Assessing Respiratory Immune Responses to Haemophilus Influenzae

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.

1. Subcellular components
   1. Obtain subcellular components from sources, including commercial preparations, in-house developed components and/or from other investigators.
      ​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.
2. Killed/inactivated bacteria
   1. 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).
   2. 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 °C in glycerol broth in microcentrifuge tubes.
      NOTE: In this experiment, ten distinct strains were used.
   3. Thaw the strains (one microcentrifuge tube at a time) out onto chocolate agar plates and grow overnight in a 37 °C incubator with 5% CO₂. Add the bacteria to 500 μL 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).
   4. Heat inactivate the bacteria by placing them in a water bath at a temperature of 56 °C for 10 min.
   5. Sonicate the bacteria using a continuous sonication setting at a low-to-mid range speed for 30 s.
   6. Aliquot the appropriate volume of samples into microcentrifuge tubes. For a sample of 10⁸ mL, use aliquots of 50 μL (i.e., 20 samples per mL), freeze them at -70 °C until required¹⁴.
3. Live bacteria
   1. 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 °C as a reserve.
   2. Grow the bacteria on enriched media such as chocolate agar plates or in broth.
      1. To use chocolate agar plates, spread the bacteria for a minimum of every 3-4 days with sterile spreaders.
      2. Alternatively, use Brain Heart Infusion (BHI) broth enriched with factors X (hemin) and V (β-nicotinamide adenine dinucleotide) to grow the bacteria. Inoculate NTHi from overnight plates in 5-10 mL of BHI broth supplemented with both hemin and β-nicotinamide adenine dinucleotide (both 10 µg/mL) and culture overnight at 37 °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.
   3. 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.
   4. 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.
      ​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.

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.

1. Mix killed, inactivated NTHi (1.2) with propidium iodide (PI) at 100 μg/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 μL of PBS. Spin down at 450 x g for 5 min and resuspend the pellet in 500 μL of PBS.
2. Incubate 450 μL of peripheral blood (from venepuncture of a human subject) with 50 μL of inactivated PI labeled H. influenzae for 20 min in a water bath at 37 °C in a 5 mL tube.
3. Remove the samples from the water bath and add 5 μL of dihydrorhodamine-1,2,3 (DHR). Vortex for 10 s, and then place it back in the water bath for a further 10 min.
4. Remove the samples from the water bath and lyse the erythrocytes (500 μL 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.
5. 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).
   1. To analyze the samples for phagocytosis, determine the proportion of cells having ingested labeled bacteria (measure the proportion of cells having the fluorescent stain).
   2. 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.
   3. Distinguish the neutrophils and macrophages morphologically from each other by their size (macrophages are larger) and granularity (neutrophils are more granular).
      ​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.
6. Modify the assay as appropriate (e.g., use live unlabeled NTHi to stimulate phagocytes and measure the ROS expression by DHR cleavage).
   1. 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.
   2. As an alternative, use BAL macrophages.
   3. 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.
   4. Add DHR to the suspension as described in step 2.3 for 10 min. Perform the analysis for ROS using flow cytometry.

3. Assessment of lymphocyte function in peripheral blood

1. Use whole blood or mononuclear cell preparations for flow cytometry assays¹⁴.
2. 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.
3. Add costimulatory antibodies (anti-CD28 and CD49d, 1 µL/mL) to both the samples. Add NTHi to the antigen sample and incubate at 37 °C and 5% CO₂ for 1 h. For the killed NTHi preparation, add 200 μL 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).
4. Add the Golgi-blocking agent Brefeldin A (10 µL/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.
5. 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 μL of 1%-2% paraformaldehyde for 1 h.
6. Count the cells by hemocytometer, permeabilize 10⁶ cells with 100 μL 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' instructions. Typically, 10⁶ cells in 100 μL of solution will be stained for 1 h. Most commercially obtained antibodies will be enough to perform 25-100 tests.
7. 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.

4. Assessment of lymphocyte function/inflammatory mediators in lung tissue

1. 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).
2. 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.
   1. 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 µL chamber before being mechanically fragmented using an appropriate disaggregator.
   2. After tissue disaggregation, lyse the red blood cells with 0.8% NH₄Cl as mentioned in step 2.4.
   3. 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 µm sterile nylon mesh. Count the number of viable cells using the trypan blue exclusion method.
3. NTHi infection assay
   1. Resuspend the lung cells to a final cell concentration of 4 x 10⁶ cells/mL per tube (control and stimulated samples).
   2. 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.
   3. Place the cells in a tube rotator and incubate them at 37 °C while rotating at 12 rpm.
   4. 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 µg/mL. Return the cell suspensions for a further 16-22 h incubation on rotation (step 4.3.3).
   5. Wash the cell suspension with 500 μL 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.
   6. 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.
   7. Incubate the cells with intracellular cytokine staining antibodies (e.g., IFN-γ and TNF-α 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.
   8. Wash the cells with 500 μL of PBS and resuspend in 100 μL of PBS before data acquisition on a flow cytometer.
4. 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 μL aliquots and store at -70 °C until ready for analysis. Alternatively, one can use multiplex chemiluminescent assays²⁰.
   1. Use multiplex bead arrays to analyze the thawed samples. Use the stored supernatant to analyze cytokine production following the manufacturer's instructions.
   2. Perform the acquisition of the multiplex bead array on a flow cytometer. Use relevant software to perform the downstream data analysis.
      ​NOTE: This bead array assay may also be performed on supernatant from peripheral blood and/or BAL samples.

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.

1. Assess direct protease expression in lung tissue (as described in step 4.2.1) using confocal microscopy and in situ zymography^15,17.
   1. Use either fresh or frozen unfixed lung tissue. Mount the sections cut to a thickness of 4 µm on superfrost/adhesion slides. Pre-warm the sections in 1x reaction buffer for 5 min.
   2. Add fluorescein-labeled gelatin substrate (30 µg/mL per section) directly on individual slides to measure proteolysis via the action of metalloproteinases.
   3. Place the slides in a horizontal position and incubate in a light-protected, humidified chamber at 37 °C for 1 h.
   4. Rinse the sections in 1x reaction buffer before being mounted.
   5. As a negative control, add only the reaction buffer without fluorescent gelatin to the sections.
   6. 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.
2. Measure the extracellular ROS expression in lung tissue by immunoreactivity for 3-nitrotyrosine (a toxic oxidative stress product)¹³.