key: cord-0002043-utsvsja0 authors: Uematsu, Takayuki; Iizasa, Ei’ichi; Kobayashi, Noritada; Yoshida, Hiroki; Hara, Hiromitsu title: Loss of CARD9-mediated innate activation attenuates severe influenza pneumonia without compromising host viral immunity date: 2015-12-02 journal: Sci Rep DOI: 10.1038/srep17577 sha: 6c982280e9ca5251ee74d0f2b84489a63a68c5bb doc_id: 2043 cord_uid: utsvsja0 Influenza virus (IFV) infection is a common cause of severe viral pneumonia associated with acute respiratory distress syndrome (ARDS), which is difficult to control with general immunosuppressive therapy including corticosteroids due to the unfavorable effect on viral replication. Studies have suggested that the excessive activation of the innate immunity by IFV is responsible for severe pathologies. In this study, we focused on CARD9, a signaling adaptor known to regulate innate immune activation through multiple innate sensor proteins, and investigated its role in anti-IFV defense and lung pathogenesis in a mouse model recapitulating severe influenza pneumonia with ARDS. We found that influenza pneumonia was dramatically attenuated in Card9-deficient mice, which showed improved mortality with reduced inflammatory cytokines and chemokines in the infected lungs. However, viral clearance, type-I interferon production, and the development of anti-viral B and T cell immunity were not compromised by CARD9 deficiency. Syk or CARD9-deficient DCs but not macrophages showed impaired cytokine but not type-I interferon production in response to IFV in vitro, indicating a possible role for the Syk-CARD9 pathway in DCs in excessive inflammation of IFV-infected lungs. Therefore, inhibition of this pathway is an ideal therapeutic target for severe influenza pneumonia without affecting viral clearance. the host's innate immune system including macrophages and dendritic cells (DCs) to secrete inflammatory cytokines/chemokines, such as IL-6, TNF, CXCL1 and CXCL10 13, 14 , which play crucial roles in the pathogenesis of ARDS 10, [15] [16] [17] . Therefore, inhibition of cytokine/chemokine production via targeting the host's innate immune system may lead to the development of effective treatment options for ARDS. Caspase recruitment domain family member 9 (CARD9) is an adaptor protein that delivers NF-κ B activating signals through multiple innate sensor proteins, such as a wide array of C-type lectin (CLR) and immunoglobulin (Ig)-superfamily receptors that are coupled to immunoreceptor tyrosine-based activation motifs (ITAMs) [18] [19] [20] , as well as cytoplasmic RNA sensors, such as RIG-I and Mda5, and the DNA sensor Rad50 21, 22 . Several lines of evidence have implied a role for CARD9-mediated innate activation pathways in influenza pathogenesis and immunity. The CLR dendritic cell-specific ICAM-3 grabbing non-integrin (DC-SIGN), which has a cytoplasmic ITAM-like sequence, was shown to bind influenza virus A 23, 24 . Additionally, CARD9 was found to be required for the production of inflammatory cytokines after infection with RNA viruses, such as vesicular stomatitis virus and encephalomyocarditis virus, by regulating NF-κ B signaling through RIG-I and Mda5 21 . However, the roles of CARD9 in influenza pneumonia, as well as in protection against IFV are yet to be elucidated. In this study, we demonstrate a role for CARD9-mediated innate activation in influenza pathogenesis and immunity. Our results showed that the CARD9 pathway was involved in fatal influenza pneumonia mediated by inflammatory cytokine/chemokine production, whereas it was dispensable for type-I interferon production as well as the development of anti-viral acquired immunity. Therefore, inhibition of this pathway may represent an ideal target for the treatment of severe influenza pneumonia without