Circadian rhythm influences induction of trained immunity by BCG vaccination

Abstract

BACKGROUNDThe antituberculosis vaccine bacillus Calmette-Guérin (BCG) reduces overall infant mortality. Induction of innate immune memory, also termed trained immunity, contributes toward protection against heterologous infections. Since immune cells display oscillations in numbers and function throughout the day, we investigated the effect of BCG administration time on the induction of trained immunity.METHODSEighteen volunteers were vaccinated with BCG at 6 pm and compared with 36 age- and sex-matched volunteers vaccinated between 8 am and 9 am. Peripheral blood mononuclear cells were stimulated with Staphylococcus aureus and Mycobacterium tuberculosis before, as well as 2 weeks and 3 months after, BCG vaccination. Cytokine production was measured to assess the induction of trained immunity and adaptive responses, respectively. Additionally, the influence of vaccination time on induction of trained immunity was studied in an independent cohort of 302 individuals vaccinated between 8 am and 12 pm with BCG.RESULTSCompared with evening vaccination, morning vaccination elicited both a stronger trained immunity and adaptive immune phenotype. In a large cohort of 302 volunteers, early morning vaccination resulted in a superior cytokine production capacity compared with later morning. A cellular, rather than soluble, substrate of the circadian effect of BCG vaccination was demonstrated by the enhanced capacity to induce trained immunity in vitro in morning- compared with evening-isolated monocytes.CONCLUSIONSBCG vaccination in the morning induces stronger trained immunity and adaptive responses compared with evening vaccination. Future studies should take vaccine administration time into account when studying specific and nonspecific effects of vaccines; early morning should be the preferred moment of BCG administration.FUNDINGThe Netherlands Organization for Scientific Research, the European Research Council, and the Danish National Research Foundation.

Keywords: Cytokines; Immunology; Innate immunity; Monocytes; Vaccines.

Figure 1. Flow diagram of individuals included in this study.

A total of 327 healthy volunteers were assessed, of which 2 did not meet inclusion criteria. Of the 325 individuals vaccinated, 18 participants were vaccinated in the evening and 307 were vaccinated in the morning. Of the 307 morning-vaccinated individuals, 5 were excluded due to medication use or lack of information. Of the 302 morning-vaccinated individuals, 36 sex- and age-matched controls were selected for further analysis with the evening-vaccinated individuals.

Figure 2. Overview of morning- and evening-vaccinated healthy volunteers, including blood counts.

(A) Two groups of healthy volunteers were vaccinated with BCG at 2 time points: 18 volunteers between 6 pm and 6:30 pm, while 36 (1:2 ratio) sex- and age-matched controls were vaccinated between 8 am and 9 am. Blood was collected in the morning at baseline, 2 weeks, and 3 months after BCG vaccination. (B) Whole blood complete blood counts and leukocyte differential (neutrophil, lymphocyte, and monocyte counts) of morning-vaccinated individuals and evening-vaccinated individuals. Mean ± SEM; morning n = 36, evening n = 18. Kruskal-Wallis test with Dunn’s multiple-comparison test.

Figure 3. BCG vaccination in the morning elicits a stronger trained immunity phenotype compared with evening vaccination.

IL-1β, IL-6, and TNF-α production in response to S. aureus stimulation 2 weeks and 3 months after BCG vaccination, and production of IFN-γ in response to M. tuberculosis (Mtb) stimulation of morning-vaccinated (A–C and G) and evening-vaccinated (D–F and H) individuals. Mean ± SEM; n = 36 morning vaccinated, n = 18 evening vaccinated. ***P < 0.001, **P < 0.01, *P < 0.05 by Friedman’s test with Dunn’s multiple-comparison test. (I) Fold changes (compared with baseline) of monocyte and lymphocyte percentages within PBMC fraction. Mean ± SEM; morning n = 36, evening n = 18. Kruskal-Wallis test with Dunn’s multiple-comparison test.

