P. aeruginosa Infected 3D Co-Culture of Bronchial Epithelial Cells and Macrophages at Air-Liquid Interface for Preclinical Evaluation of Anti-Infectives

Summary

We describe a protocol for a three-dimensional co-culture model of infected airways, using CFBE41o^- cells, THP-1 macrophages, and Pseudomonas aeruginosa, established at the air-liquid interface. This model provides a new platform to simultaneously test antibiotic efficacy, epithelial barrier function, and inflammatory markers.

Abstract

fDrug research for the treatment of lung infections is progressing towards predictive in vitro models of high complexity. The multifaceted presence of bacteria in lung models can re-adapt epithelial arrangement, while immune cells coordinate an inflammatory response against the bacteria in the microenvironment. While in vivo models have been the choice for testing new anti-infectives in the context of cystic fibrosis, they still do not accurately mimic the in vivo conditions of such diseases in humans and the treatment outcomes. Complex in vitro models of the infected airways based on human cells (bronchial epithelial and macrophages) and relevant pathogens could bridge this gap and facilitate the translation of new anti-infectives into the clinic. For such purposes, a co-culture model of the human cystic fibrosis bronchial epithelial cell line CFBE41o^- and THP-1 monocyte-derived macrophages has been established, mimicking an infection of the human bronchial mucosa by P. aeruginosa at air-liquid interface (ALI) conditions. This model is set up in seven days, and the following parameters are simultaneously assessed: epithelial barrier integrity, macrophage transmigration, bacterial survival, and inflammation. The present protocol describes a robust and reproducible system for evaluating drug efficacy and host responses that could be relevant for discovering new anti-infectives and optimizing their aerosol delivery to the lungs.

Introduction

Pseudomonas aeruginosa is a relevant pathogen in cystic fibrosis (CF) that contributes to lung tissue impairment¹. The production of polysaccharides, such as alginate and other mucoid exopolysaccharides, coordinates the progress of the disease, which leads to tenacious bacterial adherence, limits the delivery of antibiotics to bacteria and protects the bacteria against the host immune system². The transition of P. aeruginosa from the planktonic stage to biofilm formation is a critical issue in this context, also facilitating the occurrence of antibiotic tolerance.

In the context of CF, the mouse has primarily been used as a model. Mice, however, do not spontaneously develop this disease with the introduction of CF mutations³. Most of the bacterial biofilm development and drug susceptibility studies have been performed on abiotic surfaces, such as Petri dishes. However, this approach does not represent the in vivo complexity. For instance, important biological barriers are absent, including immune cells as well as the mucosal epithelium. Though P. aeruginosa is quite toxic to epithelial cells, some groups have managed to co-cultivate an earlier P. aeruginosa biofilm with human bronchial cells. These cells originated from cystic fibrosis patients with CFTR mutation (CFBE41o^- cells)⁴ and allowed to assess antibiotic efficacy⁵ or assess the correction of the CFTR protein during infection⁶. Such a model was shown to improve the predictability of drug efficacy, in addition to enabling characterization of issues with drugs that failed in later phases of drug development⁷.

However, in the lung, the mucosal epithelium is exposed to air. Moreover, immune cells present in the airways, like tissue macrophages, play an essential role against inhaled pathogens or particles⁸. Macrophages migrate through the different cell layers to reach the bronchial lumen and fight the infection. Furthermore, inhaled drugs also have to cope with the presence of mucus as an additional non-cellular element of the pulmonary air-blood barrier⁹. Indeed, several complex three-dimensional (3D) in vitro models have been developed, aiming to increase the in vivo relevance. Co-culture systems not only increase the complexity of in vitro systems for drug discovery but also enable to study cell-cell interactions. Such complexity has been addressed in studies about macrophage migration¹⁰, the release of antimicrobial peptides by neutrophils¹¹, the role of mucus in infection⁹, and the epithelial cell reaction to excessive damage¹². However, a reliable CF-infected in vitro model that features the genetic mutation in CF, that is exposed to the air (increased physiological condition), and integrates immune cells is still lacking.

