CHARACTERISATION OF LUNG DENDRITIC CELL FUNCTION IN A MOUSE MODEL OF INFLUENZA HO WEI SHIONG ADRIAN B.Sc (Hons), NUS A THESIS SUBMITTED FOR THE DEGREE OF DOCTOR OF PHILOSOPHY NUS GRADUATE SCHOOL FOR INTEGRATIVE SCIENCES AND ENGINEERING NATIONAL UNIVERSITY OF SINGAPORE 2011 Acknowledgements Firstly, a special word of thanks to Prof. Kemeny for being a fantastic supervisor, more than one could ask for. Thank you for your guidance, patience and the countless hours of consultation, especially those you willingly agreed to hold on Saturdays. You’ve taught me more than just good science – you’ve taught me how to be a good scientist (and a good fly-fisherman as well). To the influenza group – Moyar, Nayana and Richard, you all have been terrific teammates. A special word of thanks to Nayana – you’ve been a tremendous help and experiments couldn’t have gone as fast without your assistance! Thank you for lending an extra pair of hands ever so often and being a great coffee-buddy too. To Richard, you’ve been a real pal and it’s been great working with you. Thanks for being my impromptu statistics tutor and helping me hone the skill of scientific writing. ᡁᐼᵋᛘⲴॾ䈝≤ᒣнѵਾՊ∄ᡁⲴᴤྭDŽTo Moyar, it’s been great working with you ever since your honours year and I wish all success for your PhD. To the other Kemeny lab members which are just too numerous to name here, you people make all the difference to lab life. The past years would not have been as exciting and vibrant without the constant exchange of jokes, jibes, and of course, scientific ideas. There’s never a dull moment in lab with you guys. You all have been great colleagues and great friends too, and I’ll certainly miss you all. To Benson, without whom the mice colonies will fall into disarray, you’ve been instrumental to the lab’s success and thank you for looking after the mice and providing world-class animal husbandry support. To the staff of ‘the best flow cytometry unit in south-east asia’, Fei Chuin and Paul Hutchinson, no one else does cell sorting better than you guys. Thank you for being so accommodating with the sort schedules and for teaching me the fine details of flow cytometry. IP is indeed very privileged to have such people like you and I’ve benefited a great deal learning from you both. I also wish to express gratitude to my family members for their invaluable support. To my parents, thank you for your support and having the confidence in me to embark on my PhD studies. To my uncle Ku Ku D, thank you for agreeing to be the guarantor for my scholarship application. To my dear wife, you’ve really lived up to your calling to be a helpmeet! Words are truly inadequate to thank you for being a pillar of strength at home - looking after the house, the kids, and being an emotional support for me, especially when I’m downcast and experiments fail. Finally, I thank my Lord for giving me the strength to complete the long and arduous journey of working towards a PhD. Summary The uptake, transport and presentation of antigens by lung dendritic cells (DCs) is central to the initiation of CD8 T-cell responses against respiratory viruses. Although several studies have demonstrated a critical role of CD11blo/negCD103+ DCs for the initiation of cytotoxic T-cell responses against the influenza virus, the underlying mechanisms for its potent ability to prime CD8 T-cells remain poorly understood. Using a novel approach of fluorescent lipophilic dye-labelled influenza virus, we demonstrate that CD11blo/negCD103+ DCs are the dominant lung DC population transporting influenza virus to the posterior mediastinal lymph node as early as 20 hours after infection. By contrast, CD11bhiCD103neg DCs although more efficient for taking up the virus within the lung, migrate poorly to the lymph node and remain in the lung to produce pro-inflammatory cytokines instead. CD11blo/negCD103+ DCs efficiently load viral peptide onto MHC-I complexes and therefore uniquely possess the capacity to potently induce proliferation of naïve CD8 T-cells. In addition, the peptide transporter TAP1 and TAP2 is constitutively expressed at higher levels in CD11blo/negCD103+ DCs, providing first evidence of a distinct regulation of the antigen-processing pathway in these cells. Collectively, these results show that CD11blo/negCD103+ DCs are functionally specialised for the transport of antigen from the lung to the lymph node and also for efficient processing and presentation of viral antigens to CD8 T-cells. Table of Contents Chapter 1: Introduction 1.1 Influenza virus 1.1.1 The Health Threat of Influenza . 1.1.2 Clinical Symptoms of Infection and Pathology 1.1.3 Genetics and Replication of the Influenza A Virus . 1.1.4 Influenza Tropism . 10 1.2 Host Innate Immune Sensors of Influenza Virus A 11 1.2.1 TLR-mediated detection of the Influenza Virus 11 1.2.2 NLR-mediated detection of the Influenza Virus . 13 1.2.3 RLR-mediated detection of the Influenza Virus . 15 1.3 Viral evasion of immune dectection . 16 1.4 Innate Immune Responses to the influenza virus . 17 1.4.1 Mucus Secretions and Epithelial Layer 18 1.4.2 Type I Interferons . 18 1.4.3 Phagocytes 20 1.5 Adaptive Immune Responses to the influenza virus . 21 1.5.1 Humoral Immunity 21 1.5.2 CD4 T-cell response to Influenza . 22 1.5.3 CD8 T-cell response to Influenza . 24 1.6 Dendritic Cells 26 1.6.1 Origin and Function of DCs 26 1.6.2 Heterogeneity of DCs . 28 1.7 Lung Dendritic Cells . 29 1.7.1 Lung Dendritic Cell Subsets and Origin . 29 1.7.2 Lung Dendritic Cells and Tolerance . 30 1.7.3 Lung Dendritic Cells and Influenza 33 1.8 Aims Of This Study 35 Chapter 2: Materials and Methods 36 2.1 Media and buffers . 36 i 2.2 List of Antibodies Used 41 2.3 Cell Isolation . 42 2.4 Preparation of Influenza Virus 45 2.5 Flow Cytometry and Cell Sorting . 51 2.6 Culture of Dendritic cells and T-cells . 55 2.7 Reverse transcription of mRNA, RT-PCR and primers 56 2.8 Fluorescent Microscopy 58 2.9 Haematoxylin and Eosin Staining . 