Name: Assessing Respiratory Immune Responses to Haemophilus Influenzae (Video) | JoVE Journal
Uploaded: 2021-06-29T13:00:00.000+00:00
Duration: 6 min 32 s
Description: Learn methods to assess respiratory immune responses to Haemophilus influenzae using flow cytometry and confocal microscopy. This protocol enables comprehensive evaluation of innate and adaptive immunity in chronic lung disease research.

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5 minutes

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 initiation of any experiments.
<ol>
	<li>Subcellular components
	<ol>
		<li>Obtain subcellular components from sources, including commercial preparations, in-house developed components and/or from other investigators. 
		&#8203;NOTE: Subcellular components usually from the bacterial cell wall may be used and include outer-membrane proteins such as P6 and lipooligosaccharide (LOS)11,12. These subcellular components are usually derived in specialized centers. The description of how they are made is beyond the scope of this article. Direct contact with experts in this field is recommended to obtain these components.</li>
	</ol>
	</li>
	<li>Killed/inactivated bacteria
	<ol>
		<li>Obtain the bacteria from the appropriate sample (e.g., sputum or bronchoscopy). Confirm the strain as H. influenzae using an appropriate microbiology laboratory. Perform typing of the H. influenzae samples to confirm they are nontypeable (by a specialist microbiology laboratory).</li>
		<li>To obtain a representative antigen, use multiple NTHi strains (at least 5, each of approximately the same amount) to make a pooled antigen. Store the strains at -70 &#176;C in glycerol broth in microcentrifuge tubes. 
		NOTE: In this experiment, ten distinct strains were used.</li>
		<li>Thaw the strains (one microcentrifuge tube at a time) out onto chocolate agar plates and grow overnight in a 37 &#176;C incubator with 5% CO2. Add the bacteria to 500 &#956;L of phosphate-buffered saline (PBS) and wash them twice (spin at 300 x g for 5 min). Use a MacFarland standard10 or a spectrophotometer13 to aliquot NTHi to a concentration of 108 mL (using a 5 mL container or equivalent).</li>
		<li>Heat inactivate the bacteria by placing them in a water bath at a temperature of 56 &#176;C for 10 min.</li>
		<li>Sonicate the bacteria using a continuous sonication setting at a low-to-mid range speed for 30 s.</li>
		<li>Aliquot the appropriate volume of samples into microcentrifuge tubes. For a sample of 108 mL, use aliquots of 50 &#956;L (i.e., 20 samples per mL), freeze them at -70 &#176;C until required14.</li>
	</ol>
	</li>
	<li>Live bacteria
	<ol>
		<li>First, characterize live bacteria as mentioned in step 1.2.1. Use one well-characterized strain. Ensure that the strain is also stored in glycerol broth at -70 &#176;C as a reserve.</li>
		<li>Grow the bacteria on enriched media such as chocolate agar plates or in broth.
		<ol>
			<li>To use chocolate agar plates, spread the bacteria for a minimum of every 3-4 days with sterile spreaders.</li>
			<li>Alternatively, use Brain Heart Infusion (BHI) broth enriched with factors X (hemin) and V (&#946;-nicotinamide adenine dinucleotide) to grow the bacteria. Inoculate NTHi from overnight plates in 5-10 mL of BHI broth supplemented with both hemin and &#946;-nicotinamide adenine dinucleotide (both 10 &#181;g/mL) and culture overnight at 37 &#176;C in a 5% CO2 incubator. 
			NOTE: This has the advantage of reproducibility, higher colony-forming units (CFU) counts, and all bacteria being at a similar phase of log growth (on plates, bacterial viability can be greater depending on the position in the culture)13,15.</li>
		</ol>
		</li>
		<li>Use a multiplicity of infection (MOI) of 100 bacteria to one cell to elicit a strong immune response while maintaining cellular viability. Use MacFarland Standard10 or spectrophotometer13 to assess the number of bacteria.</li>
