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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P., King, P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+Comparison+of+Techniques+for+Optimal+Performance+of+Bronchoalveolar+Lavage.">A Comparison of Techniques for Optimal Performance of Bronchoalveolar Lavage.</a> Journal of Bronchology &amp; 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/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=An+optimized+disaggregation+method+for+human+lung+tumors+that+preserves+the+phenotype+and+function+of+the+immune+cells.">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/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Improving+the+quality+and+reproducibility+of+flow+cytometry+in+the+lung.+An+official+American+thoracic+society+workshop+report.">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/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+protocol+for+the+comprehensive+flow+cytometric+analysis+of+immune+cells+in+normal+and+inflamed+murine+non-lymphoid+tissues.">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><li>Duan, M., 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=Distinct+macrophage+subpopulations+characterize+acute+infection+and+chronic+inflammatory+lung+disease.">Distinct macrophage subpopulations characterize acute infection and chronic inflammatory lung disease.</a> Journal of Immunology. 189, 946-955 (2012).</li></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

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 ...

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

Research

JoVE Journal

Immunology and Infection

2.0K Views.  Monash Medical Centre. Haemophilus influenza is een belangrijke oorzaak van ontsteking bij verschillende chronische longziekten, waaronder COPD en longontsteking. Dit protocol beschrijft methoden voor het beoordelen van het effect van haemophilus op longontsteking. De voordelen van deze techniek is dat het een uitgebreide beoordeling van aangeboren en adaptieve immuunresponsen mogelijk maakt.Incubeer 450 microliter perifeer bloed met 50 microliter van een geactiveerd propidiumjodide met het label H influenza...

Video: Assessing Respiratory Immune Responses to Haemophilus Influenzae

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 ...

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 ...

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

This protocol describes noninvasive sampling of undisturbed upper airway mucosal lining fluid. It also details the extraction procedure used prior to the analysis of immune mediators in fluid eluates for the study of the airway topical immune signature, without the need for stimulation procedures (often used by other techniques). The mucosal lining fluid is sampled on a strip of filter paper placed at the anterior part of the inferior turbinate and left for 2 min of absorption. Analytes are eluted from the filter papers, and the extracted protein-based eluates are analyzed by an electrochemiluminescence-based immunoassay, allowing for the high-sensitivity quantification of low- and high-level analytes in the same sample. We measured the in vivo levels of 20 preselected immune mediators related to specific immune signaling pathways in the upper airway mucosa, but the technique is not limited to that specific panel or sampling site. The technique was first implemented in 7-year-old children from the Copenhagen Prospective Studies on Asthma in Childhood2000 (COPSAC2000) cohort with allergic rhinitis. It was thereafter used in the longitudinal COPSAC2010 birth cohort, sampled at 1 month, 2 years, and 6 years of age and at instances of acute respiratory symptoms. We successfully obtained and analyzed samples from 620 (89%) of 700 1-month-old children; a few samples were below the assay detection limit (reported as the median (Inter-Quartile Range (IQR)). The number of samples below the detection limit (i.e.&#160;from 0 to the set point for the lower limit of detection) for each mediator was 29 (7.25 - 119.5). This technique enables the quantification of the in vivo airway mucosal immune profile from birth, can be applied longitudinally, and can be applied to studies on the effect of genetics and early-life environmental exposures, pathophysiology, endotyping, and monitoring of respiratory diseases, and development and evaluation of novel therapeutics.

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

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.

This protocol describes noninvasive sampling of undisturbed upper airway mucosal lining fluid. It also details the extraction procedure used prior to the ...

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

Antibodies play a crucial role in coupling the innate and adaptive immune responses against viral pathogens through their antigen binding domains and Fc-regions. Here, we describe how to measure the activation of Fc effector functions by monoclonal antibodies targeting the influenza virus hemagglutinin with the use of a genetically engineered Jurkat cell line expressing an activating type 1 Fc-Fc&#947;R. Using this method, the contribution of specific Fc-Fc&#947;R interactions conferred by immunoglobulins can be determined using an in vitro assay.

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.

Antibodies play a crucial role in coupling the innate and adaptive immune responses against viral pathogens through their antigen binding domains and ...

Evaluation of Host-Pathogen Responses and Vaccine Efficacy in Mice

Vaccines are a 20th century medical marvel. They have dramatically reduced the morbidity and mortality caused by infectious diseases and contributed to a striking increase in life expectancy around the globe. Nonetheless, determining vaccine efficacy remains a challenge. Emerging evidence suggests that the current acellular vaccine (aPV) for Bordetella pertussis (B. pertussis) induces suboptimal immunity. Therefore, a major challenge is designing a next-generation vaccine that induces protective immunity without the adverse side effects of a whole-cell vaccine (wPV). Here we describe a protocol that we used to test the efficacy of a promising, novel adjuvant that skews immune responses to a protective Th1/Th17 phenotype and promotes a better clearance of a B. pertussis challenge from the murine respiratory tract. This article describes the protocol for mouse immunization, bacterial inoculation, tissue harvesting, and analysis of immune responses. Using this method, within our model, we have successfully elucidated crucial mechanisms elicited by a promising, next-generation acellular pertussis vaccine. This method can be applied to any infectious disease model in order to determine vaccine efficacy.

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.

Vaccines are a 20th century medical marvel. They have dramatically reduced the morbidity and mortality caused by infectious diseases and contributed to a ...

