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 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. 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.
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.
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.
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.
3. Assessment of lymphocyte function in peripheral blood
4. Assessment of lymphocyte function/inflammatory mediators in lung tissue
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.
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 for the assessment of 1) ROS production, 2) intracellular cytokine staining of human lung tissue, and 3) in situ zymography to measure lung proteolysis.
Figure 1 shows the representation of ROS production by monocytes. The measurement of ROS is by the oxidation of DHR123 to produce fluorescence. Cells are gated on, and their median fluorescence is assessed by flow cytometry. The median fluorescence of the stimulated sample is compared to the control.
Figure 2 shows the intracellular production of cytokines by lymphocytes derived from human lung tissue. The lung tissue needs to be broken down into single-cell suspension before flow cytometry assays can be performed to assess inflammatory mediator production, e.g., cytokine production by lymphocytes. Lung tissue can be broken down mechanically or digested chemically, e.g., by collagenase. The mechanical breakdown methods may produce superior results, particularly in terms of retaining cell surface staining (e.g., CD3 and CD4). Filtering of the samples is important to exclude debris that may interfere with the analysis.
Figure 3 shows the expression of protease activity as measured by in situ zymography. Unfixed tissue is used to assess protease activity. These samples are typically frozen at -70 °C until analysis. The area of the tissue that has protease fluorescence is measured, and results are compared between control and stimulated samples.
Figure 1: ROS production by monocytes. (A) The panel shows the gating strategy for peripheral blood mononuclear cells (PBMC), with forward scatter and side scatter being used to define the phagocyte population. In panel (B), the phagocyte population is further defined by CD14 expression to label the monocytes. This monocyte population is analyzed for DHR fluorescence in control (C) and stimulated samples (D). Please click here to view a larger version of this figure.
Figure 2: Cytokine production in lung tissue. Cells are first analyzed for their expression of the leukocyte marker CD45 (A) using flow cytometry. This population is then analyzed further for CD3 expression (B) and CD4/CD8 expression (C). CD3/CD4+ cells are assessed for intracellular cytokine production in control (D) and NTHi-stimulated samples (E). Please click here to view a larger version of this figure.
Figure 3: Lung in situ zymography. Panel (A) shows the expression of chromatin/DAPI in sections of lung tissue. Panel (B) shows fluorescent staining, indicating the presence of MMP activity. Panel (C) shows the merged image indicating that the MMP activity is also co-localized with the expression of chromatin. Please click here to view a larger version of this figure.
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. PI will only label dead bacteria22; other dyes such as carboxyfluorescein succinimidylester (CFSE) could be used to label live bacteria for phagocytosis assays23. A series of preliminary experiments should be undertaken to optimize the appropriate antigen. For using live NTHi, an MOI of 100:1 is optimal; a lower MOI may not induce a clear immune response, while a higher MOI may be toxic to the cells. However, a dose-response curve may give useful information and may be very valuable, particularly with the initial optimization of the technique24. As a positive control, a commercially obtained form of inactivated Staphylococcus aureus antigen may be used, which is also labeled with PI as above for the ROS/phagocytosis assay. For the T cell assays, stimulation with Staphylococcal superantigen E (SEB) may be used as a positive control19,25.
The protocols for obtaining BAL and/or lung tissue samples need to be clearly established. The BAL samples can be quite variable between different operators. A hand-held syringe for the right middle lobe lavage produces good results26. The obtaining of the lung tissue sample from lobectomy samples requires the establishment of a collaboration with a pathologist. The lung tissue sample should have some margin from the tumor (ideally at least 3-4 cm). Bigger samples (e.g., at least 25-50 g) will yield more cells, as well as samples that are more proximal without obvious emphysema. The mechanical disaggregation of the lung tissue is time-consuming and will generally take at least 2-3 h for each sample19,27.
Preliminary experiments should be done to optimize the staining of different fluorophores for both flow cytometry and confocal microscopy. Areas to concentrate on include identifying the best staining panel, staining/concentration, and treatment of cells/tissue to maximize viability28. The use of lung tissue may be associated with more tissue debris than other fluid samples such as blood or BAL, and this may have some effect on the differentiation of different cellular populations by flow cytometry. The appropriate choice of antibodies needs to be determined and optimized in preliminary experiments. For surface labeling of lymphocytes, anti-CD3 and CD4 can be used for T helper cells, anti-CD3 and CD8 for cytotoxic T cells, and anti-CD3 and CD56 for NK cells. The choice of intracellular cytokines to be studied depends on the mediators of interest and the number of parameters/colors that can be analyzed on the flow cytometer29. A specific challenge of working with macrophages in lung tissue is their high level of autofluorescence30; this problem can be dealt with by comparing stimulated cells with background control and the addition of specific markers for inflammation such as proteases and histones.
A limitation of these techniques is the requirement for appropriately trained and skilled staff to perform the experiments. Well-established flow cytometry and microscopy facilities are also required. The use of human tissue samples is associated with significant variability particularly when using lung tissue; this may require a series of preliminary experiments to optimize assays (especially with issues of background staining).
The authors have nothing to disclose.
The authors would like to thank the staff of Clinical Immunology at Monash Health for their assistance with this work.
Ammonium chloride | Sigma Aldrich | 213330 | |
Brefeldin | Sigma Aldrich | B6542 | |
CD28 | Thermofisher | 16-0289-81 | |
CD49d | Thermofisher | 534048 | |
DAPI prolong gold | Thermofisher | P36931 | |
DHR123 | Sigma Aldrich | 109244-58-8 | |
Filcon sterile nylon mesh | Becton Dickinson | 340606 | |
Gelatin substrate, Enzchek | Molecular probes | E12055 | |
MACS mix tube rotater | Miltenyi Biotec | 130-090-753 | |
Medimachine | Becton Dickinson | Catalogue number not available | |
Medicons 50 µm | Becton Dickinson | 340592 | |
Pansorbin | Sigma Aldrich | 507858 | |
Propidium iodide | Sigma Aldrich | P4170 | |
Saponin | Sigma Aldrich | 8047152 | |
Superfrost slides | Thermofisher | 11562203 |