		<li>Ensure that the media used for live NTHi assays is free of any human serum, as this will kill the bacteria16. Animal serum samples (e.g., fetal calf serum) do not generally cause any problem. 
		&#8203;NOTE: Use the methods described below to analyze standard tissue samples such as peripheral blood, bronchoscopy samples (particularly bronchoalveolar lavage (BAL)), and resected lung tissue. Incubate the samples with Hi antigen from 10 min to 24 h or more.</li>
	</ol>
	</li>
</ol>
2. Assessment of phagocytic function by flow cytometry
NOTE: This assay requires cells in solution and is typically done in whole blood or using BAL fluid. This assay is modified from a previously published protocol based on the use of inactivated Staphylococcus aureus preparation and Pansorbin17. Inactivated whole blood and fixed H. influenzae is substituted for the Pansorbin17.
<ol>
	<li>Mix killed, inactivated NTHi (1.2) with propidium iodide (PI) at 100 &#956;g/mL in phosphate-buffered saline (PBS) at a 1:1 ratio for 30 min at room temperature. Centrifuge at 450 x g for 5 min, and then wash in 500 &#956;L of PBS. Spin down at 450 x g for 5 min and resuspend the pellet in 500 &#956;L of PBS.</li>
	<li>Incubate 450 &#956;L of peripheral blood (from venepuncture of a human subject) with 50 &#956;L of inactivated PI labeled H. influenzae for 20 min in a water bath at 37 &#176;C in a 5 mL tube.</li>
	<li>Remove the samples from the water bath and add 5 &#956;L of dihydrorhodamine-1,2,3 (DHR). Vortex for 10 s, and then place it back in the water bath for a further 10 min.</li>
	<li>Remove the samples from the water bath and lyse the erythrocytes (500 &#956;L of volume as listed above in step 2.2) with 10:1 (i.e., 5 mL) of 0.8% ammonium chloride (150 mM NH4Cl, 1 mM NaHCO3, and 0.1 mM EDTA) solution. 
	NOTE: Alternatively, an automated system such as Q-prep may be used.</li>
	<li>Analyze the samples on a flow cytometer within an hour. Analyze a minimum of 3,000-5,000 cells (from each subset of interest) from each sample to obtain representative results (Figure 1).
	<ol>
		<li>To analyze the samples for phagocytosis, determine the proportion of cells having ingested labeled bacteria (measure the proportion of cells having the fluorescent stain).</li>
		<li>Quantify the ROS by the oxidation of DHR, which results in a fluorescent signal (the shift in median fluorescence is compared between baseline and stimulated samples). 
		NOTE: Neutrophils are more granular and have more side scatter, while macrophages are bigger and have more forward scatter.</li>
		<li>Distinguish the neutrophils and macrophages morphologically from each other by their size (macrophages are larger) and granularity (neutrophils are more granular). 
		&#8203;NOTE: Specific markers for neutrophils and macrophages are not generally used, although CD14 may be used to label blood monocytes. Flow cytometry may potentially be used to distinguish between different functional forms of monocytes and macrophages (e.g., M1 and M2 subsets)18.</li>
	</ol>
	</li>
	<li>Modify the assay as appropriate (e.g., use live unlabeled NTHi to stimulate phagocytes and measure the ROS expression by DHR cleavage).
	<ol>
		<li>Isolate peripheral blood mononuclear cells by density gradient centrifugation. Layer the blood over the designated polymer for density gradient centrifugation and spin at 2300 x g for 30 min. Remove the mononuclear layer using a pipette, wash it twice with PBS and resuspend the cells in PBS at 106 cells per mL.</li>
		<li>As an alternative, use BAL macrophages.</li>
		<li>Suspend the monocytes/macrophages at a concentration of 106 cells/mL in the culture medium (RPMI). Infect them with live NTHi at an MOI of 100:1 for 1 h as described in step 1.3.</li>
		<li>Add DHR to the suspension as described in step 2.3 for 10 min. Perform the analysis for ROS using flow cytometry.</li>
	</ol>
	</li>
</ol>
3. Assessment of lymphocyte function in peripheral blood
<ol>
	<li>Use whole blood or mononuclear cell preparations for flow cytometry assays14.</li>
