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`THE JOURNAL OF BIOLOGICAL CHEMISTRY
`© 2005 by The American Society for Biochemistry and Molecular Biology, Inc.
`
`Vol. 280, No. 7, Issue of February 18, pp. 5571–5580, 2005
`Printed in U.S.A.
`
`Involvement of Toll-like Receptor 3 in the Immune Response of
`Lung Epithelial Cells to Double-stranded RNA and
`Influenza A Virus*
`
`Received for publication, September 14, 2004, and in revised form, December 3, 2004
`Published, JBC Papers in Press, December 3, 2004, DOI 10.1074/jbc.M410592200
`
`Loı¨c Guillot‡§¶, Ronan Le Goffic‡§储, Sarah Bloch‡, Nicolas Escriou**, Shizuo Akira‡‡,
`Michel Chignard‡, and Mustapha Si-Tahar‡§§
`From the ‡Unite´ de De´fense Inne´e et Inflammation, INSERM E336, **Unite´ de Ge´ne´tique Mole´culaire des Virus
`Respiratoires, CNRS URA 1966, Institut Pasteur, 75015 Paris, France, and the ‡‡Department of Host Defense,
`Research Institute for Microbial Disease, Osaka University, Osaka, 565-0871, Japan
`
`Influenza A is a highly contagious single-stranded
`RNA virus that infects both the upper and lower respi-
`ratory tracts of humans. The host innate immune Toll-
`like receptor (TLR) 3 was shown previously in cells of
`myeloid origin to recognize the viral replicative, inter-
`mediate double-stranded RNA (dsRNA). Thus, dsRNA
`may be critical for the outcome of the infection. Here we
`first compared the activation triggered by either influ-
`enza A virus or dsRNA in pulmonary epithelial cells. We
`established that TLR3 is constitutively expressed in hu-
`man alveolar and bronchial epithelial cells, and we de-
`scribe its intracellular localization. Expression of TLR3
`was positively regulated by the influenza A virus and by
`dsRNA but not by other inflammatory mediators, includ-
`ing bacterial lipopolysaccharide, the cytokines tumor
`necrosis factor-␣ and interleukin (IL)-1␤, and the pro-
`tein kinase C activator phorbol 12-myristate 13-acetate.
`We also demonstrated that TLR3 contributes directly to
`the immune response of respiratory epithelial cells to
`influenza A virus and dsRNA, and we propose a molec-
`ular mechanism by which these stimuli induce epithe-
`lial cell activation. This model involves mitogen-acti-
`vated protein kinases, phosphatidylinositol 3-kinase/
`Akt
`signaling, and the TLR3-associated adaptor
`molecule TRIF but not MyD88-dependent activation of
`the transcription factors NF-␬B or interferon regulatory
`factor/interferon-sensitive response-element pathways.
`Ultimately, this signal transduction elicits an epithelial
`response that includes the secretion of the cytokines
`IL-8, IL-6, RANTES (regulated on activation normal T
`cell expressed and secreted), and interferon-␤ and the
`up-regulation of the major adhesion molecule ICAM-1.
`
`Influenza is a highly contagious, acute respiratory disease
`that affects all age groups and that can promote exacerbations
`of obstructive airways disorders, including asthma and cystic
`fibrosis. The etiological agent of the disease, the single-
`
`stranded RNA influenza viruses, are responsible for an average
`of 114,000 hospitalizations and 20,000 deaths each year, in the
`United States alone (1). Influenza viruses are classified into
`three types (A, B, and C) of which influenza A is the most
`important clinically (2). The major problem in fighting influ-
`enza is the high genetic variability of the virus, resulting in the
`rapid emergence of variants that escape the acquired immunity
`induced by the available vaccines or the resistance of the patho-
`gen to antiviral agents (1, 3). In that context, it would be
`valuable to unravel the mechanisms of virus-host cell interac-
`tions that are responsible for the “flu” syndrome. Indeed, sev-
`eral studies suggest that the inflammatory response to respi-
`ratory viral infections contributes to the pathogenesis of the
`airway symptoms. In that regard, it is of note that the viral
`replicative intermediate double-stranded RNA (dsRNA)1 is
`critical for the outcome of the infection (reviewed in Refs. 4 and
`5). For instance, synthetic dsRNA and dsRNA isolated from
`influenza virus-infected lungs are each able to induce both the
`local and systemic cytotoxic effects typical of flu (6–8).
