Pneumococcus Infection of Primary Human Endothelial Cells in Constant Flow
Summary
This study describes the microscopic monitoring of pneumococcus adherence to von Willebrand factor strings produced on the surface of differentiated human primary endothelial cells under shear stress in defined flow conditions. This protocol can be extended to detailed visualization of specific cell structures and quantification of bacteria by applying differential immunostaining procedures.
Abstract
Interaction of Streptococcus pneumoniae with the surface of endothelial cells is mediated in blood flow via mechanosensitive proteins such as the Von Willebrand Factor (VWF). This glycoprotein changes its molecular conformation in response to shear stress, thereby exposing binding sites for a broad spectrum of host-ligand interactions. In general, culturing of primary endothelial cells under a defined shear flow is known to promote the specific cellular differentiation and the formation of a stable and tightly linked endothelial layer resembling the physiology of the inner lining of a blood vessel. Thus, the functional analysis of interactions between bacterial pathogens and the host vasculature involving mechanosensitive proteins requires the establishment of pump systems that can simulate the physiological flow forces known to affect the surface of vascular cells.
The microfluidic device used in this study enables a continuous and pulseless recirculation of fluids with a defined flow rate. The computer-controlled air-pressure pump system applies a defined shear stress on endothelial cell surfaces by generating a continuous, unidirectional, and controlled medium flow. Morphological changes of the cells and bacterial attachment can be microscopically monitored and quantified in the flow by using special channel slides that are designed for microscopic visualization. In contrast to static cell culture infection, which in general requires a sample fixation prior to immune labeling and microscopic analyses, the microfluidic slides enable both the fluorescence-based detection of proteins, bacteria, and cellular components after sample fixation; serial immunofluorescence staining; and direct fluorescence-based detection in real time. In combination with fluorescent bacteria and specific fluorescence-labeled antibodies, this infection procedure provides an efficient multiple component visualization system for a huge spectrum of scientific applications related to vascular processes.
Introduction
The pathogenesis of pneumococcus infections is characterized by a multifaceted interaction with a diversity of extracellular matrix compounds and components of the human hemostasis, such as plasminogen and VWF1,2,3,4,5,6,7,8. The multidomain glycoprotein VWF serves as key regulator of a balanced hemostasis by mediating thrombocyte recruitment and fibrin incorporation at the site of vascular thrombus formation9. The importance of functional, active VWF for bleeding control and wound healing is demonstrated by von Willebrand’s disease, a common inherited bleeding disorder10.
Globular VWF circulates in the human blood system at a concentration of up to 14.0 µg/mL11,10. In response to vascular injury, the local release of VWF by endothelial Weibel Palade Bodies (WBP) is markedly increased11,12. Previous studies show that pneumococcus adherence to human endothelial cells and its production of the pore-forming toxin pneumolysin significantly stimulates luminal VWF secretion13. The hydrodynamic forces of the blood flow induce a structural opening of the mechanoresponsive VWF domains. At flow rates of 10 dyn/cm2 the VWF multimerizes to long protein strings of up to several hundred micrometers in length that remain attached to the subendothelium10,12.
To understand the function of multimerized VWF strings generated under shear stress in the interaction of pneumococcus with the endothelial surface, a microfluidic-based cell culture infection approach was established. A microfluidic device with a software-controlled air-pressure pump system was used. This enabled a continuous, unidirectional recirculation of cell culture medium with a defined flow rate. Thereby, the system applied a defined shear stress on the surface of endothelial cells, which remained attached inside specialized channel slides. This approach enabled the simulation of the shear force within the blood stream of the human vascular system, in which VWF strings are generated on differentiated endothelial cells under defined constant flow conditions. For this purpose, the endothelial cells were cultivated in specific channel slides (see Table of Materials), which were adapted for microscopic analyses during flow. The microfluidic pump system provided the highly defined and controlled shear stress situation required for the formation of extended VWF strings on the confluent endothelial cell layer. After the stimulation of VWF-secretion of confluently grown human umbilical vein endothelial cells (HUVEC) by histamine supplementation, the string formation was induced by applying a shear stress (ԏ) of 10 dyn/cm2. The shear stress is defined as the force acting on the cell layer. It is calculated approximately according to Cornish et. al.14 with equation 1:
Where ԏ = shear stress in dyn/cm2, η = viscosity in (dyn∙s)/cm2, h =half of channel height, w = half of channel width, and Φ = flowrate in mL/min.
