Name: modeling asthma and influenza co-morbidity in c57bl/6j mice
Uploaded: 2024-04-17T04:01:56.000+00:00
Description: Allergic asthma and influenza are both diseases of the pulmonary system that affect millions worldwide. During the 2009 influenza pandemic, asthma was ...

We would like to thank Ms. Amy Iverson in the Department of Infectious Diseases at St. Jude Children's Research Hospital for technical training on influenza virus culture and infections and Dr. Stacie Woolard in the Department of Flow Cytometry at St. Jude Children's Research Hospital for help in setting up the 14 color antibody cocktail for analysis. We would also like to thank Arthur Covington and Shelby Patrick of St. Jude for animal husbandry and care. These studies were supported by the National Institute of Allergy and Infectious Diseases, the National Institutes of Health, under contract number HHSN26620070005C, Centers of Excellence in Influenza Research and Surveillance (CEIRS), N 01 7005C (JAM) and Le Bonheur Junior Faculty Grant (Samarasinghe).

Ethics Statement. All experiments were approved by the Institutional Animal Use and Care Committee at St. Jude Children&#39;s Research Hospital.
Note: All described experiments in this protocol must be conducted under BSL-2 and ABSL-2 conditions since Aspergillus fumigatus and influenza A virus are BSL-2 agents.
1. Modeling Allergic Asthma in Mice (Figure 1)
<ol>
	<li>Fungal sensitization
	<ol>
		<li>Re-constitute A. fumigatus antigen in sterile PBS at 1 mg protein/mL. Aliquot and store at -20&#176;C.</li>
		<li>House animals in a micro-isolator cages with autoclaved paper bedding under specific pathogen-free conditions with ad libitum food and water in a 12 hour light:dark cycle. Obtain C57BL/6J female mice at six weeks of age and acclimatize for one week. Start mice on the asthma protocol at seven weeks of age.</li>
		<li>Dilute A. fumigatus antigen to 20 &#181;g/mL in sterile PBS and add dropwise to an equal volume of Alum while stirring. Allow antigen to be adsorbed for 30 mins at room temperature with constant stirring.</li>
		<li>Fill 1 mL luer-lock syringes with the mixture and deliver 0.1 mL intraperitoneally and 0.1 mL subcutaneously between the ears to each mouse. Administer PBS and Alum as a mock-sensitization to mice that serve as na&#239;ve controls.</li>
		<li>Two weeks after the initial sensitization, dilute A. fumigatus antigen to a concentration of 200 &#181;g/mL in sterile PBS and deliver 20 &#181;L intranasally to each mouse. Repeat once weekly for a total of three intranasal administrations. Administer PBS as a mock-sensitization to mice that serve as na&#239;ve controls.</li>
	</ol>
	</li>
	<li>Fungal challenge
	<ol>
		<li>Prepare Sabouraud dextrose agar at 65 g/L per manufacturer&#39;s guidelines and sterilize in an autoclave at 121&#176;C for 15 mins. Place in a water bath set at 50&#176;C. to temper.</li>
		<li>Once tempered, aseptically add 10 mL to 25 cm2 canted neck, vented cap cell culture flasks. Lay the flasks flat and allow agar to set. Once set, recap flasks and store at 4&#176;C until use.</li>
		<li>Re-suspend the A. fumigatus culture aseptically by adding 5 mL of sterile PBS. Store overnight at 4&#176;C. Prepare 60 &#181;L aliquots for use and store at 4&#176;C.</li>
		<li>Eight days before the final intranasal administration of A. fumigatus antigen, culture A. fumigatus on Sabouraud dextrose agar for the fungal challenge. Add one well vortexed 60 &#181;L aliquot of fungus in each agar flask and spread using a disposable inoculating loop. Incubate flasks at 37&#176;C for 8 days.</li>
		<li>Set up the inhalation challenge apparatus in a class II biological safety cabinet as described in detail by Schuh and Hoselton.7 Create holes in the fungal culture flasks, one on the rear top and one on the bottom of the flask immediately above the culture by using a cork-borer.</li>
		<li>Attach the fungal culture flask to the regulated medical grade air supply through the bottom hole and to the inhalation chamber through the top hole. Cover the inhalation ports with laboratory tape.</li>
		<li>Run medical grade air over the culture at 2 psi for 10 mins to coat the chamber. Prepare sterile anesthetic cocktail with 75 mg/kg ketamine and 25 mg/kg xylazine.</li>
		<li>Inject the drug cocktail to three mice intraperitoneally and determine anesthesia by toe pinch reflex and lack of movement. Apply a drop of lubricating ointment in each eye to prevent drying out.</li>
		<li>Turn off the air, and replace the fungal flask with a new one and open the ports of the inhalation chamber.</li>
		<li>Place the anesthetized mice supine with their noses in the ports. Turn on the air over the culture and allow the animals to inhale the liberated fungal conidia for 10 mins.</li>
		<li>Place each animal supine in a new cage and place the cage on a heating pad. Monitor animals for recovery from anesthesia marked by turning over and movement around the cage.</li>
		<li>Repeat procedure after 14 days. Anesthetize animals that serve as na&#239;ve controls and place in a new cage.</li>
	</ol>
	</li>
</ol>
2. Modeling Influenza in Mice
NOTE: Adjust the age, gender, and strain of mice and the dose of the virus according to the experimental goals.
<ol>
	<li>Obtain female C57BL/6J mice at 14 or 17 weeks of age and acclimatize to the facility in micro-isolator cages with autoclaved paper bedding and ad libitum food and water in a 12 hour light:dark cycle. Number animals in each cage by tail mark or ear notch. One week later, weigh each mouse and record weight with date and time.</li>
	<li>Thaw a stock of A/CA/04/2009 virus and dilute in sterile PBS at log104.305 TCID50/mL in a glass vial. Anesthetize mice with 2.5% isoflurane with oxygen in an anesthesia chamber within the class II biological safety cabinet.</li>
	<li>Deliver 50 &#181;L of virus intranasally to each mouse for a final dose of 1000 TCID50 pH1N1 per mouse. Hold mouse supine until conscious and place in new micro-isolator cage. Administer PBS under anesthesia to na&#239;ve controls.</li>
	<li>Record animal weights every 24 hours until experimental endpoints are reached. 
	NOTE: It is good practice to use the same stock virus tube(s) to infect all animals in the study and not use previously thawed tubes as the viral titer may decrease during repeated freeze-thaw cycles.</li>
</ol>
3. Modeling Asthma and Influenza Co-morbidity in Mice (Figures 2 and 3)
<ol>
	<li>To mimic acute asthma and influenza, infect animals one week after the second fungal challenge with pH1N1 as noted in section 2. To mimic chronic asthma and influenza, infect animals 4 weeks after the second fungal challenge.</li>
</ol>
4. Pulmonary function test
<ol>
	<li>Weigh and record each mouse weight.</li>
	<li>Perform pulmonary function test on each animal. Note: Of the various equipment available to measure pulmonary function in rodents, we have opted to use the flexiVent FX1 module and flexiware software as previously described.22
	<ol>
		<li>Open the software and go to &#34;Study Definition and Planning.&#34; Click on &#34;Create a new study&#34; to launch the Study Definition Wizard.</li>
		<li>Follow the Wizard to input details of the study: Title and Status, Objective and Hypothesis; Protocol; Subject (animal) Groups.</li>
		<li>Create subjects by clicking on the mouse icon and add all necessary information on the animal including the Group Membership which will assign subjects to Subject Groups.</li>
		<li>Verify all information and confirm to close the wizard.</li>
	</ol>
	</li>
	<li>After concluding the &#34;Study Definition and Planning&#34; session, open the &#34;Experimentation Session&#34; in the software. Follow the steps of the pop-up wizard to set-up the instrument.
	<ol>
		<li>Select the Study (see 4.2.1) and the Experiment Template which depends on the instrument.</li>
		<li>Add details as deemed necessary to Session Properties. The instrument will then be connected to the software.</li>
		<li>Select the instrument module attached to the software when promoted and click OK.</li>
