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.

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All authors declare no conflict of interest.

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...

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, V&#948;1 &#947;&#948; T cells, V&#948;2 &#947;&#948; 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.

<table><tbody><tr><td>0.5% Trypsin-EDTA</td><td>Gibco</td><td>15400-054</td><td></td></tr><tr><td>0.5 M Ethylenediaminetetraacetic acid (EDTA), pH 8.0, RNase-free</td><td>Thermofisher</td><td>AM9260G</td><td>0.5M EDTA</td></tr><tr><td>1.5 mL SafeLock Tubes</td><td>Eppendorf</td><td>0030120086</td><td>1.5mL Centrifuge Tube</td></tr><tr><td>10 mL K3EDTA Vacutainer Tubes</td><td>BD</td><td>366643</td><td>10mL Blood Collection Tubes</td></tr><tr><td>10x dPBS</td><td>Gibco</td><td>14200-075</td><td></td></tr><tr><td>10x PBS</td><td>Vivantis</td><td>PC0711</td><td></td></tr><tr><td>12-well Plate</td><td>Corning&amp;nbsp;</td><td>3513</td><td></td></tr><tr><td>12-well Transwell Insert</td><td>Corning&amp;nbsp;</td><td>3460</td><td>membrane insert</td></tr><tr><td>1x FACS Lysing Solution</td><td>BD</td><td>349202</td><td></td></tr><tr><td>2.0 mL SafeLock Tubes</td><td>Eppendorf</td><td>0030120094</td><td>2 mL centrifuge tube</td></tr><tr><td>24-well Plate</td><td>Corning&amp;nbsp;</td><td>3524</td><td></td></tr><tr><td>24-well Transwell Insert</td><td>Corning&amp;nbsp;</td><td>3470</td><td></td></tr><tr><td>3% Acetic Acid with Methylene Blue</td><td>STEMCELL Technologies</td><td>07060</td><td></td></tr><tr><td>3,3&#039;,5-triiodo-l-thyronine</td><td>Sigma</td><td>T-074</td><td></td></tr><tr><td>37% Formaldehyde Solution w 15% Methanol as Stabilizer in H&lt;sub&gt;2&lt;/sub&gt;O</td><td>Sigma</td><td>533998</td><td></td></tr><tr><td>5810R Centrifuge</td><td>Eppendorf</td><td>5811000320</td><td></td></tr><tr><td>5 mL polypropylene tubes (flow tubes)</td><td>BD</td><td>352058</td><td></td></tr><tr><td>70 &amp;micro;m Cell Strainer</td><td>Corning&amp;nbsp;</td><td>431751</td><td></td></tr><tr><td>A-4-62 Rotor</td><td>Eppendorf</td><td>5810709008</td><td></td></tr><tr><td>Accutase</td><td>Gibco</td><td>A1110501</td><td>Cell Dissociation Reagent</td></tr><tr><td>Antibiotic-Antimycotic</td><td>Gibco</td><td>15240-062</td><td></td></tr><tr><td>Avicel CL-611</td><td>FMC Biopolymer</td><td>NA</td><td>Liquid Overlay</td></tr><tr><td>Bio-Plex Manager 6.2 Standard Software</td><td>Bio-Rad Laboratories, Inc</td><td>171STND01</td><td>Multiplex Manager Software</td></tr><tr><td>Butterfly Needle 21 G</td><td>BD</td><td>367287</td><td></td></tr><tr><td>Cholera Toxin</td><td>Sigma</td><td>C8052</td><td></td></tr><tr><td>Crystal Violet&amp;nbsp;</td><td>Merck</td><td>C6158</td><td></td></tr><tr><td>Cytofix/Cytoperm Solution</td><td>BD</td><td>554722</td><td>Fixation and Permeabilization Solution</td></tr><tr><td>Dispase II</td><td>Sigma</td><td>D4693</td><td>Neutral Protease</td></tr><tr><td>DMEM/High Glucose</td><td>GE Healthcare Life Sciences</td><td>SH30243.01</td><td></td></tr><tr><td>DMEM/Nutrient Mixture F-12</td><td>Gibco-Invitrogen</td><td>11320033</td><td></td></tr><tr><td>dNTP Mix</td><td>Promega</td><td>U1515</td><td>dNTP Mix</td></tr><tr><td>EMEM (w L-Glutamine)</td><td>ATCC</td><td>30-2003</td><td></td></tr><tr><td>EVOM voltohmmeter device</td><td>WPI, Sarasota, FL, USA</td><td>300523</td><td></td></tr><tr><td>FACS Lysing Solution</td><td>BD</td><td>349202</td><td>1x Lysing Solution</td></tr><tr><td>Falcon tube 15 