Name: Studying Effects of Cigarette Smoke on Pseudomonas Infection in Lung Epithelial Cells (Video) | JoVE Journal
Uploaded: 2020-05-11T14:30:00.000+00:00
Duration: 9 min 15 s
Description: Learn how cigarette smoke extract affects Pseudomonas bacterial load in lung epithelial cells using infection assays and quantitative methods. This protocol enables researchers to study respiratory infection mechanisms.

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5 minutes

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

1. 100% CSE preparation
<ol>
	<li>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.</li>
	<li>Reversely attach an appropriately trimmed 1 mL pipette tip to the nozzle of the syringe as an adapter to hold the cigarette (3R4F).</li>
	<li>Remove the filter of the cigarette. Attach a cigarette to the tip adaptor and combust the cigarette.</li>
	<li>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).</li>
	<li>Repeat step 1.4 about 11x in ~7 min until the cigarette is completely burned out.</li>
	<li>Filter the 10 mL of smoked media with a 0.22 &#181;m 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.</li>
</ol>
2. Pseudomonas culture
<ol>
	<li>Inoculate frozen P. aeruginosa (strain PAO1) or P. fluorescens Migula (strain PAO143) into a Tryptic Soy Broth&#160;(TSB) agar plate for overnight culture at 37 &#176;C. 
	NOTE: To obtain enough bacteria for culturing, spread as much bacteria onto the TSB agar plate as possible.</li>
	<li>Collect a bacterial smear and incubate in 20 mL of TSB with 5% of glycerol as the carbon source.</li>
	<li>Shake the bacterial suspension in a 37 &#176;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 OD600 value to estimate the number of bacteria. An OD600&#160;= 1 corresponds to ~109&#160;colony forming units (CFU)/mL.</li>
</ol>
3. Human lung epithelial cell culture and CSE treatment
<ol>
	<li>Culture human&#160;BEAS-2B cells in HITES medium (500&#8197;mL of DMEM/F12, 2.5&#8197;mg insulin, 2.5&#8197;mg transferrin, 2.5&#8197;mg sodium selenite, 2.5&#8197;mg transferrin, 10&#8197;&#956;M hydrocortisone, 10&#8197;&#956;M &#946;-estradiol, 10&#8197;mM HEPES, and 2&#8197;mM L-glutamine) supplemented with 10% fetal bovine serum (FBS) as previously described24.</li>
	<li>Culture human primary small airway epithelial cells (HSAEC) in the airway epithelial cell culture medium (500 mL of Airway Cell Basal Medium, 500 &#181;g/mL HSA, 0.6 &#181;M linoleic acid, 0.6 &#181;g/mL lecithin, 6 mM L-glutamine, 0.4% extract P, 1.0 &#181;M epinephrine, 5 &#181;g/mL transferrin, 10 nM T3, 1 &#181;g/mL hydrocortisone, rh EGF 5 ng/mL, and 5 &#181;g/mL rh insulin). Incubate the cells at 37&#8239;&#176;C in 5% CO2.</li>
	<li>Dissociate the cells with 1 mL of 0.25% trypsin for 5 min until the cells completely detach from the bottom of the plate.</li>
	<li>Add 10 mL of complete HITES medium to neutralize trypsin and collect the cells in a 15 mL tube. Centrifuge at 4 &#176;C at 300 x g for 5 min. 
	CAUTION: Carefully monitor the time for trypsin digestion by microscopy, because overdigestion may cause cell death.</li>
	<li>Discard the supernatant and resuspend the cells in 2 mL of HITES medium with 10% FBS.</li>
	<li>Pipette 10 &#181;L of the above cell suspension onto the plate and insert it into an automated cell counter to obtain the concentration in cells/mL.</li>