affecting viral clearance. adapted IFV A/PR/8/34 strain (PR8) shows similar lung pathology to human ARDS 10-12 , we intratracheally infected wild-type (WT: C57BL/6) and Card9 -/mice with a lethal dose (10 4 PFU/mouse) of PR8 to determine whether CARD9-mediated innate immune responses contributed to severe influenza pneumonia. We observed that PR8-infected WT mice appeared visibly ill with ruffled fur and reduced oral intake during the 6 to 10 days after infection, whereas Card9 -/mice appeared more active than WT mice. Consistent with their activity, the final survival rate up to day 21 after infection was dramatically improved in Card9 -/mice (~80%) as compared with WT mice (~40%; Fig. 1A ). Histopathological analysis of IFV-infected lungs on days 4 and 8 revealed that lung inflammation, which was the most obvious on day 8 in WT mice, was much less severe in Card9 -/mice (Fig. 1B) . Given that inflammatory cytokines/chemokines were linked to lung damage in severe influenza pneumonia [10] [11] [12] [15] [16] [17] 25 , we analyzed infiltrated cells and cytokine/chemokine levels in bronchoalveolar lavage fluid (BALF) collected from the IFV-infected lungs of WT and Card9 -/mice. Consistent with the pathological data, the number of CD45 + BALF cells were significantly fewer in the lungs of Card9 −/− mice than in those of WT mice on day 8, with a marked decrease in the numbers of T cells and neutrophils ( Fig. 1C ) that were the two main infiltrated subpopulations at this time point (Supplementary Fig. S1 ). However, the numbers of B cells, NK cells, macrophages, cDCs, and pDCs were not significantly affected by the CARD9 deficiency. The BALF levels of inflammatory cytokines and chemokines, i.e., IL-6, TNF-α , CCL3/MIP-1α , CXCL1/KC, and CXCL10/IP-10, which have been reported to contribute to lung pathology 10, 13, 14 , peaked on day 4 and were considerably lower in the Card9 −/− mice than in the WT mice (Fig. 1D ). However, no reduction in cytokine/chemokine levels was evident on day 8, indicating that a CARD9-mediated innate response controls cytokine/chemokine production at an early time point after IFV infection and influences subsequent inflammatory cell recruitment and lung pathology at a later time point. Collectively, the loss of CARD9 attenuated severe influenza pneumonia and improved host mortality. CARD9 deficiency does not compromise anti-viral protective immunity. Activation of innate immunity in response to IFV regulates anti-viral protective immunity 26 . To evaluate the impact of CARD9 deficiency on anti-IFV protection, we first analyzed the viral burden in the IFV-infected lungs of WT and Card9 -/mice. Notably, we found that CARD9 deficiency did not increase virus titers and the amount of viral RNA in the lungs, but instead significantly accelerated viral clearance in the later stage (day 8) of infection ( Fig. 2A) . Type-I interferons (IFN-α /β ) are the primary factors that induce host resistance to IFV 26, 27 . In contrast to cytokine/chemokine production, CARD9 deficiency did not significantly affect IFN-α /β production in IFV-infected lungs (Fig. 2B) . The type II interferon IFN-γ is not essential for viral clearance 28 , but its presence during IFV infection ameliorates the severity of inflammation and lung injury 29 . IFN-γ production was significantly more in the lungs of Card9 −/− mice than in those of WT mice on day 8 but not on day 4 (Fig. 2B) ; thus, this increase might contribute to the improvement of lung pathology in Card9 −/− mice. Moreover, the expression of IP-10 encoded by an IFN-responsive gene was significantly impaired in Card9 −/− mice on day 4 (Fig. 1D) , albeit no significant reduction was seen in either Type I or Type II interferons, suggesting that synergistic signals of interferons and various