`Cells are armed with various latent mechanisms that are
`able to sense viral components and initiate intracellular signal
`transduction to respond rapidly to virus infections. Previously,
`the interferon (IFN)-inducible RNA-dependent protein kinase
`R was considered to be central in the interaction with dsRNA
`(5). However, cells from RNA-dependent protein kinase R-de-
`ficient mice still respond to polyinosinic-polycytidylic acid
`(poly(I-C)), a synthetic dsRNA analog, suggesting the existence
`of a more critical receptor involved in the sensing and signaling
`in response to this viral component. Alexopoulou et al. (9)
`demonstrated that dsRNA recognition relies on the Toll-like
`receptor (TLR) 3, a member of the conserved family of host
`innate immune receptors, essential for detecting pathogen-
`associated molecular patterns. The stimulation of TLR3 by
`dsRNA transduces signals to activate the transcription factors
`NF-␬B and interferon regulatory factor (IRF)/interferon-sensi-
`
`* This work was supported in part by “Vaincre la Mucoviscidose”
`(Paris, France) and in part by the Pasteur Institute through “Pro-
`gramme Transversal de Recherche” Grant PTR94. The costs of publi-
`cation of this article were defrayed in part by the payment of page
`charges. This article must therefore be hereby marked “advertisement”
`in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
`§ Both authors contributed equally to this work.
`¶ Supported by the Delegation General pour l’Armement.
`储 Supported by “Vaincre la Mucoviscidose” (Paris, France).
`§§ To whom correspondence and reprint requests should be ad-
`dressed: Unite´ de De´fense Inne´e et Inflammation, INSERM E336, In-
`stitut Pasteur, 25 Rue du Dr. Roux, 75015 Paris, France. Tel.: 33-1-40-
`61-32-02; Fax: 33-1-45-68-87-03; E-mail: sitahar@pasteur.fr.
`
`This paper is available on line at http://www.jbc.org
`
`1 The abbreviations used are: dsRNA, double-stranded RNA; DN,
`dominant-negative; ERK, extracellular signal-regulated kinase; IRF,
`interferon regulatory factor; ISRE, interferon-sensitive response ele-
`ment; MyD, myeloid differentiation; NS, nonstimulated; TLR, Toll-like
`receptor; poly(I-C), polyinosinic-polycytidylic acid; TIR, Toll-IL-1 recep-
`tor; TRIF, TIR domain containing adaptor inducing interferon-␤; PMA,
`phorbol 12-myristate 13-acetate; RANTES, regulated on activation nor-
`mal T cell expressed and secreted; IL, interleukin; PI3K, phosphatidyl-
`inositol 3-kinase; ELISA, enzyme-linked immunosorbent assay; IFN,
`interferon; PFU, plaque-forming units; LPS, lipopolysaccharide; FACS,
`fluorescence-activated cell sorter; RT, reverse transcription; FITC, flu-
`orescein isothiocyanate; MAPK, mitogen-activated protein kinase;
`TNF, tumor necrosis factor; PBS, phosphate-buffered saline; CRE,
`cAMP-response element; JNK, c-Jun amino-terminal kinase.
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`5572
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`TLR3 Signaling and Respiratory Epithelial Cell Activation
`
`tive response element (ISRE) via myeloid differentiation factor
`88 (MyD88)-dependent and/or independent signaling path-
`ways. The last of these involves a distinct adaptor molecule,
`namely the Toll-IL-1 receptor (TIR) domain containing adap-
`tor-inducing interferon (IFN)-␤ (TRIF), also called the TIR
`domain containing adaptor molecule (TICAM)-1 (10, 11). This
`molecule elicits an anti-viral response, especially through the
`production of type I IFN.
`In humans, TLR3 mRNA is detected in the lung, placenta,
`pancreas, liver, heart, and brain. It is expressed in dendritic
`cells (12) and in intestinal epithelial cells (13) but does not
`seem to be present in monocytes, lymphocytes, polymorphonu-
`clear leukocytes, or natural killer cells (14). Most interestingly,
`although TLR3 expression per se was not reported, a study by
`Gern et al. (15) showed recently that viral dsRNA activates
`bronchial epithelial cells. Lung epithelial cells are the primary
`target and the principal host for influenza viruses, causing
`cytopathic effects to the respiratory tract as well as shedding of
`infective viral particles. These epithelial cells also play a key
`role in the initiation of innate and subsequently adaptive im-
`mune responses to the virus (16, 17).
