Name: assessing respiratory immune responses to haemophilus influenzae
Uploaded: 2021-06-29T13:00:00
Description: Watch this Scientific Journal Video about Assessing Respiratory Immune Responses to Haemophilus Influenzae at JoVE.com

The authors would like to thank the staff of Clinical Immunology at Monash Health for their assistance with this work.

This work was approved by the human research ethics committee of Monash Health. The protocol follows the guidelines of the human research ethics committee.
1. Antigenic preparation
NOTE: Three different antigenic preparations can be used to assess the immune response to Hi. These are 1) a subcellular component (typically from the bacterial cell wall); 2) killed and inactivated bacteria; and 3) live bacteria. Determine the use of each antigenic preparation prior to the...

<ol>
	<li>Smith-Vaughan, H. C., Sriprakash, K. S., Leach, A. J., Mathews, J. D., Kemp, D. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Low+genetic+diversity+of+Haemophilus+influenzae+type+b+compared+to+nonencapsulated+H.+influenzae+in+a+population+in+which+H.+influenzae+is+highly+endemic.">Low genetic diversity of Haemophilus influenzae type b compared to nonencapsulated H. influenzae in a population in which H. influenzae is highly endemic.</a> Infection and Immunity. 66, 3403-3409 (1998).</li><li>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=Haemophilus+and+Moxarella+infections.">Haemophilus and Moxarella infections.</a> Harrisons Principles of Internal Medicine. 152, (2018).</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> Journal of Immunology Research. , 706376 (2015).</li><li>Ahearn, C. P., Gallo, M. C., 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=Insights+on+persistent+airway+infection+by+non-typeable+Haemophilus+influenzae+in+chronic+obstructive+pulmonary+disease.">Insights on persistent airway infection by non-typeable Haemophilus influenzae in chronic obstructive pulmonary disease.</a> Pathogens and Disease. 75, 9 (2017).</li><li>Brinkmann, V., 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=Neutrophil+extracellular+traps+kill+bacteria.">Neutrophil extracellular traps kill bacteria.</a> Science. 303, 1532-1535 (2004).</li><li>Brinkmann, V., Zychlinsky, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Neutrophil+extracellular+traps:+is+immunity+the+second+function+of+chromatin.">Neutrophil extracellular traps: is immunity the second function of chromatin.</a> Journal of Cell Biology. 198, 773-783 (2012).</li><li>Jorch, S. 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Z., Palaniyar, N. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=NET+balancing:+a+problem+in+inflammatory+lung+diseases.">NET balancing: a problem in inflammatory lung diseases.</a> Frontiers in Immunology. 4, 1 (2013).</li><li>Jacobs, D. M., Ochs-Balcom, H. M., Zhao, J., Murphy, T. F., Sethi, S. <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+bacterial+colonization+patterns+and+species-specific+interactions+in+chronic+obstructive+pulmonary+disease.">Lower airway bacterial colonization patterns and species-specific interactions in chronic obstructive pulmonary disease.</a> Journal of Clinical Microbiology. 56, (2018).</li><li>Barenkamp, S. J., Munson, R. S., Granoff, D. 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W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Laboratory+growth+and+maintenance+of+Haemophilus+influenzae.">Laboratory growth and maintenance of Haemophilus influenzae.</a> Current Protocols in Microbiology. , (2010).</li><li>King, P. 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=Adaptive+immunity+to+nontypeable+Haemophilus+influenzae.">Adaptive immunity to nontypeable Haemophilus influenzae.</a> American Journal of Respiratory and Critical Care Medicine. 167, 587-592 (2003).</li><li>Coleman, H. N., Daines, D. A., Jarisch, J., Smith, A. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Chemically+defined+media+for+growth+of+Haemophilus+influenzae+strains.">Chemically defined media for growth of Haemophilus influenzae strains.</a> Journal of Clinical Microbiology. 41, 4408-4410 (2003).</li><li>King, P. T., Ngui, J., Gunawardena, D., Holmes, P. W., Farmer, M. W., Holdsworth, S. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Systemic+humoral+immunity+to+non-typeable+Haemophilus+influenzae.">Systemic humoral immunity to non-typeable Haemophilus influenzae.</a> Clinical &amp; Experimental Immunology. 153, 376-384 (2008).</li><li>King, P. 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=Nontypeable+Haemophilus+influenzae+induces+sustained+lung+oxidative+stress+and+protease+expression.">Nontypeable Haemophilus influenzae induces sustained lung oxidative stress and protease expression.</a> PLoS One. 10, 0120371 (2015).</li><li>Aaron, S. D., 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=Granulocyte+inflammatory+markers+and+airway+infection+during+acute+exacerbation+of+chronic+obstructive+pulmonary+disease.">Granulocyte inflammatory markers and airway infection during acute exacerbation of chronic obstructive pulmonary disease.</a> American Journal of Respiratory and Critical Care Medicine. 163, 349-355 (2001).</li><li>King, P. 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=Lung+T-cell+responses+to+nontypeable+Haemophilus+influenzae+in+patients+with+chronic+obstructive+pulmonary+disease.">Lung T-cell responses to nontypeable Haemophilus influenzae in patients with chronic obstructive pulmonary disease.</a> The Journal of Allergy and Clinical Immunology. 131, 1314-1321 (2013).</li><li>Tsujikawa, 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=Robust+cell+detection+and+segmentation+for+image+cytometry+reveal+th17+cell+heterogeneity.">Robust cell detection and segmentation for image cytometry reveal th17 cell heterogeneity.</a> Cytometry A. 95, 389-398 (2019).</li><li>Sharma, R., O'Sullivan, K. M., Holdsworth, S. R., Bardin, P. G., King, P. 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The methods listed here use fluorescence-based flow cytometry and confocal microscopy techniques that can be used in conjunction to obtain detailed information about the inflammatory lung response to Hi.
Establishing the appropriate antigenic formulation of Hi to be used is critical, and it is advisable to have specific input from a microbiologist in this regard. Live Hi induces a stronger response, while killed Hi preparations and Hi components are more standardized and are easier to store. P...

