B75-37 Transcript Isoform Switching and Cell-Type Deconvolution Reveal Sex-Specific Lung Responses During Acute Pseudomonas Aeruginosa-induced Sepsis

Rationale

Sepsis causes 11 million deaths, annually. The lungs often serve as one of the earliest and most severely affected organs. The role of host factors, including sex-based differences, in shaping lung responses during sepsis is still under active investigation. Bacterial infections, a leading cause of sepsis can drive significant transcriptional changes and alter cell type compositions in affected tissues. Emerging evidence suggests transcript isoform switching provides a crucial layer of co- and post- transcriptional regulation. We therefore hypothesized that both cell-type dynamics and transcript isoform switching are differentially regulated between male and female lungs during sepsis, potentially contributing to sex-based differences in disease outcomes.

Methods

To address this idea, we used a murine model of Pseudomonas aeruginosa (PA103)-induced pneumonia which can progress to sepsis, and employed long-read RNA sequencing to accurately profile transcript isoforms. Cell-type composition was inferred using CIBERSORTx deconvolution of bulk RNA-seq data.

Results

With long-read transcript resolution and a robust sample size (n = 12/group), we confirmed previously reported sepsis-associated transcriptional changes, but importantly, uncovered novel sex-specific patterns of differential transcript isoform usage (DTU). We identified 209 genes in females and 169 in males which exhibited significant DTU. Strikingly, approximately 40% of DTU genes were not detected by differential gene expression (DEG) analysis in either sex, highlighting the regulatory impact of isoform-level changes beyond gene-level expression. The dominant events driving DTU were alternative transcription start and stop site usage. Males preferentially switched to isoforms with shorter 3′ UTRs, whereas females favored alternative transcription start sites. STRING-based interaction analysis of switching genes revealed three more cohesive clusters in females (bioenergetics, chromatin remodeling, and DNA repair) compared to six in males (mitochondrial energy metabolism, redox metabolism, RNA processing, ER stress, ribonuclease activity, and inflammation). Notably, some shared switches occurred in evolutionarily conserved genes essential for cellular energy homeostasis and survival. While DEGs largely overlapped between sexes, female-specific DEGs highlighted enhanced epithelial repair, oxidative metabolism, and tissue integrity. In contrast, male-specific DEGs pointed to heightened innate immune activation and inflammation. Cell-type deconvolution supported these findings, revealing a greater infiltration of immune cells and significantly reduced representation of alveolar type 2 and ciliated epithelial cells in male lungs post-infection.

Conclusion

To our knowledge, this is the first comprehensive study integrating long-read isoform profiling with cell-type deconvolution to reveal sex-based transcriptional and compositional differences in septic mouse lungs. These findings provide a foundation to understand sex-specific disease mechanisms and tailor future therapeutic strategies

This abstract is funded by: NIH: 1UL1TR001417; 1KL2TR003097. USA College of Medicine: Deans Predoctoral fellowship; Center for Lung Biology Murray Bander Faculty Development Awards. American Thoracic Society; young investigators award