Lactobacillus rhamnosus GG stimulates dietary tryptophan-dependent production of barrier-protecting methylnicotinamide

Lacticaseibacillus rhamnosus GG (LGG) is the world's most consumed probiotic but its mechanism of action on intestinal permeability and differentiation along with its interactions with an essential source of signaling metabolites, dietary tryptophan, are unclear.Untargeted metabolomic and transcriptomic analyses were performed in LGG mono-colonized germ-free (GF) mice fed tryptophan (trp)-free or -sufficient diets. LGG-derived metabolites were profiled in vitro under anaerobic and aerobic conditions. Multiomic correlations using a newly developed algorithm discovered novel metabolites tightly linked to tight junction (TJ) and cell differentiation genes whose abundances were regulated by LGG and dietary trp. Barrier-modulation by these metabolites were functionally tested in Caco2 cells, mouse enteroids, and dextran sulfate sodium (DSS) experimental colitis. The contribution of these metabolites to barrier protection is delineated at specific TJ proteins and enterocyte-promoting factors with gain and loss of function approaches.LGG, strictly with dietary trp, promotes the enterocyte program and expression of TJ genes, particularly Ocln. Functional evaluations of fecal and serum metabolites synergistically stimulated by LGG and trp revealed a novel Vitamin B3 metabolism pathway, with methylnicotinamide (MNA) unexpectedly being the most robust barrier-protective metabolite in vitro and in vivo. Reduced serum MNA is significantly associated with increased disease activity in IBD patients. Exogenous MNA enhances gut barrier in homeostasis and robustly promotes colonic healing in DSS colitis. MNA is sufficient to promote intestinal epithelial Ocln and RNF43, a master inhibitor of Wnt. Blocking trp or Vitamin B3 absorption abolishes barrier recovery in vivo.Our study uncovers a novel LGG-regulated dietary trp-dependent production of MNA that protects the gut barrier against colitis.