To bridge this gap, we describe a protocol for stable human 3D co-culture of the infected airways. The model is constituted of human CF bronchial epithelial cells and macrophages, infected with P. aeruginosa and capable of representing both a diffusional and immunological barrier. With the goal of testing anti-infectives at reasonably high throughput, this co-culture was established on the permeable filter membrane of well plate inserts, using two human cell lines: CFBE41o^- and THP-1 monocyte-derived macrophages. Moreover, to eventually study the deposition of aerosolized anti-infectives¹³, the model was established at the air-liquid interface (ALI) rather than liquid covered conditions (LCC).

As we report here, this model allows assessing not only bacterial survival upon an antibiotic treatment but also cell cytotoxicity, epithelial barrier integrity, macrophage transmigration, and inflammatory responses, which are essential parameters for drug development.

This protocol combines two relevant cell types for inhalation therapy of the pulmonary airways: macrophages and CF bronchial epithelium. These cells are seeded on opposite sides of permeable support inserts, allowing cell exposure to air (called the air-liquid interface (ALI) conditions). This co-culture of host cells is subsequently infected with P. aeruginosa. Both host cell lines are of human origin: the epithelial cells represent the cystic fibrosis bronchial epithelium, with a mutation on the CF channel (CFBE41o^-), and the THP-1¹⁴ cells are a well-characterized macrophage-like cell line. A confluent epithelial layer is first allowed to form on the upper side of well plate inserts before the macrophage-like cells are added to the opposite compartment. Once the co-culture is established at ALI, the system is inoculated with P. aeruginosa at the apical side. This infected co-culture system is then used to assess the efficacy of an antibiotic, e.g. tobramycin. The following end-points are analyzed: epithelial barrier integrity in terms of transepithelial electrical resistance (TEER), visualization of cell-cell and cell-bacteria interactions by confocal laser scanning microscopy (CLSM), bacterial survival by counting of colony-forming units (CFU), host cell survival (cytotoxicity) and cytokine release.

