INNATE IMMUNE TRAINING IN THE NEONATAL RESPONSE TO SEPSIS

Neonates, especially those born prematurely, are highly vulnerable to infection-induced mortality. Numerous observational and immunological studies in newborns have shown that live attenuated vaccines have beneficial, non-specific effects (NSEs) against secondary infections to unrelated pathogens. These beneficial effects have been attributed to trained immunity, and emergency granulopoiesis plays an essential role. However, trained immunity has been shown to affect multiple myeloid subsets and the diversity by which trained immunity influences the host protective response is still undefined. Here we show that Bacillus-Calmette-Guérin (BCG) vaccination improves mortality to polymicrobial sepsis by simultaneously reprogramming broad aspects of myelopoiesis. Specifically, BCG results in the expansion of multiple myeloid subsets, including LSKs and GMPs, and increases CD11b+GR1+ cell number, as well as their oxidative metabolism and capacity to stimulate T-cell proliferation in response to sepsis. Single-cell RNA sequencing (scRNA-seq) of cells contained in the neonatal spleen suggests that BCG-vaccination changes the broad transcriptional landscape of multiple myeloid subsets promoting the maturation of various neutrophil and monocyte subsets and stimulating antimicrobial processes, while suppressing inflammatory pathways and myeloid-derived suppressor cell (MDSC) transcription. These findings reveal that BCG administration early after birth fundamentally reorganizes the myeloid landscape to benefit the subsequent response to polymicrobial infection. Overall design: Neonatal mice received 10E6 CFUs of the BCG vaccine (TICE® strain, Merck) subcutaneously at birth. Polymicrobial intra-abdominal sepsis was induced in neonatal mice at day 7 of life using the cecal slurry model. Briefly, cecal contents were harvested from a B6 adult mouse and an 80 mg/ml of fresh CS solution was prepared using 5% dextrose. CS (1.1 – 1.3 mg/g BW) was administered intraperitoneally to achieve the desire lethality (LD30-70). Eighteen-hours after sepsis-induction, neonatal mice were euthanized following approved IACUC guidelines and spleens were collected. Gene expression and feature-barcoding data at single cell level were generated using 10X Genomics v1.1 3' chemistry and were sequenced on an Illumina HiSeq® with a target of 10,000 cells/sample. Data were processed using CellRanger followed by applying Seurat as an R package for quality control and visualization. n=4-7 per group: Naive (N), BCG-vaccinated (B), Sepsis (CS18h), BCG plus sepsis (BCG-CS18h)