Methods for Detecting Cytotoxic Amyloids Following Infection of Pulmonary Endothelial Cells by Pseudomonas aeruginosa

Podsumowanie

Simple methods are described for demonstrating the production of cytotoxic amyloids following infection of pulmonary endothelium by Pseudomonas aeruginosa.

Streszczenie

Patients who survive pneumonia have elevated death rates in the months following hospital discharge. It has been hypothesized that infection of pulmonary tissue during pneumonia results in the production of long-lived cytotoxins that can lead to subsequent end organ failure. We have developed in vitro assays to test the hypothesis that cytotoxins are produced during pulmonary infection. Isolated rat pulmonary endothelial cells and the bacterium Pseudomonas aeruginosa are used as model systems, and the production of cytoxins following infection of the endothelial cells by the bacteria is demonstrated using cell culture followed by direct quantitation using lactate dehydrogenase assays and a novel microscopic method utilizing ImageJ technology. The amyloid nature of these cytotoxins was demonstrated by thioflavin T binding assays and by immunoblotting and immunodepletion using A11 anti-amyloid antibody. Further analyses using immunoblotting demonstrated that oligomeric tau and Aβ were produced and released by endothelial cells following infection by P. aeruginosa. These methods should be readily adaptable to analyses of human clinical samples.

Wprowadzenie

Patients who survive pneumonia have elevated death rates in the months following hospital discharge^1,2,3,4,5,6. In most cases, death occurs by some type of end-organ failure including renal, pulmonary, cardiac, or liver events, as well as stroke^5,6. The reason for the elevated death rate in this patient population has never been established.

Pneumonia is classified as being either community-acquired or hospital-acquired (nosocomial), and agents that can cause pneumonia include bacteria, viruses, fungi, and chemicals. One of the major causes of nosocomial pneumonia is the bacterium Pseudomonas aeruginosa. P. aeruginosa is a gram-negative organism that uses a type III secretion system to transfer various effector molecules, termed exoenzymes, directly to the cytoplasm of target cells^7,8. During infection of pulmonary endothelial cells, the exoenzymes target various intracellular proteins, including an endothelial form of the microtubule-associated protein tau^9,10,11,12, leading to endothelial barrier breakdown resulting in severe pulmonary edema, decreased pulmonary function and, oftentimes, death.

As stated previously, patients who survive the initial pneumonia have elevated death rates in the first 12 months following hospital discharge. A potential mechanism for explaining this phenomenon is that some type of long-lived toxin is generated during the initial infection that leads to poor long-term outcome. Two observations support this possibility. First, cultured pulmonary endothelial cells that are treated initially with P. aeruginosa fail to proliferate for up to a week after the bacteria are killed by antibiotics¹³. Second, long-lived prions and agents with prion characteristics have been demonstrated in various human and animal diseases, particularly diseases associated with the nervous system^14,15.

Methods for examining the potential production of long-lived cytotoxic agents during pulmonary infection have never been described. Here a series of simple in vitro assays are outlined that can be used for investigating cytotoxin production and activity following infection using a common pneumonia causing agent, P. aeruginosa. These assays should be readily adaptable to investigate possible cytotoxin induction following infection using other agents that cause pneumonia, and the supernatants that are generated also should be useful for investigating effects of the cytotoxins in whole organs or animals. Finally, the assays that are outlined here most likely will be adaptable to test animal and human biological fluids for the production of cytotoxins during pneumonia.

Protokół

All animal procedures were reviewed and approved by the Institutional and Animal Care Committee of the University of South Alabama and were performed in accordance with all federal, state, and local regulations. Primary cultures of rat pulmonary microvascular endothelial cells (PMVECs) were obtained from the Cell Culture Core Facility at the University of South Alabama’s Center for Lung Biology. Cells were prepared using previously described procedures¹⁶.

1. Generation of Cytotoxic Supernatants

Note: Here, we use two different strains of P. aeruginosa: PA103, which has an intact type III secretion system capable of transferring the exoenzymes ExoU and ExoT to target cells during infection, and ∆PcrV, which lacks a type III secretion system and is incapable of transferring exoenzymes to target cells following inoculation.