mL</td><td>CellStar</td><td>188271</td><td>15 mL tube</td></tr><tr><td>Falcon tube 50 mL</td><td>CellStar</td><td>227261</td><td>50 mL Tube</td></tr><tr><td>Fast Start Essential DNA Probes Master</td><td>Roche</td><td>6402682001</td><td>qPCR Master Mix</td></tr><tr><td>Ficoll Paque Premium</td><td>Research Instruments</td><td>17544203</td><td>Density Gradient Media</td></tr><tr><td>H3N2 (A/Aichi/2/1968)&amp;nbsp;</td><td>ATCC</td><td>VR547</td><td></td></tr><tr><td>H3N2 M1 Forward Primer Sequence</td><td>Sigma</td><td>5&#039;- ATGGTTCTGGCCAGCACTAC-3&#039;</td><td></td></tr><tr><td>H3N2 M1 Reverse Primer Sequence</td><td>Sigma</td><td>5&#039;- ATCTGCACCCCCATTCGTTT-3&#039;</td><td></td></tr><tr><td>H3N2 NS1 Forward Primer Sequence</td><td>Sigma</td><td>5&#039;- ACCCGTGTTGGAAAGCAGAT-3&#039;</td><td></td></tr><tr><td>H3N2 NS1 Reverse Primer Sequence</td><td>Sigma</td><td>5&#039;- CCTCTTCGGTGAAAGCCCTT-3&#039;</td><td></td></tr><tr><td>Heat Inactivated Fetal Bovine Serum</td><td>Gibco</td><td>10500-064</td><td></td></tr><tr><td>hNESPCs</td><td>Human Donors</td><td>NA</td><td></td></tr><tr><td>Human Epithelial Growth Factor</td><td>Gibco-Invitrogen</td><td>PHG0314</td><td></td></tr><tr><td>Hydrocortisone</td><td>STEMCELL Techonologies</td><td>7925</td><td>Collected from nasal biopsies during septal deviation surgeries</td></tr><tr><td>Insulin</td><td>Sigma</td><td>I3536</td><td></td></tr><tr><td>Lightcycler 96</td><td>Roche</td><td>5815916001</td><td>qPCR Instrument</td></tr><tr><td>Live/DEAD Blue Cell Stain Kit *for UV Excitation</td><td>Thermofisher</td><td>L23105</td><td>Viability Stain</td></tr><tr><td>MILLIPLEX MAP Human Cytokine/Chemokine Magnetic Bead Panel II - Premixed 23 Plex</td><td>Merck Pte Ltd</td><td>HCP2MAG-62K-PX23</td><td>Immunology Multiplex Assay</td></tr><tr><td>Mitomycin C</td><td>Sigma</td><td>M4287</td><td></td></tr><tr><td>M-MLV 5x Buffer</td><td>Promega</td><td>M1705</td><td>RT-PCR 5x Buffer</td></tr><tr><td>M-MLV Reverse Transcriptase</td><td>Promega</td><td>M1706</td><td>Reverse Transcriptase</td></tr><tr><td>N-2 supplement</td><td>Gibco-Invitrogen</td><td>17502-048</td><td></td></tr><tr><td>NIH/3T3</td><td>ATCC</td><td>CRL1658</td><td></td></tr><tr><td>Perm/Wash Buffer</td><td>BD</td><td>554723</td><td>Permeabilization Wash Buffer</td></tr><tr><td>PneumaCult-ALI 10x Supplement</td><td>STEMCELL Techonologies</td><td>5001</td><td></td></tr><tr><td>PneumaCult-ALI Basal Medium</td><td>STEMCELL Techonologies</td><td>5001</td><td></td></tr><tr><td>PneumaCult-ALI Maintenance Supplement (100x)</td><td>STEMCELL Techonologies</td><td>5001</td><td></td></tr><tr><td>Random Primers</td><td>Promega</td><td>C1181</td><td>Random Primers</td></tr><tr><td>Recombinant Rnasin Rnase Inhibitor</td><td>Promega</td><td>N2511</td><td>RNase Inhibitor</td></tr><tr><td>RNA Lysis Buffer</td><td>Qiagen</td><td>Part of 52904</td><td></td></tr><tr><td>RPMI 1640 (w L-Glutamine)</td><td>ATCC</td><td>30-2001</td><td></td></tr><tr><td>STX2 electrodes</td><td>WPI, Sarasota, FL, USA</td><td>STX2</td><td>Electrode</td></tr><tr><td>T25 Flask</td><td>Corning</td><td>430639</td><td></td></tr><tr><td>T75 Flask</td><td>Corning</td><td>430641U</td><td></td></tr><tr><td>TPCK Trypsin</td><td>Sigma</td><td>T1426</td><td></td></tr><tr><td>Trypan Blue</td><td>Hyclone</td><td>SV30084.01</td><td></td></tr><tr><td>Viral RNA Extraction Kit</td><td>Qiagen</td><td>52904</td><td>Viral RNA Extraction Kit</td></tr><tr><td>V-Shaped 96-well Plate</td><td>Corning</td><td>3894</td><td></td></tr></tbody></table>