Haemophilus influenzae (Hi) is a normal commensal bacterium present in the pharynx of most healthy adults. Hi may have a polysaccharide capsule (types A-F, e.g., type B or HiB) or lack a capsule and be nontypeable (NTHi)1. Colonization of the mucosa with this bacterium begins in early childhood, and there is a turnover of different colonizing strains2. This bacterium is also capable of invasion of both the upper and lower respiratory tract; in this context, it may induce activation of the immune response and inflammation3,4. This inflammatory response may cause clinical disease and contribute to a variety of important respiratory conditions, including sinusitis, otitis media, bronchitis, cystic fibrosis, pneumonia, and chronic obstructive pulmonary disease (COPD). Most of these conditions are due to NTHi strains2. This article will describe methods to assess respiratory immune responses to Hi using flow cytometry and confocal microscopy.
The methods described below have been adapted from well-established techniques that have been modified to assess the inflammatory response to Hi. The selection of an appropriate antigenic form of Hi is a key part of this assessment. Antigenic preparations range from cell wall components to live bacteria. To establish and standardize assays, the use of peripheral blood samples may be very helpful initially.
Flow cytometry enables the measurement of a variety of parameters and functional assays from one sample at a cellular level. This technique has the advantage that specific cellular responses (e.g., production of reactive oxygen species (ROS) or intracellular cytokine production) can be assessed when compared to other more general methods such as enzyme-linked immunosorbent assay (ELISA) or ELISspot.
Extracellular traps are expressed by neutrophils (NETs)5,6,7 and by other cells such as macrophages (METs)8. They are increasingly recognized as a key inflammatory response, particularly in infection in the lung9. They may be assessed by confocal fluorescence microscopy. This technique allows definitive identification of NETs/METs and distinguishes their expression from other forms of cell death6.
Both flow cytometry and confocal microscopy are fluorescence-based assays. Their success is dependent on optimal straining protocols of biological samples. These methods do take some time to learn and require appropriate supervising expertise. The instruments involved are also expensive both to purchase and run. The optimal setting for their use includes major universities and tertiary referral hospitals.
The methods used in this protocol are transferable for the study of other similar organisms involved in respiratory disease (e.g., Moxarella catarrhalis and Streptococcus pneumoniae). NTHi also interacts with other common respiratory bacteria10.

