Inducible Major Vault Protein Plays a Pivotal Role in Double-Stranded RNA− Or Virus-Induced Proinflammatory Response

Inducible Major Vault Protein Plays a Pivotal Role in Double-Stranded RNA− or Virus-Induced Proinflammatory Response

This information is current as Nanfang Peng, Shi Liu, Zhangchuan Xia, Sheng Ren, Jian of September 28, 2021. Feng, Mingzhen Jing, Xin Gao, Erik A. C. Wiemer and Ying Zhu J Immunol 2016; 196:2753-2766; Prepublished online 3 February 2016;

doi: 10.4049/jimmunol.1501481 Downloaded from http://www.jimmunol.org/content/196/6/2753

Supplementary http://www.jimmunol.org/content/suppl/2016/02/02/jimmunol.150148 Material 1.DCSupplemental http://www.jimmunol.org/ References This article cites 53 articles, 22 of which you can access for free at: http://www.jimmunol.org/content/196/6/2753.full#ref-list-1

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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 © 2016 by The American Association of Immunologists, Inc. All rights reserved. Print ISSN: 0022-1767 Online ISSN: 1550-6606. The Journal of Immunology

Inducible Major Vault Protein Plays a Pivotal Role in Double-Stranded RNA– or Virus-Induced Proinﬂammatory Response

Nanfang Peng,*,1 Shi Liu,*,1 Zhangchuan Xia,* Sheng Ren,* Jian Feng,* Mingzhen Jing,* Xin Gao,* Erik A. C. Wiemer,† and Ying Zhu*

Pathogen invasion triggers robust antiviral cytokine production via different transcription factor signaling pathways. We have previously demonstrated that major vault protein (MVP) induces type I IFN production during viral infection; however, little is known about the role of MVP in proinﬂammatory responses. In this study, we found in vitro that expression of MVP, IL-6, and IL-8 was inducible upon dsRNA stimulation or viral infection. Moreover, MVP was essential for the induction of IL-6 and

IL-8, as impaired expression of IL-6 and IL-8 in MVP-deficient human PBMCs, human lung epithelial cells (A549), and THP- Downloaded from 1 monocytes, as well as in murine splenocytes, peritoneal macrophages, and PBMCs from MVP-knockout (MVP2/2) mice, was observed. Upon investigation of the underlying mechanisms, we demonstrated that MVP acted in synergy with AP-1 (c-Fos) and CCAAT/enhancer binding protein (C/EBP)b–liver-enriched transcriptional activating protein to activate the IL6 and IL8 promoters. Introduction of mutations into the AP-1 and C/EBPb binding sites on the IL6 and IL8 promoters resulted in the loss of synergistic activation with MVP. Furthermore, we found that MVP interacted with both c-Fos and C/EBPb. The interactions 2/2 promoted nuclear translocation and recruitment of these transcription factors to IL6 and IL8 promoter regions. In the MVP http://www.jimmunol.org/ mouse model, significantly decreased expression of early antiviral cytokines resulted in higher viral titer in the lung, higher mortality, and heavier lung damage after infection with lethal influenza A virus. Taken together, our findings help to delineate a novel role of MVP in host proinflammatory response. The Journal of Immunology, 2016, 196: 2753–2766.

ecognition of pathogen-associated molecular patterns whereas TLRs 1–13, except TLR10, are expressed in mice (2). by pattern recognition receptors (PRRs) initiates a local TLRs are expressed on both immune cells, including macro- R inflammatory response characterized by production of phages, dendritic cells, and B cells, and nonimmune cells, such as diverse proinflammatory cytokines and chemokines, and this re- fibroblasts and epithelial cells (3). The RLRs consist of RIG-I, by guest on September 28, 2021 sponse acts as a first-line defense, protecting the host from invading melanoma differentiation–associated protein (MDA)5, and LGP2. microbial pathogens. The main PRRs include membrane-bound TLR3 along with RIG-I and MDA5 have been shown to rec- TLRs and cytosolic retinoic acid–inducible gene (RIG)-I–like ognize viral dsRNA generated as a replicative intermediate receptors (RLRs) (1). To date, 13 mammalian TLR paralogs during ssRNA virus infection and the synthetic dsRNA analog have been identified. TLRs 1–10 are expressed in humans, polyinosinic-polycytidylic acid [poly(I:C)] (3–5). Although re- cently challenged by some reports, the notion that influenza A *State Key Laboratory of Virology, College of Life Sciences, Wuhan University, virus (IAV) produces replicative intermediate dsRNA after infec- Wuhan, Hubei 430072, China; and †Department of Medical Oncology, Erasmus tion is widely accepted (4). As the first sensor of viral dsRNA, University Medical Center Cancer Institute, Rotterdam 3000 CA, the Netherlands TLR3 recognizes dsRNA and signals through NF-kB and MAPKs 1 N.P. and S.L. are cofirst authors. to produce proinflammatory cytokines and chemokines via Toll/ Received for publication June 30, 2015. Accepted for publication January 4, 2016. IL-1R domain–containing adaptor inducing IFN-b–dependent This work was supported by Major State Basic Research Development Program of pathways (6). The interaction between Toll/IL-1R domain– China Grant 2013CB911102; National Natural Science Foundation of China Grants 81461130019, 81271821, 31500149, and 31570870; and by National Mega Project containing adaptor inducing IFN-b and TNFR-associated factor on Major Infectious Diseases Prevention Grant 2012ZX10004503-004. The funding (TRAF)6 activates TGF-b–activated protein kinase (TAK1), agencies had no role in study design, data collection, or analysis, decision to publish, which activates both the NF-kB signaling pathway by mediating or preparation of the manuscript. IkB kinase activation and the MAPK signaling pathway by si- Address correspondence and reprint requests to Prof. Ying Zhu, State Key Laboratory of Virology, College of Life Sciences, Wuhan University, 16 Luojia Hill Road, Wuhan, multaneously phosphorylating MAPK kinases 3 and 6 (7–9). RIG-I Hubei 430072, China. E-mail address: [email protected] and MDA5 sense the intracellular dsRNA and signal through The online version of this article contains supplemental material. mitochondrial antiviral signaling protein–dependent pathways Abbreviations used in this article: ATF, activating transcription factor; C/EPB, (10). Mitochondrial antiviral signaling protein contains multiple CCAAT/enhancer binding protein; ChIP, chromatin immunoprecipitation; h, human; TRAF-interacting motifs, which interact with TRAF family IAV, influenza A virus; IRF, IFN regulatory factor; LAP, liver-enriched transcriptional activating protein; LIP, liver-enriched transcriptional inhibitory protein; m, mouse; members, including TRAF2, TRAF3, and TRAF6 and down- MDA, melanoma differentiation–associated protein; MOI, multiplicity of infection; stream with MAP3K and TAK1. Similar to the TLR3 signaling MVP, major vault protein; PM, peritoneal macrophage; poly(I:C), polyinosinic- polycytidylic acid; PRR, pattern recognition receptor; RIG, retinoic acid–inducible pathway, TRAF6 and TAK1 play critical roles in the activation of gene; RLR, retinoic acid–inducible gene-I–like receptor; shRNA, short hairpin RNA; NF-kB and MAPK signaling pathways (10–12). TRAF6 can also TAK1, TGF-b–activated protein kinase 1; TCID50, 50% tissue culture infective dose; interact with MAPK kinase 1, which in turn contributes to the TRAF, TNFR-associated factor. activation of IkB kinase and MAPKK, leading to activation of the Copyright Ó 2016 by The American Association of Immunologists, Inc. 0022-1767/16/$30.00 NF-kB and MAPK signaling pathways (13). www.jimmunol.org/cgi/doi/10.4049/jimmunol.1501481 2754 MVP REGULATES THE EXPRESSION OF IL-6 AND IL-8