`Most surprisingly, very little information is available con-
`cerning the expression and localization of TLR3 in pulmonary
`epithelial cells. Moreover, the role of epithelial TLR3, its
`regulatory mechanisms, and the signaling pathways under-
`lying the response to influenza A virus have not been inves-
`tigated previously.
`We first studied the activation of bronchial epithelial cells
`induced by either purified dsRNA or following influenza A
`virus infection. Next, we demonstrated that TLR3 is constitu-
`tively expressed in distinct human alveolar and bronchial epi-
`thelial cells, and we described its intracellular localization.
`Finally, we determined that TLR3 and its signaling-associated
`molecule TRIF play a key role in the immune response of
`respiratory epithelial cells to both dsRNA and influenza A
`virus.
`
`EXPERIMENTAL PROCEDURES
`Reagents and Antibodies—RPMI 1640, F-12K nutrient mixture
`(Kaighn’s modification), antibiotics, glutamine, Hanks’ balanced salt
`solution, and trypsin-EDTA were from Invitrogen. Fetal calf serum was
`from Hyclone (Logan, UT). Leupeptin, aprotinin, soybean trypsin in-
`hibitor, phenylmethylsulfonyl
`fluoride, benzamidine, paraformalde-
`hyde, poly(I-C) acid, forskolin, and phorbol 12-myristate 13-acetate
`(PMA) were from Sigma. The p38 (SB203580) and ERK1/2 (PD98059)
`inhibitors were obtained from Calbiochem and Cell Signaling technol-
`ogy (Beverly, MA), respectively. The PI3K inhibitor, LY294002, was
`obtained from Cell Signaling technology. Horseradish peroxidase-con-
`jugated secondary antibody was from Pierce. Anti-TLR3 antibodies
`used included the goat polyclonal antibody N-15, directed against an
`amino-terminal region of human TLR3 (Santa Cruz Biotechnology,
`Santa Cruz, CA), and the rabbit polyclonal antibody H-125, specifically
`raised against a peptide close to the transmembrane domain (amino
`acids 653–777) of TLR3 (Santa Cruz Biotechnology). The anti-human
`␤-actin antibody was from Sigma, and the anti-NF-␬B-p65, anti-phos-
`pho-ERK1/2, and anti-phospho-JNK antibodies were from Santa Cruz
`Biotechnology. The anti-phospho-p38 was purchased from Cell Signaling.
`Fluorescein isothiocyanate (FITC)-labeled anti-goat and anti-rabbit anti-
`bodies were obtained from Dako and Rockland (Gilbertsville, PA). Anti-
`human-ICAM-1 antibody was from R&D Systems (Minneapolis, MN).
`Cell and Culture Conditions—The human promonocytic cell line
`U-937, the human alveolar epithelial cell line A549, and the human
`bronchial epithelial cell line BEAS-2B were obtained from the Ameri-
`can Type Cell Collection (Manassas, VA). The human tracheal epithe-
`lial cell lines CFT-2 and NT-1 were a kind gift from Dr. A. Paul
`(INSERM U402, Paris, France). CFT-2 was derived from primary tra-
`cheal epithelia homozygous for the common cystic fibrosis mutation
`⌬F508, and NT-1 was derived from normal primary tracheal epithelial
`cells (18). Cells were cultured as described previously (19).
`Virus Preparation and Inactivation—Influenza A/Scotland/20/
`74(H3N2) virus was grown on Madin-Darby canine kidney cells in the
`presence of 2 ␮g/ml L-1-tosylamido-2-phenylethyl chloromethyl ketone-
`
`treated trypsin. The supernatant was harvested on day 3 and clarified
`by centrifugation at 680 ⫻ g for 15 min. Viral stocks were stored in
`aliquots at ⫺80 °C. Virus titers were determined by a standard plaque
`assay using Madin-Darby canine kidney cells. A noninfected cell culture
`was used for preparation of the control inoculum. UV light-inactivated
`virus was prepared by exposing stock virus solution (0.5 ml/6-cm Petri
`dish) to a 15-watt UV light at a distance of 20 cm for 15 min.