59 2.10 Mice 60 2.11 Genotyping of Clone Mice . 61 Chapter 3: Mouse Model of Influenza Infection and Characterisation of Lung Antigen Presenting Cells . 63 3.1 Introduction . 63 3.2 Weight Loss and Bronchoalveolar Lavage . 66 3.3 Histopathology of the lung 69 3.4 Virus specific CD8 T-cell response 76 3.5 Antibody Response . 79 3.6 Surface Markers of Cells Isolated from the Alveolar Compartment 81 3.7 Surface markers of cells isolated from the Lung Parenchyma 83 3.8 Maturation status of lung dendritic cells . 88 3.9 Change in antigen presenting cell populations after influenza infection 91 3.10 Discussion 95 Chapter 4: Lipophillic Dye-Labelling of Influenza virus . 99 4.1 Introduction . 99 4.2 DiD labelling does not compromise influenza virus infectivity . 102 4.3 Comparative analysis of DiD-influenza acquisition in the lung parenchyma 106 4.4 Comparative analysis of DiD virus acquisition by lung dendritic cells 109 4.5 Comparative analysis of lung dendritic cells to endocytose the influenza virus 113 4.6 Comparative analysis of proinflammatory cytokine production by lung dendritic cells 116 4.7 Lung DCs have different capacities to migrate to the draining lymph nodes 118 ii 4.8 Antigen presentation by Lung DCs occurs in the Posterior Mediastinal Lymph Node . 121 4.9 Detection of non-replicating virus uptake using DiD-influenza 128 4.10 Poor acquisition of UV-irradiated virus by dendritic cells in the lung and subsequent poor CD8 T-cell priming . 130 4.11 Discussion . 134 Chapter 5: Antigen Presentation Capacities of Lung DC Populations . 138 5.1 Introduction . 138 5.2 Only CD103+CD11blo/neg DCs have the capacity to potently prime naive CD8 Tcells ex vivo 140 5.3 Both CD11blo/neg and CD11bhi DCs have the capacity to prime naïve CD4 T-cells 145 5.4 Infection of DCs by the influenza virus 147 5.5 Analysis of MHC-I and co-stimulatory molecule expression on lung DCs . 149 5.6 Equivalent capacity of peptide pulsed lung DC populations to prime CD8 T-cells 151 5.7 CD11blo/neg lung DCs efficiently load viral peptide onto MHC-I complexes . 153 5.8 CD11blo/neg DCs have higher mRNA transcript levels of TAP1 and TAP2 156 5.9 CD11blo/neg DCs have higher protein expression of TAP1 and TAP2 160 5.10 Discussion 164 Chapter 6: Final Discussion and Future Direction . 170 6.1 Brief Summary of Main Findings . 170 6.2 Limitations of Study . 171 6.3 The need to identify lung DC subsets in humans . 172 6.4 CD8 T-cell influenza vaccination strategy 174 6.5 Targeting antigen to DC in situ as an efficient method to stimulate host CD8 Tcell responses . 178 6.6 Future Direction 180 References . 182 iii List of Figures Figure 1.1 Schematic diagram of the influenza A virus . Figure 2.11.1 Screening of CD8 T cells from offspring from hemizygous clone transgenic mice using anti-Vbeta 8.2 TCR antibody 62 Figure 3.2.1 Percentage weight change of mice over the course of infection with PFU influenza virus. . 67 Figure 3.2.2 Levels of proinflammatory cytokines in the bronchoalveolar lavage fluid . 68 Figure 3.3.1 H&E staining of transverse section of large conducting airways in uninfected mice. 71 Figure 3.3.2 H&E staining of transverse section of large conducting airways in day p.i. mice. 72 Figure 3.3.3 H&E staining of transverse section of large conducting airways in day p.i. mice. 73 Figure 3.3.4 H&E staining of transverse section of large conducting airways in day p.i. mice. 74 Figure 3.3.5 H&E staining of transverse section of large conducting airways in day 10 p.i. mice. 75 Figure 3.4.1 Detection of virus specific CD8 T-cells using ASNENMETM tetramer after influenza infection 77 Figure 3.4.2 Total CD8 T-cells and virus-specific CD8 T-cells in the lung and BAL after influenza infection 78 Figure 3.5 Serum neutralising antibody titre 80 Figure 3.6 Surface markers and morphology of alveolar macrophages . 82 Figure 3.7.1 Enrichment of lung APCs from whole lung digest using OPTIPREP . 84 Figure 3.7.2 Surface markers of lung antigen presenting cells from the lung parenchyma . 85 Figure 3.7.3 Lung DCs not express CD8Į and CD4 . 86 iv Figure 3.7.4 Lung DCs and macrophages can be additionally distinguished by side scatter and autofluorescence . 87 Figure 3.8.1 MHC Class I and Class II expression on lymph node and lung DCs . 89 Figure 3.8.2 Lung and Lymph Node DC endocytosis of FITC Dextran 90 Figure 3.9.1 Change in DC and macrophage cell numbers in the lung after infection with influenza virus . 92 Figure 3.9.2 Analysis of co-stimulatory molecules expression on lung parenchyma CD11bhi and CD11blo/neg DCs by FACS at various time points of influenza infection. . 93 Figure 3.9.3 Analysis of co-stimulatory molecules on CD11c+ MHCIIhi DCs in the mediastinal lymph nodes at various time points of influenza infection. . 94 Figure 4.1.1 Fluorescence spectra and chemical structure of DiD . 101 Figure 4.2.1 DiD labelling does not compromise influenza virus infectivity . 103 Figure 4.2.2 DiD influenza is infectious in vivo . 104 Figure 4.2.3 Visualisation of influenza infection in mouse lungs using DiD . 105 Figure 4.3.1 Kinetics of DiD uptake by leukocyte populations in the lung after infection 107 Figure 4.3.2 Co-stimulatory molecule expression on lung DCs . 108 Figure 4.4.1 CD11bhi DCs have enhanced accumulation of DiD in vivo . 110 Figure 4.4.2 Lung DC ex vivo DiD-influenza uptake assay . 111 Figure 4.4.3 Lung DC in vivo DiD-influenza uptake assay 112 Figure 4.5.1 Surface levels of Į2-6 sialic acid receptors on the surface of lung DCs . 114 Figure 4.5.2 Relative capacities of lung DCs to endocytose of FITC Dextran . 115 Figure 4.6.1 CD11bhi DCs are potent producers of TNF-alpha 117 Figure 4.7.1 Surface expression of CCR7 on DCs subsets in the lung parenchyma 119 v Figure 4.7.2 Proportion of DC subsets that comprise total DiD+ DCs in the lymph node . 120 Figure 4.8.1 Photographs showing anatomical location of the anterior mediastinal (aMLN) and posterior (pMLN) lymph nodes within the thoracic cavity . 