H.K. and A.S.M. acknowledge funding from the Leibniz Science-Campus InfectoOptics Jena, financed by the funding line Strategic Networking of the Leibniz Association. M.A. and A.S.M. were supported by the IGF project IMPROVE funded by the Federal Ministry for Economic Affairs and Energy on the basis of a resolution of the German Bundestag. A.S.M further acknowledges financial support by the Cluster of Excellence Balance of the Microverse under Germany&#39;s Excellence Strategy - EXC 2051 - Project-ID 690 390713860.

HUVEC cells are isolated from umbilical cords and used up to passage 4. Primary monocytes are isolated from healthy donors from whole blood. The study was approved by the ethics committee of the Jena University Hospital, Jena, Germany (3939-12/13). According to the Declaration of Helsinki, all individuals donating cells for the study gave their informed consent.
1. Day 1: Preparation of the biochip
<ol>
	<li>The biochips are available in different sizes and models. For experiments here, use the model BC002. Place the biochip in a glass Petri dish, fill the dish, and flush all cavities with sterile 70% ethanol (EthOH). Incubate for 45 min at room temperature. Afterwards, flush cavities 2x with sterile ddH2O. Remove the ddH2O.</li>
	<li>Coat the biochip membrane of both cavities with sterile collagen IV solution (0.5 mg/mL in 0.5 mM acetic acid; use 1:100 concentration in Dulbecco&#39;s phosphate buffered saline containing magnesium and calcium PBS (+/+)) by gently flushing the collagen IV solution through both cavities using a 1,000 &#181;L pipet with blue tips. Incubate for 15 min at 37 &#176;C and 5% CO2. 
	NOTE: Using a 1,000 &#181;L pipet with blue filter (P1000) tips is recommended throughout the entire protocol. The diameter of the opening of the filter tip completely covers the opening of the ports connecting the channels, which avoids the insertion of air bubbles into the chip.</li>
	<li>After 30 min incubation, flush the lower and upper chambers 2x with sterile PBS (+/+) to flush out the remaining collagen and acetic acid.</li>
	<li>Prepare EC medium by adding 1:100 (v/v) Penicillin/Streptomycin (10,000 U/mL). Fill the upper chamber of each cavity with 250 &#181;L of EC medium and the lower chamber with 150 &#181;L of EC medium.</li>
	<li>Harvest HUVEC from a confluent T25 flask: Wash with 5 mL of Dulbecco&#39;s phosphate buffered saline without magnesium and calcium PBS (-/-), add 1 mL of 0.25% Trypsin + 1 mM EDTA in PBS (-/-) and incubate for 3 min at 37 &#176;C. Stop trypsin activity by adding 5 mL of PBS (+/+) + 5% FCS and centrifuge at 350 x g for 5 min at room temperature (RT) in a 50 mL conical tube. Remove the supernatant and resuspend the cell pellet in 1 mL of EC medium.</li>
	<li>Use a 1:10 serial dilution when performing cell counting. Add 10 &#181;L of the resuspended cell pellet to 90 &#181;L of cell medium. Combine the samples carefully. Use a microcentrifuge tube with 10 &#181;L of trypan blue stain. Dilute the cell solution with trypan blue (1:1) using cell counting chamber slides. Count the cells either manually with a Neubauer Chamber or with an automated cell counter. Determine cell counts and adjust concentration to 0.4 x 106 cells per 200 &#181;L.</li>
	<li>Replace the endothelial cell medium in the upper chamber of the biochip with 200 &#181;L of EC medium containing suspended HUVECs. Pipette gently to avoid the formation of air bubbles in the channels or chambers. Place the glass Petri dish with the biochips at 37 &#176;C and 5% CO2.</li>
</ol>
2. Day 2: Checking of the cell layer
<ol>
	<li>On Day 2, visually check for the confluency of the HUVEC cell layer in the upper chamber. Perform medium exchange in the upper chamber with 300 &#181;L of EC medium.&#160;Pipette gently to avoid detachment of cells from the membrane. Perform media exchange daily after 24h. Place the glass Petri dish with the biochips back at 37 &#176;C and 5% CO2.</li>
</ol>
3. Day 3: Preparation of lower chamber
<ol>
	<li>Visually check the confluency of the endothelial cells layer. At this point, determine the presence of a confluent layer and cobblestone-like morphology (Figure 1). Perform medium exchange in the upper chamber with 300 &#181;L of EC medium.</li>
	<li>Next, prepare the lower chamber for seeding the cells forming the epithelial layer. Flush the lower chamber gently with 150 &#181;L of the RPMI medium (add 1:100 (v/v) Penicillin/Streptomycin (10,000 U/mL), 5% fetal cow serum, 1 &#181;M dexamethasone).</li>
	<li>For the lower chamber, seed 0.5 x 106 H441 cells suspended in 200 &#181;L of RPMI medium (RPMI) per cavity.</li>
	<li>Harvesting H441 cells
	<ol>
		<li>Wash the cells with 5 mL of PBS (-/-) once. Add 2 mL of 0.25% Trypsin + 1 mM EDTA in PBS (-/-).</li>
		<li>Incubate the cells for 5 min at 37 &#176;C and 5% CO2. The incubation time varies depending on the confluency of the layer. Within 5-7 min, cells should detach completely.</li>
		<li>Stop the trypsin activity with 10 mL of PBS (+/+) + 5% FBS, collect the cells, and centrifuge at 200 x g for 4 min.</li>
		<li>After centrifugation, discard the supernatant, resuspend the pellet in 1 mL of RPMI, and count.</li>
		<li>Seed 0.5 x 106 cells&#160;per chamber by gently pipetting the cell solution into the lower chamber. Close all the ports and flip the biochip upside-down so the cells can attach to the PET membrane. Keep the biochip upside-down and incubate overnight in a cell culture incubator at 37 &#176;C and 5% CO2.</li>
	</ol>
	</li>
</ol>
4. Days 4 - Day7: Maintenance of cells and harvesting monocytes
<ol>
	<li>Perform daily medium exchange in the upper and lower chamber. For the upper chamber, perform the medium exchange with 300 &#181;L of EC medium.</li>
	<li>Use 200 &#181;L of RPMI+ (RPMI medium plus 1:100 (v/v) Penicillin/Streptomycin (10,000 U/mL), 10 ng/mL GM-CSF, 10% autologous human serum, 1 &#181;M dexamethasone) for the lower chamber. On day 7, flush the lower chamber with RPMI+ to prepare the cavity for seeding monocytes.</li>
	<li>Seed 0.1 x 106 monocytes suspended in 200 &#181;L RPMI+ in the lower chamber.</li>
	<li>Harvesting monocytes
	<ol>