	<li>Acquire samples from each subject by venepuncture. Collect 4 mL of blood from a peripheral vein in a lithium heparin tube and divide the samples into aliquots for control and antigen stimulation.</li>
	<li>Add costimulatory antibodies (anti-CD28 and CD49d, 1 &#181;L/mL) to both the samples. Add NTHi to the antigen sample and incubate at 37 &#176;C and 5% CO2 for 1 h. For the killed NTHi preparation, add 200 &#956;L of the antigen to 2 mL of blood. For live NTHi, add live NTHi cells at an MOI of 100:1 to the white cells (as measured by hemocytometer).</li>
	<li>Add the Golgi-blocking agent Brefeldin A (10 &#181;L/mL) to the samples and incubate them for another 5 h. 
	NOTE: Blocking the Golgi apparatus prevents the cytokines from being exported outside the cell.</li>
	<li>If whole blood is used, lyse the erythrocytes with 0.8% ammonium chloride (step 2.4) to leave only leukocytes in solution. Fix the leukocytes using 500 &#956;L of 1%-2% paraformaldehyde for 1 h.</li>
	<li>Count the cells by hemocytometer, permeabilize 106 cells with 100 &#956;L of 0.1% saponin for 15 min and incubate the cells with fluorescent-labeled antibodies. Wash the cells and analyze them using a flow cytometer. 
	NOTE: The quantity of fluorescent-labeled antibodies will be specific for each cytokine. Follow the manufacturers&#39; instructions. Typically, 106 cells in 100 &#956;L of solution will be stained for 1 h. Most commercially obtained antibodies will be enough to perform 25-100 tests.</li>
	<li>Determine the proportion of antigen responding cells by gating the relevant lymphocyte population (cells are gated on by CD45, then by CD3, and later by CD4 or CD8 expression) as shown in Figure 2. Perform background staining on non-stimulated cells for all the cytokines to be analyzed. Screen 100,000 cells to analyze each cytokine for both stimulated cells and control.</li>
</ol>
4. Assessment of lymphocyte function/inflammatory mediators in lung tissue
<ol>
	<li>It is not usually possible to obtain enough lymphocytes from bronchoscopy; therefore, use the lung tissue from lobectomy samples. The optimization of lung tissue samples requires close liaison with the anatomical pathology service. Ensure the tissue has a margin of at least 3 cm from the tumor and is the cellular tissue without significant emphysema (as determined by the pathologist).</li>
	<li>Break the lung tissue into a cellular suspension before it can be used for flow cytometry assays. Digest the lung tissue chemically (e.g., with collagenase) or disaggregate it mechanically. 
	NOTE: The mechanical disaggregation of tissue may be preferred as this is associated with better surface staining of cells (e.g., CD3/4 labeling)19.
	<ol>
		<li>Obtain lobectomy samples from a pathologist (usually obtained from patients undergoing treatment of lung cancer). Slice about 20-40 g of the sample into 3-5 mm3 sections. Place them inside a sterile 50 &#181;L chamber before being mechanically fragmented using an appropriate disaggregator.</li>
		<li>After tissue disaggregation, lyse the red blood cells with 0.8% NH4Cl as mentioned in step 2.4.</li>
		<li>Resuspend the cells in sterile RPMI (the volume will depend on the cell numbers and generally is 10-20 mL), and then filter them through a 100 &#181;m sterile nylon mesh. Count the number of viable cells using the trypan blue exclusion method.</li>
	</ol>
	</li>
	<li>NTHi infection assay
	<ol>
		<li>Resuspend the lung cells to a final cell concentration of 4 x 106 cells/mL per tube (control and stimulated samples).</li>
		<li>Use a suspension of 4 x 106-6 x 106 cells in 1 mL of RPMI for the (negative) control tube. Use the same amount for the NTHi tube (any additional sample may be used as a positive control with SEB). Infect the cells in the NTHi tube at an MOI of 100 bacteria per cell. Loosen the cap half a rotation to allow gas transfer in the tubes.</li>
		<li>Place the cells in a tube rotator and incubate them at 37 &#176;C while rotating at 12 rpm.</li>
