activates IRF3/7 and NF-κB. Many cytokines, including type I IFNs, exert their effects through the canonical JAK (Janus kinase)-STAT (signal transducers and activators of transcription) pathway (Levy and Darnell, 2002Levy D.E. Darnell Jr., J.E. Stats: transcriptional control and biological impact.Nat. Rev. Mol. Cell Biol. 2002; 3: 651-662Crossref PubMed Scopus (2492) Google Scholar). Specifically, IL-4 and IL-13 activate STAT6 (Takeda et al., 1996Takeda K. Tanaka T. Shi W. Matsumoto M. Minami M. Kashiwamura S. Nakanishi K. Yoshida N. Kishimoto T. Akira S. Essential role of Stat6 in IL-4 signalling.Nature. 1996; 380: 627-630Crossref PubMed Scopus (1266) Google Scholar) resulting in T helper cells 2 (Th2) polarization (Akimoto et al., 1998Akimoto T. Numata F. Tamura M. Takata Y. Higashida N. Takashi T. Takeda K. Akira S. Abrogation of bronchial eosinophilic inflammation and airway hyperreactivity in signal transducers and activators of transcription (STAT)6-deficient mice.J. Exp. Med. 1998; 187: 1537-1542Crossref PubMed Scopus (280) Google Scholar, Hebenstreit et al., 2006Hebenstreit D. Wirnsberger G. Horejs-Hoeck J. Duschl A. Signaling mechanisms, interaction partners, and target genes of STAT6.Cytokine Growth Factor Rev. 2006; 17: 173-188Abstract Full Text Full Text PDF PubMed Scopus (240) Google Scholar, Shimoda et al., 1996Shimoda K. van Deursen J. Sangster M.Y. Sarawar S.R. Carson R.T. Tripp R.A. Chu C. Quelle F.W. Nosaka T. Vignali D.A. et al.Lack of IL-4-induced Th2 response and IgE class switching in mice with disrupted Stat6 gene.Nature. 1996; 380: 630-633Crossref PubMed Scopus (1106) Google Scholar). IL-4 induces the phosphorylation of IL-4 receptor, which in turn recruits cytosolic STAT6 by its SH2 domain; the recruited STAT6 is phosphorylated on tyrosine 641 (Y641) by JAK1, which results in the dimerization and nuclear translocation of STAT6 to activate target genes (Mikita et al., 1996Mikita T. Campbell D. Wu P. Williamson K. Schindler U. Requirements for interleukin-4-induced gene expression and functional characterization of Stat6.Mol. Cell. Biol. 1996; 16: 5811-5820Crossref PubMed Scopus (226) Google Scholar, Mikita et al., 1998Mikita T. Daniel C. Wu P. Schindler U. Mutational analysis of the STAT6 SH2 domain.J. Biol. Chem. 1998; 273: 17634-17642Crossref PubMed Scopus (44) Google Scholar). Several cytokines, including IL-3/15, IFN-α and platelet-derived growth factor (PDGF-BB), activate STAT6 in different cell types (Bulanova et al., 2003Bulanova E. Budagian V. Orinska Z. Krause H. Paus R. Bulfone-Paus S. Mast cells express novel functional IL-15 receptor alpha isoforms.J. Immunol. 2003; 170: 5045-5055Crossref PubMed Scopus (46) Google Scholar, Masuda et al., 2000Masuda A. Matsuguchi T. Yamaki K. Hayakawa T. Kubo M. LaRochelle W.J. Yoshikai Y. Interleukin-15 induces rapid tyrosine phosphorylation of STAT6 and the expression of interleukin-4 in mouse mast cells.J. Biol. Chem. 2000; 275: 29331-29337Crossref PubMed Scopus (58) Google Scholar, Quelle et al., 1995Quelle F.W. Shimoda K. Thierfelder W. Fischer C. Kim A. Ruben S.M. Cleveland J.L. Pierce J.H. Keegan A.D. Nelms K. et al.Cloning of murine Stat6 and human Stat6, Stat proteins that are tyrosine phosphorylated in responses to IL-4 and IL-3 but are not required for mitogenesis.Mol. Cell. Biol. 1995; 15: 3336-3343Crossref PubMed Scopus (303) Google Scholar), and induce over 150 diverse targets, many of which are involved in Th2-associated processes (Elo et al., 2010Elo L.L. Jarvenpaa H. Tuomela S. Raghav S. Ahlfors H. Laurila K. Gupta B. Lund R.J. Tahvanainen J. Hawkins R.D. et al.Genome-wide profiling of interleukin-4 and STAT6 transcription factor regulation of human Th2 cell programming.Immunity. 