contact-free co-culture model for the study of innate immune cell activation during respiratory virus infection

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 na&#239;ve human populations.

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...

Watch this Scientific Journal Video about Contact-Free Co-Culture Model for the Study of Innate Immune Cell Activation During Respiratory Virus Infection at JoVE.com

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

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 ...

contact-free-co-culture-model-for-study-innate-immune-cell-activation

Research

JoVE Journal

Immunology and Infection

2.8K Views.  National University of Singapore. Dit protocol is ontworpen om overspraak van epitheelcellen en eerstelijns aangeboren immuuncellen te onderzoeken, met name aangeboren T-cellen, wat ons interesseert. Primaire cellen uit menselijke bronnen worden gebruikt in deze techniek, waardoor individueel onderzoek van fysiologische overspraak relevant is voor wat er bij mensen gebeurt. Aangeboren T-cellen zijn betrokken bij het verbeteren van de overleving van influenza-geïnfecteerde patiënten.Weten hoe ze worden geactiveerd, ka...

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

Flow Cytometric Isolation of Primary Murine Type II Alveolar Epithelial Cells for Functional and Molecular Studies

Throughout the last years, the contribution of alveolar type II epithelial cells (AECII) to various aspects of immune regulation in the lung has been increasingly recognized. AECII have been shown to participate in cytokine production in inflamed airways and to even act as antigen-presenting cells in both infection and T-cell mediated autoimmunity 1-8. Therefore, they are especially interesting also in clinical contexts such as airway hyper-reactivity to foreign and self-antigens as well as infections that directly or indirectly target AECII. However, our understanding of the detailed immunologic functions served by alveolar type II epithelial cells in the healthy lung as well as in inflammation remains fragmentary. Many studies regarding AECII function are performed using mouse or human alveolar epithelial cell lines 9-12. Working with cell lines certainly offers a range of benefits, such as the availability of large numbers of cells for extensive analyses. However, we believe the use of primary murine AECII allows a better understanding of the role of this cell type in complex processes like infection or autoimmune inflammation. Primary murine AECII can be isolated directly from animals suffering from such respiratory conditions, meaning they have been subject to all additional extrinsic factors playing a role in the analyzed setting. As an example, viable AECII can be isolated from mice intranasally infected with influenza A virus, which primarily targets these cells for replication 13. Importantly, through ex vivo infection of AECII isolated from healthy mice, studies of the cellular responses mounted upon infection can be further extended.
Our protocol for the isolation of primary murine AECII is based on enzymatic digestion of the mouse lung followed by labeling of the resulting cell suspension with antibodies specific for CD11c, CD11b, F4/80, CD19, CD45 and CD16/CD32. Granular AECII are then identified as the unlabeled and sideward scatter high (SSChigh) cell population and are separated by fluorescence activated cell sorting 3.