		<li>To prevent the extracellular export of cytokines, add a Golgi blocker (Brefeldin A) to the cell suspensions 1 h after stimulation to a final concentration of 10 &#181;g/mL. Return the cell suspensions for a further 16-22 h incubation on rotation (step 4.3.3).</li>
		<li>Wash the cell suspension with 500 &#956;L of PBS containing 1% bovine serum albumin (BSA) and 0.01% NaN3. Fix and permeabilize the cells as described in step 3.5 and step 3.6, respectively.</li>
		<li>Add the antibodies for intracellular cytokine staining (the choice of antibodies is to be determined by the investigator, as listed in the NOTE in step 3.6). Stain the cell suspension for specific human lymphocyte cell-surface markers (e.g., CD45, CD3, etc.) for 1 h. Wash the cells with PBS, fix and permeabilize them as described in steps 3.5-3.6.</li>
		<li>Incubate the cells with intracellular cytokine staining antibodies (e.g., IFN-&#947; and TNF-&#945; or IL-13 and IL-17A) for 1 h. 
		NOTE: It is recommended to stain surface markers first, then intracellular as fixation can cause conformational changes in the surface proteins to which antibodies bind.</li>
		<li>Wash the cells with 500 &#956;L of PBS and resuspend in 100 &#956;L of PBS before data acquisition on a flow cytometer.</li>
	</ol>
	</li>
	<li>A bead array assay may be used in conjunction to analyze the supernatant described in step 3.2 (Perform this without Brefeldin A). This allows analysis of a wide variety of different inflammatory mediators (potentially up to 40-50 mediators). Collect the supernatant in 100 &#956;L aliquots and store at -70 &#176;C until ready for analysis. Alternatively, one can use multiplex chemiluminescent assays20.
	<ol>
		<li>Use multiplex bead arrays to analyze the thawed samples. Use the stored supernatant to analyze cytokine production following the manufacturer&#39;s instructions.</li>
		<li>Perform the acquisition of the multiplex bead array on a flow cytometer. Use relevant software to perform the downstream data analysis. 
		&#8203;NOTE: This bead array assay may also be performed on supernatant from peripheral blood and/or BAL samples.</li>
	</ol>
	</li>
</ol>
5. Assessment of lung proteolysis by confocal microscopy
NOTE: Fluorescent confocal microscopy is complimentary to flow cytometry and can be used to assess protease and ROS inflammatory responses. Extracellular traps such as NETs and METs are composed of extracellular chromatin (DNA) with other inflammatory mediators, particularly proteases such as neutrophil elastase (NE) and matrix metalloproteinases (MMP). They can be assessed in BAL and lung tissue using confocal microscopy, and this has been described previously by Sharma, R. et al.21.
<ol>
	<li>Assess direct protease expression in lung tissue (as described in step 4.2.1) using confocal microscopy and in situ zymography15,17.
	<ol>
		<li>Use either fresh or frozen unfixed lung tissue. Mount the sections cut to a thickness of 4 &#181;m on superfrost/adhesion slides. Pre-warm the sections in 1x reaction buffer for 5 min.</li>
		<li>Add fluorescein-labeled gelatin substrate (30 &#181;g/mL per section) directly on individual slides to measure proteolysis via the action of metalloproteinases.</li>
		<li>Place the slides in a horizontal position and incubate in a light-protected, humidified chamber at 37 &#176;C for 1 h.</li>
		<li>Rinse the sections in 1x reaction buffer before being mounted.</li>
		<li>As a negative control, add only the reaction buffer without fluorescent gelatin to the sections.</li>
		<li>Use a confocal microscope to assay the lysis of the substrate by examination. Use ImageJ to determine the area of the lung with evidence of proteolysis. For this, measure the lung area that has staining above the background of the quenched sample. As another control, use the lung tissue with no fluorescent gelatin added15. 
		NOTE: Perform this on a minimum of 10 high power fields of view (FOV) for each sample.</li>
	</ol>
	</li>
	<li>Measure the extracellular ROS expression in lung tissue by immunoreactivity for 3-nitrotyrosine (a toxic oxidative stress product)13.</li>
</ol>

