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Abstract

Introduction

Protocol

Representative Results

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Acknowledgements

Materials

References

Immunology and Infection

Contact-Free Co-Culture Model for the Study of Innate Immune Cell Activation During Respiratory Virus Infection

Published: February 28th, 2021

DOI:

10.3791/62115

1Department of Otolaryngology, Yong Loo Lin School of Medicine, National University of Singapore, 2Department of Microbiology and Immunology, Yong Loo Lin School of Medicine, National University of Singapore, 3Haartman Institute, Medicum, University of Helsinki, 4Department of Otolaryngology, Head and Neck Surgery, National University Hospital, National University Health System, 5Agency for Science, Technology and Research (A*STAR), Singapore Immunology Network (SIgN), 6NUHS Infectious Diseases Translational Research Program, Yong Loo Lin School of Medicine, National University of Singapore
* These authors contributed equally

This protocol details an investigation of the early interactions between virally infected nasal epithelial cells and innate cell activation. Individual subsets of immune cells can be distinguished based on their activation in response to viral infections. They can then be further investigated to determine their effects on early antiviral responses.

The early interactions between the nasal epithelial layer and the innate immune cells during viral infections remains an under-explored area. The significance of innate immunity signaling in viral infections has increased substantially as patients with respiratory infections who exhibit high innate T cell activation show a better disease outcome. Hence, dissecting these early innate immune interactions allows the elucidation of the processes that govern them and may facilitate the development of potential therapeutic targets and strategies for dampening or even preventing early progression of viral infections. This protocol details a versatile model that can be used to study early crosstalk, interactions, and activation of innate immune cells from factors secreted by virally infected airway epithelial cells. Using an H3N2 influenza virus (A/Aichi/2/1968) as the representative virus model, innate cell activation of co-cultured peripheral blood mononuclear cells (PBMCs) has been analyzed using flow cytometry to investigate the subsets of cells that are activated by the soluble factors released from the epithelium in response to the viral infection. The results demonstrate the gating strategy for differentiating the subsets of cells and reveal the clear differences between the activated populations of PBMCs and their crosstalk with the control and infected epithelium. The activated subsets can then be further analyzed to determine their functions as well as molecular changes specific to the cells. Findings from such a crosstalk investigation may uncover factors that are important for the activation of vital innate cell populations, which are beneficial in controlling and suppressing the progression of viral infection. Furthermore, these factors can be universally applied to different viral diseases, especially to newly emerging viruses, to dampen the impact of such viruses when they first circulate in naïve human populations.

Respiratory viruses are perhaps amongst the most widespread pathogens causing severe healthcare and economic burden. From the periodic global outbreaks of emerging epidemic strains (e.g., H1N1, H5N1, H3N2, MERS, COVID-19) to the seasonal strains of influenza every year, viruses are a constant threat to public health. Although vaccines form the main bulk of the response to these global public health challenges, it is sobering to note that these countermeasures are merely responsive1,2. Furthermore, a delay between the emergence of a new infectious strain and the successful development of its vaccine is inevitab....

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NOTE: Refer to Table 1 for recipes of media used in this protocol.
NOTE: hNECS grown on 12-well transwell have been found to grow into more optimal thickness for soluble factors to reach the basal chamber readily when infected with Influenza virus. Hence use of 12-well sized transwell for co-culture is recommended. 

1. Establishment of the 3T3 feeder layer

  1. Establishment from frozen stocks
    1. Thaw a cryovial of NIH/3T3 fibroblasts from frozen sto.......

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Although conventional T cells form the main repertoire of adaptive immune response against viral infection to facilitate viral clearance, the innate T cell population works across a broader spectrum to suppress the viral load for effective clearance at a later stage. Therefore, this protocol specifically creates a robust condition to study innate T cells, their activation, and their functional population following influenza infection, without needing epithelial and immune cell samples fro.......

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Innate immune responses against viruses are an under-investigated field of study in antiviral management. The airway epithelial cells and innate immune cells work in concert to suppress viral replication during an infection, besides serving as a determinant of overactive adaptive response if the viral load is not kept in check12,13,17. However, the development of a relevant human model for the study of epithelial-innate immune c.......

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We would like to thank the research staff in NUS Department of Otolaryngology and Department of Microbiology and Immunology for their help with the hNEC culture- and viral-culture-related work. We would also like to thank the surgeons and surgical team in National University Hospital, Department of Otolaryngology, for their assistance in providing the cell and blood samples required for the study.
This study was funded by National Medical Research Council, Singapore No. NMRC/CIRG/1458/2016 (to De Yun Wang) and MOH-OFYIRG19may-0007 (to Kai Sen Tan). Kai Sen Tan is a recipient of fellowship support from European Allergy and Clinical Immunology (EAACI) Research Fel....