123 Figure 4.8.2 Kinetics of DiD+ DC accumulation in the pMLN and aMLN over time . 124 Figure 4.8.3 CD8 T-cell priming occurs in the pMLN and not the aMLN in BALBc mice . 125 Figure 4.8.4 CD8 T-cell priming occurs in the pMLN and not the aMLN in C57BL6 mice . 126 Figure 4.8.5 DiD label in lymph node DCs is due to migration of DCs and not leakage of the virus into lymphatics . 127 Figure 4.9.1 DiD-influenza is able to detect the uptake of non-replicating virus . 129 Figure 4.10.1 Comparison of the relative uptake of DiD by phagocytic cells after inoculation with either UV irradiated or non-irradiated DiD-influenza . 132 Figure 4.10.2 Poor proliferation of CD8 T-cells in pMLN of mice inoculated with UV-irradiated influenza virus . 133 Figure 5.2.1 Only CD11blo/neg DCs from have the ability to potently stimulate naïve CD8 T-cell proliferation . 142 Figure 5.2.2 Poor antigen presenting capacity of lung CD11bhi DCs . 143 Figure 5.2.3 CD11bhi DCs from pMLN of infected mice contain a small amount of CD8Į+ DCs which can induce proliferation of naïve CD8 T-cells . 144 Figure 5.3.1 Both CD11blo/neg and CD11bhi DCs have the capacity to prime naïve CD4 T-cells . 146 Figure 5.4.1 Rate of infection of CD11blo/neg and CD11bhi DCs in the lung and pMLN . 148 Figure 5.5.1 Expression of MHC I molecules on CD11bhi and CD11blo/neg DCs in the lung and pMLN . 150 Figure 5.6.1 Peptide pulsed CD11bhi DCs and CD11blo/neg DCs induce similar activation of CD8 T-cells 152 vi References CD8 dendritic cells within antibodies to Langerin, DEC205, and Clec9A." Proc Natl Acad Sci U S A 108(6): 2384-9. Ingulli, E., C. Funatake, E. L. Jacovetty and M. Zanetti (2009). "Cutting edge: antigen presentation to CD8 T cells after influenza A virus infection." J Immunol 182(1): 2933. Ito, R., Y. A. Ozaki, T. Yoshikawa, H. Hasegawa, Y. Sato, Y. Suzuki, R. Inoue, T. Morishima, N. Kondo, T. Sata, T. Kurata and S. Tamura (2003). "Roles of antihemagglutinin IgA and IgG antibodies in different sites of the respiratory tract of vaccinated mice in preventing lethal influenza pneumonia." Vaccine 21(19-20): 236271. Jakubzick, C., M. Bogunovic, A. J. Bonito, E. L. Kuan, M. Merad and G. J. Randolph (2008). "Lymph-migrating, tissue-derived dendritic cells are minor constituents within steady-state lymph nodes." J Exp Med 205(12): 2839-50. Jakubzick, C., J. Helft, T. J. Kaplan and G. J. Randolph (2008). "Optimization of methods to study pulmonary dendritic cell migration reveals distinct capacities of DC subsets to acquire soluble versus particulate antigen." J Immunol Methods 337(2): 121-31. Jakubzick, C., F. Tacke, F. Ginhoux, A. J. Wagers, N. van Rooijen, M. Mack, M. Merad and G. J. Randolph (2008). "Blood monocyte subsets differentially give rise to CD103+ and CD103- pulmonary dendritic cell populations." J Immunol 180(5): 3019-27. Jenkins, M. R., R. Webby, P. C. Doherty and S. J. Turner (2006). "Addition of a prominent epitope affects influenza A virus-specific CD8+ T cell immunodominance hierarchies when antigen is limiting." J Immunol 177(5): 2917-25. Jongbloed, S. L., A. J. Kassianos, K. J. McDonald, G. J. Clark, X. Ju, C. E. Angel, C. J. Chen, P. R. Dunbar, R. B. Wadley, V. Jeet, A. J. Vulink, D. N. Hart and K. J. Radford (2010). "Human CD141+ (BDCA-3)+ dendritic cells (DCs) represent a unique myeloid DC subset that cross-presents necrotic cell antigens." J Exp Med 207(6): 1247-60. 193 References Jurk, M., F. Heil, J. Vollmer, C. Schetter, A. M. Krieg, H. Wagner, G. Lipford and S. Bauer (2002). "Human TLR7 or TLR8 independently confer responsiveness to the antiviral compound R-848." Nat Immunol 3(6): 499. Kato, H., O. Takeuchi, S. Sato, M. Yoneyama, M. Yamamoto, K. Matsui, S. Uematsu, A. Jung, T. Kawai, K. J. Ishii, O. Yamaguchi, K. Otsu, T. Tsujimura, C. S. Koh, C. Reis e Sousa, Y. Matsuura, T. Fujita and S. Akira (2006). "Differential roles of MDA5 and RIG-I helicases in the recognition of RNA viruses." Nature 441(7089): 101-5. Kawai, T. and S. Akira (2010). "The role of pattern-recognition receptors in innate immunity: update on Toll-like receptors." Nat Immunol 11(5): 373-84. Kawai, T., K. Takahashi, S. Sato, C. Coban, H. Kumar, H. Kato, K. J. Ishii, O. Takeuchi and S. Akira (2005). "IPS-1, an adaptor triggering RIG-I- and Mda5mediated type I interferon induction." Nat Immunol 6(10): 981-8. Kees, U. and P. H. Krammer (1984). "Most influenza A virus-specific memory cytotoxic T lymphocytes react with antigenic epitopes associated with internal virus determinants." J Exp Med 159(2): 365-77. Kellermann, S. A., S. Hudak, E. R. Oldham, Y. J. Liu and L. M. McEvoy (1999). "The CC chemokine receptor-7 ligands 6Ckine and macrophage inflammatory protein-3 beta are potent chemoattractants for in vitro- and in vivo-derived dendritic cells." J Immunol 162(7): 3859-64. Kemler, I., G. Whittaker and A. Helenius (1994). "Nuclear import of microinjected influenza virus ribonucleoproteins." Virology 202(2): 1028-33. Kim, T. S. and T. J. Braciale (2009). "Respiratory dendritic cell subsets differ in their capacity to support the induction of virus-specific cytotoxic CD8+ T cell responses." PLoS One 4(1): e4204. Kittel, C., S. Sereinig, B. Ferko, J. Stasakova, J. Romanova, A. Wolkerstorfer, H. Katinger and A. Egorov (2004). "Rescue of influenza virus expressing GFP from the NS1 reading frame." Virology 324(1): 67-73. 