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>Ammonium chloride</td>
			<td>Sigma Aldrich</td>
			<td>213330</td>
			<td></td>
		</tr>
		<tr>
			<td>Brefeldin</td>
			<td>Sigma Aldrich</td>
			<td>B6542</td>
			<td></td>
		</tr>
		<tr>
			<td>CD28</td>
			<td>Thermofisher</td>
			<td>16-0289-81</td>
			<td></td>
		</tr>
		<tr>
			<td>CD49d</td>
			<td>Thermofisher</td>
			<td>534048</td>
			<td></td>
		</tr>
		<tr>
			<td>DAPI prolong gold</td>
			<td>Thermofisher</td>
			<td>P36931</td>
			<td></td>
		</tr>
		<tr>
			<td>DHR123</td>
			<td>Sigma Aldrich</td>
			<td>109244-58-8</td>
			<td></td>
		</tr>
		<tr>
			<td>Filcon sterile nylon mesh</td>
			<td>Becton Dickinson</td>
			<td>340606</td>
			<td></td>
		</tr>
		<tr>
			<td>Gelatin substrate, Enzchek</td>
			<td>Molecular probes</td>
			<td>E12055</td>
			<td></td>
		</tr>
		<tr>
			<td>MACS mix tube rotater</td>
			<td>Miltenyi Biotec</td>
			<td>130-090-753</td>
			<td></td>
		</tr>
		<tr>
			<td>Medimachine</td>
			<td>Becton Dickinson</td>
			<td></td>
			<td>Catalogue number not available</td>
		</tr>
		<tr>
			<td>Medicons 50 &#181;m</td>
			<td>Becton Dickinson</td>
			<td>340592</td>
			<td></td>
		</tr>
		<tr>
			<td>Pansorbin</td>
			<td>Sigma Aldrich</td>
			<td>507858</td>
			<td></td>
		</tr>
		<tr>
			<td>Propidium iodide</td>
			<td>Sigma Aldrich</td>
			<td>P4170</td>
			<td></td>
		</tr>
		<tr>
			<td>Saponin</td>
			<td>Sigma Aldrich</td>
			<td>8047152</td>
			<td></td>
		</tr>
		<tr>
			<td>Superfrost slides</td>
			<td>Thermofisher</td>
			<td>11562203</td>
			<td></td>
		</tr>
	</tbody>
</table>

assessing respiratory immune responses to haemophilus influenzae

Haemophilus influenzae (Hi) is a prevalent bacterium found in a range of respiratory conditions. A variety of different assays/techniques may be used to assess the respiratory immune/inflammatory response to this bacterium. Flow cytometry and confocal microscopy are fluorescence-based technologies that allow detailed characterization of biological responses.&#160;Different forms of Hi antigen can be used, including cell wall components, killed/inactivated preparations, and live bacteria. Hi is a fastidious bacterium that requires enriched media but is generally easy to grow in standard laboratory settings. Tissue samples for stimulation with Hi may be obtained from peripheral blood, bronchoscopy, or resected lung (e.g., in patients undergoing surgery for the treatment of lung cancer). Macrophage and neutrophil function may be comprehensively assessed using flow cytometry with a variety of parameters measured, including phagocytosis, reactive oxygen species, and intracellular cytokine production. Lymphocyte function (e.g., T cell and NK cell function) may be specifically assessed using flow cytometry, principally for intracellular cytokine production. Hi infection is a potent inducer of extracellular trap production, both by neutrophils (NETs) and macrophages (METs). Confocal microscopy is arguably the most optimal way to assess NET and MET expression, which may also be used to assess protease activity. Lung immunity to Haemophilus influenzae can be assessed using flow cytometry and confocal microscopy.

The representative results show how inflammatory immune responses to NTHi can be assessed/quantitated by flow cytometry and confocal microscopy. A key part of the interpretation of the results is the comparison in fluorescence between control and stimulated samples. A number of preliminary experiments are usually required to optimize the staining of samples. How many different colors can be examined simultaneously will depend on the number of channels available on the flow cytometer/confocal microscope. Results are shown...

Watch this Scientific Journal Video about Assessing Respiratory Immune Responses to Haemophilus Influenzae at JoVE.com

Assessing Respiratory Immune Responses to Haemophilus Influenzae

Haemophilus influenzae induces inflammation in the respiratory tract. This article will focus on the use of flow cytometry and confocal microscopy to define immune responses by phagocytes and lymphocytes in response to this bacterium.

Assessing Respiratory Immune Responses to Haemophilus Influenzae

Haemophilus influenzae (Hi) is a prevalent bacterium found in a range of respiratory conditions. A variety of different assays/techniques may be used to ...