1. Streak bacteria onto Vogel-Bonner (VB) agar plates¹⁷ and grow overnight at 37 °C.
   1. To prepare plates, make the following stock solution (Vogel-Bonner salts; 10x stock): measure 2 g MgSO₄, 20 g citric acid (free acid), 100 g K₂PO₄, and 35 g NaH₂PO₄. Add ddH₂O to 1 L and autoclave for 15 min with slow exhaust.
   2. Place 7.5 g agar in 450 mL ddH₂O and autoclave as above.
   3. Following autoclaving, place both solutions into a 50 °C water bath to cool.
   4. Add 50 mL of the 10x VB stock salt solution to the agar.
   5. Add carbenicillin to 400 µg/mL (for the strains of P. aeruginosa being used) and mix well by swirling the flask.
   6. Place 5 mL of the VB agar solution into individual sterile culture dishes, allow the agar to solidify, and then store at 4 °C until the day of bacterial seeding.
   7. On the day of bacterial seeding, pre-warm a plate to 37 °C and then streak bacteria onto the plate using a sterile loop. Place the plate in a 37 °C incubator overnight.
2. Verify PMVEC purity by positive staining with fluorescent Griffonia simplicifolia lectin and negative staining with fluorescent Helix pomatia lectin (a marker for arterial endothelial cells). Aliquot cells and freeze in liquid nitrogen.
3. For experiments, maintain cells in Dulbecco’s Modified Eagle’s Medium (DMEM) supplemented with 10% fetal bovine serum, and grow cells in a 37 °C incubator containing 5% CO₂. Use cells at passages 5 - 15.
   1. For generation of cytotoxic supernatants, plate 2 x 10⁶ cells into individual 150 cm dishes and grow for 3 - 4 days until confluence is achieved. Use two plates per experiment (see below).
   2. For analysis of cytotoxic supernatants, plate 1 x 10⁵ cells into individual wells of a 6-well dish and grow for four days until confluence is achieved.
4. To produce supernatant, wash one of the two 150 cm dishes of PMVECs from step 1.3.1 with phosphate-buffered saline, trypsinize, and then count the cells (we use an automated cell counter and follow the manufacturer’s protocol; see Table of Materials). Wash the other dish of PMVECs with Hank’s Balanced Salt Solution (HBSS) and then infect with either strain of P. aeruginosa at a multiplicity of infection (MOI) of 20:1. This is achieved as follows:
   1. For P. aeruginosa, OD₅₄₀ 0.25 = 2 x 10⁸ CFUs/mL. To prepare this dilution, add 1 mL HBSS to a 1 mL disposable cuvette. Scrape enough bacteria off of the plate that was prepared in step 1.1 until an OD₅₄₀ of 0.25 is achieved when measured using a spectrophotometer. The next step will provide a calculation of how much bacteria will be needed.
   2. Once the number of PMVECs in the culture dish is determined (step 1.4 above), determine the appropriate number of bacteria to be added to the second, non-trypsinized culture dish containing the PMVECs. For example, if it is determined that the culture dish of PMVECs contains 1.3 x 10⁷ cells, then prepare 1.3 x 10⁷ cells x 20 bacteria/cell x 1 mL/2 x 10⁸ CFUs = 1.3 mL bacteria.
   3. Dilute the 1.3 mL of bacteria into HBSS to a final volume of 20 mL, so that the entire surface of a 150 cm dish can be covered with fluid, then add the diluted bacteria to the plate of PMVECs.
   4. Incubate the PMVECs inoculated with the bacteria at 37 °C in a 5% CO₂ incubator for 4 - 5 h, until gaps can be seen forming in the cell monolayer when the plate is observed microscopically (See Figure 1 for proper level of gap formation).
   5. Collect the supernatant, then centrifuge for 10 min at 2,000 x g in a tabletop centrifuge to remove cellular debris.
      Note: Any table top centrifuge capable of generating sufficient g force to pellet unlysed cells and large cell fragments can be used for this step.
   6. Pour the supernatant into a syringe that has a 0.2 µm filter attached to its end, then pass the supernatant through the filter to remove bacteria.
   7. Use an aliquot of the sterile supernatant to test cytotoxicity (see 2.1) and freeze the rest of the supernatant at -80 °C for future use.