cytokines/chemokines are required for the higher expression of IP-10 at an early phase of infection. Next, we examined the induction of virus-specific CD8 + T cells using the H-2D b tetramer coupled with a viral nuclear protein (NP)-derived peptide (NP 366-374 ) 30, 31 . The percentages of NP-specific CD8 + T cells in the draining lymph nodes of the lungs on day 8 were comparable between WT and Card9 −/− mice (Fig. 2C) . To evaluate the impact of CARD9 deficiency on acquired immunity to IFV, WT and Card9 −/− mice were immunized with a sub-lethal dose (1/10 of LD 50 ) of the PR8 strain and rechallenged with a high lethal dose (100 LD 50 ) of PR8 after 4 weeks. We found that CARD9 deficiency did not alter viral clearance after the challenge (Fig. 2D ). Next, we assessed the induction of humoral and cellular acquired immunity to IFV in Card9 -/mice. We re-infected WT and Card9 -/mice on day 21 after their first infection (representing the second infection) and analyzed the production of virus-specific antibodies as well as the development of virus-specific CD8 + T cells on day 14 after the second infection. We found that the CARD9 deficiency affected neither the production of virus-specific IgG in the serum nor virus-specific IgA in the lung mucosa (Fig. 2E) . Additionally, CARD9 deficiency did not affect the IFN-γ response by splenic CD8 + T cells specific to a NP antigen epitope NP 366-374 (Fig. 2F) . Collectively, the CARD9 pathway is dispensable for the induction of protective immunity against both the primary and secondary IFV infections, and its deficiency even improves primary viral clearance upon lethal-dose infections probably because of improvement in lung damage and elevated IFN-γ production. CARD9 deficiency impairs inflammatory cytokine, but not type-I interferon, production by DCs. Because macrophages and DCs are known to be an early source of inflammatory cytokines and type-I interferons in pulmonary IFV infection 13,14 , we examined whether CARD9 was required for their production by myeloid cells in response to IFV. Thioglycolate-induced peritoneal macrophages (TG-MFs), bone marrow-derived macrophages (BMMFs), alveolar macrophages (AMFs), conventional DCs (cDCs), or Flt3 ligand-induced plasmacytoid DCs (FLT3L-DCs) prepared from WT or Card9 -/mice were brought into contact with PR8 in vitro and the production of IL-6, TNF-α , and IFN-α /β was measured. IL-6 and TNF-α produced by TG-MFs (Fig. 3A) or BMMFs (Fig. 3B ) and those mRNAs produced by AMFs (Fig. 3C ) in response to PR8 were not affected by CARD9 deficiency. As previously reported 18, 19 , CARD9 was required for a cytokine response to ox-zymosan through Dectin-1-Syk but dispensable for the response to LPS through TLR4-MyD88 in cDCs ( Supplementary Fig. S2A ). We found that Card9 -/-cDCs (Fig. 3D ) or FLT3L-DCs (Fig. 3E ) produced significantly lower levels of IL-6 and TNF-α than WT cells in response to PR8. In contrast, IFN-α /β production was not compromised in Card9 -/-cDCs or FLT3L-DCs. These findings suggest that CARD9 deficiency primarily affected DCs but not MFs regarding the production of inflammatory cytokines but not IFN-α /β in response to IFV. It has been reported that the CARD9-IPS-1 pathway mediates the cytokine response to RNA viruses through RLHs 21 , while CARD9 has been found to transmit signals from myeloid ITAM-coupled receptors via Syk [18] [19] [20] . Thus, we evaluated the contributions of these pathways. IPS-1-deficient (Ips-1 -/-) cDCs and Myd88-deficient (Myd88 -/-) FLT3L-DCs produced significantly lower levels IFN-α /β than WT cells in response to PR8 (Fig. 3D ,E), consistent with previous findings showing that the IFN-α /β response to RNA viruses is mediated mainly through RIG-I/IPS-1 in cDCs, and through TLR7/MyD88 in pDCs 32, 33 . In addition, IL-6 