Protocol

1. Growth and differentiation of cells in permeable support inserts

1. Cultivate CFBE41o^- in a T75 flask with 13 mL of minimum essential medium (MEM) containing 10% fetal calf serum (FCS), 1% non-essential amino acids and 600 mg/L glucose at 37 °C with 5 % CO₂ atmosphere. Add fresh medium to the cells every 2–3 days.
   1. Detach the cells after reaching 70% confluence in the flask with 3 mL of trypsin- Ethylenediaminetetraacetic acid (EDTA) at 37°C for 15 min. Add 7 mL of fresh MEM and centrifuge the cells at 300 x g for 4 min at room temperature (RT). Discard the supernatant and add new 10 mL of MEM while disrupting the clumps by gently pipetting up and down.
   2. Count the cells with an automated cell counter or hemocytometer chamber. Seed cells with a density of 2 x 10⁵ cells/well in 12-well plates with permeable supports (pore size of 3 μm, see Table of Materials).
      NOTE: The automated cell counter determines cell number, size distribution, and viability of the cells (see Table of Materials). Permeable supports with a pore size of 0.4 μm could be used here; however, the macrophages, in this condition, should be added directly to the apical side, and their transcellular migration will not be assessed in this case.
   3. Seed cells at liquid-liquid condition (LLC) by adding 500 µL of the cell suspension on the apical side of the permeable support and 1.5 mL of fresh medium in the basolateral side. Then incubate cells at 37°C under 5% CO₂, for 72 h.
   4. To shift to the air-liquid interface (ALI) culture, on the third day after seeding, remove the medium from the basolateral side first, then from the apical side. To the basolateral side, add 500 µL of fresh MEM and change the medium every second day until cells form a confluent monolayer.
      NOTE: For the conditions used in this protocol, the CFBE41o^- cells usually are confluent after 3-7 days in culture.
   5. Assess the epithelial barrier properties on day 7 by incubating CFBE41o^- cells with 500 μL cell medium in the apical side and 1.5 mL in the basolateral side for 1 h, at 37°C under 5% CO₂.
   6. Measure barrier properties via transepithelial electrical resistance (TEER), with an STX2 chopstick electrode and an epithelial volt-ohmmeter; after 7 days this is higher than 300 Ω×cm².
      NOTE: Eventually, in some membrane inserts, the cells have low TEER. Therefore permeable inserts with TEER < 300 Ω×cm² are not used.
2. To cultivate THP-1 cells, grow them in a T75 flask using 13 mL of Roswell Park Memorial Institute (RPMI) 1640 medium supplemented with 10% FCS, and incubate at 37°C under 5% CO₂. Split cells every second day by seeding 2 x 10⁶ cells/mL cells in a new T75 flask.
   NOTE: Non-differentiated THP-1 cells are grown as monocytes in suspension.
   1. Differentiate the THP-1 cells as follows. Centrifuge contents of a T75 at 300 x g for 4 min. Discard the supernatant, resuspend the pellet in fresh medium and put in a new T75. Add 10 ng/mL Phorbol 12-myristate 13-acetate (PMA) incubatig the cells in RPMI for 48 h at 37 °C and 5% CO₂ atmosphere¹⁵.
      NOTE: After the differentiation with PMA, cells do not proliferate anymore and attach to the flask.
   2. To detach THP-1 macrophage-like cells, wash once with phosphate-buffered saline (PBS) at 37 °C and incubate with 3 mL of cell detachment solution (e.g. accutase) containing 0.5 mM EDTA for 10 min at room temperature.
   3. Inspect the cells under an inverted microscope to look for cell detachment. Add 7 mL of fresh medium and centrifuge at 300 x g for 4 min at RT.
      NOTE: Macrophages can also be detached with trypsin-EDTA, 37 °C for 20 min. However, trypsin is harsher to macrophages than the chosen cell detachment solution (see Table of Materials).
   4. After removing the supernatant, re-suspend macrophage cells in 3 mL of THP-1 medium into a 15 mL conical tube, count the cells as described in 1.1.2. and incubate for a maximum 1 h at 37 °C under 5% CO₂ before setting up the co-culture.
      NOTE: THP-1 cells in suspension can be stained with viability dyes to image the co-culture further. At this step, use the procedure below (step 1.2.5).
   5. Stain macrophages with 10 µM of a cell viability dye (based on the conversion of acetate moieties by intracellular esterases, see Table of Materials) in which 3 µL of the cell viability dye is applied to the cell suspension. Incubate cells for 20 min at 37 °C, 5% CO₂, then wash 1x with PBS 37°C to remove the dye.
      NOTE: Centrifuge the cells to remove the dye at 300 x g for 4 min at room temperature (RT).

2. Establishment of an epithelial-macrophage co-culture on permeable supports

1. Use CFBE41o^- monolayers at ALI with TEER ≥ 300 Ω×cm² (step 1.1.6.). Remove the medium from the lower chamber, carefully invert the support inside a sterile glass Petri dish (50 mm x 200 mm), and remove the cells overgrown through the membrane pores on the bottom side of the membrane using a cell scraper.
   NOTE: Due to the pore size of 3 µm, epithelial cells tend to grow through the pores toward the basolateral side. Therefore, one needs to remove them before adding the macrophages on this side. CFBE41o^- lung epithelial cells can be stained at this step. The procedure in step 1.2.5 can be used; however, instead of a cell suspension, the dye solution in MEM is applied (500 µL apical side only) on the adhered cells on the permeable support.
2. Use 2 x 10⁵ cells/well (in 200 µL of RPMI) from the cell suspension of PMA-differentiated THP-1 macrophages and place the cells on the basolateral side of the inverted inserts.
3. Close the Petri dishes carefully and incubate for 2 h at 37 °C under 5% CO₂.
4. Place the inserts back into the 12-well microplates and add 500 µL of MEM medium in the basolateral side of the permeable insert to maintain ALI conditions. The cells are now ready for infection.