		<li>Select a subject in the Study to Site 1 and enter the current weight of the animal when prompted.</li>
		<li>Add 0.5 mL of saline to the finemist nebulizer and click OK when prompted to Prime the nebulizer (the generated mist will be visible). Attach the nebulizer to the instrument adaptor when complete.</li>
		<li>Perform Channel Calibrations when prompted. Calibrate the Cylinder Pressure and Airway Opening Pressure following the software wizard using a manometer to apply known pressures.</li>
		<li>At the conclusion of Channel Calibrations, the Tube Calibration Wizard will be initiated; attach an 18 g cannula to the Y-tubing adaptor and select the desired perturbations. Perform closed and open calibrations and verify that Calibration Results are void of errors.</li>
		<li>Once the calibrations are complete, select whether to start recording continuous data when prompted. Click &#34;Yes&#34; to start the default ventilation pattern. The instrument is ready to gather data.</li>
	</ol>
	</li>
	<li>Prepare a stock acetyl-&#946;-methylcholine (A&#946;M) at 125 mg/mL in sterile saline; freeze aliquots at -20&#176;C until use. Dilute the frozen stock of A&#946;M to the desired doses in sterile saline (3.125 mg/mL, 6.25 mg/mL, 12.5 mg/mL, and 25 mg/mL).</li>
	<li>Prepare anesthetics in sterile PBS before procedure. Administer 10 mg/kg body weight xylazine intraperitoneally and place mouse its own cage to rest for 5 min. Administer 70 mg/kg body weight pentobarbital intraperitoneally and allow mouse to rest for 5 min.</li>
	<li>Intubate mouse as previously described.23
	<ol>
		<li>Briefly, place the anesthetized mouse supine on a slanted surgical board and fix in place with plastic tape over the forelimbs and tail.</li>
		<li>Make a small incision between the upper thorax and lower jaw. Separate the salivary glands and thyroid gland to expose the trachea. Gently pass curved surgical forceps underneath the trachea and pull a piece of thread underneath.</li>
		<li>Perform tracheostomy between two tracheal rings with sharp scissors taking care not to cut through the trachea.</li>
		<li>Insert the 18 g cannula that was used to calibrate the instrument. Fasten the cannula to the trachea with thread and attach the mouse to the Y-tubing of the instrument adaptor with the cannula.</li>
	</ol>
	</li>
	<li>Perform a deep inflation to recruit the airways and help the animal succumb to the ventilator. Select the &#34;mouse inhaled dose response v7.0&#34; script with default ventilation at 150 breaths/minute.</li>
	<li>Add saline followed by each incremental dose of A&#946;M to the nebulizer when prompted to perform a dose response curve.</li>
	<li>At the conclusion of the data acquisition after the highest A&#946;M dose, disconnect the mouse from the ventilator and humanely euthanize the animal by performing a cervical dislocation. Proceed to tissue harvest.</li>
	<li>Add saline to the nebulizer and activate the nebulizer to remove any remnant high dose A&#946;M. Change the subject in the software program and proceed with next mouse beginning with a tube calibration (step 4.1-4.3).</li>
</ol>
5. Tissue harvest
<ol>
	<li>Spray euthanized mouse with 70% ethanol and open the thoracic cavity. Open diaphragm and perform bronchoalveolar lavage (BAL) with 1 mL of sterile PBS injected through the 18 g cannula and gently draw out repeating once for a total of 2 mL.24 Place the sample in ice.</li>
	<li>Harvest the cardiac and diaphragmatic lobes of the right lung for RNA work, and azygous and apical lobes for protein work, place in separate 5 mL snap-cap tubes and snap freeze in liquid nitrogen.</li>
	<li>Harvest the left lobe using blunt forceps and scissors taking care not to damage/slice through the lobe. Fix the lung ex vivo with 10% normal buffered formalin with a 30 g needle. Store in 10% normal buffered formalin until histological analyses. 
	NOTE: When animals are taken through the model for determination of viral burden in the lungs, harvest whole lungs after euthanasia and snap freeze in liquid nitrogen.</li>
</ol>
6. Tissue processing for Flow Cytometry
<ol>
	<li>Centrifuge the BAL contents at 600 g for 10 mins at 4&#176;C and save the BAL fluid for protein assays at -80&#176;C until use.</li>
	<li>Perform red blood cell lysis if necessary noting that cells are more susceptible to death after viral infection. Perform red cell lysis by adding 8 parts cold distilled water and pulse-vortexing for 30 seconds followed by a one-minute incubation at room temperature. Add one part 10 PBS, mix to neutralize the water and centrifuge as above.</li>
	<li>Re-suspend the BAL cells in 200 &#181;L of staining media (sterile PBS with 5% FBS) and enumerate cells by trypan blue dye exclusion assay to determine the cell concentration.</li>
	<li>Cytospin 50,000 cells as follows:
	<ol>
		<li>Transfer the calculated volume of cells into a tube and bring volume up to 100 &#181;L with sterile PBS. Assemble the labeled slide and cytofunnel in the metal clip and place in the centrifuge.</li>
		<li>Add the diluted cells into the cytofunnel and spin at 55 g for 5 minutes to affix the cells to the slides.</li>
		<li>Perform differential staining by sequentially dipping the slides 20 times each in methanol, xanthene, and thiazine followed by distilled water and air-drying.</li>
	</ol>
	</li>
	<li>Add 1 &#181;L of human gamma globulin for every 10,000 cells in the cell suspension in step 6.3 and incubate in ice for 30 mins. Adjust volume to 1 mL with staining media and centrifuge at 600 g for 10 mins at 4&#176;C and aspirate supernatant. Re-suspend cells in 100 &#181;L of staining media and prepare four pooled samples with 10 &#181;L from each sample. Adjust volumes to 200 &#181;L and centrifuge as above.</li>
	<li>Re-suspend cells in 50 &#181;L of antibody cocktail containing fluorochrome-conjugated antibodies diluted in staining media (see table of materials). Re-suspend one of the pooled samples in 50 &#181;L of staining media, one pooled sample in 50 &#181;L of isotype control antibodies, and the remaining pooled samples with diluted antibodies individually. Incubate samples in ice protected from light for 30 mins. 
	NOTE: The use of antibodies will be based on research interests and should be optimized previously for the experiment. Each antibody used should be used individually in a pooled sample for instrument set up. The use of bone marrow cells or splenocytes for single color controls may be necessary if the cell yield is low and is acceptable if fluorochrome-conjugated antibodies are made against an abundant marker such as B220.</li>
	<li>Add 450 &#181;L of staining media and centrifuge at 600 g for 10 mins at 4&#176;C. Aspirate supernatant and repeat.</li>
	<li>Aspirate supernatant and re-suspend cells in 50 &#181;L of freshly prepared stabilizing fixative by gentle pipetting. Add 200 &#181;L more of stabilizing fixative and strain through a 40 &#181;m nylon mesh into a polystyrene FACS tube for analysis by flow cytometry. 
	NOTE: Whole lungs may be digested to make single cell suspensions and used to analyze cell populations by flow cytometry.</li>
</ol>
7. Tissue Processing for RNA Analysis
<ol>
	<li>Place frozen lung lobes in ice within a fume hood, and perform standard Trizol extraction of RNA according to manufacturer&#39;s recommended protocol. After DNase treatment and purification of RNA, convert 1 &#181;g of RNA to cDNA.</li>
	<li>Using RNA-specific validated primer sets, measure changes in gene expression with quantitative real-time PCR and analyze data using the 2-&#916;&#916;Ct method normalized to the internal housekeeping gene25 validated for lungs.</li>
</ol>
8. Tissue processing for Viral Titration
<ol>
	<li>Place frozen lungs in ice and add 1 mL of sterile PBS and mechanically homogenize tissue until entire tissue is pulsed. Centrifuge samples at 600 g for 10 mins at 4&#176;C and store supernatants in aliquots at -80&#176;C until use.</li>
	<li>Quantify virus using standard tissue culture infectious dose 50 (TCID50) method26 with MDCK.2 cells.</li>
</ol>

