BCG vaccination in humans inhibits systemic inflammation in a sex-dependent manner

Abstract

BACKGROUNDInduction of innate immune memory, also termed trained immunity, by the antituberculosis vaccine bacillus Calmette-Guérin (BCG) contributes to protection against heterologous infections. However, the overall impact of BCG vaccination on the inflammatory status of an individual is not known; while induction of trained immunity may suggest increased inflammation, BCG vaccination has been epidemiologically associated with a reduced incidence of inflammatory and allergic diseases.METHODSWe investigated the impact of BCG (BCG-Bulgaria, InterVax) vaccination on systemic inflammation in a cohort of 303 healthy volunteers, as well as the effect of the inflammatory status on the response to vaccination. A targeted proteome platform was used to measure circulating inflammatory proteins before and after BCG vaccination, while ex vivo Mycobacterium tuberculosis- and Staphylococcus aureus-induced cytokine responses in peripheral blood mononuclear cells were used to assess trained immunity.RESULTSWhile BCG vaccination enhanced cytokine responses to restimulation, it reduced systemic inflammation. This effect was validated in 3 smaller cohorts, and was much stronger in men than in women. In addition, baseline circulating inflammatory markers were associated with ex vivo cytokine responses (trained immunity) after BCG vaccination.CONCLUSIONThe capacity of BCG to enhance microbial responsiveness while dampening systemic inflammation should be further explored for potential therapeutic applications.FUNDINGNetherlands Organization for Scientific Research, European Research Council, and the Danish National Research Foundation.

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

Figure 1. Flow chart of the study.

Flow diagram describing the number of participants who were enrolled in the study, who were excluded or dropped out of the study, or excluded from further analysis.

Figure 2. Correlations between baseline inflammatory markers and baseline whole blood counts.

(A) Spearman’s correlations between absolute whole blood counts (monocytes, total white blood cells, neutrophils, lymphocytes, eosinophils, basophils, platelets, red blood cells, and hemoglobin) and circulating inflammatory markers at baseline (before BCG vaccination). Positive correlations are depicted in red, negative correlations in blue (n = 302). Spearman’s correlations between whole blood neutrophil counts and circulating oncostatin M (OSM) (B), and between whole blood monocyte counts and circulating IL-6 (C) are shown as examples of positive correlations (n = 300).

Figure 3. Inflammation after BCG vaccination.

Fold changes in circulating inflammatory markers on day 14 versus baseline (A) and day 90 versus baseline (B). Significant changes compared with baseline are depicted in red, nonsignificant changes are depicted in gray (n = 290-fold change on day 14 versus baseline, n = 275-fold change on day 90 versus baseline; FDR < 0.05 is considered significant). Fold changes in TWEAK (C) and SIRT2 (D) are depicted as examples of significantly decreased circulating inflammatory markers after BCG vaccination (*FDR < 0.05, **FDR < 0.01).

Figure 4. Differences in fold change in circulating proteins between scar-positive and scar-negative individuals.

(A) Significant differences in fold changes in circulating proteins (significantly different in the entire cohort after FDR < 0.05 correction) between scar-positive (n = 290) and scar-negative (n = 15) individuals 90 days after vaccination. Fold changes higher than 1 depicted on a red scale, fold changes lower than 1 depicted on a blue scale. P < 0.05 by Wilcoxon’s matched-pairs signed-rank test. (B) SIRT2 was plotted as an example of a protein that is significantly lower in scar-positive individuals 3 months after BCG vaccination.

Figure 5. Validation of changes in circulating proteins after BCG vaccination.

Fold changes in circulating inflammatory markers (IL-17C, TWEAK, DNER, ADA, and NT-3) after BCG vaccination compared with baseline, validated in at least 1 of the 3 validation cohorts. The blue line represents cohort 1 (n = 15), the green line cohort 2 (n = 9), and the red line cohort 3 (n = 15). *P < 0.05 by Wilcoxon’s matched-pairs signed-rank test. Median ± range is depicted per time point.

Figure 6. Sex-specific effect of BCG vaccination on systemic inflammation.

(A) Comparison of baseline circulating inflammatory proteins plotted as fold changes between males (n = 132) and females (n = 171). Significant changes between sexes are depicted in red (FDR < 0.05). (B) Comparison of inflammatory proteins between males and females from the discovery cohort (300BCG) were plotted against the comparison between males (n = 215) and females (n = 278) from the validation cohort (500FG). Proteins that were only significantly different in the 300BCG cohort are depicted in green (n = 9), those that were only significant in the 500FG cohort are depicted in blue (n = 3), and the proteins significantly different between males and females in both cohorts are depicted in red (n = 34) and are labeled with their name (FDR < 0.05). Fold changes of circulating inflammatory markers on day 14 versus baseline (C) and day 90 versus baseline (D) in the male-only (n = 132) versus the female-only (n = 171) subset. Significant changes compared with baseline in the male-only subset are depicted in red (FDR < 0.05), and proteins that did not significantly change after BCG vaccination in either the male-only or the female-only subset are depicted in gray. There were no proteins significantly different in the female-only subset.

Figure 7. Correlations between circulating hormones and inflammatory proteins.

(A) Fold changes of proteins that significantly changed after BCG vaccination in males were correlated to baseline testosterone, adiponectin, leptin, and resistin concentrations. Only proteins with a significant correlation with one of the hormones are depicted in this figure. *P < 0.05, **P < 0.01, ***P < 0.001 by Spearman’s correlation. The color represents the strength and the direction of the correlation. (B) Spearman’s correlation between testosterone at baseline and fold change in CXCL1 2 weeks after vaccination is shown as an example.

Figure 8. Ex vivo PBMC–derived cytokine production and associations with baseline circulating inflammatory proteins.

Fold changes compared with baseline of ex vivo PBMC–derived S. aureus–induced TNF-α responses (A) and M. tuberculosis–induced IFN-γ responses (B) on day 14 versus baseline and day 90 versus baseline as examples of upregulated cytokine responses after BCG vaccination (fold change day 14 versus baseline n = 289, fold change day 90 versus baseline n = 275). *P < 0.05, **P < 0.01, ***P < 0.001 by Wilcoxon’s matched-pairs signed-rank test. (C) Fold changes in IFN-γ in response to M. tuberculosis on day 14 versus baseline separated by sex. (D) Spearman’s correlations between baseline inflammatory proteins and fold changes in PBMC-derived S. aureus–induced IL-1β, IL-6, and TNF-α responses and M. tuberculosis–induced IFN-γ responses separated by sex. Significant, positive correlations (ρ > 0) are depicted in red, significant negative correlations (ρ < 0) in blue, and nonsignificant correlations in white. Only proteins with a significant correlation with at least one of the ex vivo cytokine responses are depicted in this figure.