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. 2020 Nov 17;33(7):108387.
doi: 10.1016/j.celrep.2020.108387.

BCG Vaccination Induces Long-Term Functional Reprogramming of Human Neutrophils

Affiliations

BCG Vaccination Induces Long-Term Functional Reprogramming of Human Neutrophils

Simone J C F M Moorlag et al. Cell Rep. .

Abstract

The tuberculosis vaccine bacillus Calmette-Guérin (BCG) protects against some heterologous infections, probably via induction of non-specific innate immune memory in monocytes and natural killer (NK) cells, a process known as trained immunity. Recent studies have revealed that the induction of trained immunity is associated with a bias toward granulopoiesis in bone marrow hematopoietic progenitor cells, but it is unknown whether BCG vaccination also leads to functional reprogramming of mature neutrophils. Here, we show that BCG vaccination of healthy humans induces long-lasting changes in neutrophil phenotype, characterized by increased expression of activation markers and antimicrobial function. The enhanced function of human neutrophils persists for at least 3 months after vaccination and is associated with genome-wide epigenetic modifications in trimethylation at histone 3 lysine 4. Functional reprogramming of neutrophils by the induction of trained immunity might offer novel therapeutic strategies in clinical conditions that could benefit from modulation of neutrophil effector function.

Keywords: BCG; epigenetics; innate immune memory; neutrophil; nonspecific effects of vaccines; polymorphonuclear leukocytes; trained immunity.

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Conflict of interest statement

Declaration of Interests The authors declare no competing interests.

