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. K., Kubes, P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=An+emerging+role+for+neutrophil+extracellular+traps+in+noninfectious+disease.">An emerging role for neutrophil extracellular traps in noninfectious disease.</a> Nature Medicine. 23, 279-287 (2017).</li><li>Boe, D. M., Curtis, B. J., Chen, M. M., Ippolito, J. A., Kovacs, E. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Extracellular+traps+and+macrophages:+new+roles+for+the+versatile+phagocyte.">Extracellular traps and macrophages: new roles for the versatile phagocyte.</a> Journal of Leukocyte Biology. 97, 1023-1035 (2015).</li><li>Cheng, O. 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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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 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

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Immunology and Infection

1.9K Views.  Monash Medical Centre. 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 i...