MAPKs such as JNK, ERK, and p38 MAPK (14) target the medium. All media were supplemented with 10% heat-inactivated FBS, downstream transcription factors such as AP-1 and CCAAT/ 100 U/ml penicillin, and 100 mg/ml streptomycin sulfate and all cell enhancer binding protein (C/EBP)b (15–17). AP-1 is a hetero- cultures were maintained at 37˚C in a 5% CO2 incubator. All cell lines above were purchased from American Type Culture Collection. dimeric basic region leucine zipper (bZIP) protein comprised of members of the Jun, Fos, activating transcription factors (ATF), Virus and the Maf subfamily (18). The Jun proteins (c-Jun, JunB, and The influenza virus A/Hong Kong/498/97 (H3N2) strain used in this study JunD) both homodimerize and heterodimerize with Fos, whereas was provided by the China Center for Type Culture Collection. The final the Fos proteins (c-Fos, FosB, Fra-1, and Fra-2) only hetero- concentration of H3N2 virus infection used in this study was a multiplicity dimerize with members of the Jun family (14). C/EBPb, which is a of infection (MOI) of 1. The influenza virus A/FM/1/47 (H1N1) used for member of the C/EBP transcription factor family, is widely in- in vivo experiments was adapted for lethality in mice through serial lung passage. volved in cytokine production in response to a variety of stimuli Stock virus was propagated in 10-d-old embryonated chicken eggs (19). C/EBPb binding motifs are found in the functional regula- (Shijun Li laboratory at Central China Agricultural University) for 36–48 h tory region of genes encoding IL-6, IL-8, and TNF-a (20). The at 37˚C. The allantoic fluid was then harvested, and aliquots were stored at 2 C/ebpb gene is translated into at least three different protein 80˚C before being used. isoforms, namely the liver-enriched transcriptional activating Reagent proteins (LAPs), the 38 kDa (full) and the 34 kDa (LAP), and the 20-kDa liver-enriched transcriptional inhibitory proteins Synthetic poly(I:C) (InvivoGen, San Diego, CA), recombinant human (h) IFN-g (PeproTech, London, U.K.), and recombinant mouse (m)IFN-g (LIPs). Thus, the outcome of C/EBPb binding can be either (ProSpec, Rehovot, IL) were dissolved in endotoxin-free water. Pento- activating or repressing depending on which C/EBPb-binding barbital sodium salt (Sigma-Aldrich, Oakville, CA) was dissolved in sterile Downloaded from partner is involved. saline. Abs against human b-actin (cw0096A) and GAPDH (cw0100A) The major vault protein (MVP) is the predominant component of were purchased from ComWin Biotech (Beijing, China), and Abs specific the largest ribonucleoprotein particle present in eukaryotic cells for human MVP (sc-18701), C/EBPb (sc-150), c-Fos (sc-52), IRF3 (sc- 9082), lamin A (sc-20680), and c-Myc tag (sc-789) were purchased from that is named the vault complex. MVP/vaults have been associated Santa Cruz Biotechnology (Santa Cruz, CA). Anti-Flag (F1804) was with multidrug resistance (21–23), nuclear–cytoplasmic transport purchased from Sigma-Aldrich. Abs specific for C/EBPb (sc-150X) and

(24), innate immunity (25), autophagy (26), and signal trans- c-Fos (sc-52X) used in chromatin immunoprecipitation (ChIP) assays http://www.jimmunol.org/ duction pathways (27–31); however, the precise function of MVP/ were also purchased from Santa Cruz Biotechnology. vault remains enigmatic. Our previous work indicating MVP- Isolation and transfection of human PBMCs induced type I IFN production upon hepatitis C virus infection uncovers a new role of MVP in modulating innate immunity Human PBMCs were isolated from venous blood samples from healthy volunteers by density centrifugation using Histopaque as per the man- during viral infection (32). That most PRRs signal through NF- ufacturer’s instructions (Sigma-Aldrich, St. Louis, MO). PBMCs were kB, IFN regulatory factors (IRFs), and MAPK cascades to induce transfected with plasmid DNA by electroporation with an Amaxa Nucle- production of both type I IFNs and proinﬂammatory cytokines ofector II device according to the manufacturer’s protocol and then raises the question whether MVP is involved in the PRR-mediated resuspended in RPMI 1640 supplemented with penicillin (100 U/ml) and

