Name: intra-tracheal administration of haemophilus influenzae in mouse models to study airway inflammation
Uploaded: 2016-03-02T15:00:00
Description: Watch this Scientific Journal Video about Intra-tracheal Administration of Haemophilus influenzae in Mouse Models to Study Airway Inflammation at JoVE.com

We thank Dr. Carson Harrod for critical reading of the manuscript. We also thank Mr. Minghui Zeng and Drs. Mahesh Kathania and Prashant Khare for their contributions. This work was supported by grants from the American Cancer Society (Research Scholar grant, 122713-RSG-12-260-01-LIB) and the Sammons Cancer Center (Pilot Project grant) to K. Venuprasad.

All experiments were performed in accordance with the guidelines of the Institutional Animal Care and Use Committee (IACUC) of Baylor Research Institute.
1. Culturing Non-typeable Haemophilus influenzae (NTHi) and Preparing the Inoculum
<ol>
	<li>Plate NTHi on a chocolate agar plate and keep the plate upside down in a humidified CO2 incubator overnight at 37 &#176;C and 5% CO2. The following day, culture the bacteria in brain heart infusion broth. Then, add 1 ...

<ol>
	<li>Medzhitov, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Origin+and+physiological+roles+of+inflammation.">Origin and physiological roles of inflammation.</a> Nature. 454, 428-435 (2008).</li><li>Grivennikov, S. I., Greten, F. R., Karin, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Immunity,+inflammation,+and+cancer.">Immunity, inflammation, and cancer.</a> Cell. 140, 883-899 (2010).</li><li>Barnes, P. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Immunology+of+asthma+and+chronic+obstructive+pulmonary+disease.">Immunology of asthma and chronic obstructive pulmonary disease.</a> Nat Rev Immunol. 8, 183-192 (2008).</li><li>Sethi, S., Murphy, T. F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Infection+in+the+pathogenesis+and+course+of+chronic+obstructive+pulmonary+disease.">Infection in the pathogenesis and course of chronic obstructive pulmonary disease.</a> N Engl J Med. 359, 2355-2365 (2008).</li><li>King, P. T., Sharma, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+Lung+Immune+Response+to+Nontypeable+Haemophilus+influenzae+(Lung+Immunity+to+NTHi).">The Lung Immune Response to Nontypeable Haemophilus influenzae (Lung Immunity to NTHi).</a> J Immunol Res. 2015, 706376 (2015).</li><li>Kapur, N., Grimwood, K., Masters, I. B., Morris, P. S., Chang, A. B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Lower+airway+microbiology+and+cellularity+in+children+with+newly+diagnosed+non-CF+bronchiectasis.">Lower airway microbiology and cellularity in children with newly diagnosed non-CF bronchiectasis.</a> Pediatr Pulmonol. 47, 300-307 (2012).</li><li>King, P. T., Holdsworth, S. R., Freezer, N. J., Villanueva, E., Holmes, P. W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Microbiologic+follow-up+study+in+adult+bronchiectasis.">Microbiologic follow-up study in adult bronchiectasis.</a> Respir Med. 101, 1633-1638 (2007).</li><li>Shuto, T., 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=Activation+of+NF-kappa+B+by+nontypeable+Hemophilus+influenzae+is+mediated+by+toll-like+receptor+2-TAK1-dependent+NIK-IKK+alpha+/beta-I+kappa+B+alpha+and+MKK3/6-p38+MAP+kinase+signaling+pathways+in+epithelial+cells.">Activation of NF-kappa B by nontypeable Hemophilus influenzae is mediated by toll-like receptor 2-TAK1-dependent NIK-IKK alpha /beta-I kappa B alpha and MKK3/6-p38 MAP kinase signaling pathways in epithelial cells.</a> Proc Natl Acad Sci U S A. 98, 8774-8779 (2001).</li><li>Moghaddam, S. 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=Haemophilus+influenzae+lysate+induces+aspects+of+the+chronic+obstructive+pulmonary+disease+phenotype.">Haemophilus influenzae lysate induces aspects of the chronic obstructive pulmonary disease phenotype.</a> Am J Respir Cell Mol Biol. 38, 629-638 (2008).</li><li>Rajagopalan, 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=Intranasal+exposure+to+bacterial+superantigens+induces+airway+inflammation+in+HLA+class+II+transgenic+mice.">Intranasal exposure to bacterial superantigens induces airway inflammation in HLA class II transgenic mice.</a> Infect Immun. 74, 1284-1296 (2006).</li><li>Ganesan, 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=Elastase/LPS-exposed+mice+exhibit+impaired+innate+immune+responses+to+bacterial+challenge:+role+of+scavenger+receptor+A.">Elastase/LPS-exposed mice exhibit impaired innate immune responses to bacterial challenge: role of scavenger receptor A.</a> Am J Pathol. 180, 61-72 (2012).</li><li>Hogner, K., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Macrophage-expressed+IFN-beta+contributes+to+apoptotic+alveolar+epithelial+cell+injury+in+severe+influenza+virus+pneumonia.">Macrophage-expressed IFN-beta contributes to apoptotic alveolar epithelial cell injury in severe influenza virus pneumonia.</a> PLoS Pathog. 9, e1003188 (2013).</li><li>Karisola, 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=Invariant+Natural+Killer+T+Cells+Play+a+Role+in+Chemotaxis,+Complement+Activation+and+Mucus+Production+in+a+Mouse+Model+of+Airway+Hyperreactivity+and+Inflammation.">Invariant Natural Killer T Cells Play a Role in Chemotaxis, Complement Activation and Mucus Production in a Mouse Model of Airway Hyperreactivity and Inflammation.</a> PloS one. 10, e0129446 (2015).</li><li>Theivanthiran, B., 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=The+E3+ubiquitin+ligase+Itch+inhibits+p38alpha+signaling+and+skin+inflammation+through+the+ubiquitylation+of+Tab1.">The E3 ubiquitin ligase Itch inhibits p38alpha signaling and skin inflammation through the ubiquitylation of Tab1.</a> Sci Signal. 8, 22 (2015).</li><li>Venuprasad, K., Zeng, M., Baughan, S. L., Massoumi, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Multifaceted+role+of+the+ubiquitin+ligase+Itch+in+immune+regulation.">Multifaceted role of the ubiquitin ligase Itch in immune regulation.</a> Immunol Cell Biol. , (2015).</li><li>Decramer, M., Janssens, W., Miravitlles, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Chronic+obstructive+pulmonary+disease.">Chronic obstructive pulmonary disease.</a> Lancet. 379, 1341-1351 (2012).</li></ol>

