We would like to thank Patrick Hayden (MatTek) for helpful discussions, and Robert Smith and Libby Perry of Georgia Health Sciences University for their EM skills. This study was funded by NIDCD grant DC010187 to D.A.D.

1. Preparing the biosafety cabinet for the EpiAirway tissues
<ol>
<li>Wearing a dedicated lab coat, with hair tied back and gloves on, start the laminar flow. After 5 minutes, move any tools in the cabinet (pipettors, tips, centrifuge tubes, etc.) to one side, spray the biosafety cabinet interior and sash with 70% ethanol and wipe down with clean paper towels. Spray the cleaned interior again with 70% ethanol and leave to dry. Move the tools to the clean side and repeat the ethanol procedure.</li>
<li>Use aerosol barrier pipette tips and dedicated pipettors in the cabinet. To use individually-wrapped sterile serological pipettes, pass the plugged end through the laminar flow, break open, and slide the pipette into the cabinet, discarding the packaging outside.</li>
<li>The EpiAirway inserts must be handled with sterile forceps. Place 6-inch fine-tip curved dissecting forceps into self-sealing sterilization pouches and autoclave. Prepare enough to have at least six per day ready for use.</li>
</ol>
2. Unpacking the EpiAirway tissues
<ol>
<li>Spray the work surface in the biosafety cabinet with 70% ethanol and wipe down with clean paper towels. Spray again and allow the surface to dry prior to use. Open the EpiAirway box and discard the Styrofoam and packing material.</li>
<li>The EpiAirway antibiotic-free maintenance media, AIR-100-MM ABF (MM), is proprietary to MatTek and is sent in the kit with the inserts. In the biosafety cabinet, label six sterile 50 ml centrifuge tubes with the media type and date, and loosen the caps. Spray the media bottle with 70% ethanol and open inside the cabinet. Aliquot 25 ml of MM into each tube using a new sterile serological pipette for each tube, tighten the caps, and store at 4&deg;C. Use a fresh aliquot of MM each day.</li>
<li>Repeat 2.2 above using a fresh bottle of 1 X Dulbecco's phosphate-buffered saline (D-PBS) with calcium and magnesium. Repeat again using D-PBS without calcium and magnesium. Store all aliquots at 4&deg;C.</li>
<li>The EpiAirway inserts AIR-100 arrive in a 24-well plate at 4&deg;C on solid media-containing agarose, and must be placed into 6-well plates for use. Label each plate with the date and description using an ethanol-resistant marker. Place 1 milliliter of 4&deg;C EpiAirway MM into each well. Remove the transport plate from its packaging, spray with 70% ethanol, and place in the biosafety cabinet. Remove the tape and open the plate. Using autoclaved forceps, remove the damp gauze over the EpiAirway inserts.</li>
<li>Pick up an insert with autoclaved forceps using a twisting motion to aid in releasing it from the agarose, and place into a well of a 6-well plate. Some agarose may adhere to the insert, particularly as the temperature of the transport plate increases to ambient. Use a sterile cotton swab to carefully remove the agarose from the insert. Avoid touching the membrane, and check that no air bubbles are trapped beneath the inserts. Place the plate into a humidified 37&deg;C incubator with 5% CO2.</li>
<li>Allow at least 24 hours for the tissues to equilibrate in the incubator before starting co-cultures.</li>
</ol>
3. Maintenance of EpiAirway tissues
<ol>
<li>Using autoclaved forceps, pick up an insert and gently pipette 200 microliters of pre-warmed D-PBS with calcium and magnesium onto the tissue and rock the insert. Tilt the insert at an angle and place your sterile P1000 pipette tip against the plastic ring that holds the membrane at the bottom of the insert. Do not touch the tissue. Aspirate the D-PBS rinse and place into a labeled sterile cryovial. Samples can be frozen at -20&deg;C and assayed for mucin and/or cytokine expression at a later date.</li>
<li>Change the basal MM daily by aspirating and replacing with 1 milliliter of fresh MM. Use a new barrier pipette tip for each well. Treat the used media with 10% bleach overnight and discard.</li>
</ol>
4. Inoculation of EpiAirway tissues
<ol>
<li>Streak out a fresh culture of nontypeable Haemophilus influenzae (NTHi) from frozen glycerol stock onto chocolate agar. Grow overnight in a humidified 5% CO2 incubator.</li>
<li>The next day, resuspend single colonies of NTHi in pre-warmed D-PBS with calcium and magnesium to an OD (600 nm) of approximately 0.7. Vortex vigorously prior to measuring the optical density. The relationship of optical density to colony-forming units per ml (CFU/ml) of NTHi is that an OD (600 nm) of 0.2 approximately equals 1.0 x 108 CFU/ml. Therefore, an OD (600 nm) of 0.7 is about 3.5 x 108 CFU/ml. This algorithm should be validated for each NTHi strain prior to inoculation.</li>
<li>The EpiAirway tissues AIR-100 are grown on inserts with a surface area of 0.6 cm2 and a 0.4 micron membrane. The tissues are composed of ~8.0 x 105 to ~1.0 x 106 cells. Therefore, an inoculation of 1.0 x 107 CFU corresponds to a multiplicity of infection of about 10 - 12. To inoculate, rinse each EpiAirway insert as in 3.1, then add 28 microliters of the bacterial suspension prepared in 4.2 to the apical surface of each EpiAirway insert in the biosafety cabinet. Do not inoculate the basal MM. Return each plate to the incubator.</li>
</ol>
5. Quantification of the NTHi inoculum
<ol>
<li>Prepare a phosphate-buffered saline (PBS) solution with 0.1% gelatin (PBS-G), autoclave to sterilize. Label sterile 1.5 ml Eppendorf tubes with the desired 1:10 dilutions based on the OD (600 nm) of the inoculum and aliquot 900 microliters of the sterile PBS-G into each tube.</li>
<li>Determine the number of chocolate agar plates required to enumerate the inoculated NTHi. Place these plates in an air incubator upside down with the bottom of the plate resting on half of the top for one hour. This will allow the plate to both warm to 37&deg;C and the surface to dry, facilitating the drop-plating of NTHi.</li>