		<li>Wash the cells with 1 mL of PBS (-/-) and incubate for 7 min at 37 &#176;C in pre-warmed PBS (-/-) containing 4 mg/mL Lidocaine + 5 mM EDTA at 37 &#176;C and 5% CO2.</li>
		<li>Collect the cells and centrifuge at 350 x g for 7 min, resuspend the cells in 1mL of RPMI+, and count. Place the biochip ports facing downwards so the cells attach to the membrane.</li>
	</ol>
	</li>
</ol>
5. Day 8: Biochip perfusion
<ol>
	<li>At this point, ensure the biochips are ready to be connected to the flow and perfused. Before starting the perfusion, prepare an empty incubator and place the peristaltic pump inside.</li>
	<li>Perfusion of the biochips
	<ol>
		<li>Before attaching the sterilized tubing to the biochip, flush the tubing with 200 &#181;L of PBS (+/+), followed by 200 &#181;L of EC medium. The tubing has an inner diameter of 0.5 mm, consisting of longer (20 cm) and shorter (12 cm) sides divided by two peristaltic pump stoppers. Put the tubing aside and prepare the reservoirs for the medium.</li>
		<li>Perform medium exchange in the upper and lower chambers by flushing them with the respective freshly prepared cell culture medium.</li>
		<li>Insert the reservoir on the outlet side of the biochip and connect the reservoir with the tubing to the inlet of the biochip (Figure 2). Connect the tubing to the reservoir and the biochip so that there is a circular medium perfusion between the reservoir and the biochip.</li>
		<li>Once everything is connected to the ports, fill in the reservoir with a 500 &#181;L of EC medium. Connect the biochip with the tubing on a perfusion with a flow rate of 21 &#181;L/min (Figure 2). Observe closely during the first 15 min of perfusion for the insertion of bubbles. Perform medium exchange daily in both chambers.</li>
	</ol>
	</li>
</ol>
6. Day 9 - Day 11: Establishing an air-liquid interface
<ol>
	<li>Stop the perfusion, empty the reservoirs, and refill with freshly prepared medium under a sterile safety cabinet. Pipette 500 &#181;L of EC medium into the reservoirs and flush gently the lower chamber with 200 &#181;L of RPMI+. Start the perfusion again with a perfusion rate of 21 &#181;L/min.</li>
	<li>Air Liquid Interface (ALI)
	<ol>
		<li>Establish ALI from day 12 until the end of the experiment (day 14). During ALI culture, expose the epithelial cells to air.</li>
		<li>To establish the ALI culture, prepare a new vascular medium EC (add 1:100 (v/v) Penicillin/Streptomycin (10,000 U/mL), 10 ng/ml GM-CSF, 20% autologous human serum, 1&#181;M dexamethasone) to maintain all cell types in the chip. At this point, the medium contains a higher concentration of AS (20%).</li>
		<li>Open the plugs at the lower chamber under a sterile safety cabinet and carefully soak up the medium until an air bubble is visible, as shown in Figure 3. Ensure the entire liquid from the channel and the chamber is removed and close the ports carefully.</li>
		<li>Fill in the reservoirs with EC (-/-) medium and continue perfusion culture with a flow rate of 21 &#181;L/min. Exchange medium only at the upper chamber until day 14 and keep the epithelial cells in the lower chamber with air only.</li>
	</ol>
	</li>
</ol>
7. Day 14: Tissue harvesting and analysis
<ol>
	<li>Disconnect the biochip from the flow. Plug out the tubing and check on the microscope for cell confluency. Due to the air in the lower chamber, the cell layers might appear fuzzy and difficult to visualize. At this point, the model is ready for further experimentation.</li>
	<li>Fixation and staining of tissue in biochip
	<ol>
		<li>Wash the cavities with PBS (+/+) to remove the cell culture medium. Pipette 300 &#181;L of 4% paraformaldehyde (PFA) dissolved in PBS (-/-) to the upper chamber and 200 &#181;L in the lower chamber and incubate for 10 min at RT. 
		NOTE: When working with fixation chemicals such as PFA, use a fume hood. Collect and dispose of the waste accordingly.</li>
		<li>Wash the chambers 3x with PBS (+/+). After fixation, store the biochips at 4 &#176;C or use them directly for immunofluorescence staining of cells.</li>
		<li>For permeabilization, pipette 300 &#181;L of 0.25% Triton X-100 in PBS (+/+). Incubate for 30 min at RT.</li>
		<li>After incubation, wash both chambers with PBS (+/+) and pipette 300 &#181;L of 3% Bovine serum albumin (BSA) in PBS (+/+) in both chambers. Incubate for 1 h at RT.</li>
		<li>In the meantime, prepare the antibody panel for immunofluorescence staining. To prepare the antibody panel for immunofluorescence staining, a tube with a sufficient amount of 3% BSA was set up to dilute the antibodies. Use 50 &#181;L of antibody dilutions for each membrane. Use a dilution ratio of 1:100 for primary antibody panels and 1:200 for secondary antibody panels. Additionally, for the secondary antibody panel, prepare DAPI at a dilution ratio of 1:2,000. For corresponding antibodies, see (Table 1).</li>
		<li>To directly access the cells within the biochip, the bonding foil must be removed. To open the microfluidic chambers, make an exact cut across the outside of the cavity and remove the bonding foil. Cut the membrane out by removing the edges sealed to the biochip with the scalpel. 
		NOTE: Work precisely and accurately during this step to avoid any membrane damage and injuries due to the sharpness of the scalpel.</li>
		<li>After detaching the membrane from the biochip, divide the membrane into two pieces. Collect the membrane pieces with a tweezer and place them on a microscopic slide for staining.</li>
		<li>Pipette 50 &#181;L of antibody solution per membrane piece. Incubate overnight at 4 &#730;C, protected from light.</li>
		<li>After incubation, place the membrane in 500 &#181;L of PBS (-/-) in a 24 well-plate and wash the membrane 3x for 5 min each in PBS (+/+). After washing, pipette 50 &#181;L of secondary antibody solution and incubate for 1 h at RT protected from light.</li>
		<li>Wash the membrane with PBS (+/+) 3x for 5 min each. Meanwhile, prepare and label the microscopic slides for the experiment. Place a drop of mounting medium on the slide and place the corresponding membrane. Repeat this step for all membranes. At the end, place one more drop of the mounting medium on the top of the membrane and use a cover glass. Store at 4 &#730;C.</li>
	</ol>
	</li>
</ol>