streptomycin (100 mg/ml). by guest on September 28, 2021 proinflammatory response. In this study, we demonstrate that MVP is involved in the Plasmids proinflammatory response to dsRNA and virus. Results from 2/2 pCMV-tag2B-MVP, Flag-tagged C/EBPb-LAP, Flag-tagged C/EBPb-LIP, in vitro experiments and the MVP mouse model suggested that the luciferase reporter vector (pGL3) containing the IL6 promoter region MVP deficiency impaired expression of proinflammatory cyto- (2651/0) and its site-specific mutants, and the IL8 promoter region (21534/ kines and chemokines, such as IL-6 and IL-8, upon dsRNA 227) and its site-specific mutants were reported previously (19, 32–34). To stimulation or virus infection. Furthermore, we uncovered a pre- generate GFP-tagged MVP and Myc-tagged MVP expression vector, human viously unrecognized mechanism wherein MVP interacts selec- MVP fragment was amplified from pCMV-tag2B-MVP and inserted to pEGFP-C1 and pcDNA3.1/myc-His(2) B. Flag-tagged c-Fos, c-Jun, CREB1, tively with c-Fos and C/EBPb, which are important downstream and CREB2 were purchased from Addgene (Cambridge, MA). Human p50 transcription factors involved in the proinflammatory signaling. and p65 cDNA were amplified from A549 cells and cloned into pCMV- MVP promoted the nuclear translocation of c-Fos and C/EBPb tag2B to generate Flag-p50 and -p65 expression vectors; all constructs and increased their recruitment to the promoters of target genes, were confirmed by DNA sequencing. Four plasmids involved in producing recombinant lentivirus, that is, ultimately leading to expression of proinflammatory cytokines and lentiviral vector PLKO.1-TRC control, PLKO.1-scramble short hairpin chemokines. RNA (shRNA), and two packaging plasmids (psPAX2 and pMD2.G), were obtained from Dr. Yingliang Wu (Wuhan University). To construct PLKO.1-shMVP shRNA, we designed four oligonucleotide pairs listed in Materials and Methods Supplemental Table I by utilizing online tools (http://www.addgene.org/ Ethics statement tools/protocols/plko/), and then the paired oligonucleotides were annealed and cloned into AgeI and BamHI sites of PLKO.1-TRC control vector. The The human PBMC collection from blood samples for research was con- constructs were confirmed by DNA sequencing. MVP shRNA (shRNA- ducted according to the principles of the Declaration of Helsinki and was MVP) and negative control (shRNA-Ctrl) were described previously (32). approved by the Institutional Review Board of the College of Life Sciences, Wuhan University, in accordance with guidelines for the protection of Generation of stable MVP-knockdown cells human subjects. All study participants provided written informed consent The lentiviral vector PLKO.1-scramble shRNA or pLKO.1-MVP shRNA for the collection of samples and subsequent analysis. no. 4, along with lentivirus packaging plasmids psPAX2 and pMD2.G, were All animal experiments were undertaken in accordance with the National cotransfected into HEK 293T cells at a ratio of 3:2:2 using Lipofectamine Guide for the Care and Use of Laboratory Animals Institutes of Health . 2000 reagent (Invitrogen, Carlsbad, CA). After 24 h, the culture media were The protocol was approved by the Institutional Animal Care and Use replaced with fresh medium. The viruses in conditioned medium were Committee of Wuhan University (project license no. WDSKY0201302). harvested on 2 consecutive days and filtered with low-protein binding filters Cell culture (Millex-HV, 0.45-mm polyvinylidene difluoride; Millipore) before they were aliquoted and stored at 280˚C. Cells of interest were seeded into Human lung epithelial cells (A549) were cultured in F12K media, HEK 35-mm cell culture dishes and changed to fresh culture media containing 293T cells and Madin-Darby canine kidney cells were cultured in DMEM, 8 mg/ml Polybrene when cells were ∼70% confluent, after which 0.1 ml and human THP-1 monocytes were maintained in RPMI 1640 culture PLKO.1-scramble shRNA or pLKO.1-MVP shRNA no. 4 lentivirus-containing The Journal of Immunology 2755 media, which were thawed completely at room temperature before infec- formaldehyde crosslink. DNA was obtained by phenol-chloroform ex- tion, were added. After 24 h, the virus-infected cells were selected by traction and ethanol precipitation. Pellets were resuspended in 100 ml culture with puromycin (0.7 mg/ml for A549 cells and 1.0 mg/ml for THP- TE buffer. 1 monocytes) for at least 3 d and subjected to required assays. Real-time PCR was performed using the primer pairs listed in Supplemental Table I. IL6 and IL8 promoter abundances were first nor- Transient transfection and luciferase reporter gene assay malized by dividing them by the abundances in the input control. Incre- ment folds are defined as the ratio of the normalized abundances for the A549 cells that were grown to 80% confluence in 24-well plates were experimental group to those for the control group. cotransfected with the luciferase reporter plasmid and relevant recombinant expression plasmids at an appropriate ratio using Lipofectamine 2000 Mice (Invitrogen). The reporter gene activities were measured using a Dual- 2 2 Luciferase reporter assay, according to the manufacturer’s instructions Mice homozygous for MVP deficiency (C57BL/6 MVP / ) were reported (Promega, Madison, WI). previously (36). Wild-type C57BL/6 mice were purchased from the Ani- mal Facility at Wuhan University ZhongNan Hospital. All mice were Real-time PCR maintained under specific pathogen-free conditions. When mice met cer- tain clinical criteria or at the end of the experiments, they were humanely Total RNAwas extracted using TRIzol reagent (Invitrogen) according to the euthanized. manufacturer’s instructions. Quantitative PCR assays were performed using the ABI StepOne real-time PCR system (Applied Biosystems, Waltham, Preparation of primary mouse cells MA) and SYBR RT-PCR kits (Applied Biosystems). Primers specific to 2 2 either human or murine genes are listed in Supplemental Table I or were Female C57BL/6 MVP / mice (5–8 wk old) and wild-type mice matched described previously (35). for age and sex were used for preparation of splenocytes and All data are presented as a relative quantitation with efficiency correction thioglycollate-elicited peritoneal macrophages (PMs) as described previ- based on the relative expression of target genes versus b-actin (for human ously (37). Both splenocytes and mouse PMs were cultured in endotoxin- Downloaded from genes) or Hprt (for murine genes) as a reference gene. free DMEM with 10% FCS. Mouse PBMCs were isolated from blood using mouse lymphocyte separation medium (TBD, Tianjin, China) and Immunoblot analysis cultured in RPMI 1640. For our experiments, 2 3 106 splenocytes/ml or 2 3 105 PMs and mPBMCs per milliliter were used. Cells were lysed in PBS (pH 7.4) containing 0.01% Triton X-100, 0.01% EDTA, and 10% protease inhibitor mixture (Roche, Mannheim, Germany). Viral infection model Protein concentration was determined using the Bradford assay (Bio-Rad, Hercules, CA). Cell lysates (100 mg) were electrophoresed on a 12% SDS- Before infection, a frozen aliquot of mouse-adapted influenza virus A/FM/1/ http://www.jimmunol.org/ PAGE gel and transferred to a nitrocellulose membrane (Bio-Rad). Non- 47 (H1N1) was diluted in sterile PBS containing 144 tissue culture infective 2/2 specific sites were blocked with 5% (mass/v) nonfat dried milk in PBS and dose at 50% (TCID50)in40ml. Female C57BL/6 MVP mice (7–10 wk 0.1% (v/v) Tween 20 at room temperature for 1 h before incubation with old) and wild-type mice matched for age and sex were lightly anesthetized the indicated Abs. Protein bands were detected using the SuperSignal with 1% pentobarbital sodium salt and infected by intranasal instillation of chemiluminescent reagent (Pierce, Rockford, IL). 20 ml/nostril. In the meantime, the control group of each genotype was challenged with an equivalent amount of PBS. Mice were euthanized on Coimmunoprecipitation indicated days after infection except in the mortality study, where mice were observed for 2 wk and mortality was recorded daily. Cells were cultured in 10-cm dishes and lysed in 800 ml lysis buffer (20 mM Tris-HCl [pH 7.4–7.5], 150 mM NaCl, 1 mM EDTA, and 1% [v/v] Nonidet Lung/body index P-40) supplemented with 0.1% protease inhibitor mixture. Lysates were by guest on September 28, 2021 clarified by centrifugation and were incubated overnight at 4˚C with Mice were euthanized and weighed on days 0, 2, and 6 postinfection, lung constant agitation with 0.5 mg of the indicated Ab crosslinked to 30 ml tissues were harvested and weighed, and the corresponding ratio was protein G–agarose. After extensive washing with lysis buffer, immuno- calculated. complexes were resuspended in 20 ml13 SDS sample buffer for analysis by SDS-PAGE. Pulmonary virus quantitation On days 0, 2, and 6 of infection, right lobes of lungs were homogenized (9% Nuclear extraction w/v) in influenza virus growth medium (DMEM supplemented with 0.2% Cells were washed with ice-cold PBS and collected by centrifugation, and BSA solution, 100 U/ml penicillin, 100 mg/ml streptomycin, 2 mM pellets were resuspended in 10 packed cell volumes of hypotonic buffer A L-glutamine, 25 mM HEPES buffer, 2 mg/ml TPCK trypsin). The homoge- 3 (10 mM Tris-HCl [pH 7.4], 5 mM MgCl , 10 mM NaCl, 1 mM DTT, 10% nates were centrifuged at 12,000 g for 20 min to remove cell debris and the 2 2 protease inhibitor mixture) for 15 min on ice prior to incubation with 0.5% supernatants were stored at 80˚C until assay. Lung viral titers were deter- Nonidet P-40 on ice for 1 min. Nuclei pellets were collected by centri- mined by a modified TCID50 assay reported by the World Health Organiza- fugation and incubated in a buffer B (20 mM HEPES-KOH [pH 7.9], tion (38). Briefly, confluent monolayers of Madin-Darby canine kidney cells on 96-well plates were incubated, in octuplicate, with 100 ml 10-fold serially 1.5 mM MgCl2, 0.5 mM NaCl, 1 mM DTT, 0.2 mM EDTA, 10% protease inhibitor mixture, 1% Nonidet P-40) for 30 min on ice. Nuclear protein– diluted lung homogenates. After 3 d of incubation in 37˚C, the wells with containing supernatants were collected by centrifugation at 13,000 3 g for cytopathic effect were observed and counted. The TCID50 was calculated 30 min. Cytoplasmic and nuclear extracts were snap frozen in liquid ni- according to the Reed–Muench method. 2 trogen and stored at 70˚C until use. Quantitation of cytokines in lungs Immunofluorescence Mice were euthanized on days 0, 2, 4, and 6 postinfection, and right-lung A549 cells that were grown at 60% confluence in 15-mm cell culture dishes lobes were homogenized for RNA isolation. The mRNA levels of cytokines were washed three times with PBS for 10 min and fixed with 4% in lungs were determined by real-time PCR. paraformaldehyde solution at room temperature for 10 min. After per- Histopathologic examination meabilization with PBS buffer containing 0.1% Triton X-100 on ice for 20 min, cells were blocked in 5% BSA for 1 h at room temperature and then For histopathologic analysis, mice were euthanized on days 0, 2, 4, and 6 incubated with the indicated primary Abs for 1 h at 37˚C followed by postinfection, and the left lobes of lungs were fixed in 10% neutral buffered incubation with different dye-conjugated secondary Abs at 37˚C for 1 h. formalin, embedded in paraffin, sectioned, and stained with H&E. Ob- Subsequently, cells were rinsed and stained with DAPI dissolved in servations were made using an inverted light microscope at 3200. methanol at a ratio of 1:100 (v/v) for 5 min at 37˚C. Images were acquired using a laser scanning confocal microscope. Semiquantitative RT-PCR ChIP assays Total RNA from murine splenocytes was isolated using TRIzol reagent and reverse transcribed with a random primer, then amplified with ChIP assays were performed using reagents that were obtained from Upstate specific primers listed in Supplemental Table I by PCR. PCR products Biotechnology (Lake Placid, NY) according to the manufacturer’s in- were analyzed by electrophoresis on 2.5% agarose gel containing structions with slight modification of DNA purification after a reversed ethidium bromide. 2756 MVP REGULATES THE EXPRESSION OF IL-6 AND IL-8

