This study is comprised of experiments on P. aeruginosa culture, human airway epithelial cells stimulated with P. aeruginosa-culture filtrates, mouse models of P. aeruginosa lung infection, and bronchoalveolar lavage fluid (BALF) of bronchiectasis patients with first-time detection of P. aeruginosa infection. Figure 1 provides an overview of the study design and procedures.

Fig. 1

Flow chart of the study design and procedures. PMA palmitoleic acid, QS quorum sensing, BALF bronchoalveolar lavage fluid

Bacterial strains and PMA treatment

Wildtype P. aeruginosa PAO1 strains were grown in Lysogeny Broth (LB) medium with 50 mM 3-(N-morpholino) propanesulfonic acid at 37 °C with 250 rpm shaking overnight. When indicated, cultures were supplemented with different doses of PMA (0.01 mg/mL, 0.05 mg/mL, 0.5 mg/mL).

AHL and pyocyanin measurements

Signaling molecules were extracted using ethyl acetate, and both 3OC12-HSL and C4-HSL concentrations were measured using reporter strains as previously described [9, 10]. Pyocyanin was extracted from 4 mL culture fluid with 2 mL chloroform, and was then extracted from the chloroform with 1 mL 0.1 mol/L hydrochloric acid–water. At 520 nm, the absorbance was measured and multiplied by 17.072 to get the concentration of pyocyanin [11].

Biofilm detection

Bacterial biofilm was quantitatively detected using a 96-well microplate. Briefly, 100 μL LB medium with or without 0.5 mg/mL PMA was added to each well and incubated with 10 μL overnight PAO1 culture at 37 °C for 36 h. The plate wells were washed and fixed with methanol for 15 min. Add 100 μL 1% crystal violet solution to each well and dye at room temperature for 5 min, and then the wells were rinsed and dried. After complete drying, add 100 μL of 33% glacial acetic acid solution to each well and incubate at 37 °C for 30 min to dissolve crystal violet. Measure the optical density (OD) value of the solution in the culture well using a microplate reader (BioTek Epoch, BioTek Instruments, Winooski, VT, US) under 590 nm conditions.

Bronchial epithelial cell stimulation experiments

Pseudomonas aeruginosa filtrates were prepared as reported in a previous study [12]. Briefly, PAO1 cultures were grown in LB medium supplemented with PMA (0.5 mg/mL) for 24 h. Cultures grown in LB medium without PMA were used as controls. Cultures were centrifuged and filtered with 0.22-μm cellulose acetate filters, and the filtrates were collected. Human bronchial epithelial BEAS-2B cells (2.0 × 105 cells/well) were incubated in a starvation medium for 16 h, and then stimulated with 60 μL of sterile filtrates or LB medium for the time-points of 3 h, 6 h, 12 h, and 24 h.

Cell Counting Kit-8 kit (Signalway Antibody, Greenbelt, MD, USA) was utilized to assess cell viability. After stimulation, cells were incubated with 10 μL kit solution in 96-well plates for one hour. Optical density was measured at 450 nm. For cell apoptosis assay, cells were collected and stained with Annexin V-FITC (Beyotime Biotechnology, Shanghai, China). To analyse the cell cycle, cells were treated with propidium iodide (PI) and RNase A for 30 min. The proportion of cells in each phase was determined using the FlowJo software (FlowJo LLC, Ashland, OR, USA). Human interleukin (IL)-6, and IL-8 in the epithelial cell cultures were determined by the use of enzyme-linked immunosorbent assay kits (R&D Systems, Inc., Minneapolis, MN, USA). The mRNA levels of IL-6 and IL-8 were determined by qRT-PCR. Each experiment was performed in triplicate.

Prophylactic administration of PMA in mice with P. aeruginosa infection

Male C57BL/6 mice (specifically pathogen-free, 6 weeks of age) were purchased from SLAC (Shanghai, China). Experimental Animal Care and Ethics Committee of Shanghai General Hospital approved all experimental protocols used in this study. Mice were inoculated intratracheally with 50 μL P. aeruginosa-laden agarose beads (2.0 × 106 CFU/mL) as previously described [13]. Mice were prophylactically administered with PMA (300 mg/kg) by gavage daily starting 2 weeks before inoculation. Those administered with saline were used as controls. The treatment continued after inoculation until mice were euthanized at indicated times.

The lungs were lavaged, and the BALFs were collected and centrifuged at 3000 rpm for 10 min at 4 °C. BALF cells were counted using a hemocytometer by two independent observers. Lungs were aseptically homogenized in sterile saline for CFU enumeration, and lung myeloperoxidase (MPO) activities were measured by using a commercial kit (Nanjing Jiancheng Bioengineering Institute, Nanjing, China). Hematoxylin/eosin-stained sections were blindly scored for inflammatory infiltrates using the scoring system [14].

RNA isolation and qRT-PCR

For PAO1 cultures, diluted cultures were grown at 37 °C, and when reaching the indicated OD600nm, cells were pelleted and preserved in RNA Protect Bacteria reagent (Qiagen, Hilden, Germany). For mouse lung tissues, the left lungs were excised aseptically and kept in liquid nitrogen. Cell pellets or lung tissues were lysed in QIAzol Lysis Reagent. Then the total RNA was extracted and purified using the RNeasy MinElute cleanup kit (Qiagen, Hilden, Germany). The expression of target genes was analyzed by following the protocol for the iQ SYBR Green SuperMix (Bio-Rad Laboratories, Hercules, CA, USA) on a ViiA 7 PCR System (Applied Biosystems, CA, USA). rplU was used as the reference gene, and the primers used in the study are listed in Table S1.

P. aeruginosa QS signal measurements and spirometry tests in bronchiectasis patients

Bronchiectasis patients with first-time detection of P. aeruginosa infection were recruited between January 2020 to December 2023 from Shanghai General Hospital, China. At the time of admission, all patients presented with purulent sputum, and underwent spirometry tests, including forced vital capacity (FVC) and forced expiratory volume in the first second (FEV1.0). Then the bronchoscopy examination was performed, and all patients were positive for P. aeruginosa (> 104 CFU/mL) in the samples of BALF. The measurements of P. aeruginosa 3OC12-HSL and C4-HSL were performed using BALF samples as above described. The Medical Ethics Committee of Shanghai General Hospital approved the protocol.

PMA measurement in BALF

BALF sample mixed with chloroform methanol (2:1 v/v) had an ultrasonication of 30 min. Fatty-acid methyl esterification was achieved by 30 min of 80 °C water bath and later mixed with n-hexane. The supernatant was subjected to an Agilent Model 7890A/5975C GC–MS system. An Agilent DB-WAX capillary GC column was used to separate the samples. PMA quantification (μg/mL) was performed by making a calibration curve using the Supelco 37-component FAME mix (Sigma-Aldrich). The stability and repeatability of the system was verified by using a quality-control sample.

Statistical analysis

Data were analyzed by GraphPad Prism (version 9; GraphPad Software, San Diego, CA, USA), and graphs were drawn accordingly. For normally distributed quantitative variables, t-test or variance was used for data analysis; while for non-normally distributed variables, Mann–Whitney U test was used. Correlation between two nonparametric variables was performed using Spearman correlation coefficient (rs). P-values less than 0.05 were considered statistically significant.