Haemophilus influenza is a major cause of inflammation in various chronic lung diseases including COPD and pneumonia. This protocol describes methods for assessing the effect of haemophilus on lung inflammation. The advantages of this technique is it allows for a comprehensive assessment of innate and adaptive immune responses.Incubate 450 microliters of peripheral blood with 50 microliters of an activated propidium iodide labeled H influenzae in a five milliliter tube for 20 minutes in a water bath at 37 degrees Celsius. Remove the sample from the water bath, add five microliters of DHR and vortex for 10 seconds. Then place it back in the water bath for another 10 minutes.After removing the sample from the water bath, lyse the erythrocytes with five milliliters of 0.8%ammonium chloride solution and analyze the samples on a flow cytometer as described in the text. Divide the peripheral blood sample into aliquots for control and antigen stimulation. Add costimulatory antibodies to both samples.Then add the non typable H influenzae to the antigen sample and incubate at 37 degrees Celsius and 5%carbon dioxide for one hour. Next, add the Golgi blocking agent brefeldin A to the samples and incubate them for another five hours. After lysing the erythrocytes as demonstrated previously, fix the leukocytes using 500 microliters of one to 2%para-formaldehyde for one hour.Count the cells using a hemocytometer, then permeabilize 1 million cells with 100 microliters of 0.1%saponin for 15 minutes. Next, incubate the cells with appropriate fluorescent labeled antibodies. Wash the cells, then analyze them using a flow cytometer.Determine the proportion of antigen responding cells by getting the relevant lymphocyte populations. Perform background staining on non stimulated cells for all the cytokines to be analyzed. Slice about 20 to 40 grams of the lobectomy sample into three to five cubic millimeter sections.Place them inside a sterile 50 microliter chamber and mechanically fragment the tissue using an appropriate dis-aggregator. After tissue disaggregation, lyse the red blood cells as demonstrated and resuspend the cells in sterile RPMI. Then filter the cells through a 100 micrometer sterile nylon mesh and count the of viable cells using the trypan blue exclusion method.For the infection assay, resuspend the lung cells in RPMI to a final concentration of 4 million cells per milliliter per tube. Then infect the cells at an MOI of 100 bacteria per cell. Loosen the cap half a rotation to allow gas transfer in the tubes.Place the cells in the tube rotator and incubate them at 37 degrees Celsius while rotating at 12 RPM. One hour after stimulation, add grefeldin A to prevent the extracellular export of cytokines and incubate the cell suspensions for a further 16 to 22 hours with rotation. The following day, wash the cell suspension with 500 microliters of PBS containing 1%bovine serum albumin and 0.01%sodium azide.Next, stain the cell suspension for specific human lymphocytes cell surface markers for one hour. Then wash the cells with PBS and fix and permeabilize them as demonstrated previously. Next, incubate the cells with intracellular cytokine staining antibodies for one hour.Then wash the cells and resuspend them in 100 microliters of PBS before data acquisition on a flow cytometer. Measure proteolysis via the action of metalloproteinases, add fluorescein labeled gelatin substrate directly on the lung tissue section slides. Place the slides horizontally and incubate them in a light protected humidified chamber at 37 degrees Celsius for one hour.To prepare negative controls, add one x reaction buffer without fluorescent gelatin to the sections for further analysis. Peripheral blood mononuclear cells were gated using forward and side scattered to define the phagocyte population. The phagocyte population was further defined by CD14 expression to label the monocytes.ROS measurement was performed via oxidation of DHR 123 to produce fluorescence. The median fluorescence of the stimulated sample was compared to the control. Intracellular cytokine production by lymphocytes was measured using flow cytometry.Cells were analyzed first for their expression of the leukocyte marker CD45 and then for CD three and CD four, CD eight. The CD three, CD four positive cells were assessed for intracellular cytokine production in control and stimulated samples. Protease activity was measured by NC two zymography in unfixed lung tissue sections.Fluorescent staining indicated the presence of MMP activity, which was also co-localized with the expression of chromatin. Adding antibodies to the lung tissue samples requires concentration and staining of the cells will require optimization in preliminary experiments. The supernatant of the lung tissue samples can be further analyzed for other inflammatory mediators using techniques such as ELISA and These techniques can be used to assess the effect of potential therapies on lung inflammation.

assessing-respiratory-immune-responses-to-haemophilus-influenzae

Research

JoVE Journal

Immunology and Infection

Measuring Bacterial Load and Immune Responses in Mice Infected with Listeria monocytogenes