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W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+Role+of+RSV+Infection+in+Asthma+Initiation+and+Progression:+Findings+in+a+Mouse+Model.">The Role of RSV Infection in Asthma Initiation and Progression: Findings in a Mouse Model.</a> Pulm Med. 2011, 748038 (2011).</li><li>Hsu, H. C., 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=Age-related+thymic+involution+in+C57BL/6J+x+DBA/2J+recombinant-inbred+mice+maps+to+mouse+chromosomes+9+and+10.">Age-related thymic involution in C57BL/6J x DBA/2J recombinant-inbred mice maps to mouse chromosomes 9 and 10.</a> Genes Immun. 4, 402-410 (2003).</li><li>Chang, Y. J., 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=Influenza+infection+in+suckling+mice+expands+an+NKT+cell+subset+that+protects+against+airway+hyperreactivity.">Influenza infection in suckling mice expands an NKT cell subset that protects against airway hyperreactivity.</a> J Clin Invest. 121, 57-69 (2011).</li><li>Wohlleben, G., 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=Influenza+A+virus+infection+inhibits+the+efficient+recruitment+of+Th2+cells+into+the+airways+and+the+development+of+airway+eosinophilia.">Influenza A virus infection inhibits the efficient recruitment of Th2 cells into the airways and the development of airway eosinophilia.</a> J Immunol. 170, 4601-4611 (2003).</li><li>Mori, 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=Differences+in+respiratory+syncytial+virus+and+influenza+infection+in+a+house-dust-mite-induced+asthma+mouse+model:+consequences+for+steroid+sensitivity.">Differences in respiratory syncytial virus and influenza infection in a house-dust-mite-induced asthma mouse model: consequences for steroid sensitivity.</a> Clin Sci (Lond). 125, 565-574 (2013).</li><li>Myles, P. R., 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=Predictors+of+clinical+outcome+in+a+national+hospitalised+cohort+across+both+waves+of+the+influenza+A/H1N1+pandemic+2009-2010+in+the+UK.">Predictors of clinical outcome in a national hospitalised cohort across both waves of the influenza A/H1N1 pandemic 2009-2010 in the UK.</a> Thorax. 67, 709-717 (2012).</li><li>van der Zalm, M. 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=Highly+frequent+infections+with+human+rhinovirus+in+healthy+young+children:+a+longitudinal+cohort+study.">Highly frequent infections with human rhinovirus in healthy young children: a longitudinal cohort study.</a> J Clin Virol. 52, 317-320 (2011).</li></ol>

