Studying Effects of Cigarette Smoke on <em>Pseudomonas</em> Infection in Lung Epithelial Cells

Tiao Li; Chen Long; Kristen  V. Fanning; Chunbin Zou

doi:10.3791/61163

JoVE Journal > Immunology and Infection

免疫学と感染

Studying Effects of Cigarette Smoke on Pseudomonas Infection in Lung Epithelial Cells

Published: May 11, 2020

doi:

10.3791/61163

Tiao Li¹, Chen Long¹, Kristen V. Fanning¹, Chunbin Zou¹

¹Acute Lung Injury Center of Excellence, Pulmonary, Allergy, Critical Care Medicine, Department of Medicine,University of Pittsburgh School of Medicine

概要

Described here is a protocol to study how cigarette smoke extract affects bacterial colonization in lung epithelial cells.

Abstract

Cigarette smoking is the major etiological cause for lung emphysema and chronic obstructive pulmonary disease (COPD). Cigarette smoking also promotes susceptibility to bacterial infections in the respiratory system. However, the effects of cigarette smoking on bacterial infections in human lung epithelial cells have yet to be thoroughly studied. Described here is a detailed protocol for the preparation of cigarette smoking extracts (CSE), treatment of human lung epithelial cells with CSE, and bacterial infection and infection determination. CSE was prepared with a conventional method. Lung epithelial cells were treated with 4% CSE for 3 h. CSE-treated cells were, then, infected with Pseudomonas at a multiplicity of infection (MOI) of 10. Bacterial loads of the cells were determined by three different methods. The results showed that CSE increased Pseudomonas load in lung epithelial cells. This protocol, therefore, provides a simple and reproducible approach to study the effect of cigarette smoke on bacterial infections in lung epithelial cells.

Introduction

Cigarette smoking affects the public health of millions of people worldwide. Many deleterious diseases, including lung cancer and chronic obstructive pulmonary disease (COPD), are reported to be related to cigarette smoking¹^,². Cigarette smoking increases susceptibility to acute microbial infections in the respiratory system³^,⁴^,⁵. Furthermore, mounting evidence proves that cigarette smoking enhances the pathogenesis of many chronic disorders⁶^,⁷^,⁸. For instance, cigarette smoking may increase viral or bacterial infections that cause COPD exacerbation⁹. Among the bacterial pathogens that etiologically contribute to acute exacerbation of COPD, an opportunistic gram-negative bacillus pathogen, Pseudomonas aeruginosa, causes infections that correlate with poor prognoses and higher mortalities¹⁰^,¹¹. COPD exacerbation worsens the disease by accelerating pathological progression. There are no effective therapies against COPD exacerbation except for the antisymptomatic management¹². COPD exacerbation promotes patient mortality, decreases quality of life, and increases economic burden on society¹³.

The respiratory airway is an open system, continuously subjected to various microbial pathogens present externally. Opportunistic bacterial pathogens are usually detected in the upper airways but sometimes are observed in the lower airways¹⁴^,¹⁵. In animal models P. aeruginosa can be detected in alveolar sacs as soon as 1 h after infection¹⁶. As a major defense mechanism, immune cells such as macrophages or neutrophils eliminate the bacteria in the airways. Lung epithelial cells, as the first physiological barrier, perform a unique role in the host defense against microbial infections. Lung epithelial cells may regulate microbial invasion, colonization, or replication independent of immune cells¹⁷. Some molecules found in epithelial cells, including PPARg, exert antibacterial functions, thereby regulating bacterial colonization and replication in lung epithelial cells¹⁸. Cigarette smoking may alter the molecules and impair normal defense function in lung epithelial cells¹⁹^,²⁰. Recent studies reported direct exposure of cigarette smoke to lung epithelial cells using robot smoking apparatus²¹^,²². Exposure to smoke can be performed in other ways, however, including application of CSE. Preparation of CSE is a reproducible approach with potential applications in other cell types, including vascular endothelial cells that are indirectly exposed to cigarette smoke.