`RT-PCR—Total RNA was extracted by using an RNeasy kit (Qiagen,
`Courtaboeuf, France). RT was performed using 0.5 ␮g of total RNA
`extracted as described previously (19). PCR was performed using spe-
`cific primers (Proligo, Evry, France) for human TLR3 (sense, 5⬘-AAA
`TTG GGC AAG AAC TCA CAG G-3⬘; and antisense, 5⬘-GTG TTT CCA
`GAG CCG TGC TAA-3⬘). As an internal control, we used primers for
`human ␤-actin (sense, 5⬘-AAG GAG AAG CTG TGC TAC GTC GC-3⬘;
`and antisense, 5⬘-AGA CAG CAC TGT GTT GGC GTA CA-3⬘). Ampli-
`fications were performed in a Peltier thermal cycler apparatus (MJ
`Research, Watertown, MA) using the Qbiotaq polymerase (Qbiogene,
`Illkirch, France). To detect TLR3, the thermocycling protocol was as
`follows: 95 °C for 3 min, 36 cycles of denaturation at 95 °C for 45 s,
`annealing at 56 °C for 45 s, and extension at 72 °C for 1 min. To detect
`␤-actin, only 30 cycles were used, and the annealing temperature was
`62 °C. Amplification products were resolved on 1.5% agarose gel con-
`taining ethidium bromide. Band intensities on gels were recorded after
`amplification with an Ultra-Lum system (Ultra-Lum, Claremont, CA).
`Samples for each point were serially diluted to verify that PCR was
`performed in the linear phase of the amplification reaction (data not
`shown).
`Immunoblotting—Epithelial cell extracts were prepared and solubi-
`lized as described previously (19). Aliquots (15 ␮g of protein) were run
`on SDS-10% PAGE, and the proteins were then electrotransfered to a
`nitrocellulose membrane (Optitran BA-S 85, Schleicher and Schuell)
`and probed with specific antibodies, as specified in the figure legends.
`Bound antibodies were detected using ECL⫹ immunoblotting detection
`system (Amersham Biosciences), according to the manufacturer’s in-
`structions. Molecular masses were estimated from calibration stand-
`ards included in each gel.
`Flow Cytometry Analysis—Epithelial and monocytic cells were dis-
`pensed (1 ⫻ 106 cells/ml) into conical bottomed 96-well plastic plates
`(Nunc A/S, Roskilde, Denmark) and were centrifuged at 100 ⫻ g at 4 °C
`for 10 min. Cells were washed with Hanks’ balanced salt solution, 0.5%
`bovine serum albumin supplemented with 1 mM Ca2⫹ and Mg2⫹, and a
`saturating concentration of anti-TLR3 antibodies (5 ␮g/ml), anti-
`ICAM-1 antibody (1 ␮g/ml), or nonimmune IgG as controls was then
`added, and the samples were incubated for 30 min at 4 °C. Cells were
`washed and incubated for 30 min at 4 °C with the corresponding sec-
`ondary FITC-conjugated antibody (5 ␮g/ml). For intracellular staining,
`cells were fixed and permeabilized by incubation for 90 min on ice with
`a solution of PBS, 3.2% paraformaldehyde, 0.2% Tween 20; they were
`then incubated with an anti-TLR3 antibody (5 ␮g/ml) and FITC-conju-
`gated secondary antibodies (5 ␮g/ml). FACScan flow cytometer (Immuno-
`cytometry Systems) was used for cytometric analysis.
`NF-␬B Immunostaining—BEAS-2B epithelial cells were cultured on
`22-mm glass cell culture coverslips (CML France, Nemours, France).
`Cells were washed three times with PBS and then fixed for 15 min in
`PBS, 3.2% paraformaldehyde. After washing with gentle shaking, cells
`were permeabilized for 5 min with 0.1% Triton X-100 and washed, as
`before, prior to incubation with an anti-NF-␬B-p65 antibody (0.4 ␮g/ml)
`and a specific secondary antibody (5 ␮g/ml). In control experiments,
`cells were incubated with nonimmune IgG as control isotypes. Finally,
`the cells were washed extensively with PBS, and the coverslips were
`mounted in fluorescence mounting medium. Fluorescence microscopy
`was performed with a 63⫻/1.4 oil objective lens on a confocal microscope
`(model LSM 510; Carl Zeiss France, Le Pecq, France), using laser
`excitation at 488 nm.
`Epithelial Cell Transfection and Reporter Gene Studies—BEAS-2B
`cells were seeded at 5 ⫻ 104 on 24-well plates (Costar, New York) 96 h
`before transfection using FuGENE 6 transfection reagent (Roche Ap-
`plied Science), according to the manufacturer’s instructions. Each sam-
`ple contained 200 ng of an NF-␬B-luciferase- (kindly provided by Dr. A.
