C54-03 Sepsis Precipitates Dynamic Changes in Distinct Monocyte States: Insights From Serial Single-cell Profiling in Pigs

Rationale Transcriptional studies of immune cells during sepsis have revealed dysfunctional myeloid subpopulations which are hypothesized to mediate poor outcomes and predict response to clinical therapies. The mechanisms underlying these subpopulations and their causal roles in sepsis remain poorly understood due to the cross-sectional nature of human studies and variable translatability of murine sepsis models, which limit the development of targeted immunotherapies. To address this gap, we used a high-fidelity porcine model of sepsis that permits serial sampling to define the temporal trajectories of myeloid subpopulations and their relationship with sepsis severity. Methods Sepsis was induced in mixed-Yorkshire pigs (n = 5) via intrarenal injection of Escherichia coli. Blood was collected at baseline, 8, 16, and 24 hours. scRNAseq was performed on peripheral blood mononuclear cells (>136,000 total). Cell types were annotated using a porcine reference. Myeloid cells (monocytes, dendritic cells) were reclustered. Differentially expressed genes and pathway analysis were performed. Dynamic changes in cluster proportion were compared across time and by severity of organ dysfunction (SOFA) using linear mixed-effects models, leveraging pairwise comparisons of estimated marginal means for hypothesis testing. Results Amongst the 11 myeloid subclusters identified (Fig. A), monocytes exhibited the highest variability following sepsis induction. Three monocyte subclusters (1, 5, and 6) expanded significantly relative to the total myeloid population during early sepsis (p < 0.05) but displayed different temporal trajectories (Fig. B). Clusters 1 and 5 were minor clusters at baseline, expanded ∼2-fold by 8 hours, and were characterized by NF-κB signaling (NFKBIA, RELB) and lysosome/phagocytosis (LGMN, CTSB, MERTK) pathways, respectively. Cluster 6 was nearly absent at baseline, increased >10-fold with sepsis, and tended to correlate with peak organ dysfunction (SOFA >5, n = 3 v. SOFA <5, n = 2, p = 0.18). This cluster was enriched for cell cycle and proliferation markers (including CEP55, KIF15, and PRC1) that are present in dysregulated myeloid populations identified as predictors of poor outcomes in patients with sepsis. Conclusions We successfully traced the evolution of myeloid states in a large-animal model that faithfully recapitulates human sepsis physiology. We found several monocyte subclusters enriched in sepsis, including a proliferating monocyte subcluster, that correlates with disease severity. Additional studies will determine the temporal relationship of subcluster expansion with circulating inflammatory mediators in the porcine model and whether they are conserved or distinct from subclusters induced in humans during sepsis. This abstract is funded by: University of Michigan, Office of the Vice President of Research Grant; NIH/NIGMS R35GM136312; NIH/NHLBI K08 HL153799