Figures

None
Graphical abstract
Figure 1
Figure 1
BCG Vaccination Alters the Phenotype of Circulating Neutrophils (A) Schematic representation of the BCG vaccination trial. Neutrophils were isolated from 25 healthy volunteers before (D0, n = 25), 2 weeks after (D14, n = 25) and 3 months after (D90, n = 23) vaccination with BCG. (B) Absolute counts of CD10+ mature and CD10− immature neutrophils before, 2 weeks after, and 3 months after BCG vaccination (mean ± SEM, n = 25, p < 0.05, Wilcoxon signed-rank test). (C) CITRUS hierarchy plot with stratifying features using FDR of <1%. (D) Overlay of the five parental stratifying CITRUS clusters on the viSNE plot. (E) Relative abundance of the clusters before, 2 weeks after, and 3 months after vaccination. (F) Heatmaps showing the differential expression of the surface markers in the clusters of neutrophils (clusters 1, 2, and 5) and eosinophils (clusters 3 and 4) (normalized MFI clustered with Ward’s method). See also Figures S1 and S2 and Data S1.
Figure 2
Figure 2
Neutrophil Phenotype upon Exposure to Unrelated Pathogens Is Modified by BCG Cell-surface expression molecules associated with neutrophil activation were analyzed by flow cytometry upon ex vivo stimulation with M. tuberculosis, C. albicans, or LPS before, 2 weeks after, and 3 months after vaccination. (A) CD66b, (B) MPO, (C) CD11b, and (D) CD62L. Fold change of MFI as compared to medium control, upon vaccination versus baseline (mean ± SEM, n = 22 [D14], n = 18 [D90], CD62L_Mtb_D14 [n = 23], CD62L_Candida/LPS_D14 [n = 17]; p < 0.05, ∗∗p < 0.01, ∗∗∗p < 0.001, Wilcoxon signed-rank test). See also Figures S3 and S4.
Figure 3
Figure 3
BCG Vaccination Induces Trained Immunity In Vivo in Neutrophils (A–C) Neutrophils were isolated before (n = 25) and after vaccination (n = 23) with BCG and restimulated ex vivo. Production of IL-8, elastase, and lactate was measured in the supernatants. Fold increases, as compared to medium control upon vaccination versus baseline, of IL-8 (A), elastase (B), and lactate (C) to sonicated M. tuberculosis, heat-killed C. albicans, LPS, and PMA are shown. (D) Quantification of C. albicans colony-forming units (CFUs) after 17 h ex vivo incubation with neutrophils before and after BCG vaccination (mean ± SEM, ∗∗∗p < 0.001, Wilcoxon signed-rank test). (E) Fold change (compared to levels before BCG vaccination) in C. albicans-induced NET formation (n = 12). (F) Fold change in the production of ROS in response to C. albicans before and after vaccination. (G) Correlation plot showing the relationship between C. albicans-induced lactate production and the remaining number of C. albicans CFU after 17 h ex vivo incubation with neutrophils. (H) BCG vaccination increases the killing capacity of neutrophils, which may be partially mediated by enhanced glycolysis and the release of antimicrobial molecules. (I) Wild-type (WT) mice were injected with BCG or PBS, and ROS production and phagocytosis were assessed in splenic neutrophils 7 days later. (J) ROS production in CD11b+ Ly6g+ cells. (K) Phagocytosis of S. aureus BioParticles in CD11b+ Ly6g+ cells. Data are presented as mean ± SEM, p < 0.05, ∗∗p < 0.01, n = 10 mice per group, unpaired t test. See also Figure S4.
Figure 4
Figure 4
MAPKAPK2, p38, CREB, and NF-κB Phosphorylation in Neutrophils before and 3 Months after BCG Vaccination (A) Endogenous signal for the indicated phosphoproteins before BCG vaccination (D0) and 3 months post-BCG vaccination (D90). (B) Phosphoprotein phosphorylation in response to the indicated stimuli. (C) pMAPKAPK2 and pp38 co-expression upon LPS restimulation. Data are presented as mean ± SEM, p < 0.05, ∗∗p < 0.01, ∗∗∗p < 0.001, n = 11, Wilcoxon signed-rank test. See Figures S5 and S6 for gating strategy.
Figure 5
Figure 5
Vaccination with BCG Induces Epigenetic Reprogramming of Neutrophils (A) PCA of ChIP-seq data of human neutrophils isolated before and 3 months after BCG vaccination (p < 0.05, fold change > 1.5, n = 5). (B) Heatmap of genome-wide H3K4me3 changes before and 3 months after BCG vaccination. (C) Tracks of H3K4me3 peaks near STAT4. (D) Pathways associated with genes near the 230 differential peaks. (E and F) Tracks of H3K4me3 peaks near CXCL8 and IL-1β (E) and mTOR and PFKB (F). See also Figure S7.
Figure 6
Figure 6
Transcriptomic Analysis of Neutrophils Trained by BCG (A) Heatmap showing genes that are differentially expressed in human neutrophils upon BCG vaccination (p < 0.2, fold change > 1.5, n = 3). (B) Pathways associated with genes that are differentially expressed 3 months after BCG vaccination as compared to baseline. (C) Fold change in the expression of HK1 (3 months upon BCG as compared to baseline, mean ± SEM, n = 3). (D) Fold change in the expression of IPLs UMLILO and IPL-IL1 after BCG vaccination as compared to levels before vaccination in high responders and low responders (mean ± SEM, n = 6 high responders, n = 4 low responders, p < 0.05, ∗∗p < 0.01, Wilcoxon signed-rank test). (E) In contrast to low responders, high responders display increased IPL expression upon BCG vaccination, leading to higher levels of H3K4me3 on target genes and enhanced gene transcription upon secondary exposure.
Figure 7
Figure 7
Schematic Overview of the Mechanisms That May Underlie Enhanced Neutrophil Effector Function upon BCG Vaccination BCG-induced trained immunity results in an increased responsiveness of neutrophils, with effector functions such as cytokine production and killing capacity being increased upon secondary stimulation with non-related pathogens. Changes in the inflammatory profile of neutrophils upon BCG are associated with changes in epigenetic profile and cellular metabolism. Increased expression of lncRNAs, termed IPLs, might lead to elevated H3K4me3 accumulation at genes coding for antimicrobial molecules. Together with the upregulation of signaling pathways and glycolysis, epigenetic changes at the level of histone methylation result in enhanced transcription of antimicrobial proteins upon exposure to a secondary stimulus.

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