<li>Start the serial 1:10 dilutions of the inoculum by adding 100 microliters of the bacterial suspension from 4.2 to the first tube. Vortex each dilution vigorously for 5 seconds prior to making the next. Drop 10 microliter aliquots onto the pre-warmed and surface-dried chocolate agar plates, using half the plate for each dilution. Five to six 10 microliter aliquots can fit on one half of each 100 mm plate without running together. When dry, place upside down in a humidified 37&deg;C CO2 incubator overnight.</li>
<li>The next day, determine the CFU/ml by counting the dilutions that have 15-30 distinct colonies in each drop and calculating back to your actual viable inoculum of NTHi.</li>
</ol>
6. Long-term co-culture with NTHi
<ol>
<li>Every 24 hours, wash the inserts as in 3.1, then pool the washings of each tissue that has been inoculated with the same strain of bacteria and freeze at -20&deg;C in a labeled cryovial for future analysis. These samples can be assayed for the presence of mucin by dot blots or for the expression of cytokines by ELISA. Alternatively, tissues can be harvested for mRNA and qPCR can be performed on genes of interest. Change the basal MM daily as in 3.2. Continue for the duration of the co-culture.</li>
</ol>
7. Harvesting of EpiAirway tissues
<ol>
<li>Prepare a fresh 1% solution of saponin in D-PBS without calcium and magnesium, filter-sterilize and warm to 37&deg;C.</li>
<li>Warm a 50 ml tube of MM and another of D-PBS without calcium and magnesium to 37&deg;C.</li>
<li>Prepare dilution tubes as in 5.1, as well as one empty sterile 1.5 ml Eppendorf tube per insert, labeled with the insert number and NTHi strain.</li>
<li>The EpiAirways can be harvested for either total cell-associated bacteria, which includes both adherent and internalized organisms, or for internalized bacteria only.</li>
<li> Determine the number of chocolate agar plates needed to drop-plate the cell-associated or invaded NTHi, label and dry for one hour in an air incubator as in 5.2.</li>
<li>To harvest total cell-associated bacteria, rinse the apical surface of the EpiAirways three times with 200 microliters of D-PBS without calcium or magnesium, then tilt the insert and rinse any remaining MM from the basal membrane. The first wash can be frozen for later analysis; discard the following washes.</li>
<li>Place the washed insert in a fresh 6-well plate without MM, and pipette 250 microliters of the sterile 1% saponin solution from 7.1 onto the apical surface of each tissue. Return to the incubator for 10 minutes.</li>
<li>Remove from the incubator, and using a sterile P1000 pipette tip, scrub the tissues from the membrane using a back-and-forth motion, followed by a circular motion to remove the tissue from the edges of the insert. Using a large-bore sterile P1000 pipette tip, place the suspension into the empty labeled tube prepared in 7.3. Add 250 microliters of D-PBS without calcium or magnesium onto the apical surface of the insert.</li>
<li>Examine the insert using an inverted phase-contrast microscope to determine if there is any remaining tissue to be harvested. If needed, scrub again with another sterile P1000 pipette tip, then add this suspension to the tube from 7.8 using a large-bore sterile P1000 pipette tip. Bring the total volume of the tube to 1 milliliter using D-PBS without calcium or magnesium.</li>
<li>Vigorously vortex the cell suspension at top speed for one minute. Using a sterile 1 ml syringe fitted with a 26 gauge needle, aspirate the suspension into the syringe through the needle. Slowly pass the suspension through the needle 3 times, taking care not to create bubbles. Vortex vigorously again for one minute.</li>
<li>Using a large-bore pipette tip, place 100 microliters of the suspension into the first dilution tube prepared in 7.3. Vortex vigorously for five seconds, then make serial 1:10 dilutions. Drop-plate the desired dilutions onto the surface-dried chocolate agar plates.</li>
<li>To harvest internalized bacteria only, wash each insert 3 times with 200 microliters of D-PBS without calcium or magnesium. Retain and freeze the first wash. Add gentamicin sulfate to pre-warmed MM to a final concentration of 100 micrograms/ml. Prepare 1.5 ml of media per insert to be harvested. Replace the basal MM with the gentamicin-containing MM, and add 300 microliters of gentamicin-containing MM onto the apical surface. Place the plate back into the CO2 incubator for one hour.</li>
<li>Remove the gentamicin-containing MM from the apical surface and wash the apical surface and basal membrane of the insert extensively with pre-warmed D-PBS without calcium and magnesium. Proceed as in 7.7-7.11.</li>
</ol>
8. Representative results: 
Scanning electron micrographs of (A.) uninfected EpiAirway tissue and (B.) tissue after a 5-day co-culture with NTHi are shown in Figure 1. Long-term co-culture with NTHi does not result in significant damage to the apical tissues, underscoring the utility of the EpiAirway model. A graph depicting the number of internalized bacteria quantified over time inside tissues is shown in Figure 2. Both results are quite reproducible, making the EpiAirway tissues a consistent and biologically relevant in vitro model of the human upper airway in which to study NTHi host-pathogen interactions.
<img src="/files/ftp_upload/3261/3261fig1.jpg" alt="Figure 1"/> Figure 1. Scanning electron microscopy of EpiAirway tissues. A. Uninfected control tissue. B. Tissue after five days of co-culture with NTHi. No significant damage to the apical surface is observed in the infected tissues.
<img src="/files/ftp_upload/3261/3261fig2.jpg" alt="Figure 2"/> Figure 2. Number of NTHi internalized over time during EpiAirway co-culture. Strain R2866 was inoculated at approximately 1.0 x 107 CFU/insert (Day 0), then harvested for internalized bacteria at each indicated time point. Bars represent n=3 replicates in at least duplicate. Error bars are SD.