Establishing a Lung-on-Chip Model for Respiratory Infection Studies

<ol>
	<li>Mettelman, R. C., Allen, E. K., Thomas, P. G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Mucosal+immune+responses+to+infection+and+vaccination+in+the+respiratory+tract.">Mucosal immune responses to infection and vaccination in the respiratory tract.</a> Immunity. 55 (5), 749-780 (2022).</li><li>Artzy-Schnirman, A., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Advanced+in+vitro+lung-on-chip+platforms+for+inhalation+assays:+From+prospect+to+pipeline.">Advanced in vitro lung-on-chip platforms for inhalation assays: From prospect to pipeline.</a> Eur J Pharma Biopharma. 144, 11-17 (2019).</li><li>Huh, D., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Reconstituting+organ-level+lung+functions+on+a+chip.">Reconstituting organ-level lung functions on a chip.</a> Science. 328 (5986), 1662-1668 (2010).</li><li>Benam, K. H., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Small+airway-on-a-chip+enables+analysis+of+human+lung+inflammation+and+drug+responses+in+vitro.">Small airway-on-a-chip enables analysis of human lung inflammation and drug responses in vitro.</a> Nat Meth. 13 (2), 151-157 (2016).</li><li>Ronaldson-Bouchard, K., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+multi-organ+chip+with+matured+tissue+niches+linked+by+vascular+flow.">A multi-organ chip with matured tissue niches linked by vascular flow.</a> Nat Biomed Eng. 6 (4), 351 (2022).</li><li>Deinhardt-Emmer, S., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Co-infection+with+staphylococcus+aureus+after+primary+influenza+virus+infection+leads+to+damage+of+the+endothelium+in+a+human+alveolus-on-a-chip+model.">Co-infection with staphylococcus aureus after primary influenza virus infection leads to damage of the endothelium in a human alveolus-on-a-chip model.</a> Biofabrication. 12 (2), 025012 (2020).</li><li>Schicke, E., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Staphylococcus+aureus+lung+infection+results+in+down-regulation+of+surfactant+protein-a+mainly+caused+by+pro-inflammatory+macrophages.">Staphylococcus aureus lung infection results in down-regulation of surfactant protein-a mainly caused by pro-inflammatory macrophages.</a> Microorganisms. 8 (4), 577 (2020).</li><li>King, S. D., Chen, S. Y. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Recent+progress+on+surfactant+protein+a:+Cellular+function+in+lung+and+kidney+disease+development.">Recent progress on surfactant protein a: Cellular function in lung and kidney disease development.</a> Am J Physiol Cell Physiol. 319 (2), C316-C320 (2020).</li><li>Hoang, T. N. M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Invasive+aspergillosis-on-chip:+A+quantitative+treatment+study+of+human+aspergillus+fumigatus+infection.">Invasive aspergillosis-on-chip: A quantitative treatment study of human aspergillus fumigatus infection.</a> Biomaterials. 283, 121420 (2022).</li><li>Yuksel, H., Ocalan, M., Yilmaz, O. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=E-cadherin:+An+important+functional+molecule+at+respiratory+barrier+between+defence+and+dysfunction.">E-cadherin: An important functional molecule at respiratory barrier between defence and dysfunction.</a> Front Physiol. 12, 720227 (2021).</li><li>Van Roy, F., Berx, G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+cell-cell+adhesion+molecule+e-cadherin.">The cell-cell adhesion molecule e-cadherin.</a> Cell Mol Life Sci. 65 (23), 3756-3788 (2008).</li></ol>

K.R. is CEO of Dynamic42 GmbH and holds equity in the company. A.S.M. is a scientific advisor to Dynamic 42 GmbH and holds equity in the company. All the other authors have no conflicts of interest.

The alveolus-on-chip model represents a multilayered tissue model of the human alveolus, integrating essential cell types of the lower respiratory tract, including lung epithelial cells, endothelial cells, and macrophages, cultured in an organotypic arrangement at an ALI with medium perfusion of the endothelial lining. Cells of different layers express specific cell marker proteins such as E-cadherin, a calcium-dependent adhesion molecule of lung epithelial cells, which is central in establishing intercellular epithelial...

The human lung has a remarkable role in respiration and immune defense, with complex interactions between the immune responses of the alveolar mucosa1. The ability of the alveoli to create an efficient immune response is vital for preventing lung infections and securing pulmonary health. Since the lungs are constantly exposed to a wide range of potential risks, including bacteria, viruses, fungi, allergies, and particulate matter,&#160;understanding the complexities of alveolar mucosal immune responses is critical for discovering the mechanisms behind respiratory infections, inflammatory disorders and treating pulmonary diseases1.
To study infection and inflammation-related processes of the respiratory tract in vitro, models that could faithfully mimic the alveolar milieu and the immune responses are required. 2D cell culture and animal modules have been used for decades as essential tools for biomedical research on lung immune response. However, they often have limitations in their translational potential to human situations. Lung-on-chip models can contribute to filling the gap between traditional in vitro and in vivo models and provide a novel approach to studying human-specific immune responses2,3. Lung-on-chip models can mimic the air-liquid interface, which is required for lung cells to recapitulate physiological conditions of the respiratory tract and to develop a more accurate and robust tissue model. This culture technique enables a precise examination of cell differentiation, functioning, and responses to drugs or disease-related stimuli in vitro2.
In this study, we present a microfluidic-based model of the human alveolus as an effective tool to recapitulate the human alveolar milieu by applying perfusion to mimic blood flow and biomechanical stimulation of endothelial cells and incorporating an air-liquid interface with epithelial cells exposed towards an air phase4. We have developed a microfluidic perfused alveolus-on-chip that mimics the physical structure and biological interactions of the human alveolus, with a particular focus on the air-liquid interface. This interface plays a crucial role in the differentiation of respiratory epithelial cells, which is essential for accurately modeling the pulmonary environment. The model uses human distal lung epithelial cells (H441) and human umbilical vein endothelial cells (HUVECs), separated by porous polyethylene terephthalate (PET) membranes, with primary monocyte-derived macrophages positioned between the cell layers. This setup replicates the intricate cellular arrangement of the alveolus and is critical for accurately simulating the air-liquid interface, which is a significant factor in the physiological function of lung tissue.
The rationale behind the model development extends to integrating both circulating and tissue-resident immune cells. This approach is designed to accurately mimic the inflammatory host response to human respiratory infections, providing a dynamic environment to study pathogen-host interactions. The presence of macrophages allows for the examination of immediate immune responses and their interaction with pathogens, reflecting the first line of defense against respiratory infections. Furthermore, the design of the biochip platform facilitates convenient and precise manipulation of both biophysical and biochemical cues, which is crucial for replicating alveolus function in vitro. This flexibility is instrumental in dissecting the contributing factors to human infections, enabling researchers to adjust conditions to reflect various disease states or to test potential therapeutic interventions. The compatibility of the platform with multiple readout technologies, including advanced microscopy, microbiological analyses, and biochemical effluent analysis, enhances its utility. These capabilities allow for a comprehensive assessment of the tissue response to infections, including evaluating cellular behavior, pathogen proliferation, and the effectiveness of immune responses.
We present a detailed protocol and techniques to create and utilize a human alveolus-on-chip model focused on replicating the air-liquid interface and integrating immune cells to study human infections in vitro.

