Toll-like receptor pre-stimulation protects mice against lethal infection with highly pathogenic influenza viruses
© Shinya et al; licensee BioMed Central Ltd. 2011
Received: 3 December 2010
Accepted: 4 March 2011
Published: 4 March 2011
Since the beginning of the 20th century, humans have experienced four influenza pandemics, including the devastating 1918 'Spanish influenza'. Moreover, H5N1 highly pathogenic avian influenza (HPAI) viruses are currently spreading worldwide, although they are not yet efficiently transmitted among humans. While the threat of a global pandemic involving a highly pathogenic influenza virus strain looms large, our mechanisms to address such a catastrophe remain limited. Here, we show that pre-stimulation of Toll-like receptors (TLRs) 2 and 4 increased resistance against influenza viruses known to induce high pathogenicity in animal models. Our data emphasize the complexity of the host response against different influenza viruses, and suggest that TLR agonists might be utilized to protect against lethality associated with highly pathogenic influenza virus infection in humans.
During the 20th century, humans experienced three influenza pandemics, each resulting in significant global mortality: 20 to 40 million deaths in 1918 (Spanish influenza), 1 to 4 million deaths in 1957 (Asian influenza), and 1 to 4 million deaths in 1968 (Hong Kong influenza) . In addition, the pandemic (H1N1) 2009 virus has spread rapidly around the world since spring of 2009; and HPAI viruses have been circulating worldwide since late 2003. Although HPAI viruses are not yet efficiently transmitted to or among humans, their sustained proliferation and continued genetic evolution in avian species, combined with parallel infections in humans, makes this an eventual possibility.
Some patients infected with either 2009 pandemic H1N1 or HPAI viruses develop acute respiratory distress syndrome and severe alveolar damage [2–5]. This pathologic condition is associated with a strong upregulation of cytokines and chemokines: in particular, interferon-induced protein 10 (IP-10; CXCL10), monokine induced by interferon gamma (MIG; CXCL9), monocyte chemotactic protein 1 (MCP-1; CCL2), interleukin (IL)-8, IL-10, IL-6, interferon γ (IFN-γ), and tumor necrosis factor α (TNF-α) [6–10]. In the macaque model, infection with the 1918 Spanish influenza virus markedly increased serum levels of IL-6, IL-8, MCP-1, and RANTES (RANTES; CCL5) . Thus, it has been suggested that the severity of influenza is associated with the aberrant induction of innate immunity.
Pre-stimulation of innate immunity has been shown to confer resistance against lethal influenza infection. Specifically, influenza A virus titers decreased in cells pre-treated with TNF-α, and inoculation of mice with bacterial lysates before viral infection protects against lethal influenza pneumonia [12–15]. Moreover, the general stimulation of innate immunity with interferon α, as well as the stimulation of specific Toll-like receptors (TLRs), promotes survival in mouse models of lethal influenza pneumonia [16–19]. However, the ability of innate immunity pre-stimulation to attenuate disease associated with HPAI viruses has not been explored. In the present study, we aimed to determine the protective effects of TLR pre-stimulation in mice inoculated with influenza A viruses.
Materials and methods
Madin-Darby canine kidney (MDCK) cells were maintained in Dulbecco's modified Eagle's medium (DMEM) supplemented with 10% fetal calf serum, and 293T human embryonic kidney cells were maintained in minimal essential medium (MEM) with 5% newborn calf serum. All cells were maintained at 37°C in an atmosphere of 5% CO2.
Influenza A/Puerto Rico/8/34 (PR8; H1N1) and A/Vietnam/1203/04 (VN1203; H5N1) stock viruses were prepared in 10-day-old embryonated chicken eggs or MDCK cells, respectively. After MDCK cells were inoculated with influenza virus, they were grown in MEM containing 0.3% BSA with TPCK-trypsin (0.5 ug/ml) to propagate PR8 or without TPCK-trypsin to propagate VN1203. One reassortant virus was generated from plasmids by reverse genetics, as described previously [20, 21]. The reassortant possessed the hemagglutinin (HA) segment from influenza A/South Carolina/1918 (H1N1) and the remaining seven viral RNA segments from influenza A/WSN/33 (H1N1), and was designated SpHA/WSN. The SpHA/WSN transfectant produced in 293T cells were used to inoculate MDCK cells for stock virus production. Stock virus titers were determined by the median egg infectious dose (EID50) or plaque assay. Experiments using VN1203 or SpHA/WSN were conducted in an enhanced biosafety level 3 (BSL3+) containment laboratory approved for such use by the Centers for Disease Control and Prevention and the United States Department of Agriculture.