and TNF-α production was impaired in Ips-1 -/-cDCs and Myd88 -/-FLT3L-DCs. Lower levels of cytokine production were also observed in Myd88 -/-cDCs, likely due to impaired activation through several TLRs (i.e. TLR3, 7, 8 and 9) that sense virus nucleic acids 34, 35 . We found that inducible Syk-deficient (Syk del/del ) cDCs and FLT3L-DCs produced significantly lower levels of inflammatory cytokines than WT cells similar to the responses of Card9 -/-cDCs and FLT3L-DCs, whereas the Syk deficiency did not affect cytokine production in response to LPS (Supplementary Fig. S2A ). Consistent with these findings, treatment of WT cDCs (Fig. 4A ) and FLT3L-DCs (Fig. 4B) with the Syk inhibitor BAY61-3606 reduced the production of inflammatory cytokines but not type-I interferons in response to PR8 in a dose-dependent manner. The treatment with BAY61-3606 reduced the cytokine response to ox-zymosan but not to LPS, indicating that the effect of the inhibitor on cytokine expression depends on the stimulus but cannot be attributed to its general effect on cytokine gene expression ( Supplementary Fig. S2B ). Overall, these results suggest that the attenuation of influenza pneumonia by Card9 deficiency was likely attributable to the reduced cytokine/chemokine production by pulmonary DCs through the Syk-CARD9 pathway in response to IFV. We have shown that CARD9-mediated activation of the innate immune system exacerbates influenza pneumonia in mice. CARD9 deficiency resulted in the reduction of inflammatory cytokine/chemokines and the infiltration of inflammatory cells in IFV-infected lungs, resulting in improved mouse mortality rates. Given the pathological similarity between the mouse intratracheal infection model and influenza-associated ARDS in human 10-12 , we postulate that the CARD9 pathway contributes to the exacerbation of human influenza pneumonia. Although the Syk-CARD9-mediated innate immune BALF were analyzed on day 0 and 14 after the second infection. (F) IFV-specific adaptive T cell response. Splenocytes from WT and Card9 -/mice (n = 6 per group) 14 days after infection as in (E) were stimulated in vitro with IFV nucleoprotein peptide (NP 366-374 ) for 3 days. The culture supernatants were analyzed for IFN-γ production. Data are presented as mean ± SD of triplicates. Data are representative of three independent experiments. *P < 0.05 by Student's t-test. Scientific RepoRts | 5:17577 | DOI: 10.1038/srep17577 response is crucial for anti-fungal acquired immunity [36] [37] [38] [39] , our data showed that CARD9 was dispensable for anti-IFV immunity, as demonstrated by the findings that CARD9 deficiency did not alter viral burden, the elevation of IFN-α /β , or the induction of anti-viral adaptive T and B cell responses in the IFV-infected mice. Thus, other innate mechanisms independent of the CARD9 pathway may play a more dominant role in protective immunity against IFV. Innate immune sensors for IFV and its downstream signaling pathway have been well illustrated 26, 40, 41 . In our study, CARD9 deficiency resulted in a selective reduction in inflammatory cytokines, but not IFN-α /β production, by both cDCs and FLT3L-DCs in response to IFV. This pattern of impairment was observed in Ips-1 -/-cDCs, and was consistent with results from previous reports indicating that CARD9 selectively regulates the cytokine response through RIG-I-IPS-I in cDCs 21 . Additionally, we found that Syk del/del and Syk inhibitor-treated cDCs showed the same pattern of impairment as Card9 -/or Ips-1 -/-cDCs. On the other hand, in FLT3L-DCs known to release large amounts of inflammatory cytokines and type-I interferons upon recognition of IFV 32,33 , IPS-1 deficiency affected neither cytokine or type-I interferon responses. However, Syk deficiency in FLT3L-DCs, similar