An Automated Culture System for Use in Preclinical Testing of Host-Directed Therapies for Tuberculosis

Podsumowanie

Rapid and efficient quantification of intracellular M. tuberculosis growth is crucial for pursuing improved therapies against tuberculosis (TB). This protocol describes a broth-based colorimetric detection assay using an automated liquid culture system to quantify Mtb growth in macrophages treated with candidate host-directed therapies.

Streszczenie

Mycobacterium tuberculosis (Mtb), the causative agent of tuberculosis (TB), was the most significant infectious disease killer globally until the advent of COVID-19. Mtb has evolved to persist in its intracellular environment, evade host defenses, and has developed resistance to many anti-tubercular drugs. One approach to solving resistance is identifying existing approved drugs that will boost the host immune response to Mtb. These drugs could then be repurposed as adjunctive host-directed therapies (HDT) to shorten treatment time and help overcome antibiotic resistance.

Quantification of intracellular Mtb growth in macrophages is a crucial aspect of assessing potential HDT. The gold standard for measuring Mtb growth is counting colony-forming units (CFU) on agar plates. This is a slow, labor-intensive assay that does not lend itself to rapid screening of drugs. In this protocol, an automated, broth-based culture system, which is more commonly used to detect Mtb in clinical specimens, has been adapted for preclinical screening of host-directed therapies. The capacity of the liquid culture assay system to investigate intracellular Mtb growth in macrophages treated with HDT was evaluated. The HDTs tested for their ability to inhibit Mtb growth were all-trans Retinoic acid (AtRA), both in solution and encapsulated in poly(lactic-co-glycolic acid) (PLGA) microparticles and the combination of interferon-gamma and linezolid. The advantages of this automated liquid culture-based technique over the CFU method include simplicity of setup, less labor-intensive preparation, and faster time to results (5-12 days compared to 21 days or more for agar plates).

Wprowadzenie

Mycobacterium tuberculosis (Mtb), the causative agent of TB, was the most significant infectious disease killer globally in 2019¹. To evade host defenses, Mtb subverts the mycobactericidal activity of innate immune cells such as macrophages and dendritic cells (DCs), allowing it to persist intracellularly and replicate². The lack of an effective vaccine to prevent adult pulmonary TB and the increasing emergence of drug-resistant strains highlight the urgent need for new therapies.

Adjunctive host-directed therapies (HDT) could shorten treatment time and help overcome resistance³. Preclinical assessment of HDT candidates in vitro to determine mycobactericidal activity within macrophages often relies on the quantification of Mtb growth by colony-forming units (CFU) on solid agar plates. This is a slow, labor-intensive assay that does not lend itself to rapid screening of drugs. Commercially available automated, broth-based microbial detection systems are more commonly used in clinical microbiology laboratories for detection and drug susceptibility testing of Mtb and other mycobacterial species in clinical specimens⁴. These instruments measure growth indirectly based on the bacterial metabolic activity leading to physical changes in the culture media (change in CO₂ or O₂ levels or pressure) monitored over time⁵. The readout is time to positivity (TPP), which has previously been shown to correlate with Mtb CFU in sputum specimens of TB patients in response to treatment^6,7 and in lysates of infected murine lung and spleen⁸. In addition, liquid culture detection systems have been used to measure the effect of conventional pathogen-directed therapies on the growth of mycobacteria in axenic culture and cultured macrophages^9,10. The instrument has also been used to investigate the innate ability of dendritic cells and of alveolar macrophages to control intracellular growth of Mtb^11,12. This experimental protocol demonstrates that a liquid culture diagnostic system can be adapted to perform preclinical screening of HDT for TB in cultured macrophages. Compared to CFU enumeration, the main advantage of this technique is that it considerably reduces the experimental labor and time required to quantify intracellular mycobacterial growth/survival. This technique relies on access to an automated culture instrument that can be used to assess intracellular mycobacterial survival in immune cells treated with a broad range of pharmacological reagents targeting cellular functions to boost host immunity.