Lethal dose studies in mice
BALB/c mice (6-week-old) used in this study were maintained in a specific pathogen-free environment. All manipulations (TLR pre-treatments and virus inoculations) were performed in mice anesthetized with sevoflurane. Pre-treatments with the indicated TLR ligands were carried out by intranasal administration of 100 μl phosphate-buffered saline (PBS) containing these ligands at the indicated times before infection. Control mice were inoculated with PBS only. To determine the mouse lethal dose 50 (MLD50) following pre-stimulation, anesthetized BALB/c mice were intranasally inoculated with 10-fold serial dilutions of virus in 50 μl PBS (each group, n = 3). Mice were monitored daily over 14 days for disease symptoms and survival, and the MLD50 value was calculated according to the method of Reed and Muench . To minimize the number of animals used for these experiments, we performed each MLD50 titration once. Animal care and experimental procedures were approved by the Animal Research Committees at Tohoku University and the University of Wisconsin-Madison.
TLR ligand pre-treatment in mice
For initial MLD50 determination with PR8, we used LPS from Escherichia coli (E. coli) serotype O26:B6 (Sigma, Tokyo; catalog #L8274) at 2.5, 1.25, 0.625, 0.3125, or 0.15625 mg/kg. For subsequent MLD50 experiments comparing the antiviral effects of stimulating different TLRs, we used the following TLR ligands from InvivoGen (San Diego, CA, USA): synthetic mycoplasmal lipoprotein (FSL-1, 50 μg/kg; cat# tlrl-fsl) as the TLR2 ligand; analog of dsRNA (Poly(I:C), 1.25 mg/kg; cat# tlrl-pic) as the TLR3 ligand; LPS from E. coli K12 msbB strain (2.5 mg/kg; cat# tlrl-mklps) and LPS from E. coli serotype O111:B4 (2.5 mg/kg; cat# tlrl-pelps) as TLR4 ligands; and a guanosine analog (loxoribine, 2.5 mg/kg; cat# tlrl-lox) as the TLR7 ligand.
TLR4 prestimulation with lipopolysaccharide protects mice against lethal influenza virus challenge
Antiviral effect of intranasal prestimulation by lipopolysaccharide against influenza A/Puerto Rico/8/34.
Ratio of MLD50(log10EID50) between LPS-pretreated and mock-treated miceb
Protective effects of pre-stimulation of various TLRs
Antiviral effect of prestimulation with Toll-like receptor specific ligands against influenza A/Puerto Rico/8/34.
Ratio of MLD50(log10EID50) between LPS-pretreated and mock-treated mice
Synthetic mycoplasmal lipoprotein (FSL-1)
Synthetic analog of dsRNA (Poly(I:C))
LPS from E. coli K12 msbB
LPS from E. coli serotype O111:B4
Guanosine analog (loxoribine)
Differential effects of TLR prestimulation on lethal pneumonia associated with highly pathogenic influenza viruses in mice
Antiviral effect of prestimulation by Toll-like receptor-specific ligands.
Ratio of MLD50(log10EID50) between LPS-pretreated and mock-treated mice
Sp HA/WSN c
Sp HA/WSN c
We observed a difference in the ability of TLR2 and TLR4 pre-stimulation to protect against highly pathogenic viruses. Consistent with protection against PR8 in LPS-pretreated mice, TLR4 stimulation with msbB LPS (weak agonist) protected against lethality induced by VN1203 (10-fold increase in MLD50 compared to untreated control). In contrast, TLR2 stimulation with FSL-1 did not protect against VN1203 (Table 3). However, we observed the opposite pattern with SpHA/WSN: TLR2 pre-stimulation conferred better protection against lethality compared to TLR4 pre-stimulation. These results suggest that the best method of stimulating innate immunity depends on the particular virus in question.
Our results indicate that TLR pre-stimulation protects mice from lethal challenge with highly pathogenic influenza viruses. Although previous reports have suggested the efficacy of TLR pre-stimulation in promoting survival after influenza challenge[15, 17], we provide the first evidence that TLR activation is effective against HPAI and other highly pathogenic influenza strains.
