444 scRNA seq analysis of lower respiratory tract immune cells to uncover immuno-endotypes in SA-PARDS

Objectives/Goals: This study aims to uncover immuno-endotypes in sepsis-associated pediatric acute respiratory distress syndrome (SA-PARDS) by using single-cell RNA sequencing (scRNA seq) to analyze the immune cell populations of the lower respiratory tract of intubated pediatric subjects with SA-PARDS. Methods/Study Population: Inclusion criteria are age less than 18 years, admission to the PICU, 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. After informed consent is obtained, a tracheal aspirate and blood sample will be obtained on days 1, 3, and 7. Both samples will be processed for single-cell RNA seq via the HIVE platform per manufacturer protocol. cDNA libraries will then be sent for 150 base pair paired-end sequencing. Sequences will be aligned to a reference genome, and count matrices will be generated. 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 expect to find that subjects with nonresolving SA-PARDS, defined as intubation and mechanical ventilation, will have a monocyte/macrophage transcriptome characterized by continued hyper-inflammation (M1-like phenotype) that does not transition over time to an anti-inflammatory and pro-repair phenotype (M2-like). Additionally, we expect to see that subjects with non-resolving SA-PARDS will have evidence of continued inflammation driven by hyper-inflammatory neutrophils. Finally, we expect that subjects with non-resolving SA-PARDS will have epithelial cells characterized by continued upregulation of canonical pathways of innate immunity including interferon signaling and the damage associated molecular pattern recognition pathway. Discussion/Significance of Impact: The discovery of immuno-endotypes in SA-PARDS would represent a major step toward developing precision medicine therapies for this group of patients. It would simultaneously provide a strategy to reduce biological heterogeneity and identify novel pathways and targets for therapy.