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Name Company Catalog Number Comments
0.5% Trypsin-EDTA Gibco 15400-054
0.5 M Ethylenediaminetetraacetic acid (EDTA), pH 8.0, RNase-free Thermofisher AM9260G 0.5M EDTA
1.5 mL SafeLock Tubes Eppendorf 0030120086 1.5mL Centrifuge Tube
10 mL K3EDTA Vacutainer Tubes BD 366643 10mL Blood Collection Tubes
10x dPBS Gibco 14200-075
10x PBS Vivantis PC0711
12-well Plate Corning  3513
12-well Transwell Insert Corning  3460 membrane insert
1x FACS Lysing Solution BD 349202
2.0 mL SafeLock Tubes Eppendorf 0030120094 2 mL centrifuge tube
24-well Plate Corning  3524
24-well Transwell Insert Corning  3470
3% Acetic Acid with Methylene Blue STEMCELL Technologies 07060
3,3',5-triiodo-l-thyronine Sigma T-074
37% Formaldehyde Solution w 15% Methanol as Stabilizer in H2O Sigma 533998
5810R Centrifuge Eppendorf 5811000320
5 mL polypropylene tubes (flow tubes) BD 352058
70 µm Cell Strainer Corning  431751
A-4-62 Rotor Eppendorf 5810709008
Accutase Gibco A1110501 Cell Dissociation Reagent
Antibiotic-Antimycotic Gibco 15240-062
Avicel CL-611 FMC Biopolymer NA Liquid Overlay
Bio-Plex Manager 6.2 Standard Software Bio-Rad Laboratories, Inc 171STND01 Multiplex Manager Software
Butterfly Needle 21 G BD 367287
Cholera Toxin Sigma C8052
Crystal Violet  Merck C6158
Cytofix/Cytoperm Solution BD 554722 Fixation and Permeabilization Solution
Dispase II Sigma D4693 Neutral Protease
DMEM/High Glucose GE Healthcare Life Sciences SH30243.01
DMEM/Nutrient Mixture F-12 Gibco-Invitrogen 11320033
dNTP Mix Promega U1515 dNTP Mix
EMEM (w L-Glutamine) ATCC 30-2003
EVOM voltohmmeter device WPI, Sarasota, FL, USA 300523
FACS Lysing Solution BD 349202 1x Lysing Solution
Falcon tube 15 mL CellStar 188271 15 mL tube
Falcon tube 50 mL CellStar 227261 50 mL Tube
Fast Start Essential DNA Probes Master Roche 6402682001 qPCR Master Mix
Ficoll Paque Premium Research Instruments 17544203 Density Gradient Media
H3N2 (A/Aichi/2/1968)  ATCC VR547
H3N2 M1 Forward Primer Sequence Sigma 5'- ATGGTTCTGGCCAGCACTAC-3'
H3N2 M1 Reverse Primer Sequence Sigma 5'- ATCTGCACCCCCATTCGTTT-3'
H3N2 NS1 Forward Primer Sequence Sigma 5'- ACCCGTGTTGGAAAGCAGAT-3'
H3N2 NS1 Reverse Primer Sequence Sigma 5'- CCTCTTCGGTGAAAGCCCTT-3'
Heat Inactivated Fetal Bovine Serum Gibco 10500-064
hNESPCs Human Donors NA
Human Epithelial Growth Factor Gibco-Invitrogen PHG0314
Hydrocortisone STEMCELL Techonologies 7925 Collected from nasal biopsies during septal deviation surgeries
Insulin Sigma I3536
Lightcycler 96 Roche 5815916001 qPCR Instrument
Live/DEAD Blue Cell Stain Kit *for UV Excitation Thermofisher L23105 Viability Stain
MILLIPLEX MAP Human Cytokine/Chemokine Magnetic Bead Panel II - Premixed 23 Plex Merck Pte Ltd HCP2MAG-62K-PX23 Immunology Multiplex Assay
Mitomycin C Sigma M4287
M-MLV 5x Buffer Promega M1705 RT-PCR 5x Buffer
M-MLV Reverse Transcriptase Promega M1706 Reverse Transcriptase
N-2 supplement Gibco-Invitrogen 17502-048
NIH/3T3 ATCC CRL1658
Perm/Wash Buffer BD 554723 Permeabilization Wash Buffer
PneumaCult-ALI 10x Supplement STEMCELL Techonologies 5001
PneumaCult-ALI Basal Medium STEMCELL Techonologies 5001
PneumaCult-ALI Maintenance Supplement (100x) STEMCELL Techonologies 5001
Random Primers Promega C1181 Random Primers
Recombinant Rnasin Rnase Inhibitor Promega N2511 RNase Inhibitor
RNA Lysis Buffer Qiagen Part of 52904
RPMI 1640 (w L-Glutamine) ATCC 30-2001
STX2 electrodes WPI, Sarasota, FL, USA STX2 Electrode
T25 Flask Corning 430639
T75 Flask Corning 430641U
TPCK Trypsin Sigma T1426
Trypan Blue Hyclone SV30084.01
Viral RNA Extraction Kit Qiagen 52904 Viral RNA Extraction Kit
V-Shaped 96-well Plate Corning 3894

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