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 inevitable3, leading to a period when measures available to curb the spread of the virus are highly limited.
These delays are further emphasized by the costs that are inflicted upon society-economically and socially. The seasonal flu alone is responsible for approximately $8 billion in indirect costs, $3.2 billion in medical costs, and 36.3 thousand deaths in the United States of America annually4. This is before consideration of the research costs that are necessary to fund vaccine development. Epidemic outbreaks have even more severe effects on society, compounded by the increasing rate of globalization every year, as evidenced by the global disruptions caused by the emergence and rapid spread of severe acute respiratory syndrome coronovirus 2 (SARS-CoV-2)5,6,7.
Recent studies have shown that infected patients having a greater population of activated innate T cells tend to have a better disease outcome8,9,10. Furthermore, the innate T cell population is categorized into multiple subgroups: the mucosal-associated invariant T (MAIT) cells, Vδ1 γδ T cells, Vδ2 γδ T cells, and the natural killer T (NKT) cells. These subgroups of innate T cells also exhibit heterogeneity within their populations, increasing the complexity of the interactions between the cell populations involved in the innate immune response11. Hence, the mechanism that activates these innate T cells and the knowledge of the specific subgroups of innate T cells may provide a different avenue of research to curtail the infectious effects of these viruses on the human host, especially during the period of vaccine development.
Epithelial cells infected by influenza produce factors that activate innate T cells rapidly12,13,14. Building upon that finding, this contact-free Air-Liquid Interface (ALI) co-culture model aims to mimic the early chemical interactions (mediated by soluble factors released by the infected epithelial layer) between the infected nasal epithelial layer and the PBMCs during early infection. The physical separation between the nasal epithelial layer (cultured on membrane inserts) and the PBMCs (in the chamber underneath) and the epithelial integrity prevent direct infection of the PBMCs by the virus, allowing a detailed study of the effects of epithelial-derived soluble factors on the PBMCs. The identified factors can therefore be further investigated for their therapeutic potential in inducing the appropriate innate T cell population that may protect against influenza infection. This paper therefore has detailed the methods of establishing a co-culture for the study of innate T-cell activation from epithelium-derived soluble factors.
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
2. Establishment of human nasal epithelial cell (hNEC) culture
NOTE: Clinical samples should be obtained from patients who are free of symptoms of upper respiratory tract infection.
3. Transepithelial electrical resistance (TEER) measurement
NOTE: Confirmation of epithelial integrity is important to ensure that an intact and healthy epithelial layer is obtained. An intact epithelial layer is determined through TEER measurement performed on 4 random wells using a voltohmmeter.
4. Isolation of peripheral blood monocytes and NK cells
NOTE: Blood samples should be obtained from healthy volunteers and used on the same day of isolation.
5. hNEC Viral infection and transition to hNEC:PBMC co-culture
NOTE: H3N2 (A/Aichi/2/1968) is used as the representative strain of infection in this protocol. Multiplicity of infection (MOI) of 0.1 is used as the representative MOI in this protocol.
6. Flow cytometry
NOTE: This section of the protocol is continued directly from the previous section using the PBMC cell suspension from step 5.3.3.2. Ensure minimal light exposure during the following steps in this section. A sample panel of surface staining markers can be found in Table 2.
7. Determination of cytokine and chemokine levels
8. Assessment of viral contamination
9. Plaque assay
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 from the same donor. This protocol can also be applied to other viruses, although it may be limited to viruses with apical release, i.e., no virus should enter the basal layer to come into contact with the PBMC compartment.
Based on the representative results in Figure 1, this protocol can help to obtain hNESPC populations grown from a primary cell suspension in a 3T3 feeder layer. Figure 1 provides a sample of the expected progression of the hNESPCs as they grow on the 3T3 feeder layer. These cells will be used for differentiation in the ALI culture to obtain multilayered hNECs, complete with functional ciliated and goblet cells (Figure 4). Using the hNECs, innate T-cell activation can be investigated using flow cytometry. The results shown in Figure 5 show the detection of MAIT cell, γδ-T cell, and NK cell populations, which were significantly increased in co-culture involving hNECs infected with influenza virus. This setup can then be applied to other strains of the influenza virus to tease out the universal population across strains, as well as other viruses and their ability to activate innate T cell populations. In addition, the detection panel can also be customized according to the innate immune cell population of interest to observe their respective activation under co-culture conditions with infected epithelial cells.
Figure 1: hNESPCs grown on a 3T3 feeder layer 2/5/10 days from seeding. Day 2: Note the islets of hNESPCs (an example is demarcated with a white arrow) that should be observed 2 days after seeding on the 3T3 feeder layer. Day 5: The islets observed on Day 2 should now be larger (examples of islands of hNESPCs are demarcated by green circles), and the 3T3 layer should be observed to be degenerating. Day 10: The hNECPSs should be dominating the entire plate with little or no 3T3 cells visible. Scale bars for Day 2 and Day 5 = 50 µm based on a magnification of 200x, scale bar for Day 10 = 100 µm based on a magnification of 100x. Abbreviation: hNESPCs = human nasal epithelium stem/progenitor cells. Please click here to view a larger version of this figure.