2. Analysis of Cytotoxic Supernatants

1. Cytotoxicity Assay
   1. Grow PMVECs in 6-well dishes until confluence. Wash wells once with HBSS, then add 1.5 mL of filter-sterilized supernatant (collected from either PA103- or ∆PcrV-inoculated cells) to individual wells. Add sterile HBSS to another well as a negative control.
   2. Place the plate into the CO₂ incubator for 21 - 24 h, then observe for cell killing/cytotoxicity (see next step). Use supernatants that exhibit cytotoxicity for further experimentation, and discard those that do not show cell killing.
   3. Quantitation of cell killing
      1. Measure lactate dehydrogenase (LDH) release from dead cells using commercially available LDH Assay Kits and manufacturer’s recommended procedures.
      2. Alternatively, quantify cell killing microscopically using ImageJ software. For this, record microscopic images and areas of the culture dish containing intact cells, then quantify regions lacking cells (indicative of cell death in a monolayer) using a custom ImageJ (National Institutes of Health) macro (see Supplementary Coding File). If desired, carry out the macro steps manually as follows:
         1. Input images (RGB or Tiff format) into the macro and adjust contrast to 15% saturated pixels. Duplicate contrast-adjusted images and perform the “subtract background” command to obtain a high-contrast image of both cell and gap area within the field of view.
         2. Subtract this image from the original image and combine the resulting image with the original image using the image calculator “AND” function. Convert the resultant image to black (gaps) and white (cells) mask with the threshold function (minimum 0, maximum 5), and use the “binary erode” function to remove noise from the image.
         3. Measure the ratio of black to white pixels within the resultant image using the “area fraction” measurement. Plot and express fractional areas for each treatment time point as percent of maximal gap area.
         4. Calculate means and compare using one-way ANOVA with Tukey’s post hoc test, with p values less than 0.05 considered significant.
   4. Use immunoblot analysis to establish the presence of amyloid, oligomeric tau, and Aβ in culture supernatants. Perform immunoblot analysis using standard procedures^10,11,12, except for concentrating culture supernatants at least 10-fold prior to electrophoresis.
      1. To concentrate supernatant, place 1 - 2 mL of supernatant into a centrifugation filter unit whose membrane has a molecular weight cut-off of 10 kDa. Then, place the filter unit into a table top centrifuge and centrifuge at 2,000 x g until the required degree of concentration is achieved (usually 45 - 60 min).
      2. Determine the amount of protein in the concentrated supernatant¹¹ and load equal amounts of samples into individual wells of the gel.
      3. Run gels, transfer the proteins to nitrocellulose, then probe blots with either A11 anti-amyloid antibody, T22 anti-oligomeric tau antibody, or MOAB2 anti-Aβ antibody followed by appropriate secondary antibodies. Develop the blots using standard chemiluminescence procedures.
   5. To do a thioflavin T Assay, use thioflavin T fluorescence (ThT) to quantify amyloids in supernatants. For these measurements, use filter-sterilized supernatants.
      1. Prepare a stock solution (50x) of thioflavin T by suspending 8 mg thioflavin T (ThT) into 10 mL phosphate buffered saline (PBS). After mixing, filter the solution through a 0.22 µm filter to remove particulates.
      2. For measurements, add 20 µL of ThT stock to 1 mL of PBS in a 1 mL spectrophotometer cuvette. Place the diluted sample into a spectrofluorimeter.
      3. Measure the baseline fluorescence emission using 425 nm excitation and scanning the fluorescence emission from 450-575 nm in 2 nm increments.
      4. Perform a time-lapse scan using 425 nm excitation and 482 nm emission, with data acquired every 0.2 s for 60 s.
      5. The initial 20 s of the time-lapse scan measures fluorescence in the blank cuvette. At 20 s, pause the scan and add 10 µL of the filter-sterilized supernatant to the cuvette. Mix the cuvette by inversion and then place back into the spectrofluorimeter.
      6. Resume the time-based scan, acquiring the final 40 s of data.
      7. Upon completion of the time-lapse scan, perform a final fluorescence emission spectrum scan using identical settings, as detailed in 2.1.5.3.

Wyniki

A simple in vitro assay has been developed to assay for the presence of cytotoxins in supernatants of cells infected with the bacterium P. aeruginosa. Basically, culture medium from infected cells is collected 4 h after bacterial addition, the bacteria are removed by filter sterilization of the culture supernatant, and then the sterile supernatant is added to a new population of cells. The cells are then observed 21 - 24 h after the addition of supernatant and cell killi...

Dyskusje

Here, simple in vitro methods are outlined which allow demonstration of the generation of cytotoxic amyloids during infection with a pneumonia causing organism. These methods include a cell culture cytotoxicity assay, immunoblotting, quantitation of cell killing using a novel microscopic method, and ThT binding. Analyses of the cytotoxic agents have demonstrated that they are amyloid in nature (Figure 2 and Figure 4) and exhibit charact...

Materiały

Name	Company	Catalog Number	Comments
Rabbit anti beta amyloid	Thermo	71-5800
A11 amyloid oligomer antibody	StressMarq	SPC-506D
T22 anti-tau oligomer antibody	EMD Millipore	ABN454
Thioflavin T	Sigma Aldrich	T3516
HBSS	Gibco	14025-092
PBS	Gibco	10010-023
0.22 micron syringe filters	Millipore	SLGP033RS
DMEM	Gibco	11965-092
HRP Goat antirabbit IgG	Abcam	ab6721-1
Strains PA103 and ΔPcrV			These strains of P. aeruginosa were obtained from Dr. Dara Frank, University of Wisconsin Medical College, Milwaukee, WI
FITC Griffonia lectin	Sigma Aldrich	L9381
TRITC Helix pomatia lectin	Sigma Aldrich	L1261
Agar	Fisher	BP1423-500
0.22 micron nitrocellulose	BioRad	162-0112
Type 2 collagenase	Worthington	LS004176
Fetal bovine serum	Hyclone	SH30898.03IH
PenStrep	Gibco	15070063
Carbenicillin Disodium salt	Sigma	C1389
Microcetrifugation concentrators- 10,000 MW cut-off	Millipore	UFC801008
Potassium phosphate dibasic	Sigma	795496-500G
Magnesium phosphate heptahydrate	MP Biomedicals	MP021914221
Citric Acid	G Biosciences	50-103-5801
Sodium phosphate dibasic heptahydrate	Sigma	S9506-500G
Countess Automated Cell Counter	Invitrogen	C10277