Statistical analysis these data demonstrate that MVP, IL6, and IL8 mRNA can be Each set of experiments was repeated at least three times with similar induced via activation of the TLR3/RLR signaling pathway trig- results, and a representative example is shown. Statistical analyses were ged by IAV or poly(I:C) plus IFN-g and that MVP deficiency performed using Origin 7.5 software. Differences between two groups were impairs this expression of IL6 and IL8. determined with a Student t test or ANOVA, and were considered statistically significant at a value p , 0.05. Kaplan–Meier survival curves were IL6 and IL8 mRNA expression is impaired in established stable analyzed using a log-rank test. MVP-knockdown cells To further explore whether TLR3/RLR-mediated proinflammatory Results signaling is dependent on MVP, we performed additional knock- MVP is involved in the proinflammatory response trigged by down experiments using a lentiviral vector system. Four recombi- IAV or a combination of poly(I:C) and IFN-g nant lentiviral vectors targeting MVP (pLKO.1-MVP shRNA nos. To investigate the involvement of MVP in the proinflammatory 1–4) were constructed. The pLKO.1-MVP shRNA no. 4 displayed signaling, a time-course analysis of MVP induction in human lung the highest knockdown efficiency (Fig. 2A), and thus we chose this epithelial A549 cells infected with IAV was performed by real-time construct to establish stable MVP-knockdown A549 and THP-1 PCR. As shown in Fig. 1A, MVP mRNA increased gradually and cells. Morphological observation revealed that A549 and THP-1 reached its peak at 24 h postinfection. Simultaneously, IAV in- cells with stable MVP knockdown were phenotypically similar to duced significant expression of IL6 and IL8 (Fig. 1A). A combi- parental cells (data not shown). As shown by immunoblot, MVP nation of synthetic dsRNA analogs poly(I:C) and IFN-g mimicked expression was significantly reduced in MVP-knockdown cells

IAV infection, initiating an immune response as reported previ- relative to scramble-knockdown cells and was partially restored Downloaded from ously (4). We further investigated the expression of MVP, IL6, and after poly(I:C) plus IFN-g treatment (Fig. 2B, 2C). Furthermore, IL8 mRNA levels in PBMCs following stimulation with poly(I:C) stable MVP-knockdown A549 cells exhibited decreased IL6 and plus IFN-g and found similar results to those observed following IL8 mRNA levels following poly(I:C) plus IFN-g stimulation IAV infection (Fig. 1B). (Fig. 2D), and similar results were obtained using stable MVP- Furthermore, we studied the effects of MVP deﬁciency on the knockdown human THP-1 monocytes (Fig. 2E). Finally, to verify

proinﬂammatory response triggered by TLR3/RLR ligands by the speciﬁcity of pLKO.1-MVP shRNA no. 4 for endogenous MVP, http://www.jimmunol.org/ measuring IL6 and IL8 mRNA levels in A549 cells and PBMCs MVP was ectopically overexpressed in stable MVP-knockdown A549 transfected with shRNA-MVP or shRNA control. As expected, cells and then cells were treated with poly(I:C) plus IFN-g. IL6 and mRNA levels of IL6 and IL8 were decreased upon knockdown of IL8 expression was successfully rescued after restoration of MVP MVP in A549 cells (Fig. 1C) and PBMCs (Fig. 1D) compared expression in these knockdown cells (Fig. 2F). Taken together, our with those in cells treated with shRNA control. Taken together, results indicate that TLR3/RLR-mediated expression of IL6 and by guest on September 28, 2021

FIGURE 1. Identiﬁcation of MVP as a regulator in the proinﬂammatory response. (A and B) Expression of MVP, IL6, and IL8 is inducible upon stimulation. (A) A549 cells were infected by IAV (MOI of 1) for 0–24 h. (B) Freshly isolated PBMCs were stimulated with poly(I:C) (50 mg/ml) plus IFN-g (150 IU/ml) for 0–24 h. The mRNA levels of MVP, IL6, and IL8 were measured by real-time PCR. (C and D) Effects of shRNA-MVP plasmids on IL6 and IL8 expression. (C) A549 cells were transfected with indicated plasmids for 24 h and then infected with IAV (MOI of 1) for another 24 h. (D) PBMCs were transfected by electroporation with indicated plasmids for 36 h and then stimulated by poly(I:C) (50 mg/ml) plus IFN-g (150 IU/ml) for another 12 h. The total RNA was used to analyze MVP, IL6, and IL8 mRNA levels by real-time PCR. Data are representative of three experiments with similar results and are shown as mean 6 SD of triplicates. *p , 0.05, **p , 0.01 (Student t test). The Journal of Immunology 2757

FIGURE 2. Impaired IL6 and IL8 expression in MVP-deﬁ- cient A549 cells and THP-1 monocytes upon stimulation with poly(I:C) plus IFN-g.(A) Effects of pLKO.1-MVP shRNAs on endogenous MVP. A549 cells were transfected with the indicated plasmids for 48 h. Then, the total RNA was used for real- time PCR (upper panel), and the cell lysates were analyzed by immunoblot analysis (lower panel). (B and C) MVP restoration in stable MVP-knockdown (KD) cells upon stimulation with poly(I:C) plus IFN-g. Stable MVP-KD A549 cells (B), THP-1 monocytes (C), and control cells (scramble [Scr.]-KD) were treated with poly(I:C) (50 mg/ml) plus IFN-g (150 IU/ml) for 0–48 h prior to immunoblot analysis. (D and E) IL6 and IL8 expression in stable MVP-KD cells. Stable MVP-KD A549 cells (D) or THP-1 monocytes (E) and control cells were stimulated with poly(I:C) (50 mg/ml) plus IFN-g (150 IU/ml) for 0–12 h, and then IL6 and IL8 mRNA levels were determined by real-time PCR. (F) Ectopically expressed MVP Downloaded from successfully rescues the impaired IL6 and IL8 expression in MVP-KD cells. MVP-KD A549 and control cells were transfected with the indicated plasmids for 36 h and stimulated with poly(I:C) (50 mg/ml) plus IFN-g (150 IU/ml) for 12 h prior to immunoblot analysis (bottom) and real-time PCR analysis (top). Data represent one of three experiments with similar A C results ( – ) or are representative of three experiments with http://www.jimmunol.org/ similar results [mean 6 SD of triplicates in (D)–(F)]. *p , 0.05, **p , 0.01 (Student t test). KD, knockdown; Scr., scramble.

IL8 is impaired following knockdown of MVP and that these defects reporters of IL6 and IL8 expression (Fig. 3A), resulting in up- can be successfully rescued by ectopically overexpressing MVP. regulated mRNA levels (Fig. 3B). Activation of the IL6 and IL8 by guest on September 28, 2021 promoters relies on binding of transcription factors to the con- Synergistic activation of IL6 and IL8 promoters by MVP and sensus cis elements, leading us to hypothesize that MVP activates c-Fos or C/EBPb upon TLR3/RLR activation the IL6 and IL8 promoters by acting synergistically with tran- The observation that MVP deﬁciency disrupted IL6 and IL8 ex- scription factors. IL6 promoter activity was upregulated signiﬁ- pression prompted us to further investigate the effects of MVP on cantly upon coexpression of MVP with different transcriptional their promoters. As shown in a reporter assay, overexpression of factors, such as c-Fos, C/EBPb-LAP, p65, and p50, in A549 cells MVP potentiated the poly(I:C) plus IFN-g–triggered activation of stimulated with poly(I:C) plus IFN-g. In this experiment, both p65

