Mycobacterium manresensis induces trained immunity in vitro

Abstract

The COVID-19 pandemic posed a global health crisis, with new severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) variants weakening vaccine-driven protection. Trained immunity could help tackle COVID-19 disease. Our objective was to analyze whether heat-killed Mycobacterium manresensis (hkMm), an environmental mycobacterium, induces trained immunity and confers protection against SARS-CoV-2 infection. To this end, THP-1 cells and primary monocytes were trained with hkMm. The increased secretion of tumor necrosis factor alpha (TNF-α), interleukin (IL)-6, IL-1β, and IL-10, metabolic activity, and changes in epigenetic marks suggested hkMm-induced trained immunity in vitro. Healthcare workers at risk of SARS-CoV-2 infection were enrolled into the MANRECOVID19 clinical trial (NCT04452773) and were administered Nyaditum resae (NR, containing hkMm) or placebo. No significant differences in monocyte inflammatory responses or the incidence of SARS-CoV-2 infection were found between the groups, although NR modified the profile of circulating immune cell populations. Our results show that M. manresensis induces trained immunity in vitro but not in vivo when orally administered as NR daily for 14 days.

Keywords: Biological sciences; Immunology; Microbiology; Molecular biology.

Graphical abstract

Figure 1

Increased inflammatory responses of THP-1 cells upon heat-killed M. manresensis training (A) Schematic overview of trained immunity methodology in THP-1 cells. Production of cytokines after training and the secondary stimulation with (B) 10 ng/mL LPS, (C) 5x10⁶ cfu/mL hkS. aureus, and (D) 0.218 ng nucleocapsid/mL InSARS-CoV-2. (E) Levels of L(+)-Lactate in untrained (PBS) and hkBCG- and hkMm-trained monocytes. (n = 6, ∗p < 0.05, Wilcoxon matched-pairs signed-rank test, mean ± SEM). hkMm, heat-killed M. manresensis; hkBCG, heat-killed BCG; hkS. aureus, heat-killed S. aureus; InSARS-CoV-2, inactivated SARS-CoV-2.

Figure 2

Increased cytokine responses of primary monocytes upon heat-killed M. manresensis training (A–E) Cytokines from the supernatant of cultured trained monocytes were measured by ELISA after the secondary stimulation with (A) 10 ng/mL LPS, (B) 5x10⁶ cfu/mL hkS. aureus, (C) 10⁶ cfu/mL hkMtb, (D) 0.218 ng nucleocapsid/mL InSARS-CoV-2, and (E) 10^4.29 TCID₅₀/mL InSIV H1N1. (F) Levels of L(+)-Lactate in untrained (PBS) and hkBCG- and hkMm-trained primary monocytes. (n = 5–11, ∗p < 0.05, ∗∗p < 0.01, ∗∗∗p < 0.001, Wilcoxon matched-pairs signed-rank test, mean ± SEM; Spearman’s ρ and p values are shown in the right corner of the correlation plot). hkMm, heat-killed M. manresensis; hkBCG, heat-killed BCG; hkS. aureus, heat-killed S. aureus; hkMtb, heat-killed M. tuberculosis; InSARS-CoV-2, inactivated SARS-CoV-2; InSIV H1N1, inactivated SIV H1N1.

Figure 3

Trained immunity by heat-killed M. manresensis generates epigenetic modifications (A and B) Enrichment in (A) H3K4me3 and (B) H3K27ac in TNF-α, IL-6, and IL-1β gene promoters in untrained and hkBCG- and hkMm-trained primary monocytes. Results are reported as enrichment of immunoprecipitated DNA relative to the input (n = 3, Wilcoxon matched-pairs signed-rank test, mean ± SEM). (C) Correlation between H3K4me3 and H3K27ac epigenetic modifications in TNF-α, IL-6, and IL-1β gene promoters. (Spearman’s ρ and p values are shown in the right corner of the plot). hkMm, heat-killed M. manresensis; hkBCG, heat-killed BCG.

Figure 4

Flowchart of study participants A total of 320 individuals were assessed for eligibility to participate in the study; 317 were randomized to receive Nyaditum resae (n = 214) or placebo (n = 103), and they composed the ITT sample. The PP sample was composed of 198 participants, 131 in the Nyaditum resae group and 67 in the placebo group.

Figure 5

Heat-killed M. manresensis does not protect against SARS-CoV-2 infection (A and B) Kaplan-Meier curves and patients at risk data for the time to COVID-19 infection in each treatment arm for the (A) ITT and the (B) PP population. Follow-up days truncated at 150, but there are patients with up to 175 days of follow-up.

Figure 6

Heat-killed M. manresensis does not modify the inflammatory responses of monocytes from healthy volunteers (A–E) Fold change of TNF-α, IL-6, and IL-1β after stimulating with (A) 100 ng/mL LPS, (B) 10⁶ cfu/mL hkMtb, (C) 10⁷ cfu/mL hkS. aureus, (D) 22.5 pfu/mL InSARS-CoV-2, or (E) 10^3.99 TCID₅₀/mL InSIV H1N1 (day 15/day 0). (n = 57, n = 18 Placebo, n = 39 Nyaditym resae, Wilcoxon Mann-Whitney test, boxplot shows the median, quartiles, and minimum and maximum values). Raw data are detailed in Table S8 hkMtb, heat-killed M. tuberculosis; hkS. aureus, heat-killed S. aureus; InSARS-CoV-2, inactivated SARS-CoV-2; InSIV H1N1, inactivated SIV H1N1.

Figure 7

Heat-killed M. manresensis changes the profile of circulating immune cell populations from healthy volunteers (A–Q) Cell populations were analyzed at baseline and 15 days post-treatment administration, and the corresponding fold change (FC) was calculated. Fold change of the frequency of (A) lymphocytes, (B) B cells, (C) T cells, (D) CD8⁺ T cells, (E) CD4⁺ T cells, (F) CD4⁺CD25⁺ T cells, (G) Treg cells, (H) NK cells, (I) CD56⁺CD16⁻ NK cells, (J) CD56⁺CD16⁺ NK cells, (K) CD56⁻CD16⁺ NK cells, (L) neutrophils, (M) monocytes, (N) classical monocytes, (O) intermediate monocytes, (P) non-classical monocytes, and (Q) CD16⁻CD14⁻cells (day 15/day 0), (n = 57, n = 18 Placebo, n = 39 Nyaditym resae, Wilcoxon Mann-Whitney test, boxplot shows the median, quartiles, and minimum and maximum values). Raw data are detailed in Table S9.