In comparison to alternative methods of isolating primary epithelial cells from mouse lungs, our protocol for flow cytometric isolation of AECII by negative selection yields untouched, highly viable and pure AECII in relatively short time. Additionally, and in contrast to conventional methods of isolation by panning and depletion of lymphocytes via binding of antibody-coupled magnetic beads 14, 15, flow cytometric cell-sorting allows discrimination by means of cell size and granularity. Given that instrumentation for flow cytometric cell sorting is available, the described procedure can be applied at relatively low costs. Next to standard antibodies and enzymes for lung disintegration, no additional reagents such as magnetic beads are required. The isolated cells are suitable for a wide range of functional and molecular studies, which include in vitro culture and T-cell stimulation assays as well as transcriptome, proteome or secretome analyses 3, 4.

We describe the rapid isolation of primary murine type II alveolar epithelial cells (AECII) by flow cytometric negative selection. These AECII show high viability and purity and are suitable for a wide range of functional and molecular studies regarding their role in respiratory conditions such as autoimmune or infectious diseases.

Throughout  the last years, the contribution of alveolar type II epithelial cells (AECII)  to various aspects of immune regulation in the lung has been ...

Establishing a Liquid-covered Culture of Polarized Human Airway Epithelial Calu-3 Cells to Study Host Cell Response to Respiratory Pathogens In vitro

The apical and basolateral surfaces of airway epithelial cells demonstrate directional responses to pathogen exposure in vivo. Thus, ideal in vitro models for examining cellular responses to respiratory pathogens polarize, forming apical and basolateral surfaces. One such model is differentiated normal human bronchial epithelial cells (NHBE). However, this system requires lung tissue samples, expertise isolating and culturing epithelial cells from tissue, and time to generate an air-liquid interface culture.
Calu-3 cells, derived from a human bronchial adenocarcinoma, are an alternative model for examining the response of proximal airway epithelial cells to respiratory insult1, pharmacological compounds2-6, and bacterial7-9 and viral pathogens, including influenza virus, rhinovirus and severe acute respiratory syndrome - associated coronavirus10-14. Recently, we demonstrated that Calu-3 cells are susceptible to respiratory syncytial virus (RSV) infection in a manner consistent with NHBE15,16 . Here, we detail the establishment of a polarized, liquid-covered culture (LCC) of Calu-3 cells, focusing on the technical details of growing and culturing Calu-3 cells, maintaining cells that have been cultured into LCC, and we present the method for performing respiratory virus infection of polarized Calu-3 cells.
To consistently obtain polarized Calu-3 LCC, Calu-3 cells must be carefully subcultured before culturing in Transwell inserts. Calu-3 monolayer cultures should remain below 90% confluence, should be subcultured fewer than 10 times from frozen stock, and should regularly be supplied with fresh medium. Once cultured in Transwells, Calu-3 LCC must be handled with care. Irregular media changes and mechanical or physical disruption of the cell layers or plates negatively impact polarization for several hours or days. Polarization is monitored by evaluating trans-epithelial electrical resistance (TEER) and is verified by evaluating the passive equilibration of sodium fluorescein between the apical and basolateral compartments17,18 . Once TEER plateaus at or above 1,000 &Omega;&times;cm2, Calu-3 LCC are ready to use to examine cellular responses to respiratory pathogens.