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<li>Ueckert, J. E., Nebe von-Caron, G., Bos, A. P., ter Steeg, P. F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Flow+cytometric+analysis+of+Lactobacillus+plantarum+to+monitor+lag+times%2C+cell+division+and+injury%5BTitle%5D)+AND+%22Letters+in+Applied+Microbiology%22%5BJournal%5D)">Flow cytometric analysis of Lactobacillus plantarum to monitor lag times, cell division and injury.</a> Letters in Applied Microbiology. 25, 295-299 (1997).</li>
<li>Essilfie, A. T., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Combined+Haemophilus+influenzae+respiratory+infection+and+allergic+airways+disease+drives+chronic+infection+and+features+of+neutrophilic+asthma%5BTitle%5D)+AND+%22Thorax%22%5BJournal%5D)">Combined Haemophilus influenzae respiratory infection and allergic airways disease drives chronic infection and features of neutrophilic asthma.</a> Thorax. 67, 588-599 (2012).</li>
<li>Huvenne, W., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Exacerbation+of+cigarette+smoke-induced+pulmonary+inflammation+by+Staphylococcus+aureus+enterotoxin+B+in+mice%5BTitle%5D)+AND+%22Respiratory+Research%22%5BJournal%5D)">Exacerbation of cigarette smoke-induced pulmonary inflammation by Staphylococcus aureus enterotoxin B in mice.</a> Respiratory Research. 12, 69(2011).</li>
<li>Radhakrishna, N., Farmer, M., Steinfort, D. P., King, P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((A+Comparison+of+Techniques+for+Optimal+Performance+of+Bronchoalveolar+Lavage%5BTitle%5D)+AND+%22Journal+of+Bronchology+%26+Interventional+Pulmonology%22%5BJournal%5D)">A Comparison of Techniques for Optimal Performance of Bronchoalveolar Lavage.</a> Journal of Bronchology & Interventional Pulmonology. 22, 300-305 (2015).</li>
<li>Quatromoni, J. G., Singhal, S., Bhojnagarwala, P., Hancock, W. W., Albelda, S. M., Eruslanov, E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((An+optimized+disaggregation+method+for+human+lung+tumors+that+preserves+the+phenotype+and+function+of+the+immune+cells%5BTitle%5D)+AND+%22Journal+of+Leukocyte+Biology%22%5BJournal%5D)">An optimized disaggregation method for human lung tumors that preserves the phenotype and function of the immune cells.</a> Journal of Leukocyte Biology. 97, 201-209 (2015).</li>
<li>Tighe, R. M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Improving+the+quality+and+reproducibility+of+flow+cytometry+in+the+lung.+An+official+American+thoracic+society+workshop+report%5BTitle%5D)+AND+%22American+Journal+of+Respiratory+and+Critical+Care+Medicine%22%5BJournal%5D)">Improving the quality and reproducibility of flow cytometry in the lung. An official American thoracic society workshop report.</a> American Journal of Respiratory and Critical Care Medicine. 61, 150-161 (2019).</li>
<li>Yu, Y. R., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((A+protocol+for+the+comprehensive+flow+cytometric+analysis+of+immune+cells+in+normal+and+inflamed+murine+non-lymphoid+tissues%5BTitle%5D)+AND+%22PLoS+One%22%5BJournal%5D)">A protocol for the comprehensive flow cytometric analysis of immune cells in normal and inflamed murine non-lymphoid tissues.</a> PLoS One. 11, 0150606(2016).</li>
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</ol>

