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Probiotic Limosilactobacillus reuteri DSM 17938 (DSM 17938) prolonges the survival of Treg-deficient scurfy (SF) mice and reduces multiorgan inflammation by a process requiring adenosine receptor 2A (A2A) on T cells. We hypothesized that L. reuteri-derived ecto-5’-nucleotidase (ecto-5’NT) activity acts to generate adenosine, which may be a central mediator for L. reuteri protection in SF mice. We evaluated DSM 17938-5’NT activity and the associated adenosine and inosine levels in plasma, gut and liver of SF mice. We examined orally fed DSM 17938, DSM 17938Δ5NT (with a deleted 5’NT gene), and DSM 32846 (BG-R46) (a naturally selected strain derived from DSM 17938). Results showed that DSM 17938 and BG-R46 produced adenosine while “exhausting” AMP, whereas DSM 17938∆5NT did not generate adenosine in culture. Plasma 5’NT activity was increased by DSM 17938 or BG-R46, but not by DSM 17938Δ5NT in SF mice. BG-R46 increased both adenosine and inosine levels in the cecum of SF mice. DSM 17938 increased adenosine levels, whereas BG-R46 increased inosine levels in the liver. DSM 17938Δ5NT did not significantly change the levels of adenosine or inosine in the GI tract or the liver of SF mice. Although regulatory CD73+CD8+ T cells were decreased in spleen and blood of SF mice, these regulatory T cells could be increased by orally feeding DSM 17938 or BG-R46, but not DSM 17938Δ5NT. In conclusion, probiotic-5’NT may be a central mediator of DSM 17938 protection against autoimmunity. Optimal 5’NT activity from various probiotic strains could be beneficial in treating Treg-associated immune disorders in humans.

When new-born mice are subjected to acute maternal separation stress, cow-milk based formula feeding, and brief recurrent hypoxia with cold stress, they develop gut inflammation similar to the phenotype of neonatal necrotizing enterocolitis, characterised by an increase in gut mucosal effector T (Teffs) and reduced Foxp3+ regulatory T (Tregs) cells. The imbalance can be prevented by probiotic Limosilactobacillus reuteri DSM 17938 (LR 17938). We hypothesised that LR 17938 could potentiate a tolerogenic function of Tregs. To analyse whether LR 17938 can educate Tregs to improve their tolerogenic potency during neonatal stress, we isolated T cells (Tregs and Teffs) from ‘donor’ mice fed with either LR 17938 (107 cfu) or control media. The cells were adoptively transferred (AT) by intraperitoneal injection (5 × 105 cells/mouse) to new-born (d5) recipient mice. Mice were then separated from their dams, fed formula by gavage, and exposed to hypoxia and cold stress (NeoStress) for 4 days. We analysed the percentage of Tregs in CD4+T helper cells in the intestine (INT) and mesenteric lymph nodes (MLN) of recipient mice. We found that: (1) the percentage of Tregs in the INT and MLN following NeoStress were significantly reduced compared to dam-fed unstressed mice; (2) AT of either naïve Tregs or LR-educated Tregs to mice with Neostress increased the percentage of Tregs in the INT and MLN compared to the percentage in NeoStress mice without Treg treatment; however, LR-educated Tregs increased the Tregs significantly more than naïve Tregs; and (3) AT of LR-educated Tregs reduced pro-inflammatory CD44+Foxp3-NonTregs and inflammatory CX3CR1+ dendritic cells in the intestinal mucosa of NeoStress mice. In conclusion, adoptive transfer of Tregs promotes the generation of and/or migration of endogenous Tregs in the intestinal mucosa of recipient mice. Importantly, probiotic-educated Tregs are more potent than naïve Tregs to enhance immune tolerance following neonatal stress.

The combination of CD39 and CD73 degrade ATP to adenosine that shifts ATP-driven pro-inflammatory immune cell activity toward an anti-inflammatory state. This immunological switch is a major mechanism for regulatory T cells (Tregs) to control inflammation. However, in Treg-deficiency-induced autoimmune pathologies, exemplified by IPEX syndrome (immune dysregulation, polyendocrinopathy, enteropathy, with X-linked inheritance, as modeled by the scurfy (SF) mouse), the expression of these molecules on T cells is unknown. We analyzed homeostatic markers CD39 and CD73 on T cells in spleen and peripheral blood by flow cytometry in SF males and their sibling WT male mice at d21. Among Foxp3+ Tregs in WT mice, ~68% cells had CD39 and 85% cells had CD73 expression. In the spleens of SF mice, the % of CD39+CD4+ T cells was higher than the % in WT mice, while no difference was observed in the CD73+CD4+ T cells. In addition, the data showed ~70% of NonTregs expressed CD39, 2-fold higher than both % of CD73+ NonTregs in SF, and % of CD39+ NonTregs in WT. Only ~10% of these CD39+ and CD73+ NonTregs had the CD25 expression. The proportion of CD8+ T cells in splenocytes and blood of SF mice were significantly higher than those in WT mice. Amongst CD8+ T cells, the % of CD39+ T cells was significantly increased, conversely, the % of CD73+ T cells was significantly decreased in SF compared to WT mice. We conclude that normally, CD73 is a more highly expressed Treg surface marker than CD39; however, in Treg-deficiency, there is a higher expression by CD4+ T and CD8+ T cells of CD39 than CD73; and CD39 and CD73 expression may not be due to cell activation. This study provides data to aid further discovery of potential biomarkers and therapeutic targets in Treg deficiency diseases. Supported by grants from NIH/NIAID (R03AI153725)

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