322 - Modeling bronchopulmonary dysplasia ex vivo: transcriptomic analysis of Lipopolysaccharide-treated preterm rabbit precision-cut lung slices

Publication Number: 1306.322

Carlotta Boggi, University of Parma, Parma, Emilia-Romagna, Italy; Enrica Scalera, CHIESI FARMACEUTICI S.P.A., Parma, Emilia-Romagna, Italy; Marco Morselli, University of Parma, Parma, Emilia-Romagna, Italy; Aurora Radicati, Chiesi S.p.A, Parma, Emilia-Romagna, Italy; Giorgio Aquila, Chiesi Farmaceutici Spa, Parma, Emilia-Romagna, Italy; Simone De Meo, University of Siena, Formia, Lazio, Italy; Xabier Murguia Esteve, Consultant in Biomedical Science (Freelance), Bilbao, Pais Vasco, Spain; Francesca Ricci, Chiesi Farmaceuitci, PARMA, Emilia-Romagna, Italy; Barbara Montanini, University of Parma, Parma, Emilia-Romagna, Italy; Matteo Storti, Chiesi Farmaceutici, Parma, Emilia-Romagna, Italy

Background: Precision-cut lung slices (PCLS) from preterm-born rabbits exposed to lipopolysaccharide (LPS) are a useful ex vivo model to study how anti-inflammatory drugs may work in bronchopulmonary dysplasia.

Objective: In this study, we employed bulk RNA sequencing to characterize the inflammatory response induced by LPS in PCLS.

Design/Methods: Lungs were collected from preterm rabbits delivered by C-section at 28 days of gestation (saccular stage; term = 31 days). PCLS were prepared, and punches were kept under normoxic conditions. On day 1, they were exposed to LPS, while control samples were not treated. Samples from 5 independent experiments were collected 4 and 24 hours after exposure (n = 7 per condition). Differentially expressed genes (DEGs) were identified with DESeq2 (|log2FC| > 1; adjusted p-value < 0.05), and pathway enrichment analysis was performed using GSEA.

Results: Principal component analysis (Figure 1), performed after batch effect removal, showed clear clustering by treatment and exposure time, clearly separating LPS-exposed samples at 4 hours from the controls. After 4 hours, 335 DEGs were found between LPS-exposed and control PCLS (Figure 2). LPS exposure activated immune-related pathways, such as response to bacteria and cellular response to LPS, showing an innate immune reaction. Pathways related to DNA replication and chromosome segregation were downregulated, suggesting reduced cell cycle activity (Figure 3). After 24 hours, only 25 DEGs were detected, so pathway enrichment analysis was not performed.

Conclusion(s): LPS exposure caused strong immune activation and reduced cell cycle activity in PCLS from preterm rabbit lungs. These effects were seen only at 4 hours after exposure, while no differences were detected at the 24 hours time point.

Figure 1. Principal component analysis of gene expression profiles from PCLS exposed or not to LPS for 4 or 24 hours.
The analysis was performed after removing batch effects. Each point represents one biological replicate, and colors indicate treatment and time conditions. Samples cluster according to treatment and exposure time, showing clear separation between LPS-exposed and untreated PCLS at 4 hours.

Figure 2. Volcano plots showing differentially expressed genes (DEGs) in PCLS exposed to LPS compared to untreated controls.
The upper panel represents the 4h exposure, and the lower panel the 24h exposure. Genes significantly up- or downregulated (|log₂FC| > 1, adjusted p < 0.05) are highlighted in red. LPS exposure at 4h induced strong upregulation of inflammatory genes such as TNF, IL1A, and SELE, while only a few DEGs were detected after 24h.

Figure 3. Gene Set Enrichment Analysis (GSEA) results comparing different conditions.
The bar plots display the top 10 upregulated and downregulated pathways when comparing LPS-treated and untreated samples at 4 hours. The normalized enrichment score (NES) is shown on the x-axis, indicating pathway activation or suppression; the scale gradient color indicates the adjusted p-value. Analyses were performed in R using GSEA.