Establishing a Liquid-covered Culture of Polarized Human Airway Epithelial Calu-3 Cells to Study Host Cell Response to Respiratory Pathogens In vitro

The findings and conclusions in this report are those of the authors and do not necessarily represent the views of the Centers for Disease Control and Prevention.

The  apical and basolateral surfaces of airway epithelial cells demonstrate  directional responses to pathogen exposure in  vivo. Thus, ideal in vitro ...

An In vitro Model to Study Immune Responses of Human Peripheral Blood Mononuclear Cells to Human Respiratory Syncytial Virus Infection

Human respiratory syncytial virus (HRSV) infections present a broad spectrum of disease severity, ranging from mild infections to life-threatening bronchiolitis. An important part of the pathogenesis of severe disease is an enhanced immune response leading to immunopathology.	Here, we describe a protocol used to investigate the immune response of human immune cells to an HRSV infection. First, we describe methods used for culturing, purification and quantification of HRSV. Subsequently, we describe a human in vitro model in which peripheral blood mononuclear cells (PBMCs) are stimulated with live HRSV. This model system can be used to study multiple parameters that may contribute to disease severity, including the innate and adaptive immune response. These responses can be measured at the transcriptional and translational level. Moreover, viral infection of cells can easily be measured using flow cytometry. Taken together, stimulation of PBMC with live HRSV provides a fast and reproducible model system to examine mechanisms involved in HRSV-induced disease.

An In vitro Model to Study Immune Responses of Human Peripheral Blood Mononuclear Cells to Human Respiratory Syncytial Virus Infection

Human respiratory syncytial virus (HRSV) can cause severe bronchiolitis in young infants. Part of the pathogenesis of severe HRSV disease is caused by the host immune response. Stimulation of primary human immune cells with HRSV provides a fast and reproducible model system to study activation of inflammatory pathways and infection.

Human respiratory syncytial  virus (HRSV) infections present a broad spectrum of disease severity, ranging  from mild infections to life-threatening ...

Assessing the Innate Sensing of HIV-1 Infected CD4+ T Cells by Plasmacytoid Dendritic Cells Using an Ex vivo Co-culture System.

HIV-1 innate sensing requires direct contact of infected CD4+ T cells with plasmacytoid dendritic cells (pDCs). In order to study this process, the protocols described here use freshly isolated human peripheral blood mononuclear cells (PBMCs) or plasmacytoid dendritic cells (pDCs) to sense infections in either T cell line (MT4) or heterologous primary CD4+ T cells. In order to ensure proper sensing, it is essential that PBMC are isolated immediately after blood collection and that optimal percentage of infected T cells are used. Furthermore, multi-parametric flow cytometric staining can be used to confirm that PBMC samples contain the different cell lineages at physiological ratios. A number of controls can also be included to evaluate viability and functionality of pDCs. These include, the presence of specific surface markers, assessing cellular responses to known agonist of Toll-Like Receptors (TLR) pathways, and confirming a lack of spontaneous type-I interferon (IFN) production. In this system, freshly isolated PBMCs or pDCs are co-cultured with HIV-1 infected cells in 96 well plates for 18-22 hr. Supernatants from these co-cultures are then used to determine the levels of bioactive type-I IFNs by monitoring the activation of the ISGF3 pathway in HEK-Blue IFN-&#945;/&#946; cells. Prior and during co-culture conditions, target cells can be subjected to flow cytometric analysis to determine a number of parameters, including the percentage of infected cells, levels of specific surface markers, and differential killing of infected cells. Although, these protocols were initially developed to follow type-I IFN production, they could potentially be used to study other imuno-modulatory molecules released from pDCs and to gain further insight into the molecular mechanisms governing HIV-1 innate sensing.

Assessing the Innate Sensing of HIV-1 Infected CD4+ T Cells by Plasmacytoid Dendritic Cells Using an Ex vivo Co-culture System.

