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

الملخص

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.

البروتوكول

1. Human lung epithelial cell culture and cigarette smoke extract (CSE) treatment

1. 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.
2. 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.
3. Dissociate the cells with 1 mL of 0.25% trypsin for 5 min until the cells completely detach from the bottom of the plate.
4. 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.
5. Discard the supernatant and resuspend the cells in 2 mL of HITES medium with 10% FBS.
6. 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.
7. 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.
8. Treat the cells at approximately 80% confluency, or 5 x 105 cells/mL, with 4% cigarette smoke extract (CSE) for 3 h. Before CSE treatment, change the medium with HITES medium with 1% of FBS.

2. Bacterial infection

1. 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.
2. 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.
3. 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.

3. Determination of bacterial concentration using the drop plate method

1. To determine bacterial load in infected cells with the drop plate method, wash the gentamycin-treated cells 2x with 2 mL of cold PBS.
2. Add 1 mL of cell lysis buffer (0.5% triton X-100 in PBS) to each well.
3. 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.
4. After 16 h of incubation, obtain the results of CFU by counting the bacterial colonies.

المواد

قائمة المواد المستخدمة في هذه المقالة

الاسم	الشركة	رقم فهرسي	التعليقات
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 cell	ATCC	ATCC CRL-9609
Human primary small airway epithelial cells	ATCC	ATCC PCS-300-030
OD reader	USA Scientific
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
Tryptic Soy Broth	BD Biosciences