BCG vaccination-induced emergency granulopoiesis provides rapid protection from neonatal sepsis

Abstract

Death from sepsis in the neonatal period remains a serious threat for millions. Within 3 days of administration, bacille Calmette-Guérin (BCG) vaccination can reduce mortality from neonatal sepsis in human newborns, but the underlying mechanism for this rapid protection is unknown. We found that BCG was also protective in a mouse model of neonatal polymicrobial sepsis, where it induced granulocyte colony-stimulating factor (G-CSF) within hours of administration. This was necessary and sufficient to drive emergency granulopoiesis (EG), resulting in a marked increase in neutrophils. This increase in neutrophils was directly and quantitatively responsible for protection from sepsis. Rapid induction of EG after BCG administration also occurred in three independent cohorts of human neonates.

Fig. 1.. BCG protected neonatal mice from infectious, but not sterile septic death.

(A) BCG-vaccinated mice demonstrated a significant increase in survival from neonatal polymicrobial sepsis (solid blue line) compared with controls (dashed black line) (n=67 per group, P < 0.0001). (B) BCG reduced tumor necrosis factor-α (TNF-α) production 24 hours after septic challenge, just before the maximal mortality period in this model (n = 10). (C) BCG vaccination reduced bacterial load across blood, organs, and peritoneal wash (P.Wash) (n = 26 to 27) 24 hours after sepsis. (D) Principal components analysis (PCA) of all cytokines prechallenge (circles, 3 days after vaccination), post–CS-induced sepsis (post-CS, squares), and post-LPS challenge (post-LPS, triangles), colored by vaccination status (BCG, blue; control, black; n = 10 to 16 per group; individual cytokines in fig. S2 and summarized roles in table S1). (E) BCG did not protect mice from endotoxin shock (n = 42 to 45, P = 0.42). (F) BCG did not reduce TNF-α production 12 hours after LPS challenge, just before the maximal mortality period in this model (n = 14 to 16, P = 0.96). Statistical analysis by log-rank test (A and D); unpaired two-sided t test, Benjamini-Hochberg (BH) adjusted (B and E); and Wilcoxon rank-sum test BH adjusted (C). *P < 0.05, **P < 0.01, ***P < 0.001, and ****P < 0.0001.

Fig. 2. BCG-induced increase in neutrophil numbers was necessary and sufficient for protection.

(A) BCG increased total neutrophils in the spleen, with immature and mature neutrophils peaking on days 2 and 3, respectively, after vaccination (BCG, solid blue line; control, dashed black line). Mature neutrophils mobilized from the spleen within 24 hours of CS challenge (vertical dotted line) (n = 6 to 7, 10, 13 to 14, and 7 to 8 on days 1, 2, 3, and 4, respectively, with 95% CI presented in gray). (B) Adoptive transfer of splenocytes from BCG-vaccinated donors (“BCG WS”) versus control splenocytes (“Control WS”) reduced bacterial burden (n = 55 and 32), but not if mature neutrophils were depleted from BCG-vaccinated spleens before adoptive transfer (“BCG WS – Neuts”; n = 9). (C) Mature neutrophils purified from BCG-vaccinated donors and transferred at expected BCG-vaccinated numbers (“BCG Neut @ [BCG]”; n = 40) reduced bacterial burden, whereas transfer of fewer cells, specifically the expected cell numbers in a control mouse (“BCG Neut @ [Ctrl]”; n = 9), impaired bacterial clearance. Control donor neutrophils artificially increased to match BCG numbers (“Ctrl Neut @ [BCG]”; n = 23) were similarly protective as BCG-purified neutrophils (colored by donor mouse vaccination status; control, black; BCG, blue). Statistical analysis by BH-adjusted, unpaired two-sided t test (A) and Kruskal-Wallis rank-sum test unpaired Wilcoxon rank-sum test in (B) and (C) with *P < 0.05, **P < 0.01, ***P < 0.001, and ****P < 0.0001.

Fig. 3.. BCG vaccination–induced EG.

(A) BCG (blue solid line) versus control (black dashed line) induced production of EG-supporting cytokines within 8 to 12 hours after vaccination (n = 10 per group and time point). (B) Antibody-mediated blocking inhibited EG when targeting G-CSF, but not GM-CSF (n = 8 to 14). N.S., not significant. (C) Recombinant G-CSF (rG-CSF) given at 0.1 μg rG-CSF/g mouse induced BCG-like protection against sepsis. (D) Cebp-β RNA expression increased in the spleens of BCG-vaccinated mice 12 hours after vaccination presented as relative gene expression compared with the 18S housekeeping gene (calculated by 2^ΔΔCT, n = 17 per group). (E) GMP numbers increased in the spleens 2 days after vaccination, as determined by in vitro culture (n = 11 per group). (F) Kinetics of BCG-induced splenocyte GMP increase after vaccination, identified by flow cytometry (n = 6 to 7 per group and time point; gating strategy in fig. S11). Statistical analysis by unpaired two-sided t test for (A), (B), and (D) to (F), with BH adjustment for (A) and (F), and log-rank test for (C). *P < 0.05, **P < 0.01, ***P < 0.001, and ****P < 0.0001.

Fig. 4.. BCG vaccination induced EG in murine and human newborns.

Transcriptional changes after BCG vaccination were identified by NanoString nCounter using sparse partial least squares discriminant analysis (sPLS-DA) in neonatal mouse (A) and human (B) peripheral blood 24 hours after vaccination (n = 12 mice, 60 transcripts; n = 20 humans, 100 transcripts; visualized using variate plots). (C) The top 10 pathways most affected by neonatal BCG at 24 hours after vaccination (determined through gene enrichment analysis) substantially overlapped between mouse (left) and human (right) (neutrophils marked in blue; other overlaps marked in black). DC, dendritic cell; NK, natural killer; FDR, false discovery rate. (D) Validation of BCG-induced EG signatures in two independent human newborn cohorts (The Gambia, West Africa; PNG, Australasia). The PCA depicts signatures for GM-CSF–induced changes in neutrophils, contrasting vaccinated (blue) versus control (black) separated by day after vaccination, with distinctness of the clusters tested using PERMANOVA. These gene signatures of GM-CSF–induced changes in neutrophils explained 89% of the variance observed between control and BCG-vaccinated newborns (calculated with 10 PCs). Similar patterns emerged for other EG-related gene sets shown in fig. S12. DPV, days postvaccination. (E) BCG increased human neutrophil numbers present on day 3 after vaccination in neonates from The Gambia and PNG (EPIC cohort, n = 6 to 8 per group per time point, *P = 0.02, Wilcoxon rank-sum test, BH adjusted). Data are presented as medians with 25 to 75% IQR.