2010; 32: 852-862Abstract Full Text Full Text PDF PubMed Scopus (110) Google Scholar, Wei et al., 2010Wei L. Vahedi G. Sun H.W. Watford W.T. Takatori H. Ramos H.L. Takahashi H. Liang J. Gutierrez-Cruz G. Zang C. et al.Discrete roles of STAT4 and STAT6 transcription factors in tuning epigenetic modifications and transcription during T helper cell differentiation.Immunity. 2010; 32: 840-851Abstract Full Text Full Text PDF PubMed Scopus (238) Google Scholar). A thorough understanding of biological consequences of STAT6 signaling awaits additional studies. It is known that NF-κB, AP-1 and IRFs are responsible for the induction of many IFN-stimulated genes (ISGs), however, the role of STAT6 in anti-viral response is unclear. Here we report a STAT6-dependent antiviral innate immune signaling event that leads to the induction of chemokines, including CCL2, CCL20, and CCL26, and these chemokines recruit immune cells to combat viral infection. More importantly, virus induces STAT6 activation independently of JAK, but instead relies on STING and TBK1, as well as MAVS in the case of RNA virus. The physiological significance of the novel pathway is reflected by a higher susceptibility of Stat6–/– mice to viral infections; moreover, unlike other cell type-specific STAT6 signaling pathways, virus-induced STAT6 activation is ubiquitously detected, implying a fundamental requirement of this mechanism in the defense against viral infections. Using C-terminal STING (aa 178–379) as bait in the yeast 2-hybrid screen, we identified an STING-STAT6 interaction and confirmed it in 293 cells by coimmunoprecipitation (coIP) (Figure S1A available online). Specifically, the DNA-binding domain (DBD) of STAT6 and STING C terminus (aa 317–379) were required for this interaction (Figures S1B–S1D). We next examined this interaction at endogenous protein levels. Analysis with confocal microscope showed a dispersed pattern of STAT6 in the cytosol of unstimulated HeLa cells; upon infection with Sendai virus (SeV, an RNA virus), STAT6 redistributes to the perinuclear regions, colocalizes with STING, and eventually translocates into the nucleus (Figure 1A ). CoIP analyses also revealed an inducible interaction of endogenous STAT6 with STING, as well as MAVS and TBK1, in SeV-infected primary MEFs, 2fTGH and THP-1 cells (Figure 1B). Consistent with these observations, endogenous STAT6 co-fractionate with STING in HeLa cell lysates after Herpes simplex virus 1 (HSV-1, a DNA virus) infection (Figure 1C). Similar results were obtained from SeV-infected HeLa cells, with an additional location to a mixed fraction containing MAVS-resident mitochondria-associated ER membrane (MAM) and also MAVS-resident peroxisomal membrane (Dixit et al., 2010Dixit E. Boulant S. Zhang Y. Lee A.S. Odendall C. Shum B. Hacohen N. Chen Z.J. Whelan S.P. Fransen M. et al.Peroxisomes are signaling platforms for antiviral innate immunity.Cell. 2010; 141: 668-681Abstract Full Text Full Text PDF PubMed Scopus (588) Google Scholar, Ishikawa et al., 2009Ishikawa H. Ma Z. Barber G.N. STING regulates intracellular DNA-mediated, type I interferon-dependent innate immunity.Nature. 2009; 461: 788-792Crossref PubMed Scopus (1664) Google Scholar, Zhong et al., 2008Zhong B. Yang Y. Li S. Wang Y.Y. Li Y. Diao F. Lei C. He X. Zhang L. Tien P. et al.The adaptor protein MITA links virus-sensing receptors to IRF3 transcription factor activation.Immunity. 