This report describes a protocol to generate cigarette smoke extract to alter bacterial load in lung epithelial cells. CSE increases the bacterial load of P. aeruginosa, and it may contribute to the recurrence of bacterial infections usually seen in COPD exacerbation. A conventional method is used for the preparation of CSE. Lung epithelial cells, at their exponential growth stage, are treated with 4% CSE for 3 h. Alternatively, monolayer-cultured lung epithelial cells can be directly exposed to cigarette smoke in an air-liquid interface. CSE-treated cells are then challenged with Pseudomonas at a multiplicity of infection (MOI) of 10. The bacteria are propagated at a particular shaking speed to ensure the morphology of their flagella remains intact to retain their full invasive capacity. Gentamycin is employed to kill the bacteria left in the culture medium, thereby reducing the potential contamination during the subsequent determination of the bacterial load. The protocol also uses GFP-labeled Pseudomonas, which has been utilized as a powerful tool in studying Pseudomonas infection in different models. A representative strain is P. fluorescens Migula²³. The degree of infection or bacterial load after CSE treatment is determined in three ways: the drop plate method with colony counting, quantitative PCR using Pseudomonas 16S rRNA-specific primers, or flow cytometry in cells infected with fluorescent Pseudomonas. This protocol is a simple and reproducible approach to study the effect of cigarette smoke on bacterial infections in lung epithelial cells.

Protocol

1. 100% CSE preparation

Draw 10 mL of serum-free cell culture media (DMEM/F12 for BEAS-2B cells; airway epithelial cell basal medium for HSAEC cells) into a 60 mL syringe.
Reversely attach an appropriately trimmed 1 mL pipette tip to the nozzle of the syringe as an adapter to hold the cigarette (3R4F).
Remove the filter of the cigarette. Attach a cigarette to the tip adaptor and combust the cigarette.
Draw 40 mL of smoke-containing air into 10 mL of serum-free media. Mix the smoke with the medium by vigorously shaking (30 s per draw).
Repeat step 1.4 about 11x in ~7 min until the cigarette is completely burned out.
Filter the 10 mL of smoked media with a 0.22 µm filter to exclude any microorganisms and insoluble particles. Transfer to a closed sterile tube. Prepare the 100% CSE no more than 30 min before the subsequent assay.

2. Pseudomonas culture

Inoculate frozen P. aeruginosa (strain PAO1) or P. fluorescens Migula (strain PAO143) into a Tryptic Soy Broth (TSB) agar plate for overnight culture at 37 °C.
NOTE: To obtain enough bacteria for culturing, spread as much bacteria onto the TSB agar plate as possible.
Collect a bacterial smear and incubate in 20 mL of TSB with 5% of glycerol as the carbon source.
Shake the bacterial suspension in a 37 °C incubator at 200 rpm for 1 h until the OD₆₀₀ value = 0.6.
CAUTION: Do not let the shaking speed exceed 200 rpm. Higher shaking speeds may damage the morphology of the bacterial flagella and impact the bacterial invasion into lung epithelia. Likewise, limit the shaking time to 1 h to obtain highly invasive bacteria. Measure the OD₆₀₀value to estimate the number of bacteria. An OD₆₀₀ = 1 corresponds to ~10⁹ colony forming units (CFU)/mL.