The authors have nothing to disclose.

The high incidences of allergic asthma and influenza increase the likelihood that these two diseases would occur simultaneously in the same individual. While some viruses are clearly demonstrated to be causative agents initiating the pathogenesis of asthma,28,29 the impact of influenza virus infections on either the development or exacerbation of asthma is unclear. The association and the importance of studying influenza viruses in the context of allergic disease became evident when asthma was identified as a ...

Commonly referred to as a disease, asthma is a syndrome1 that affects over 235 million of the world's population.2 Allergic asthma is the most prevalent type of asthma and results from exposures to environmental factors such as pollen, house dust mite and cockroach antigens, and fungi.3 Fungal antigens are common sensitizing allergens affecting 25% of persistent asthmatics and can cause life threatening asthma attacks.4 The development of an accurate model of allergic asthma is challenging because mice do not naturally develop asthma and there are differences between mouse and human respiratory systems.5 The selection of the mouse asthma model system is largely dependent on the experimental question since the output may vary with the antigen used. A fungal asthma model using Aspergillus fumigatus conidia used in native form and natural route of entry into the airways of mice, described in detail elsewhere,6-8 has been shown to elicit all the hallmarks of human disease including airways hyperresponsiveness, eosinophilic inflammation, mucus hypersecretion, and airway wall remodeling events.9
Influenza affects millions worldwide every year. Genetic alterations that occur in influenza viruses by antigenic drifts and shifts, and re-assortments in common hosts can result in a novel virus with the potential to cause a pandemic. The influenza pandemic of 1918 (Spanish Flu) claimed over 50 million lives mostly of young adults.10 The first influenza pandemic of the 21st century occurred in 2009. Although the 2009 influenza pandemic was not as severe as that of 1918, approximately 90 million people were infected11 of which a little over 200,000 died.12 Hospitalized patients included those with underlying diseases such as obesity, cardiovascular and metabolic disease, asthma, COPD, and diabetes.13-16 Although asthma was identified as a risk factor for severe influenza morbidity,17,18 asthmatics were less likely to be admitted to the intensive care unit and die compared to patients without asthma.19,20 The reasons for these seemingly counterintuitive findings are unknown. Therefore, a reliable animal model system was necessary to study the intersection of these two diseases. 
Since other respiratory viruses such as rhinovirus and respiratory syncytial virus (RSV) have been shown to induce asthma, existing animal models of asthma and influenza explored whether influenza virus infections induces the development of asthma.21 The airways, lung parenchymal tissue, and the immune responses are different during acute allergic asthma exacerbations and chronic stable asthma. Therefore, models that can explore how influenza virus infections intersect allergic asthma in each situation are necessary to understand the underlying mechanisms of asthma and influenza comorbidities. This protocol describes a mouse model that can recapitulate these two scenarios.