2008; 29: 538-550Abstract Full Text Full Text PDF PubMed Scopus (1010) Google Scholar) (Figure 1D). These data demonstrate that STAT6 interacts with STING during virus infection.Figure 1Virus-Induced STAT6-STING Interaction and STAT6 TranslocationShow full caption(A) STAT6 translocates and colocalizes with STING after virus infection. Confocal microscopy of endogenous STING (red), STAT6 (green) and the merge in HeLa cells infected with Sendai virus (SeV) for the indicated hours. Nuclei were stained with DAPI. All images are representative of at least three independent experiments in which >95% of the cells displayed similar staining. Scale bars represent 10 μm.(B) STAT6 interacts with STING and TBK1 after virus infection. Primary MEFs, 2fTGH and THP-1 cells were infected with SeV for the indicated hours. Cell lysates were immunoprecipitated (IP) with α-STING and blotted (IB) with STAT6, TBK1 and MAVS antibodies. WCL, whole cell lysates.(C and D) Virus infection induces STAT6 translocation. Western blot analyses of fractionated HeLa cells infected with Herpes simplex virus 1 (HSV-1) (C) or SeV (D) for the indicated hours. Cyt, cytosolic; ER, endoplasmic reticulum-rich; Nuc, nuclear; and Mit, mitochondrial; were revealed by Caspase 3, Calnexin, Histone H3, and COX IV, respectively.See also Figure S1.View Large Image Figure ViewerDownload Hi-res image Download (PPT) (A) STAT6 translocates and colocalizes with STING after virus infection. Confocal microscopy of endogenous STING (red), STAT6 (green) and the merge in HeLa cells infected with Sendai virus (SeV) for the indicated hours. Nuclei were stained with DAPI. All images are representative of at least three independent experiments in which >95% of the cells displayed similar staining. Scale bars represent 10 μm. (B) STAT6 interacts with STING and TBK1 after virus infection. Primary MEFs, 2fTGH and THP-1 cells were infected with SeV for the indicated hours. Cell lysates were immunoprecipitated (IP) with α-STING and blotted (IB) with STAT6, TBK1 and MAVS antibodies. WCL, whole cell lysates. (C and D) Virus infection induces STAT6 translocation. Western blot analyses of fractionated HeLa cells infected with Herpes simplex virus 1 (HSV-1) (C) or SeV (D) for the indicated hours. Cyt, cytosolic; ER, endoplasmic reticulum-rich; Nuc, nuclear; and Mit, mitochondrial; were revealed by Caspase 3, Calnexin, Histone H3, and COX IV, respectively. See also Figure S1. 293 cells lack a functional endogenous STAT6 but express the other components of the IL-4 signaling pathway (Mikita et al., 1996Mikita T. Campbell D. Wu P. Williamson K. Schindler U. Requirements for interleukin-4-induced gene expression and functional characterization of Stat6.Mol. Cell. Biol. 1996; 16: 5811-5820Crossref PubMed Scopus (226) Google Scholar, Mikita et al., 1998Mikita T. Daniel C. Wu P. Schindler U. Mutational analysis of the STAT6 SH2 domain.J. Biol. Chem. 1998; 273: 17634-17642Crossref PubMed Scopus (44) Google Scholar). Taking advantage of this property, we first established a 293 cell-line stably expressing Flag-STAT6 (293-STAT6) and confirmed its normal responsiveness to IL-4/13 with intact Y641 phosphorylation (data not shown). Virus infection resulted in the nuclear translocation of STAT6, suggesting that STAT6 may serve as a transcriptional activator under this situation. To confirm this hypothesis, we assessed Y641 phosphorylation of STAT6, since it is required for STAT6 activation in response to cytokines. We found that STAT6 was indeed phosphorylated on Y641 in SeV-infected and poly (I:C)/poly dAdT-transfected cells, and this STAT6 phosphorylation takes place prior to the phosphorylation of IRF3 and other STATs (Figures 2A, 2D, and 2H , and Figures S2B and S2H ). A STAT6-responsive luciferase reporter (E3-Luc) (Yuan et al., 2006Yuan Q. Campanella G.S. Colvin R.A. Hamilos D.L. Jones K.J. Mathew A. Means T.K. Luster A.D. Membrane-bound eotaxin-3 mediates eosinophil transepithelial migration in IL-4-stimulated epithelial cells.Eur. J. Immunol. 