Listeria monocytogenes (Listeria) is a Gram-positive facultative intracellular pathogen1. Mouse studies typically employ intravenous injection of Listeria, which results in systemic infection2. After injection, Listeria quickly disseminates to the spleen and liver due to uptake by CD8&alpha;+ dendritic cells and Kupffer cells3,4. Once phagocytosed, various bacterial proteins enable Listeria to escape the phagosome, survive within the cytosol, and infect neighboring cells5. During the first three days of infection, different innate immune cells (e.g. monocytes, neutrophils, NK cells, dendritic cells) mediate bactericidal mechanisms that minimize Listeria proliferation. CD8+ T cells are subsequently recruited and responsible for the eventual clearance of Listeria from the host, typically within 10 days of infection6.
Successful clearance of Listeria from infected mice depends on the appropriate onset of host immune responses6	. There is a broad range of sensitivities amongst inbred mouse strains7,8. Generally, mice with increased susceptibility to Listeria infection are less able to control bacterial proliferation, demonstrating increased bacterial load and/or delayed clearance compared to resistant mice. Genetic studies, including linkage analyses and knockout mouse strains, have identified various genes for which sequence variation affects host responses to Listeria infection6,8-14. Determination and comparison of infection kinetics between different mouse strains is therefore an important method for identifying host genetic factors that contribute to immune responses against Listeria. Comparison of host responses to different Listeria strains is also an effective way to identify bacterial virulence factors that may serve as potential targets for antibiotic therapy or vaccine design.
We describe here a straightforward method for measuring bacterial load (colony forming units [CFU] per tissue) and preparing single-cell suspensions of the liver and spleen for FACS analysis of immune responses in Listeria-infected mice. This method is particularly useful for initial characterization of Listeria infection in novel mouse strains, as well as comparison of immune responses between different mouse strains infected with Listeria. We use the Listeria monocytogenes EGD strain15 that, when cultured on blood agar, exhibits a characteristic halo zone around each colony due to &beta;-hemolysis1 (Figure 1). Bacterial load and immune responses can be determined at any time-point after infection by culturing tissue homogenate on blood agar plates and preparing tissue cell suspensions for FACS analysis using the protocols described below. We would note that individuals who are immunocompromised or pregnant should not handle Listeria, and the relevant institutional biosafety committee and animal facility management should be consulted before work commences.

Measuring Bacterial Load and Immune Responses in Mice Infected with Listeria monocytogenes

Listeria monocytogenes is a model organism for studying immune responses and genetic susceptibility to intracellular bacteria in mice. This method enables one to measure bacterial load and generate single-cell suspensions of the liver and spleen from mice for FACS analysis to determine changes in immune cells due to Listeria infection.

Listeria monocytogenes (Listeria) is a Gram-positive facultative intracellular pathogen1. Mouse studies typically employ intravenous injection of ...

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 ...

Quantification of the Respiratory Burst Response as an Indicator of Innate Immune Health in Zebrafish

The phagocyte respiratory burst is part of the innate immune response to pathogen infection and involves the production of reactive oxygen species (ROS). ROS are toxic and function to kill phagocytized microorganisms. In vivo quantification of phagocyte-derived ROS provides information regarding an organism's ability to mount a robust innate immune response. Here we describe a protocol to quantify and compare ROS in whole zebrafish embryos upon chemical induction of the phagocyte respiratory burst. This method makes use of a non-fluorescent compound that becomes fluorescent upon oxidation by ROS. Individual zebrafish embryos are pipetted into the wells of a microplate and incubated in this fluorogenic substrate with or without a chemical inducer of the respiratory burst. Fluorescence in each well is quantified at desired time points using a microplate reader. Fluorescence readings are adjusted to eliminate background fluorescence and then compared using an unpaired t-test. This method allows for comparison of the respiratory burst potential of zebrafish embryos at different developmental stages and in response to experimental manipulations such as protein knockdown, overexpression, or treatment with pharmacological agents. This method can also be used to monitor the respiratory burst response in whole dissected kidneys or cell preparations from kidneys of adult zebrafish and some other fish species. We believe that the relative simplicity and adaptability of this protocol will complement existing protocols and will be of interest to researchers who seek to better understand the innate immune response.

The innate immune response protects organisms against pathogen infection. A critical component of the innate immune response, the phagocyte respiratory burst, generates reactive oxygen species that kill invading microorganisms. We describe a respiratory burst assay that quantifies reactive oxygen species produced when the innate immune response is chemically induced.