3. Human lung epithelial cell culture and CSE treatment

Culture human BEAS-2B cells in HITES medium (500 mL of DMEM/F12, 2.5 mg insulin, 2.5 mg transferrin, 2.5 mg sodium selenite, 2.5 mg transferrin, 10 μM hydrocortisone, 10 μM β-estradiol, 10 mM HEPES, and 2 mM L-glutamine) supplemented with 10% fetal bovine serum (FBS) as previously described²⁴.
Culture human primary small airway epithelial cells (HSAEC) in the airway epithelial cell culture medium (500 mL of Airway Cell Basal Medium, 500 µg/mL HSA, 0.6 µM linoleic acid, 0.6 µg/mL lecithin, 6 mM L-glutamine, 0.4% extract P, 1.0 µM epinephrine, 5 µg/mL transferrin, 10 nM T3, 1 µg/mL hydrocortisone, rh EGF 5 ng/mL, and 5 µg/mL rh insulin). Incubate the cells at 37 °C in 5% CO₂.
Dissociate the cells with 1 mL of 0.25% trypsin for 5 min until the cells completely detach from the bottom of the plate.
Add 10 mL of complete HITES medium to neutralize trypsin and collect the cells in a 15 mL tube. Centrifuge at 4 °C at 300 x g for 5 min.
CAUTION: Carefully monitor the time for trypsin digestion by microscopy, because overdigestion may cause cell death.
Discard the supernatant and resuspend the cells in 2 mL of HITES medium with 10% FBS.
Pipette 10 µL of the above cell suspension onto the plate and insert it into an automated cell counter to obtain the concentration in cells/mL.
Plate BEAS-2B cells at a concentration of 3 × 10⁵ cells/mL into 6 well plates in a total volume of 2 mL in HITES medium supplemented with 10% of FBS for overnight culture.
Treat the cells at approximately 80% confluency, or 5 x 10⁵cells/mL, with 4% CSE for 3 h. Before CSE treatment, change the medium with HITES medium with 1% of FBS.

4. Bacterial infection

Add P. aeruginosa or P. fluorescens Migula (~1 × 10⁷ CFU/mL) to each well of the CSE-treated cells and incubate for 1 h at 37 °C in 5% CO₂.
Aspirate the supernatants and replace with 2 mL of fresh HITES medium to treat with 4% CSE and 100 µg/mL gentamicin.
NOTE: Gentamicin is used because it is unable to penetrate human lung epithelial cellular membranes. Thus, it can kill all the bacteria in the medium but not those that invaded the lung epithelial cells.
After 1 h of CSE/gentamicin treatment at 37 °C in 5% CO₂, aspirate the supernatants and wash the cells 3x with PBS for the subsequent bacterial concentration determination.
NOTE: To confirm the internalized bacteria, cells infected with GFP-labeled P. fluorescens Migula were observed under fluorescent microscopy.

5. Determination of bacterial concentration using the drop plate method

To determine bacterial load in infected cells with the drop plate method, wash the gentamycin-treated cells 2x with 2 mL of cold PBS.
Add 1 mL of cell lysis buffer (0.5% triton X-100 in PBS) to each well.
Dilute the cell lysates containing the internalized bacteria in a gradient (1:10, 1:100, 1:1,000, and 1:10,000) for the following inoculation to the TSB agar plate.
After 16 h of incubation, obtain the results of CFU by counting the bacterial colonies.

6. RT-qPCR detection of bacterial 16S rRNA

Treat the Pseudomonas-infected lung epithelial cells (~1 x 10⁶ cells/mL) with gentamycin as described above. Aspirate the medium and wash the cells 2x with 2 mL of cold PBS.
Add 0.35 mL of the guanidium thiocyanate lysis buffer per well of a 6 well plate. Collect the cells with a cell scraper. Pipette the lysate into a microcentrifuge tube and mix gently with the pipette.
Add the same volume (0.35 mL) of freshly prepared 70% ethanol into the lysate and mix well. Transfer all samples to a spin column placed in a 2 mL collection tube. Centrifuge at 10,000 x g for 30 s at 20–25 °C. Then, discard the buffer in the collection tube.
Wash the column with 0.7 mL of wash buffer 1. Centrifuge the column at 10,000 x g for 30 s. Wash the column 2x with 0.5 mL of buffer to wash the membrane-bound RNA. Repeat the centrifugation at 10,000 x g for 2 min.
Place the column into a new 1.5 mL collection tube. Add 30–50 µL RNase-free water. Centrifuge at 10,000 x g for 1 min. Collect the flow-through and measure the RNA concentration.
Perform a reverse transcription reaction according to the manufacturer's protocol. Mix 1 µg of total RNA with 10 mL of reaction buffer, 1 µL of reverse transcriptase, and RNase-free water for a 20 µL reaction. Conduct the reverse transcription reaction at 37 °C for 1 h and then 95 °C for 5 min.
Mix together the cDNA templates (1 µL of each reverse transcription reaction above), 5 µL of the Master Mix containing SYBR dye, 1 µL of each 200 nM specific primers, and water in a 20 µL mixture for the following PCR analysis, according to the manufacturer's recommendations.
NOTE: The following are the primers targeting the 16S rRNA of P. aeruginosa: forward 5′-CAAAACTACTGAGCTAGAGTACG-3′; reverse 5′-TAAGATCTCAAG GATCCCAACGGC-3′. GAPDH was used as a loading control with the following primers: forward: 5′-GGCATGGACTGGTCATGA-3′; reverse: 5’-TTCACCATGGAGAAGGC-3′.
Use the comparative CT method to determine the expression.