2006; 36: 2700-2714Crossref PubMed Scopus (35) Google Scholar) was activated in 293-STAT6 cells upon virus infection and poly (I:C)/poly dAdT transfection, whereas a nonresponsive control reporter (mutated at the STAT6-binding site, E3-Luc-M) was not affected (Figure 2C and Figure S2A). By contrast, neither reporter was activated in 293 cells, indicating a transactivation function of STAT6 in response to virus. These findings imply a previously unknown pathway of STAT6 activation in response to viral infection and cytoplasmic dsRNA/DNA.Figure S2STAT6 Is Phosphorylated and Activated upon Viral Infection, Related to Figure 2Show full caption(A) 293-STAT6 or 293 cells transfected with E3-Luc were treated with IL-4/IL-13 for 12 hr, or transfected with poly (I:C) (pIC) (left); or 293-STAT6 cells transfected with E3-Luc were transfected with poly (I:C) (pIC-trans) or treated with poly (I:C) (pIC-add) for indicated hours (right), STAT6 activation was analyzed using luciferase assay (fold induction).(B) 293-STAT6 cells were transfected with poly dAdT (upper) or poly (I:C) (lower) for indicated hours. Cell lysates were prepared and phosphorylation of proteins were analyzed by western blot using indicated phospho-antibodies.(C) Mouse primary MEF (left) or U5A (right) cells were infected with SeV for indicated hours, supernatants from infected cells were collected and production of indicated cytokines were analyzed by ELISA or type I-IFN bioassay.(D) Left: the table of the cell lines used and their missing proteins; Right: indicated cells were treated with IL-13 (10 ng/ml) for indicated times, phosphorylation of STAT6 was analyzed with phospho-STAT6 (Tyr641) antibody (upper). The expression of endogenous STAT6 was blotted with STAT6 antibody (bottom).(E) JAK1 deficiency abrogates the tyrosine-phosphorylation of STAT6 induced by IL-4/13 treatment. U4A (Jak1−/−) and U5A (Ifnar2−/−) cells were treated with IL-4 or IL-13 (10 ng/ml) for indicated times. Phosphorylation of STAT6 was analyzed with phospho-STAT6 (Tyr641) antibody (upper), and expression of STAT6 with STAT6 antibody (bottom).(F) 2fTGH and U5A cells were treated with IFNα (500 unit/ml) plus IFNβ (500 unit/ml) for indicated times, STAT1/6 phosphorylation was analyzed with phospho-STAT1 (Tyr701) and phosphor-STAT6 (Tyr641) antibody, and protein loading was monitored with STAT6 antibody.(G) JAK1 deficient U4A cell shows normal response to viral infection but no response to IL-4. Indicated cells transfected with E3-Luc were left untreated (Vec), infected with SeV, transfected with poly (I:C) (pIC) for 24 hr, or treated with IL-4 for 12 hr, followed by luciferase assay.(H) STAT6 is phosphorylated in HeLa cells after SeV infection. HeLa cells were infected with SeV for indicated hours, or treated with IL-4 for 2 hr. Phosphorylation of proteins was analyzed with indicated phospho-antibodies.(I) Leu-551of STAT6 is one of the most conserved amino acid in SH2 domain of STAT proteins. Sequence alignment of the SH2 domain of STAT6 from different species. The arrow indicates Leu-551. Red lines indicate amino acids mutated in this study.(J) The mutants of SH2 domain have different effects on STAT6 activation in response to viral infection or IL-4 treatment. 