<ol>
	<li>Murphy, T. F., Apicella, M. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Nontypeable+Haemophilus+influenzae:+a+review+of+clinical+aspects,+surface+antigens,+and+the+human+immune+response+to+infection.">Nontypeable Haemophilus influenzae: a review of clinical aspects, surface antigens, and the human immune response to infection.</a> Rev. Infect. Dis. 9, 1-15 (1987).</li><li>Murphy, T. F., Faden, H., Bakaletz, L. O., Kyd, J. M., Forsgren, A., Campos, J., Virji, M., Pelton, S. I. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Nontypeable+Haemophilus+influenzae+as+a+pathogen+in+children.">Nontypeable Haemophilus influenzae as a pathogen in children.</a> Ped. Infect. Dis. J. 28, 43-48 (2009).</li><li>Hotomi, M., Arai, J., Billal, D. S., Takei, S., KIkeda, Y., Ogami, M., Kono, M., Beder, L. B., Toya, K., Kimura, M., Yamanaka, N. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Nontypeable+Haemophilus+influenzae+isolated+from+intractable+acute+otitis+media+internalized+into+cultured+human+epithelial+cells.">Nontypeable Haemophilus influenzae isolated from intractable acute otitis media internalized into cultured human epithelial cells.</a> Auris Nasus Larynx. 37, 137-144 (2010).</li><li>Chemuturi, N. V., Hayden, P., Kalausner, M., Donovan, M. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Comparison+of+human+tracheal/bronchial+epithelial+cell+culture+and+bovine+nasal+respiratory+explants+for+nasal+drug+transport+studies.">Comparison of human tracheal/bronchial epithelial cell culture and bovine nasal respiratory explants for nasal drug transport studies.</a> J. Pharm. Sci. 94, 1976-1985 (2005).</li><li>Sharma, M., Schoop, R., Hudson, J. B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+efficacy+of+Echinacea+in+a+3-D+tissue+model+of+human+airway+epithelium.">The efficacy of Echinacea in a 3-D tissue model of human airway epithelium.</a> Phytother. Res. 24, 900-904 (2010).</li><li>Sexton, K., Balharry, D., BeruBe, K. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Genomic+biomarkers+of+pulmonary+exposure+to+tobacco+smoke+components.">Genomic biomarkers of pulmonary exposure to tobacco smoke components.</a> Pharmacogenet Genomics. 10, 853-860 (2008).</li><li>Babu, R. J., Dayal, P., Singh, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Effect+of+cyclodextrins+on+the+complexation+and+nasal+permeation+of+melatonin.">Effect of cyclodextrins on the complexation and nasal permeation of melatonin.</a> Drug Deliv. 6, 381-388 (2008).</li><li>Balharry, D., Sexton, K., BeruBe, K. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=An+in+vitro+approach+to+assess+the+toxicity+of+inhaled+tobacco+smoke+components:+nicotine,+cadmium,+formaldehyde+and+urethane.">An in vitro approach to assess the toxicity of inhaled tobacco smoke components: nicotine, cadmium, formaldehyde and urethane.</a> Toxicology. 244, 66-76 (2008).</li><li>Fahy, J. V., Dickey, M. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Airway+mucus+function+and+dysfunction.">Airway mucus function and dysfunction.</a> N. Engl. J. Med. 363, 2233-2247 (2010).</li><li>Huang, Y., Mikami, F., Jono, H., Zhang, W., Weng, X., Koga, T., Xu, H., Yan, C., Kai, H., Li, J. -. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Opposing+roles+of+PAK2+and+PAK4+in+synergistic+induction+of+MUC5AC+mucin+by+bacterium+NTHi.">Opposing roles of PAK2 and PAK4 in synergistic induction of MUC5AC mucin by bacterium NTHi.</a> 359, 691-696 (2007).</li></ol>

