Characterisation of lung dendritic cell function in a mouse model of influenza

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." 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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.