293 cells transfected with E3-Luc, and WT or indicated mutants of STAT6 were left untreated (Con), infected with SeV, or transfected with poly (I:C) (pIC) for 24 hr, or treated with IL-4 for 12 hr before the luciferase assay was performed. The expression of STAT6 proteins was shown by western blot (bottom).(K) Leu-551 is essential for STAT6 activation in response to viral infection. The experiments were performed as in (J) except that the different L551 mutants were used.(L) The experiments were performed as in (J) except that the reversed mutants were used to verify that the aforementioned phenotype was caused by the amino acid replacement.(M) STAT6 dimerizes after viral infection. 293 cells transfected with Flag- and HA- STAT6 were infected with SeV for indicated hours, cell lysates were IP-ed with α-Flag and blotted with α-HA. The expression of HA-, Flag- proteins was analyzed in WCL.(N) STAT6-L551A is not dimerized after virus infection. 293-STAT6 (WT) and 293-STAT6-L551A (L551A) cells untreated (C), infected with SeV for 12 hr or treated with IL-4 for 2 hr. Cell lysates were analyzed by native PAGE and western analyses of STAT6 dimers (top) and IRF3 dimers (bottom).(O) STAT6-L551A is not phosphorylated at Y641 after virus infection. The experiments were performed same as in (N) except that phosphorylation of STAT6 (top) and type I-IFN production (bottom) were analyzed.View Large Image Figure ViewerDownload Hi-res image Download (PPT) (A) 293-STAT6 or 293 cells transfected with E3-Luc were treated with IL-4/IL-13 for 12 hr, or transfected with poly (I:C) (pIC) (left); or 293-STAT6 cells transfected with E3-Luc were transfected with poly (I:C) (pIC-trans) or treated with poly (I:C) (pIC-add) for indicated hours (right), STAT6 activation was analyzed using luciferase assay (fold induction). (B) 293-STAT6 cells were transfected with poly dAdT (upper) or poly (I:C) (lower) for indicated hours. Cell lysates were prepared and phosphorylation of proteins were analyzed by western blot using indicated phospho-antibodies. (C) Mouse primary MEF (left) or U5A (right) cells were infected with SeV for indicated hours, supernatants from infected cells were collected and production of indicated cytokines were analyzed by ELISA or type I-IFN bioassay. (D) Left: the table of the cell lines used and their missing proteins; Right: indicated cells were treated with IL-13 (10 ng/ml) for indicated times, phosphorylation of STAT6 was analyzed with phospho-STAT6 (Tyr641) antibody (upper). The expression of endogenous STAT6 was blotted with STAT6 antibody (bottom). (E) JAK1 deficiency abrogates the tyrosine-phosphorylation of STAT6 induced by IL-4/13 treatment. U4A (Jak1−/−) and U5A (Ifnar2−/−) cells were treated with IL-4 or IL-13 (10 ng/ml) for indicated times. Phosphorylation of STAT6 was analyzed with phospho-STAT6 (Tyr641) antibody (upper), and expression of STAT6 with STAT6 antibody (bottom). (F) 2fTGH and U5A cells were treated with IFNα (500 unit/ml) plus IFNβ (500 unit/ml) for indicated times, STAT1/6 phosphorylation was analyzed with phospho-STAT1 (Tyr701) and phosphor-STAT6 (Tyr641) antibody, and protein loading was monitored with STAT6 antibody. (G) JAK1 deficient U4A cell shows normal response to viral infection but no response to IL-4. Indicated cells transfected with E3-Luc were left untreated (Vec), infected with SeV, transfected with poly (I:C) (pIC) for 24 hr, or treated with IL-4 for 12 hr, followed by luciferase assay. (H) STAT6 is phosphorylated in HeLa cells after SeV infection. HeLa cells were infected with SeV for indicated hours, or treated with IL-4 for 2 hr. Phosphorylation of proteins was analyzed with indicated phospho-antibodies. (I) Leu-551of STAT6 is one of the most conserved amino acid in SH2 domain of STAT proteins. Sequence alignment of the SH2 domain of STAT6 from different species. The arrow indicates Leu-551. Red lines indicate amino acids mutated in this study. (J) The mutants of SH2 domain have different effects on STAT6 activation in response to viral infection or IL-4 treatment. 