to that in cDCs, resulted in a selective reduction in inflammatory cytokines but not IFN-α /β . Thus, it is likely that the Syk-CARD9 pathway controls detrimental cytokine production by pulmonary DCs upon acute influenza infection. Alternatively, because CARD9 deficiency impairs cytokine production by DCs following stimulation with several TLR ligands 18 , it may be important to examine whether the Syk-CARD9 pathway is involved in the TLR7-meditated response to IFV. The involvement of the Syk-CARD9 pathway implies the presence of ITAM-coupled receptors that sense IFV and stimulate cytokine production by DCs. It has been reported that an interaction between DC-SIGN, which possesses an ITAM-like "hemITAM" motif, and the carbohydrate of IFV hemagglutinin induces maturation of DCs and promotes endocytosis of the virus 42 , indicating the signal-activating capacity of DC-SIGN upon virus binding. However, it is unclear whether the DC-SIGN hemITAM is capable of transmitting sufficient signals to produce inflammatory cytokines. With regard to other viruses, recognition of the dengue virus by the DAP12-associated CLR CLEC5A results in increased vascular permeability due to the overproduction of TNF-α , resulting in fatal outcomes 43 . However, its ability to recognize the IFV remains unknown. Thus, it is worth examining whether these CLRs are involved in the cytokine response to IFV. It is also conceivable that damage-associated molecular patterns (DAMPs), generated due to lung damage by IFV infection, may be ligands for some ITAM-coupled receptors. Indeed, the FcRγ -associated CLR Mincle was shown to recognize SAP130, a component of the ribonucloprotein released from dead cells 44 . In conclusion, CARD9-mediated innate immune activation in pulmonary DCs acts to exacerbate severe influenza pneumonia, but is dispensable for host protection against IFV. Thus, inhibiting the CARD9 pathway may represent a promising therapeutic target for the control of PIVP without affecting viral clearance. Mice. Card9 -/-, Syk flox/flox , Ips-1 -/and Myd88 -/mice have been previously described 18, [45] [46] [47] . These mice were backcrossed at least 8 times onto C57BL/6 mice. C57BL/6 mice were purchased from CLEA Japan, Inc. (Tokyo, Japan). The animals were housed in specific pathogen-free conditions. All experiments were approved by the Institutional Animal Care and Use Committee for Kitasato University Medical Center and animals were treated in accordance with the Regulations for Animal Experiments in Kitasato University. All surgeries were performed under ketamine hydrochloride/xylazine anesthesia, and all efforts were made to minimize suffering. Antibodies and reagents. Fluorescein isothiocyanate (FITC)-conjugated anti-F4/80 (clone BM8), FITC-conjugated anti-CD45R/B220 (clone RA3-6B2), FITC-conjugated CD19 (clone 6D5), Phycoerythrin (PE)-conjugated anti-Ly-6G (clone 1A8), PE-conjugated anti-SiglecH (clone 551), biotin-conjugated anti-NK1.1 (clone PK136), biotin-conjugated anti-BST2/PDCA-1 (clone 927), peridinin chlorophyll protein/Cy5.5 (PerCP/Cy5.5)-conjugated anti-CD45 (clone 30-F11), phycoerythrin-Cy7 (PC7)-conjugated anti-CD3ε (clone 145-2C11) and PC7-conjugated anti-CD11c (clone N418) monoclonal antibodies were purchased from Biolegend (San Diego, CA). FITC-conjugated anti-CD8 (clone KT15) monoclonal antibodies, PE-conjugated H-2D b tetramer coupled with a viral NP-derived ASNENMETM peptide (NP 366-374 : ASNENMETM), H-2D b -restricted influenza NP 366-374 peptide were purchased from MBL (Nagoya, Japan). Syk inhibitor IV (BAY 61-3606) was purchased from Merck Millipore (Billerica, MA). LPS from Escherichia coli 0111:B4 was purchased from Sigma-Aldrich (St. Louis, MO). NaClO-oxidized zymosan was prepared as described 48 . IFV infection in mice. C57BL/6 and