7. Detection of fluorescent Pseudomonas with flow cytometry

Treat the above fluorescent Pseudomonas-infected lung epithelial cells (~1 x 10⁶cells/mL) with gentamycin as previously described. Aspirate the medium and wash the cells 2x with 2 mL of cold PBS.
Analyze the samples with a flow cytometer at a wavelength of 509 nm for the detection of GFP. Terminate each read at 100,000 counts. Analyze the acquired data with related software.

Representative Results

A diagram is used to illustrate the protocol in Figure 1. Lung epithelial BEAS-2B cells were treated with CSE and challenged with Pseudomonas. Pseudomonas in the culture medium were killed by the added gentamycin and the cells were subjected to the drop plate assay, RT-qPCR detection of Pseudomonas ribosome 16S RNA, and flow cytometry. Compared with control, CSE treatment substantially increased bacterial infection in drop plate methods (Figure 2). Correspondingly, CSE affected bacterial load in HSAEC (Figure 3). Cell viability did not change considerably after 3 h of 4% CSE treatment, in 1 h of Pseudomonas infection, or in 1 h of gentamycin treatment (Figure 4 Figure 5 Figure 6). The 16S rRNA-target RT-qPCR method (Figure 7) and flow cytometry (Figure 8) demonstrated similar results. Results from fluorescent microscopy showed that GFP-labeled bacteria colocalized with BEAS-2B cells in a P. fluorescens Migula infection experiment (Figure 9). These results suggest that cigarette smoking increased Pseudomonas load in BEAS-2B cells.

Figure 1: Schematic presentation of the protocol to study cigarette smoke effects on Pseudomonas infection in lung epithelial cells. Lung epithelial cells grown in cell culture inserts or conventional culture plates or lung organoids were exposed to cigarette smoke for 16 min via smoking robot or treated with prepared 4% CSE for 3 h. These cells were then infected with P. aeruginosa for 1 h (MOI = 10). Gentamycin was used to eliminate live Pseudomonas in the culture medium. The above cells were subject to the drop plate method, qRT-PCR, or flow cytometry approaches to determine the bacterial load. Please click here to view a larger version of this figure.

Figure 2: Drop plate method to determine bacterial load in lung epithelial BEAS-2B cells. BEAS-2B cells were treated with 4% CSE for 3 h. Cells were then subjected to P. aeruginosa (strain PAO1) infection for 1 h followed by gentamycin treatment for another 1 h. Cells were lysed and the cell lysates were diluted to inoculate TSB plates for 16 h. Colonies were counted; the CFU numbers are illustrated in the plot. Graph shows mean ± SD, and “*” denotes P < 0.05. Results are representative of n = 3 experiments. Two-way unpaired Student t-test was used for smoke-treated and untreated groups. P < 0.05 indicates statistical significance. Please click here to view a larger version of this figure.

Figure 3: Drop plate method to determine bacterial load in HSAEC cells. Human primary small airway epithelial cells were treated with 4% CSE for 3 h. Cells were then subjected to P. aeruginosa (strain PAO1) infection for 1 h followed by gentamycin treatment for another 1 h. The cells were lysed and the cell lysates were diluted to inoculate on TSB plates for 16 h. Colonies were counted; the CFU numbers are illustrated in the plot. Graph shows mean ± SD, and “*” denotes P < 0.05. Results are representative of n = 3 experiments. Two-way unpaired Student t-test was used for smoke-treated and untreated groups. P < 0.05 indicates statistical significance. Please click here to view a larger version of this figure.