Herein, we describe a unique and minimally invasive procedure to inoculate the lungs of mice with a bacterial lung pathogen. We demonstrate that this procedure can be used to study the function of different genes using mice that are deficient in genes of inflammatory signaling pathways. This procedure can also be used to study the inflammatory responses to viral and fungal lung infections. The advantages of this procedure over other methods such as intranasal or aerosol inhalation are (1) in this procedure, the pathogeni...

Inflammation is a fundamental immune mechanism of defense against infectious agents. It promotes pathogen eradication and repair of damaged tissue. It also facilitates the recovery to a normal healthy state1. However, dysregulated inflammation often leads to chronic inflammatory diseases2. Airway inflammation is an initial trigger for different pulmonary diseases such as chronic obstructive pulmonary disease (COPD), asthma and pulmonary fibrosis3.
The non-typeable (unencapsulated) Haemophilus influenzae (NTHi) is associated with chronic upper and lower lung inflammatory diseases4,5. It is the dominant species isolated from the lower airways of children and adults with chronic obstructive pulmonary disease4,6,7. The inflammatory response after NTHi infection is characterized by the upregulation of proinflammatory cytokines (such as TNF and IL-1&#946;), and it is mediated by mitogen activated protein kinase (MAPK) and nuclear factor-&#954;B (NF-&#954;B) through toll-like receptors (TLRs)8.
Mouse models are very useful tools for analyzing the underlying pathology of lung inflammatory disease because of the availability of different gene-deficient lines. Several methods have been used to inoculate live/attenuated bacteria and bacterial products, including intranasal instillation and aerosolized inhalation9,10. Here, we demonstrate intra-tracheal instillation. Although used less frequently, this approach is more efficient and highly reproducible because of the direct delivery of the inoculum to the lower respiratory tract.