Inactivated H5N1 virus has been shown to induce acute lung injury through the TLR4-TRIF pathway , suggesting that viral surface glycoproteins HA and/or neuraminidase (NA), may be sensed by TLR4 on the cell surface. TLR4 responds to molecular signatures of microbial origin (i.e., LPS), but more recent evidence suggests that TLR4 can also be activated by viral infection [27–29]. Thus, robust replication and expression of viral antigens may induce hyperactivation of the TLR4 signaling pathway and lead to lung injury; the H5N1 virus is not likely to be an exception. Our data showed that TLR4 pre-stimulation could also offer protection against lethal HPAI infections. It is interesting that a molecule associated with influenza severity can also protect against influenza-induced lethality when given prophylactically.
While pre-stimulation of both TLR2 and TLR4 signaling pathways protected against the PR8 virus, protection against the VN1203 virus could only be achieved by TLR4 pre-stimulation. Contrastingly, a virus carrying the 1918 Spanish influenza HA molecule was not inhibited by a TLR4 agonist, but rather was more affected by pre-stimulation of TLR2. TLR2 and TLR4 induce overlapping and unique signaling associated with innate immunity, and these pathways may be differentially required for protection against different influenza viruses. We suggest that TLR4-mediated signaling may have a principle role in protection against HPAI viruses, while control of Spanish influenza may involve TLR2-related mechanisms. Our data highlights the complex nature of innate signaling in response to infection with different influenza virus strains, and emphasizes the need for further dissection of the pathways that are involved in controlling influenza infection and promoting influenza pathogenesis. Further, we suggest that this information could be utilized in the development of countermeasures against highly pathogenic influenza virus infections in humans.
This work was supported by Precursory Research for Embryonic Science and Technology (PRESTO), ERATO, and Global Center of Excellence (G-COE) for Education and Research on Signal Transduction (Japan Science and Technology Agency); by a grant-in-aid for Specially Promoted Research; by a contract research fund for the Program of Founding Research Centers for Emerging and Reemerging Infectious Diseases from the Ministries of Education, Culture, Sports, Science, and Technology; by grants-in-aid from the Ministry of Health, Labor, and Welfare of Japan; and by National Institute of Allergy and Infectious Disease Public Health Service research grants.
- Wright P, Neumann G, Kawaoka Y: Orthomyxoviruses. In Fields virology. Edited by: Knipe D, Howley P, Griffin D, Martin M, Lamb R. Philadelphia: Lippincott Williams & Wilkins; 2009:1691-1740.Google Scholar
- Bauer TT, Ewig S, Rodloff AC, Muller EE: Acute respiratory distress syndrome and pneumonia: a comprehensive review of clinical data. Clin Infect Dis 2006, 43: 748-756. 10.1086/506430View ArticlePubMedGoogle Scholar
- Venkata C, Sampathkumar P, Afessa B: Hospitalized patients with 2009 H1N1 influenza infection: the Mayo Clinic experience. Mayo Clin Proc 2010, 85: 798-805. 10.4065/mcp.2010.0166PubMed CentralView ArticlePubMedGoogle Scholar
- Domínguez-Cherit G, Lapinsky SE, Macias AE, Pinto R, Espinosa-Perez L, de la Torre A, Poblano-Morales M, Baltazar-Torres JA, Bautista E, Martinez A, Martinez MA, Rivero E, Valdez R, Ruiz-Palacios G, Hernández M, Stewart TE, Fowler RA: Critically Ill patients with 2009 influenza A(H1N1) in Mexico. JAMA 2009, 302: 1880-1887.View ArticlePubMedGoogle Scholar
