PROJECT SUMMARY/ABSTRACT Oral feeding of a probiotic (Lactobacillus reuteri DSM 17938) to mice with 2 experimental autoimmune diseases can reprogram the gut microbes and markedly reduce disease severity. One mouse model is essentially the same as a condition in humans called IPEX syndrome (immunodeficiency and polyendocrinopathy, with x-linked inheritance). LR 17938 reduced the severity of the mouse’s skin lesions, improved breathing and lung inflammation, and prolonged the mouse’s life span from less than 1 month to > 4 months. Our studies identified a novel mechanism in which an adenosine, a rapid-acting anti-inflammatory molecule released from ATP, by interacting with its receptor on white blood cells (T cells) was responsible for the probiotic LR 17938 to improve the animal’s health. We also recently found that LR 17938 improves health in a mouse model of multiple sclerosis. Adenosine generated from ATP is broken down by 2 intestinal enzymes, CD39 and CD73, called ectonucleotidases. CD 39 and CD 73 are present on white blood cells and intestinal cells, but certain probiotics can also convert ATP to adenosine. Adenosine is transported into gut mucosa by nucleoside transporters (NTs). After absorption, adenosine and its active product inosine interact with a receptor (A2A) on T cells to inhibit inflammation in the body. A major gap in probiotic biology is our lack of understanding of how LR 17938 affects the adenosine pathway during Treg deficiency. We found that probiotic LR 17938 contains 5NTE (CD73) gene and that LR 17938 would be able to generate adenosine from AMP when anaerobically cultured the laboratory. However, a LR 17938 strain with a 5NTE mutation (LR 179385NTE) could not generate adenosine from AMP. Aim 1 is to assess the effects of LR 17938 to compare with LR 179385NTE on clinical outcome and the adenosine pathway in SF mice. Aim 2 is to define the critical role of probiotic 5NTE (CD73) in autoimmune protection by depleting host CD73 in SF mice. This study will lead to further investigate the mechanism of adenosine- producing probiotic strain in interaction with host immune system, as well as in modulation of microbial associated metabolites and transcriptomics in Treg deficiency. The long-term goal of these studies is to determine how to choose the best probiotic to relieve primary autoimmune diseases in humans. These conditions include IPEX syndrome due to Foxp3 gene mutation/deletion, and IPEX-like syndrome due to other single gene defects.