`Israel, Pasteur Institute, Paris, France), an ISRE-luciferase-, or a CRE-
`luciferase-reporter plasmid (Clontech) and 500 ng of vector expressing
`a dominant-negative form of either MyD88 (DN-Myd88; a kind gift from
`Dr. M. Muzio, Mario Negri Institute, Milan, Italy) or TRIF (DN-TRIF).
`The TLR3 construct from which the TIR domain is deleted (pZERO-
`hTLR3) and encoding a nonfunctional TLR3 molecule were purchased
`from InvivoGen (San Diego, CA). The empty plasmids, pcDNA3 (In-
`vitrogen) and pCMV (Clontech), were used as controls as appropriate.
`After 24 h, cells were left untreated or stimulated for 6 or 24 h at 37 °C
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`TLR3 Signaling and Respiratory Epithelial Cell Activation
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`5573
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`with influenza A virus or 1 ␮g/ml poly(I-C). Luciferase activity was
`measured in the cell lysates as described previously (19), using an
`EGNG Berthold luminometer. Results are expressed as relative lucif-
`erase units.
`Cytokine Measurements—Human IL-8, IL-6, RANTES, and IFN-␤
`concentrations in cell culture supernatants were determined using Duo-
`Set ELISA kits obtained from R&D Systems (Minneapolis, MN).
`Statistical Analysis—Each point corresponds to the mean ⫾ S.D. of
`the indicated number of experiments. The statistical significance of
`differences between groups was tested using the unpaired Student’s t
`test with a threshold of p ⬍ 0.05.
`
`RESULTS
`Comparison of the Activation of Bronchial Respiratory Cells
`by dsRNA and Influenza A Virus—dsRNA is known to accu-
`mulate within infected cells, and it has the required physical
`and biological properties needed to induce antiviral responses
`and pathological inflammatory processes (4, 5). Thus, it ap-
`pears important to examine whether influenza A virus-induced
`activation of pulmonary epithelial cells shares any character-
`istics with that stimulated by a synthetic dsRNA such as
`poly(I-C). Fig. 1 reports a comparison of the effects from two
`inducers in the human bronchial epithelial cell line BEAS-2B.
`Poly(I-C) triggered a strong secretion of IL-8, in a concentra-
`tion- (Fig. 1A) and time-dependent manner (Fig. 1B). The se-
`cretion started within 3 h and IL-8 accumulated in the culture
`medium up to 24 h (Fig. 1B). We also observed a time-depend-
`ent accumulation of IL-6 (Fig. 1C). RANTES was also induced
`by poly(I-C) but ⬃3 h after the start of production of the other
`cytokines. This suggests an autocrine/paracrine activation by
`another mediator; for example IFN-␤ might feed back on
`RANTES production (Fig. 1D). In that regard, IFN-␤ produc-
`tion peaked 6 h post-stimulation but was not detected at 24 h,
`suggesting a reprocessing of this cytokine by BEAS-2B cells
`(Fig. 1E). To exclude any stimulatory effect associated with
`contamination of poly(I-C) by bacterial endotoxin, experiments
`were also performed using poly(I-C) supplemented with 20
`␮g/ml polymyxin B, a well characterized LPS inhibitor. Under
`these experimental conditions, IL-8 secretion by BEAS-2B cells
`was not modified (not shown).
`When BEAS-2B cells were infected with influenza A virus,
`IL-8 secretion was clearly detected 24 h later and was dose-de-
`pendent (Fig. 1, G and H). RANTES and IL-6 secretion were
`also dose-dependent up to a maximum at 5 ⫻ 104 PFU/multi-
`plicity of infection of 0.25) (Fig. 1, I and K). Cytokine secretion
`was delayed by 20 h with respect to that by poly(I-C)-stimu-
`lated BEAS-2B cells. This delay may be consistent with the
`time required to generate dsRNA within the infected cell, dur-
`ing the replication of the virus (Fig. 1, H and J, and data not
`shown).
`Epithelial cells in the lung express ICAM-1, which is in-
`volved in the recruitment and the local accumulation of inflam-
`matory cells through the binding to lymphocyte function-asso-
`ciated antigen (LFA)-1 (20). Previous studies have shown that
`pro-inflammatory mediators including LPS and TNF-␣ induce
`ICAM-1 expression on pulmonary epithelial cells (21). We con-
`firmed these findings by flow cytometric analysis, and we ex-
`tended them to dsRNA and influenza A virus by demonstrating
`a potent up-regulation of ICAM-1 expression, which was as
`strong as that obtained with TNF-␣ (Fig. 1F and not
`illustrated).