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>Consumables</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Cellcounting chamber slides (Countess)</td>
			<td>Invitrogen</td>
			<td>C10283</td>
		</tr>
		<tr>
			<td>Cell culture Multiwell Plates, 24 Well, steril</td>
			<td>Greiner Bio-One</td>
			<td>662 160</td>
		</tr>
		<tr>
			<td>Cell culture Multiwell Plates, 6 Well, steril</td>
			<td>Greiner Bio-One</td>
			<td>657 160</td>
		</tr>
		<tr>
			<td>Coverslips (24x40mm; #1.5)</td>
			<td>Menzel-Gl&#228;ser</td>
			<td>15747592</td>
		</tr>
		<tr>
			<td>Eco wipes</td>
			<td>Dr. Schuhmacher</td>
			<td>00-915-REW10003-01</td>
		</tr>
		<tr>
			<td>Eppies 2.0</td>
			<td>Sarstedt</td>
			<td>72.691</td>
		</tr>
		<tr>
			<td>Eppis 0.5</td>
			<td>Sarstedt</td>
			<td>72.699</td>
		</tr>
		<tr>
			<td>Eppis 1.5</td>
			<td>Sarstedt</td>
			<td>72.690.001</td>
		</tr>
		<tr>
			<td>Falcons 15mL</td>
			<td>Greiner Bio-One</td>
			<td>188 271-TRI</td>
		</tr>
		<tr>
			<td>Falcons 50mL</td>
			<td>Greiner Bio-One</td>
			<td>227 261-TRI</td>
		</tr>
		<tr>
			<td>Gauze swab</td>
			<td>Noba</td>
			<td>PZN 2417767</td>
		</tr>
		<tr>
			<td>Gloves Nitril 3000</td>
			<td>Meditrade</td>
			<td>1280</td>
		</tr>
		<tr>
			<td>Microscope slides</td>
			<td>Menzel-Gl&#228;ser</td>
			<td>AAAA000001##12E</td>
		</tr>
		<tr>
			<td>Multiwell Plates 24 Well, sterile</td>
			<td>Greiner Bio-One</td>
			<td>662 160</td>
		</tr>
		<tr>
			<td>Pasteur pipettes (glass) 150mm</td>
			<td>Assistent</td>
			<td>40567001</td>
		</tr>
		<tr>
			<td>Pasteur pipettes (glass) 230mm</td>
			<td>Assistent</td>
			<td>40567002</td>
		</tr>
		<tr>
			<td>Round-bottom tubes (PS, 5mL)</td>
			<td>Falcon</td>
			<td>352052</td>
		</tr>
		<tr>
			<td>Safety-Multifly-Set, 20G, 200mm</td>
			<td>Sarstedt</td>
			<td>85.1637.235</td>
		</tr>
		<tr>
			<td>Scalpels</td>
			<td>Dahlhausen</td>
			<td>11.000.00.715</td>
		</tr>
		<tr>
			<td>Serological pipettes 10mL</td>
			<td>Greiner Bio-One</td>
			<td>607 160-TRI</td>
		</tr>
		<tr>
			<td>Serological pipettes 25mL</td>
			<td>Greiner Bio-One</td>
			<td>760 160-TRI</td>
		</tr>
		<tr>
			<td>Serological pipettes 2mL</td>
			<td>Greiner Bio-One</td>
			<td>710 160-TRI</td>
		</tr>
		<tr>
			<td>Serological pipettes 50mL</td>
			<td>Greiner Bio-One</td>
			<td>768 160-TRI</td>
		</tr>
		<tr>
			<td>Serological pipettes 5mL</td>
			<td>Greiner Bio-One</td>
			<td>606 160-TRI</td>
		</tr>
		<tr>
			<td>S-Monovette, 7,5ml Z-Gel</td>
			<td>Sarstedt</td>
			<td>1.1602</td>
		</tr>
		<tr>
			<td>S-Monovette, 9,0ml K3E</td>
			<td>Sarstedt</td>
			<td>02.1066.001</td>
		</tr>
		<tr>
			<td>Softasept N</td>
			<td>Braun</td>
			<td>3887138</td>
		</tr>
		<tr>
			<td>T25 flask</td>
			<td>Greiner Bio-One</td>
			<td>690 960</td>
		</tr>
		<tr>
			<td>Tips sterile 10&#181;L</td>
			<td>Greiner Bio-One</td>
			<td>771 261</td>
		</tr>
		<tr>
			<td>Tips sterile 1250&#181;L</td>
			<td>Greiner Bio-One</td>
			<td>750 261</td>
		</tr>
		<tr>
			<td>Tips sterile 300&#181;L</td>
			<td>Greiner Bio-One</td>
			<td>738 261</td>
		</tr>
		<tr>
			<td>Tips unsterile 10&#181;L</td>
			<td>Greiner Bio-One</td>
			<td>765 290</td>
		</tr>
		<tr>
			<td>Tips unsterile 1000&#181;L</td>
			<td>Greiner Bio-One</td>
			<td>739 291</td>
		</tr>
		<tr>
			<td>Tips unsterile 200&#181;L</td>
			<td>Greiner Bio-One</td>
			<td>686 290</td>
		</tr>
		<tr>
			<td>Tweezers (Pr&#228;zisionspinzette DUMONT abgewinkelt Inox08, 5/45, 0,06 mm)</td>
			<td>Roth</td>
			<td>K343.1</td>
		</tr>
		<tr>
			<td>Chemicals</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Descosept AF</td>
			<td>Dr. Schuhmacher</td>
			<td>N-20338</td>
		</tr>
		<tr>
			<td>Ethanol 96%</td>
			<td>Nordbrand-Nordhausen</td>
			<td>410</td>
		</tr>
		<tr>
			<td>Fluorescein isothiocyanate (FITC)-dextran (3-5kDa)</td>
			<td>Sigma Aldrich</td>
			<td>FD4-100MG</td>
		</tr>
		<tr>
			<td>Fluorescent Mounting Medium</td>
			<td>Dako</td>
			<td>S3023</td>
		</tr>
		<tr>
			<td>Methanol</td>
			<td>VWR</td>
			<td>20847.295</td>
		</tr>
		<tr>
			<td>Saponin</td>
			<td>Fluka</td>
			<td>47036</td>
		</tr>
		<tr>
			<td>Tergazyme</td>
			<td>Alconox</td>
			<td>1304-1</td>
		</tr>
		<tr>
			<td>Cell culture</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Collagen IV</td>
			<td>Sigma-Aldrich</td>
			<td>C5533-5MG</td>
		</tr>
		<tr>
			<td>Dexametason</td>
			<td>Sigma-Aldrich</td>
			<td>D4902</td>
		</tr>
		<tr>
			<td>DPBS (-/-)</td>
			<td>Lonza</td>
			<td>BE17-516F</td>
		</tr>
		<tr>
			<td>DPBS (+/+)</td>
			<td>Lonza</td>
			<td>BE17-513F</td>
		</tr>
		<tr>
			<td>EDTA solution</td>
			<td>Sigma-Aldrich</td>
			<td>E788S</td>
		</tr>
		<tr>
			<td>Endothelial Cell Growth Medium</td>
			<td>Promocell</td>
			<td>C-22020</td>
		</tr>
		<tr>
			<td>Endothelial Cell Growth Medium supplement mix</td>
			<td>Promocell</td>
			<td>C-39225</td>
		</tr>
		<tr>
			<td>Fetal bovine Serum</td>
			<td>Sigma-Aldrich</td>
			<td>E2129-10g</td>
		</tr>
		<tr>
			<td>H441</td>
			<td>ATCC</td>
			<td></td>
		</tr>
		<tr>