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Protokół

The experiments outlined in this protocol were carried out using the attenuated H37Ra strain of Mtb, which can be handled in a Containment Level 2 laboratory. All manipulations of live mycobacteria were carried out in Class II biological safety cabinet (BSC). Experimental procedures were designed to minimize the generation of aerosols. Eukaryotic cell culture (THP-1 cells) was also carried out in a Class II BSC. The laboratory carried out a risk assessment and ensured that all procedures were carried out in line with institutional and national biological safety regulations. The human monocytic THP-1 cell line was used to perform the method as described (step 1). Cells are differentiated into macrophages following stimulation with phorbol 12-myristate 13-acetate (PMA) before infection with mycobacteria.

1. Cell culture

1. Propagate H37Ra seed stock to log phase in Middlebrook 7H9 (MB) broth supplemented with albumin-dextrose-catalase (ADC) enrichment (10%) and 0.05% polysorbate 80. Store H37Ra stock in 1 mL aliquots in a -80 °C freezer for up to 1 year.
2. Thaw a 1 mL vial of Mtb-H37Ra and transfer it to a T25 flask with a filter cap containing 9 mL of MB supplemented broth approximately 1 week before the planned experiment. Incubate at 37 °C for 5-7 days in a static incubator.
3. Grow THP-1 cells in RPMI-1640 supplemented with non-heat killed 10% fetal calf serum (complete (c)RPMI) in a T75 flask in a CO₂ humidified incubator at 5% CO₂/37 °C and subculture twice per week to maintain a density of less than 1 x 10⁶ cells/mL.
4. Differentiate THP-1 cells into macrophages 3 days before infection by gently pipetting cells several times using a serological pipette in T75 flasks to disperse any clumps and place them in a 50 mL conical tube.
5. Centrifuge cells at 300 x g for 10 min at room temperature, decant off the supernatant, and gently resuspend the pellet in 2 mL of RPMI. Perform cell count to estimate cells/mL.
6. Seed 2 mL of THP-1 macrophages in 12-well tissue culture plates at a density of 100,000 cells/mL in cRPMI with 100 ng/mL PMA for 72 h. Remove PMA-containing medium from cells and replenish with fresh cRPMI before Mtb infection.
7. Set up individual plates for each time point required.
8. Seed cells at the same density (100,000 cells/mL) in 2-well glass chamber slides to determine the multiplicity of infection (MOI).
9. Place in a 5% CO₂ humidified incubator for 3 days at 37 °C.