3. Infection by P. aeruginosa

NOTE: All following steps from here must be done in a biosafety level 2 (BSL2) laboratory.

1. Inoculate 15 mL of lysogeny broth (LB) supplemented with 300 μg/mL ampicillin in an Erlenmeyer flask (50 mL) with a single colony of P. aeruginosa PAO1-GFP.
   NOTE: Other strains of P. aeruginosa could also be used here, for instance, PAO1 wild type, PA14, or clinical strains, following their own cultivation protocols.
2. Incubate the bacteria for 18 h at 37°C, shaking at 180 rpm.
3. Transfer the contents after the 18 h to a 50 mL conical tube and centrifuge at 3850 x g for 5 min. Discard the supernatant and add 10 mL of sterile PBS at 37°C.
4. Measure optical density on a spectrophotometer at wavelength 600 nm and adjust the concentration of bacteria using the cell culture medium to a final concentration of 2 x 10⁵ CFU/mL. This corresponds to a multiplicity of infection (MOI) of one bacterium per epithelial cell.
5. Add 100 μL of bacterial suspension to the apical side of the permeable support (step 2.4.) and incubate at 37 °C under 5% CO₂ for 1 h, to allow bacteria attachment to the cells. Then, remove apical liquid carefully with a pipette to restore ALI conditions. Keep some samples uninfected as a control.
   NOTE: At this stage, the bacteria attached should be plated in LB agar (see steps 5.4/5.5) to determine the initial bacteria inoculum.
6. Incubate the drug of interest after bacterial adhesion in the cells. For treatment experiments, add 500 μL of a drug solution diluted in cell medium (in this protocol tobramycin 6 μg/mL was used) to the apical side. Add 1,500 µL of cell medium without drug on the basolateral side.
   NOTE: Instead of instilling the drugs as a solution, the model can also be adapted to aerosol deposition. For such purposes, the cells at ALI are fed from the basolateral side with 500 µL of cell medium. The drug is then first nebulized and allowed to deposit in the apical compartment by an appropriate device (not described here). The infected and treated sample can be checked for the endpoints outlined in the sections 4–7. From this step on, permeable supports can be used to create either images (section 4) or to get results of bacteria growth and mammalian cell viability, amongst others (sections 5–7).

4. Sample preparation for confocal laser-scanning microscopy (CLSM)

1. After the establishment of the co-culture, infection and drug treatment, remove all medium from the apical and basolateral side. Wash 1x with PBS at 37°C and then fix the cells with 3% paraformaldehyde (PFA) for 1 h at RT (300 µL on apical/600 µL on basolateral). Cell nuclei are stained with 5 µg/mL of DAPI-PBS for 30 min at room temperature.
   CAUTION: PFA is hazardous.
2. Cut the membranes using a scalpel and place them between two 12 mm microscopy cover slides using a mounting medium (see Table of Materials). Let it dry inside the flow bench for 30 min before storage at 4 °C. Visualize by confocal scanning microscopy.
   NOTE: After the co-culture and before the mounting, tight junctions immunostaining can be performed. For that, cells are fixed with paraformaldehyde 3% for 30 min, washed again with PBS, and permeabilized with saponin 0.05%/BSA 1% in PBS. In this protocol, the zonula occludens protein (ZO-1) was detected via mouse anti-human ZO-1 antibody (1:400, incubation at 4 °C overnight). The samples were then incubated for 2 h at RT with goat anti-mouse IgG antibody Alexa Fluor 633 (1:2000 in red). Nuclei were stained with DAPI (1 µg/mL) and mounted with mounting medium on coverslips.
3. Use a confocal microscope for imaging the stored membranes. Choose 25x or 63x water-immersion objectives and lasers at 405, 488, 505 or 633 nm for detection. Images should have a 1024 x 1024 pixel resolution.
   NOTE: The lasers are chosen according to the stain used.
4. Acquire apical and cross-section views, and use zeta-stack mode (10–15 stacks) for the construction of a three-dimensional model using imaging software.