FIGURE 3. MVP acts in synergy with c-Fos and C/EBPb- LAP to activate the IL6 and IL8 promoters. (A) Effects of overexpressed MVP on activation of IL6 and IL8 promoters. A549 cells were transfected with reporter plasmids for IL6 or IL8, together with the indicated plasmids for 36 h, and then treated with poly(I:C) (50 mg/ml) plus IFN-g (150 IU/ml) for 12 h before the luciferase assay was performed. The normalized fold of firefly luciferase activity for each gene is given relative to the empty control vector. (B) Effects of overexpressed MVP on IL6 and IL8 expression. A549 cells were transfected with the indicated plasmids for 36 h prior to stimulation with poly(I:C) (50 mg/ml) plus IFN-g (150 IU/ml) for 12 h prior to analysis with real-time PCR. (C) MVP aug- ments c-Fos– and C/EBPb-LAP–mediated activation of the IL6 and IL8 promoters. A549 cells were transfected with reporter plasmids for IL6 or IL8, together with the indicated plasmids, and empty vector was added to ensure that each transfection received the same amount of total DNA. After 36 h, cells were stimulated with poly(I:C) (50 mg/ml) plus IFN-g (150 IU/ml) for 12 h before the luciferase assay was performed. The normalized fold of firefly luciferase activity for each gene is given relative to the control. Data are representative of three experiments with similar results (mean 6 SD of triplicates). *p , 0.05, **p , 0.01 (Student t test). 2758 MVP REGULATES THE EXPRESSION OF IL-6 AND IL-8

FIGURE 4. MVP interacts with c-Fos and C/EBPb.(A and B) MVP interacts with c-Fos and C/EBPb-LAP but not C/ EBPb-LIP in transfected cells. HEK 293 cells were transfected for 48 h with Myc-tagged MVP together with Flag-tagged (A) c-Fos, (B) C/EBPb-LAP, or C/EBPb-LIP. Cell lysates were immunoprecipitated with anti–Flag-conjugated agarose or control IgG agarose and analyzed by immunoblot analysis with anti-Myc (top panel). The expression of the transfected proteins was analyzed by immunoblot analysis with anti-Myc and anti-Flag (middle and bottom panels), respectively. (C and D) MVP interacts with endogenous c-Fos and C/EBPb. A549 cells were stimulated with poly(I:C) (50 mg/ml) plus IFN-g (150 IU/ml; left) or IAV (MOI of 1; right) for 0–48 h. Cell lysates were assessed before immunoprecipitation (input, bottom) or after immunoprecipitation (top) with indicated Abs. Rabbit IgG served as an immunoprecipitation control. (E and F) Colocal- Downloaded from ization of intracellular MVP with c-Fos and C/EBPb. A549 cells were stimulated by poly(I:C) (50 mg/ml) plus IFN-g (150 IU/ml; left)orIAV(MOIof1;right) or left untreated for 24 h. Then cells were ﬁxed, permeabilized, and stained with (E) anti-MVP (green) and anti–c-Fos (red) Abs or (F) anti- MVP (green) and anti-C/EBPb (red) Abs before observation

using confocal microscopy. Scale bars, 20 mm. Data are from http://www.jimmunol.org/ one of three experiments with similar results. IB, immunoblot; IP, immunoprecipitation.

and p50, which were pivotal in expression of cytokines and che- MVP upregulated the activity of the IL6 and IL8 promoters via mokines, were used as the positive control. Similar results were c-Fos, C/EBPb-LAP, and NF-kB, we proposed that these tran- by guest on September 28, 2021 obtained for the IL8 promoter, indicating that the activation of the scriptional factors may be targets of MVP in the TLR3/RLR- IL8 promoter was upregulated significantly by coexpression of mediated proinflammatory signaling pathway. We performed c-Fos, C/EBPb-LAP, p65, or p50 with MVP (Fig. 3C). These findings coimmunoprecipitation assays in HEK 293T cells cotransfected demonstrate that MVP acts in synergy with c-Fos and C/EBPb-LAP with Myc-tagged MVP and Flag-tagged c-Fos, LAP, or LIP. This to activate the IL6 and IL8 promoters and induce their expression. experiment showed that MVP interacted with c-Fos and LAP but not LIP (Fig. 4A, 4B). Furthermore, MVP interacted with p65 MVP interacts with c-Fos and C/EBPb-LAP and p50, but not CREB1/2, in transfected HEK 293T cells We sought to elucidate the underlying mechanisms by which MVP (Supplemental Fig. 1A, 1B). Interactions between MVP and regulates the expression of IL6 and IL8. Given the observation that c-Fos and LAP were confirmed by analysis following coexpression

FIGURE 5. MVP translocates from cytoplasm to nucleus. (A) Immunoblot analysis of MVP translocation. A549 cells were stimulated by poly(I:C) (50 mg/ml) plus IFN-g (150 IU/ml; top panel)orIAV(MOIof1;bottom panel) for indicated time points, and cytoplasmic and nuclear extracts were examined by immunoblot analysis for the indicated proteins. GAPDH and lamin A were used as cytoplasmic and nuclear markers, respectively, to assess the equivalent loading. (B) Detection of MVP translocation by confocal immunoﬂuorescence. A549 cells were stimulated by poly(I:C) (50 mg/ml) plus IFN-g (150 IU/ml; left panel) or IAV (MOI of 1; right panel) or left untreated for 24 h. Then cells were ﬁxed, permeabilized, and labeled with DAPI for nucleus staining (blue) and anti-MVP Ab (green) before observation using confocal microscopy. Scale bars, 20 mm. Data are from one of three experiments with similar results. The Journal of Immunology 2759 Downloaded from http://www.jimmunol.org/

FIGURE 6. MVP mediates nuclear translocation of c-Fos and C/EBPb but does not affect their expression. (A–C) Effects of MVP on nuclear translocation of c-Fos and C/EBPb. MVP-knockdown (KD) and control A549 cells were transfected with the indicated plasmids for 36 h. Twelve hours after stimulation without (A) or with (B) poly(I:C) (50 mg/ml) plus IFN-g (150 IU/ml) or (C) IAV (MOI of 1), cytoplasmic and nuclear extracts were examined by by guest on September 28, 2021 immunoblot analysis for the indicated proteins. GAPDH and lamin A were used as cytoplasmic and nuclear markers, respectively, to assess equivalent loading. (D) The bands indicating nuclear c-Fos, C/EBPb, and lamin A were quantified by ImageJ software; each column represents the average of three replicates, and error bars represent the SEM. Results are normalized to the nuclear lamin A levels and are presented relative to the results obtained from control cells with the value set as 1. (E and F) c-Fos and C/EBPb expression in MVP-KD cells. MVP-KD and control A549 cells were stimulated with (E) poly(I:C) (50 mg/ml) plus IFN-g (150 IU/ml) or (F) IAV (MOI of 1) for the indicated times and then analyzed by immunoblot. Data are from one of three experiments with similar results. KD, knockdown; Scr., scramble. of GFP-tagged MVP and Flag-tagged c-Fos and LAP; however, the cytoplasm, although a small amount of these proteins colo- also in this setup, MVP failed to interact with LIP (Supplemental calized in the nucleus of A549 cells either stimulated with poly(I:C) Fig. 1C, 1D). To exclude the possibility that MVP or c-Fos plus IFN-g or infected with IAV for 24 h (Fig. 4E). Similar interacted with the tag of the candidate interacting protein, we colocalization patterns were observed for MVP and C/EBPb in examined this interaction in HEK 293T cells that were cotrans- A549 cells (Fig. 4F). Collectively, these results provide evidence that fected with Flag-tagged MVP and Myc-tagged c-Fos, Flag-tagged MVP functions as a regulator in the TLR3/RLR-mediated proin- MVP and Myc-tagged empty vector, or Myc tagged-c-Fos and flammatory signaling pathway, possibly by interacting with tran- Flag-tagged empty vector. These experiments revealed that MVP scription factors, including c-Fos and C/EBPb-LAP. specifically interacted with c-Fos (Supplemental Fig. 1E). The interaction between MVP and LAP was further verified in similar MVP translocates from the cytoplasm to the nucleus experiments (Supplemental Fig. 1F). To determine whether en- The observation that MVP interacts with c-Fos and C/EBPb-LAP dogenous MVP associates with c-Fos or C/EBPb in untransfected prompted us to investigate the downstream events. Indeed, as shown cells, we performed coimmunoprecipitation of endogenous proin the confocal images in Fig. 4, both c-Fos and C/EBPb displayed teins in A549 cells at various time points after stimulation with nuclear and cytoplasmic distribution in unstimulated A549 cells. poly(I:C) plus IFN-g or after IAV infection. Consistent with the After stimulation with poly(I:C) plus IFN-g or infection with IAV, results above, we found that MVP interacted with c-Fos and some translocation of c-Fos and C/EBPb from the cytoplasm to the C/EBPb as early as 12 h after stimulation (Fig. 4C, 4D). Because nucleus was observed. Surprisingly, nuclear translocation of MVP IRF3 acts as a transcription factor that regulates type I IFN expression was observed as well. To better understand the cytoplasmic–nuclear in the TLR3/RLR signaling pathway, we used IRF3 as a negative translocation of MVP in response to dsRNA or IAV, we assessed the control in this experiment. As expected, coimmunoprecipitation in MVP distribution in A549 cells stimulated with poly(I:C) plus IAV-infected A549 cells revealed that MVP does not interact with IFN-g or cells infected with IAV for different intervals. Immunoblot IRF3 (Supplemental Fig. 1G). Confocal microscopy also showed analysis revealed that MVP protein levels were elevated in the that most of the intracellular MVP localized together with c-Fos in nucleus at 6 h poststimulation with poly(I:C) plus IFN-g and 12 h 2760 MVP REGULATES THE EXPRESSION OF IL-6 AND IL-8 Downloaded from http://www.jimmunol.org/ by guest on September 28, 2021