No conflicts of interest declared.

This method allows the investigation of long-term host-pathogen interactions in a biologically relevant background of primary human respiratory tissues at the air-liquid interface. Here we have used NTHi as the infecting organism, but the interaction of any bacterium that does not introduce unacceptable cytotoxicity over time can be quantified with this method. The EpiAirway model can also be used for the study of viruses, drugs, or chemicals that impact the human upper airway5; 6; 7; 8. We have maintained uni...

<table border="1">
	<tbody>
		<tr>
			<th>
				Name of reagent</th>
			<th>
				Company</th>
			<th>
				Catalogue#</th>
			<th>
				Comments</th>
		</tr>
		<tr>
			<td>Saponin</td>
			<td>Calbiochem</td>
			<td>558255-25GM</td>
			<td>1% in D-PBS without calcium or magnesium, filter sterilize</td>
		</tr>
		<tr>
			<td>1 X Dulbecco's phosphate-buffered saline with calcium and magnesium</td>
			<td>Lonza</td>
			<td>17-513Q</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>1 X Dulbecco's phosphate-buffered saline without calcium or magnesium</td>
			<td>Lonza</td>
			<td>17-515Q</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>EpiAirway antibiotic-free tissues</td>
			<td>MatTek</td>
			<td>AIR-100-ABF</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>EpiAirway antibiotic-free maintenance media</td>
			<td>MatTek</td>
			<td>AIR-100-MM-ABF</td>
			<td>Supplied with kit</td>
		</tr>
		<tr>
			<td>10X phosphate-buffered saline solution</td>
			<td>EMD</td>
			<td>6506</td>
			<td>Dilute to 1X before use</td>
		</tr>
		<tr>
			<td>Gelatin</td>
			<td>JT Baker</td>
			<td>2124-01</td>
			<td>Add to a final concentration of 0.1% in 1 X PBS and autoclave</td>
		</tr>
		<tr>
			<td>Difco GC Medium Base (chocolate agar)</td>
			<td>VWR</td>
			<td>90002-016</td>
			<td>Autoclave 36 g in 500 ml ddH2O and cool to 60&deg;C</td>
		</tr>
		<tr>
			<td>BBL Hemoglobin (chocolate agar)</td>
			<td>VWR</td>
			<td>90000-662</td>
			<td>Autoclave 10 g in 500 ml ddH2O, cool to 60&deg;C and mix with the GC medium base above</td>
		</tr>
		<tr>
			<td>BD BBL IsoVitaleX enrichment (chocolate agar)</td>
			<td>VWR</td>
			<td>90000-414</td>
			<td>Cool the mixture of GC medium base and hemoglobin to 55&deg;C and add 10 ml of rehydrated IsoVitaleX, pour chocolate agar plates</td>
		</tr>
		<tr>
			<td>Dissecting forceps, fine tip, curved</td>
			<td>VWR</td>
			<td>82027-406</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>Self-sealing sterilization pouches</td>
			<td>VWR</td>
			<td>89140-802</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>Gentamicin sulfate, 10 mg/ml</td>
			<td>Lonza</td>
			<td>17-519Z</td>
			<td>Add 10 microliters/ml to EpiAirway MM for the gentamicin kill</td>
		</tr>
	</tbody>
</table>

use of the epiairway model for characterizing long-term host-pathogen interactions