194 References Kleijmeer, M. J., A. Kelly, H. J. Geuze, J. W. Slot, A. Townsend and J. Trowsdale (1992). "Location of MHC-encoded transporters in the endoplasmic reticulum and cis-Golgi." Nature 357(6376): 342-4. Klenk, H. D., R. Rott, M. Orlich and J. Blodorn (1975). "Activation of influenza A viruses by trypsin treatment." Virology 68(2): 426-39. Komar, N. and B. Olsen (2008). "Avian influenza virus (H5N1) mortality surveillance." Emerg Infect Dis 14(7): 1176-8. Kris, R. M., R. Asofsky, C. B. Evans and P. A. Small, Jr. (1985). "Protection and recovery in influenza virus-infected mice immunosuppressed with anti-IgM." J Immunol 134(2): 1230-5. La Gruta, N. L., K. Kedzierska, J. Stambas and P. C. Doherty (2007). "A question of self-preservation: immunopathology in influenza virus infection." Immunol Cell Biol 85(2): 85-92. Lakadamyali, M., M. J. Rust, H. P. Babcock and X. Zhuang (2003). "Visualizing infection of individual influenza viruses." Proc Natl Acad Sci U S A 100(16): 9280-5. Lamb, R. A., S. L. Zebedee and C. D. Richardson (1985). "Influenza virus M2 protein is an integral membrane protein expressed on the infected-cell surface." Cell 40(3): 627-33. Lambrecht, B. N., M. De Veerman, A. J. Coyle, J. C. Gutierrez-Ramos, K. Thielemans and R. A. Pauwels (2000). "Myeloid dendritic cells induce Th2 responses to inhaled antigen, leading to eosinophilic airway inflammation." J Clin Invest 106(4): 551-9. Lambrecht, B. N. and H. Hammad (2010). "The role of dendritic and epithelial cells as master regulators of allergic airway inflammation." Lancet 376(9743): 835-43. Lawrence, C. W., R. M. Ream and T. J. Braciale (2005). "Frequency, specificity, and sites of expansion of CD8+ T cells during primary pulmonary influenza virus infection." J Immunol 174(9): 5332-40. 195 References Lay, M., B. Callejo, S. Chang, D. K. Hong, D. B. Lewis, T. D. Carroll, S. Matzinger, L. Fritts, C. J. Miller, J. F. Warner, L. Liang and J. Fairman (2009). "Cationic lipid/DNA complexes (JVRS-100) combined with influenza vaccine (Fluzone) increases antibody response, cellular immunity, and antigenically drifted protection." Vaccine 27(29): 3811-20. Le Goffic, R., V. Balloy, M. Lagranderie, L. Alexopoulou, N. Escriou, R. Flavell, M. Chignard and M. Si-Tahar (2006). "Detrimental contribution of the Toll-like receptor (TLR)3 to influenza A virus-induced acute pneumonia." PLoS Pathog 2(6): e53. Lee, L. Y., L. A. Ha do, C. Simmons, M. D. de Jong, N. V. Chau, R. Schumacher, Y. C. Peng, A. J. McMichael, J. J. Farrar, G. L. Smith, A. R. Townsend, B. A. Askonas, S. Rowland-Jones and T. Dong (2008). "Memory T cells established by seasonal human influenza A infection cross-react with avian influenza A (H5N1) in healthy individuals." J Clin Invest 118(10): 3478-90. Legge, K. L. and T. J. Braciale (2003). "Accelerated migration of respiratory dendritic cells to the regional lymph nodes is limited to the early phase of pulmonary infection." Immunity 18(2): 265-77. Li, P., M. Lu, M. T. Nguyen, E. J. Bae, J. Chapman, D. Feng, M. Hawkins, J. E. Pessin, D. D. Sears, A. K. Nguyen, A. Amidi, S. M. Watkins, U. Nguyen and J. M. Olefsky (2010). "Functional heterogeneity of CD11c-positive adipose tissue macrophages in diet-induced obese mice." J Biol Chem 285(20): 15333-45. Li, Q., Z. Guo, X. Xu, S. Xia and X. Cao (2008). "Pulmonary stromal cells induce the generation of regulatory DC attenuating T-cell-mediated lung inflammation." Eur J Immunol 38(10): 2751-61. Lim, A. P., C. E. Chan, S. K. Wong, A. H. Chan, E. E. Ooi and B. J. Hanson (2008). "Neutralizing human monoclonal antibody against H5N1 influenza HA selected from a Fab-phage display library." Virol J 5: 130. Lin, M. L., Y. Zhan, A. I. Proietto, S. Prato, L. Wu, W. R. Heath, J. A. Villadangos and A. M. Lew (2008). "Selective suicide of cross-presenting CD8+ dendritic cells by 196 References cytochrome c injection shows functional heterogeneity within this subset." Proc Natl Acad Sci U S A 105(8): 3029-34. Lopez, C. B., B. Moltedo, L. Alexopoulou, L. Bonifaz, R. A. Flavell and T. M. Moran (2004). "TLR-independent induction of dendritic cell maturation and adaptive immunity by negative-strand RNA viruses." J Immunol 173(11): 6882-9. Lowen, A. C., S. Mubareka, T. M. Tumpey, A. Garcia-Sastre and P. Palese (2006). "The guinea pig as a transmission model for human influenza viruses." Proc Natl Acad Sci U S A 103(26): 9988-92. Ludwig, S., X. Wang, C. Ehrhardt, H. Zheng, N. Donelan, O. Planz, S. Pleschka, A. Garcia-Sastre, G. Heins and T. Wolff (2002). "The influenza A virus NS1 protein inhibits activation of Jun N-terminal kinase and AP-1 transcription factors." J Virol 76(21): 11166-71. Lumeng, C. N., J. L. Bodzin and A. R. Saltiel (2007). "Obesity induces a phenotypic switch in adipose tissue macrophage polarization." J Clin Invest 117(1): 175-84. Manicassamy, B., S. Manicassamy, A. Belicha-Villanueva, G. Pisanelli, B. Pulendran and A. Garcia-Sastre (2010). "Analysis of in vivo dynamics of influenza virus infection in mice using a GFP reporter virus." Proc Natl Acad Sci U S A 107(25): 11531-6. Mariathasan, S. and D. M. Monack (2007). "Inflammasome adaptors and sensors: intracellular regulators of infection and inflammation." Nat Rev Immunol 7(1): 31-40. Martinon, F., K. Burns and J. Tschopp (2002). "The inflammasome: a molecular platform triggering activation of inflammatory caspases and processing of proILbeta." Mol Cell 10(2): 417-26. Masten, B. J. and M. F. Lipscomb (1999). "Comparison of lung dendritic cells and B cells in stimulating naive antigen-specific T cells." J Immunol 162(3): 1310-7. Matrosovich, M. N., T. Y. Matrosovich, T. Gray, N. A. Roberts and H. D. Klenk (2004). "Human and avian influenza viruses target different cell types in cultures of human airway epithelium." Proc Natl Acad Sci U S A 101(13): 4620-4. 