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Protocol

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Materials

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Immunology and Infection

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

Published: June 15th, 2020

DOI:

10.3791/61069

1Department of Drug Delivery, Helmholtz Institute for Pharmaceutical Research Saarland (HIPS), 2Department of Pharmacy, Saarland University, 3Department of Vaccinology and Applied Microbiology, Helmholtz Centre for Infection Research, 4Department of Internal Medicine V - Pulmonology, Allergology, Respiratory Intensive Care Medicine, Saarland University Hospital
* These authors contributed equally

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.

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.

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 cont....

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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 mi.......

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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-

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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........

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This work received funding from the European Union’s HORIZON 2020 Program for research, technological development, and demonstration under grant agreement no. 642028 H2020-MSCA-ITN-2014, NABBA - Design, and Development of advanced Nanomedicines to overcome Biological Barriers and to treat severe diseases. We thank Dr. Ana Costa and Dr. Jenny Juntke for the great support on the development of the co-culture, Olga Hartwig, for the scientific illustration, Anja Honecker, for ELISA assays, Petra König, Jana Westhues and Dr. Chiara De Rossi for the support on cell culture, analytics, and microscopy. We also thank Chelsea Thorn for proofreading our manuscript.

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NameCompanyCatalog NumberComments
AccutaseAccutaseAT104
AmpicillinCarl Roth, GermanyHP62.1
CASY TT Cell Counter and AnalyzerOLS Omni Life Sciences-
CellTrace Far RedThermo FischerC34564
Centrifuge Universal 320RHettich, Germany1406
CFBE41o- cells1. Gruenert Cell Line Distribution Program
2. Sigma-Aldrich
1. gift from Dr. Dieter C. Gruenert
2. SCC151
Chopstick Electrode Set for EVOM2, 4mmWorld Precision Instruments, Sarasota, USASTX2
Confocal Laser-Scanning Microscope CLSMLeica, Mannheim, GermanyTCS SP 8
Cytokines ELISA Ready-SET-Go kitsAffymetrix eBioscience, USA15541037
Cytokines Panel I and IILEGENDplex Immunoassay (Biolegend, USA).740102
Cytotoxicity Detection Kit (LDH)Roche11644793001
D-(+) GlucoseMerck47829
Dako Fluorescence Mounting MediumDAKOS3023
DAPI (4′,6-diamidino-2-phenylindole)Thermo FischerD1306
Epithelial voltohmmeterWorld Precision Instruments, Sarasota, USAEVOM2
Falcon Permeable Support for 12 Well Plate with 3.0μm Transparent PET Membrane, SterileCorning, Amsterdam, Netherlands353181
Fetal calf serumLonza, Basel, SwitzerlandDE14-801F
Goat anti-mouse (H+L) Cross-adsorbed secondary Antibody, Alexa Fluor 633InvitrogenA-21050
L-Lactate Dehydrogenase (LDH), rabbit muscleRoche, Mannheim, Germany10127230001
LB brothSigma-Aldrich, GermanyL2897-1KG
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 PAO1American Type Culture Collection47085
P. aeruginosa strain PAO1-GFPAmerican Type Culture Collection10145GFP
Paraformaldehyde Aqueous Solution -16%EMS DIASUM15710-S
Phosphate buffer solution bufferThermo Fischer10010023
Petri dishesGreiner664102
Phorbol 12-myristate 13-acetate (PMA)Sigma, GermanyP8139-1MG
Precision Cover GlassesThorLabsCG15KH
Purified Mouse anti-human ZO-1 IgG antibodyBD Transduction Laboratories610966
Roswell Park Memorial Institute (RPMI) 1640 mediumGibco by Lifetechnologies, Paisley, UK11875093
Soda-lime glass Petri dish, 50 x 200 mm (height x outside diameter)Normax, Portugal5058561
SaponinSigma-Aldrich, GermanyS4521
T75 culture flasksThermo Fischer156499
THP-1 cellsDeutsche Sammlung von Mikroorganismen und Zellkulturen (DSMZ; Braunschweig, Germany)No. ACC-16
Tobramycin sulfate saltSigmaT1783-500MG
Trypsin-EDTA 0.05%Thermo Fischer25300054
Tween80Sigma-Aldrich, GermanyP1754