	<li>Plate BEAS-2B cells at a concentration of 3 &#215; 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.</li>
	<li>Treat the cells at approximately 80% confluency, or 5 x 105 cells/mL, with 4% CSE for 3 h. Before CSE treatment, change the medium with HITES medium with 1% of FBS.</li>
</ol>
4. Bacterial infection
<ol>
	<li>Add P. aeruginosa or P. fluorescens Migula&#160;(~1 &#215; 107&#160;CFU/mL) to each well of the CSE-treated cells and incubate for 1 h at 37 &#176;C in 5% CO2.</li>
	<li>Aspirate the supernatants and replace with 2 mL of fresh HITES medium to treat with 4% CSE and 100 &#181;g/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.</li>
	<li>After 1 h of CSE/gentamicin treatment at 37 &#176;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&#160;were observed under fluorescent microscopy.</li>
</ol>
5. Determination of bacterial concentration using the drop plate method
<ol>
	<li>To determine bacterial load in infected cells with the drop plate method, wash the gentamycin-treated cells 2x with 2 mL of cold PBS.</li>
	<li>Add 1 mL of cell lysis buffer (0.5% triton X-100 in PBS) to each well.</li>
	<li>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.</li>
	<li>After 16 h of incubation, obtain the results of CFU by counting the bacterial colonies.</li>
</ol>
6. RT-qPCR detection of bacterial 16S rRNA
<ol>
	<li>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.</li>
	<li>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.</li>
	<li>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&#8211;25 &#176;C. Then, discard the buffer in the collection tube.</li>
	<li>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.</li>
	<li>Place the column into a new 1.5 mL collection tube. Add 30&#8211;50 &#181;L RNase-free water. Centrifuge at 10,000 x g for 1 min. Collect the flow-through and measure the RNA concentration.</li>
	<li>Perform a reverse transcription reaction according to the manufacturer&#39;s protocol. Mix 1 &#181;g of total RNA with 10 mL of reaction buffer, 1 &#181;L of reverse transcriptase, and RNase-free water for a 20 &#181;L reaction. Conduct the reverse transcription reaction at 37 &#176;C for 1 h and then 95 &#176;C for 5 min.</li>
	<li>Mix together the cDNA templates (1 &#181;L of each reverse transcription reaction above), 5 &#181;L of the Master Mix containing SYBR dye, 1 &#181;L of each 200 nM specific primers, and water in a 20 &#181;L mixture for the following PCR analysis, according to the manufacturer&#39;s recommendations. 
	NOTE: The following are the primers targeting the 16S rRNA of P. aeruginosa: forward 5&#8242;-CAAAACTACTGAGCTAGAGTACG-3&#8242;; reverse 5&#8242;-TAAGATCTCAAG GATCCCAACGGC-3&#8242;. GAPDH was used as a loading control with the following primers: forward: 5&#8242;-GGCATGGACTGGTCATGA-3&#8242;; reverse: 5&#8217;-TTCACCATGGAGAAGGC-3&#8242;.</li>
	<li>Use the comparative CT method to determine the expression.</li>
</ol>
7. Detection of fluorescent Pseudomonas with flow cytometry
<ol>
	<li>Treat the above fluorescent Pseudomonas-infected lung epithelial cells (~1 x 106 cells/mL) with gentamycin as previously described. Aspirate the medium and wash the cells 2x with 2 mL of cold PBS.</li>
	<li>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.</li>
</ol>