The result of equation 1 depends on the different heights and widths of the different slides used (see Table of Materials). In this study a Luer channel slide of 0.4 µm resulting in a chamber slide factor of 131.6 was used (see formula 2).
Viscosity of the medium at 37 °C is 0.0072 dyn∙s/cm² and a shear stress of 10 dyn/cm² was used. This resulted in a flow rate of 10.5 mL/min (see formula 3).
Here, the adaptation and advancement of a microfluidic cell culturing procedure using a unidirectional laminar flow system for the investigation and visualization of bacterial infection mechanisms in the host vasculature is described in detail. The generation of VWF strings on endothelial layers can also be stimulated by using other pump systems that are able to apply a continuous and steady shear stress15.
After cultivation of primary endothelial cells to confluence in flow and stimulation of VWF string formation, pneumococci expressing red fluorescence protein (RFP)16 were added to the endothelial cell layer under constant microscopic control. The attachment of bacteria to VWF strings on the surface of endothelial cells was microscopically visualized and monitored for up to three hours in real time by using VWF-specific fluorescent-labelled antibodies. With this approach, the role of VWF as an adhesion cofactor promoting bacterial attachment to the vascular endothelium was determined8.
In addition to the microscopic visualization of protein secretion and conformational changes, this method could be used to monitor single steps of bacterial infection processes in real time and to quantify the amount of attached bacteria at different time points of infection. The specific software-controlled pump system also provides the possibility to culture the endothelial cells in defined constant flow conditions for up to several days and enables a defined pulsed medium flow incubation. Moreover, this method can be applied using different cell types. Adapting the staining protocol also enables the detection and visualization of bacteria internalized into eukaryotic cells.
This manuscript describes this advanced experimental protocol that can be used as a defined, reliable, and reproducible approach for an efficient and versatile characterization of pathophysiological processes.
Protocol
Representative Results
Discussion
The simulation of bacterial interaction with mechanosensitive host proteins such as VWF requires a perfusable cell culture system that enables the generation of a defined, unidirectional, and continuous flow of liquids, thereby generating reliable shear stress. Several microfluidic pump systems have already been described. A comprehensive review from Bergmann et al. summarizes the key aspects of different two- and three-dimensional cell culture models17.
The microfluidi…
Disclosures
The authors have nothing to disclose.
Acknowledgements
The project was funded by the DFG (BE 4570/4-1) to S.B.
Materials
| 1 mL Luer-syringe | Fisher Scientific | 10303002 | with 1 mL volume for gelatin injection using the luer-connection of the slides |
| 2 mL Luer-syringe | Sarstedt | 9077136 | For pieptting/injecting fluids into the luer connections of the channel chamber slides |
| Accutase | eBioscience now thermo fisher | 00-4555-56 | protease mix used for gentle detachment of endothelial cells |
| AlexaFluor350-conjugated Phalloidin | Abcam | ab176751 | no concentration available from the manufacturer, stock solution is sufficient for 300 tets, company recommends to use 100 µl of a 1:1000 dilution, blue fluorescence (DAPI-filter settings) |
| AlexaFluor488-conjugated goat-derived anti-mouse antibody | Thermo Fisher Sientific | A11001 | stock concentration: 2 mg/mL for immunostaining of human VWF in microfluidic slide after PFA fixation, green fluorescence |