Unlike cell-free HIV-1 particles, infected CD4+ T cells are effectively sensed by plasmacytoid dendritic cells (pDCs). This manuscript describes a method where peripheral blood mononuclear cells (PBMCs) or isolated pDCs are co-cultured with HIV-1 infected T cells to evaluate innate sensing by pDCs as assessed by release of type-I IFN.

HIV-1 innate sensing requires direct contact of infected CD4+ T cells with plasmacytoid dendritic cells (pDCs). In order to study this process, the ...

Airway Model for Studying Lung Barrier Integrity, Disease, and Drug Discovery

iPSC-Derived Epithelial, Mesenchymal, Endothelial, and Immune Cell Co-Culture to Model Airway Barrier Integrity in Lung Health and Disease

Human lung tissue is composed of an interconnected network of epithelium, mesenchyme, endothelium, and immune cells from the upper airway of the nasopharynx to the smallest alveolar sac. Interactions between these cells are crucial in lung development and disease, acting as a barrier against harmful chemicals and pathogens. Current in vitro co-culture models utilize immortalized cell lines with different biological backgrounds, which may not accurately represent the cellular milieu or interactions of the lung. We differentiated human iPSCs into 3D lung organoids (containing both epithelium and mesenchyme), endothelial cells, and macrophages. These were co-cultured in an air-liquid interface (ALI) format to form an epithelial/mesenchymal apical barrier invested with macrophages and a basolateral endothelial barrier (iAirway). iPSC-derived iAirways showed a reduction in barrier integrity in response to infection with respiratory viruses and cigarette toxins. This multi-lineage lung co-culture system provides a platform for studying cellular interactions, signaling pathways, and molecular mechanisms underlying lung development, homeostasis, and disease progression. iAirways closely mimic human physiology and cellular interactions, can be generated from patient-derived iPSC&#39;s, and can be customized to include different cell types of the airway. Overall, iPSC-derived iAirway models offer a versatile and powerful tool for studying barrier integrity to better understand genetic drivers for disease, pathogen response, immune regulation, and drug discovery or repurposing in vitro, with the potential to advance our understanding and treatment of airway diseases.

Author Spotlight: Modeling Human Airway Remodeling and Viral Responses Using Isogenic Epithelial, Endothelial, and Immune Cells

This article describes the generation of a complex, multi-cellular airway barrier model composed of induced pluripotent stem cell (iPSC)-derived lung epithelium, mesenchyme, endothelial cells, and macrophages in an air-liquid interface culture.

Human lung tissue is composed of an interconnected network of epithelium, mesenchyme, endothelium, and immune cells from the upper airway of the ...

Developmental Biology

Imaging Cell Interaction in Tracheal Mucosa During Influenza Virus Infection Using Two-photon Intravital Microscopy

The analysis of cell-cell or cell-pathogen interaction in vivo is an important tool to understand the dynamics of the immune response to infection. Two-photon intravital microscopy (2P-IVM) allows the observation of cell interactions in deep tissue in living animals, while minimizing the photobleaching generated during image acquisition. To date, different models for 2P-IVM of lymphoid and non-lymphoid organs have been described. However, imaging of respiratory organs remains a challenge due to the movement associated with the breathing cycle of the animal.
Here, we describe a protocol to visualize in vivo immune cell interactions in the trachea of mice infected with influenza virus using 2P-IVM. To this purpose, we developed a custom imaging platform, which included the surgical exposure and intubation of the trachea, followed by the acquisition of dynamic images of neutrophils and dendritic cells (DC) in the mucosal epithelium. Additionally, we detailed the steps needed to perform influenza intranasal infection and flow cytometric analysis of immune cells in the trachea. Finally, we analyzed neutrophil and DC motility as well as their interactions during the course of a movie. This protocol allows for the generation of stable and bright 4D images necessary for the assessment of cell-cell interactions in the trachea.

