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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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 mechanisms25,26. 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&#8217;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.

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

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 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.
<img alt="Figure 1" class="xfigimg" src="/files/ftp_upload/61163/61163fig1.jpg" /> 
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. <a href="https://www-jove-com.bdigitaluss.remotexs.co/files/ftp_upload/61163/61163fig1large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 2" class="xfigimg" src="/files/ftp_upload/61163/61163fig2.jpg" /> 
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 &#177; SD, and &#8220;*&#8221; denotes P &#60; 0.05. Results are representative of n = 3 experiments. Two-way unpaired Student t-test was used for smoke-treated and untreated groups. P&#8239;&#60;&#8239;0.05 indicates statistical significance. <a href="https://www-jove-com.bdigitaluss.remotexs.co/files/ftp_upload/61163/61163fig2large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 3" class="xfigimg" src="/files/ftp_upload/61163/61163fig3.jpg" /> 
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 &#177; SD, and &#8220;*&#8221; denotes P &#60; 0.05. Results are representative of n = 3 experiments. Two-way unpaired Student t-test was used for smoke-treated and untreated groups. P&#8239;&#60;&#8239;0.05 indicates statistical significance. <a href="https://www-jove-com.bdigitaluss.remotexs.co/files/ftp_upload/61163/61163fig3large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 4" class="xfigimg" src="/files/ftp_upload/61163/61163fig4.jpg" /> 
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 &#177; SD. Results are representative of n = 3 experiments. <a href="https://www-jove-com.bdigitaluss.remotexs.co/files/ftp_upload/61163/61163fig4large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 5" class="xfigimg" src="/files/ftp_upload/61163/61163fig5.jpg" /> 
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 &#177; SD. Results are representative of n = 3 experiments. <a href="https://www-jove-com.bdigitaluss.remotexs.co/files/ftp_upload/61163/61163fig5large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 6" class="xfigimg" src="/files/ftp_upload/61163/61163fig6.jpg" /> 
Figure 6: Determination of cell viability in gentamycin-treated lung epithelial BEAS-2B cells. Lung epithelial BEAS-2B cells were treated with 100 &#181;g/mL gentamycin for 1 h. Cells were stained with trypan blue and cell viability was measured with a cell counter. Graph shows mean &#177; SD. Results are representative of n = 3 experiments. <a href="https://www-jove-com.bdigitaluss.remotexs.co/files/ftp_upload/61163/61163fig6large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 7" class="xfigimg" src="/files/ftp_upload/61163/61163fig7.jpg" /> 
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 &#177; SD, and &#8220;*&#8221; denotes P &#60; 0.05. Results are representative of n = 3 experiments. Two-way unpaired Student t-test was used for smoke-treated and untreated groups. P&#8239;&#60;&#8239;0.05 indicates statistical significance. <a href="https://www-jove-com.bdigitaluss.remotexs.co/files/ftp_upload/61163/61163fig7large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 8" class="xfigimg" src="/files/ftp_upload/61163/61163fig8.jpg" /> 
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 &#177; SD, and &#8220;*&#8221; denotes P&#8239;&#60;&#8239;0.05. Results are representative of n = 3 experiments. Two-way unpaired Student t-test was used for smoke treated and untreated groups. P&#8239;&#60;&#8239;0.05 indicates statistical significance. <a href="https://www-jove-com.bdigitaluss.remotexs.co/files/ftp_upload/61163/61163fig8large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 9" class="xfigimg" src="/files/ftp_upload/61163/61163fig9.jpg" /> 
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 &#181;m. <a href="https://www-jove-com.bdigitaluss.remotexs.co/files/ftp_upload/61163/61163fig9large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>

Watch this Scientific Journal Video about Studying Effects of Cigarette Smoke on Pseudomonas Infection in Lung Epithelial Cells at JoVE.com

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

Described here is a protocol to study how cigarette smoke extract affects bacterial colonization 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 ...

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

Universidad San Sebastian

Research

JoVE Journal

Immunology and Infection

5.8K Views.  University of Pittsburgh School of Medicine. Described here is a protocol to study how cigarette smoke extract affects bacterial colonization in lung epithelial cells.

