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>
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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. 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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.

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	<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 ...

The overall goal of this experiment is to study the immune responses in the lung during bacterial infection. This matter can help in answering some key questions in the field of lung inflammation such as what are the key regulatory mechanisms and genes that inhibit or promote lung inflammation. The major advantage of this technique is that it is very efficient and highly reproducable because of the direct delivery of the bacterial inoculum into the lower respiratory tract.To begin, plate the NTHi on a chocolate agar plate. Incubate the plate upside down over night, at 37 degrees Celsius with 5%carbon dioxide. The following day, culture the bacteria in a brain-heart infusion broth.Then, in a microcentrifuge tube, add one milliliter of the broth. Spin the mix down at 4, 000 g's for five minutes at room temperature. Then, discard the supernatant and resuspend the pellet in one milliliter of PBS.Repeat this wash step one more time. Next, dilute 100 microliters of resuspended solution in 900 microliters of PBS, and measure the culture's absorbance at 600 nanometers. Use this information to adjust the inoculum to 20 million CFUs per milliliter.Next, determine the viability and CFUs of the inoculum. Culture one to ten serial dilutions of the broth on chocolate agar plates, and incubate the plates overnight. Begin with anesthetizing the mouse, and verifying it is adequately anesthetized using a toe pinch.Then, position the mouse supine and shave the ventral area of the neck. Disinfect the exposed skin. Now, lift the skin of the upper ventral part of the neck with rat tooth forceps, and using a scalpel, make a one half to one centimeter incision above the thymus.Then, carefully separate the muscle to expose the trachea. Into the trachea, slowly inject 50 microliters of air, followed by 50 microliters of prepared NTHi, followed by another 50 microliters of air. For a negative control procedure, inject saline instead of NTHi.After the injection, keep the animal vertical for around a minute. Then, close the incision with tissue adhesive and keep the mouse laterally recumbent and warm for about 10 minutes. 24 hours after infection, euthanize the infected animal and open the abdominal cavity with anatomical scissors.Then, cut the ventral aorta to bleed the animal. Next, expose the ventral part of the lung to access the trachea. Intubate the trachea with a 20 gauge catheter.Using the catheter, instill 0.8 milliliters of BALF buffer and collect the lavage by dental aspiration. Repeat this procedure three more times, pooling the four lavage collections into a single suspension. Take a 100 microliter aliquot and spin down for two minutes at 2, 000 RPM.Remove the supernatant and resuspend in RBC lysis buffer. Spin it down and resuspend the pellet with 1x PBS. Now, count the total number of cells in 100 microliters of the suspension using a hemocytometer under 10x magnification with trypan blue staining.Then, prepare slides to stain the cells. Aliquot 100 microliters of BALF suspension into the appropriate wells of cytospin slides, and spin the slides at 500 g's for five minutes. Then, dry the slides for 10 minutes at room temperature, and stain the cells using a modified Giemsa stain according to the manufacturer's protocols.Use the remaining cells for subsequent analyses, such as facts, confocal microscopy, and so forth. After collecting lavage from the infected lung, take a 20-40 milligram piece of lung and incubate it in RNA stabilizing reagent at four degrees Celsius overnight, followed by long term storage at 80 degrees Celsius. When ready, wash the lung tissue samples in cold PBS to remove the RNA stabilization reagent.Then, place the tissue into a 1.5 milliliter centrifuge tube containing lysis buffer with beta mercaptoethanol. Next, with small scissors, chop the tissue into pieces and homogenize the pieces using a motorized pestle for 20-40 seconds. Keep the lysate on ice for five minutes, then spin them down at 18, 000 g's for three minutes.Carefully remove the supernatant and transfer it to a new tube. Then add one volume of 70%ethanol and triturate the mixture. Let the mixture rest at 25 degrees Celsius for two to four minutes.Then, transfer up to 700 microliters to a spin column on a two milliliter collection tube and centrifuge the column at 9600 g's for 15 seconds. Discard the flow-through, add 350 microliters of wash buffer and repeat the spin. Next, incubate the membrane with 80 microliters of DNase I Solution for 15 minutes at room temperature.Then, add 350 microliters of wash buffer to the column and repeat the spin. Next, add 500 microliters of RNA wash buffer. Repeat the short spin, and discard the flow-through.Do two washes with RNA wash buffer. Finally, over a new collection tube, add 30-50 microliters of RNase-free water directly to the column and spin it for one minute to collect the RNA sample in the flow-through. The procedure was performed as described with normal, healthy, mice.Intratracheal installation resulted in a markedly increased number of leukocytes in the bronchoalveolar lavage fluid of mice innoculated with NTHi compared to saline treatment controls. The differential count analysis of the leukocytes clearly shows increased neutrophil infiltration. Facts analysis of the cells in the BALF further confirms the increased number of neutrophils.HNE stained sections of the lung tissue show increased airway inflammation. Collectively, these data suggest that intratracheal installation induces airway inflammation in mice. In a similar experiment with mutant and control mice, lung tissue RNA was analyzed by whole transcriptome sequencing.Compared to wild-type controls, Itch deficiency resulted in differential expression of several genes. Itch is thought to be a regulator of inflammatory signalling pathways, so this result can be used to foster new studies. All through its development this technique paved ways for researchers in the field of lung diseases to explore cell signalling in immune cells during lung infection and inflammation.While attempting this procedure, it is important to remember to take extra precautions during procedures like exposure of trachea and installation of bacteria into trachea. Once mastered, intratracheal installation can be performed within ten minutes and vial fluid can be collected within 10-15 minutes. After this procedure, additional matters such as RNA sequencing, flow cytometry, and immunoblocking can be performed to address additional questions such as, which are the genes that are upregulated or downregulated.

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