			<td>Human recombinant GM-CSF</td>
			<td>Peprotech</td>
			<td>300-30</td>
		</tr>
		<tr>
			<td>Lidocain</td>
			<td>Sigma-Aldrich</td>
			<td>L5647-15G</td>
		</tr>
		<tr>
			<td>Penicillin-Streptomycin (10,000 U/mL)</td>
			<td>Gibco</td>
			<td>15140-122 /-163</td>
		</tr>
		<tr>
			<td>RPMI</td>
			<td>Gibco</td>
			<td>72400047</td>
		</tr>
		<tr>
			<td>Trypane blue stain 0.4%</td>
			<td>Invitrogen</td>
			<td>T10282</td>
		</tr>
		<tr>
			<td>Trypsin</td>
			<td>Gibco</td>
			<td>15090-046</td>
		</tr>
		<tr>
			<td>Primary antibodies</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Cadherin-5 / VE-Cadherin (goat)</td>
			<td>BD</td>
			<td>610252</td>
		</tr>
		<tr>
			<td>CD68 (rabbit)</td>
			<td>CellSignaling</td>
			<td>76437</td>
		</tr>
		<tr>
			<td>E-Cadherin (goat)</td>
			<td>R&#38;D</td>
			<td>AF748</td>
		</tr>
		<tr>
			<td>SP-A (mouse)</td>
			<td>Abcam</td>
			<td>ab51891</td>
		</tr>
		<tr>
			<td>Secondary antibodies</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>AF488 (donkey anti mouse)</td>
			<td>Invitrogen</td>
			<td>R37114</td>
		</tr>
		<tr>
			<td>AF647 (donkey anti mouse)</td>
			<td>invitrogen</td>
			<td>A31571</td>
		</tr>
		<tr>
			<td>AF647 (donkey anti rabbit)</td>
			<td>Invitrogen</td>
			<td>A31573</td>
		</tr>
		<tr>
			<td>Cy3 (donkey anti goat)</td>
			<td>jackson research</td>
			<td>705-165-147</td>
		</tr>
		<tr>
			<td>DAPI (4&#39;,6-Diamidino-2-Phenylindole, Dilactate)</td>
			<td>Invitrogen</td>
			<td>D3571</td>
		</tr>
		<tr>
			<td>Microfluidic</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Chip</td>
			<td>Dynamic 42</td>
			<td>BC002</td>
		</tr>
		<tr>
			<td>Male Luer Lock (small)</td>
			<td>ChipShop</td>
			<td>09-0503-0270-09</td>
		</tr>
		<tr>
			<td>Male mini luer plugs, row of four,PP, green</td>
			<td>Microfluidic chipshop</td>
			<td>09-0558-0336-11</td>
		</tr>
		<tr>
			<td>Male mini luer plugs, row of four,PP, opaque</td>
			<td>Microfluidic chipshop</td>
			<td>09-0556-0336-09</td>
		</tr>
		<tr>
			<td>Male mini luer plugs, row of four,PP, red</td>
			<td>Microfluidic chipshop</td>
			<td>09-0557-0336-10</td>
		</tr>
		<tr>
			<td>Plugs</td>
			<td>Cole Parmer</td>
			<td>GZ-45555-56</td>
		</tr>
		<tr>
			<td>Reservoir 4.5mL</td>
			<td>ChipShop</td>
			<td>16-0613-0233-09</td>
		</tr>
		<tr>
			<td>Tubing</td>
			<td>Dynamic 42</td>
			<td>ST001</td>
		</tr>
		<tr>
			<td>Equipment</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Autoclave</td>
			<td>Tuttnauer</td>
			<td>5075 ELV</td>
		</tr>
		<tr>
			<td>Centrifuge</td>
			<td>Eppendorf</td>
			<td>5424</td>
		</tr>
		<tr>
			<td>CO2 Incubator</td>
			<td>Heracell</td>
			<td>150i</td>
		</tr>
		<tr>
			<td>Countess automated cell counter</td>
			<td>Invitrogen</td>
			<td>C10227</td>
		</tr>
		<tr>
			<td>Flowcytometer</td>
			<td>BD</td>
			<td>FACS Canto II</td>
		</tr>
		<tr>
			<td>Freezer (-20 &#176;C)</td>
			<td>Liebherr</td>
			<td>LCexv 4010</td>
		</tr>
		<tr>
			<td>Freezer (-80 &#176;C)</td>
			<td>Heraeus</td>
			<td>Herafreeze HFU 686</td>
		</tr>
		<tr>
			<td>Fridge</td>
			<td>Liebherr</td>
			<td>LCexv 4010</td>
		</tr>
		<tr>
			<td>Heraeus Multifuge</td>
			<td>Thermo Scientific</td>
			<td>X3R</td>
		</tr>
		<tr>
			<td>Microscope</td>
			<td>Leica</td>
			<td>DM IL LED</td>
		</tr>
		<tr>
			<td>Orbital shaker</td>
			<td>Heidolph</td>
			<td>Reax2000</td>
		</tr>
		<tr>
			<td>Peristaltic pump</td>
			<td>REGLO Digital MS-4/12</td>
			<td>ISM597D</td>
		</tr>
		<tr>
			<td>Pipettes 10&#181;L</td>
			<td>Eppendorf Research plus</td>
			<td>3123000020</td>
		</tr>
		<tr>
			<td>Pipettes 100&#181;L</td>
			<td>Eppendorf Research plus</td>
			<td>3123000047</td>
		</tr>
		<tr>
			<td>Pipettes 1000&#181;L</td>
			<td>Eppendorf Research plus</td>
			<td>3123000063</td>
		</tr>
		<tr>
			<td>Pipettes 2.5&#181;L</td>
			<td>Eppendorf Research plus</td>
			<td>3123000012</td>
		</tr>
		<tr>
			<td>Pipettes 20&#181;L</td>
			<td>Eppendorf Research plus</td>
			<td>3123000039</td>
		</tr>
		<tr>
			<td>Pipettes 200&#181;L</td>
			<td>Eppendorf Research plus</td>
			<td>3123000055</td>
		</tr>
		<tr>
			<td>Scale</td>
			<td>Sartorius</td>
			<td>6101</td>
		</tr>
		<tr>
			<td>Scale</td>
			<td>Sartorius</td>
			<td>TE1245</td>
		</tr>
		<tr>
			<td>Sterile bench</td>
			<td>Kojair</td>
			<td>Biowizard SL-130</td>
		</tr>
		<tr>
			<td>Waterbath</td>
			<td>Julabo</td>
			<td>SW-20C</td>
		</tr>
		<tr>
			<td>Fluorescence Microscope Setup</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Apotome.2</td>
			<td>Zeiss</td>
			<td></td>
		</tr>
		<tr>
			<td>Illumination device</td>
			<td>Zeiss</td>
			<td>HXP 120 C</td>
		</tr>
		<tr>
			<td>Microscope</td>
			<td>Zeiss</td>
			<td>Axio Observer 5</td>
		</tr>
		<tr>
			<td>Optical Sectioning</td>
			<td>Zeiss</td>
			<td>ApoTome</td>
		</tr>
		<tr>
			<td>Power Supply Microscope</td>
			<td>Zeiss</td>
			<td>Eplax Vp232</td>
		</tr>
		<tr>
			<td>Software</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>ZEN Blue Edition</td>
			<td>Zeiss</td>
			<td></td>
		</tr>
	</tbody>
</table>