FIGURE 7. Effects of MVP on c-Fos and C/EBPb binding to the IL6 and IL8 promoters. (A) Functional analysis of MVP-regulated cis-regulatory elements in the IL6 and IL8 promoters. Schematic diagram of individual IL6 and IL8 cis-regulatory elements and the introduced truncations or site-specific mutations (left) is shown. The results from the luciferase activity assay are shown (right). A549 cells were transfected with indicated plasmids for 36 h and then stimulated with poly(I:C) (50 mg/ml) plus IFN-g (150 IU/ml) for 12 h prior to the luciferase assay. The MVP induction relative to wild-type and mutant promoters was compared. The results are presented relative to the luciferase activity of cells cotransfected with empty control vector and intact promoter and given a set value of 1. (B and C) ChIP analysis of MVP effects on the recruitment of C/EBPb to the IL6 and IL8 promoters. (B) A549 cells were transfected with the indicated plasmids for 36 h prior to stimulation with poly(I:C) (50 mg/ml) plus IFN-g (150 IU/ml; left) or IAV (MOI of 1; right) for 12 h. (C) MVP-knockdown (KD) and control A549 cells were stimulated with poly(I:C) (50 mg/ml) plus IFN-g (150 IU/ml; left) or IAV infection (MOI of 1; right) for 12 h. ChIP assays were performed with anti–C/EBPb-conjugated agarose or rabbit IgG-conjugated agarose (as a (Figure legend continues) The Journal of Immunology 2761 postinfection with IAV (Fig. 5A). The translocation of MVP was AP-1, and C/EBPb bindingsitesintheIL6 and IL8 promoter further verified in these cells using immunofluorescence with a regions were indispensable for their activation and, furthermore, partial merge of MVP indirectly conjugated with FITC and nuclei that MVP regulates expression of IL6 and IL8 in an NF-kB–, AP- stained with DAPI following stimulation of A549 cells (Fig. 5B). 1–, and C/EBPb-dependent manner. Taken together, these findings provide direct evidence that MVP We then used a ChIP assay to assess the effects of MVP on the translocates from the cytoplasm to the nucleus in response to recruitment of NF-kB, c-Fos, and C/EBPb to the IL6 and IL8 dsRNA stimulation or virus infection. promoters. Indeed, C/EBPb recruitment to the promoter regions of IL6 and IL8 was increased significantly by overexpression of MVP MVP enhances nuclear translocation of c-Fos and C/EBPb in A549 cells and inhibited in stable MVP-knockdown A549 cells Colocalization of MVP with c-Fos and C/EBPb in the cytoplasm stimulated with poly(I:C) plus IFN-g or IAV infection (Fig. 7B, and nucleus prompted us to determine whether MVP contributed 7C). Similarly, recruitment of c-Fos to the IL6 and IL8 promoters to the nuclear translocation of c-Fos and C/EBPb, as such trans- was enhanced by MVP overexpression, whereas it was inhibited location is the hallmark of transcription factor activation. To this in MVP knockdown in A549 cells (Fig. 7D, 7E). Furthermore, we end, we first detected nuclear translocation of several transcrip- found that overexpressed MVP promoted NF-kB occupancy on tional factors in response to poly(I:C) plus IFN-g or IAV infection both IL6 and IL8 promoters, but not the b-actin promoter, which is and found that overexpressed MVP promoted the translocation of a housekeeper gene and acts as a negative control. The above data c-Fos and C/EBPb-LAP but not C/EBPb-LIP into the nucleus. were confirmed by performing ChIP semiquantitative PCR assays, p65 and p50 served as positive controls (Supplemental Fig. 2). and similar results were observed (Fig. 7F).

Then, we further examined the nuclear translocation of c-Fos and Combining the results showing MVP enhanced occupancy of Downloaded from C/EBPb-LAP in stable MVP-knockdown A549 cells with or NF-kB, c-Fos, and C/EBPb to the IL6 and IL8 promoters and without stimulation. As expected, basal and stimulation-induced nuclear translocation of MVP itself upon stimulation of dsRNA or nuclear translocation of c-Fos and C/EBPb was reduced in stable IAV, we hypothesized that MVP may bind to the promoter regions MVP-knockdown A549 cells; however, these defects were rescued as a member of transcriptional machinery consisting of NF-kB, by ectopic expression of MVP (Fig. 6A–D). To exclude the pos- c-Fos, and C/EBPb to regulate the transcription of IL6 and IL8.

sibility that the decreased nuclear fractions of c-Fos and C/EBPb However, our observations indicated that IAV-induced occupancy http://www.jimmunol.org/ with MVP knockdown were simply the result of lower c-Fos and of MVP on both IL6 and IL8 promoters was not detectable C/EBPb expression, we measured the total expression of c-Fos (Fig. 7G). and C/EBPb in stable MVP-knockdown and control A549 cells. The above results lend further support to the proposed tethering MVP knockdown did not affect c-Fos or C/EBPb expression scheme, which requires site occupancy of the IL6 and IL8 pro- levels upon stimulation with poly(I:C) plus IFN-g or IAVinfection moters with NF-kB, c-Fos, and C/EBPb upon TLR3/RLR ac- (Fig. 6E, 6F). Taken together, these data suggest that MVP reg- tivation. Moreover, these findings indicate that MVP regulates ulates TLR3/RLR signaling by facilitating nuclear translocation of activation of the IL6 and IL8 promoters by affecting recruitment of c-Fos and C/EBPb as opposed to intracellular expression of these NF-kB, c-Fos, and C/EBPb to the relevant regions. by guest on September 28, 2021 transcription factors. However, MVP affects the nuclear translo- 2/2 cation of c-Fos more significantly than that of C/EBPb. Impaired proinflammatory response in MVP murine cells To further confirm the involvement of MVP in the TLR3/RLR- MVP enhances recruitment of c-Fos and C/EBPb to the IL6 mediated proinflammatory response, we employed the Mvp knock- and IL8 promoters out (MVP2/2) mouse model and confirmed MVP deficiency by IL6 and IL8 expression relies on many cis elements, including NF- semiquantitative RT-PCR and immunoblot analysis of poly(I:C) kB, AP-1, and C/EBPb binding sites on the promoters. Therefore, plus mIFN-g–stimulated splenocytes (Fig. 8A, 8B). We initially we investigated whether MVP activated IL6 and IL8 expression by assessed the mRNA levels of proinflammatory cytokines from enhancing the recruitment of transcription factors to the relevant IAV-infected or poly(I:C) plus mIFN-g–stimulated splenocytes promoter regions. Initially, to determine the localization of NF- from either wild-type C57BL/6 mice or MVP2/2 mice. MVP in- kB, AP-1, and C/EBPb at the endogenous IL6 gene locus, the duction upon poly(I:C) plus mIFN-g stimulation was impaired intact core IL6 promoter together with site-directed mutated NF- in MVP2/2 splenocytes. Significantly suppressed expression of kB, AP-1, or C/EBPb binding sites were used in the luciferase mIL6,mIL8 orthologs (mCxcl1,mCxcl2,mCxcl5), mIL1-b, and assay. Mutations in the NF-kB, AP-1, or C/EBPb binding sites mTNF-a were detected in MVP2/2 mouse splenocytes compared significantly abrogated IL6 promoter activity as well as the syn- with those in wild-type splenocytes. The induction of mIL6 and ergistic activation of expression by MVP and NF-kB, AP-1, or mIL8 orthologs after IAV infection was nearly completely abol- C/EBPb triggered by poly(I:C) plus IFN-g in A549 cells (Fig. 7A). ished in MVP2/2 mouse splenocytes (Fig. 8C, 8D). Impaired Similar results were also obtained with site-directed mutations in expression of mIL6 and mIL8 orthologs were also observed in NF-kB, AP-1, or C/EBPb binding sites and with truncated mutants thioglycollate-elicited mouse PMs upon poly(I:C) plus mIFN-g of the IL8 promoter (Fig. 7A). These data indicated that NF-kB, stimulation; however, in our experimental system, PMs failed to

