Opsonophagocytic Killing Assay to Assess Immunological Responses Against Bacterial Pathogens

Summary

This opsonophagocytic killing assay is used to compare the ability of phagocytic immune cells to respond to and kill bacteria based on different treatments and/or conditions. Classically, this assay serves as the gold standard for assessing effector functions of antibodies raised against a bacterium as opsonin.

Abstract

A key aspect of the immune response to bacterial colonization of the host is phagocytosis. An opsonophagocytic killing assay (OPKA) is an experimental procedure in which phagocytic cells are co-cultured with bacterial units. The immune cells will phagocytose and kill the bacterial cultures in a complement-dependent manner. The efficiency of the immune-mediated cell killing is dependent on a number of factors and can be used to determine how different bacterial cultures compare with regard to resistance to cell death. In this way, the efficacy of potential immune-based therapeutics can be assessed against specific bacterial strains and/or serotypes. In this protocol, we describe a simplified OPKA that utilizes basic culture conditions and cell counting to determine bacterial cell viability after co-culture with treatment conditions and HL-60 immune cells. This method has been successfully utilized with a number of different pneumococcal serotypes, capsular and acapsular strains, and other bacterial species. The advantages of this OPKA protocol are its simplicity, versatility (as this assay is not limited to antibody treatments as opsonins), and minimization of time and reagents to assess basic experimental groups.

Introduction

The opsonophagocytic killing assay (OPKA) is a critical tool for linking alterations in bacterial structure or function to subsequent changes in immune response and function. As such, it is frequently used as a complementary assay to determine immune-based efficacy of antibody treatments, vaccine candidates, enzyme optimization, etc. While in vivo assays are necessary to determine effective clearance or protection in a bacterial infection model, the OPKA can be used to assess immune contribution to bacterial cell death at the most basic components: bacteria, immune cells, and experimental treatments. Previous studies have shown that OPKAs can be modified and used for a variety of bacteria and serotypes, including Streptococcus pneumoniae¹, Staphylococcus aureus², Pseudomonas aeruginosa³. Furthermore, these optimized assays can be used to assess different experimental treatments, including the ability of an enzyme to make the bacterium more accessible to complement-mediated immune cells⁴ and antibody treatments to improve opsonization⁵. Classically, OPKA assay has been successfully used in basic and clinical research settings as a powerful indicator for protection induced by pathogen-specific antibodies^6,7,8,9.

Different types of immune cells may be used for assessment of opsonophagocytic killing. One commonly used phagocytic population is the HL-60 human leukemic cell line. This cell line can be kept as inactivated promyelocytes in culture; however, they can be differentiated into various activated states via different drug treatments^10,11. Treatment of HL60 with N,N-dimethylformamide differentiates the cell line into activated neutrophils with strong phagocytic activity¹¹. While HL-60 cells have been optimized and are frequently used for these phagocytosis assays¹⁰, other primary polymorphonuclear leukocytes can be used as the immune arm of the experiment¹².

Additionally, these assays can be simplified¹³ or multiplexed¹⁴ to look at multiple antibiotic-resistant strains of the bacteria to be tested. The multiplexed method has been made more feasible through the development of software that can efficiently count bacterial colony forming units (CFUs) per spot on an agar plate¹⁵. Here, we describe a streamlined method using one bacterial strain, HL-60 cells, baby rabbit complement, and blood agar plates. With this method, multiple treatments can be assessed quickly to address specific research questions on how the innate immune response to bacterial infection can be modulated.

