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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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 ...

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.

intra-tracheal-administration-haemophilus-influenzae-mouse-models-to

Research

JoVE Journal

Immunology and Infection

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 surfaces. In this paper, we describe protocols for forming Pseudomonas aeruginosa biofilms on human airway epithelial cells (CFBE cells) grown in culture. In the first method (termed the Static Co-culture Biofilm Model), P. aeruginosa is incubated with CFBE cells grown as confluent monolayers on standard tissue culture plates. Although the bacterium is quite toxic to epithelial cells, the addition of arginine delays the destruction of the monolayer long enough for biofilms to form on the CFBE cells. The second method (termed the Flow Cell Co-culture Biofilm Model), involves adaptation of a biofilm flow cell apparatus, which is often used in biofilm research, to accommodate a glass coverslip supporting a confluent monolayer of CFBE cells. This monolayer is inoculated with P. aeruginosa and a peristaltic pump then flows fresh medium across the cells. In both systems, bacterial biofilms form within 6-8 hours after inoculation. Visualization of the biofilm is enhanced by the use of P. aeruginosa strains constitutively expressing green fluorescent protein (GFP). The Static and Flow Cell Co-culture Biofilm assays are model systems for early P. aeruginosa infection of the Cystic Fibrosis (CF) lung, and these techniques allow different aspects of P. aeruginosa biofilm formation and virulence to be studied, including biofilm cytotoxicity, measurement of biofilm CFU, and staining and visualizing the biofilm.

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

This paper describes different methods of growing Pseudomonas aeruginosa biofilms on cultured human airway epithelial cells. These protocols can be adapted to study different aspects of biofilm formation, including visualization of the biofilm, staining of the biofilm, measuring the colony forming units (CFU) of the biofilm, and studying biofilm cytotoxicity.

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 peripheral tissues in steady state; however in response to antigen stimulation, DCs take up the antigen and rapidly migrate to the draining lymph nodes where they initiate T cell response against the antigen1,2. Additionally, DCs also play a key role in initiating autoimmune as well as allergic immune response3.
DCs play an essential role in both initiation of immune response and induction of tolerance in the setting of lung environment4. Lung environment is largely tolerogenic, owing to the exposure to vast array of environmental antigens5. However, in some individuals there is a break in tolerance, which leads to induction of allergy and asthma. In this study, we describe a strategy, which can be used to monitor airway DC maturation and migration in response to the antigen used for sensitization. The measurement of airway DC maturation and migration allows for assessment of the kinetics of immune response during airway allergic inflammation and also assists in understanding the magnitude of the subsequent immune response along with the underlying mechanisms.
Our strategy is based on the use of ovalbumin as a sensitizing agent. Ovalbumin-induced allergic asthma is a widely used model to reproduce the airway eosinophilia, pulmonary inflammation and elevated IgE levels found during asthma6,7. After sensitization, mice are challenged by intranasal delivery of FITC labeled ovalbumin, which allows for specific labeling of airway DCs which uptake ovalbumin. Next, using several DC specific markers, we can assess the maturation of these DCs and can also assess their migration to the draining lymph nodes by employing flow cytometry.

We describe a strategy to monitor maturation and migration of pulmonary dendritic cells in response to ovalbumin in the setting of ovalbumin induced allergic airway inflammation. This strategy can be modified to assess migration of pulmonary dendritic cells in settings of infection.