Nontypeable Haemophilus influenzae (NTHi) are human-adapted Gram-negative bacteria that can cause recurrent and chronic infections of the respiratory mucosa 1; 2. To study the mechanisms by which these organisms survive on and inside respiratory tissues, a model in which successful long-term co-culture of bacteria and human cells can be performed is required. We use primary human respiratory epithelial tissues raised to the air-liquid interface, the EpiAirway model (MatTek, Ashland, MA). These are non-immortalized, well-differentiated, 3-dimensional tissues that contain tight junctions, ciliated and nonciliated cells, goblet cells that produce mucin, and retain the ability to produce cytokines in response to infection. 
This biologically relevant in vitro model of the human upper airway can be used in a number of ways; the overall goal of this method is to perform long-term co-culture of EpiAirway tissues with NTHi and quantitate cell-associated and internalized bacteria over time. As well, mucin production and the cytokine profile of the infected co-cultures can be determined. This approach improves upon existing methods in that many current protocols use submerged monolayer or Transwell cultures of human cells, which are not capable of supporting bacterial infections over extended periods3. For example, if an organism can replicate in the overlying media, this can result in unacceptable levels of cytotoxicity and loss of host cells, arresting the experiment. The EpiAirway model allows characterization of long-term host-pathogen interactions. Further, since the source for the EpiAirway is normal human tracheo-bronchial cells rather than an immortalized line, each is an excellent representation of actual human upper respiratory tract tissue, both in structure and in function4.
For this method, the EpiAirway tissues are weaned off of anti-microbial and anti-fungal compounds for 2 days prior to delivery, and all procedures are performed under antibiotic-free conditions. This necessitates special considerations, since both bacteria and primary human tissues are used in the same biosafety cabinet, and are co-cultured for extended periods.

Watch this Scientific Journal Video about Use of the EpiAirway Model for Characterizing Long-term Host-pathogen Interactions at JoVE.com

Use of the EpiAirway Model for Characterizing Long-term Host-pathogen Interactions

This method allows characterization of extended bacterial co-culture with EpiAirways, primary human respiratory epithelial tissue grown at the air-liquid interface, a biologically relevant in vitro model. The approach can be used with any microbe that is amenable to long-term co-culture.

Nontypeable Haemophilus influenzae  (NTHi) are human-adapted Gram-negative bacteria that can cause recurrent and chronic infections of the respiratory ...

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Immunology and Infection

11.6K Views.  Mercer University School of Medicine. This method allows characterization of extended bacterial co-culture with EpiAirways, primary human respiratory epithelial tissue grown at the air-liquid interface, a biologically relevant in vitro model. The approach can be used with any microbe that is amenable to long-term co-culture.

Video: Use of the EpiAirway Model for Characterizing Long-term Host-pathogen Interactions

Use of an Optical Trap for Study of Host-Pathogen Interactions for Dynamic Live Cell Imaging

Dynamic live cell imaging allows direct visualization of real-time interactions between cells of the immune system1, 2; however, the lack of spatial and temporal control between the phagocytic cell and microbe has rendered focused observations into the initial interactions of host response to pathogens difficult. Historically, intercellular contact events such as phagocytosis3 have been imaged by mixing two cell types, and then continuously scanning the field-of-view to find serendipitous intercellular contacts at the appropriate stage of interaction. The stochastic nature of these events renders this process tedious, and it is difficult to observe early or fleeting events in cell-cell contact by this approach. This method requires finding cell pairs that are on the verge of contact, and observing them until they consummate their contact, or do not. To address these limitations, we use optical trapping as a non-invasive, non-destructive, but fast and effective method to position cells in culture.
Optical traps, or optical tweezers, are increasingly utilized in biological research to capture and physically manipulate cells and other micron-sized particles in three dimensions4. Radiation pressure was first observed and applied to optical tweezer systems in 19705, 6, and was first used to control biological specimens in 19877. Since then, optical tweezers have matured into a technology to probe a variety of biological phenomena8-13.
We describe a method14 that advances live cell imaging by integrating an optical trap with spinning disk confocal microscopy with temperature and humidity control to provide exquisite spatial and temporal control of pathogenic organisms in a physiological environment to facilitate interactions with host cells, as determined by the operator. Live, pathogenic organisms like Candida albicans and Aspergillus fumigatus, which can cause potentially lethal, invasive infections in immunocompromised individuals15, 16 (e.g. AIDS, chemotherapy, and organ transplantation patients), were optically trapped using non-destructive laser intensities and moved adjacent to macrophages, which can phagocytose the pathogen. High resolution, transmitted light and fluorescence-based movies established the ability to observe early events of phagocytosis in living cells. To demonstrate the broad applicability in immunology, primary T-cells were also trapped and manipulated to form synapses with anti-CD3 coated microspheres in vivo, and time-lapse imaging of synapse formation was also obtained. By providing a method to exert fine spatial control of live pathogens with respect to immune cells, cellular interactions can be captured by fluorescence microscopy with minimal perturbation to cells and can yield powerful insight into early responses of innate and adaptive immunity.

A method is described to individually select, manipulate, and image live pathogens using an optical trap coupled to a spinning disk microscope. The optical trap provides spatial and temporal control of organisms and places them adjacent to host cells. Fluorescence microscopy captures dynamic intercellular interactions with minimal perturbation to cells.

Dynamic live cell imaging allows direct visualization of real-time interactions between cells of the immune system1, 2; however, the lack of spatial and ...