negative control). (D and E) ChIP analysis of the effects of MVP on the recruitment of c-Fos to the IL8 promoter. (D) A549 cells were treated as in (B); (E) MVP-KD and control A549 cells were treated as in (C). ChIP assays were performed with anti–c-Fos-conjugated agarose or rabbit IgG-conjugated agarose. (F and G) ChIP analysis of the effects of overexpressed MVP on the recruitment of NF-kB and MVP to the IL6, IL8, and b-actin promoters. A549 cells were transfected with the indicated plasmids for 36 h prior to infection with IAV (MOI of 1) for 12 h. ChIP assays were performed with (F) anti-p50–, anti- p65–, or rabbit IgG–conjugated agarose and (G) anti-MVP– or goat IgG–conjugated agarose. IL6, IL8, and b-actin promoter sequences in the input DNA and the DNA recovered from Ab-bound chromatin segments were detected by real-time PCR or semiquantitative PCR. Results are normalized to the corresponding input control and are presented relative to the results obtained from empty control vector transfection (B, D, F, and G) or control cells (C and E) with the value set as 1. Data are representative of three experiments with similar results (mean 6 SD of triplicate experiments). *p , 0.05, **p , 0.01 (Student t test). KD, knockdown; Scr., scramble. 2762 MVP REGULATES THE EXPRESSION OF IL-6 AND IL-8 Downloaded from http://www.jimmunol.org/ by guest on September 28, 2021

FIGURE 8. Impaired murine IL6 and mIL8 orthologs expression in MVP2/2 murine cells. (A and B) MVP is absent in splenocytes from MVP2/2 mice. MVP expression in splenocytes stimulated with poly(I:C) (20 mg/ml) plus murine IFN-g (60 IU/ml) was detected by semiquantification PCR (A) and immunoblot (B) at the indicated time points. (C)mMVP,mIL1-b, and mTNF-a expression in MVP2/2 mouse–derived cells. Murine splenocytes and PBMCs were stimulated without or with poly(I:C) (20 mg/ml) plus murine IFN-g (60 IU/ml) for 12 h. Then, expression of mMVP,mIL1-b, and mTNF-a was examined by real-time PCR. (D) Splenocytes, (E) PMs, and (F) PBMCs from either wild-type (WT) or MVP2/2 mice were stimulated with IAV (MOI of 1; left) or poly(I:C) (20 mg/ml) plus murine IFN-g (60 IU/ml; right) for the indicated times. Then, mIL6 and mIL8 ortholog (mCxcl1,mCxcl2,mCxcl5) mRNA levels were examined by real-time PCR. Results are normalized to the expression of Hprt and are presented relative to expression in cells from wild- type mice treated for 0 h, with a value set as 1. Data are representative of three experiments with similar results (mean 6 SD of triplicate experiments). *p , 0.05, **p , 0.01 (Student t test). ND, not detected; WT, wild-type. respond to IAV infection, and this is consistent with a previous Antiviral activity of MVP in mouse model 2/2 report (39). Additionally, these MVP PMs showed lower basal To establish the potential function for MVP in vivo, wild-type expression of mIL6 and mIL8 orthologs relative to wild-type PMs and MVP2/2 mice were intranasally infected with lethal mouse- (Fig. 8E). Finally, we found that poly(I:C) plus mIFN-g stimula- adapted influenza virus A/FM/1/47 (H1N1). At the indicated dose 2/2 tion failed to induce MVP expression in MVP mouse PBMCs, infected, MVP2/2 mice were much less viable than wild-type and MVP deficiency in murine PBMCs significantly impaired mice, namely, MVP2/2 mice died within 9 d after infection, expression of mIL6 and mIL8 orthologs triggered by IAV infection whereas wild-type mice had a prolonged survival time and higher or poly(I:C) plus mIFN-g stimulation (Fig. 8C, 8F). Overall, these survival rate (Fig. 9A). 2 2 results from the MVP / mouse model indicate that MVP is es- The observation that MVP protected mice against lethal IAV sential for IL6 and IL8 expression in response to dsRNA or IAV infection prompted us to discover the underlying mechanism. First, infection in murine cells as well. we found that on day 6 postinfection, MVP2/2 mice had a higher The Journal of Immunology 2763 Downloaded from http://www.jimmunol.org/ by guest on September 28, 2021

FIGURE 9. MVP delays death and lung lesions during infection with lethal influenza A virus and inhibits viral replication in vivo. (A) Survival rate of mice intranasally infected with lethal H1N1 influenza A virus. Wild-type (WT) (black square, n = 9) and MVP2/2 (red circle, n = 9) mice were intranasally infected with 144 TCID50 of influenza virus A/FM/1/47 (H1N1) and observed every day. Survival curves show data until day 14 postinfection. Statistical analysis was performed using a log-rank test. (B) Lung/body weight was evaluated on days 0, 2, and 6 after influenza viral infection at a dose of 144