Protocol

1. Culture, Differentiation, and Validation of HL-60 Cells

1. Prepare HL-60 cell culture media composed of 500 mL RPMI with L-glutamine and 50 mL heat-inactivated fetal bovine serum. Do not add antibiotics as this may affect the differentiation of the HL-60 cells.
2. For propagation/maintenance of HL-60 cells, culture 5 x 10⁶ cells in 10 mL of HL-60 cell culture media in 75 cm² vented flasks at 37 °C and 5% CO₂. Passage cells every 3−4 days to maintain optimal cell concentrations.
   NOTE: The cell concentration should not exceed 5 x 10⁶/mL.
3. Generate working stocks of HL-60 cells by aliquoting approximately 1 x 10⁶ cells/mL in HL60 culture media with 10% dimethyl sulfoxide (DMSO) into 1 mL cryogenic tubes.
   NOTE: Working stocks may be stored at -80 °C. The master stock should be stored at -120 °C.
4. Differentiate HL-60 cells by culturing 1.5 x 10⁷ cells in 15 mL of HL-60 cell culture media with 0.6% N,N-dimethylformamide (DMF) at 37 °C and 5% CO₂ in sterile filter-capped 75 cm² flasks for 3 days prior to OPKA.
5. Validate that the HL-60 cells have been successfully differentiated and are appropriate for use in the OPKA assay by testing viability and cell surface markers according to established flow cytometry protocols^16,17,18,19. After differentiation, harvest HL-60 cells and stain approximately 1 x 10⁴ cells with fluorescently-conjugated antibodies/stains for CD71, CD35, annexin V, and propidium iodide.
   NOTE: Differentiated cells should be ≥65% viable, ≥55% CD35⁺, and ≤20% CD71⁺ as determined through established validation protocols¹⁴ (Figure 1).

2. Preparation of OPKA Buffers and Reagents

1. Prepare 50 mL of sterile opsonization buffer B (OBB) by mixing 42.5 mL of sterile 1x phosphate-buffered saline (PBS) with Ca²⁺/Mg²⁺, 5 mL of heat-inactivated fetal bovine serum, and 2.5 mL of 0.1% sterile gelatin. Store at 4 °C.
2. Obtain baby rabbit complement and store at -80 °C.
3. Obtain or prepare bacterial culture plates (i.e., 15 x 100 mm² 5% sheep’s blood agar plates).

3. Preparation of Bacterial Stock Samples

1. Obtain a stock of the bacterial strain(s) to be tested.
   NOTE: For this protocol, serotype 3 Streptococcus pneumoniae (WU2, generously provided by Dr. Moon Nahm) is used.
2. Grow the bacterial strain in an appropriate broth (i.e., Todd-Hewitt broth + 0.5% yeast extract for this WU2 strain) for approximately 2−4 h at 37 °C.
   NOTE: The optical density at 600 nm (OD₆₀₀) of the culture should be between 0.6 and 0.8.
3. Pellet the bacteria by centrifugation at 6,000 x g for 2 min and resuspend the cells in 10−30 mL of 15% glycerol in the appropriate broth. Aliquot the bacterial culture (500 µL per aliquot) into sterile 1.5 mL centrifuge tubes and store at -80 °C.
4. Thaw out one vial of bacterial stock in a 37 °C water bath. Pellet the bacterial cells and resuspend in 500 µL of OBB under sterile conditions.
5. Prepare different dilutions of the bacterial stock in OBB (i.e., 10 µL of no dilution, 10 µL of 1:10, 10 µL of 1:100, etc.). Perform the OPKA assay (sections 4–6, including HL-60/complement co-culture) as described below using various dilutions of the untreated bacterial stock. Culture the plates overnight at 30 °C (no CO₂).
   NOTE: The temperature 30 °C is specific for WU2 to prevent overgrowth; other strains/serotypes may grow optimally at 37 °C.
6. Count the colonies for each dilution of untreated bacterial stock co-cultured with HL-60 cells and complement. Determine which dilution of bacteria yields the optimal number of countable colonies (approximately 80−120 CFUs for untreated bacteria co-cultured with HL-60 cells). Note this dilution for future OPKAs involving this bacterial stock.

4. Bacterial Treatment and Culture

1. Thaw one tube of bacterial stock prepared in step 3.3. Pellet bacteria (6,000 x g for 2 min) and resuspend cell pellet in OBB at optimal dilution as determined in step 3.6.
2. Pipette 10 µL of resuspended bacterial dilution per well in a round-bottom 96-well cell culture plate.
3. Add 20 µL of appropriate antibody or drug treatment to each experimental well in duplicate.
   NOTE: In this protocol, a serotype-specific antibody generated in mice is added as treatment X and a glycoside hydrolase enzyme known to degrade the serotype 3 polysaccharide capsule is added as treatment Y (Figure 2)^4,20. For control wells, use 1x PBS or OBB, depending on the buffer used for treatment wells.
4. Shake the sample plate at approximately 90 rpm for 1 h at room temperature. Adjust these conditions depending on the optimal temperature or shaking conditions of the treatments being tested.