  1. Cutting, G. R. Cystic fibrosis genetics: from molecular understanding to clinical application. Nature Reviews Genetics. 16 (1), 45-56 (2015).
  2. Lyczak, J. B., Cannon, C. L., Pier, G. B. Establishment of Pseudomonas aeruginosa infection: Lessons from a versatile opportunist. Microbes and Infection. 2 (9), 1051-1060 (2000).
  3. Wilke, M., Buijs-Offerman, R. M., et al. Mouse models of cystic fibrosis: Phenotypic analysis and research applications. Journal of Cystic Fibrosis. 10, 152-171 (2011).
  4. Moreau-Marquis, S., Stanton, B. A., O'Toole, G. A. Pseudomonas aeruginosa biofilm formation in the cystic fibrosis airway. Pulmonary Pharmacology and Therapeutics. 21 (4), 595-599 (2008).
  5. Yu, Q., et al. In vitro evaluation of tobramycin and aztreonam versus Pseudomonas aeruginosa biofilms on cystic fibrosis-derived human airway epithelial cells. Journal of Antimicrobial Chemotherapy. 67 (11), 2673-2681 (2012).
  6. Moreau-Marquis, S., Redelman, C. V., Stanton, B. a., Anderson, G. G. Co-culture models of Pseudomonas aeruginosa biofilms grown on live human airway cells. Journal of visualized experiments : JoVE. (44), e2186 (2010).
  7. Stanton, B. A., Coutermarsh, B., Barnaby, R., Hogan, D. Pseudomonas aeruginosa reduces VX-809 stimulated F508del-CFTR chloride secretion by airway epithelial cells. PLoS ONE. 10 (5), 1-13 (2015).
  8. Lambrecht, B. N., Prins, J., Hoogsteden, H. C. Lung dendritic cells and host immunity to infection. European Respiratory Journal. (18), 692-704 (2001).
  9. Murgia, X., De Souza Carvalho, C., Lehr, C. M. Overcoming the pulmonary barrier: New insights to improve the efficiency of inhaled therapeutics. European Journal of Nanomedicine. 6 (3), 157-169 (2014).
  10. Ding, P., Wu, H., Fang, L., Wu, M., Liu, R. Transmigration and phagocytosis of macrophages in an airway infection model using four-dimensional techniques. American Journal of Respiratory Cell and Molecular Biology. 51 (1), 1-10 (2014).
  11. Hartl, D., Tirouvanziam, R., et al. Innate Immunity of the Lung: From Basic Mechanisms to Translational Medicine. Journal of Innate Immunity. 10 (5-6), 487-501 (2018).
  12. Esposito, S., et al. Manipulating proteostasis to repair the F508del-CFTR defect in cystic fibrosis. Molecular and cellular pediatrics. 3 (1), 13 (2016).
  13. Hein, S., Bur, M., Schaefer, U. F., Lehr, C. M. A new Pharmaceutical Aerosol Deposition Device on Cell Cultures (PADDOCC) to evaluate pulmonary drug absorption for metered dose dry powder formulations. European Journal of Pharmaceutics and Biopharmaceutics. 77 (1), 132-138 (2011).
  14. Kletting, S., Barthold, S., et al. Co-culture of human alveolar epithelial (hAELVi) and macrophage (THP-1) cell lines. Altex. 35 (2), 211-222 (2018).
  15. Schwende, H., Fitzke, E., Ambs, P., Dieter, P. Differences in the state of differentiation of THP-1 cells induced by phorbol ester and 1,25-dihydroxyvitamin D3. Journal of leukocyte biology. 59 (4), 555-561 (1996).
  16. Castoldi, A., Empting, M., et al. Aspherical and Spherical InvA497-Functionalized Nanocarriers for Intracellular Delivery of Anti-Infective Agents. Pharmaceutical Research. 36 (1), 1-13 (2019).
