Fig. 7: Circulating microbial metabolites in BCG-vaccinated hosts leads to innate immune training of AMs.

a, Experimental schema of in vitro innate training of AM. b, Representative bright-field microscopic images of AM after training with BCG-S or treatment with control serum or after restimulation. Representative of two independent experiments (n = 4 wells per condition). c–e, Increased median fluorescence intensity (MFI) of MHC II (*P = 0.0132 and *P = 0.0182) (c) and frequencies of IL-6 (*P = 0.0362) (d), but not TNF (e), producing AM trained with BCG-S and upon re-stimulation after 24-h or 3-d resting. Each data point represents n = 3 wells per PBS serum and n = 4 wells per BCG serum. f,g, Cytokine/chemokine protein contents in culture supernatants of AM trained with BCG-S and upon restimulation after 3-d resting. Each data point represents n = 4 wells per condition. TNF, **P = 0.0047; KC, *P = 0.0491. h–j, Inhibition of innate immune training of AM by BCG-S upon histone methylation and acetylation blockade with methyltransferase inhibitor MTA or acetyltransferase inhibitor EGCG. Data shown are IL-1β (***P = 0.0008) (h), IL-6 (***P = 0.001) (i) and TNF (****P < 0.0001) (j) protein contents produced by AM upon restimulation. Each data point represents n = 4 wells per condition. Data in bar graphs are presented as mean ± s.e.m. Statistical analysis was determined by two-tailed t-test for data in c–e, comparing PBS serum (US) versus BCG serum (US) and PBS serum (S) versus BCG serum (S) within each time point poststimulation for data in f and g, comparing PBS serum (S) versus BCG serum (S) for h–j, comparing to BCG-S.