2. Quantification of Mtb uptake

1. Determination of Mtb uptake by macrophages (MOI)
   1. Set up the Class II biological safety cabinet (BSC) on the day of infection and work on two layers of tissue paper to catch any spills. Set up waste discard containers according to local regulations.
   2. Remove 6-8 mL of mycobacterial culture from the T25 flask and transfer it into a 15 mL polypropylene tube.
      NOTE: Smaller volume tubes can be used for smaller experiments.
   3. Centrifuge the tube in a benchtop centrifuge at room temperature for 10 min at 2890 x g.
   4. Carefully remove the tube from the centrifuge and transfer it to the biological safety cabinet. Wait 1 min to allow the bacteria to settle.
      1. Pour off the supernatant into the disinfectant discard container, recap tube, and resuspend the bacteria in the remaining medium by tapping the side of the tube. Wait 1 min to allow the bacteria to settle.
   5. Add 2 mL of pre-warmed cRPMI, mix gently, and transfer to a 50 mL conical tube.
   6. Resuspend the mycobacteria very carefully using a 25 G needle and 5 mL syringe. To resuspend, draw up the mycobacteria suspension into the syringe and eject very gently down the sidewall of the tube to minimize aerosol production. Repeat 6-8 times.
      NOTE: Exercise utmost caution as this is a high-density culture of mycobacteria. To avoid the risk of needle stick injury, use blunt needles where possible, and Luer lock syringes.
   7. Dispose of the needle and syringe in a sharps container in the BSC.
   8. Transfer the suspension to a 2 mL microfuge tube (with screw-on cap) and centrifuge at room temperature for 3 min at 100 x g to pellet any remaining clumps. Return the tube to the safety cabinet and wait 1 min to allow the bacteria to settle.
   9. Transfer the top 1-1.5 mL of the supernatant to a new tube. Discard the original tube in the waste bucket containing disinfectant. Mix well and add various amounts of the mycobacterial suspension (e.g., 5, 25, 50, 150 µL) to the 2-well glass chamber slides and incubate for 3 h in a CO₂ incubator at 37 °C.
2. Staining for acid fast bacteria (AFB)
   NOTE: After 3 h incubation, the macrophages are washed and fixed with paraformaldehyde to inactivate mycobacteria. The slides are then stained using a Modified Auramine O kit (see Table of Materials) to estimate mycobacteria phagocytosed per cell. Due to their waxy cell wall, mycobacteria retain the Auramine dye after an acid-alcohol wash. The macrophage nuclei are then counter-stained with Hoechst. This method allows for the number of phagocytosed bacteria per cell to be counted and is used to determine the multiplicity of infection (MOI) of the macrophages.
   1. Remove the medium from the glass chamber slide after pipetting up and down three times to dislodge bacteria that have not been phagocytosed.
   2. Wash once with 2 mL of room temperature PBS.
   3. Store stocks of 4% paraformaldehyde, dissolved in PBS in aliquots at -20 °C for up to 6 months. Thaw an aliquot of 4% paraformaldehyde immediately before use. Dilute to 2% paraformaldehyde with PBS and add 2 mL per well.
   4. Incubate for 10 min at room temperature. The glass chamber slide can be removed from the safety cabinet at this stage for staining.
   5. Wash the slide under a gentle stream of tap water.
   6. Dispense enough Auramine onto the slide to cover the cells using a plastic transfer pipette and incubate for 1 min at room temperature in the dark (cover with aluminum foil).
   7. Wash excess dye off the slide with tap water and add the decolorizer/quencher for 1 min in the dark.
   8. Wash off the excess with tap water and incubate for 15 min at room temperature with Hoechst 33342 (10 µg/mL in PBS) in the dark.
   9. Wash off the Hoechst stain with tap water, remove the chambers, drain excess water from the slide, add a drop of antifade and coverslip, and air dry.
   10. Examine the slide under the fluorescent microscope using the 100x oil objective. Mycobacteria will fluoresce green under the FITC filter. The nuclei fluoresce blue under the DAPI filter (Figure 1C).
   11. Determine the MOI by counting the number of mycobacteria phagocytosed per cell and the percentage of cells infected.
   12. Calculate the volume of mycobacterial suspension needed to achieve the required MOI based on the surface area of a well in the plate; for example, the surface area of the glass chamber slides used in this experiment is 4 cm². Low MOI (approximately 1-2 bacilli/cell) is desirable for experiments conducted over several days (e.g., 5 days).
3. Infection of macrophages
   1. Mix the mycobacteria suspension well and add the amount needed to the cells on 12-well plates once the volume required to achieve the desired MOI has been determined.
   2. Incubate at 37 °C for 3 h to allow mycobacteria to be phagocytosed.
   3. Remove extracellular bacteria by washing the wells with either warm RPMI or HBSS several times.
   4. Lyse the macrophages in one well (3 h sample) to determine the percentage time to positivity (TTP) of the initial inoculum (3 h sample) as outlined in step 3 below.
   5. Add fresh cRPMI and the required drug doses or vehicle control to the remaining wells, incubate the plates in the CO₂ incubator at 37 °C for the time necessary (dependent on the experimental design but usually at several intervals between 1 to 8 days).

3. Harvesting samples for the liquid culture detection system

NOTE: On the day of infection, extracellular mycobacteria are removed by washing, and intracellular mycobacteria are harvested by lysis of one well of macrophages (3 h sample) to determine the initial amount phagocytosed as a baseline control for infection. At subsequent times both the medium, cell lysate, and washes are combined to measure total mycobacterial growth. Extracellular and intracellular growth can also be assessed separately if desired.