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

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

Pulmonary epithelial cells can be isolated from the respiratory tract of mice and cultured at air-liquid interface (ALI) as a model of differentiated respiratory epithelium. A protocol is described for isolating and exposing these cells to mainstream cigarette smoke (CS), in order to study epithelial cell responses to CS exposure. The protocol consists of three parts: the isolation of airway epithelial cells from mouse trachea, the culturing of these cells at air-liquid interface (ALI) as fully differentiated epithelial cells, and the delivery of calibrated mainstream CS to these cells in culture. The ALI culture system allows the culture of respiratory epithelia under conditions that more closely resemble their physiological setting than ordinary liquid culture systems. The study of molecular and lung cellular responses to CS exposure is a critical component of understanding the impact of environmental air pollution on human health. Research findings in this area may ultimately contribute towards understanding the etiology of chronic obstructive pulmonary disease (COPD), and other tobacco-related diseases, which represent major global health problems.

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

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.

Pulmonary epithelial cells can be isolated from the respiratory tract of mice and cultured at air-liquid interface (ALI) as a model of differentiated ...

Medicine

Use of Artificial Sputum Medium to Test Antibiotic Efficacy Against Pseudomonas aeruginosa in Conditions More Relevant to the Cystic Fibrosis Lung

There is growing concern about the relevance of in vitro antimicrobial susceptibility tests when applied to isolates of P. aeruginosa from cystic fibrosis (CF) patients. Existing methods rely on single or a few isolates grown aerobically and planktonically. Predetermined cut-offs are used to define whether the bacteria are sensitive or resistant to any given antibiotic1. However, during chronic lung infections in CF, P. aeruginosa populations exist in biofilms and there is evidence that the environment is largely microaerophilic2. The stark difference in conditions between bacteria in the lung and those during diagnostic testing has called into question the reliability and even relevance of these tests3.
 Artificial sputum medium (ASM) is a culture medium containing the components of CF patient sputum, including amino acids, mucin and free DNA. P. aeruginosa growth in ASM mimics growth during CF infections, with the formation of self-aggregating biofilm structures and population divergence4,5,6. The aim of this study was to develop a microtitre-plate assay to study antimicrobial susceptibility of P. aeruginosa based on growth in ASM, which is applicable to both microaerophilic and aerobic conditions.
 An ASM assay was developed in a microtitre plate format. P. aeruginosa biofilms were allowed to develop for 3 days prior to incubation with antimicrobial agents at different concentrations for 24 hours. After biofilm disruption, cell viability was measured by staining with resazurin. This assay was used to ascertain the sessile cell minimum inhibitory concentration (SMIC) of tobramycin for 15 different P. aeruginosa isolates under aerobic and microaerophilic conditions and SMIC values were compared to those obtained with standard broth growth. Whilst there was some evidence for increased MIC values for isolates grown in ASM when compared to their planktonic counterparts, the biggest differences were found with bacteria tested in microaerophilic conditions, which showed a much increased resistance up to a &gt;128 fold, towards tobramycin in the ASM system when compared to assays carried out in aerobic conditions.
 The lack of association between current susceptibility testing methods and clinical outcome has questioned the validity of current methods3. Several in vitro models have been used previously to study P. aeruginosa biofilms7, 8. However, these methods rely on surface attached biofilms, whereas the ASM biofilms resemble those observed in the CF lung9 . In addition, reduced oxygen concentration in the mucus has been shown to alter the behavior of P. aeruginosa2 and affect antibiotic susceptibility10. Therefore using ASM under microaerophilic conditions may provide a more realistic environment in which to study antimicrobial susceptibility.

Use of Artificial Sputum Medium to Test Antibiotic Efficacy Against Pseudomonas aeruginosa in Conditions More Relevant to the Cystic Fibrosis Lung

Current diagnostic antimicrobial susceptibility testing relies on the planktonic growth of isolates in nutrient rich, aerobic conditions. Here, we employ an alternative artificial sputum medium to study antimicrobial susceptibility of Pseudomonas aeruginosa biofilms under both aerobic and microaerophilic conditions more representative of the cystic fibrosis lung.

There  is growing concern about the relevance of in vitro antimicrobial  susceptibility tests when applied to isolates of P. aeruginosa from  cystic ...