197 References Matsui, M., S. Kohyama, T. Suda, S. Yokoyama, M. Mori, A. Kobayashi, M. Taneichi and T. Uchida (2010). "A CTL-based liposomal vaccine capable of inducing protection against heterosubtypic influenza viruses in HLA-A*0201 transgenic mice." Biochem Biophys Res Commun 391(3): 1494-9. Matsuoka, Y., E. W. Lamirande and K. Subbarao (2009). "The ferret model for influenza." Curr Protoc Microbiol Chapter 15: Unit 15G 2. McGeoch, D., P. Fellner and C. Newton (1976). "Influenza virus genome consists of eight distinct RNA species." Proc Natl Acad Sci U S A 73(9): 3045-9. McGill, J. and K. L. Legge (2009). "Cutting edge: contribution of lung-resident T cell proliferation to the overall magnitude of the antigen-specific CD8 T cell response in the lungs following murine influenza virus infection." J Immunol 183(7): 4177-81. McGill, J., N. Van Rooijen and K. L. Legge (2008). "Protective influenza-specific CD8 T cell responses require interactions with dendritic cells in the lungs." J Exp Med 205(7): 1635-46. McGill, J., N. Van Rooijen and K. L. Legge (2010). "IL-15 trans-presentation by pulmonary dendritic cells promotes effector CD8 T cell survival during influenza virus infection." J Exp Med 207(3): 521-34. McMichael, A. J., F. M. Gotch, G. R. Noble and P. A. Beare (1983). "Cytotoxic Tcell immunity to influenza." N Engl J Med 309(1): 13-7. Merad, M. and M. G. Manz (2009). "Dendritic cell homeostasis." Blood 113(15): 3418-27. Miles, P. R., L. Bowman, A. Rengasamy and L. Huffman (1998). "Constitutive nitric oxide production by rat alveolar macrophages." Am J Physiol 274(3 Pt 1): L360-8. Morens, D. M., J. K. Taubenberger and A. S. Fauci (2008). "Predominant role of bacterial pneumonia as a cause of death in pandemic influenza: implications for pandemic influenza preparedness." J Infect Dis 198(7): 962-70. 198 References Nguyen-Van-Tam, J. S. and A. W. Hampson (2003). "The epidemiology and clinical impact of pandemic influenza." Vaccine 21(16): 1762-8. Nijman, H. W., M. J. Kleijmeer, M. A. Ossevoort, V. M. Oorschot, M. P. Vierboom, M. van de Keur, P. Kenemans, W. M. Kast, H. J. Geuze and C. J. Melief (1995). "Antigen capture and major histocompatibility class II compartments of freshly isolated and cultured human blood dendritic cells." J Exp Med 182(1): 163-74. O'Neill, R. E., R. Jaskunas, G. Blobel, P. Palese and J. Moroianu (1995). "Nuclear import of influenza virus RNA can be mediated by viral nucleoprotein and transport factors required for protein import." J Biol Chem 270(39): 22701-4. Onai, N., A. Obata-Onai, M. A. Schmid, T. Ohteki, D. Jarrossay and M. G. Manz (2007). "Identification of clonogenic common Flt3+M-CSFR+ plasmacytoid and conventional dendritic cell progenitors in mouse bone marrow." Nat Immunol 8(11): 1207-16. Palucka, A. K., J. Gatlin, J. P. Blanck, M. W. Melkus, S. Clayton, H. Ueno, E. T. Kraus, P. Cravens, L. Bennett, A. Padgett-Thomas, F. Marches, M. Islas-Ohlmayer, J. V. Garcia and J. Banchereau (2003). "Human dendritic cell subsets in NOD/SCID mice engrafted with CD34+ hematopoietic progenitors." Blood 102(9): 3302-10. Pang, I. K. and A. Iwasaki (2011). "Inflammasomes as mediators of immunity against influenza virus." Trends Immunol 32(1): 34-41. Parvin, J. D., A. Moscona, W. T. Pan, J. M. Leider and P. Palese (1986). "Measurement of the mutation rates of animal viruses: influenza A virus and poliovirus type 1." J Virol 59(2): 377-83. Peperzak, V., Y. Xiao, E. A. Veraar and J. Borst (2010). "CD27 sustains survival of CTLs in virus-infected nonlymphoid tissue in mice by inducing autocrine IL-2 production." J Clin Invest 120(1): 168-78. Pichlmair, A., O. Schulz, C. P. Tan, T. I. Naslund, P. Liljestrom, F. Weber and C. Reis e Sousa (2006). "RIG-I-mediated antiviral responses to single-stranded RNA bearing 5'-phosphates." Science 314(5801): 997-1001. 199 References Pirhonen, J., T. Sareneva, M. Kurimoto, I. Julkunen and S. Matikainen (1999). "Virus infection activates IL-1 beta and IL-18 production in human macrophages by a caspase-1-dependent pathway." J Immunol 162(12): 7322-9. Pollard, A. M. and M. F. Lipscomb (1990). "Characterization of murine lung dendritic cells: similarities to Langerhans cells and thymic dendritic cells." J Exp Med 172(1): 159-67. Raue, H. P., J. D. Brien, E. Hammarlund and M. K. Slifka (2004). "Activation of virus-specific CD8+ T cells by lipopolysaccharide-induced IL-12 and IL-18." J Immunol 173(11): 6873-81. Reading, P. C., L. S. Morey, E. C. Crouch and E. M. Anders (1997). "Collectinmediated antiviral host defense of the lung: evidence from influenza virus infection of mice." J Virol 71(11): 8204-12. Rehwinkel, J. and C. Reis e Sousa (2011). "RIGorous detection: exposing virus through RNA sensing." Science 327(5963): 284-6. Renegar, K. B., P. A. Small, Jr., L. G. Boykins and P. F. Wright (2004). "Role of IgA versus IgG in the control of influenza viral infection in the murine respiratory tract." J Immunol 173(3): 1978-86. Rust, M. J., M. Lakadamyali, F. Zhang and X. Zhuang (2004). "Assembly of endocytic machinery around individual influenza viruses during viral entry." Nat Struct Mol Biol 11(6): 567-73. Sagerstrom, C. G., E. M. Kerr, J. P. Allison and M. M. Davis (1993). "Activation and differentiation requirements of primary T cells in vitro." Proc Natl Acad Sci U S A 90(19): 8987-91. Saha, S. K., E. M. Pietras, J. Q. He, J. R. Kang, S. Y. Liu, G. Oganesyan, A. Shahangian, B. Zarnegar, T. L. Shiba, Y. Wang and G. Cheng (2006). "Regulation of antiviral responses by a direct and specific interaction between TRAF3 and Cardif." EMBO J 25(14): 3257-63. 200 References Sallusto, F., M. Cella, C. Danieli and A. Lanzavecchia (1995). "Dendritic cells use macropinocytosis and the mannose receptor to concentrate macromolecules in the major histocompatibility complex class II compartment: downregulation by cytokines and bacterial products." J Exp Med 182(2): 389-400. Salomon, R., P. Staeheli, G. Kochs, H. L. Yen, J. Franks, J. E. Rehg, R. G. Webster and E. Hoffmann (2007). "Mx1 gene protects mice against the highly lethal human H5N1 influenza virus." Cell Cycle 6(19): 2417-21. Satoh, T., H. Kato, Y. Kumagai, M. Yoneyama, S. Sato, K. Matsushita, T. Tsujimura, T. Fujita, S. Akira and O. Takeuchi (2010). "LGP2 is a positive regulator of RIG-Iand MDA5-mediated antiviral responses." Proc Natl Acad Sci U S A 107(4): 1512-7. Schnell, J. R. and J. J. Chou (2008). "Structure and mechanism of the M2 proton channel of influenza A virus." Nature 451(7178): 591-5. Schon-Hegrad, M. A., J. Oliver, P. G. McMenamin and P. G. Holt (1991). "Studies on the density, distribution, and surface phenotype of intraepithelial class II major histocompatibility complex antigen (Ia)-bearing dendritic cells (DC) in the conducting airways." J Exp Med 173(6): 1345-56. Schultz-Cherry, S. and V. S. Hinshaw (1996). "Influenza virus neuraminidase activates latent transforming growth factor beta." J Virol 70(12): 8624-9. Scott, B., R. Liblau, S. Degermann, L. A. Marconi, L. Ogata, A. J. Caton, H. O. McDevitt and D. Lo (1994). "A role for non-MHC genetic polymorphism in susceptibility to spontaneous autoimmunity." Immunity 1(1): 73-83. Seo, S. H. and R. G. Webster (2002). "Tumor necrosis factor alpha exerts powerful anti-influenza virus effects in lung epithelial cells." J Virol 76(3): 1071-6. Sertl, K., T. Takemura, E. Tschachler, V. J. Ferrans, M. A. Kaliner and E. M. Shevach (1986). "Dendritic cells with antigen-presenting capability reside in airway epithelium, lung parenchyma, and visceral pleura." J Exp Med 163(2): 436-51. 