- Perez-Padilla R, de la Rosa-Zamboni D, Ponce de Leon S, Hernandez M, Quiñones-Falconi F, Bautista E, Ramirez-Venegas A, Rojas-Serrano J, Ormsby CE, Corrales A, Higuera A, Mondragon E, Cordova-Villalobos JA, INER Working Group on Influenza: Pneumonia and respiratory failure from swine-origin influenza A (H1N1) in Mexico. N Engl J Med 2009, 361: 680-689. 10.1056/NEJMoa0904252View ArticlePubMedGoogle Scholar
- Yuen KY, Chan PK, Peiris M, Tsang DN, Que TL, Shortridge KF, Cheung PT, To WK, Ho ET, Sung R, Cheng AF: Clinical features and rapid viral diagnosis of human disease associated with avian influenza A H5N1 virus. Lancet 1998, 351: 467-471. 10.1016/S0140-6736(98)01182-9View ArticlePubMedGoogle Scholar
- To KF, Chan PK, Chan KF, Lee WK, Lam WY, Wong KF, Tang NL, Tsang DN, Sung RY, Buckley TA, Tam JS, Cheng AF: Pathology of fatal human infection associated with avian influenza A H5N1 virus. J Med Virol 2001, 63: 242-246. 10.1002/1096-9071(200103)63:3<242::AID-JMV1007>3.0.CO;2-NView ArticlePubMedGoogle Scholar
- Fouchier RA, Schneeberger PM, Rozendaal FW, Broekman JM, Kemink SA, Munster V, Kuiken T, Rimmelzwaan GF, Schutten M, Van Doornum GJ, Koch G, Bosman A, Koopmans M, Osterhaus AD: Avian influenza A virus (H7N7) associated with human conjunctivitis and a fatal case of acute respiratory distress syndrome. Proc Natl Acad Sci USA 2004, 101: 1356-1361. 10.1073/pnas.0308352100PubMed CentralView ArticlePubMedGoogle Scholar
- Peiris JS, Yu WC, Leung CW, Cheung CY, Ng WF, Nicholls JM, Ng TK, Chan KH, Lai ST, Lim WL, Yuen KY, Guan Y: Re-emergence of fatal human influenza A subtype H5N1 disease. Lancet 2004, 363: 617-619. 10.1016/S0140-6736(04)15595-5View ArticlePubMedGoogle Scholar
- de Jong MD, Simmons CP, Thanh TT, Hien VM, Smith GJ, Chau TN, Hoang DM, Chau NV, Khanh TH, Dong VC, Qui PT, Cam BV, Ha dQ, Guan Y, Peiris JS, Chinh NT, Hien TT, Farrar J: Fatal outcome of human influenza A (H5N1) is associated with high viral load and hypercytokinemia. Nat Med 2006, 12: 1203-1207. 10.1038/nm1477PubMed CentralView ArticlePubMedGoogle Scholar
- Kobasa D, Jones SM, Shinya K, Kash JC, Copps J, Ebihara H, Hatta Y, Kim JH, Halfmann P, Hatta M, Feldmann F, Alimonti JB, Fernando L, Li Y, Katze MG, Feldmann H, Kawaoka Y: Aberrant innate immune response in lethal infection of macaques with the 1918 influenza virus. Nature 2007, 445: 319-323. 10.1038/nature05495View ArticlePubMedGoogle Scholar
- Van Campen H: Influenza A virus replication is inhibited by tumor necrosis factor-alpha in vitro. Arch Virol 1994, 136: 439-446. 10.1007/BF01321073View ArticlePubMedGoogle Scholar
- Obi N, Hayashi K, Miyahara T, Shimada Y, Terasawa K, Watanabe M, Takeyama M, Obi R, Ochiai H: Inhibitory Effect of TNF-alpha Produced by Macrophages Stimulated with Grifola frondosa Extract (ME) on the Growth of Influenza A/Aichi/2/68 Virus in MDCK Cells. Am J Chin Med 2008, 36: 1171-1183. 10.1142/S0192415X08006508View ArticlePubMedGoogle Scholar
- Sadler AJ, Williams BR: Interferon-inducible antiviral effectors. Nat Rev Immunol 2008, 8: 559-568. 10.1038/nri2314PubMed CentralView ArticlePubMedGoogle Scholar
- Tuvim MJ, Evans SE, Clement CG, Dickey BF, Gilbert BE: Augmented lung inflammation protects against influenza A pneumonia. PLoS One 2009, 4: e4176. 10.1371/journal.pone.0004176PubMed CentralView ArticlePubMedGoogle Scholar
- Abe T, Takahashi H, Hamazaki H, Miyano-Kurosaki N, Matsuura Y, Takaku H: Baculovirus induces an innate immune response and confers protection from lethal influenza virus infection in mice. J Immunol 2003, 171: 1133-1139.View ArticlePubMedGoogle Scholar
- Cluff CW, Baldridge JR, Stover AG, Evans JT, Johnson DA, Lacy MJ, Clawson VG, Yorgensen VM, Johnson CL, Livesay MT, Hershberg RM, Persing DH: Synthetic toll-like receptor 4 agonists stimulate innate resistance to infectious challenge. Infect Immun 2005, 73: 3044-3052. 10.1128/IAI.73.5.3044-3052.2005PubMed CentralView ArticlePubMedGoogle Scholar