| AlexaFluor568-conjugated goat-derived anti-rabbit-antibody | Thermo Fisher Scientific | A-11011 | stock concentration: 2 mg/mL for immunostaining of pneumococci in microfluidic slide after pFA fixation, red fluorescence |
| Bacto Todd-Hewitt-Broth | Becton Dickinson GmbH | BD 249210 | complex bacterial culture medium |
| Bovine Serum Albumin (BSA) | Sigma Aldrich | A2153-25G | solubilized, for preparation of blocking buffer |
| Cell culture flasks with filter | TPP | 90026 | subcultivation of HUVEC in non-coated cell culture flasks of 25 cm2 surface |
| Centrifuge Allegra X-12R | Beckman Coulter Life Sciences | 392304 | spinning down of bacteria (volumes of > 2mL) |
| Centrifuge Allegra X-30 | Beckman Coulter Life Sciences | B06314 | spinning down of eukaryotic cells |
| Centrifuge Z 216 MK | Hermle | 305.00 V05 – Z 216 M | spinning down of bacteria (volumes of less than 2 mL) |
| Chloramphenicol | Carl Roth GmbH + Co. KG, Karlsruhe | 3886.2 | used in a concentration of 0.2 /mL for cultivation of pneumococci transformed with genetic construct carrying red fluorescent protein and chloramphenicol resistance cassette |
| Clamp for perfusion tubing | ibidi | 10821 | for holding the liquid in the tube bevor connecting the slide to the pump system |
| CO2-Incubator | Fisher Scientific | MIDI 40 | incubator size is perfectly adapted to teh size of the fluidic unit with connected channel slide and was used for flow cultivation at 37°C and 5% CO2 |
| CO2-Incubator | Sanyo | MCO-18 AIC | for incubation of bacteria and cells in a defined atmosphere at 5% CO2 and 37°C |
| Colombia blood agar plates | Becton Dickinson GmbH | PA-254005.06 | agar-based complex culture medium for S. pneumoniae supplemented with 7% sheep blood |
| Computer | Dell | Latitude 3440 | Comuter with pressure pump software |
| Confocal Laser Scanning Microscope (CLSM) | Leica | DMi8 | An inverse microscope with a stage covered by a heatable chamber and with a fluorescence unit equipped with fluorescence filter, Xenon-light source (SP8, DMi8) and DFC 365 FX Kamera (1392 x 1040, 1.4 Megapixel) |
| Di Potassium hydrogen phosphate (KH2PO4) | Carl Roth GmbH + Co. KG, Karlsruhe | 3904.1 | used for PBS buffer |
| Drying material | Merck | 101969 | orange silica beads for drying used in a glass bottle with a tubing adaptor |
| ECGM supplement Mix | Promocell | C-39215 | supplement mix for ECGM -medium, required for precultivation of endothelial cells: 0.02 mL/mL Fetal calf serum, 0.004 mL/ mL endothelial cell growth supplement, 0.1 ng / mL epidermal growth factor, 1 ng / mL basic fibroblast growth factor, 90 µg / mL heparin, 1 µg / mL Hydrocortisone |
| ECGMS | Promocell | C-22010 | ECGM supplemented with 5 % [w/w] FCS and 1 mM MgSO4 to increase cell adhesion |
| Endothelial Cell growth medium (ECGM, ready to use) | Promocell | C-22010 | culture medium of HUVECs, already supplemented with all components of the supplement mix |
| Fetal Calf Serum (FCS) | biochrome now Merck | S 0415 | supplement for cell culture, used for infection analyses |
| FITC-conjugated goat anti-human VWF antibody | Abcam | ab8822 | stock concentration: 10 mg/mL, for immunodetection of globular and multimerized VWF in flow |
| Fluidic Unit | ibidi | 10903 | fluidic unit for flow cultivation |
| Gelatin (porcine) | Sigma Aldrich | G-1890-100g | for precoating of microslide channel surface |
| histamine dihydrochloride | Sigma Aldrich | H-7250-10MG | for induction of VWF secretion from endothelial Weibel Palade Bodies |
| Human Umbilical Vein Endothelial Cell (HUVEC) | Promocell | C-12203 Lot-Nr. 396Z042 | primary endothelial cells from pooled donor, stored crypcoserved in liquid nitrogen |
| Human VWF-specific antibody derived from mouse (monoclonal) | Santa Cruz | sc73268 | stock concentration: 200 µg/mL for immunostaining of VWF in microfluidic slide after PFA fixation |