1. Harvesting 3 h sample to determine TTP
   1. Wash off extracellular mycobacteria from all the wells after the initial 3 h of infection as outlined in step 2.3.3. Add 1 mL of fresh media to the 3 h control well to equalize the lysate volume with those of later time points.
      ​NOTE: See step 3.2.7 if extracellular mycobacteria are to be excluded from the analysis.
2. Sample collection
   1. Warm MB broth and instrument culture bottles to bring them to room temperature.
   2. Transfer the medium from the 12-well plate to the corresponding labeled conical tubes.
   3. Add 500 µL of sterile lysis buffer (0.1% Triton x-100 in PBS filtered through a sterile 0.2 µm filter) to each well for 10 min.
   4. Gently scrape the cells from the well with a sterile scraper and combine with the medium in the appropriate conical tube.
   5. Wash the well with 0.5 mL of sterile PBS and transfer to the appropriate tube.
   6. Gently pass each sample through a needle and syringe (25 G) 6-8 times to break up the clumps. Dilute samples 1:10 in MB broth; 100 µL sample + 900 µL MB medium.
   7. At the required times/days (usually between 1 to 8 days), harvest the remaining samples by following steps 3.2.1-3.2.6 above.
      ​NOTE: Investigators may prefer to exclude extracellular mycobacteria from their analysis, in which case, in step 3.2.2 above, the medium from each well is discarded, and macrophages are washed several times before adding lysis buffer.
3. Inoculating and loading instrument culture bottles
   NOTE: Details of the liquid culture instrument and related consumables are provided in the Table of Materials.
   1. Sterilize the rubber cap of the instrument culture bottle with tissue paper soaked in 70% alcohol and allow it to air dry.
      NOTE: This step needs to be carried out in the BSC.
   2. Prepare bottles by transferring enough Nutrient supplements for all the samples into a conical tube (0.5 mL/bottle). Use a needle and syringe to inject 0.5 mL of Nutrient supplement into the instrument culture bottle.
   3. Pipette 500 µL of the diluted sample (1:10) into a 1 mL V-bottomed tube.
   4. Use a needle and syringe to inject the 500 µL of sample into the assigned instrument culture bottle.
   5. Sterilize the rubber cap of the instrument culture bottle with tissue paper soaked in 70% alcohol and allow it to air dry. Wipe the bottles with tissue paper soaked in 70% alcohol before removal from the BSC.
   6. Carefully transport bottles from the biosafety cabinet to the instrument for loading.
   7. Press the loading button on the automated microbial detection system.
   8. Scan the barcodes on instrument culture bottles and place the bottles into the detection system incubator at 37 °C for up to 42 days. Read and record the time taken to reach positivity from the instrument screen.
      NOTE: The barcode allows the instrument to identify the bottle and link reflectance readings with a particular bottle.
   9. Calculate percentage time to positivity (TTP) by comparing the TTP of the initial intracellular inoculum (Day 0) to that of macrophages cultured for the indicated times. A positive change in percentage TTP means mycobacterial growth¹³.
      For example, for day 3:
      Percentage change in time to positivity =

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Wyniki

The automated liquid culture instrument used in this study monitors CO₂ levels every 10 min. A color change in the sensor at the bottom of the instrument bottle is measured colorimetrically and expressed as reflectance units. The instrument software then applies detection algorithms to calculate time to positivity (TTP), i.e., the number of days from inoculation until cultures are flagged as positive (Figure 1A). An inverse relationship between TTP and log₁₀CFU (determi...

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Dyskusje

The authors have used the liquid culture method described in this protocol to monitor Mtb growth in monocyte-derived macrophages and alveolar macrophages and THP-1 cells differentiated with PMA^11,16,17. This technique can also be modified for use with non-adherent cells¹². More recently, the instrument was also used in preclinical studies to evaluate inhaled all-trans retinoic acid (AtRA) as an HDT for TB...