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

The apical and basolateral surfaces of airway epithelial cells demonstrate directional responses to pathogen exposure in vivo. Thus, ideal in vitro models for examining cellular responses to respiratory pathogens polarize, forming apical and basolateral surfaces. One such model is differentiated normal human bronchial epithelial cells (NHBE). However, this system requires lung tissue samples, expertise isolating and culturing epithelial cells from tissue, and time to generate an air-liquid interface culture.
Calu-3 cells, derived from a human bronchial adenocarcinoma, are an alternative model for examining the response of proximal airway epithelial cells to respiratory insult1, pharmacological compounds2-6, and bacterial7-9 and viral pathogens, including influenza virus, rhinovirus and severe acute respiratory syndrome - associated coronavirus10-14. Recently, we demonstrated that Calu-3 cells are susceptible to respiratory syncytial virus (RSV) infection in a manner consistent with NHBE15,16 . Here, we detail the establishment of a polarized, liquid-covered culture (LCC) of Calu-3 cells, focusing on the technical details of growing and culturing Calu-3 cells, maintaining cells that have been cultured into LCC, and we present the method for performing respiratory virus infection of polarized Calu-3 cells.
To consistently obtain polarized Calu-3 LCC, Calu-3 cells must be carefully subcultured before culturing in Transwell inserts. Calu-3 monolayer cultures should remain below 90% confluence, should be subcultured fewer than 10 times from frozen stock, and should regularly be supplied with fresh medium. Once cultured in Transwells, Calu-3 LCC must be handled with care. Irregular media changes and mechanical or physical disruption of the cell layers or plates negatively impact polarization for several hours or days. Polarization is monitored by evaluating trans-epithelial electrical resistance (TEER) and is verified by evaluating the passive equilibration of sodium fluorescein between the apical and basolateral compartments17,18 . Once TEER plateaus at or above 1,000 &Omega;&times;cm2, Calu-3 LCC are ready to use to examine cellular responses to respiratory pathogens.

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

The findings and conclusions in this report are those of the authors and do not necessarily represent the views of the Centers for Disease Control and Prevention.

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

Inflammation is a fundamental aspect of many human diseases. In this video report, we demonstrate non-invasive bioluminescence imaging techniques that distinguish acute and chronic inflammation in mouse models. With tissue damage or pathogen invasion, neutrophils are the first line of defense, playing a major role in mediating the acute inflammatory response. As the inflammatory reaction progresses, circulating monocytes gradually migrate into the site of injury and differentiate into mature macrophages, which mediate chronic inflammation and promote tissue repair by removing tissue debris and producing anti-inflammatory cytokines. Intraperitoneal injection of luminol (5-amino-2,3-dihydro-1,4-phthalazinedione, sodium salt) enables detection of acute inflammation largely mediated by tissue-infiltrating neutrophils. Luminol specifically reacts with the superoxide generated within the phagosomes of neutrophils since bioluminescence results from a myeloperoxidase (MPO) mediated reaction. Lucigenin (bis-N-methylacridinium nitrate) also reacts with superoxide in order to generate bioluminescence. However, lucigenin bioluminescence is independent of MPO and it solely relies on phagocyte NADPH oxidase (Phox) in macrophages during chronic inflammation. Together, luminol and lucigenin allow non-invasive visualization and longitudinal assessment of different phagocyte populations across both acute and chronic inflammatory phases. Given the important role of inflammation in a variety of human diseases, we believe this non-invasive imaging method can help investigate the differential roles of neutrophils and macrophages in a variety of pathological conditions.

In vivo Imaging Method to Distinguish Acute and Chronic Inflammation

We describe a non-invasive imaging method for distinguishing inflammatory stages. Systemic delivery of luminol reveals areas of acute inflammation dependent upon MPO activity in neutrophils. In contrast, injection of lucigenin allows for visualization of chronic inflammation dependent upon Phox activity in macrophages.

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 physiological mechanisms that mediate immune responses to vaccines perhaps due to the overall success of vaccination, which has reduced interest into the molecular and physiological mechanisms of vaccine immunity. However, several important human pathogens including influenza virus still pose a challenge for vaccination, and may benefit from immune-based strategies. 
Although influenza reverse genetics has been successfully applied to the generation of live-attenuated influenza vaccines (LAIVs), the addition of molecular tools in vaccine preparations such as tracer components to follow up the kinetics of vaccination in vivo, has not been addressed. In addition, the recent generation of mouse models that allow specific depletion of leukocytes during kinetic studies has opened a window of opportunity to understand the basic immune mechanisms underlying vaccine-elicited protection. Here, we describe how the combination of reverse genetics and chimeric mouse models may help to provide new insights into how vaccines work at physiological and molecular levels, using as example a recombinant, cold-adapted, live-attenuated influenza vaccine (LAIV). We utilized laboratory-generated LAIVs harboring cell tracers as well as competitive bone marrow chimeras (BMCs) to determine the early kinetics of vaccine immunity and the main physiological mechanisms responsible for the initiation of vaccine-specific adaptive immunity. In addition, we show how this technique may facilitate gene function studies in single animals during immune responses to vaccines. We propose that this technique can be applied to improve current prophylactic strategies against pathogens for which urgent medical countermeasures are needed, for example influenza, HIV, Plasmodium, and hemorrhagic fever viruses such as Ebola virus.