201 References Shinya, K., Y. Fujii, H. Ito, T. Ito and Y. Kawaoka (2004). "Characterization of a neuraminidase-deficient influenza a virus as a potential gene delivery vector and a live vaccine." J Virol 78(6): 3083-8. Snelgrove, R. J., L. Edwards, A. J. Rae and T. Hussell (2006). "An absence of reactive oxygen species improves the resolution of lung influenza infection." Eur J Immunol 36(6): 1364-73. Sokol, C. L., N. Q. Chu, S. Yu, S. A. Nish, T. M. Laufer and R. Medzhitov (2009). "Basophils function as antigen-presenting cells for an allergen-induced T helper type response." Nat Immunol 10(7): 713-20. Song, S. K., Z. Moldoveanu, H. H. Nguyen, E. H. Kim, K. Y. Choi, J. B. Kim and J. Mestecky (2007). "Intranasal immunization with influenza virus and Korean mistletoe lectin C (KML-C) induces heterosubtypic immunity in mice." Vaccine 25(34): 6359-66. Srivastava, B., P. Blazejewska, M. Hessmann, D. Bruder, R. Geffers, S. Mauel, A. D. Gruber and K. Schughart (2009). "Host genetic background strongly influences the response to influenza a virus infections." PLoS One 4(3): e4857. Stegmann, T., J. M. Delfino, F. M. Richards and A. Helenius (1991). "The HA2 subunit of influenza hemagglutinin inserts into the target membrane prior to fusion." J Biol Chem 266(27): 18404-10. Steinhauer, D. A. and J. J. Skehel (2002). "Genetics of influenza viruses." Annu Rev Genet 36: 305-32. Steinman, R. M. (1991). "The dendritic cell system and its role in immunogenicity." Annu Rev Immunol 9: 271-96. Steinman, R. M. and Z. A. Cohn (1973). "Identification of a novel cell type in peripheral lymphoid organs of mice. I. Morphology, quantitation, tissue distribution." J Exp Med 137(5): 1142-62. 202 References Steinman, R. M., B. Gutchinov, M. D. Witmer and M. C. Nussenzweig (1983). "Dendritic cells are the principal stimulators of the primary mixed leukocyte reaction in mice." J Exp Med 157(2): 613-27. Strutt, T. M., K. K. McKinstry, J. P. Dibble, C. Winchell, Y. Kuang, J. D. Curtis, G. Huston, R. W. Dutton and S. L. Swain (2010). "Memory CD4+ T cells induce innate responses independently of pathogen." Nat Med 16(5): 558-64, 1p following 564. Sun, J., R. Madan, C. L. Karp and T. J. Braciale (2009). "Effector T cells control lung inflammation during acute influenza virus infection by producing IL-10." Nat Med 15(3): 277-84. Sun, J. C. and M. J. Bevan (2003). "Defective CD8 T cell memory following acute infection without CD4 T cell help." Science 300(5617): 339-42. Sung, S. S., S. M. Fu, C. E. Rose, Jr., F. Gaskin, S. T. Ju and S. R. Beaty (2006). "A major lung CD103 (alphaE)-beta7 integrin-positive epithelial dendritic cell population expressing Langerin and tight junction proteins." J Immunol 176(4): 216172. Swanson, K. A., Y. Zheng, K. M. Heidler, Z. D. Zhang, T. J. Webb and D. S. Wilkes (2004). "Flt3-ligand, IL-4, GM-CSF, and adherence-mediated isolation of murine lung dendritic cells: assessment of isolation technique on phenotype and function." J Immunol 173(8): 4875-81. Szretter, K. J., S. Gangappa, J. A. Belser, H. Zeng, H. Chen, Y. Matsuoka, S. Sambhara, D. E. Swayne, T. M. Tumpey and J. M. Katz (2009). "Early control of H5N1 influenza virus replication by the type I interferon response in mice." J Virol 83(11): 5825-34. Talon, J., C. M. Horvath, R. Polley, C. F. Basler, T. Muster, P. Palese and A. GarciaSastre (2000). "Activation of interferon regulatory factor is inhibited by the influenza A virus NS1 protein." J Virol 74(17): 7989-96. Tan, W. C., X. Xiang, D. Qiu, T. P. Ng, S. F. Lam and R. G. Hegele (2003). "Epidemiology of respiratory viruses in patients hospitalized with near-fatal asthma, 203 References acute exacerbations of asthma, or chronic obstructive pulmonary disease." Am J Med 115(4): 272-7. Taubenberger, J. K. and D. M. Morens (2008). "The pathology of influenza virus infections." Annu Rev Pathol 3: 499-522. Teijaro, J. R., D. Verhoeven, C. A. Page, D. Turner and D. L. Farber (2010). "Memory CD4 T cells direct protective responses to influenza virus in the lungs through helper-independent mechanisms." J Virol 84(18): 9217-26. Theodoratos, A., B. Whittle, A. Enders, D. C. Tscharke, C. M. Roots, C. C. Goodnow and A. M. Fahrer (2009). "Mouse strains with point mutations in TAP1 and TAP2." Immunol Cell Biol 88(1): 72-8. Thomas, P. G., S. A. Brown, M. Y. Morris, W. Yue, J. So, C. Reynolds, R. J. Webby and P. C. Doherty (2010). "Physiological numbers of CD4+ T cells generate weak recall responses following influenza virus challenge." J Immunol 184(4): 1721-7. Thomas, P. G., P. Dash, J. R. Aldridge, Jr., A. H. Ellebedy, C. Reynolds, A. J. Funk, W. J. Martin, M. Lamkanfi, R. J. Webby, K. L. Boyd, P. C. Doherty and T. D. Kanneganti (2009). "The intracellular sensor NLRP3 mediates key innate and healing responses to influenza A virus via the regulation of caspase-1." Immunity 30(4): 56675. Thompson, W. W., D. K. Shay, E. Weintraub, L. Brammer, N. Cox, L. J. Anderson and K. Fukuda (2003). "Mortality associated with influenza and respiratory syncytial virus in the United States." JAMA 289(2): 179-86. Topham, D. J. and P. C. Doherty (1998). "Clearance of an influenza A virus by CD4+ T cells is inefficient in the absence of B cells." J Virol 72(1): 882-5. Topham, D. J., R. A. Tripp and P. C. Doherty (1997). "CD8+ T cells clear influenza virus by perforin or Fas-dependent processes." J Immunol 159(11): 5197-200. Tsoumakidou, M., N. Tzanakis, H. A. Papadaki, H. Koutala and N. M. Siafakas (2006). "Isolation of myeloid and plasmacytoid dendritic cells from human bronchoalveolar lavage fluid." Immunol Cell Biol 84(3): 267-73. 