immunocompetent alveolus-on-chip model for studying alveolar mucosal immune responses

We introduce an advanced immunocompetent lung-on-chip model designed to replicate the human alveolar structure and function. This innovative model employs a microfluidic-perfused biochip that supports an air-liquid interface mimicking the environment in the human alveoli. Tissue engineering is used to integrate key cellular components, including endothelial cells, macrophages, and epithelial cells, to create a representative tissue model of the alveolus. The model facilitates in-depth examinations of the mucosal immune responses to various pathogens, including viruses, bacteria, and fungi, thereby advancing our understanding of lung immunity. The primary goal of this protocol is to provide details for establishing this alveolus-on-chip model as a robust in vitro platform for infection studies, enabling researchers to closely observe and analyze the complex interactions between pathogens and the host's immune system within the pulmonary environment. This is achieved through the application of microfluidic-based techniques to simulate key physiological conditions of the human alveoli, including blood flow and biomechanical stimulation of endothelial cells, alongside maintaining an air-liquid interface crucial for the realistic exposure of epithelial cells to air. The model system is compatible with a range of standardized assays, such as immunofluorescence staining, cytokine profiling, and colony-forming unit (CFU)/plaque analysis, allowing for comprehensive insights into immune dynamics during infection. The Alveolus-on-chip is composed of essential cell types, including human distal lung epithelial cells (H441) and human umbilical vein endothelial cells (HUVECs) separated by porous polyethylene terephthalate (PET) membranes, with primary monocyte-derived macrophages strategically positioned between the epithelial and endothelial layers. The tissue model enhances the ability to dissect and analyze the nuanced factors involved in pulmonary immune responses in vitro. As a valuable tool, it should contribute to the advancement of lung research, providing a more accurate and dynamic in vitro model for studying the pathogenesis of respiratory infections and testing potential therapeutic interventions.

An examination of morphological alterations and the expression of marker proteins could be performed using immunofluorescence staining. After co-culturing for 14 days, the vascular and epithelial sides are analyzed for expression of respective cell markers. This method is useful for studying the interactions and integrity of vascular and epithelial components, which is essential for disease modeling as a functional biological readout related to infection. Immunofluorescence staining could be supported by quantifying anal...

Author Spotlight: Developing a Microfluidic Lung-on-Chip Model for In-Depth Study of Human Immune Response and Infection Mechanisms

Lung-on-chip models surpass traditional 2D cultures by mimicking the air-liquid interface and endothelial cell perfusion, simulating blood flow and nutrient exchange crucial for lung physiology studies. This enhances lung research relevance, offering a dynamic, physiologically accurate environment to advance the understanding and treatment of respiratory infections.

Immunocompetent Alveolus-on-Chip Model for Studying Alveolar Mucosal Immune Responses

We introduce an advanced immunocompetent lung-on-chip model designed to replicate the human alveolar structure and function. This innovative model employs ...

immunocompetent-alveolus-on-chip-model-for-studying-alveolar-mucosal

Research

JoVE Journal

Immunology and Infection

927 Views.  Jena University Hospital. Lung-on-chip models surpass traditional 2D cultures by mimicking the air-liquid interface and endothelial cell perfusion, simulating blood flow and nutrient exchange crucial for lung physiology studies. This enhances lung research relevance, offering a dynamic, physiologically accurate environment to advance the understanding and treatment of respiratory infections.

Video: Author Spotlight: Developing a Microfluidic Lung-on-Chip Model for In-Depth Study of Human Immune Response and Infection Mechanisms

An Orthotopic Model of Serous Ovarian Cancer in Immunocompetent Mice for in vivo Tumor Imaging and Monitoring of Tumor Immune Responses

Background: Ovarian cancer is generally diagnosed at an advanced stage where the case/fatality ratio is high and thus remains the most lethal of all gynecologic malignancies among US women 1,2,3. Serous tumors are the most widespread forms of ovarian cancer and 4,5 the Tg-MISIIR-TAg transgenic represents the only mouse model that spontaneously develops this type of tumors. Tg-MISIIR-TAg mice express SV40 transforming region under control of the Mullerian Inhibitory Substance type II Receptor (MISIIR) gene promoter 6. Additional transgenic lines have been identified that express the SV40 TAg transgene, but do not develop ovarian tumors. Non-tumor prone mice exhibit typical lifespan for C57Bl/6 mice and are fertile. These mice can be used as syngeneic allograft recipients for tumor cells isolated from Tg-MISIIR-TAg-DR26 mice.
Objective: Although tumor imaging is possible 7, early detection of deep tumors is challenging in small living animals. To enable preclinical studies in an immunologically intact animal model for serous ovarian cancer, we describe a syngeneic mouse model for this type of ovarian cancer that permits in vivo imaging, studies of the tumor microenvironment and tumor immune responses.
Methods: We first derived a TAg+ mouse cancer cell line (MOV1) from a spontaneous ovarian tumor harvested in a 26 week-old DR26 Tg-MISIIR-TAg female. Then, we stably transduced MOV1 cells with TurboFP635 Lentivirus mammalian vector that encodes Katushka, a far-red mutant of the red fluorescent protein from sea anemone Entacmaea quadricolor with excitation/emission maxima at 588/635 nm 8,9,10. We orthotopically implanted MOV1Kat in the ovary 11,12,13,14 of non-tumor prone Tg-MISIIR-TAg female mice. Tumor progression was followed by in vivo optical imaging and tumor microenvironment was analyzed by immunohistochemistry.
Results: Orthotopically implanted MOV1Kat cells developed serous ovarian tumors. MOV1Kat tumors could be visualized by in vivo imaging up to three weeks after implantation (fig. 1) and were infiltrated with leukocytes, as observed in human ovarian cancers 15 (fig. 2).
Conclusions: We describe an orthotopic model of ovarian cancer suitable for in vivo imaging of early tumors due to the high pH-stability and photostability of Katushka in deep tissues. We propose the use of this novel syngeneic model of serous ovarian cancer for in vivo imaging studies and monitoring of tumor immune responses and immunotherapies.

An Orthotopic Model of Serous Ovarian Cancer in Immunocompetent Mice for in vivo Tumor Imaging and Monitoring of Tumor Immune Responses

To study in vivo tumor growth and tumor microenvironment, we used a syngeneic and orthotopic mouse model of ovarian cancer in immunocompetent animals. We transduced a mouse tumor cell line (MOV1) with Katushka fluorescent protein (MOV1KAT) and here we show its orthotopic implantation in ovary and in vivo imaging.