5. Measurement of bacterial proliferation via colony-forming units (CFU)

1. Collect the apical and basolateral medium (containing bacteria) to assess CFU of non-attached bacteria. Collect 500 µL from the apical and basolateral sides and pool them.
   NOTE: Use this suspension directly to count bacteria (step 5.4) or centrifuge at 21,250 x g for 10 min to evaluate lactate dehydrogenase (LDH) from the supernatant (section 6) and/or re-suspended in PBS to count extracellular bacteria (step 5.4).
2. Assess survival of bacteria attached and/or internalized in the cells by adding 500 µL of sterile deionized cold water in each compartment of the permeable support. Incubate cells for 30 min at room temperature.
   NOTE: The samples can either be plated on LB agar (see step 5.4) or frozen (as whole insert plate) at -20 °C for plating later on.
3. For assessing CFU of adherent/internalized bacteria, thaw samples at 37°C for 10 min (if frozen). Using pipette tips for each well, scrape the membrane surface and pipette up and down to remove all adhered content.
   NOTE: At this step, all the epithelial cells are lysed and adherent/internalized bacteria are available as a suspension to be plated.
4. With the bacterial suspension from both fractions, perform a 1/10 serial dilution using Tween-80 0.05% in PBS and plate the bacteria on LB agar plates.
   NOTE: Dilutions in between 1 to 10 are recommended. The bacteria should be counted in the highest dilution, where single colonies are first identified.
5. Incubate agar plates at 30°C for 16–72 h to count colonies, and calculate CFU accordingly.
   NOTE: A temperature of 30°C at the time of plate incubation is essential for treated-samples and to observe delayed-growth of colonies.

6. Evaluation of cell cytotoxicity via lactate dehydrogenase assay

1. Use the supernatant of infected cells containing bacteria (from step 5.1) for cell viability assessment for LDH assay¹⁶. Centrifuge the supernatant at 21,250 x g for 10 min to pellet the bacteria and eventually rest of the cells. Use the bacteria-free supernatant to measure LDH release.
   NOTE: The supernatant should not be frozen before measuring LDH by this assay.
2. Transfer 100 µL of the supernatant to a 96-well plate, and add 100 µL of the LDH assay solution (see the Table of Materials). Incubate at room temperature for 5 min in the dark, then read absorbance at 492 nm.

7. Assessing the release of human cytokines

1. For cytokine quantification, use either ELISA or cytometric bead array immunoassay¹⁷(see the Table of Materials). For this, centrifuge supernatant from step 5.1 at 21,250 x g for 10 min and measure either immediately or store -80 °C for up to 15 days until analysis.
2. Evaluate supernatants with a commercially available ELISA kit.
   NOTE: The procedure follows the manufacture instructions, which include the coating of plates with the capture antibody, addition of the samples (100 µL) and cytokine standards, incubation, washing, and addition of detection antibody to provide a colorimetric measurement of cytokine presence. Alternatively, flow cytometry can be used to measure cytokines via commercially available kits (see Table of Materials).