| Injection Port | ibidi | 10820 | for injection of histamin or bacteria into the reservoir tubing during the flow circulation |
| Light microscope | Zeiss | Axiovert 35M | inverse light microscope for control of eukaryotic cell detachement and cell counting using a 40 x water objective allowing 400 x magnification |
| Luer-slides I0.4 (ibiTreat472microslides) | ibidi | 80176 | physically modified slides for fludic cultivation (μ–Slide I0.4Luer with a channel hight of 0.4 mm, a channel volume of 100 μl, a growth area of 2.5 cm and a coating area of 25.4 cm2) suitable for all kinds of flow assay, the physical treatment generates a hydrophilic and adhesive surface. |
| Magnesium sulfate (MgSO4, unhydrated) | Sigma Aldrich | M7506-500G | For preparation of ECGMS medium |
| Microfluidic Pump | ibidi | 10905 | air pressure pump |
| Neubauer cell counting chamber | Karl Hecht GmbH&Co KG | 40442002 | microscopic counting chamber for HUVECs |
| Paraformaldehyde 16% (PFA) | Electron Microscopy Sciences | 15710-S | for cross linking of samples |
| Perfusion Set | ibidi | 10964 | Perfusion Set Yellow/Green has a tubing diameter of 1.6 mm, a tube length of 50 cm, a total working volume of 13.6 mL, a dead tube volume of 2.8 mL and a reservoir size of 10 mL. combined with the µ-slide L0.4Luer, at 37°C and a viscosity of 0.0072 dyn x s/cm2 a flow rate range of 3.8mL/min up to 33.9 mL/min and shear stress between 3.5 dyn/cm2 and 31.2 dyn/cm2 can be reached. with 50 cm lenght for microfluidic |
| Phosphate-buffered saline (PBS) | the solution was prepared using the following chemicals: 0.2 g/L KCl, 1.44 g/L Na2HPO4, 0.24 g/L KH2PO4 , pH 7.4 | ||
| Plastic cuvettes | Sarstedt | 67,741 | (2 x optic) for OD measurement at 600 nm |
| Pneumococcus-specific antiserum | Pineda | raised in rabbit using heat-inactivated Streptococcus pneumoniae NCTC10319 and D39, IgG-purified using proteinA-sepharose column. | |
| Polystyrene or Styrofoam plate | this is a precaution step to avoid cold stress on the cells seeded in the channel slides. Any type of styrofoam such as packaging box-material can be used. The plate might by 0.5 cm thick and should have a size of 20 cm2. | ||
| Potassium chloride (KCl) | Carl Roth GmbH + Co. KG, Karlsruhe | 6781.1 | used for PBS buffer |
| Pump Control Software (PumpControl v1.5.4) | ibidi | v1.5.4 | Computer software for controlling the pressure pump, setting the flow conditions and start/end the flow |
| reaction tubes 1.5/ 2.0mL | Sarstedt | 72.706/ 72.695.500 | required for antibody dilutions |
| reaction tubes with 50 mL volume | Sarstedt | 6,25,48,004 | |
| RFP-expressing pneumococci | National Collection of Type Cultures, Public Health England | 10,319 | Streptococcus pneumoniae serotype 47 expressing RFP fused to ahistone-like protein integrated into the genome |
| serological pipets 5, 10 mL | Sarstedt | 86.1253.025/ 86.1254.025 | for pipeting larger volumes |
| Sodium Carbonate (Na2CO3, water free) | Sigma Aldrich | 451614-25G | for preparation of 100 mM Sodium Carbonate buffer |
| Sodium dihydrogen phosphate (NaH2PO4) | Carl Roth GmbH + Co. KG, Karlsruhe | P030.2 | used for PBS buffer |
| Spectral Photometer Libra S22 | Biochrom | 80-2115-20 | measurement of optical density (OD) of bacterial suspension at 600 nm |
| Sucrose | Sigma Aldrich | S0389-500G | for preparation of blocking buffer |
| Triton X-100 | Sigma Aldrich | T9284-500ML | Used in 0.1% end concentration diluted in dH20 for eukaryotic cell permeabilization after PFA fixation |
| Yeast extract | oxoid | LP0021 | bacteria are cultivated in THB supplement with 1% [w/w] yeast extract = complete bacterial cultivation medium THY |
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