Card9 -/mice were anesthetized and infected with 10 4 plaque forming units (PFU) (unless otherwise indicated) of a mouse-adapted IFV (A/PR/8/34 strain: H1N1 isotype, kindly provided by the Kitasato Institute, Tokyo, Japan) by intratracheal administration as described 10-12 . Histology. Whole lungs were collected at 0, 4, and 8 days after IFV infection. Paraffin embedding and hematoxylin and eosin staining of tissues were performed using standard methodologies. . BAL was carried out as described previously 25, 49 . In Brief, tracheas of mice were cannulated with 1.2-mm diameter polyethylene catheters. Lungs were instilled with 1 mL of pre-warmed PBS containing 5 mM EDTA, followed by the retrieval of lavage fluid aliquots. Cells in the BAL fluid (BALF) were counted after red blood cell lysis and subjected to flow cytometric analysis. The supernatants of the BALF were subjected to multi cytokine/chemokine expression analysis and cytokine ELISA. Cytomix Cytokine Bead Assay (Bender MedSystems, Vienna, Austria), with the exception of IFN-β , which was measured by a VeriKine TM Mouse Interferon-β ELISA kit (PBL InterferonSource, Piscataway, NJ). Cell culture supernatants from splenocytes were assayed using a specific ELISA kit for IFN-γ (Biolegend). Cell culture supernatants from TG-MFs, BMMFs, cDCs, and FLT3L-DCs were assayed using specific ELISA kits for IL-6, TNF-α (Biolegend), IFN-α and IFN-β (PBL InterferonSource). All measurements were performed in triplicate. For quantitative PCR (qPCR) analysis for cytokine mRNAs, total RNA was extracted from cells with ReliaPrep RNA Cell Miniprep System (Promega, Madison, WI) and cDNA was synthesized with PrimeScript RT reagent Kit (Takara Bio, Shiga, Japan) according to the manufacturer's instructions. qPCR was performed with the Applied Biosystems 7900HT Fast Real Time PCR System (Life Technologies). Primer sequences were as follows: mouse Il6, forword, 5′-CCA CTT CAC AAG TCG GAG GCT TA -3′, and reverse, 5′-GCA AGT GCA TCA TCG TTG TTC ATA C -3′; and Tnf forword, 5′-GTT CTA TGG CCC AGA CCC TCA C -3′, and reverse, 5′-GGC ACC ACT AGT TGG TTG TCT TTG -3′. Viral copy numbers. Mice were euthanized by intraperitoneal administration of sodium pentobarbital at 0, 4, or 8 days after IFV infection. Lung tissues were homogenized using a gentleMACS dissociator (Miltenyi Biotec) and RNA was extracted with an Isogen II RNA extraction kit (Nippon Gene, Tokyo, Japan). Reverse transcription was conducted with the Uni-12 primer (5′-AGC AAA AGC AGG -3′) 50 and qPCR was preformed with primers specific for NP (Forward: 5′-GAT TGG TGG AAT TGG ACG AT -3′; Reverse: 5′-AGA GCA CCA TTC TCT CTA TT -3′) using the Applied Biosystems 7900HT Fast Real Time PCR System (Life Technologies, Carlsbad, CA). The standard calibration curve for qPCR was obtained by stepwise dilution of the cloned NP gene fragment with a known copy number. IFV rechallenge. WT and Card9 -/mice were left uninfected or infected intratracheally with sublethal dose (10 3 PFU/mouse: 1/10 LD 50 ) of PR8. Twenty-eight days after the first infection, mice were challenged with a high lethal dose (10 6 PFU/mouse: 100 LD 50 ) of PR8. Two days postchallenge, virus titers in the lungs were measured in a plaque assay in MDCK cells. Antigen-specific B cell responses. B cell-mediated humoral responses were measured as virion-specific immunoglobulin production by ELISA, as previously described 32 . Briefly, 96-well ELISA plates (Corning) were coated with ultrasonicated influenza virion (A/PR/8/34 strain) at 5 × 10 6 PFU/ mL in a carbonate buffer (pH 9.6), and incubated overnight at 4 °C. Plates were then washed with PBS containing 0.05% Tween 20 (Wako Pure Chemical Industries). Serum and BALF collected from mice at day 14 after the secondary infection were serially diluted with