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

Antibody titers are commonly used as surrogate markers for serological protection against influenza and other pathogens. Detailed knowledge of antibody production pre- and post-vaccination is required to understand vaccine-induced immunity. This article describes a reliable point-by-point protocol to determine influenza-specific antibody titers. The first protocol describes a method to specify the antigen amounts required for hemagglutination, which standardizes the concentrations for subsequent usage in the second protocol (hemagglutination assay, HA assay). The second protocol describes the quantification of influenza-specific antibody titers against different viral strains by using a serial dilution of human serum or cell culture supernatants (hemagglutination inhibition assay, HI assay).
As an applied example, we show the antibody response of a healthy cohort, which received a trivalent inactivated influenza vaccine. Additionally, the cross-reactivity between the different influenza viruses is shown and methods to minimize cross-reactivity by using different types of animal red blood cells (RBCs) are explained. The discussion highlights advantages and disadvantages of the presented assays and how the determination of influenza-specific antibody titers can improve the understanding of vaccine-related immunity.

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

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.

Antibody titers are commonly used as surrogate markers for serological protection against influenza and other pathogens. Detailed knowledge of antibody ...

Medicine

Absorption of Nasal and Bronchial Fluids: Precision Sampling of the Human Respiratory Mucosa and Laboratory Processing of Samples

The methods of nasal absorption (NA) and bronchial absorption (BA) use synthetic absorptive matrices (SAM) to absorb the mucosal lining fluid (MLF) of the human respiratory tract. NA is a non-invasive technique which absorbs fluid from the inferior turbinate, and causes minimal discomfort. NA has yielded reproducible results with the ability to frequently repeat sampling of the upper airway. 
By comparison, alternative methods of sampling the respiratory mucosa, such as nasopharyngeal aspiration (NPA) and conventional swabbing, are more invasive and may result in greater data variability. Other methods have limitations, for instance, biopsies and bronchial procedures are invasive, sputum contains many dead and dying cells and requires liquefaction, exhaled breath condensate (EBC) contains water and saliva, and lavage samples are dilute and variable.
BA can be performed through the working channel of a bronchoscope in clinic. Sampling is well tolerated and can be conducted at multiple sites in the airway. BA results in MLF samples being less dilute than bronchoalveolar lavage (BAL) samples.
This article demonstrates the techniques of NA and BA, as well as the laboratory processing of the resulting samples, which can be tailored to the desired downstream biomarker being measured. These absorption techniques are useful alternatives to the conventional sampling techniques used in clinical respiratory research.

This manuscript describes the use of nasal and bronchial absorption techniques, specifically using synthetic absorptive matrices (SAM) to sample the mucosal lining fluid (MLF) of the upper and lower airway. These methods provide better standardization and tolerability than existing respiratory sampling techniques.

The methods of nasal absorption (NA) and bronchial absorption (BA) use synthetic absorptive matrices (SAM) to absorb the mucosal lining fluid (MLF) of the ...

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

Air volume changes created by a conscious subject breathing spontaneously within a body box are at the basis of plethysmography, a technique used to non-invasively assess some features of the respiratory function in humans as well as in laboratory animals. The present article focuses on the application of the double-chamber plethysmography (DCP) in small animals. It provides background information on the methodology as well as a detailed step-by-step procedure to successfully assess respiratory function in conscious, spontaneously breathing animals in a non-invasive manner. The DCP can be used to monitor the respiratory function of multiple animals in parallel, as well as to identify changes induced by aerosolized substances over a chosen time period and in a repeated manner. Experiments on control and allergic mice are used herein to demonstrate the utility of the technique, explain the associated outcome parameters, as well as to discuss the related advantages and shortcomings. Overall, the DCP provides valid and theoretically sound readouts that can be trusted to evaluate the respiratory function of conscious small animals both at baseline and after challenges with aerosolized substances.

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.

Air volume changes created by a conscious subject breathing spontaneously within a body box are at the basis of plethysmography, a technique used to ...

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

The early interactions between the nasal epithelial layer and the innate immune cells during viral infections remains an under-explored area. The significance of innate immunity signaling in viral infections has increased substantially as patients with respiratory infections who exhibit high innate T cell activation show a better disease outcome. Hence, dissecting these early innate immune interactions allows the elucidation of the processes that govern them and may facilitate the development of potential therapeutic targets and strategies for dampening or even preventing early progression of viral infections. This protocol details a versatile model that can be used to study early crosstalk, interactions, and activation of innate immune cells from factors secreted by virally infected airway epithelial cells. Using an H3N2 influenza virus (A/Aichi/2/1968) as the representative virus model, innate cell activation of co-cultured peripheral blood mononuclear cells (PBMCs) has been analyzed using flow cytometry to investigate the subsets of cells that are activated by the soluble factors released from the epithelium in response to the viral infection. The results demonstrate the gating strategy for differentiating the subsets of cells and reveal the clear differences between the activated populations of PBMCs and their crosstalk with the control and infected epithelium. The activated subsets can then be further analyzed to determine their functions as well as molecular changes specific to the cells. Findings from such a crosstalk investigation may uncover factors that are important for the activation of vital innate cell populations, which are beneficial in controlling and suppressing the progression of viral infection. Furthermore, these factors can be universally applied to different viral diseases, especially to newly emerging viruses, to dampen the impact of such viruses when they first circulate in na&#239;ve human populations.

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.

The early interactions between the nasal epithelial layer and the innate immune cells during viral infections remains an under-explored area. The ...

An In Vitro Technique to Stimulate Lymphocytes via Pathogenic Bacteria

Source: Dousha, L., et al. <a href="https://app.jove.com/v/62572">Assessing Respiratory Immune Responses to Haemophilus Influenzae.</a> J. Vis. Exp. (2021)
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.

Source: Dousha, L., et al. Assessing Respiratory Immune Responses to Haemophilus Influenzae. J. Vis. Exp. (2021)
This video demonstrates an in vitro ...

Assessing Respiratory Immune Responses to Haemophilus Influenzae

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