`Previous studies have shown that production of inflamma-
`tory mediators in response to a viral infection can occur in the
`presence or absence of multiplication of the pathogen (22). We
`tested whether viral contact with the plasma membrane and/or
`viral penetration of the host cell was sufficient to trigger the
`inflammatory response. Bronchial epithelial cells were infected
`with UV-treated, and therefore nonreplicative, virus. Also,
`
`BEAS-2B cells were treated with an intact virus in the pres-
`ence of 1 ␮g/ml amantadine, an influenza-specific inhibitor
`blocking the early release of the viral genome into the cyto-
`plasm but not the endocytosis of the viral particle into the cell
`(1). Neither UV-treated virus nor virus in the presence of
`amantadine induced IL-8 release (Fig. 1L). Thus, components
`generated during viral replication are required for the inflam-
`matory response. Similarly, there was no RANTES or IL-6
`secretion in the absence of viral replication (data not shown).
`Hence, the inflammatory response to influenza A virus infec-
`tion is replication-dependent and is not mediated solely by the
`initial virus-host cell interaction or by any artifact possibly
`present in the infectious inoculum.
`Expression and Localization of TLR3 in Pulmonary Epithe-
`lial Cells—Because the foregoing results suggested that
`dsRNA is required for the immunostimulatory activity of influ-
`enza A virus, we examined the expression of TLR3, a recently
`described dsRNA sensor, in pulmonary epithelial cells (9). We
`first used RT-PCR to test for the presence of TLR3 mRNA in
`unstimulated human respiratory epithelial cells. As shown in
`Fig. 2A, TLR3 mRNA was detected in PMA-differentiated
`U-937 cells and in both human alveolar (A549) and tracheo-
`bronchial (BEAS-2B, NT-1, and CFT-2) epithelial cells lines.
`RT-PCR analysis of ␤-actin mRNA confirmed the quality of all
`RNA preparations.
`We then used two antibodies, N-15 and H-125, to test for
`TLR3 protein in human pulmonary epithelial cells. The speci-
`ficity of these antibodies was confirmed by Western blotting
`with a recombinant TLR3-glycosylated peptide, consisting of
`amino acids 21–711 (not illustrated). Protein expression level
`of TLR3 was analyzed by flow cytometry in A549 and BEAS-2B
`cells (Fig. 2B). No TLR3 signal was detected at the cell surface
`of these respiratory cells (left panels), but abundant intracellu-
`lar TLR3 was revealed using a mild fixation and permeabili-
`zation protocol (middle and right panels).
`Epithelial TLR3 Expression Is Up-regulated by Poly(I-C) and
`Influenza A Virus—Next we examined whether different stim-
`uli regulate the epithelial TLR3 mRNA. BEAS-2B cells were
`exposed to an optimal concentration of poly(I-C) (1 ␮g/ml), LPS
`(1 ␮g/ml), TNF-␣ (50 ng/ml), IL-1␤ (20 ng/ml), or the potent
`protein kinase C activator PMA (15 nM) and to influenza A
`virus (5 ⫻ 104 PFU) for 24 h. Under these conditions, cell
`activation was fairly equipotent, as assessed by the measure-
`ment of IL-8 secretion induced by each stimulus (not shown).
`Expression of TLR3 mRNA was normalized to that of ␤-actin
`and is reported in Fig. 3B, histogram bars. This semi-quanti-
`tative densitometric measurement clearly shows that only in-
`fluenza A virus infection and cell stimulation by the viral RNA
`mimetic component specifically up-regulated TLR3 mRNA; the
`other treatments had no significant effect (Fig. 3, A and B).
`IL-8 but Not RANTES Secretion Triggered by dsRNA or
`Influenza A Virus Shares a Common Signaling Pathway—
`Virus infection of susceptible cells activates multiple signaling
`pathways, including dynamic protein phosphorylations, that
`orchestrate the induction of genes contributing to the innate
`immune response. The kinases involved include p38, extracel-
`lular signal-regulated kinase (ERK)1/2, and JNK. Little is
`known concerning the involvement of PI3K in virus-induced
`and/or TLR signaling. PI3K catalyzes the production of phos-
`phatidylinositol 3,4,5-trisphosphate, which allows the recruit-
`ment of signaling proteins, including the serine-threonine ki-
`nase Akt (23). Therefore, we examined whether dsRNA
`activates p38, JNK, ERK1/2, and Akt in bronchial epithelial
`cells. Treatment with poly(I-C) strongly up-regulated phospho-
`rylation of these signaling components. The maximum level of
`phosphorylation was between 15 and 60 min and declined
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`TLR3 Signaling and Respiratory Epithelial Cell Activation
`
`FIG. 1. Comparison of the activa-
`tion of bronchial epithelial cells in-
`duced by dsRNA and influenza A
`virus. Monolayers of BEAS-2B cells were
`stimulated for 24 h with a series of con-
`centrations of poly(I-C) (0.01, 0.1, 1, and
`10 ␮g/ml (A)) or with 1 ␮g/ml poly(I-C) for
`various times (1, 3, 6, and 24 h; B–E).
