Fig. 8: Water supplemented with microbial metabolites in BCG-vaccinated hosts leads to innate immune training in AMs in naive mice.

a, Experimental schema of in vivo continuous supplementation of circulating SCFAs butyrate and l-carnitine via (DW + M) given to naïve mice for 3 weeks. Mice were then placed on regular DW for 1 week. The control animals were on regular DW. b,c, IL-6 (*P = 0.0071) (b) and MIP-1α (*P = 0.0381) (c) protein production by airway AM isolated from animals on DW + M or DW, with (S) or without (US) re-stimulation. n = 3 mice per group. d, PCA of gene expression in airway AM from metabolite-supplemented (DW + M) and control (DW) hosts with (S) or without (US) stimulation. n = 3 mice per group. e, Signature scores of genes involved in Ag processing and presentation in airway AM comparing control (DW) with metabolite-supplemented (DW + M) animals with (S) and without (US) stimulation (P = 4.34 × 10⁻⁶). Horizontal lines in violin plots denote medians and dotted lines denote lower and upper quartiles. f, Numbers of M. tuberculosis CFU in the lung tissue of naive animals given metabolite-supplemented water (DW + M) compared to BCG-vaccinated (DW + M versus BCG, *P = 0.0203) (BCG versus PBS *P = 0.0115) n = 3 mice in PBS group, n = 4 mice in BCG group, 4/DW + M. g–j, Comparing MFI of MHC II expression in airway AM from WT (**P = 0078 and *P = 0.0118) (g), TLR2KO (**P = 0.0043) (h), TLR4KO (i) and NOD2-KO (**P = 0.0062) (j) mice trained ex vivo with BCG-conditioned or control serum and upon restimulation after 3-d resting. Data in bar graphs are presented as mean ± s.e.m. Statistical analysis was done by two-tailed t-test for data in b, c and f. Adjusted P values are presented for violin plots (e) and obtained using limma package and BH correction (Methods). Statistical analysis was determined by two-tailed t-test for data in g–j, comparing PBS serum (US) versus BCG serum (US) and PBS serum (S) versus BCG serum (S).