The phagocyte respiratory burst is part of the innate immune response to  pathogen infection and involves the production of reactive oxygen species (ROS). ...

An In vitro Model to Study Immune Responses of Human Peripheral Blood Mononuclear Cells to Human Respiratory Syncytial Virus Infection

Human respiratory syncytial virus (HRSV) infections present a broad spectrum of disease severity, ranging from mild infections to life-threatening bronchiolitis. An important part of the pathogenesis of severe disease is an enhanced immune response leading to immunopathology.	Here, we describe a protocol used to investigate the immune response of human immune cells to an HRSV infection. First, we describe methods used for culturing, purification and quantification of HRSV. Subsequently, we describe a human in vitro model in which peripheral blood mononuclear cells (PBMCs) are stimulated with live HRSV. This model system can be used to study multiple parameters that may contribute to disease severity, including the innate and adaptive immune response. These responses can be measured at the transcriptional and translational level. Moreover, viral infection of cells can easily be measured using flow cytometry. Taken together, stimulation of PBMC with live HRSV provides a fast and reproducible model system to examine mechanisms involved in HRSV-induced disease.

An In vitro Model to Study Immune Responses of Human Peripheral Blood Mononuclear Cells to Human Respiratory Syncytial Virus Infection

Human respiratory syncytial virus (HRSV) can cause severe bronchiolitis in young infants. Part of the pathogenesis of severe HRSV disease is caused by the host immune response. Stimulation of primary human immune cells with HRSV provides a fast and reproducible model system to study activation of inflammatory pathways and infection.

Human respiratory syncytial  virus (HRSV) infections present a broad spectrum of disease severity, ranging  from mild infections to life-threatening ...

An In Vitro Model for Measuring Immune Responses to Malaria in the Context of HIV Co-infection

Malaria and HIV co-infection is a growing health priority. However, most research on malaria or HIV currently focuses on each infection individually. Although understanding the disease dynamics for each of these pathogens independently is vital, it is also important that the interactions between these pathogens are investigated and understood. 
We have developed a versatile in vitro model of HIV-malaria co-infection to study host immune responses to malaria in the context of HIV infection. Our model allows the study of secreted factors in cellular supernatants, cell surface and intracellular protein markers, as well as RNA expression levels. The experimental design and methods used limit variability and promote data reliability and reproducibility. 
All pathogens used in this model are natural human pathogens (Plasmodium falciparum and HIV-1), and all infected cells are naturally infected and used fresh. We use human erythrocytes parasitized with P. falciparum and maintained in continuous in vitro culture. We obtain freshly isolated peripheral blood mononuclear cells from chronically HIV-infected volunteers. Every condition used has an appropriate control (P. falciparum parasitized vs. normal erythrocytes), and every HIV-infected donor has an HIV uninfected control, from which cells are harvested on the same day. This model provides a realistic environment to study the interactions between malaria parasites and human immune cells in the context of HIV infection.

An In Vitro Model for Measuring Immune Responses to Malaria in the Context of HIV Co-infection

Human co-infection is difficult to replicate in vitro. However, human malaria parasites can readily be cultured in vitro, as can freshly isolated human peripheral blood mononuclear cells naturally infected with HIV. This provides an excellent model for studying early immune responses to malaria parasites in the context of HIV co-infection.

Malaria and HIV co-infection is a growing health priority. However, most research on malaria or HIV currently focuses on each infection individually. ...

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 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.

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

Using Zebrafish Models of Human Influenza A Virus Infections to Screen Antiviral Drugs and Characterize Host Immune Cell Responses