A Comparative Approach to Characterize the Landscape of Host-Pathogen Protein-Protein Interactions

Significant efforts were gathered to generate large-scale comprehensive protein-protein interaction network maps. This is instrumental to understand the pathogen-host relationships and was essentially performed by genetic screenings in yeast two-hybrid systems. The recent improvement of protein-protein interaction detection by a Gaussia luciferase-based fragment complementation assay now offers the opportunity to develop integrative comparative interactomic approaches necessary to rigorously compare interaction profiles of proteins from different pathogen strain variants against a common set of cellular factors.
This paper specifically focuses on the utility of combining two orthogonal methods to generate protein-protein interaction datasets: yeast two-hybrid (Y2H) and a new assay, high-throughput Gaussia princeps protein complementation assay (HT-GPCA) performed in mammalian cells.
A large-scale identification of cellular partners of a pathogen protein is performed by mating-based yeast two-hybrid screenings of cDNA libraries using multiple pathogen strain variants. A subset of interacting partners selected on a high-confidence statistical scoring is further validated in mammalian cells for pair-wise interactions with the whole set of pathogen variants proteins using HT-GPCA. This combination of two complementary methods improves the robustness of the interaction dataset, and allows the performance of a stringent comparative interaction analysis. Such comparative interactomics constitute a reliable and powerful strategy to decipher any pathogen-host interplays.

This article focuses on the identification of high-confident interaction datasets between host and pathogen proteins using a combination of two orthogonal methods: yeast two-hybrid followed by a high-throughput interaction assay in mammalian cells called HT-GPCA.

Significant efforts were gathered to generate  large-scale comprehensive protein-protein interaction network maps. This is  instrumental to understand the ...

Long Term Chronic Pseudomonas aeruginosa Airway Infection in Mice

A mouse model of chronic airway infection is a key asset in cystic fibrosis (CF) research, although there are a number of concerns regarding the model itself. Early phases of inflammation and infection have been widely studied by using the Pseudomonas aeruginosa agar-beads mouse model, while only few reports have focused on the long-term chronic infection in vivo. The main challenge for long term chronic infection remains the low bacterial burden by P. aeruginosa and the low percentage of infected mice weeks after challenge, indicating that bacterial cells are progressively cleared by the host.
This paper presents a method for obtaining efficient long-term chronic infection in mice. This method is based on the embedding of the P. aeruginosa clinical strains in the agar-beads in vitro, followed by intratracheal instillation in C57Bl/6NCrl mice. Bilateral lung infection is associated with several measurable read-outs including weight loss, mortality, chronic infection, and inflammatory response. The P. aeruginosa RP73 clinical strain was preferred over the PAO1 reference laboratory strain since it resulted in a comparatively lower mortality, more severe lesions, and higher chronic infection. P. aeruginosa colonization may persist in the lung for over three months. Murine lung pathology resembles that of CF patients with advanced chronic pulmonary disease.
This murine model most closely mimics the course of the human disease and can be used both for studies on the pathogenesis and for the evaluation of novel therapies.

Long Term Chronic Pseudomonas aeruginosa Airway Infection in Mice

We describe the agar-beads method to establish persistent long-term chronic Pseudomonas aeruginosa airway infection in the mouse model.

A mouse model of chronic airway infection is a key asset in cystic fibrosis (CF) research, although there are a number of concerns regarding the model ...

Use of Shigella flexneri to Study Autophagy-Cytoskeleton Interactions

Shigella flexneri is an intracellular pathogen that can escape from phagosomes to reach the cytosol, and polymerize the host actin cytoskeleton to promote its motility and dissemination. New work has shown that proteins involved in actin-based motility are also linked to autophagy, an intracellular degradation process crucial for cell autonomous immunity. Strikingly, host cells may prevent actin-based motility of S. flexneri by compartmentalizing bacteria inside &#8216;septin cages&#8217; and targeting them to autophagy. These observations indicate that a more complete understanding of septins, a family of filamentous GTP-binding proteins, will provide new insights into the process of autophagy. This report describes protocols to monitor autophagy-cytoskeleton interactions caused by S. flexneri in vitro using tissue culture cells and in vivo using zebrafish larvae. These protocols enable investigation of intracellular mechanisms that control bacterial dissemination at the molecular, cellular, and whole organism level.

Use of Shigella flexneri to Study Autophagy-Cytoskeleton Interactions

To counteract pathogen dissemination, host cells reorganize their cytoskeleton to compartmentalize bacteria and induce autophagy. Using Shigella infection of tissue culture cells, host and pathogen determinants underlying this process are identified and characterized. Using zebrafish models of Shigella infection, the role of discovered molecules and mechanisms are investigated in vivo.

Shigella flexneri is an intracellular pathogen that can escape from phagosomes to reach the cytosol, and polymerize the host actin cytoskeleton to promote ...