PBS/Tween 20 containing 5% skim milk, applied onto the virion-coated plates, and incubated for 2 h at room temperature. After washing, goat anti-mouse total IgG or IgA conjugated to horseradish peroxidase (Jackson Immunoresearch, Baltimore Pike, PA) was applied and incubated for 2 h at room temperature. After washing, the plates were stained with a TMB Substrate Set (Biolegend). The reaction was terminated with 1 M H 2 SO 4 (Wako Pure Chemical Industries) and the absorbance was measured. Antigen-specific T cell responses. IFV-specific T cell responses were measured as viral NP-specific IFN-γ secretion by splenocytes, as described previously 32, 51 . Briefly, 5 × 10 5 splenocytes extracted from mice 14-days after the secondary infection were seeded on 96-well cell culture plates (Corning) and then stimulated with 10 μ g/mL of NP 366-374 peptides. After 3 days of culture, supernatants were collected and analyzed for IFN-γ production by ELISA (Biolegend). TG-MFs were prepared as described 36 . AMFs were isolated from BALF of WT or Card9 -/mice. BMMFs, cDCs, or FLT3L-DCs were prepared by culturing bone marrow cells for 5-8 days with RPMI1640 medium (Wako Pure Chemical Industries) supplemented with 10% fetal bovine serum (Life Technologies) and antibiotics (100 IU/mL penicillin and 100 μ g/mL streptomycin; Sigma-Aldrich) containing M-CSF (25 ng/mL, Peprotech, Rocky Hill, NJ), GM-CSF (20 ng/mL, Peprotech) or human Flt3-Ligand (100 ng/mL, Peprotech), respectively. The FLT3L-DCs derived from the culture contain constantly 10-13% of PDCA-1 + SiglecH + plasmacytoid DCs (pDCs) irrespective of deficiency in Syk, Card9, Ips-1 or MyD88. For Syk deletion in vitro, cultured cells derived from Syk flox/flox mice were incubated with 0.6 μ M of the active metabolite of tamoxifen, 4-hydroxytamoxifen (Sigma-Aldrich) for 3 d. For stimulation of MFs/DCs of, 1 × 10 5 cells were seeded on 24-well culture plates (Corning) and incubated overnight, followed by replacement of 200 μ L of serum-free medium containing 10 6 PFU (multiplicity of infection [M.O.I] = 10) of PR8. After 1 h of incubation, unabsorbed viruses were removed and the cells were incubated for a further 24 h in serum-containing medium. DCs were also stimulated with LPS (100 ng/mL) or NaClO-oxidized zymosan (10 μ g/mL) for 24 h in serum-containing medium. The culture supernatants were assayed for IL-6, TNF-α , IFN-α , and IFN-β by ELISA. For stimulation of AMFs, 1 × 10 4 cells were seeded on 96-well culture plate (Corning), stimulated as above with 10 5 PFU (M.O.I = 10) of PR8 for 6 h, and total RNAs were extracted for qPCR analysis for cytokine expression. For treatment of cells with a chemical Syk inhibitor, WT DCs were incubated with 0.25 or 1 μ M BAY 61-3606 for 1 h prior to virus stimulation. Cell viability. Cell viability was determined by the MTS assay using the CellTiter 96 AQueousOne Solution Cell Proliferation Assay kit (Promega). In brief, 20 μ L of MTS solution was added to cells in 96-well plates after in vitro IFV stimulation and the plates were incubated for 2 h at 37 °C, then the absorbance at 490 nm was measured using a Benchmark microplate reader (Bio-Rad, Hercules, CA). Statistical analysis. Survival curves were generated by the Kaplan-Meier method, and statistical analyses were performed using the log-rank test. The statistical significance was assessed by Student's t-tests. 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We also thank Dr. Yumiko Imai and Dr. Keiji Kuba (Akita University) for helpful discussions, and Dr. Shinobu Suzuki (Nippon Boehringer Ingelheim Co., Ltd) and Dr. Shizuo Akira (Osaka University) for kindly providing the . The funders had no role in the study design, data collection or analysis, the decision to publish, or the preparation of the manuscript.