Each year, seasonal influenza outbreaks profoundly affect societies worldwide. In spite of global efforts, influenza remains an intractable healthcare burden. The principle strategy to curtail infections is yearly vaccination. In individuals who have contracted influenza, antiviral drugs can mitigate symptoms. There is a clear and unmet need to develop alternative strategies to combat influenza. Several animal models have been created to model host-influenza interactions. Here, protocols for generating zebrafish models for systemic and localized human influenza A virus (IAV) infection are described. Using a systemic IAV infection model, small molecules with potential antiviral activity can be screened. As a proof-of-principle, a protocol that demonstrates the efficacy of the antiviral drug Zanamivir in IAV-infected zebrafish is described. It shows how disease phenotypes can be quantified to score the relative efficacy of potential antivirals in IAV-infected zebrafish. In recent years, there has been increased appreciation for the critical role neutrophils play in the human host response to influenza infection. The zebrafish has proven to be an indispensable model for the study of neutrophil biology, with direct impacts on human medicine. A protocol to generate a localized IAV infection in the Tg(mpx:mCherry) zebrafish line to study neutrophil biology in the context of a localized viral infection is described. Neutrophil recruitment to localized infection sites provides an additional quantifiable phenotype for assessing experimental manipulations that may have therapeutic applications. Both zebrafish protocols described faithfully recapitulate aspects of human IAV infection. The zebrafish model possesses numerous inherent advantages, including high fecundity, optical clarity, amenability to drug screening, and availability of transgenic lines, including those in which immune cells such as neutrophils are labeled with fluorescent proteins. The protocols detailed here exploit these advantages and have the potential to reveal critical insights into host-IAV interactions that may ultimately translate into the clinic.

Systemic and localized zebrafish infection models for human influenza A virus are demonstrated. Using a systemic infection model, zebrafish can be used to screen antiviral drugs. Using a localized infection model, zebrafish can be used to characterize host immune cell responses.

Each year, seasonal influenza outbreaks profoundly affect societies worldwide. In spite of global efforts, influenza remains an intractable healthcare ...

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.

The investigation of the immunogenic potential of commensal bacteria on the host immune system is one essential component when studying intestinal ...

Isolation of Tonsillar Mononuclear Cells to Study Ex Vivo Innate Immune Responses in a Human Mucosal Lymphoid Tissue

Studying isolated cells from mucosa-associated lymphoid tissues (MALT) allows understanding of immune cells response in pathologies involving mucosal immunity, because they can model host-pathogen interactions in the tissue. While isolated cells derived from tissues were the first cell culture model, their use has been neglected because tissue can be hard to obtain. In the present protocol, we explain how to easily process and culture tonsillar mononuclear cells (TMCs) from healthy human tonsils to study innate immune responses upon activation, mimicking viral infection in mucosal tissues. Isolation of TMCs from the tonsils is quick, because the tonsils barely have any epithelium and yield up to billions of all major immune cell types. This method allows detection of cytokine production using several techniques, including immunoassays, qPCR, microscopy, flow cytometry, etc., similar to the use of peripheral mononuclear cells (PBMCs) from blood. Furthermore, TMCs show a higher sensitivity to drug testing than PBMCs, which needs to be considered for future toxicity assays. Thus, ex vivo TMCs cultures are an easy and accessible mucosal model.

In the present protocol, we explain how to easily process and culture tonsillar mononuclear cells from healthy humans undergoing partial surgical tonsillectomy to study innate immune responses upon activation, mimicking viral infection in mucosal tissues.

Studying isolated cells from mucosa-associated lymphoid tissues (MALT) allows understanding of immune cells response in pathologies involving mucosal ...

Xenograft Skin Model to Manipulate Human Immune Responses In Vivo

The human skin xenograft model, in which human donor skin is transplanted onto an immunodeficient mouse host, is an important option for translational research in skin immunology. Murine and human skin differ substantially in anatomy and immune cell composition. Therefore, traditional mouse models have limitations for dermatological research and drug discovery. However, successful xenotransplants are technically challenging and require optimal specimen and mouse graft site preparation for graft and host survival. The present protocol provides an optimized technique for transplanting human skin onto mice and discusses necessary considerations for downstream experimental aims. This report describes the appropriate preparation of a human donor skin sample, assembly of a surgical setup, mouse and surgical site preparation, skin transplantation, and post-surgical monitoring. Adherence to these methods allows for maintenance of xenografts for over 6 weeks post-surgery. The techniques outlined below allow maximum grafting efficiency due to the development of engineering controls, sterile technique, and pre- and post-surgical conditioning. Appropriate performance of the xenograft model results in long-lived human skin graft samples for experimental characterization of human skin and preclinical testing of compounds in vivo.

Xenograft Skin Model to Manipulate Human Immune Responses In Vivo

The present protocol describes how to graft human skin onto non-obese diabetic (NOD)-scid interleukin-2 gamma chain receptor (NSG) mice. A detailed description of the preparation of human skin for transplant, preparation of mice for transplant, transplantation of split-thickness human skin, and post-transplantation recovery procedure are included in the report.

The human skin xenograft model, in which human donor skin is transplanted onto an immunodeficient mouse host, is an important option for translational ...