419 Sepsis-associated PARDS is associated with an exhausted neutrophil phenotype in the lower respiratory tract

Objectives/Goals: This study aims to uncover immuno-endotypes in sepsis-associated pediatric acute respiratory distress syndrome (SA-PARDS) using single-cell RNA sequencing (scRNA seq) to analyze the immune cell populations of the respiratory tract of intubated pediatric subjects with SA-PARDS. Methods/Study Population: The inclusion criteria are an age of less than 18 years, admission to the PICU, a diagnosis of SA-PARDS, and intubation. Both sepsis and PARDS will be defined using the most recent consensus definitions. Exclusion criteria include an order of limited resuscitation and clinician discretion. A tracheal aspirate and blood sample will be obtained on days 1, 3, and 7. Both samples will be processed for single-cell RNA sequencing via the HIVE platform according to the manufacturer’s protocol. cDNA libraries will be generated and undergo 150 bp sequencing, quality control, and alignment. The Seurat package in R will be used for cell-type annotation and analysis of differential gene expression. Clinical variables, labs, and outcomes will be recorded in REDCap. Results/Anticipated Results: We evaluated five samples from two subjects. After quality control, 31,330 cells remained for analysis. UMAP projections were created, and we annotated cell types. We found that most of the cells were immune cells with very few epithelial cells. There were two groups of neutrophils, which we termed classic and migratory. Classical neutrophils expressed classic neutrophil markers, and migratory neutrophils expressed the classical markers plus increased adhesion markers. We identified a third group of cells that were neutrophil-like, and these were isolated for further analysis. These cells exhibited characteristics of exhausted neutrophils, including downregulation of CXCR1 and CXCR2, and upregulation of PI3 and SLIPI. In each subject, the proportion of exhausted neutrophils increased over time. Discussion/Significance of Impact: We found that in our subjects with SA-PARDS, there were a subset of neutrophils characterized by down-regulation of classical neutrophil markers and up-regulation of neutrophil stress markers, suggested an exhausted phenotype.