TCID50. Mice were weighed (grams) and euthanized. Whole lungs were taken and weighed (grams), and the corresponding lung/body index was calculated. Error bars represent SEM from four to five different mice in each group at indicated time point. (C) Viral titers in lung tissues. The mouse lungs from WT 2/2 and MVP mice infected with 144 TCID50 of influenza virus A/FM/1/47 (H1N1) intranasally were taken on days 0, 2, and 6. The homogenates were used for quantifying the virus titers. Error bars represent SEM from three to five different mice in each group at indicated time point. (D) mRNA levels of 2/2 cytokines in lung tissue. Lung tissues from WT and MVP mice intranasally infected with 144 TCID50 of influenza virus A/FM/1/47 (H1N1) were harvested on days 0, 2, 4, and 6. Total RNA from lung tissue was extracted and used for detecting expression of mIL6,mCxcl1,mCxcl2,mCxcl5,mTNF-a, mIL1-b, and mIFN-a. Error bars represent SEM from three to five different mice in each group at indicated time point. Data represent mean 6 SEM. Statistical analysis was performed by ANOVA (*p , 0.05, **p , 0.01, and NS, not significant, indicating p . 0.05). (E) Histology of lung tissue stained with H&E on days 0, 2, 4, and 6 after intranasal infection with 144 TCID50 influenza virus A/FM/1/47 (H1N1). Intact columnar epithelial cells and clear alveoli are illustrated in lung tissues from uninfected WT and MVP2/2 mice. At day 2 postinfection, lung tissues from WT and MVP2/2 mice show pulmonary hemorrhage and edema, different from WT lung tissue presenting a peribronchiolar lymphoid cuff; MVP2/2 lung tissue shows restricted inflammatory cell exudates in alveoli and mild necrosis of bronchial epithelium. Dense granulocytes and lymphocytic cell infiltration in the alveoli and mild necrosed bronchial epithelium are present in lung tissue from WT mice on day 4 postinfection, whereas MVP2/2 lung tissue demonstrates inflammatory cell infiltrates around vessels and airways, as well as the severe necrosis of bronchiolar epithelium. At day 6 postinfection, totally eroded columnar epithelial cells and an airway with totally denuded lamina propria, of which lumen is filled with granulocytic and lymphocytic exudate (bottom right, red arrowhead), are visible in lung tissue from MVP2/2 mice. WT lung tissue shows delayed complete necrosis of columnar epithelial cells. Asterisk indicates edema, black arrow indicates necrotic debris, and black arrowhead shows lymphoplasmacytic, histiocytic prebronchial, and vascular cuffs. Representative figures are shown at original magnification 3200. Each group contained four to five mice at indicated time point. WT, wild-type. lung/body index, which indicated worse lung tissue swelling and mice was observed by days 2 and 6 postinfection (Fig. 9C), which the more production of exudate, but the difference between two correlated with impaired expression of early innate antiviral cytokines, groups was not statistically significant (Fig. 9B). Then, a signifi- such as mIL6 and mIL8 orthologs, mIL1-b,mTNF-a, and mIFN-a cant increase of pulmonary influenza A virus titer in MVP2/2 on day 2 postinfection (Fig. 9D). Results from histopathologic 2764 MVP REGULATES THE EXPRESSION OF IL-6 AND IL-8 examination demonstrated that lethal H1N1 influenza A infection in vivo that protects mice from mortality and lung lesions asso- was associated with lung lesions characterized with the occurrence ciated with lethal influenza A virus. Collectively, our results from of inflammatory cellular infiltration, bronchitis, interstitial in- human-derived cells and the MVP2/2 mouse model provide strong flammation, endothelialitis, pulmonary hemorrhage and edema, evidence that MVP is essential in the TLR3/RLR signaling epithelial hyperplasia, and epithelial necrosis in both wild-type pathway and crucial for virus-triggered proinflammatory cytokine and MVP2/2 mice. Different from wild-type mice, MVP2/2 and chemokine production. mice displayed delayed formation of lymphoplasmacytic, Both TLR3 and RLRs commonly signal through NF-kB and perivascular/peribronchial cuffs on day 4 postinfection, but more MAPKs to activate expression of downstream proinflammatory severe epithelial necrosis and desquamation since day 4 postin- cytokines and chemokines. Given that our previous study indi- fection (Fig. 9E). Overall, our results indicate that MVP contrib- cated that MVP induces type I IFN expression by promoting nu- utes to production of early innate antiviral cytokines in vivo, by clear translocation of NF-kB, we speculated that MVP also which the hosts establish an antiviral state against viral replication regulated proinflammatory cytokines and chemokines via the NF- in the lung and relieve lung lesion and influenza-mediated mor- kB signaling pathway. Our present study suggests that MVP can tality, and they further confirm our conclusion based on cellular augment NF-kB–mediated activation of the IL6 and IL8 pro- experiments (Fig. 10). moters. In fact, previous reports have indicated that proinflammatory cytokine and chemokine expression are dependent on Discussion some other transcription factors in addition to NF-kB (42). In this MVP is linked to multidrug resistance in cancer, although the exact study, we identified c-Fos and C/EBPb-LAP as candidate tran- mechanisms are not yet completely clear (23, 40). The observation scription factors that act in synergy with MVP to activate the IL6 Downloaded from that MVP is upregulated in HepG2.2.15 cells, which stably pro- and IL8 promoters in response to stimulation with poly(I:C) plus duce hepatitis B virus, compared with the parental HepG2 cells IFN-g or IAV infection. Furthermore, introduction of mutations (41) suggests new functions of MVP. We have previously dem- into the AP-1 and C/EBPb binding sites on the IL6 and IL8 onstrated that MVP is an effector in IFN signaling pathways upon promoters resulted in the loss of synergistic activation with MVP. viral infection (32). It is well known that pathogen-triggered Our findings help to delineate the role of MVP in classical immune responses contribute to robust expression of proin- proinflammatory signaling, including the AP-1 pathway, as well as http://www.jimmunol.org/ flammatory cytokines and chemokines, in addition to type I IFNs. the nonclassical C/EBPb-mediated proinflammatory signaling The role of MVP in proinflammatory signaling pathways requires pathway as complementing complexes involved in TLR3/RLR further clarification. In the present study, we demonstrate in vitro signaling. that MVP is inducible in A549 cells, PBMCs, and mouse-derived We previously proposed a mechanism to account for enhanced splenocytes exposed to TLR3/RLR ligands. MVP deficiency im- type I IFN expression by MVP upon HCV infection wherein MVP pairs IAV infection– or poly(I:C) plus IFN-g–triggered expression promotes nuclear translocation of NF-kB and IRF7; however, how of IL6 and IL8 in human PBMCs, A549 cells, and THP-1 mono- MVP promotes nuclear translocation was unclear. A previous re- cytes, as well as in mouse-derived MVP2/2 splenocytes, PMs, and port suggested the synergistic activation of IL6 and IL8 promoters PBMCs. Further studies in the MVP2/2 mouse model confirm that by transcription factors NF-kB and NF–IL-6 and demonstrated a by guest on September 28, 2021 MVP is critical in establishing an antiviral microenvironment direct interaction between p65 and NF–IL-6, reminiscent of the direct interaction between MVP and transcription factors (43). In this study, we have clarified the upstream events of nuclear translocation of NF-kB, c-Fos, and C/EBPb-LAP. We demonstrated that MVP selectively interacts with NF-kB, c-Fos, and C/EBPb-LAP, but not with C/EBPb-LIP or CREB1/2. MVP has previously been reported to interact with SHP-2 and ERK as a scaffold protein (27) and with PTEN as a nuclear–cytoplasmic transporter (44). Besides, we excluded the possibility that c-Fos acted in synergy with C/EBPb-LAP, as C/EBPb-LAP–mediated activation of the IL6 and IL8 promoters did not significantly increase in the presence of c-Fos (Supplemental Fig. 3). As a putative downstream transcription factor in MAPK signaling pathways, the classical AP-1 involved in TLR/RLR signaling is a heterodimer composed of c-Jun and ATF-2 (2, 45). The observations that MVP acted synergistically and directly interacted with c-Fos rather than other classical AP-1 components seemingly contradicted these earlier reports. In fact, two expla- nations will support our results. First, in addition to the classical heterodimer, nonclassical and variable AP-1 heterodimers have been reported as well (16, 18, 46, 47), and this may account for the interaction and synergy between MVP and c-Fos but not ATF-2. Second, we cannot exclude the possibility that c-Jun participates in IL6 and IL8 activation as a heterodimer with c-Fos, of which physical interaction is unique to the particular family member. FIGURE 10. A working model of MVP. IAV infection or dsRNA in- M20A/M20A duces MVP expression, and MVP interacts with c-Fos and C/EBPb-LAP Another report suggested that C/ebpb mice, in which and promotes the nuclear translocation of c-Fos and C/EBPb-LAP. These the expression of the 34-kDa LAP is abolished, display impaired interactions also recruit them to the target gene promoter regions, leading inflammatory cytokine and chemokine expression (20). In this to expression of downstream cytokines and chemokines such as IL-6 and study, we demonstrated a direct interaction between MVP and IL-8. LAP but not LIP, which lacks the transactivation domain and The Journal of Immunology 2765 regulates C/EBPb-targeting genes negatively; however, the mech- 6. Yamamoto, M., S. Sato, H. Hemmi, K. Hoshino, T. Kaisho, H. Sanjo, O. Takeuchi, M. Sugiyama, M. Okabe, K. Takeda, and S. Akira. 2003. Role of anisms by which MVP interacts with c-Fos and LAP still need to be adaptor TRIF in the MyD88-independent Toll-like receptor signaling pathway. elucidated. Science 301: 640–643. Our present study provides direct evidence for the nuclear 7. Akira, S., and K. Takeda. 2004. Toll-like receptor signalling. Nat. Rev. Immunol. 4: 499–511. translocation of MVP upon TLR3/RLR activation. This observa- 8. Kawai, T., and S. Akira. 2006. TLR signaling. Cell Death Differ. 13: 816–825. tion is supported by a previous report indicating that MVP contains 9. 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