<table border="0" cellpadding="0" cellspacing="0" width="693">
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		<tr height="21">
			<td height="21" style="height:21px;width:227px;">Aspergillus fumigatus antigen</td>
			<td style="width:124px;">Greer Labs</td>
			<td style="width:131px;">XPM3D3A25</td>
			<td style="width:212px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:227px;">Aspergillus fumigatus</td>
			<td style="width:124px;">ATCC</td>
			<td style="width:131px;">NIH 5233</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:227px;">Imject Alum</td>
			<td style="width:124px;">Pierce</td>
			<td style="width:131px;">77161</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:227px;">Difco Sabouraud dextrose agar</td>
			<td style="width:124px;">BD&#160;</td>
			<td style="width:131px;">210950</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:227px;">Spore-Klenz</td>
			<td style="width:124px;">Fisher Scientific</td>
			<td style="width:131px;">04-324-20</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:227px;">Ketamine</td>
			<td style="width:124px;">Hospira</td>
			<td style="width:131px;">0409-2051-05</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:227px;">Xylazine</td>
			<td style="width:124px;">Akorn Inc.</td>
			<td style="width:131px;">59399-110-20</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:227px;">Isothesia (Isoflurane)</td>
			<td style="width:124px;">Butler Schein</td>
			<td>05260-04-09</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:227px;">Euthasol</td>
			<td style="width:124px;">Virbac</td>
			<td style="width:131px;">710101</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:227px;">Acetyl-&#946;-methylcholine</td>
			<td style="width:124px;">Sigma Aldrich</td>
			<td style="width:131px;">A2251</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:227px;">flexiVent FX1</td>
			<td style="width:124px;">SCIREQ</td>
			<td style="width:131px;">FV-FXM1</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:227px;">Nebulizer (standard mist)</td>
			<td style="width:124px;">SCIREQ</td>
			<td style="width:131px;">ANP-1100</td>
			<td></td>
		</tr>
		<tr height="24">
			<td height="24" style="height:24px;width:227px;">flexiWare Software</td>
			<td style="width:124px;">SCIREQ</td>
			<td style="width:131px;">version 7.0.1 rel B</td>
			<td></td>
		</tr>
		<tr height="24">
			<td height="24" style="height:24px;width:227px;">Quik-Dip 3 Part Differential Stain</td>
			<td style="width:124px;">Mercedes Medical</td>
			<td style="width:131px;">MER 1002</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:227px;">Human gamma globulin</td>
			<td style="width:124px;">Sigma Aldrich</td>
			<td style="width:131px;">G4386</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:227px;">Rat anti-mouse CD3e-PE-Cy7</td>
			<td style="width:124px;">BD Biosciences</td>
			<td style="width:131px;">552774</td>
			<td>Clone: 145-2C11; Diluted 1:50</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:227px;">Hamster anti-mouse CD8&#945;-FITC</td>
			<td style="width:124px;">BD Biosciences</td>
			<td style="width:131px;">553031</td>
			<td>Clone: 53-6.7, Diluted 1:50</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:227px;">FITC Rat IgG2a</td>
			<td style="width:124px;">BD Biosciences</td>
			<td style="width:131px;">553929</td>
			<td>Diluted 1:50</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:227px;">PE-Cy7 Armenian Hamster IgG</td>
			<td style="width:124px;">BD Biosciences</td>
			<td style="width:131px;">400922</td>
			<td>Clone: HTK888; Diluted 1:50</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:227px;">Stabilizing fixative</td>
			<td style="width:124px;">BD Biosciences</td>
			<td style="width:131px;">338036</td>
			<td>Freshly prepare&#160;</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:227px;">MDCK.2</td>
			<td style="width:124px;">ATCC</td>
			<td style="width:131px;">CRL-2936</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:227px;">TRIzol Reagent</td>
			<td style="width:124px;">Life technologies</td>
			<td style="width:131px;">15596-026</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:227px;">Chloroform</td>
			<td style="width:124px;">IBI Scientific</td>
			<td style="width:131px;">IB05040</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:227px;">iScript</td>
			<td style="width:124px;">Bio-Rad</td>
			<td style="width:131px;">170-8891</td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:227px;">QuantiTect Primer Assays: HPRT1</td>
			<td style="width:124px;">Qiagen</td>
			<td style="width:131px;">QT00166768</td>
			<td></td>
		</tr>
	</tbody>
</table>

modeling asthma and influenza co-morbidity in c57bl/6j mice

Allergic asthma and influenza are both diseases of the pulmonary system that affect millions worldwide. During the 2009 influenza pandemic, asthma was identified as an underlying disease in hospitalized patients, although reasons for this increased susceptibility were unknown. Animal models are necessary to explore mechanisms of disease pathogenesis. However, models that could be used to study influenza virus infections in existing asthma are lacking. This protocol describes the development of mouse model systems of asthma and influenza comorbidity using an Aspergillus fumigatus-induced asthma model and the 2009 pandemic H1N1 influenza strain A/CA/04/2009. The host responses that occur in acute and chronic asthma can be explored by changing the timing of the influenza virus infection. These models suggest that the allergic host response to influenza virus depends on the state of the allergic airways and level of allergic inflammation.