Long Term Chronic Pseudomonas aeruginosa Airway Infection in Mice

A mouse model of chronic airway infection is a key asset in cystic fibrosis (CF) research, although there are a number of concerns regarding the model itself. Early phases of inflammation and infection have been widely studied by using the Pseudomonas aeruginosa agar-beads mouse model, while only few reports have focused on the long-term chronic infection in vivo. The main challenge for long term chronic infection remains the low bacterial burden by P. aeruginosa and the low percentage of infected mice weeks after challenge, indicating that bacterial cells are progressively cleared by the host.
This paper presents a method for obtaining efficient long-term chronic infection in mice. This method is based on the embedding of the P. aeruginosa clinical strains in the agar-beads in vitro, followed by intratracheal instillation in C57Bl/6NCrl mice. Bilateral lung infection is associated with several measurable read-outs including weight loss, mortality, chronic infection, and inflammatory response. The P. aeruginosa RP73 clinical strain was preferred over the PAO1 reference laboratory strain since it resulted in a comparatively lower mortality, more severe lesions, and higher chronic infection. P. aeruginosa colonization may persist in the lung for over three months. Murine lung pathology resembles that of CF patients with advanced chronic pulmonary disease.
This murine model most closely mimics the course of the human disease and can be used both for studies on the pathogenesis and for the evaluation of novel therapies.

Long Term Chronic Pseudomonas aeruginosa Airway Infection in Mice

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

A mouse model of chronic airway infection is a key asset in cystic fibrosis (CF) research, although there are a number of concerns regarding the model ...

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

Cigarette smoke (CS) has a major impact on lung biology and may result in the development of lung diseases such as chronic obstructive pulmonary disease or lung cancer. To understand the underlying mechanisms of disease development, it would be important to examine the impact of CS exposure directly on lung tissues. However, this approach is difficult to implement in epidemiological studies because lung tissue sampling is complex and invasive. Alternatively, tissue culture models can facilitate the assessment of exposure impacts on the lung tissue. Submerged 2D cell cultures, such as normal human bronchial epithelial (NHBE) cell cultures, have traditionally been used for this purpose. However, they cannot be exposed directly to smoke in a similar manner to the in vivo exposure situation. Recently developed 3D tissue culture models better reflect the in vivo situation because they can be cultured at the air-liquid interface (ALI). Their basal sides are immersed in the culture medium; whereas, their apical sides are exposed to air. Moreover, organotypic tissue cultures that contain different type of cells, better represent the physiology of the tissue in vivo. In this work, the utilization of an in vitro exposure system to expose human organotypic bronchial and nasal tissue models to mainstream CS is demonstrated. Ciliary beating frequency and the activity of cytochrome P450s (CYP) 1A1/1B1 were measured to assess functional impacts of CS on the tissues. Furthermore, to examine CS-induced alterations at the molecular level, gene expression profiles were generated from the tissues following exposure. A slight increase in CYP1A1/1B1 activity was observed in CS-exposed tissues compared with air-exposed tissues. A network-and transcriptomics-based systems biology approach was sufficiently robust to demonstrate CS-induced alterations of xenobiotic metabolism that were similar to those observed in the bronchial and nasal epithelial cells obtained from smokers.

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.

Cigarette smoke (CS) has a major impact on lung biology and may result in the development of lung diseases such as chronic obstructive pulmonary disease ...

Bioengineering

Pseudomonas aeruginosa Induced Lung Injury Model

In order to study human acute lung injury and pneumonia, it is important to develop animal models to mimic various pathological features of this disease. Here we have developed a mouse lung injury model by intra-tracheal injection of bacteria Pseudomonas aeruginosa (P. aeruginosa or PA). Using this model, we were able to show lung inflammation at the early phase of injury. In addition, alveolar epithelial barrier leakiness was observed by analyzing bronchoalveolar lavage (BAL); and alveolar cell death was observed by Tunel assay using tissue prepared from injured lungs. At a later phase following injury, we observed cell proliferation required for the repair process. The injury was resolved 7 days from the initiation of P. aeruginosa injection. This model mimics the sequential course of lung inflammation, injury and repair during pneumonia. This clinically relevant animal model is suitable for studying pathology, mechanism of repair, following acute lung injury, and also can be used to test potential therapeutic agents for this disease.

Pseudomonas aeruginosa Induced Lung Injury Model

We have developed a mouse lung injury model by intra-tracheal injection of bacteria Pseudomonas aeruginosa. This model mimics lung injury during pneumonia and is clinically relevant.

In order to study human acute lung injury and pneumonia, it is important to develop animal models to mimic various pathological features of this disease. ...