In this study, we present a protocol to perform two-photon intravital imaging and cell interaction analysis in the murine tracheal mucosa after infection with influenza virus. This protocol will be relevant for researchers studying immune cell dynamics during respiratory infections.

The analysis of cell-cell or cell-pathogen interaction in vivo is an important tool to understand the dynamics of the immune response to infection. ...

P. aeruginosa Infected 3D Co-Culture of Bronchial Epithelial Cells and Macrophages at Air-Liquid Interface for Preclinical Evaluation of Anti-Infectives

fDrug research for the treatment of lung infections is progressing towards predictive in vitro models of high complexity. The multifaceted presence of bacteria in lung models can re-adapt epithelial arrangement, while immune cells coordinate an inflammatory response against the bacteria in the microenvironment. While in vivo models have been the choice for testing new anti-infectives in the context of cystic fibrosis, they still do not accurately mimic the in vivo conditions of such diseases in humans and the treatment outcomes. Complex in vitro models of the infected airways based on human cells (bronchial epithelial and macrophages) and relevant pathogens could bridge this gap and facilitate the translation of new anti-infectives into the clinic. For such purposes, a co-culture model of the human cystic fibrosis bronchial epithelial cell line CFBE41o- and THP-1 monocyte-derived macrophages has been established, mimicking an infection of the human bronchial mucosa by P. aeruginosa at air-liquid interface (ALI) conditions. This model is set up in seven days, and the following parameters are simultaneously assessed: epithelial barrier integrity, macrophage transmigration, bacterial survival, and inflammation. The present protocol describes a robust and reproducible system for evaluating drug efficacy and host responses that could be relevant for discovering new anti-infectives and optimizing their aerosol delivery to the lungs.

P. aeruginosa Infected 3D Co-Culture of Bronchial Epithelial Cells and Macrophages at Air-Liquid Interface for Preclinical Evaluation of Anti-Infectives

We describe a protocol for a three-dimensional co-culture model of infected airways, using CFBE41o- cells, THP-1 macrophages, and Pseudomonas aeruginosa, established at the air-liquid interface. This model provides a new platform to simultaneously test antibiotic efficacy, epithelial barrier function, and inflammatory markers.

fDrug research for the treatment of lung infections is progressing towards predictive in vitro models of high complexity. The multifaceted presence of ...

Assessing Respiratory Immune Responses to Haemophilus Influenzae

Haemophilus influenzae (Hi) is a prevalent bacterium found in a range of respiratory conditions. A variety of different assays/techniques may be used to assess the respiratory immune/inflammatory response to this bacterium. Flow cytometry and confocal microscopy are fluorescence-based technologies that allow detailed characterization of biological responses.&#160;Different forms of Hi antigen can be used, including cell wall components, killed/inactivated preparations, and live bacteria. Hi is a fastidious bacterium that requires enriched media but is generally easy to grow in standard laboratory settings. Tissue samples for stimulation with Hi may be obtained from peripheral blood, bronchoscopy, or resected lung (e.g., in patients undergoing surgery for the treatment of lung cancer). Macrophage and neutrophil function may be comprehensively assessed using flow cytometry with a variety of parameters measured, including phagocytosis, reactive oxygen species, and intracellular cytokine production. Lymphocyte function (e.g., T cell and NK cell function) may be specifically assessed using flow cytometry, principally for intracellular cytokine production. Hi infection is a potent inducer of extracellular trap production, both by neutrophils (NETs) and macrophages (METs). Confocal microscopy is arguably the most optimal way to assess NET and MET expression, which may also be used to assess protease activity. Lung immunity to Haemophilus influenzae can be assessed using flow cytometry and confocal microscopy.

Assessing Respiratory Immune Responses to Haemophilus Influenzae

Haemophilus influenzae induces inflammation in the respiratory tract. This article will focus on the use of flow cytometry and confocal microscopy to define immune responses by phagocytes and lymphocytes in response to this bacterium.

Haemophilus influenzae (Hi) is a prevalent bacterium found in a range of respiratory conditions. A variety of different assays/techniques may be used to ...