The development and characterization of mouse models that recapitulated patient subsets with asthma and influenza is described. Such models are important in understanding host-pathogen interactions that occur during influenza virus infections in hosts that have underlying disease in the pulmonary system which alters the immune response to the invading virus.
Fungal asthma model
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Asthma and influenza are diseases affecting the pulmonary system that affects millions worldwide. The aim of this study was to develop a mouse model of asthma and influenza comorbidity to study the intersection of these two diseases in the same host.

Modeling Asthma and Influenza Co-morbidity in C57BL/6J Mice

Allergic asthma and influenza are both diseases of the pulmonary system that affect millions worldwide. During the 2009 influenza pandemic, asthma was ...

modeling-asthma-and-influenza-co-morbidity-in-c57bl6j-mice

Research

JoVE Journal

Biology

1.5K Views.  University of Tennessee Health Science Center. Asthma and influenza are diseases affecting the pulmonary system that affects millions worldwide. The aim of this study was to develop a mouse model of asthma and influenza comorbidity to study the intersection of these two diseases in the same host. 

Video: Modeling Asthma and Influenza Co-morbidity in C57BL/6J Mice

Murine Model of Allergen Induced Asthma

Asthma is a major cause of morbidity and mortality, affecting some 300 million people throughout the world.1 More than 8% of the US population has asthma, with the prevalence increasing.2 As with other diseases, animal models of allergic airway disease greatly facilitate understanding of the underlying pathophysiology, help identify potential therapeutic targets, and allow preclinical testing of possible new therapies. Models of allergic airway disease have been developed in several animal species, but murine models are particularly attractive due to the low cost, ready availability, and well-characterized immune systems of these animals.3 Availability of a variety of transgenic strains further increases the attractiveness of these models.4 Here we describe two murine models of allergic airway disease, both employing ovalbumin as the antigen. Following initial sensitization by intraperitoneal injection, one model delivers the antigen challenge by nebulization, the other by intratracheal delivery. These two models offer complementary advantages, with each mimicking the major features of human asthma.5
The major features of acute asthma include an exaggerated airway response to stimuli such as methacholine (airway hyperresponsiveness; AHR) and eosinophil-rich airway inflammation. These are also prominent effects of allergen challenge in our murine models,5,6 and we describe techniques for measuring them and thus evaluating the effects of experimental manipulation. Specifically, we describe both invasive7 and non-invasive8 techniques for measuring airway hyperresponsiveness as well as methods for assessing infiltration of inflammatory cells into the airways and the lung. Airway inflammatory cells are collected by bronchoalveolar lavage while lung histopathology is used to assess markers of inflammation throughout the organ. These techniques provide powerful tools for studying asthma in ways that would not be possible in humans.

Experimental mouse models of allergic asthma offer new possibilities for studying disease pathogenesis and developing new therapeutics. These models are well suited to measuring factors governing the allergic immune response, airway inflammation, and pulmonary pathophysiology.

Asthma is a major cause of morbidity and mortality, affecting some 300 million people throughout the world.1 More than 8% of the US population has asthma, ...

Immunology and Infection

Studying Microbial Communities In Vivo: A Model of Host-mediated Interaction Between Candida Albicans and Pseudomonas Aeruginosa in the Airways

Studying host-pathogen interaction enables us to understand the underlying mechanisms of the pathogenicity during microbial infection. The prognosis of the host depends on the involvement of an adapted immune response against the pathogen1. Immune response is complex and results from interaction of the pathogens and several immune or non-immune cellular types2. In vitro studies cannot characterise these interactions and focus on cell-pathogen interactions. Moreover, in the airway3, particularly in patients with suppurative chronic lung disease or in mechanically ventilated patients, polymicrobial communities are present and complicate host-pathogen interaction. Pseudomonas aeruginosa and Candida albicans are both problem pathogens4, frequently isolated from tracheobronchial samples, and associated to severe infections, especially in intensive care unit5. Microbial interactions have been reported between these pathogens in vitro but the clinical impact of these interactions remains unclear6. To study the interactions between C. albicans and P. aeruginosa, a murine model of C. albicans airways colonization, followed by a P. aeruginosa-mediated acute lung infection was performed.

Studying Microbial Communities In Vivo: A Model of Host-mediated Interaction Between Candida Albicans and Pseudomonas Aeruginosa in the Airways

While in vitro study of host-pathogen interactions allow the characterization of specific immune responses, in vivo models are required to observe the effects of complex responses. Using Candida albicans exposure followed by Pseudomonas aeruginosa-mediated lung infection, we established a murine model of microbial interactions involved in ventilator-associated pneumonia pathogenicity.

Studying host-pathogen interaction enables us to understand the underlying mechanisms of the pathogenicity during microbial infection. The prognosis of ...