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

Studying host-pathogen interaction enables us to understand the underlying mechanisms of the pathogenicity during microbial infection. The prognosis of the host depends on the involvement of an adapted immune response against the pathogen1. Immune response is complex and results from interaction of the pathogens and several immune or non-immune cellular types2. In vitro studies cannot characterise these interactions and focus on cell-pathogen interactions. Moreover, in the airway3, particularly in patients with suppurative chronic lung disease or in mechanically ventilated patients, polymicrobial communities are present and complicate host-pathogen interaction. Pseudomonas aeruginosa and Candida albicans are both problem pathogens4, frequently isolated from tracheobronchial samples, and associated to severe infections, especially in intensive care unit5. Microbial interactions have been reported between these pathogens in vitro but the clinical impact of these interactions remains unclear6. To study the interactions between C. albicans and P. aeruginosa, a murine model of C. albicans airways colonization, followed by a P. aeruginosa-mediated acute lung infection was performed.

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

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 host-pathogen interaction enables us to understand the underlying mechanisms of the pathogenicity during microbial infection. The prognosis of ...

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

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 A&#946; were produced and released by endothelial cells following infection by P. aeruginosa. These methods should be readily adaptable to analyses of human clinical samples.

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

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

Patients who survive pneumonia have elevated death rates in the months following hospital discharge. It has been hypothesized that infection of pulmonary ...

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

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.

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

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 ...

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

Close to 14% of adults in the United States were reported to smoke cigarettes in 2018. The effects of cigarette smoke (CS) on lungs and cardiovascular diseases have been widely studied, however, the impact of CS in other tissues and organs such as blood and bone marrow remain incompletely defined. Finding the appropriate system to study the effects of CS in rodents can be prohibitively expensive and require the purchase of commercially available systems. Thus, we set out to build an affordable, reliable, and versatile system to study the pathologic effects of CS in mice. This whole-body inhalation exposure system (WBIS) set-up mimics the breathing and puffing of cigarettes by alternating exposure to CS and clean air. Here we show that this do-it-yourself (DIY) system induces airway inflammation and lung emphysema in mice after 4-months of cigarette smoke exposure. The effects of whole-body inhalation (WBI) of CS on hematopoietic stem and progenitor cells (HSPCs) in the bone marrow using this apparatus are also shown.

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.

Close to 14% of adults in the United States were reported to smoke cigarettes in 2018. The effects of cigarette smoke (CS) on lungs and cardiovascular ...

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

The airway epithelial cell layer forms the first barrier between lung tissue and the outside environment and is thereby constantly exposed to inhaled substances, including infectious agents and air pollutants. The airway epithelial layer plays a central role in a large variety of acute and chronic lung diseases, and various treatments targeting this epithelium are administered by inhalation. Understanding the role of epithelium in pathogenesis and how it can be targeted for therapy requires robust and representative models. In vitro epithelial culture models are increasingly being used and offer the advantage of performing experiments in a controlled environment, exposing the cells to different kinds of stimuli, toxicants, or infectious agents. The use of primary cells instead of immortalized or tumor cell lines has the advantage that these cells differentiate in culture to a pseudostratified polarized epithelial cell layer with a better representation of the epithelium compared to cell lines.
Presented here is a robust protocol, that has been optimized over the past decades, for the isolation and culture of airway epithelial cells from lung tissue. This procedure allows successful isolation, expansion, culture, and mucociliary differentiation of primary bronchial epithelial cells (PBECs) by culturing at the air-liquid interface (ALI) and includes a protocol for biobanking. Furthermore, the characterization of these cultures using cell-specific marker genes is described. These ALI-PBEC cultures can be used for a range of applications, including exposure to whole cigarette smoke or inflammatory mediators, and co-culture/infection with viruses or bacteria. 
The protocol provided in this manuscript, illustrating the procedure in a step-by-step manner, is expected to provide a basis and/or reference for those interested in implementing or adapting such culture systems in their laboratory.

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.

The airway epithelial cell layer forms the first barrier between lung tissue and the outside environment and is thereby constantly exposed to inhaled ...

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

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Chapters in this video

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

Use of Artificial Sputum Medium to Test Antibiotic Efficacy Against Pseudomonas aeruginosa in Conditions More Relevant to the Cystic Fibrosis Lung

Long Term Chronic Pseudomonas aeruginosa Airway Infection in Mice

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

Pseudomonas aeruginosa Induced Lung Injury Model

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

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

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

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

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