Quantification of Inflammatory Mediators in Infected Human Tissue-Derived Cells

All procedures involving human participants have been performed in compliance with the institutional, national, and international guidelines for human welfare and have been reviewed by the local institutional review board.

1. Antigenic preparation

1. Live bacteria
   1. First, characterize live bacteria. Obtain the bacteria from the appropriate sample (e.g., sputum or bronchoscopy). Confirm the strain as Haemophilus influenzae using an appropriate microbiology laboratory. Perform typing of the H. influenzae samples to confirm they are nontypeable (by a specialist microbiology laboratory). 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 non-typeable H. influenzae (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% carbon dioxide, 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).
   3. Use a multiplicity of infection (MOI) of 100 bacteria in one cell to elicit a strong immune response while maintaining cellular viability. Use MacFarland Standard or spectrophotometer 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 bacteria. Animal serum samples (e.g., fetal calf serum) do not generally cause any problems.        
      ​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 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).
   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 erythrocytes with 10:1 (i.e., 5 mL) of 0.8% ammonium chloride (150 mM ammonium chloride (NH₄Cl), 1 mM sodium bicarbonate (NaHCO₃), and 0.1 mM ethylenediaminetetraacetic acid (EDTA) solution.     
      NOTE: Alternatively, an automated system such as Q-prep may be used.
   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 Staphylococcal superantigen E (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.
5. Wash the cell suspension with 500 μL of phosphate-buffered saline (PBS) containing 1% bovine serum albumin (BSA) and 0.01% sodium azide, NaN₃.
6. Add the antibodies for intracellular cytokine staining (the choice of antibodies is to be determined by the investigator). 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.
7. Fix the cells using 500 μL of 1%-2% paraformaldehyde for 1 h.
8. 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 manufacturer's 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.
9. Incubate the cells with intracellular cytokine staining antibodies (e.g., interferon-gamma, IFN-γ and tumor necrosis factor beta, TNF-α or interleukin, 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.
10. Wash the cells with 500 μL of PBS and resuspend in 100 μL of PBS before data acquisition on a flow cytometer.