Background: Ovarian cancer is generally diagnosed at an advanced stage where the case/fatality ratio is high and thus remains the most lethal of all ...

Measuring Bacterial Load and Immune Responses in Mice Infected with Listeria monocytogenes

Listeria monocytogenes (Listeria) is a Gram-positive facultative intracellular pathogen1. Mouse studies typically employ intravenous injection of Listeria, which results in systemic infection2. After injection, Listeria quickly disseminates to the spleen and liver due to uptake by CD8&alpha;+ dendritic cells and Kupffer cells3,4. Once phagocytosed, various bacterial proteins enable Listeria to escape the phagosome, survive within the cytosol, and infect neighboring cells5. During the first three days of infection, different innate immune cells (e.g. monocytes, neutrophils, NK cells, dendritic cells) mediate bactericidal mechanisms that minimize Listeria proliferation. CD8+ T cells are subsequently recruited and responsible for the eventual clearance of Listeria from the host, typically within 10 days of infection6.
Successful clearance of Listeria from infected mice depends on the appropriate onset of host immune responses6	. There is a broad range of sensitivities amongst inbred mouse strains7,8. Generally, mice with increased susceptibility to Listeria infection are less able to control bacterial proliferation, demonstrating increased bacterial load and/or delayed clearance compared to resistant mice. Genetic studies, including linkage analyses and knockout mouse strains, have identified various genes for which sequence variation affects host responses to Listeria infection6,8-14. Determination and comparison of infection kinetics between different mouse strains is therefore an important method for identifying host genetic factors that contribute to immune responses against Listeria. Comparison of host responses to different Listeria strains is also an effective way to identify bacterial virulence factors that may serve as potential targets for antibiotic therapy or vaccine design.
We describe here a straightforward method for measuring bacterial load (colony forming units [CFU] per tissue) and preparing single-cell suspensions of the liver and spleen for FACS analysis of immune responses in Listeria-infected mice. This method is particularly useful for initial characterization of Listeria infection in novel mouse strains, as well as comparison of immune responses between different mouse strains infected with Listeria. We use the Listeria monocytogenes EGD strain15 that, when cultured on blood agar, exhibits a characteristic halo zone around each colony due to &beta;-hemolysis1 (Figure 1). Bacterial load and immune responses can be determined at any time-point after infection by culturing tissue homogenate on blood agar plates and preparing tissue cell suspensions for FACS analysis using the protocols described below. We would note that individuals who are immunocompromised or pregnant should not handle Listeria, and the relevant institutional biosafety committee and animal facility management should be consulted before work commences.

Measuring Bacterial Load and Immune Responses in Mice Infected with Listeria monocytogenes

Listeria monocytogenes is a model organism for studying immune responses and genetic susceptibility to intracellular bacteria in mice. This method enables one to measure bacterial load and generate single-cell suspensions of the liver and spleen from mice for FACS analysis to determine changes in immune cells due to Listeria infection.

Listeria monocytogenes (Listeria) is a Gram-positive facultative intracellular pathogen1. Mouse studies typically employ intravenous injection of ...

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 ...

An In Vitro Model for Measuring Immune Responses to Malaria in the Context of HIV Co-infection

Malaria and HIV co-infection is a growing health priority. However, most research on malaria or HIV currently focuses on each infection individually. Although understanding the disease dynamics for each of these pathogens independently is vital, it is also important that the interactions between these pathogens are investigated and understood. 
We have developed a versatile in vitro model of HIV-malaria co-infection to study host immune responses to malaria in the context of HIV infection. Our model allows the study of secreted factors in cellular supernatants, cell surface and intracellular protein markers, as well as RNA expression levels. The experimental design and methods used limit variability and promote data reliability and reproducibility. 
All pathogens used in this model are natural human pathogens (Plasmodium falciparum and HIV-1), and all infected cells are naturally infected and used fresh. We use human erythrocytes parasitized with P. falciparum and maintained in continuous in vitro culture. We obtain freshly isolated peripheral blood mononuclear cells from chronically HIV-infected volunteers. Every condition used has an appropriate control (P. falciparum parasitized vs. normal erythrocytes), and every HIV-infected donor has an HIV uninfected control, from which cells are harvested on the same day. This model provides a realistic environment to study the interactions between malaria parasites and human immune cells in the context of HIV infection.

An In Vitro Model for Measuring Immune Responses to Malaria in the Context of HIV Co-infection

Human co-infection is difficult to replicate in vitro. However, human malaria parasites can readily be cultured in vitro, as can freshly isolated human peripheral blood mononuclear cells naturally infected with HIV. This provides an excellent model for studying early immune responses to malaria parasites in the context of HIV co-infection.

Malaria and HIV co-infection is a growing health priority. However, most research on malaria or HIV currently focuses on each infection individually. ...

Experimental Infection with Listeria monocytogenes as a Model for Studying Host Interferon-&#947; Responses

L. monocytogenes is a gram-positive bacterium that is a cause of food borne disease in humans. Experimental infection of mice with this pathogen has been highly informative on the role of innate and adaptive immune cells and specific cytokines in host immunity against intracellular pathogens. Production of IFN-&#947; by innate cells during sublethal infection with L. monocytogenes is important for activating macrophages and early control of the pathogen1-3. In addition, IFN-&#947; production by adaptive memory lymphocytes is important for priming the activation of innate cells upon reinfection4. The L. monocytogenes infection model thus serves as a great tool for investigating whether new therapies that are designed to increase IFN-&#947; production have an impact on IFN-&#947; responses in vivo and have productive biological effects such as increasing bacterial clearance or improving mouse survival from infection. Described here is a basic protocol for how to conduct intraperitoneal infections of C57BL/6J mice with the EGD strain of L. monocytogenes and to measure IFN-&#947; production by NK cells, NKT cells, and adaptive lymphocytes by flow cytometry. In addition, procedures are described to: (1) grow and prepare the bacteria for inoculation, (2) measure bacterial load in the spleen and liver, and (3) measure animal survival to endpoints. Representative data are also provided to illustrate how this infection model can be used to test the effect of specific agents on IFN-&#947; responses to L. monocytogenes and survival of mice from this infection.

Experimental Infection with &lt;em&gt;Listeria monocytogenes&lt;/em&gt; as a Model for Studying Host Interferon-&amp;#947; Responses

This protocol describes how to inoculate C57BL/6J mice with the EGD strain of Listeria monocytogenes (L. monocytogenes) and to measure interferon-&#947; (IFN-&#947;) responses by natural killer (NK) cells, natural killer T (NKT) cells, and adaptive T lymphocytes post-infection. This protocol also describes how to conduct survival studies in mice after infection with a modified LD50 dose of the pathogen.

L. monocytogenes is a gram-positive bacterium that is a cause of food borne disease in humans. Experimental infection of mice with this pathogen has been ...