- Seo SU, Lee KH, Byun YH, Kweon MN, Seong BL: Immediate and broad-spectrum protection against heterologous and heterotypic lethal challenge in mice by live influenza vaccine. Vaccine 2007, 25: 8067-8076. 10.1016/j.vaccine.2007.09.012View ArticlePubMedGoogle Scholar
- Wong JP, Christopher ME, Viswanathan S, Dai X, Salazar AM, Sun LQ, Wang M: Antiviral role of toll-like receptor-3 agonists against seasonal and avian influenza viruses. Curr Pharm Des 2009, 15: 1269-1274. 10.2174/138161209787846775View ArticlePubMedGoogle Scholar
- Kobasa D, Takada A, Shinya K, Hatta M, Halfmann P, Theriault S, Suzuki H, Nishimura H, Mitamura K, Sugaya N, Usui T, Murata T, Maeda Y, Watanabe S, Suresh M, Suzuki T, Suzuki Y, Feldmann H, Kawaoka Y: Enhanced virulence of influenza A viruses with the haemagglutinin of the 1918 pandemic virus. Nature 2004, 431: 703-707. 10.1038/nature02951View ArticlePubMedGoogle Scholar
- Neumann G, Watanabe T, Ito H, Watanabe S, Goto H, Gao P, Hughes M, Perez DR, Donis R, Hoffmann E, Hobom G, Kawaoka Y: Generation of influenza A viruses entirely from cloned cDNAs. Proc Natl Acad Sci USA 1999, 96: 9345-9350. 10.1073/pnas.96.16.9345PubMed CentralView ArticlePubMedGoogle Scholar
- Reed LJ, Muench H: A simple method of estimating fifty percent endpoints. Am J Hygiene 1938, 27: 493-497.Google Scholar
- Wong JP, Christopher ME, Salazar AM, Dale RM, Sun LQ, Wang M: Nucleic acid-based antiviral drugs against seasonal and avian influenza viruses. Vaccine 2007, 25: 3175-3178. 10.1016/j.vaccine.2007.01.051View ArticlePubMedGoogle Scholar
- Armstrong L, Medford AR, Uppington KM, Robertson J, Witherden IR, Tetley TD, Millar AB: Expression of functional toll-like receptor-2 and -4 on alveolar epithelial cells. Am J Respir Cell Mol Biol 2004, 31: 241-245. 10.1165/rcmb.2004-0078OCView ArticlePubMedGoogle Scholar
- Hatta M, Hatta Y, Kim JH, Watanabe S, Shinya K, Nguyen T, Lien PS, Le QM, Kawaoka Y: Growth of H5N1 influenza A viruses in the upper respiratory tracts of mice. PLoS Pathog 2007, 3: 1374-1379. 10.1371/journal.ppat.0030133PubMedGoogle Scholar
- Imai Y, Kuba K, Neely GG, Yaghubian-Malhami R, Perkmann T, van LG, Ermolaeva M, Veldhuizen R, Leung YH, Wang H, Liu H, Sun Y, Pasparakis M, Kopf M, Mech C, Bavari S, Peiris JS, Slutsky AS, Akira S, Hultqvist M, Holmdahl R, Nicholls J, Jiang C, Binder CJ, Penninger JM: Identification of oxidative stress and Toll-like receptor 4 signaling as a key pathway of acute lung injury. Cell 2008, 133: 235-249. 10.1016/j.cell.2008.02.043View ArticlePubMedGoogle Scholar
- Kurt-Jones EA, Popova L, Kwinn L, Haynes LM, Jones LP, Tripp RA, Walsh EE, Freeman MW, Golenbock DT, Anderson LJ, Finberg RW: Pattern recognition receptors TLR4 and CD14 mediate response to respiratory syncytial virus. Nat Immunol 2000, 1: 398-401. 10.1038/80833View ArticlePubMedGoogle Scholar
- Haynes LM, Moore DD, Kurt-Jones EA, Finberg RW, Anderson LJ, Tripp RA: Involvement of toll-like receptor 4 in innate immunity to respiratory syncytial virus. J Virol 2001, 75: 10730-10737. 10.1128/JVI.75.22.10730-10737.2001PubMed CentralView ArticlePubMedGoogle Scholar
- Haeberle HA, Takizawa R, Casola A, Brasier AR, Dieterich HJ, Van RN, Gatalica Z, Garofalo RP: Respiratory syncytial virus-induced activation of nuclear factor-kappaB in the lung involves alveolar macrophages and toll-like receptor 4-dependent pathways. J Infect Dis 2002, 186: 1199-1206. 10.1086/344644View ArticlePubMedGoogle Scholar
This article is published under license to BioMed Central Ltd. This is an Open Access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/2.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.