`Supernatant fluids were tested for IL-8 (A
`and B), IL-6 (C), RANTES (D), and IFN-␤
`(E) by ELISA. BEAS-2B cells were stim-
`ulated for 24 h or not stimulated (NS)
`with 1 ␮g/ml LPS, 1 ␮g/ml poly(I-C), or 20
`ng/ml TNF-␣, and ICAM-1 expression
`was assayed by FACS analysis (F). Re-
`sults are expressed as mean fluorescence
`intensity (MFI). Monolayers of BEAS-2B
`cells were stimulated for 24 h by increas-
`ing concentrations of influenza A virus
`(0.5, 1, 5, and 10 ⫻ 104 PFU/ml (G, I, and
`K)) or with 5 ⫻ 104 PFU/ml for various
`times (6, 12, 24, 48, and 72 h (H and J)).
`Supernatant fluids were tested for IL-8 (G
`and H), RANTES (I and J), and IL-6 (K)
`by ELISA. L, BEAS-2B cells were stimu-
`lated or not for 24 h with 5 ⫻ 104 PFU/ml
`of influenza A virus, of UV-treated influ-
`enza A virus or of virus after cell pretreat-
`ment with 1 ␮g/ml of amantadine. Super-
`natant fluids were tested for IL-8 by
`ELISA. Data are means ⫾ S.D. of tripli-
`cate determinations of a representative
`experiment performed three times. Black
`diamonds are poly(I-C)- or virus-treated
`cells, and open boxes represent non-
`treated samples.
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`FIG. 3. TLR3 gene regulation by
`pro-inflammatory stimuli. BEAS-2B
`cells were stimulated or not stimulated
`(NS) with 1 ␮g/ml LPS, 1 ␮g/ml poly(I-C),
`5 ⫻ 104 PFU/ml of influenza A virus, 20
`ng/ml TNF-␣, 50 ng/ml IL-1␤, or 15 nM
`PMA for 24 h. Total RNA was extracted,
`and TLR3 mRNA was analyzed by RT-
`PCR. A, representative result out of three
`is shown. B, histogram bars show the
`value for TLR3 normalized against that
`for ␤-actin and are the means ⫾ S.D. of
`three
`independent
`experiments per-
`formed in triplicate.
`
`FIG. 2. Expression and localization
`of TLR3 in pulmonary epithelial
`cells. A, representative RT-PCR showing
`TLR3 expression in human alveolar
`(A549) and tracheobronchial (BEAS-2B,
`NT-1, and CFT-2) epithelial cell lines.
`The human macrophage cell line U-937, a
`known source of TLR3, was used as a
`positive control. B, representative FACS
`analysis of TLR3 expression in two differ-
`ent lung epithelial cell lines. A549 and
`BEAS-2B were permeabilized ((⫹)perm,
`right panels) or not ((⫺) perm, left panels)
`and immunostained with N-15 or H-125
`as described under “Experimental Proce-
`dures.” The light and dark line histo-
`grams represent the fluorescence signal
`obtained using isotype-control antibody
`and TLR3-specific antibody, respectively.
`Fluorescence tracings are representative
`of three independent experiments.
`
`thereafter, except for p38 phosphorylation that increased until
`360 min post-stimulation (Fig. 4A). To delineate specifically the
`role of these kinases in the production of epithelial cytokines,
`BEAS-2B cells were pretreated with the specific inhibitors
`PD98059 for ERK1/2, SB203580 for p38, and LY294002 for
`PI3K (Fig. 4B). None of these inhibitors caused cytotoxic effects
`on BEAS-2B cells at the concentrations used in these experi-
`ments. However, all
`inhibitors significantly reduced the
`dsRNA-induced production of IL-8 (Fig. 4B) and IL-6 (not il-
`lustrated). By contrast, only the PI3K/Akt pathway inhibitor
`reduced RANTES production.