Infection of Primary Nasal Epithelial Cells Grown at an Air-Liquid Interface to Characterize Human Coronavirus-Host Interactions

Three highly pathogenic human coronaviruses (HCoVs) - SARS-CoV (2002), MERS-CoV (2012), and SARS-CoV-2 (2019) - have emerged and caused significant public health crises in the past 20 years. Four additional HCoVs cause a significant portion of common cold cases each year (HCoV-NL63, -229E, -OC43, and -HKU1), highlighting the importance of studying these viruses in physiologically relevant systems. HCoVs enter the respiratory tract and establish infection in the nasal epithelium, the primary site encountered by all respiratory pathogens. We use a primary nasal epithelial culture system in which patient-derived nasal samples are grown at an air-liquid interface (ALI) to study host-pathogen interactions at this important sentinel site. These cultures recapitulate many features of the in vivo airway, including&#160;the cell types present, ciliary function, and mucus production. We describe methods to characterize viral replication, host cell tropism, virus-induced cytotoxicity, and innate immune induction in nasal ALI cultures following HCoV infection, using recent work comparing lethal and seasonal HCoVs as an example1. An increased understanding of host-pathogen interactions in the nose has the potential to provide novel targets for antiviral therapeutics against HCoVs and other respiratory viruses that will likely emerge in the future.

The nasal epithelium is the primary barrier site encountered by all respiratory pathogens. Here, we outline methods to use primary nasal epithelial cells grown as air-liquid interface (ALI) cultures to characterize human coronavirus-host interactions in a physiologically relevant system.

Three highly pathogenic human coronaviruses (HCoVs) - SARS-CoV (2002), MERS-CoV (2012), and SARS-CoV-2 (2019) - have emerged and caused significant public ...

Optimized Whole Blood Immunoassays for Analyzing Fungal and Viral Immunity

Whole Blood Assay with Dual Co-Stimulation for Antigen-Specific Analysis of Host Immunity to Fungal and Viral Pathogens

Rapid and resource-efficient sample processing, high throughput, and high robustness are critical for effective scientific and clinical application of advanced antigen-specific immunoassays. Traditionally, such immunoassays, especially antigen-specific T-cell analysis by flow cytometry or enzyme-linked immunosorbent spot assays, often rely on the isolation of peripheral blood mononuclear cells. This process is time-consuming, subject to many pre-analytic confounders, and requires large blood volumes. Whole blood-based assays provide a facile alternative with increased pre-analytic robustness and lower blood volume requirements. Furthermore, whole blood-based assays allow for the preservation of inter-cellular interactions that are not captured by assays using isolated cell subsets. Recently, a refined whole blood immunoassay with dual anti-CD28 and anti-CD49d co-stimulation for comprehensive analysis of both antigen-specific T-cell functions and complex intercellular interactions in response to various fungal and viral antigens has been proposed. This protocol provides guidance for the preparation of stimulation tubes, blood stimulation, and downstream sample processing for flow cytometry, cytokine secretion assays, and transcriptional analyses. This includes a validated and functionally equivalent, previously unpublished, low-volume protocol (250 &#181;L) to make flow cytometric and cytokine-based T-cell monitoring more accessible for studies in pediatric patients or preclinical studies in small animals (e.g., mice). Altogether, these protocols provide a versatile toolbox for complex antigen-specific immune analysis in both clinical and translational research settings.

Author Spotlight: Advancing Immune Monitoring in Critical Care Patients Using Whole Blood Assays

Whole blood-based immunoassays provide a facile and resource-efficient tool to analyze antigen-specific immunity for diagnostic and research purposes. This article provides an optimized whole blood-based protocol with dual co-stimulation for comprehensive analysis of host immunity to fungal and viral pathogens, including a low-volume version for pediatric patients and small animals.

Rapid and resource-efficient sample processing, high throughput, and high robustness are critical for effective scientific and clinical application of ...

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

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