Figure 4: Determination of cell viability in CSE-treated lung epithelial BEAS-2B cells. Lung epithelial BEAS-2B cells were treated with 4% CSE for 3 h. Cells were stained with trypan blue and cell viability was measured with cell counter. Graph shows mean ± SD. Results are representative of n = 3 experiments. Please click here to view a larger version of this figure.

Figure 5: Determination of cell viability in Pseudomonas-infected lung epithelial BEAS-2B cells. Lung epithelial BEAS-2B cells were infected with P. aeruginosa (MOI = 10) for 1 h. Cells were stained with trypan blue and cell viability was measured with a cell counter. Graph shows mean ± SD. Results are representative of n = 3 experiments. Please click here to view a larger version of this figure.

Figure 6: Determination of cell viability in gentamycin-treated lung epithelial BEAS-2B cells. Lung epithelial BEAS-2B cells were treated with 100 µg/mL gentamycin for 1 h. Cells were stained with trypan blue and cell viability was measured with a cell counter. Graph shows mean ± SD. Results are representative of n = 3 experiments. Please click here to view a larger version of this figure.

Figure 7: qRT-PCR to determine bacterial load in lung epithelial cells. Treated cells in Figure 2 were subjected to total RNA extraction. An equivalent amount of RNA from each sample was reverse transcribed into cDNA, and the amount of 16S RNA of P. aeruginosa was determined with quantitative PCR using specific primer pairs. Results from qPCR are plotted in the graph. Graph shows mean ± SD, and “*” denotes P < 0.05. Results are representative of n = 3 experiments. Two-way unpaired Student t-test was used for smoke-treated and untreated groups. P < 0.05 indicates statistical significance. Please click here to view a larger version of this figure.

Figure 8: Flow cytometry to determine bacterial load in lung epithelial cells. BEAS-2B cells were treated with CSE and infected with P. fluorescens Migula (strain PAO143). Infected cells were treated with gentamycin and digested with trypsin to make a cell suspension. Cell suspensions were passed through flow cytometer and fluorescent Pseudomonas-positive cells were determined at a wavelength of 509 nm. Results from flow cytometry are plotted in the graph. Graph shows mean ± SD, and “*” denotes P < 0.05. Results are representative of n = 3 experiments. Two-way unpaired Student t-test was used for smoke treated and untreated groups. P < 0.05 indicates statistical significance. Please click here to view a larger version of this figure.

Figure 9: Observation of bacterial infection with fluorescent microscopy. BEAS-2B cells were infected with P. fluorescens Migula (strain PAO143) for 1 h. The cells were treated with gentamycin for another 1 h and washed 2x with cold PBS. The GFP-labeled bacteria were observed under a fluorescent microscope at a wavelength of 480 nm and BEAS-2B cells were visualized with a phase image. The images were merged, and the representative result is shown. Scale bar = 10 µm. Please click here to view a larger version of this figure.

Discussion

Bacterial invasion into lung epithelial cells is a crucial step in the pathogenesis of bacterial infections. The process of bacterial invasion into the cells can be broken down into the following three steps: First, the bacteria contact and adhere to the surface of the epithelial cell using their flagella. Second, the bacteria either undergo internalization or penetrate the cellular membrane. Finally, the bacteria replicate and colonize the cells if they successfully escape cellular defense mechanisms²⁵^,²⁶. Approaches for the observation of bacterial infections in lung microphages have long been developed but with limited knowledge of lung epithelial cells²⁷^,²⁸. This study determined bacterial load in the lung epithelial cells via three approaches: a drop plate assay, RT-qPCR for Pseudomonas ribosome 16S RNA, and flow cytometry. All three approaches work well with similar sensitivities. Choosing the approaches to determine bacterial load in lung epithelial cells depends on the availability of equipment and time. In these experiments, keeping the bacterial flagella undamaged and intact is crucial for successful lung epithelial cell infection²⁹. A shorter shaking time with limited speed may help to further promote bacterial invading capacity³⁰.