Results

Figure 1A shows the morphology of the resulting co-culture of human bronchial epithelial cells and macrophages after growing both for 24 h on the apical and basolateral side of permeable supports, respectively. The epithelial barrier integrity is shown by higher TEER (834 Ω×cm²) and CLSM by immunostaining for the tight junction protein ZO-1 (Figure 1B). The same results observed in terms of barrier integrity of uninfected CFBE41o^-

Discussion

This paper describes a protocol for a 3D co-culture of the infected airways, constituted by the human cystic fibrosis bronchial epithelial cell line CFBE41o- and the human monocyte-derived macrophage cell line THP-1. The protocol allows the assessment of epithelial barrier integrity, macrophage transmigration, bacteria survival, and inflammation, which are important parameters when testing drug efficacy and host-responses simultaneously. The novelty in the model lies within the incorporation of epithelial cells (i.e....

Materials

Name	Company	Catalog Number	Comments
Accutase	Accutase	AT104
Ampicillin	Carl Roth, Germany	HP62.1
CASY TT Cell Counter and Analyzer	OLS Omni Life Sciences	-
CellTrace Far Red	Thermo Fischer	C34564
Centrifuge Universal 320R	Hettich, Germany	1406
CFBE41o- cells	1. Gruenert Cell Line Distribution Program 2. Sigma-Aldrich	1. gift from Dr. Dieter C. Gruenert 2. SCC151
Chopstick Electrode Set for EVOM2, 4mm	World Precision Instruments, Sarasota, USA	STX2
Confocal Laser-Scanning Microscope CLSM	Leica, Mannheim, Germany	TCS SP 8
Cytokines ELISA Ready-SET-Go kits	Affymetrix eBioscience, USA	15541037
Cytokines Panel I and II	LEGENDplex Immunoassay (Biolegend, USA).	740102
Cytotoxicity Detection Kit (LDH)	Roche	11644793001
D-(+) Glucose	Merck	47829
Dako Fluorescence Mounting Medium	DAKO	S3023
DAPI (4′,6-diamidino-2-phenylindole)	Thermo Fischer	D1306
Epithelial voltohmmeter	World Precision Instruments, Sarasota, USA	EVOM2
Falcon Permeable Support for 12 Well Plate with 3.0μm Transparent PET Membrane, Sterile	Corning, Amsterdam, Netherlands	353181
Fetal calf serum	Lonza, Basel, Switzerland	DE14-801F
Goat anti-mouse (H+L) Cross-adsorbed secondary Antibody, Alexa Fluor 633	Invitrogen	A-21050
L-Lactate Dehydrogenase (LDH), rabbit muscle	Roche, Mannheim, Germany	10127230001
LB broth	Sigma-Aldrich, Germany	L2897-1KG
MEM (Minimum Essential Medium)	Gibco Thermo Fisher Scientific Inc.	11095072
Non-Essential Amino Acids Solution (100X)	Gibco Thermo Fisher Scientific Inc.	11140050
P. aeruginosa strain PAO1	American Type Culture Collection	47085
P. aeruginosa strain PAO1-GFP	American Type Culture Collection	10145GFP
Paraformaldehyde Aqueous Solution -16%	EMS DIASUM	15710-S
Phosphate buffer solution buffer	Thermo Fischer	10010023
Petri dishes	Greiner	664102
Phorbol 12-myristate 13-acetate (PMA)	Sigma, Germany	P8139-1MG
Precision Cover Glasses	ThorLabs	CG15KH
Purified Mouse anti-human ZO-1 IgG antibody	BD Transduction Laboratories	610966
Roswell Park Memorial Institute (RPMI) 1640 medium	Gibco by Lifetechnologies, Paisley, UK	11875093
Soda-lime glass Petri dish, 50 x 200 mm (height x outside diameter)	Normax, Portugal	5058561
Saponin	Sigma-Aldrich, Germany	S4521
T75 culture flasks	Thermo Fischer	156499
THP-1 cells	Deutsche Sammlung von Mikroorganismen und Zellkulturen (DSMZ; Braunschweig, Germany)	No. ACC-16
Tobramycin sulfate salt	Sigma	T1783-500MG
Trypsin-EDTA 0.05%	Thermo Fischer	25300054
Tween80	Sigma-Aldrich, Germany	P1754