There is an overall lack of knowledge about how vaccines work. Here we propose the combined use of reverse genetics and bone marrow chimeric mice to gain insight into the early host immune responses to vaccines with a special focus on dendritic cells and T cell 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 of proteins with other macromolecules in a complex sample is called co-immunoprecipitation. The described co-immunoprecipitation protocol allows to demonstrate and further investigate the interaction between the antiviral myxovirus resistance protein 1 (Mx1) and one of its viral targets, the influenza A virus nucleoprotein (NP). The protocol starts with transfected mammalian cells, but it is also possible to use influenza A virus infected cells as starting material. After cell lysis, the viral NP protein is pulled-down with a specific antibody and the resulting immune-complexes are precipitated with protein G beads. The successful pull-down of NP and the co-immunoprecipitation of the antiviral Mx1 protein are subsequently revealed by western blotting. A prerequisite for successful co-immunoprecipitation of Mx1 with NP is the presence of N-ethylmaleimide (NEM) in the cell lysis buffer. NEM alkylates free thiol groups. Presumably this reaction stabilizes the weak and/or transient NP&#8211;Mx1 interaction by preserving a specific conformation of Mx1, its viral target or an unknown third component. An important limitation of co-immunoprecipitation experiments is the inadvertent pull-down of contaminating proteins, caused by nonspecific binding of proteins to the protein G beads or antibodies. Therefore, it is very important to include control settings to exclude false positive results. The described co-immunoprecipitation protocol can be used to study the interaction of Mx proteins from different vertebrate species with viral proteins, any pair of proteins, or of a protein with other macromolecules. The beneficial role of NEM to stabilize weak and/or transient interactions needs to be tested for each interaction pair individually.

This co-immunoprecipitation protocol allows to study the interaction between the influenza A virus nucleoprotein and the antiviral Mx1 protein in human cells. The protocol emphasizes the importance of N-ethylmaleimide for successful co-immunoprecipitation of Mx1 and 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 are therefore characterized as an innate T cell population capable of activating and steering adaptive immune responses. The development of improved techniques for the culture and expansion of murine iNKT cells facilitates the study of iNKT cell biology in in vitro and in vivo model systems. Here we describe an optimized procedure for the isolation and expansion of murine splenic iNKT cells.
Spleens from C57Bl/6 mice are removed, dissected and strained and the resulting cellular suspension is layered over density gradient media. Following centrifugation, splenic mononuclear cells (MNCs) are collected and CD5-positive (CD5+) lymphocytes are enriched for using magnetic beads. iNKT cells within the CD5+ fraction are subsequently stained with &#945;GalCer-loaded CD1d tetramer and purified by fluorescence activated cell sorting (FACS). FACS sorted iNKT cells are then initially cultured in vitro using a combination of recombinant murine cytokines and plate-bound T cell receptor (TCR) stimuli before being expanded in the presence of murine recombinant IL-7. Using this technique, approximately 108 iNKT cells can be generated within 18-20 days of culture, after which they can be used for functional assays in vitro, or for in vivo transfer experiments in mice.

Here we present an adapted protocol that can be used to generate a large number of murine invariant natural killer T cells from mouse spleen. The protocol outlines an approach by which splenic iNKT cells can be enriched for, isolated and expanded in vitro using a limited number of animals and reagents.