293 cells transfected with E3-Luc, and WT or indicated mutants of STAT6 were left untreated (Con), infected with SeV, or transfected with poly (I:C) (pIC) for 24 hr, or treated with IL-4 for 12 hr before the luciferase assay was performed. The expression of STAT6 proteins was shown by western blot (bottom). (K) Leu-551 is essential for STAT6 activation in response to viral infection. The experiments were performed as in (J) except that the different L551 mutants were used. (L) The experiments were performed as in (J) except that the reversed mutants were used to verify that the aforementioned phenotype was caused by the amino acid replacement. (M) STAT6 dimerizes after viral infection. 293 cells transfected with Flag- and HA- STAT6 were infected with SeV for indicated hours, cell lysates were IP-ed with α-Flag and blotted with α-HA. The expression of HA-, Flag- proteins was analyzed in WCL. (N) STAT6-L551A is not dimerized after virus infection. 293-STAT6 (WT) and 293-STAT6-L551A (L551A) cells untreated (C), infected with SeV for 12 hr or treated with IL-4 for 2 hr. Cell lysates were analyzed by native PAGE and western analyses of STAT6 dimers (top) and IRF3 dimers (bottom). (O) STAT6-L551A is not phosphorylated at Y641 after virus infection. The experiments were performed same as in (N) except that phosphorylation of STAT6 (top) and type I-IFN production (bottom) were analyzed. STAT6 can be activated by several cytokines. To clarify a potential role of cytokines in STAT6 activation during viral challenges, we first monitored cytokine production in virus-infected cells. Neither IL-4 nor IL-13 was induced by virus (Figure 2B and Figure S2C), thus excluding their involvement in STAT6 activation after virus infection. Strikingly, other cytokines including type I IFNs, IL-8 and STAT6-induced genes (CCL2 and CCL20, see below), displayed similar kinetics post infection. Therefore, CCL2/20 is unlikely regulated by cytokines like type I IFNs or IL-8. In fact, when media of SeV-infected 293-STAT6 cells were used to treat naive 293-STAT6 cells, STAT6 phosphorylation was only detected in virus-infected but not media-treated cells, whereas phosphorylation of STAT1/2/3/5 was detected in media-treated cells (Figure 2D and data not shown), excluding any STAT6-activating cytokines in the media within these time points. Furthermore, STAT6 phosphorylation was intact upon SeV and poly (I:C) stimulation when production of cytokines including IL-8 and type I IFNs was inhibited by cycloheximide (CHX) pretreatment (Figures 2E and 2F). These data collectively indicate a cytokine –independent pathway of STAT6 activation upon virus infection. Next we used 2fTGH and its derivative cell lines (Kumar et al., 1997Kumar A. Commane M. Flickinger T.W. Horvath C.M. Stark G.R. Defective TNF-alpha-induced apoptosis in STAT1-nu" @default.
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• W2157428298 title "Activation of STAT6 by STING Is Critical for Antiviral Innate Immunity" @default.
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