A Non-invasive and Technically Non-intensive Method for Induction and Phenotyping of Experimental Bacterial Pneumonia in Mice

Although community-acquired pneumonia remains a major public health problem, murine models of bacterial pneumonia have recently facilitated significant preclinical advances in our understanding of the underlying cellular and molecular pathogenesis. In vivo mouse models capture the integrated physiology and resilience of the host defense response in a manner not revealed by alternative, simplified ex vivo approaches. Several methods have been described in the literature for intrapulmonary inoculation of bacteria in mice, including aerosolization, intranasal delivery, peroral endotracheal cannulation under &#39;blind&#39; and visualized conditions, and transcutaneous endotracheal cannulation. All methods have relative merits and limitations. Herein, we describe in detail a non-invasive, technically non-intensive, inexpensive, and rapid method for intratracheal delivery of bacteria that involves aspiration (i.e., inhalation) by the mouse of an infectious inoculum pipetted into the oropharynx while under general anesthesia. This method can be used for pulmonary delivery of a wide variety of non-caustic biological and chemical agents, and is relatively easy to learn, even for laboratories with minimal prior experience with pulmonary procedures. In addition to describing the aspiration pneumonia method, we also provide step-by-step procedures for assaying the subsequent in vivo pulmonary innate immune response of the mouse, in particular, methods for quantifying bacterial clearance and the cellular immune response of the infected airway. This integrated and simple approach to pneumonia assessment allows for rapid and robust evaluation of the effect of genetic and environmental manipulations upon pulmonary innate immunity.

Several methods have been described in the literature for modeling bacterial pneumonia in mice. Herein, we describe a non-invasive, inexpensive, rapid method for inducing pneumonia via aspiration (i.e., inhalation) of a bacterial inoculum pipetted into the oropharynx. Downstream methods for assessment of the pulmonary innate immune response are also detailed.

Although community-acquired pneumonia remains a major public health problem, murine models of bacterial pneumonia have recently facilitated significant ...

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 ...

Influenza A Virus Studies in a Mouse Model of Infection

Influenza viruses cause over 500,000 deaths worldwide1 and are associated with an annual cost of 12 - 14 billion USD in the United States alone considering direct medical and hospitalization expenses and work absenteeism2. Animal models are crucial in Influenza A virus (IAV) studies to evaluate viral pathogenesis, host-pathogen interactions, immune responses, and the efficacy of current and/or novel vaccine approaches as well as antivirals. Mice are an advantageous small animal model because their immune system is evolutionarily similar to that found in humans, they are available from commercial vendors as genetically identical subjects, there are multiple strains that can be exploited to evaluate the genetic basis of infections, and they are relatively inexpensive and easy to manipulate. To recapitulate IAV infection in humans via the airways, mice are first anesthetized prior to intranasal inoculation with infectious IAVs under proper biosafety containment. After infection, the pathogenesis of IAVs is determined by monitoring daily the morbidity (body weight loss) and mortality (survival) rate. In addition, viral pathogenesis can also be evaluated by assessing virus replication in the upper (nasal mucosa) or lower (lungs) respiratory tract of infected mice. Humoral responses upon IAV infection can be rapidly evaluated by non-invasive bleeding and secondary antibody detection assays aimed at detecting the presence of total or neutralizing antibodies. Here, we describe the common methods used to infect mice intranasally (i.n) with IAV and evaluate pathogenesis, humoral immune responses and protection efficacy.

Influenza A viruses (IAVs) are important human respiratory pathogens. To understand the pathogenicity of IAVs and to perform preclinical testing of novel vaccine approaches, animal models mimicking human physiology are required. Here, we describe techniques to evaluate IAV pathogenesis, humoral responses and vaccine efficacy using a mouse model of infection.

Influenza viruses cause over 500,000 deaths worldwide1 and are associated with an annual cost of 12 - 14 billion USD in the United States alone ...

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

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

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

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

Generation of a Chronic Obstructive Pulmonary Disease Model in Mice by Repeated Ozone Exposure

Chronic obstructive pulmonary disease (COPD) is characterized by persistent airflow limitation and lung parenchymal destruction. It has a very high incidence in aging populations. The current conventional therapies for COPD focus mainly on symptom-modifying drugs; thus, the development of new therapies is urgently needed. Qualified animal models of COPD could help to characterize the underlying mechanisms and can be used for new drug screening. Current COPD models, such as lipopolysaccharide (LPS) or the porcine pancreatic elastase (PPE)-induced emphysema model, generate COPD-like lesions in the lungs and airways but do not otherwise resemble the pathogenesis of human COPD. A cigarette smoke (CS)-induced model remains one of the most popular because it not only simulates COPD-like lesions in the respiratory system, but it is also based on one of the main hazardous materials that causes COPD in humans. However, the time-consuming and labor-intensive aspects of the CS-induced model dramatically limit its application in new drug screening. In this study, we successfully generated a new COPD model by exposing mice to high levels of ozone. This model demonstrated the following: 1) decreased forced expiratory volume 25, 50, and 75/forced vital capacity (FEV25/FVC, FEV50/FVC, and FEV75/FVC), indicating the deterioration of lung function; 2) enlarged lung alveoli, with lung parenchymal destruction; 3) reduced fatigue time and distance; and 4) increased inflammation. Taken together, these data demonstrate that the ozone exposure (OE) model is a reliable animal model that is similar to humans because ozone overexposure is one of the etiological factors of COPD. Additionally, it only took 6 - 8 weeks, based on our previous work, to create an OE model, whereas it requires 3 - 12 months to induce the cigarette smoke model, indicating that the OE model might be a good choice for COPD research.

This study describes the successful generation of a new chronic obstructive pulmonary disease (COPD) animal model by repeatedly exposing mice to high concentrations of ozone.

Chronic obstructive pulmonary disease (COPD) is characterized by persistent airflow limitation and lung parenchymal destruction. It has a very high ...