204 References Tumpey, T. M., A. Garcia-Sastre, A. Mikulasova, J. K. Taubenberger, D. E. Swayne, P. Palese and C. F. Basler (2002). "Existing antivirals are effective against influenza viruses with genes from the 1918 pandemic virus." Proc Natl Acad Sci U S A 99(21): 13849-54. Tumpey, T. M., A. Garcia-Sastre, J. K. Taubenberger, P. Palese, D. E. Swayne, M. J. Pantin-Jackwood, S. Schultz-Cherry, A. Solorzano, N. Van Rooijen, J. M. Katz and C. F. Basler (2005). "Pathogenicity of influenza viruses with genes from the 1918 pandemic virus: functional roles of alveolar macrophages and neutrophils in limiting virus replication and mortality in mice." J Virol 79(23): 14933-44. Turan, K., M. Mibayashi, K. Sugiyama, S. Saito, A. Numajiri and K. Nagata (2004). "Nuclear MxA proteins form a complex with influenza virus NP and inhibit the transcription of the engineered influenza virus genome." Nucleic Acids Res 32(2): 643-52. van Heijst, J. W., C. Gerlach, E. Swart, D. Sie, C. Nunes-Alves, R. M. Kerkhoven, R. Arens, M. Correia-Neves, K. Schepers and T. N. Schumacher (2009). "Recruitment of antigen-specific CD8+ T cells in response to infection is markedly efficient." Science 325(5945): 1265-9. Van Kaer, L., P. G. Ashton-Rickardt, H. L. Ploegh and S. Tonegawa (1992). "TAP1 mutant mice are deficient in antigen presentation, surface class I molecules, and CD48+ T cells." Cell 71(7): 1205-14. van Rijt, L. S., S. Jung, A. Kleinjan, N. Vos, M. Willart, C. Duez, H. C. Hoogsteden and B. N. Lambrecht (2005). "In vivo depletion of lung CD11c+ dendritic cells during allergen challenge abrogates the characteristic features of asthma." J Exp Med 201(6): 981-91. Vermaelen, K. Y., I. Carro-Muino, B. N. Lambrecht and R. A. Pauwels (2001). "Specific migratory dendritic cells rapidly transport antigen from the airways to the thoracic lymph nodes." J Exp Med 193(1): 51-60. Vignola, A. M., P. Chanez, G. Chiappara, A. Merendino, E. Zinnanti, J. Bousquet, V. Bellia and G. Bonsignore (1996). "Release of transforming growth factor-beta (TGF- 205 References beta) and fibronectin by alveolar macrophages in airway diseases." Clin Exp Immunol 106(1): 114-9. Vitalis, T. Z., Q. J. Zhang, J. Alimonti, S. S. Chen, G. Basha, A. Moise, J. Tiong, M. M. Tian, K. B. Choi, D. Waterfield, A. Jeffries and W. A. Jefferies (2005). "Using the TAP component of the antigen-processing machinery as a molecular adjuvant." PLoS Pathog 1(4): e36. von Garnier, C., L. Filgueira, M. Wikstrom, M. Smith, J. A. Thomas, D. H. Strickland, P. G. Holt and P. A. Stumbles (2005). "Anatomical location determines the distribution and function of dendritic cells and other APCs in the respiratory tract." J Immunol 175(3): 1609-18. von Itzstein, M., W. Y. Wu, G. B. Kok, M. S. Pegg, J. C. Dyason, B. Jin, T. Van Phan, M. L. Smythe, H. F. White, S. W. Oliver and et al. (1993). "Rational design of potent sialidase-based inhibitors of influenza virus replication." Nature 363(6428): 418-23. Vremec, D., J. Pooley, H. Hochrein, L. Wu and K. Shortman (2000). "CD4 and CD8 expression by dendritic cell subtypes in mouse thymus and spleen." J Immunol 164(6): 2978-86. Walsh, J. J., L. F. Dietlein, F. N. Low, G. E. Burch and W. J. Mogabgab (1961). "Bronchotracheal response in human influenza. Type A, Asian strain, as studied by light and electron microscopic examination of bronchoscopic biopsies." Arch Intern Med 108: 376-88. Wang, J. P., G. N. Bowen, C. Padden, A. Cerny, R. W. Finberg, P. E. Newburger and E. A. Kurt-Jones (2008). "Toll-like receptor-mediated activation of neutrophils by influenza A virus." Blood 112(5): 2028-34. Wang, X., M. Li, H. Zheng, T. Muster, P. Palese, A. A. Beg and A. Garcia-Sastre (2000). "Influenza A virus NS1 protein prevents activation of NF-kappaB and induction of alpha/beta interferon." J Virol 74(24): 11566-73. 206 References Ward, A. C. (1997). "Virulence of influenza A virus for mouse lung." Virus Genes 14(3): 187-94. Waskow, C., K. Liu, G. Darrasse-Jeze, P. Guermonprez, F. Ginhoux, M. Merad, T. Shengelia, K. Yao and M. Nussenzweig (2008). "The receptor tyrosine kinase Flt3 is required for dendritic cell development in peripheral lymphoid tissues." Nat Immunol 9(6): 676-83. Watanabe, Y., Y. Hashimoto, A. Shiratsuchi, T. Takizawa and Y. Nakanishi (2005). "Augmentation of fatality of influenza in mice by inhibition of phagocytosis." Biochem Biophys Res Commun 337(3): 881-6. Webster, R. G., W. J. Bean, O. T. Gorman, T. M. Chambers and Y. Kawaoka (1992). "Evolution and ecology of influenza A viruses." Microbiol Rev 56(1): 152-79. Wileman, T. E., M. R. Lennartz and P. D. Stahl (1986). "Identification of the macrophage mannose receptor as a 175-kDa membrane protein." Proc Natl Acad Sci U S A 83(8): 2501-5. Wong, K. L., F. C. Lew, P. A. MacAry and D. M. Kemeny (2008). "CD40Lexpressing CD8 T cells prime CD8alpha(+) DC for IL-12p70 production." Eur J Immunol 38(8): 2251-62. Wong, K. L., L. F. Tang, F. C. Lew, H. S. Wong, Y. L. Chua, P. A. MacAry and D. M. Kemeny (2009). "CD44high memory CD8 T cells synergize with CpG DNA to activate dendritic cell IL-12p70 production." J Immunol 183(1): 41-50. 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 adaptor TRIF in the MyD88-independent toll-like receptor signaling pathway." Science 301(5633): 640-3. Yanagihara, S., E. Komura, J. Nagafune, H. Watarai and Y. Yamaguchi (1998). "EBI1/CCR7 is a new member of dendritic cell chemokine receptor that is upregulated upon maturation." J Immunol 161(6): 3096-102. 