442 - Bacille Calmette-Guérin (BCG) stimulation of cultured human monocytes in vitro reshapes the extracellular metabolome in an age-dependent manner

To characterize age-specific shifts in extracellular metabolism of human monocytes (Mos) induced by BCG reprogramming in vitro.


Design/Methods: We used a human Mo training platform to detect metabolites significantly perturbed after BCG stimulation and reprogramming. Newborn (NB) and adult (AD) CD33+ Mos were stimulated with RPMI (control) or BCG (SSI, Denmark) for 24h, washed and cultured for 6 days prior to stimulation with the Toll-like Receptor-4 agonist lipopolysaccharide (LPS) for 24h. Day 1 (D1, post-BCG stim) and D7 (BCG reprogrammed followed by LPS stim) supernatants (sups) were subjected to mass-spectrometry based global metabolomic analysis.

Results: 1249 metabolites were detected in BCG-conditioned Mo sups. Principal component analysis demonstrated a clear separation between NB and AD. NB vs AD conditions at D0, 1, and 7 demonstrated significant changes with 6.4% of metabolites being differentially regulated on D7. Random forest analysis indicated that the 30 top-ranked metabolites distinguishing NB vs AD exposed to BCG were dominated by lipids, both on D1 and 7. BCG- vs RPMI-stimulated NB Mo sups (D1) showed significant increases in saturated free fatty acids, consistent with published in vivo and in vitro observations in whole blood of BCG vaccinated NB. D7 vs D1 AD Mo sups demonstrated significantly elevated pyruvate and lactate concentrations, while NB Mo sups did not, suggesting activation of glycolysis only in AD. Top enriched pathways in sups from BCG-reprogrammed NB Mos included steroid hormones (pregnenolones, p=0.008; progestins, p=0.047), lipid metabolism (sphingomyelins, p< 0.001; phosphatidylcholines, p=0.001) and glutathione synthesis (g-glutamyl amino acids, p< 0.001), all involved in innate immune regulation. BCG-trained AD vs NB Mos showed increased tryptophan catabolism, evidenced by significant sup increases in kynurenine and anthranilate metabolites, possibly reflecting induction of indoleamine 2,3-dioxygenase expression (enzyme converting tryptophan to kynurenine) by key cytokines involved in AD trained immunity (i.e. TNF, IL-1b). γ-glutamyl amino acids and 5-oxoproline were significantly elevated in BCG-trained NB Mo sups, consistent with a robust oxidative stress response compared to AD.

Conclusion(s): BCG induces distinct immunometabolic shifts that may contribute to age-specific immunogenicity and off-target effects.