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Materiały

Name	Company	Catalog Number	Comments
IX51 Fluorescent Microscope	Olympus, Japan	N/A	AFB detection and imaging
2 mL microtube, flat bottom, screw cap, sterile	Sarstedt, North Carolina, USA	72.694.006	Mtb infection of macropahges
5 mL syringe, Luer lock	BD Biosciences, San Jose, CA, USA	SZR-150-031K	Mtb infection of macropahges/CFU
50 mL tube, sterile	Sarstedt, North Carolina, USA	62.547.254	Mtb infection of macropahges
all-trans-Retinoic Acid (ATRA) ≥98% (HPLC)	Sigma Aldrich, Missouri, USA	R2625	Host directed therapy candidate
BacT/ALERT 3D Microbial Detection System	Biomerieux ( Hampshire, UK)	247001	Broth-based colormetric detection system
BACT/ALERT MP  BACT/ALERT MP Nutrient Supplement	Biomerieux ( Hampshire, UK)	414997	Broth-based colormetric detection assay
BACT/ALERT MP culture bottles	Biomerieux ( Hampshire, UK)	419744	Broth-based colormetric detection assay
BD BBL Middlebrook ADC Enrichment, 20 mL	BD Biosciences, San Jose, CA, USA	M0553	Mycobacterium liquid culture
BD BBL Middlebrook OADC Enrichment, 20mL	BD Biosciences, San Jose, CA, USA	M0678	Colony Forming Units
Cell scraper, 25 cm	Sarstedt, North Carolina, USA	83.1830	Harvest of lmacrophage lysates
Corning Syringe Filter, 0.2 µm	Corning Incorporated, Germany	431219	Sterilization of lysis buffer
Cover glass (borosilicate), 24 x 50 mm, #1.5 thickness	VWR International Limited	631 - 0147
Cycloheximide, from microbial	Sigma Aldrich, Missouri, USA	C7698	Colony Forming Units
Dako Fluorescent Mounting Medium	Agilent Technologies Ireland Limited	S3023	Antifade mounting medium
Dulbecco’s Phosphate Buffered Saline	Sigma Aldrich, Missouri, USA	D8537	Mtb infection of macropahges
Fetal Bovine Serum, Gibco	Thermo Fisher, Massachusetts, USA	10270106	Macrophage cell culture
Glycerol, Difco	BD Biosciences, San Jose, CA, USA	228220	Colony Forming Units
Hoescht 33342 (bisBenzimide H 33342 trihydrochloride)	Sigma Aldrich, Missouri, USA	B2261	Nuclear stain
IFNγ, recombinant human	R&D Systems Inc, Minnesota, USA	285-IF	Host directed therapy candidate
Labtek 2-well chamber slide, sterile, Nunc	Thermo Fisher, Massachusetts, USA	TKT-210-150R	Mtb infection of macropahges
L-Asparagine, anhydrous	Sigma Aldrich, Missouri, USA	A4159	Colony Forming Units
Linezolid	Sigma Aldrich, Missouri, USA	PZ0014	Antibiotic
Microlance Hypodermic Needle 25 G	BD Biosciences, San Jose, CA, USA	300400	Mtb infection of macropahges/CFU
Middlebrook 7H10 Agar Base	BD Biosciences, San Jose, CA, USA	M0303	Colony Forming Units
Middlebrook 7H9 Broth Base	BD Biosciences, San Jose, CA, USA	M0178	Mycobacterium liquid culture
Modified Auramine O Stain and Decolourizer	Scientific Device Laboratory, IL, USA	345-250	AFB stain
Paraformaldehyde	Sigma Aldrich, Missouri, USA	158127	Mtb infection of macropahges
Petri dishes, 92 x 16mm (20/bag)	Sarstedt, North Carolina, USA	82.1473.001	Colony Forming Units
Phorbol 12-myristate 13-acetate (PMA)	Sigma Aldrich, Missouri, USA	P8139	Macrophage cell culture
Polysorbate 80, Difco	BD Biosciences, San Jose, CA, USA	231181	Mycobacterium liquid culture
RPMI-1640, Gibco	Thermo Fisher, Massachusetts, USA	52400025	Macrophage cell culture
Sterile Cell Spreader, L-Shaped	Fisherbrand, Thermo Fisher, MA, USA	RB-44103	Colony Forming Units
T25 TC flask, angled neck, filter cap, sterile, Nunc	Thermo Fisher, Massachusetts, USA	156367	Mycobacterium liquid culture
THP-1 cell line	ATCC, Virginia, USA	ATCC TIB-202	Macrophage cell culture