  17. Ebensen, T., Delandre, S., Prochnow, B., Guzmán, C. A., Schulze, K. The Combination Vaccine Adjuvant System Alum/c-di-AMP Results in Quantitative and Qualitative Enhanced Immune Responses Post Immunization. Frontiers in cellular and infection microbiology. 9, 31 (2019).
  18. Brockman, S. M., Bodas, M., Silverberg, D., Sharma, A., Vij, N. Dendrimer-based selective autophagy-induction rescues δF508-CFTR and inhibits Pseudomonas aeruginosa infection in cystic fibrosis. PLoS ONE. 12 (9), 1-17 (2017).
  19. Anderson, G. G., Moreau-Marquis, S., Stanton, B. A., O'Toole, G. A. In vitro analysis of tobramycin-treated Pseudomonas aeruginosa biofilms on cystic fibrosis-derived airway epithelial cells. Infection and Immunity. 76 (4), 1423-1433 (2008).
  20. Moreau-Marquis, S., Bomberger, J. M., et al. The DeltaF508-CFTR mutation results in increased biofilm formation by Pseudomonas aeruginosa by increasing iron availability. American journal of physiology. Lung cellular and molecular physiology. 295, 25-37 (2008).
  21. Braakhuis, H. M., Kloet, S. K., et al. Progress and future of in vitro models to study translocation of nanoparticles. Archives of Toxicology. 89 (9), 1469-1495 (2015).
  22. Bosshart, H., Heinzelmann, M. THP-1 cells as a model for human monocytes. Annals of Translational Medicine. 4 (21), 4-7 (2016).
  23. Daigneault, M., Preston, J. a., Marriott, H. M., Whyte, M. K. B., Dockrell, D. H. The identification of markers of macrophage differentiation in PMA-stimulated THP-1 cells and monocyte-derived macrophages. PLoS ONE. 5 (1), (2010).
  24. Bismarck, P. V., Schneppenheim, R., Schumacher, U. Successful treatment of pseudomonas aeruginosa respiratory tract infection with a sugar solution - A case report on a lectin based therapeutic principle. Klinische Padiatrie. 213 (5), 285-287 (2001).
  25. Klinger-Strobel, M., Lautenschläger, C., et al. Aspects of pulmonary drug delivery strategies for infections in cystic fibrosis - where do we stand. Expert Opinion on Drug Delivery. 5247, 1-24 (2015).
  26. Ehrhardt, C., Collnot, E. -. M., et al. Towards an in vitro model of cystic fibrosis small airway epithelium: characterisation of the human bronchial epithelial cell line CFBE41o-. Cell and tissue research. 323 (3), 405-415 (2006).
  27. Anderson, G. G., Kenney, T. F., Macleod, D. L., Henig, N. R., O'Toole, G. A. Eradication of Pseudomonas aeruginosa biofilms on cultured airway cells by a fosfomycin/tobramycin antibiotic combination. Pathogens and Disease. 67 (1), 39-45 (2013).
  28. Cavalieri, F., Tortora, M., Stringaro, A., Colone, M., Baldassarri, L. Nanomedicines for antimicrobial interventions. Journal of Hospital Infection. 88 (4), 183-190 (2014).
  29. Savla, R., Minko, T. Nanotechnology approaches for inhalation treatment of fibrosis. Journal of Drug Targeting. 21 (10), 914-925 (2013).
  30. Ho, D. -. K., et al. Challenges and strategies in drug delivery systems for treatment of pulmonary infections. European Journal of Pharmaceutics and Biopharmaceutics. 144, 110-124 (2019).

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