Figure 2: Well diagrams for membrane inserts in 24-well and 12-well plates. Note the medium volume to be used for each compartment. hNESPCs are seeded in the apical chambers of the membrane inserts. Abbreviation: hNESPCs = human nasal epithelium stem/progenitor cells. Please click here to view a larger version of this figure.
Figure 3: Well diagrams for membrane inserts in 24-well and 12-well plates for ALI co-culture establishment. Note the medium volume to be used for each compartment. Note the differences in medium volume to be used for the different intervals (2 days/3 days) between medium changes. Abbreviation: ALI = air-liquid interface. Please click here to view a larger version of this figure.
Figure 4: β4-Tubulin and MUC5AC co-stain of an hNEC layer. β4-Tubulin is stained in green, while MUC5AC is stained in red. The nuclei are stained in blue with DAPI. MUC5AC indicates the presence of mucus-producing goblet cells, while β4-tubulin indicates the presence of cilia on ciliated cells. Scale bar = 20 µm based on 600x magnification. Abbreviations: hNEC = human nasal epithelial cell; MUC5AC = mucin 5AC; DAPI = 4′,6-diamidino-2-phenylindole. Please click here to view a larger version of this figure.
Figure 5: Representative results of PBMCs incubated with or without nasal epithelium or influenza-infected epithelium for 24 h. Activation of MAIT, Vδ1 T cells, Vδ2 T cells, and NK cells was determined by cell-type-specific markers including Vα 7.2 TCR, Vδ1 TCR, Vδ2 TCR, CD56, and CD69 staining. The values above the gates indicate the percentage of CD69-positive cells. Abbreviations: PBMCs = peripheral blood mononuclear cells; Epith = nasal epithelium; FLU-Epith = influenza-infected epithelium; MAIT = mucosal-associated invariant T cells; NK = natural killer; TCR = T-cell receptor; CD = cluster of differentiation. Please click here to view a larger version of this figure.
Medium | Recipe | Composition | Yorumlar |
Medium 3 | DMEM/Nutrient Mixture F-12 | 500 mL | |
Human Epithelial Growth Factor | 5 ng/mL | ||
Insulin | 2.5 µg/mL | ||
Cholera Toxin | 0.1 nM | ||
Hydrocortisone | 0.5 µg/mL | ||
3,3',5-triiodo-l-thyronine | 2 nM | ||
N-2 supplement | 5 mL | 10 µL/mL | |
Antibiotic-Antimycotic | 5 mL | ||
Differentiation Media | PneumaCult-ALI Basal Medium | 441 mL | |
PneumaCult-ALI 10x Supplement | 50 mL | ||
Hydrocortisone Solution (200x) | 2.5 mL | ||
0.2% (2 mg/mL; 1000 IU/mL) Heparin Sodium Salt in Phosphate-Buffered Saline | 1 mL | ||
Antibiotic-Antimycotic (100x) | 5 mL | ||
PneumaCult-ALI Maintenance Supplement (100x) | 500 µL | Only to be added right before use | |
Complete Dulbecco's Minimal Essential Medium (DMEM) | DMEM/High Glucose | 450 mL | |
Heat-inactivated Fetal Bovine Serum | 50 mL | ||
Antibiotic-Antimycotic (100x) | 5 mL | ||
Complete Roswell Park Memorial Institute (RPMI) Medium | RPMI 1640 (w L-Glutamine) | 445 mL | |
Heat-inactivated Fetal Bovine Serum | 50 mL | ||
Antibiotic-Antimycotic (100x) | 5 mL | ||
Complete Eagle's Minimal Essential Medium (EMEM) | EMEM (w L-Glutamine) | 450 mL | |
Heat-inactivated Fetal Bovine Serum | 50 mL | ||
Infection Medium | EMEM (w L-Glutamine) | 4 mL | |
TPCK Trypsin (500 µg/mL) | 8 µL | Final TPCK Trypsin Concentration of 1 µg/mL | |
Magnetic-Activated Cell Sorting Buffer | 1x PBS | 498 mL | |
0.5 M EDTA | 2 mL | ||
BSA (Tissue Culture Grade) | 2.5 g | ||
Mitomycin C-Supplemented Complete DMEM | Complete DMEM | 10 mL | |
Mitomycin C | 500 μL | Mitomycin C (10 µg/mL) |
Table 1: Recipe for media used.