<table border="0" cellpadding="0" cellspacing="0" width="705">
	<tbody>
		<tr height="21">
			<td height="21" style="height:21px;width:293px;">Chocolate agar plate</td>
			<td style="width:127px;">Fisher Scientific</td>
			<td style="width:119px;">CAS50-99-7</td>
			<td style="width:167px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:293px;">Dextrose Anhydrous</td>
			<td style="width:127px;">Themo Scientific</td>
			<td style="width:119px;">R01300</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:293px;">Heparin</td>
			<td style="width:127px;">Hospira,Inc</td>
			<td style="width:119px;">RL-3010</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:293px;">Deft quick solution</td>
			<td style="width:127px;">Sigma</td>
			<td>GS500-500ML</td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:293px;">Syringe needle 20/26G&#160;</td>
			<td style="width:127px;">BD</td>
			<td style="width:119px;">(REF305115/175)</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:293px;">Iml syringe</td>
			<td style="width:127px;">BD</td>
			<td style="width:119px;">REF 309602</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:293px;">Catheter 20GA</td>
			<td style="width:127px;">BD</td>
			<td style="width:119px;">REF 381433</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Dissecting Scissors, straight, 10 cm long</td>
			<td>kentscientific</td>
			<td>INS600393</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Iris Forceps, serrated, 10cm long</td>
			<td>kentscientific</td>
			<td>INS650915</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Tweezer #5 Stainless steel, 11cm long</td>
			<td>kentscientific</td>
			<td>INS600095</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:293px;">10% Formalin</td>
			<td style="width:127px;">Fisher Scientific</td>
			<td style="width:119px;">CAS 67-56-1</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:293px;">Agarose</td>
			<td style="width:127px;">peqlab</td>
			<td style="width:119px;">35-1020</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:293px;">5ml polystyrene round-bottom tubes</td>
			<td style="width:127px;">BD</td>
			<td style="width:119px;">REF 352058</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:293px;">1.5 ml Microcentrifuge tubes</td>
			<td style="width:127px;">Light Labs</td>
			<td style="width:119px;">A-7001-R</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:293px;">Reasy Mini&#160; kit&#160;</td>
			<td style="width:127px;">Qiagen</td>
			<td align="right" style="width:119px;">74104</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:293px;">Pellet pestile motor (Tissue homoginizer)</td>
			<td style="width:127px;">Sigma</td>
			<td style="width:119px;">Z359971-1EA</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:293px;">96 well microtiter plates V bottom</td>
			<td style="width:127px;">Thermo</td>
			<td align="right" style="width:119px;">2605</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:293px;">1X PBS</td>
			<td style="width:127px;">Gibco</td>
			<td style="width:119px;">10010-023</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:293px;">OneComp eBeads</td>
			<td style="width:127px;">eBioscience</td>
			<td style="width:119px;">01-1111-42</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:293px;">CD45.2-APC</td>
			<td style="width:127px;">eBioscience</td>
			<td style="width:119px;">17-0454-81</td>
			<td>Working dilution 1:100</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:293px;">Ly-6G-eFlor 450</td>
			<td style="width:127px;">eBioscience</td>
			<td style="width:119px;">48-5931-82</td>
			<td>Working dilution 1:100</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:293px;">BSA</td>
			<td style="width:127px;">HyClone</td>
			<td style="width:119px;">SH30574.03</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:293px;">RBC Lysis Buffer (10X)</td>
			<td style="width:127px;">Biolegend</td>
			<td style="width:119px;">420301</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:293px;">Live/Dead fixable aqua dead cell stain kit</td>
			<td style="width:127px;">Invitrogen</td>
			<td style="width:119px;">L-34957</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:293px;">EDTA (0.5M)</td>
			<td style="width:127px;">lifetechnologies</td>
			<td style="width:119px;">15575-020</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:293px;">CD16/CD32 FcBlock</td>
			<td style="width:127px;">BD</td>
			<td style="width:119px;">553142</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:293px;">Facs tubes polystyrene round bottom tube</td>
			<td style="width:127px;">BD</td>
			<td style="width:119px;">352052</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:293px;">Formaldehyde</td>
			<td style="width:127px;">Polyscience</td>
			<td style="width:119px;">4018</td>
			<td></td>
		</tr>
	</tbody>
</table>

intra-tracheal administration of haemophilus influenzae in mouse models to study airway inflammation