Medicine

Assessment of Respiratory Function in Conscious Mice by Double-chamber Plethysmography

Air volume changes created by a conscious subject breathing spontaneously within a body box are at the basis of plethysmography, a technique used to non-invasively assess some features of the respiratory function in humans as well as in laboratory animals. The present article focuses on the application of the double-chamber plethysmography (DCP) in small animals. It provides background information on the methodology as well as a detailed step-by-step procedure to successfully assess respiratory function in conscious, spontaneously breathing animals in a non-invasive manner. The DCP can be used to monitor the respiratory function of multiple animals in parallel, as well as to identify changes induced by aerosolized substances over a chosen time period and in a repeated manner. Experiments on control and allergic mice are used herein to demonstrate the utility of the technique, explain the associated outcome parameters, as well as to discuss the related advantages and shortcomings. Overall, the DCP provides valid and theoretically sound readouts that can be trusted to evaluate the respiratory function of conscious small animals both at baseline and after challenges with aerosolized substances.

The objective of the present article is to provide a detailed description of the recommended procedures to evaluate respiratory function in conscious mice by double-chamber plethysmography.

Air volume changes created by a conscious subject breathing spontaneously within a body box are at the basis of plethysmography, a technique used to ...

A Mouse Model for the Transition of Streptococcus pneumoniae from Colonizer to Pathogen upon Viral Co-Infection Recapitulates Age-Exacerbated Illness

Streptococcus pneumoniae (pneumococcus) is an asymptomatic colonizer of the nasopharynx in most individuals but can progress to a pulmonary and systemic pathogen upon influenza A virus (IAV) infection. Advanced age enhances host susceptibility to secondary pneumococcal pneumonia and is associated with worsened disease outcomes. The host factors driving those processes are not well defined, in part due to a lack of animal models that reproduce the transition from asymptomatic colonization to severe clinical disease. 
This paper describes a novel mouse model that recreates the transition of pneumococci from asymptomatic carriage to disease upon viral infection. In this model, mice are first intranasally inoculated with biofilm-grown pneumococci to establish asymptomatic carriage, followed by IAV infection of both the nasopharynx and lungs. This results in bacterial dissemination to the lungs, pulmonary inflammation, and obvious signs of illness that can progress to lethality. The degree of disease is dependent on the bacterial strain and host factors. 
Importantly, this model reproduces the susceptibility of aging, because compared to young mice, old mice display more severe clinical illness and succumb to disease more frequently. By separating carriage and disease into distinct steps and providing the opportunity to analyze the genetic variants of both the pathogen and the host, this S. pneumoniae/IAV co-infection model permits the detailed examination of the interactions of an important pathobiont with the host at different phases of disease progression. This model can also serve as an important tool for identifying potential therapeutic targets against secondary pneumococcal pneumonia in susceptible hosts.

A Mouse Model for the Transition of Streptococcus pneumoniae from Colonizer to Pathogen upon Viral Co-Infection Recapitulates Age-Exacerbated Illness

This paper describes a novel mouse model for the transition of pneumococcus from an asymptomatic colonizer to a disease-causing pathogen during viral infection. This model can be readily adapted to study polymicrobial and host-pathogen interactions during the different phases of disease progression and across various hosts.

Streptococcus pneumoniae (pneumococcus) is an asymptomatic colonizer of the nasopharynx in most individuals but can progress to a pulmonary and systemic ...

Phenotyping Mouse Pulmonary Function In Vivo with the Lung Diffusing Capacity

The mouse is now the primary animal used to model a variety of lung diseases. To study the mechanisms that underlie such pathologies, phenotypic methods are needed that can quantify the pathologic changes. Furthermore, to provide translational relevance to the mouse models, such measurements should be tests that can easily be done in both humans and mice. Unfortunately, in the present literature few phenotypic measurements of lung function have direct application to humans. One exception is the diffusing capacity for carbon monoxide, which is a measurement that is routinely done in humans. In the present report, we describe a means to quickly and simply measure this diffusing capacity in mice. The procedure involves brief lung inflation with tracer gases in an anesthetized mouse, followed by a 1 min gas analysis time. We have tested the ability of this method to detect several lung pathologies, including emphysema, fibrosis, acute lung injury, and influenza and fungal lung infections, as well as monitoring lung maturation in young pups. Results show significant decreases in all the lung pathologies, as well as an increase in the diffusing capacity with lung maturation. This measurement of lung diffusing capacity thus provides a pulmonary function test that has broad application with its ability to detect phenotypic structural changes with most of the existing pathologic lung models.

Phenotyping Mouse Pulmonary Function In Vivo with the Lung Diffusing Capacity

We describe a means to quickly and simply measure the lung diffusing capacity in mice and show that it is sufficiently sensitive to phenotype changes in multiple common lung pathologies. This metric thus brings direct translational relevance to the mouse models, since diffusing capacity is also easily measured in humans.

The mouse is now the primary animal used to model a variety of lung diseases. To study the mechanisms that underlie such pathologies, phenotypic methods ...

Modeling Asthma and Influenza Co-morbidity in C57BL/6J Mice

Summary

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Assessment of Respiratory Function in Conscious Mice by Double-chamber Plethysmography

A Mouse Model for the Transition of Streptococcus pneumoniae from Colonizer to Pathogen upon Viral Co-Infection Recapitulates Age-Exacerbated Illness

Phenotyping Mouse Pulmonary Function In Vivo with the Lung Diffusing Capacity