The authors have nothing to disclose.

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><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 &amp;micro;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

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...

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.

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

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

Research

JoVE Journal

Immunology and Infection

This video demonstrates an in vitro technique to stimulate lymphocytes via non-typeable Haemophilus influenzae (NTHi) and to assess the immune response. Incubating isolated peripheral blood mononuclear cells (PBMCs) with NTHi causes the bacterial cells to stimulate T lymphocytes, leading to cytokine production. Upon inhibiting the secretion of cytokines, immunofluorescence staining is performed to detect the accumulated intracellular cytokines in stimulated T lymphocytes.

An In Vitro Technique to Stimulate Lymphocytes via Pathogenic Bacteria

This video demonstrates the quantification of inflammatory mediators during infection by incubating human lung-tissue-derived immune cells with Haemophilus influenzae. Flow cytometry is used to measure cytokine fluorescence in labeled lymphocytes, correlating with the production of pro-inflammatory cytokines in response to bacterial infection.

Quantification of Inflammatory Mediators in Infected Human Tissue-Derived Cells

This video demonstrates an assay for measuring neutralizing antibodies against respiratory syncytial virus (RSV). Pre-incubation with serum containing RSV-neutralizing antibodies neutralizes the virus particles, reducing cellular infection and plaque formation. Virus-infected cells are then subjected to fixation, blocking, and fluorophore-conjugated antibody labeling for plaque visualization. This process enables the identification of the highest serum dilution that neutralizes fifty percent of...

An Assay to Measure the Neutralizing Antibody Titer Specific for Respiratory Syncytial Virus

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.

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

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

This protocol describes a noninvasive technique for the sampling of undisturbed mucosal lining fluid from the upper airways. It can be used to perform the quantification of in vivo levels of protein mediators, such as cytokines and chemokines, in subjects of all ages.

Noninvasive Sampling of Mucosal Lining Fluid for the Quantification of In Vivo Upper Airway Immune-mediator Levels

The presented protocols describe how to perform a hemagglutination inhibition assay to quantify influenza-specific antibody titers from serum samples of influenza vaccine recipients. The first assay determines optimal viral antigen concentrations by hemagglutination. The second assay quantifies influenza-specific antibody titers by hemagglutination inhibition.

An Optimized Hemagglutination Inhibition (HI) Assay to Quantify Influenza-specific Antibody Titers

We describe a method to measure the activation of Fc-mediated effector functions by antibodies that target the influenza virus hemagglutinin. This assay can also be adapted to assess the ability of monoclonal antibodies or polyclonal sera targeting other viral surface glycoproteins to induce Fc-mediated immunity.

A Method to Assess Fc-mediated Effector Functions Induced by Influenza Hemagglutinin Specific Antibodies

The objective of the present article is to provide a detailed description of the recommended procedures to evaluate respiratory function in conscious mice by double-chamber plethysmography.

Assessment of Respiratory Function in Conscious Mice by Double-chamber Plethysmography

Here we present an elegant protocol for in vivo evaluation of vaccine effectiveness and host immune responses. This protocol can be adapted for vaccine models that study viral, bacterial, or parasitic pathogens.

Evaluation of Host-Pathogen Responses and Vaccine Efficacy in Mice

This protocol details an investigation of the early interactions between virally infected nasal epithelial cells and innate cell activation. Individual subsets of immune cells can be distinguished based on their activation in response to viral infections. They can then be further investigated to determine their effects on early antiviral responses.

Contact-Free Co-Culture Model for the Study of Innate Immune Cell Activation During Respiratory Virus Infection

Learn methods to assess respiratory immune responses to Haemophilus influenzae using flow cytometry and confocal microscopy. This protocol enables comprehensive evaluation of innate and adaptive immunity in chronic lung disease research.

Assessing Respiratory Immune Responses to Haemophilus Influenzae

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