207 References Yang, Y., K. Fruh, J. Chambers, J. B. Waters, L. Wu, T. Spies and P. A. Peterson (1992). "Major histocompatibility complex (MHC)-encoded HAM2 is necessary for antigenic peptide loading onto class I MHC molecules." J Biol Chem 267(17): 1166972. Yasuda, J., S. Nakada, A. Kato, T. Toyoda and A. Ishihama (1993). "Molecular assembly of influenza virus: association of the NS2 protein with virion matrix." Virology 196(1): 249-55. Yewdell, J. W. (2005). "Immunoproteasomes: regulating the regulator." Proc Natl Acad Sci U S A 102(26): 9089-90. Yu, C. I., M. Gallegos, F. Marches, G. Zurawski, O. Ramilo, A. Garcia-Sastre, J. Banchereau and A. K. Palucka (2008). "Broad influenza-specific CD8+ T-cell responses in humanized mice vaccinated with influenza virus vaccines." Blood 112(9): 3671-8. Zaks, K., M. Jordan, A. Guth, K. Sellins, R. Kedl, A. Izzo, C. Bosio and S. Dow (2006). "Efficient immunization and cross-priming by vaccine adjuvants containing TLR3 or TLR9 agonists complexed to cationic liposomes." J Immunol 176(12): 733545. Zhang, X., R. Goncalves and D. M. Mosser (2008). "The isolation and characterization of murine macrophages." Curr Protoc Immunol Chapter 14: Unit 14 1. Zhou, B., M. R. Comeau, T. De Smedt, H. D. Liggitt, M. E. Dahl, D. B. Lewis, D. Gyarmati, T. Aye, D. J. Campbell and S. F. Ziegler (2005). "Thymic stromal lymphopoietin as a key initiator of allergic airway inflammation in mice." Nat Immunol 6(10): 1047-53. 208 [...]... results in diffuse inflammation of the larynx, trachea and bronchi accompanied by lymphocyte and histiocyte cellular infiltrate (Walsh et al 1961) Infection of the ciliated epithelium results in initial shrinkage and vacuolaization of the cells, culminating in necrosis and eventual desquamation of these cells into the luminal space The lung interstitium may show congestion and edema and the air spaces... differences in the genetic structure of the virus Influenza A viruses are classified by their surface hemagglutinin (HA) and neuraminidase (NA) proteins, of which there are currently 16 known HA subtypes and 9 NA subtypes 1.1.1 The Health Threat of Influenza The success of the influenza A virus is attributed to its ability to reassort viral RNAs in a host cell infected with more than one strain of influenza A. .. influenza vRNA, the CARD domains initiate a signalling cascade by associating with the adaptor protein, IFN-ȕ promoter stimulator 1 (IPS-1) (Kawai et al 2005), which in turn binds to TNF-receptor-associated factor 3 (TRAF3) (Saha et al 2006) and initiates multiple downstream signalling pathways that ultimately results in the transcription of type I IFNs and pro-inflammatory cytokines 15 Chapter 1: Introduction... RANTES and IFN-ȕ (Guillot et al 2005) In a clinical setting, a missense mutation of the TLR3 12 Chapter 1: Introduction gene resulting in a loss -of- function was associated with influenza- associated encephalopathy in one patient, proving additional evidence that TLR3 plays an important role to control influenza viral replication (Hidaka et al 2006) Interestingly although TLR3 is essential for driving... pro-IL-18 into their active form The inflammasome plays an 13 Chapter 1: Introduction essential role in host defense against the influenza virus and mice lacking components of the inflammasome such as ASC1, caspase-1 and NLRP3 have compromised survival following infection (Allen et al 2009; Ichinohe et al 2009; Thomas et al 2009) The ability of the influenza virus to activate the inflammasome was discovered... components of the viral RNA dependent RNA polymerase, PA, PB1 and PB2 The 8 viral RNA segments encode a total of 11 proteins and the details of each protein and their function are summarised in the table below (Table 1) 7 Chapter 1: Introduction Figure 1.1 Schematic diagram of the influenza A virus The two major surface glycoproteins of the influenza virus are hemagglutinin (HA), neuraminidase (NA), which form... Į2,3linked sialic acid linkages predominate in the upper airways Of note, Į2,3-linked sialic acid residues are also expressed in non-ciliated cells in the human tracheal epithelium, but these cells constitute a minority and the density of sialic-acid expression on the cell surface is also lower, which may explain the relatively poor transmissibility of avian influenza strains to a human host (Matrosovich... Although influenza infection results in activation of the inflammasome in both lung stromal and hematopoietic cells, only inflammasome activation in hematopoietic cells was necessary for the induction of adaptive T -cell responses 14 Chapter 1: Introduction 1.2.3 RLR-mediated detection of the Influenza Virus RIG-I-like receptors, also known as RIG-I-like helicases, are a family of cytoplasmic RNA helicases... the cell surface In human strains (H1 to H3), the virus via the HA molecule preferentially binds to residues terminating with Į2,6-linked sialic acid In contrast, avian strains of the virus (H4 to H16) bind preferentially to Į2,3-linked sialic acid residues Accordingly, Į2,6-linked sialic acid linkages are mainly expressed on the apical surface of ciliated cells in the tracheal epithelium, whereas in. .. delayed viral clearance, secrete lower levels of pro-inflammatory cytokines and have attenuated cellular infiltrate into the alveolar spaces (Le Goffic et al 2006) In addition to immune cells, TLR3 is also expressed on human bronchial and alveolar epithelial cells and the infection of these cells results in the the upregulation of TLR3 expression as well as the release of soluble mediators such as . staining of TAP1 and TAP2 in DCs from lung and pMLN 161  Figure 5.9.1 Intracellular staining of TAP1 and TAP2 in DCs from lung and pMLN (continued) 162  Figure 5.9.2 Validation of TAP1 and TAP2. 3.3.1 H&E staining of transverse section of large conducting airways in uninfected mice. 71 Figure 3.3.2 H&E staining of transverse section of large conducting airways in day 3 p.i 3.3.3 H&E staining of transverse section of large conducting airways in day 5 p.i. mice. 73  Figure 3.3.4 H&E staining of transverse section of large conducting airways in day 7 p.i.