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</ol>

The authors have nothing to disclose.

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...

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&#160;GFP-labeled Pseudomonas, which has been utilized&#160;as&#160;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.

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

studying effects of cigarette smoke on pseudomonas infection in lung epithelial cells

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

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.

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...

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

Cigarette smoking is the major etiological cause for lung emphysema and chronic obstructive pulmonary disease (COPD). Cigarette smoking also promotes ...

studying-effects-cigarette-smoke-on-pseudomonas-infection-lung

Research

JoVE Journal

Immunology and Infection

Pulmonary epithelial cells can be isolated from the respiratory tract of mice and cultured at air-liquid interface as a model of differentiated respiratory epithelium.  A protocol is described for isolating, culturing and exposing these cells to mainstream cigarette smoke, in order to study molecular responses to this environmental toxin.

Isolation of Mouse Respiratory Epithelial Cells and Exposure to Experimental Cigarette Smoke at Air Liquid Interface  

Source: Li, T., et al. Studying Effects of Cigarette Smoke on Pseudomonas Infection in Lung Epithelial Cells. J. Vis. Exp. (2020).This video demonstrates how cigarette smoke extract weakens antimicrobial defense in lung epithelial cells by reducing AMP production, leading to increased bacterial invasion. The procedure measures intracellular bacterial load to assess the impact of smoke exposure on host-pathogen interactions.

Analyzing the Effects of Cigarette Smoke on Antimicrobial Defense in Lung Epithelial Cells

We describe the agar-beads method to establish persistent long-term chronic Pseudomonas aeruginosa airway infection in the mouse model.

Long Term Chronic Pseudomonas aeruginosa Airway Infection in Mice

The goal of this protocol is to provide automated methods to quantify chronic lung pathologies in a murine model of COPD. The protocol includes exposing mice to cigarette smoke (CS), measuring pulmonary function, inflating the lungs, and using morphometry methods to measure emphysema and small airway remodeling in mice.

Automated Measurement of Pulmonary Emphysema and Small Airway Remodeling in Cigarette Smoke-exposed Mice

The aim of this protocol is to expose human organotypic 3D bronchial and nasal tissue models to mainstream cigarette smoke (CS) at the air-liquid interface. The impact of CS on the tissues is then investigated using a cytochrome P450 activity assay, a cilia beating measurement, and a systems biology approach.

Impact Assessment of Repeated Exposure of Organotypic 3D Bronchial and Nasal Tissue Culture Models to Whole Cigarette Smoke

Using optimized human peripheral blood mononuclear cell (PBMC) ex vivo assays, we showed that a combustible tobacco product preparation markedly suppresses receptor-mediated intracellularly secreted cytokines and cytolytic ability of effector PBMCs. These rapid assays may be useful in product evaluation and understanding the potential long-term effects of tobacco exposure.

Methods to Evaluate Cytotoxicity and Immunosuppression of Combustible Tobacco Product Preparations

While in vitro study of host-pathogen interactions allow the characterization of specific immune responses, in vivo models are required to observe the effects of complex responses. Using Candida albicans exposure followed by Pseudomonas aeruginosa-mediated lung infection, we established a murine model of microbial interactions involved in ventilator-associated pneumonia pathogenicity.

Studying Microbial Communities In Vivo: A Model of Host-mediated Interaction Between Candida Albicans and Pseudomonas Aeruginosa in the Airways

Presented here is a useful protocol for lung fixation that creates a stable condition for histological evaluate of lung specimens from a mouse model of emphysema. The main advantage of this model is that it can fix many lungs with the same constant pressure without lung collapse or deflation.

Lung Fixation under Constant Pressure for Evaluation of Emphysema in Mice

This protocol demonstrates the study of the pathophysiologic effects of cigarette smoke (CS) with a whole-body inhalation (WBI) exposure system (WBIS) built in-house. This system can expose animals to CS under controlled repeatable conditions for research of CS-mediated effects on lung emphysema and hematopoiesis.

Cigarette Smoke Exposure in Mice using a Whole-Body Inhalation System

Presented here is a reproducible, affordable, and robust method for the isolation and expansion of primary bronchial epithelial cells for long-term biobanking and the generation of differentiated epithelial cells by culture at the air-liquid interface.

Isolating Bronchial Epithelial Cells from Resected Lung Tissue for Biobanking and Establishing Well-Differentiated Air-Liquid Interface Cultures

Here, we describe an in vitro model for isolating and differentiating murine airway epithelial cells, focusing on their acclimation to chronic cigarette smoke extract (CSE). The model could be utilized to comprehensively characterize the multi-omics impact of CSE, which possibly provides insights into the cellular responses under chronic smoke exposure.

In Vitro Model for Studying Differentiation and Changes of Multi-Omics on Murine Airway Epithelial Cells Stimulated with Cigarette Smoke Extract

Learn how cigarette smoke extract affects Pseudomonas bacterial load in lung epithelial cells using infection assays and quantitative methods. This protocol enables researchers to study respiratory infection mechanisms.

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

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