A major obstacle in determination of the bacterial concentration in lung epithelial cells is the bacterial contamination from the culture medium. In cell infection experiments, the bacteria are added into the culture medium for a specific time period (i.e., 1 h). Within that time, part of that bacterial load successfully invades the cytoplasmic compartment, but residues may attach to the outer membrane of the cells. An antibiotic, gentamycin, is used to kill the bacteria in the culture medium and the residues that attached to the outer membrane of the cells³¹. Gentamycin is considered not permeable to the cellular membrane and thus will not affect the bacteria already within the cells. Treatment with gentamycin makes it ideal for this system to exclude the potential contamination from outside the infected lung epithelial cells.

To study the effects of cigarette smoke on bacterial infection in lung epithelial cells, lung cells must be exposed to cigarette smoke prior to bacterial infection in cellular models. A conventional approach is preparing fresh CSE to treat cells. Generation of CSE is a cost-effective method for studies. CSE is easy to handle, the process of making CSE is simple, and CSE intratracheal injection is effective in the generation of emphysema in animal models in a short time period³². Approaches of direct cigarette exposure have also been developed for both in vitro cellular models and in vivo rodent models. Six months of daily cigarette smoke exposure to mice is widely used to generate emphysema³³. Direct cigarette smoke exposure requires complicated equipment that combines cigarette combusting and cell culture systems. Cigarette combusting produces cigarette smoke, but also generates a sum amount of heat that may affect the culture chamber’s temperature and humidity. Fortunately, recent techniques make direct cigarette smoke exposure easier²¹. An International Organization for Standardization (ISO) protocol has been implemented for direct cigarette smoke exposure experiments²². Exposure to the cigarette smoke can be performed once or multiple times. In addition, along with the progress of cell culture techniques, lung primary epithelial cells could also be grown on transparent inserts to obtain a confluent lung epithelial cell monolayer³⁴. Lung epithelial cell monolayers structurally mimic the physiological conditions in lung tissues. Cells can then be exposed to apical gaseous smoke or air at the air-liquid interface³⁵. Furthermore, culture of lung organoids has emerged in current lung studies³⁶^,³⁷. It will be interesting to know how cigarette smoke affects bacterial infection in lung organoids. The approach described may mimic human infection in tissues instead of cultured cells and may make possible further insights in lower respiratory bacterial infection.

開示

The authors have nothing to disclose.

Acknowledgements

This work was supported in part by a National Institutes of Health R01 grants HL125435 and HL142997 (to CZ).

Materials

50mL syringe	BD Biosciences
airway epithelial cell basal medium	ATCC	PCS-300-030
Bacteria shaker	ThermoFisher Scientific
bronchial epithelial cell growth kit	ATCC	PCS-300-040
Cell Counter	Bio-Rad
CFX96 Real-Time PCR System	Bio-Rad
High-Capacity RNA-to-DNA KIT	ThermoFisher Scientific	4387406
HITES medium	ATCC	ATCC 30-2004
human BEAS-2B cells	ATCC	ATCC CRL-9609
human primary small airway epithelial cells	ATCC	ATCC PCS-300-030
LSRII flow cytometer	BD Biosciences
Nikkon confocal microscope	Nikkon
OD reader	USA Scientific
PCR primers	ITD
Pseudomonas aeruginosa	ATCC	ATCC 47085	PAO1-LAC
Pseudomonas fluorescens Migula	ATCC	ATCC 27853	P.aeruginosa GFP
Research-grade cigarettes (3R4F)	University of Kentucky	TP-7-VA
RNeasy Mini Kit	Qiagen	74106
Transprent PET Transwell Insert	Corning Costar
Tryptic Soy Broth	BD Biosciences

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Li, T., Long, C., Fanning, K. V., Zou, C. Studying Effects of Cigarette Smoke on Pseudomonas Infection in Lung Epithelial Cells. J. Vis. Exp. (159), e61163, doi:10.3791/61163 (2020).

Studying Effects of Cigarette Smoke on Pseudomonas Infection in Lung Epithelial Cells