`When pulmonary epithelial cells were infected by influenza
`A virus in the presence of 10 ␮M of the above inhibitors, IL-8
`
`secretion was similar to that induced by synthetic dsRNA un-
`der the same experimental conditions: ⬇45% inhibition in the
`presence of PD98059, and a secretion almost abolished in the
`presence of SB203580 or LY294002. By contrast, RANTES
`release triggered by either stimulus does not exhibit a similar
`inhibitory pattern. Remarkably, although the p38 inhibitor did
`not affect dsRNA-induced RANTES secretion, it strongly inhib-
`ited RANTES secretion following influenza A virus infection
`(Fig. 4C).
`The TLR3/TRIF Pathway Is Essential for dsRNA and Influ-
`enza A Virus-induced NF-␬B and IRF/ISRE Activation in Pul-
`monary Epithelial Cells—Activation of transcription factors is
`pivotal to many signal transduction pathways. For instance,
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`FIG. 4. Signal transduction induced
`by dsRNA and influenza A virus. A,
`time course of the activation of MAPKs
`p38, JNK, ERK1/2, and the PI3K-depend-
`ent kinase Akt. BEAS-2B cells were stim-
`ulated or not stimulated (NS) with 1
`␮g/ml poly(I-C) for different time inter-
`vals and lysed, and phosphorylation of
`these enzymes was determined by immu-
`noblotting with specific antibodies (all di-
`luted 1:2000). To confirm equal loading,
`membranes were reprobed with an anti-
`␤-actin antibody (diluted 1:15,000). Data
`are representative of three independent
`experiments. B and C, the role of MAPK-
`and PI3K/Akt-dependent signaling in the
`activation of epithelial cells by poly(I-C)
`and influenza A virus. B, BEAS-2B cells
`were pretreated for 1 h with inhibitors of
`ERK1/2 (PD98059), p38 (SB203580), and
`PI3K (LY294002) and then stimulated
`with 1 ␮g/ml poly(I-C)
`for 6 h. C,
`BEAS-2B cells were pretreated for 1 h
`with the same inhibitors used at 10 ␮M
`and then stimulated with 5 ⫻ 104 PFU/ml
`of influenza A virus for 24 h. Supernatant
`fluids were tested for IL-8 (left panels)
`and RANTES (right panels) by ELISA.
`Data are means ⫾ S.D. of triplicate deter-
`minations of a representative experiment
`performed three times.
`
`NF-␬B can be activated in response to many different stress
`conditions, including infection, inflammation, and tissue re-
`pair. IRFs consist of a growing family of related transcription
`proteins initially identified as regulators of the IFN-␣/␤ gene
`promoters and the ISRE of various IFN-stimulated genes. Ac-
`tivators of the cyclic AMP-response element (CRE) contribute
`to diverse physiological processes, including the control of cel-
`lular metabolism and cell survival. We assessed the involve-
`ment of these regulatory signaling elements in the antiviral
`
`immune response induced by dsRNA and influenza A virus. In
`that purpose, BEAS-2B cells were transfected with a set of
`vectors each of which contains a different cis-acting enhancer
`element (NF-␬B, ISRE, or CRE) upstream from a luciferase
`reporter gene. As shown in Fig. 5A, NF-␬B and IRF/ISRE were
`strongly activated upon dsRNA challenge in bronchial epithe-
`lial cells. In contrast, CRE was not activated under the same
`experimental conditions, even though forskolin (10 ␮M), a po-
`tent activator of the cAMP signaling pathway, confirmed the
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`FIG. 5. Involvement of TLR3 and TRIF pathway in dsRNA and influenza A virus-induced NF-␬B and IRF/ISRE activation in
`pulmonary epithelial cells. A, BEAS-2B cells were transfected with 200 ng of an NF-␬B or IRF/ISRE luciferase reporter construct and then
`stimulated with 1 ␮g/ml poly(I-C) for 6 h. Cell lysates were prepared and assayed for luciferase activity. Results are expressed as relative
`luciferase units (RLU) and are means ⫾ S.D. of triplicate determinations of a representative experiment performed three times. B, BEAS-2B
`cells were stimulated with 1 ␮g/ml poly(I-C) for 30 and 90 min. The cells were then immunostained with an anti-NF-␬B antibody and examined
`by confocal microscopy as described under “Experimental Procedures.” C and D, BEAS-2B cells were co-transfected with 200 ng of an NF-␬B-
`or IRF/ISRE luciferase reporter construct and with 500 ng of the expression plasmid encoding DN forms of MyD88 or TRIF, or wi