Long Term Intravital Multiphoton Microscopy Imaging of Immune Cells in Healthy and Diseased Liver Using CXCR6.Gfp Reporter Mice

Liver inflammation as a response to injury is a highly dynamic process involving the infiltration of distinct subtypes of leukocytes including monocytes, neutrophils, T cell subsets, B cells, natural killer (NK) and NKT cells. Intravital microscopy of the liver for monitoring immune cell migration is particularly challenging due to the high requirements regarding sample preparation and fixation, optical resolution and long-term animal survival. Yet, the dynamics of inflammatory processes as well as cellular interaction studies could provide critical information to better understand the initiation, progression and regression of inflammatory liver disease. Therefore, a highly sensitive and reliable method was established to study migration and cell-cell-interactions of different immune cells in mouse liver over long periods (about 6 hr) by intravital two-photon laser scanning microscopy (TPLSM) in combination with intensive care monitoring.
The method provided includes a gentle preparation and stable fixation of the liver with minimal perturbation of the organ; long term intravital imaging using multicolor multiphoton microscopy with virtually no photobleaching or phototoxic effects over a time period of up to 6 hr, allowing tracking of specific leukocyte subsets; and stable imaging conditions due to extensive monitoring of mouse vital parameters and stabilization of circulation, temperature and gas exchange.
To investigate lymphocyte migration upon liver inflammation CXCR6.gfp knock-in mice were subjected to intravital liver imaging under baseline conditions and after acute and chronic liver damage induced by intraperitoneal injection(s) of carbon tetrachloride (CCl4).
CXCR6 is a chemokine receptor expressed on lymphocytes, mainly on Natural Killer T (NKT)-, Natural Killer (NK)- and subsets of T lymphocytes such as CD4 T cells but also mucosal associated invariant (MAIT) T cells1. Following the migratory pattern and positioning of CXCR6.gfp+ immune cells allowed a detailed insight into their altered behavior upon liver injury and therefore their potential involvement in disease progression.

Stable intravital high-resolution imaging of immune cells in the liver is challenging. Here we provide a highly sensitive and reliable method to study migration and cell-cell-interactions of immune cells in mouse liver over long periods (about 6 hours) by intravital multiphoton laser scanning microscopy in combination with intensive care monitoring.

Liver inflammation as a response to injury is a highly dynamic process involving the infiltration of distinct subtypes of leukocytes including monocytes, ...

Evaluation of the Efficacy And Toxicity of RNAs Targeting HIV-1 Production for Use in Gene or Drug Therapy

Small RNA therapies targeting post-integration steps in the HIV-1 replication cycle are among the top candidates for gene therapy and have the potential to be used as drug therapies for HIV-1 infection. Post-integration inhibitors include ribozymes, short hairpin (sh) RNAs, small interfering (si) RNAs, U1 interference (U1i) RNAs and RNA aptamers. Many of these have been identified using transient co-transfection assays with an HIV-1 expression plasmid and some have advanced to clinical trials. In addition to measures of efficacy, small RNAs have been evaluated for their potential to affect the expression of human RNAs, alter cell growth and/or differentiation, and elicit innate immune responses. In the protocols described here, a set of transient transfection assays designed to evaluate the efficacy and toxicity of RNA molecules targeting post-integration steps in the HIV-1 replication cycle are described. We have used these assays to identify new ribozymes and optimize the format of shRNAs and siRNAs targeting HIV-1 RNA. The methods provide a quick set of assays that are useful for screening new anti-HIV-1 RNAs and could be adapted to screen other post-integration inhibitors of HIV-1 replication.

Methods to evaluate the efficacy and toxicity of RNA molecules targeting post-integration steps of the HIV-1 replication cycle are described. These methods are useful for screening new molecules and optimizing the format of existing ones.

Small RNA therapies targeting post-integration steps in the HIV-1 replication cycle are among the top candidates for gene therapy and have the potential ...

An IL-8 Transiently Transgenized Mouse Model for the In Vivo Long-term Monitoring of Inflammatory Responses

Airway inflammation is often associated with bacterial infections and represents a major determinant of lung disease. The in vivo determination of the pro-inflammatory capabilities of various factors is challenging and requires terminal procedures, such as bronchoalveolar lavage and the removal of lungs for in situ analysis, precluding longitudinal visualization in the same mouse. Here, lung inflammation is induced through the intratracheal instillation of Pseudomonas aeruginosa culture supernatant (SN) in transiently transgenized mice expressing the luciferase reporter gene under the control of a heterologous IL-8 bovine promoter. Luciferase expression in the lung is monitored by in vivo bioluminescent image (BLI) analysis over a 2.5- to 48-h timeframe following the instillation. The procedure can be repeated multiple times within 2 - 3 months, thus permitting the evaluation of the inflammatory response in the same mice without the need to terminate the animals. This approach permits the monitoring of pro- and anti-inflammatory factors acting in the lung in real time and appears suitable for functional and pharmacological studies.

An IL-8 Transiently Transgenized Mouse Model for the In Vivo Long-term Monitoring of Inflammatory Responses

The method described here allows for the visualization of IL-8 promoter-dependent inflammation activation in the lungs of mice through non-invasive bioluminescence imaging (BLI). The same animal can be subjected to BLI multiple times for up to two months from the time of delivery of the luciferase reporter construct.

Airway inflammation is often associated with bacterial infections and represents a major determinant of lung disease. The in vivo determination of the ...

