Identifying immune signatures of sepsis in very preterm babies

Bacterial infections are a major cause of mortality in preterm babies, yet our understanding of early-life disease-associated immune dysregulation remains limited. Here, we combined multi-parameter flow cytometry, single-cell RNA sequencing (scRNAseq) and targeted plasma analysis to prospectively and longitudinally profile blood from very preterm babies (<32 weeks gestation) across episodes of invasive bacterial infection (sepsis). We identified a dynamically changing peripheral blood immune signature of sepsis, including lymphopenia, reduced dendritic cell frequencies and monocyte HLA-DR expression, which characterized sepsis even when the common clinical marker of inflammation, C-reactive protein (CRP) was not elevated. Furthermore, scRNAseq showed upregulation of amphiregulin in different leukocyte populations during sepsis, which we validated as a plasma analyte that correlated with clinical signs of disease, even when CRP was normal. This study provides new insights into immune pathways associated with early-life sepsis and identifies potential immune analytes as diagnostic adjuncts for sepsis to guide targeted antibiotic prescribing. Paired blood samples were drawn from five preterm babies on the intensive care unit (n=10 samples). For each pair, one sample was taken contemporaneous with onset (or close to onset) of bacterial sepsis or necrotising enterocolitis, whereas the second sample was taken approximately one week either side of the clinical event. We then applied single cell RNA sequencing to determine gene expression changes within peripheral blood mononuclear cells (PBMC), between the paired timepoints. PBMC were stained with Cite-seq antibodies, and in some cases, anti-human Hashtag reagents, to allow better differentiation of key immune subsets, and multiplexing of multiple samples together respectively. In 4/5 babies we performed scRNAseq on total PBMC, whilst in 1 baby, we first sorted CD3+ T cells, prior to scRNA-seq.