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

Take cultures of human lung epithelial cells. These cells secrete antimicrobial peptides (AMPs), which act as a defense barrier against respiratory pathogens.

Treat one group with cigarette smoke extract (CSE); add media to the untreated.

CSE components enter the cells, bind to intracellular receptors, and trigger signaling that reduces AMP production, weakening the cells’ defense.

Incubate both cultures with a respiratory pathogenic bacterium.

In the untreated group, AMPs kill most bacteria, keeping infection levels low.

In the CSE-treated group, fewer AMPs allow more bacteria to survive and invade the epithelial cells.

Add an antibiotic to eliminate the extracellular bacteria and wash to remove the antibiotic.

Add a lysis buffer to disrupt the host cells and release the internalized bacteria. Plate the diluted lysates and incubate to allow bacterial growth.

A higher colony count in the CSE-treated group indicates that cigarette smoke exposure impairs the AMP-mediated bacterial killing.

Plate the lung epithelial cells at a concentration of three times 10 to the fifth cells per milliliter, in a total volume of two milliliters of medium per well. Incubate the plates overnight. When the cells reach approximately 80% confluency, replace the medium with Heights medium, plus 1% FBS.

Then, add 4% CSE to the wells and incubate the plates for three hours. Add a Pseudomonas to each well of CSE-treated lung epithelial cells. Then, incubate the plates for one hour at 37 degrees Celsius with 5% carbon dioxide.

Next, replace the medium in the six-well plates with two milliliters of fresh Heights medium, containing 4% CSE and 100 micrograms per milliliter of gentamycin. After incubating the plates for one hour at 37 degrees Celsius with 5% carbon dioxide, aspirate the medium from the wells. Wash the gentamycin-treated cells twice with two milliliters of cold PBS.

Bacterial load in the lung epithelial cells can then be detected by the drop plate method, qRT-PCR, or flow cytometry. Add one milliliter of cell lysis buffer to each well of lung epithelial cells. Repair serial dilutions of the cell lysate, ranging from 1 to 10 to 1 to 10,000.

Then inoculate the TSB agar plate. After 16 hours of incubation, determine how many colony-forming units were present in the lung epithelial cells by counting the number of bacterial colonies.