Probiotics have been used to prevent/treat a variety of digestive diseases including alcoholic liver disease (ALD). Live probiotics need to colonize the gut to exert their function. Unfortunately, underlying disease states provide an unfavorable environment for probiotic bacterial gut colonization, which diminishes probiotics’ function. In last few years, we showed that LGG culture supernatant (LGGs, without live bacteria) was effective in the prevention of ALD in experimental models of acute and chronic alcohol exposure in mice. However, how LGG supernatant exerts its therapeutic effects is not fully understood. Exosomes are nanoparticles (NPs) derived from cell endocytosis which act as transmitters between cells. Recent studies show that bacteria, both Gram-negative and Gram-positive, produce NPs. The NPs derived from “bad” bacteria have been demonstrated to be pathogenic. However, “good” bacteria-, probiotics-derived NPs have not been studied. Our preliminary study showed that administration of LGG-derived exosome-like NPs (LDNPs) effectively reversed ALD in binge-on- chronic alcohol exposure mouse model, suggesting that probiotic LGGs may exert its function through LDNPs in ALD. LDNPs administration markedly increased intestinal AhR activity, IL-22, regenerating islet-derived 3 (Reg3) beta and gamma expression, which play a key role in maintaining gut microbiota homeostasis and preventing bacterial intestinal transcytosis. In addition, LDNPs administration significantly increased intestinal epithelial cell (IEC) tight junctions and decreased circulating LPS concentration, associated with upregulation of intestinal Nrf2 signaling, which is known for protecting intestinal barrier junctions against oxidative stress-induced damage by alcohol. Metabolomic analysis revealed that LDNPs contain high levels of microbial metabolites of tryptophan, which are AhR ligands, indicating LDNPs may activate intestinal AhR signaling. Furthermore, we demonstrated that ginger exosome-like nanoparticles (GDNPs) are preferentially taken up by LGG, suggesting that GDNP may serve as a prebiotic to enhance the effects of LGG. These preliminary studies provide the groundwork for our central hypothesis that, by activating intestinal AhR-Nrf2 signaling, LDNPs increase intestinal expression of Il-22, Reg3 and tight junctions, and modulate gut microbiota homeostasis and enhance intestinal barrier function, leading to the suppression of ALD. We will test our hypothesis in following three specific aims: (1) Determine the role of bacteria-derived NPs in ALD; (2) Define the mechanisms of the beneficial effect of LDNPs in ALD; (3) Determine whether ginger-derived exosome-like nanoparticles (GDNPs) treatment enhances LDNP production and AhR agonist enrichment that lead to improved effects of LDNPs against ALD. Completion of this study is expected to significantly impact the development of LGG-based probiotic therapeutics in the treatment of alcohol-associated liver diseases.

The goals of this project are to investigate the mechanisms of probiotic Lactobacillus rhamnosus GG action on alcoholic liver disease. This study will have a major impact on the development of a probiotics-based new therapeutic strategy for the prevention and treatment of alcoholic liver disease. PHS 398/2590 (Rev. 05/01) Page 1 Continuation Format Page