B75-38 Single-cell Transcriptomics Reveals Cell-type Specific Anti-inflammatory Responses to Trek-1 K+ Channel Activation in an In-vitro Human Triple-hit ARDS Model

Rationale

Acute Respiratory Distress Syndrome (ARDS) lacks targeted therapies, with mortality rates stagnant at 35-45%. We previously demonstrated that TREK-1 K⁺ channel activation reverses lung inflammation in experimental mouse ARDS models. At the cellular level, we found a decrease in inflammatory mediator secretion from alveolar epithelial and endothelial cells. However, the molecular pathways responsible for these anti-inflammatory effects remain unknown. We hypothesized that TREK-1 activation with the novel compound ML335 induces distinct transcriptional programs in human epithelial and endothelial cells under ARDS-like conditions that are critical mediators of the observed anti-inflammatory effects.

Methods

Primary human alveolar epithelial cells and human lung microvascular endothelial cells were infected with Pseudomonas aeruginosa (PA; MOI 10) for 1 hour, followed by hyperoxia (HO; FiO2 0.9) and cyclic mechanical stretch (ST; 5% stretch, 15 cycles/min) exposure for 1 additional hour to create a clinically-relevant “triple-hit” injury model characteristic of human ARDS. Cells were treated with ML335 (100μM) or vehicle at the time of HO+ST initiation as a clinically feasible approach. For single-cell RNA sequencing, 12 independent samples (approximately 130,000 cells in total) were processed using Seurat (v5) with log-normalization, variable features (2000), and PCA/UMAP dimensionality reduction. Differential expression analysis was performed using a Wilcoxon test by cell type. Significant genes (Bonferroni p < 0.05, |log2FC|>0.25) were used for gene ontology analysis.

Results

Transcriptomic analysis of changes in gene expression following TREK-1 activation (Figure 1) revealed 908 differentially expressed genes in epithelial cells (119 upregulated, 789 downregulated) and 136 differentially expressed genes in endothelial cells (16 upregulated, 120 downregulated). In epithelial cells, TREK-1 activation predominantly suppressed genes critical for immune cell regulation (ARL14, CLEC2D, EREG, ISG15) and inflammatory cytokine/chemokine production (CXCL10, CXCL11, CCL5, IL-24). In endothelial cells, the top 5 genes downregulated by TREK-1 activation are known to regulate barrier function/cell adhesion/migration/cell-to-cell contact (PRICKLE1), cytoskeletal remodeling (EDN1, DLC1), oxidative stress (PLK2), and immune cell/host defense mechanisms (ZF36L1) (Figure 1). Notably, in both cell types by TREK-1 activation was one pathway was accompanied by downregulation of (EFNA5, NRP2, NRG1) all involved in negative chemotaxis.

Conclusions

TREK-1 activation triggers distinct, cell-type specific transcriptional responses in primary human alveolar epithelial and endothelial cells exposed to ARDS-like conditions by predominantly downregulating cellular inflammation in epithelial cells vs. barrier function in endothelial cells. Detailed characterization of the mechanisms underlying TREK-1-mediated protection will accelerate targeted drug development against ARDS and ultimately allow for testing of TREK-1 activating compounds in Phase-1 trials.

This abstract is funded by: NHLBI