Figure 4. Trained immunity responses induced by BCG vaccination are most profound in the early morning.

(A) A total of 302 healthy volunteers were BCG vaccinated between 8 am and 12 pm and blood was collected before, 2 weeks after, and 3 months after BCG vaccination. Fold changes (compared with baseline) 2 weeks and 3 months after BCG vaccination of PBMC-produced IL-1β (B), IL-6 (C), and TNF-α (D) in response to S. aureus stimulation, and IFN-γ production in response to M. tuberculosis stimulation (E). Mean ± SEM; n = 68 vaccinated between 8 am and 9 am, n = 80 vaccinated between 9 am and 10 am, n = 84 vaccinated between 10 am and 11 am, n = 66 vaccinated between 11 am and 12 pm. ***P < 0.001, **P < 0.01, *P < 0.05 by Kruskal-Wallis test with Dunn’s multiple-comparison test.

Figure 5. Percentages of monocytes and lymphocytes in PBMC fraction.

(A and B) Spearman’s correlation plots of lymphocyte percentages (P = 0.004) (A) and monocyte percentages (P = 0.002) (B) within the PBMC fraction against time of blood collection at baseline visit. (C and D) Comparisons of fold changes (FC) in lymphocyte (C) and monocyte percentages (D) between morning-vaccinated subgroups. Mean ± SEM; n = 68 vaccinated between 8 am and 9 am, n = 80 vaccinated between 9 am and 10 am, n = 84 vaccinated between 10 am and 11 am, n = 66 vaccinated between 11 am and 12 pm. Kruskal-Wallis test with Dunn’s multiple-comparison test.

Figure 6. Trained immunity responses in vitro are affected by time of the day.

(A) Blood was collected from healthy volunteers for isolation of serum and Percoll purification of monocytes during the morning (8 am) and evening (6 pm) on the same day. (B and C) Fold changes (compared with medium-primed, LPS-restimulated conditions) in IL-6 (B) and TNF-α (C) production of BCG-primed monocytes supplemented with morning-derived serum versus evening-derived serum. (D–F) Fold changes (relative to medium-primed, LPS-restimulated conditions) in IL-6 (D), TNF-α (E), and IL-10 (F) production after LPS restimulation of BCG-trained monocytes derived after morning blood donation versus evening blood donation. Mean ± SEM; n = 18 morning evening serum, n = 23 morning evening monocytes. ***P < 0.001, **P < 0.01 by Wilcoxon’s matched-pairs signed-rank test.

Figure 7. mRNA expression of CLOCK and ARNTL in monocytes from individuals before and 1 month after BCG vaccination.

Individuals were divided into responders, which were protected from subsequent yellow fever viremia (maximum yellow fever viremia CT > 36 [n = 3]), and nonresponders (CT < 36 [n = 3]). H3K27ac levels at gene promoters (Z score) of CLOCK and ARNTL before and 1 month after BCG vaccination in monocytes (n = 2, both groups).

Figure 8. The effect of BCG vaccination on epigenome remodeling in PBMCs differs based on the time of vaccination.

Differential chromatin accessibility (DA) analysis of an interaction effect between BCG training (3 months after BCG compared with baseline) and time of vaccination (evening compared with morning). (A) Open chromatin regions were assigned to genes based on proximity. (B) KEGG pathways enrichment of regions for which remodeling 3 months after BCG differs based on the time of vaccination (showing top 10 pathways for each direction, all displayed pathways passed FDR of 0.02). (C and D) JASPAR and CODEX transcription factor binding sites (TFBSs) enrichment of regions for which remodeling 3 months after BCG differs based on the time of vaccination (showing top 10 TFs for each direction, all displayed TFs passed FDR of 0.005). n = 36 morning vaccinated, n = 18 evening vaccinated; DA was performed with LIMMA, which computes P values with a moderated t test; enrichment analysis was performed with a Fisher’s exact test; Benjamini-Hochberg procedure was used to control the FDR.