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 cytokines and other mediators of inflammation. For a long time, humoral and cellular mechanisms have been studied for their role in regulating the immune response, but recent advances in the field of immunology and neuroscience have also unraveled specific neural mechanisms with interesting therapeutic potential. The so-called cholinergic anti-inflammatory pathway (CAP) has been described to control innate immune responses and inflammation in a very potent manner. In the early 2000s, Tracey and collaborators developed a technique that stimulates the vagus nerve and mimics the effect of the pathway. The methodology is based on the electrical stimulation of the vagus nerve at low voltage and frequency, in order to avoid any side effects of overstimulation, such as deregulation of heart rate variability. Electrical devices for stimulation are now available, making it easy to set up the methodology in the laboratory. The goal of this research was to investigate the potential involvement of prostaglandins in the CAP. Unfortunately, based on earlier attempts, we failed to use the original protocol, as the induced inflammatory response either was too high or was not suitable for enzymatic metabolism properties. The different settings of the original surgery protocol remained mostly unchanged, but the conditions regarding inflammatory induction and the time point before sacrifice were improved to fit our purposes (i.e., to investigate the involvement of the CAP in more limited inflammatory responses). 
The modified version of the original protocol, presented here, includes a longer time range between vagus nerve stimulation and analysis, which is associated with a lower induction of inflammatory responses. Additionally, while decreasing the level of lipopolysaccharides (LPS) to inject, we also came across new observations regarding mechanistic properties in the spleen.

Vagus nerve stimulation has proven to have a strong efficacy for decreasing peripheral inflammation. Here, we present a modified vagus nerve stimulation protocol that allows for further examinations of the cholinergic anti-inflammatory 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 quantitative analysis of cell surface proteins on immune cells, but it provides no information on the localization and migration patterns of these cells within the lung. Similarly,&#160;chemotaxis assays can be performed to study the potential of cells to respond to chemotactic factors in vitro, but these assays do not reproduce the complex environment of the intact lung. In contrast to these aforementioned techniques, the location of individual cell types within the lung can be readily visualized by generating Precision-cut Lung Slices (PCLS), staining them with commercially available, fluorescently tagged antibodies, and visualizing the sections by confocal microscopy. PCLS can be used for both live and fixed lung tissue, and the slices can encompass areas as large as a cross section of an entire lobe. We have used this protocol to successfully visualize the location of a wide variety of cell types in the lung, including distinct types of dendritic cells, macrophages, neutrophils, T cells and B cells, as well as structural cells such as lymphatic, endothelial, and epithelial cells. The ability to visualize cellular interactions, such as those between dendritic cells and T cells, in live, three-dimensional lung tissue, can reveal how cells move within the lung and interact with one another at steady state and during inflammation. Thus, when used in combination with other procedures, such as flow cytometry and quantitative PCR, PCLS can contribute to a comprehensive understanding of cellular events that underlie allergic and inflammatory diseases of the lung.

We describe a method for generating Precision-cut Lung Slices (PCLS) and immunostaining them to visualize the localization of various immune cell types in the lung. Our protocol can be extended to visualize the location and function of many different cell types under a variety of conditions.

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 ...

A Galleria mellonella Oral Administration Model to Study Commensal-Induced Innate Immune Responses

The investigation of the immunogenic potential of commensal bacteria on the host immune system is one essential component when studying intestinal host-microbe interactions. It is well established that different commensals exhibit a different potential to stimulate the host intestinal immune system. Such investigations involve vertebrate animals, especially rodents. Since increasing ethical concerns are linked with experiments involving vertebrates, there is a high demand for invertebrate replacements models. 
Here, we provide a Galleria mellonella oral administration model using commensal non-pathogenic bacteria and the possible assessment of the immunogenic potential of commensals on the G. mellonella immune system. We demonstrate that G. mellonella is a useful alternative invertebrate replacement model that allows the analysis of commensals with different immunogenic potential such as Bacteroides vulgatus and Escherichia coli. Interestingly, the bacteria exhibited no killing effect on the larvae, which is similar to mammals. The immune responses of G. mellonella were comparable with vertebrate innate immune responses and involve recognition of the bacteria and production of antimicrobial molecules. We propose that G. mellonella was able to restore previous microbiota balance, which is well known from healthy mammalian individuals. Although providing comparable innate immune responses in both G. mellonella and vertebrates, G. mellonella does not harbor an adaptive immune system. Since the investigated components of the innate immune system are evolutionary conserved, the model allows a prescreening and first analysis of bacterial immunogenic properties.

A Galleria mellonella Oral Administration Model to Study Commensal-Induced Innate Immune Responses

Here, we provide a detailed protocol for an oral administration model using Galleria mellonella larvae and how to characterize induced innate immune responses. Using this protocol, researchers without practical experience will be able to use the G. mellonella force-feeding method.