Noninvasive Sampling of Mucosal Lining Fluid for the Quantification of In Vivo Upper Airway Immune-mediator Levels

This protocol describes noninvasive sampling of undisturbed upper airway mucosal lining fluid. It also details the extraction procedure used prior to the analysis of immune mediators in fluid eluates for the study of the airway topical immune signature, without the need for stimulation procedures (often used by other techniques). The mucosal lining fluid is sampled on a strip of filter paper placed at the anterior part of the inferior turbinate and left for 2 min of absorption. Analytes are eluted from the filter papers, and the extracted protein-based eluates are analyzed by an electrochemiluminescence-based immunoassay, allowing for the high-sensitivity quantification of low- and high-level analytes in the same sample. We measured the in vivo levels of 20 preselected immune mediators related to specific immune signaling pathways in the upper airway mucosa, but the technique is not limited to that specific panel or sampling site. The technique was first implemented in 7-year-old children from the Copenhagen Prospective Studies on Asthma in Childhood2000 (COPSAC2000) cohort with allergic rhinitis. It was thereafter used in the longitudinal COPSAC2010 birth cohort, sampled at 1 month, 2 years, and 6 years of age and at instances of acute respiratory symptoms. We successfully obtained and analyzed samples from 620 (89%) of 700 1-month-old children; a few samples were below the assay detection limit (reported as the median (Inter-Quartile Range (IQR)). The number of samples below the detection limit (i.e.&#160;from 0 to the set point for the lower limit of detection) for each mediator was 29 (7.25 - 119.5). This technique enables the quantification of the in vivo airway mucosal immune profile from birth, can be applied longitudinally, and can be applied to studies on the effect of genetics and early-life environmental exposures, pathophysiology, endotyping, and monitoring of respiratory diseases, and development and evaluation of novel therapeutics.

Noninvasive Sampling of Mucosal Lining Fluid for the Quantification of In Vivo Upper Airway Immune-mediator Levels

This protocol describes a noninvasive technique for the sampling of undisturbed mucosal lining fluid from the upper airways. It can be used to perform the quantification of in vivo levels of protein mediators, such as cytokines and chemokines, in subjects of all ages.

This protocol describes noninvasive sampling of undisturbed upper airway mucosal lining fluid. It also details the extraction procedure used prior to the ...

A Galleria mellonella Oral Administration Model to Study Commensal-Induced Innate Immune Responses

The investigation of the immunogenic potential of commensal bacteria on the host immune system is one essential component when studying intestinal host-microbe interactions. It is well established that different commensals exhibit a different potential to stimulate the host intestinal immune system. Such investigations involve vertebrate animals, especially rodents. Since increasing ethical concerns are linked with experiments involving vertebrates, there is a high demand for invertebrate replacements models. 
Here, we provide a Galleria mellonella oral administration model using commensal non-pathogenic bacteria and the possible assessment of the immunogenic potential of commensals on the G. mellonella immune system. We demonstrate that G. mellonella is a useful alternative invertebrate replacement model that allows the analysis of commensals with different immunogenic potential such as Bacteroides vulgatus and Escherichia coli. Interestingly, the bacteria exhibited no killing effect on the larvae, which is similar to mammals. The immune responses of G. mellonella were comparable with vertebrate innate immune responses and involve recognition of the bacteria and production of antimicrobial molecules. We propose that G. mellonella was able to restore previous microbiota balance, which is well known from healthy mammalian individuals. Although providing comparable innate immune responses in both G. mellonella and vertebrates, G. mellonella does not harbor an adaptive immune system. Since the investigated components of the innate immune system are evolutionary conserved, the model allows a prescreening and first analysis of bacterial immunogenic properties.

A Galleria mellonella Oral Administration Model to Study Commensal-Induced Innate Immune Responses

Here, we provide a detailed protocol for an oral administration model using Galleria mellonella larvae and how to characterize induced innate immune responses. Using this protocol, researchers without practical experience will be able to use the G. mellonella force-feeding method.

The investigation of the immunogenic potential of commensal bacteria on the host immune system is one essential component when studying intestinal ...

Studying Organelle Dynamics in B Cells During Immune Synapse Formation

Recognition of surface-tethered antigens&#160;by the B cell receptor (BCR)&#160;triggers the formation of an immune synapse (IS), where both signaling&#160;and antigen uptake are coordinated. IS formation involves dynamic actin remodeling accompanied by the polarized recruitment to the synaptic membrane of the centrosome and associated intracellular organelles such as lysosomes and the Golgi apparatus. Initial stages of actin remodeling allow B cells to increase their cell surface and maximize the quantity of antigen-BCR complexes gathered at the synapse. Under certain conditions, when B cells recognize antigens associated to rigid surfaces, this process is coupled to the local recruitment and secretion of lysosomes, which can facilitate antigen extraction. Uptaken antigens&#160;are internalized into specialized endo-lysosome compartments for processing into peptides, which are loaded onto major histocompatibility complex II (MHC-II) molecules for further presentation to T helper cells. Therefore, studying organelle dynamics associated with the formation of an IS is crucial to understanding how B cells are activated. In the present article we will discuss both imaging and a biochemical technique used to study changes in intracellular organelle positioning and cytoskeleton rearrangements that are associated with the formation of an IS in B cells.

Herein we describe two approaches to characterize cell polarization events in B lymphocytes during the formation of an IS. The first, involves quantification of organelle recruitment and cytoskeleton rearrangements at the synaptic membrane. The second is a biochemical approach, to characterize changes in composition of the centrosome, which undergoes polarization to the immune synapse.

Recognition of surface-tethered antigens&#160;by the B cell receptor (BCR)&#160;triggers the formation of an immune synapse (IS), where both ...

Isolation of Tonsillar Mononuclear Cells to Study Ex Vivo Innate Immune Responses in a Human Mucosal Lymphoid Tissue

Studying isolated cells from mucosa-associated lymphoid tissues (MALT) allows understanding of immune cells response in pathologies involving mucosal immunity, because they can model host-pathogen interactions in the tissue. While isolated cells derived from tissues were the first cell culture model, their use has been neglected because tissue can be hard to obtain. In the present protocol, we explain how to easily process and culture tonsillar mononuclear cells (TMCs) from healthy human tonsils to study innate immune responses upon activation, mimicking viral infection in mucosal tissues. Isolation of TMCs from the tonsils is quick, because the tonsils barely have any epithelium and yield up to billions of all major immune cell types. This method allows detection of cytokine production using several techniques, including immunoassays, qPCR, microscopy, flow cytometry, etc., similar to the use of peripheral mononuclear cells (PBMCs) from blood. Furthermore, TMCs show a higher sensitivity to drug testing than PBMCs, which needs to be considered for future toxicity assays. Thus, ex vivo TMCs cultures are an easy and accessible mucosal model.

In the present protocol, we explain how to easily process and culture tonsillar mononuclear cells from healthy humans undergoing partial surgical tonsillectomy to study innate immune responses upon activation, mimicking viral infection in mucosal tissues.

Studying isolated cells from mucosa-associated lymphoid tissues (MALT) allows understanding of immune cells response in pathologies involving mucosal ...

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 ...

Methods Article

Immunocompetent Alveolus-on-Chip Model for Studying Alveolar Mucosal Immune Responses