Long-term In Vivo Tracking of Inflammatory Cell Dynamics Within Drosophila Pupae

During the rapid inflammatory response to tissue damage, cells of the innate immune system are quickly recruited to the injury site. Once at the wound, innate immune cells perform a number of essential functions, such as fighting infection, clearing necrotic debris, and stimulating matrix deposition. In order to fully understand the diverse signaling events that regulate this immune response, it is crucial to observe the complex behaviors of (and interactions that occur between) multiple cell lineages in vivo, and in real-time, with the high spatio-temporal resolution. The optical translucency and the genetic tractability of Drosophila embryos have established Drosophila as an invaluable model to live-image and dissect fundamental aspects of inflammatory cell behavior, including mechanisms of developmental dispersal, clearance of apoptotic corpses&#160;and/or microbial pathogens, and recruitment to wounds. However, more recent work has now demonstrated that employing a much later stage in the Drosophila lifecycle &#8211; the Drosophila pupa &#8211; offers a number of distinct advantages, including improved RNAi efficiency, longer imaging periods, and significantly greater immune cell numbers. Here we describe a protocol for imaging wound repair and the associated inflammatory response at the high spatio-temporal resolution in live Drosophila pupae. To follow the dynamics of both re-epithelialization and inflammation, we use a number of specific in vivo fluorescent markers for both the epithelium and innate immune cells. We also demonstrate the effectiveness of photo-convertible fluorophores, such as Kaede, for following the specific immune cell subsets, to track their behavior as they migrate to, and resolve from, the injury site.

Long-term In Vivo Tracking of Inflammatory Cell Dynamics Within Drosophila Pupae

Here we present a protocol for live-imaging wound repair and the associated inflammatory response at high spatio-temporal resolution in vivo. This method utilizes the pupal stage of Drosophila development to enable long-term imaging and tracking of specific cell populations over time and is compatible with efficient RNAi-mediated gene inactivation.

During the rapid inflammatory response to tissue damage, cells of the innate immune system are quickly recruited to the injury site. Once at the wound, ...

Complementary Use of Microscopic Techniques and Fluorescence Reading in Studying Cryptococcus-Amoeba Interactions

To simulate Cryptococcus infection, amoeba, which is the natural predator of cryptococcal cells in the environment, can be used as a model for macrophages. This predatory organism, similar to macrophages, employs phagocytosis to kill internalized cells. With the aid of a confocal laser-scanning microscope, images depicting interactive moments between cryptococcal cells and amoeba are captured. The resolution power of the electron microscope also helps to reveal the ultrastructural detail of cryptococcal cells when trapped inside the amoeba food vacuole. Since phagocytosis is a continuous process, quantitative data is then integrated in the analysis to explain what happens at the timepoint when an image is captured. To be specific, relative fluorescence units are read in order to quantify the efficiency of amoeba in internalizing cryptococcal cells. For this purpose, cryptococcal cells are stained with a dye that makes them fluoresce once trapped inside the acidic environment of the food vacuole. When used together, information gathered through such techniques can provide critical information to help draw conclusions on the behavior and fate of cells when internalized by amoeba and, possibly, by other phagocytic cells.

Complementary Use of Microscopic Techniques and Fluorescence Reading in Studying Cryptococcus-Amoeba Interactions

This paper details a protocol for preparing a co-culture of cryptococcal cells and amoebae that is studied using still, fluorescent images and high-resolution transmission electron microscope images. Illustrated here is how quantitative data can complement such qualitative information.

To simulate Cryptococcus infection, amoeba, which is the natural predator of cryptococcal cells in the environment, can be used as a model for ...

Evaluation of Host-Pathogen Responses and Vaccine Efficacy in Mice

Vaccines are a 20th century medical marvel. They have dramatically reduced the morbidity and mortality caused by infectious diseases and contributed to a striking increase in life expectancy around the globe. Nonetheless, determining vaccine efficacy remains a challenge. Emerging evidence suggests that the current acellular vaccine (aPV) for Bordetella pertussis (B. pertussis) induces suboptimal immunity. Therefore, a major challenge is designing a next-generation vaccine that induces protective immunity without the adverse side effects of a whole-cell vaccine (wPV). Here we describe a protocol that we used to test the efficacy of a promising, novel adjuvant that skews immune responses to a protective Th1/Th17 phenotype and promotes a better clearance of a B. pertussis challenge from the murine respiratory tract. This article describes the protocol for mouse immunization, bacterial inoculation, tissue harvesting, and analysis of immune responses. Using this method, within our model, we have successfully elucidated crucial mechanisms elicited by a promising, next-generation acellular pertussis vaccine. This method can be applied to any infectious disease model in order to determine vaccine efficacy.

Here we present an elegant protocol for in vivo evaluation of vaccine effectiveness and host immune responses. This protocol can be adapted for vaccine models that study viral, bacterial, or parasitic pathogens.

Vaccines are a 20th century medical marvel. They have dramatically reduced the morbidity and mortality caused by infectious diseases and contributed to a ...