Cell surface marker | Fluorophore |
Vδ1 T-cell receptor (TCR) | Fluorescein isothiocyanate (FITC) |
Vδ2 TCR | Peridinin-cholorphyll-protein (PerCP) |
CD3 | V500 |
CD8 | Allophycocyanin-Cyanine 7 dye (APC-Cy7) |
CD14 | Phycoerythrin (PE)-CF594 |
CD56 | Phycoerythrin (PE)-Cyanine 7 (Cy7) |
CD69 | Brilliant Violet 421 (BV421) |
CD83 | Allophycocyanin (APC) |
CD161 | Brilliant Violet 605 (BV605) |
Vα 7.2 | Phycoerythrin (PE) |
CD38 | Brilliant UltraViolet 395 (BUV395) |
Table 2: Sample surface staining markers.
qPCR Reaction Mix | qPCR Master Mix | 5 µL |
Nuclease-free Water | 3 µL | |
Forward Primer (1 mM) | 0.5 µL | |
Reverse Primer (1 mM) | 0.5 µL | |
cDNA (12.5 ng/µL) | 1 µL | |
Total Reaction Volume | 10 µL | |
RT-PCR Reaction Mix | RT-PCR 5x Buffer | 2.5 µL |
Random Primers (500 ng/µL) | 0.2 µL | |
RNase Inhibitor | 0.625 µL | |
dNTP Mix | 2.5 µL | |
Reverse Transcriptase | 0.5 µL | |
RNA (200 ng/µL) | 1 µL | |
Nuclease-Free Water | 12.675 µL | |
Total Reaction Volume | 20 µL |
Table 3: Recipe for reaction mixes of reverse-transcription polymerase chain reaction (RT-PCR) and quantitative PCR (qPCR).
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 crosstalk to investigate the activation of innate immune cells to confer an appropriate antiviral response remains a challenge. Hence, this ALI co-culture model represents a versatile technique that can be used to assess a whole host of interactions between the nasal epithelial layer and the immune cells. As this model combines in-vitro-differentiated hNECs, PBMC activation analysis via flow cytometry, and viral infection, many of the crucial steps have been clearly demarcated to ensure the success of this protocol. In addition, further modification can also be done to the part of the airway involved where in-vitro–differentiated cells from both the upper and lower airways can be used, adding another layer of versatility to the protocol.
However, when working with epithelial-immune cell crosstalk in viral infection, it is critical that the viruses do not interact directly with the PBMCs to identify early local epithelial-derived soluble factors released by the hNECs. Therefore, this model is more suitable for examining viruses with polarized viral release, wherein viruses only bud out from the apical surface into the apical chamber, e.g., influenza viruses12 and SARS-CoV-2 virus19. In addition, to prevent leakage of apical-release viruses into the basal chamber compromising the experiment, an intact epithelial layer of sufficient thickness is vital. Therefore, it is important that TEER measurement and viral RNA quantification be performed to ensure that the results are free of viral leakages into the basal chamber13,16,20. A TEER reading of >1000 implies an intact multilayer of cells suitable for viruses with polarized release; the basal media should be free of any viral RNA contamination13,15,16. However, the utility of the model for bidirectional release viruses, such as rhinoviruses, remains to be explored16. Such viruses are not limited to releasing their progeny in a polarized manner and may bidirectionally release new viruses into both apical and basal regions of the epithelium. Further optimization is required before this model can be applied to viruses with non-polarized release.
As this protocol involves working with human samples from different individuals, no two samples of hNECs will exhibit the same properties and responses12,16. For example, the viscosity of the mucus produced by the terminally differentiated hNEC layer could differ greatly. The speed of cilia development may also be different. It is important, therefore, to exercise some level of flexibility when adhering to the guidelines laid out in this protocol. While it is certainly possible to utilize epithelial cell lines, this would remove the complexity (mucus, interactions between different cell types) of the interactions between the different cell types of the epithelial layer, which would be ideal for investigation of how epithelial crosstalk influences the responses of the PBMCs. A primary cell line is necessary to mimic the physiology of the nasal tissues, where the cells are multilayered, and the different cell types are localized to their own individual niche, although variability might be an issue. Variability in this respect can be overcome by utilizing single-cell RNA sequencing to differentiate and separate the heterogenous population of cells.
The types of interactions that can be assessed are indeed limited by the origin of the PBMCs and the hNECs. When the PBMCs and hNECs are obtained from different donors, ensuring epithelial integrity and separation is crucial. When PBMCs come into contact with hNECs, allogenic immune reactions could occur. Hence, the only interactions that are relevant are interactions mediated by soluble factors that can pass through the membrane inserts, as has been described in the protocol above. However, when both populations of cells originate from the same individual, this model has an added layer of utility as conventional immune cell reactions between the hNECs and the PBMC population can now be assessed, including T-cell-mediated cytotoxicity and antibody-mediated responses. In addition, epigenetic studies can also be performed to examine how modifications to the genome may affect cytokine gene/protein expression/secretion.
Furthermore, different cell populations can be added to the basal chamber to further investigate a specific population. This can be performed by isolating the cell populations of interest (T cells, NK cells, monocytes) and introducing them to the basal chamber instead of the PBMCs. However, this model cannot be used to investigate cellular interactions that require direct contact owing to the separation of the two cell populations by the membrane. As such, the investigation of adaptive immune responses may be limited by this detail. In conclusion, this ALI co-culture model offers a versatile starting point for the in vitro investigation of the crosstalk between nasal tissues and immune cells. The protocol described in this manuscript attempts to provide a guideline that will be helpful even if the populations/conditions are altered.
The authors have nothing to disclose.
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 Fellowship 2019.
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 |