5. HL-60 bacterial Co-culture

1. Prepare HL-60 cells by harvesting the HL-60 differentiated cells that are treated with DMF three days prior (see step 1.4) into 15 mL conical tubes. Pellet the cells (500 x g, 3 min), discard the supernatant, and wash with at least 10 mL of 1x PBS.
2. Pellet the washed cells (500 x g, 3 min), discard the supernatant, and resuspend the cells in OBB (start with 1 mL OBB and adjust for a final concentration of 1 x 10⁷/mL after cell counting).
3. Add baby rabbit complement (sterile, undiluted baby rabbit serum, age 3−4 weeks) at a 1:5 final volume.
   NOTE: The final concentration of the HL-60-complement mixture should be 1 x 10⁷/mL. If testing complement dependency, a second solution containing active HL-60 cells with heat-inactivated complement may be used (complement may be inactivated by incubating in a water bath at >55 °C for at least 30 min).
4. After 1 h bacterial culture is complete (step 4.4), divide each sample (i.e., 10 µL of each 30 µL sample well into two new wells) into duplicate wells for two groups (i.e., use only 20 µL of the original 30 µL co-culture to account for pipetting error): one set will be co-cultured with HL-60-complement and one will include bacteria only. Add 50 µL of the HL-60-complement mixture (from step 5.3) to each experimental set of wells (delegated +HL-60); add 50 µL of OBB alone to the wells of bacteria only (delegated -HL-60).
   NOTE: For this example, approximately 800 bacterial CFUs are used for the initial co-culture with 5 x 10⁵/50 µL HL-60 cells. If this multiplicity of infection is too high or too low as indicated by final colony numbers, adjust the initial bacterial dilution as opposed to the HL-60 cell count.
5. Shake the 96-well plate at 37 °C for 1 h (no CO₂).

6. Sample Plating and Overnight Incubation

1. Dilute each well 1:5 with OBB, so that each sample has a volume of at least 50 µL.
2. Pipette 50 µL of each sample directly onto a designated area of a bacterial culture plate, ensuring adequate spacing between samples. For 15 x 100 mm² round agar plates, pipet approximately 4 samples onto one plate.
3. Cover and allow samples to dry for approximately 15 min at room temperature.
4. Invert plates and culture overnight at 30 °C (no CO₂). Alternatively, culture plates in anaerobic jars to test whether anoxic conditions affect the bacterial growth or to control for morphology.
5. After overnight culture, count the colonies in each designated sample area. Analyze data by comparing the number of live cells in each set to the corresponding control and/or samples that do not receive HL-60 cell co-culture (indicative of 100% cell survival, 0% cell killing).

Results

Validation of HL-60 differentiation should be performed before starting the OPKA. This can be accomplished using flow cytometry to determine the extracellular expression of CD11b, CD35, CD71, and annexin V (Figure 1). Propidium iodide can also be used as a viability marker. After being treated with DMF for 3 days, expression of CD35 should be increased (≥55% of all cells) and expression of CD71 should be decreased (≤20% of all cells). The percentage of annexin V+ and propidium io...

Discussion

OPKAs serve essential roles in assessing antibody mediated immune responses induced by vaccinations^6,8. The main significance of this simplified OPKA is the adaptability in the conditions to be tested (i.e., antibodies, enzyme treatments, etc.). In this sense, while this assay can be used to test the contribution of opsonins (i.e., antibodies) in phagocytosis, it can also be used to assess ways to overcome virulence factors (i.e., capsular polysaccharides) that n...

Materials

Name	Company	Catalog Number	Comments
Annexin V (APC conjugated)	BioLegend	640919
anti-CD35, human (PE conjugated)	BioLegend	333405
anti-CD71, human (PE conjugated)	BioLegend	334105
bacterial strain to be used (ie, Streptococcus pneumoniae, WU2)	Bacterial Respiratory Reference Laboratory (Dr. Moon Nahm)
blood agar plates	Hardy Diagnostic	A10
Fetal Clone serum	HyClone	SH30080.03
glycerol	Sigma	G9012-1L
HL-60 cells	ATCC	CCL-240
IgG Isotype Control (PE conjugated)	BioLegend	400907
N,N-dimethylformamide (DMF)	Fisher Chemical	UN2265
propidium iodide	Sigma	P4864
RPMI media with L-glutamine	Corning	10-040-CV