Here, we describe a detailed procedure to efficiently and directly deliver Haemophilus influenzae into the lower respiratory tracts of mice. We demonstrate the procedure for preparing H. influenzae inoculum, intra-tracheal instillation of H. influenzae into the lung, collection of broncho-alveolar lavage fluid (BALF), analysis of immune cells in the BALF, and RNA isolation for differential gene expression analysis. This procedure can be used to study the lung inflammatory response to any bacteria, virus or fungi. Direct tracheal instillation is mostly preferred over intranasal or aerosol inhalation procedures because it more efficiently delivers the bacterial inoculum into the lower respiratory tract with less ambiguity.

Intra-tracheal instillation resulted in a markedly increased number of leukocytes in the BALF (Figure 1A, left panel) than installation with saline. The differential count analysis of the leukocytes clearly showed increased neutrophil infiltration (Figure 1, right panel). The FACS analysis of the cells in the BALF further confirmed the increased number of neutrophils (Figure 1B). Histological analysis of H&#38;E-stained sections of the lu...

Watch this Scientific Journal Video about Intra-tracheal Administration of Haemophilus influenzae in Mouse Models to Study Airway Inflammation at JoVE.com

Intra-tracheal Administration of Haemophilus influenzae in Mouse Models to Study Airway Inflammation

We demonstrate the procedure for intra-tracheal inoculation of Haemophilus influenzae into the lower respiratory tracts of mice. This is a very useful tool to study signaling pathways that regulate airway inflammation in mouse models.

Intra-tracheal Administration of Haemophilus influenzae in Mouse Models to Study Airway Inflammation

Here, we describe a detailed procedure to efficiently and directly deliver Haemophilus influenzae into the lower respiratory tracts of mice. We ...

Research

JoVE Journal

Immunology and Infection

Co-culture Models of Pseudomonas aeruginosa Biofilms Grown on Live Human Airway Cells

Co-culture Models of Pseudomonas aeruginosa Biofilms Grown on Live Human Airway Cells

Bacterial biofilms have been associated with a number of different human diseases, but biofilm development has generally been studied on non-living ...

Analysis of Pulmonary Dendritic Cell Maturation and Migration during Allergic Airway Inflammation

Dendritic cells (DCs) are the key players involved in initiation of adaptive immune response by activating antigen-specific T cells. DCs are present in ...

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

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

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

In vivo Imaging Method to Distinguish Acute and Chronic Inflammation

In vivo Imaging Method to Distinguish Acute and Chronic Inflammation

Inflammation is a fundamental aspect of many human diseases. In this video report, we demonstrate non-invasive bioluminescence imaging techniques that ...

Intranasal Administration of Recombinant Influenza Vaccines in Chimeric Mouse Models to Study Mucosal Immunity

Vaccines are one of the greatest achievements of mankind, and have saved millions of lives over the last century. Paradoxically, little is known about the ...

Co-immunoprecipitation of the Mouse Mx1 Protein with the Influenza A Virus Nucleoprotein

Studying the interaction between proteins is key in understanding their function(s). A very powerful method that is frequently used to study interactions ...

An Optimized Method for Isolating and Expanding Invariant Natural Killer T Cells from Mouse Spleen

The ability to rapidly secrete cytokines upon stimulation is a functional characteristic of the invariant natural killer T (iNKT) cell lineage. iNKT cells ...

Increased Recovery Time and Decreased LPS Administration to Study the Vagus Nerve Stimulation Mechanisms in Limited Inflammatory Responses

Inflammation is a local response to infection and tissue damage mediated by activated macrophages, monocytes, and other immune cells that release ...

Precision-cut Mouse Lung Slices to Visualize Live Pulmonary Dendritic Cells

Inhalation of allergens and pathogens elicits multiple changes in a variety of immune cell types in the lung. Flow cytometry is a powerful technique for ...