A103-24 High-dimensional Immune Profiling of Critically Ill Sepsis Patients Identifies Distinct Inflammatory Programs Within Hyperinflammatory Sepsis

Rationale

Latent class analysis applied to clinical parameters and protein biomarkers identified two molecular subphenotypes in sepsis. The hyperinflammatory subphenotype is consistently defined by higher plasma levels of inflammatory biomarkers (IL-6, IL-8, TNFr-1), higher mortality and preferential response to therapies in secondary analyses of randomized controlled trials. Additional work to understand biologic heterogeneity within subphenotypes is critical to guide treatment.

Methods

We performed paired single-cell RNA sequencing (scRNA-seq) of circulating leukocytes and spectral flow cytometry on peripheral blood mononuclear cells (PBMCs) isolated via ficoll gradient from 27 patients with sepsis within 24 hours of admission to the ICU. ScRNAseq was performed using the 10X FLEX RNA kit to preserve neutrophils. Patients were classified into molecular subphenotypes using a previously validated parsimonious model. Immune cell proportions were quantified from spectral flow cytometry using manual gating based on canonical markers. We performed hierarchical clustering of immune cell proportions to identify immune groups. ScRNAseq data were analyzed using Seurat. We then performed differential gene expression within neutrophils and monocytes using MAST, and pathway enrichment using Enrichr. We derived immune group gene signatures from pseudobulked scRNA-seq data using limma and applied ssGSEA to a validation cohort of 303 bulk RNA-seq samples from septic patients. Clinical and microbiologic variables were compared between the 3 immune groups in the larger validation cohort.

Results

Hierarchical clustering of PBMC cell proportions identified three immune groups in septic patients. (Fig 1A). Patients classified as hyperinflammatory segregated into two distinct groups: a low-density neutrophil-dominant group (Immune 1) and a monocyte-dominant group (Immune 2), while hypoinflammatory patients were predominantly T lymphocyte enriched (Immune 3). Neutrophils in Immune group 1 exhibited increased expression of genes associated with emergency granulopoiesis and IL-1B signaling (Fig 1B). In contrast, neutrophils and monocytes in Immune group 2 showed enrichment of TNFalpha and NF-kB signaling pathways (Fig 1C). Application of immune group gene signatures to an independent bulk RNA-seq cohort recapitulated these three groups, validating their presence beyond the discovery cohort (Fig 1D). Immune group 1 was associated with more severe organ injury, including greater shock severity, higher ARDS prevalence and increased representation of Enterobacteriaceae pathogens (Fig 1E).

Conclusion

High-dimensional immune profiling of critically ill septic patients using a neutrophil preserving protocol identified additional heterogeneity within the hyperinflammatory subphenotype. Distinct neutrophilic/IL-1B and monocytic/TNFalpha immune profiles are associated with divergent clinical outcomes and pathogen profiles, supporting the potential for IL-1B targeted therapies in this subset of hyperinflammatory sepsis patients.

This abstract is funded by: 5T32HL007185-47