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 an...

<ol>
	<li>Borish, L., Culp, J. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Asthma:+a+syndrome+composed+of+heterogeneous+diseases.">Asthma: a syndrome composed of heterogeneous diseases.</a> Ann Allergy Asthma Immunol. 101, 1-11 (2008).</li><li>Denning, D. W., O'Driscoll, B. R., Hogaboam, C. M., Bowyer, P., Niven, R. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+link+between+fungi+and+severe+asthma:+a+summary+of+the+evidence.">The link between fungi and severe asthma: a summary of the evidence.</a> Eur Respir. J. 27, 615-626 (2006).</li><li>Wenzel, S., Holgate, S. T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+mouse+trap:+It+still+yields+few+answers+in+asthma.">The mouse trap: It still yields few answers in asthma.</a> Am J Respir Crit Care Med. 174, 1173-1178 (2006).</li><li>Hoselton, S. A., Samarasinghe, A. E., Seydel, J. M., Schuh, J. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=An+inhalation+model+of+airway+allergic+response+to+inhalation+of+environmental+Aspergillus+fumigatus+conidia+in+sensitized+BALB/c+mice.">An inhalation model of airway allergic response to inhalation of environmental Aspergillus fumigatus conidia in sensitized BALB/c mice.</a> Med Mycol. 48, 1056-1065 (2010).</li><li>Schuh, J. M., Hoselton, S. 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+inhalation+model+of+allergic+fungal+asthma:+Aspergillus+fumigatus-induced+inflammation+and+remodeling+in+allergic+airway+disease.">An inhalation model of allergic fungal asthma: Aspergillus fumigatus-induced inflammation and remodeling in allergic airway disease.</a> Methods Mol Biol. 1032, 173-184 (2013).</li><li>Samarasinghe, A. E., Hoselton, S. A., Schuh, J. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+absence+of+the+VPAC(2)+receptor+does+not+protect+mice+from+Aspergillus+induced+allergic+asthma.">The absence of the VPAC(2) receptor does not protect mice from Aspergillus induced allergic asthma.</a> Peptides. 31, 1068-1075 (2010).</li><li>Samarasinghe, A. E., Hoselton, S. A., Schuh, J. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+comparison+between+intratracheal+and+inhalation+delivery+of+Aspergillus+fumigatus+conidia+in+the+development+of+fungal+allergic+asthma+in+C57BL/6+mice.">A comparison between intratracheal and inhalation delivery of Aspergillus fumigatus conidia in the development of fungal allergic asthma in C57BL/6 mice.</a> Fungal Biol. 115, 21-29 (2011).</li><li>Taubenberger, J. K., Morens, D. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Influenza:+the+mother+of+all+pandemics.">Influenza: the mother of all pandemics.</a> Emerg Infect Dis. 12, 15-22 (1918).</li><li>. Pandemic Flu History Available from: <a target="_blank" href="https://www.cdc.gov/flu/">https://www.cdc.gov/flu/</a> (2016)</li><li>Dawood, F. S., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Estimated+global+mortality+associated+with+the+first+12+months+of+2009+pandemic+influenza+A+H1N1+virus+circulation:+a+modelling+study.">Estimated global mortality associated with the first 12 months of 2009 pandemic influenza A H1N1 virus circulation: a modelling study.</a> Lancet Infect Dis. 12, 687-695 (2012).</li><li>Jain, S., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Hospitalized+patients+with+2009+H1N1+influenza+in+the+United+States,+April-June+2009.">Hospitalized patients with 2009 H1N1 influenza in the United States, April-June 2009.</a> N Engl J Med. 361, 1935-1944 (2009).</li><li>Jhung, M. A., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Epidemiology+of+2009+pandemic+influenza+A+(H1N1)+in+the+United+States.">Epidemiology of 2009 pandemic influenza A (H1N1) in the United States.</a> Clin Infect Dis. 52, S13-S26 (2011).</li><li>Chowell, 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=Epidemiological+characteristics+and+underlying+risk+factors+for+mortality+during+the+autumn+pandemic+wave+in+Mexico.">Epidemiological characteristics and underlying risk factors for mortality during the autumn pandemic wave in Mexico.</a> PLoS One. 7, e41069 (2009).</li><li>Kusznierz, 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=Clinical+features+of+the+hospitalized+patients+with+2009+pandemic+influenza+A+(H1N1)+in+Santa+Fe,+Argentina.">Clinical features of the hospitalized patients with 2009 pandemic influenza A (H1N1) in Santa Fe, Argentina.</a> Influenza Other Respir Viruses. 7, 410-417 (2013).</li><li>Nguyen-Van-Tam, J. S., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Risk+factors+for+hospitalisation+and+poor+outcome+with+pandemic+A/H1N1+influenza:+United+Kingdom+first+wave+(May-September+2009).">Risk factors for hospitalisation and poor outcome with pandemic A/H1N1 influenza: United Kingdom first wave (May-September 2009).</a> Thorax. 65, 645-651 (2009).</li><li>Gilca, 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=Risk+factors+for+hospitalization+and+severe+outcomes+of+2009+pandemic+H1N1+influenza+in+Quebec,+Canada.">Risk factors for hospitalization and severe outcomes of 2009 pandemic H1N1 influenza in Quebec, Canada.</a> Influenza Other Respi Viruses. 5, 247-255 (2011).</li><li>McKenna, J. 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=Asthma+in+patients+hospitalized+with+pandemic+influenza+A(H1N1)pdm09+virus+infection-United+States,+2009.">Asthma in patients hospitalized with pandemic influenza A(H1N1)pdm09 virus infection-United States, 2009.</a> BMC Infect Dis. 13, 57 (2013).</li><li>Myles, P., 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+between+asthmatics+and+nonasthmatics+hospitalised+with+influenza+A+infection.">Differences between asthmatics and nonasthmatics hospitalised with influenza A infection.</a> Eur Respir J. 41, 824-831 (2013).</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=Innate+lymphoid+cells+mediate+influenza-induced+airway+hyper-reactivity+independently+of+adaptive+immunity.">Innate lymphoid cells mediate influenza-induced airway hyper-reactivity independently of adaptive immunity.</a> Nat Immunol. 12, 631-638 (2011).</li><li>McGovern, T. K., Robichaud, A., Fereydoonzad, L., Schuessler, T. F., Martin, J. G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Evaluation+of+respiratory+system+mechanics+in+mice+using+the+forced+oscillation+technique.">Evaluation of respiratory system mechanics in mice using the forced oscillation technique.</a> J Vis Exp. , e50172 (2013).</li><li>Moldestad, O., Karlsen, P., Molden, S., Storm, J. 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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">
	<tbody>
		<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 ...

Research

JoVE Journal

Biology

1.5K ビュー.  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. 