Thomas K. Hoang

Resetting microbiota by Lactobacillus reuteri inhibits T reg deficiency–induced autoimmunity via adenosine A2A receptors

Regulatory T (T reg) cell deficiency causes lethal, CD4+ T cell–driven autoimmune diseases. Stem cell transplantation is used to treat these diseases, but this procedure is limited by the availability of a suitable donor. The intestinal microbiota drives host immune homeostasis by regulating the differentiation and expansion of T reg, Th1, and Th2 cells. It is currently unclear if T reg cell deficiency–mediated autoimmune disorders can be treated by targeting the enteric microbiota. Here, we demonstrate that Foxp3+ T reg cell deficiency results in gut microbial dysbiosis and autoimmunity over the lifespan of scurfy (SF) mouse. Remodeling microbiota with Lactobacillus reuteri prolonged survival and reduced multiorgan inflammation in SF mice. L. reuteri changed the metabolomic profile disrupted by T reg cell deficiency, and a major effect was to restore levels of the purine metabolite inosine. Feeding inosine itself prolonged life and inhibited multiorgan inflammation by reducing Th1/Th2 cells and their associated cytokines. Mechanistically, the inhibition of inosine on the differentiation of Th1 and Th2 cells in vitro depended on adenosine A2A receptors, which were also required for the efficacy of inosine and of L. reuteri in vivo. These results reveal that the microbiota–inosine–A2A receptor axis might represent a potential avenue for combatting autoimmune diseases mediated by T reg cell dysfunction.

Lactobacillus reuteri for Infants with Colic: A Double-Blind, Placebo-Controlled, Randomized Clinical Trial

Objective To assess the safety of probiotic Lactobacillus reuteri strain Deutsche Sammlung von Mikroorganismen (DSM) 17938 with daily administration to healthy infants with colic and to determine the effect of L reuteri strain DSM 17938 on crying, fussing, inflammatory, immune, and microbiome variables. Study design We performed a controlled, double‐blinded, phase 1 safety and tolerability trial in healthy breast‐fed infants with colic, aged 3 weeks to 3 months, randomly assigned to L reuteri strain DSM 17938 (5 × 108 colony‐forming units daily) or placebo for 42 days and followed for 134 days. Results Of 117 screened infants, 20 were randomized to L reuteri strain DSM 17938 or placebo (sunflower oil) (in a 2:1 ratio) with 80% retention. Eleven of the 20 (55%) presented with low absolute neutrophil counts (<1500/mm3), which resolved in all subjects by day 176. L reuteri strain DSM 17938 produced no severe adverse events and did not significantly change crying time, plasma bicarbonate, or inflammatory biomarkers. Fecal calprotectin decreased rapidly in both groups. In the infants with dominant fecal gram negatives (Klebsiella, Proteus, and Veillonella), resolution of colic was associated with marked decreases in these organisms. Conclusions Daily administration of L reuteri strain DSM 17938 appears to be safe in newborn infants with colic, including those with neutropenia, which frequently coexists. A placebo response of 66% suggests that many infants with colic will have resolution within 3 weeks. Trial registration ClinicalTrials.gov: NCT01849991.

Protective effect of Lactobacillus reuteri DSM 17938 against experimental necrotizing enterocolitis is mediated by Toll-like receptor 2

Lactobacillus reuteri DSM 17938 (LR 17938) has been shown to reduce the incidence and severity of necrotizing enterocolitis (NEC). It is unclear if preventing NEC by LR 17938 is mediated by Toll-like receptor 2 (TLR2), which is known to mediate proinflammatory responses to bacterial cell wall components. NEC was induced in newborn TLR2-/- or wild-type (WT) mice by the combination of gavage-feeding cow milk-based formula and exposure to hypoxia and cold stress. Treatment groups were administered formula supplemented with LR 17938 or placebo (deMan-Rogosa-Sharpe media). We observed that LR 17938 significantly reduced the incidence of NEC and reduced the percentage of activated effector CD4+T cells, while increasing Foxp3+ regulatory T cells in the intestinal mucosa of WT mice with NEC, but not in TLR2-/- mice. Dendritic cell (DC) activation by LR 17938 was mediated by TLR2. The percentage of tolerogenic DC in the intestine of WT mice was increased by LR 17938 treatment during NEC, a finding not observed in TLR2-/- mice. Furthermore, gut levels of proinflammatory cytokines IL-1β and IFN-γ were decreased after treatment with LR 17938 in WT mice but not in TLR2-/- mice. In conclusion, the combined in vivo and in vitro findings suggest that TLR2 receptors are involved in DC recognition and DC-priming of T cells to protect against NEC after oral administration of LR 17938. Our studies further clarify a major mechanism of probiotic LR 17938 action in preventing NEC by showing that neonatal immune modulation of LR 17938 is mediated by a mechanism requiring TLR2. NEW & NOTEWORTHY Lactobacillus reuteri DSM 17938 (LR 17938) has been shown to protect against necrotizing enterocolitis (NEC) in neonates and in neonatal animal models. The role of Toll-like receptor 2 (TLR2) as a sensor for gram-positive probiotics, activating downstream anti-inflammatory responses is unclear. Our current studies examined TLR2 -/- mice subjected to experimental NEC and demonstrated that the anti-inflammatory effects of LR 17938 are mediated via a mechanism requiring TLR2.

Adenosine A2A receptor deletion blocks the beneficial effects of Lactobacillus reuteri in regulatory T-Deficient scurfy mice

The lack of a functional Foxp3 transcription factor and regulatory T (Treg) cells causes lethal, CD4+ T cell-driven autoimmune diseases in scurfy (SF) mice and humans. Recent studies have shown that adenosine A2A receptor activation limits inflammation and tissue damage, thereby playing an anti-inflammatory role. However, the role of the adenosine A2A receptor in the development of disease in SF mice remains unclear. Using a genetic approach, we found that adenosine A2A receptor deletion in SF mice (SF⋅A2A-/-) does not affect early life events, the development of a lymphoproliferative disorder, or hyper-production of pro-inflammatory cytokines seen in the Treg-deficiency state. As shown previously, Lactobacillus reuteri DSM 17938 treatment prolonged survival and reduced multiorgan inflammation in SF mice. In marked contrast, A2A receptor deletion completely blocked these beneficial effects of L. reuteri in SF mice. Altogether, these results suggest that although absence of the adenosine A2A receptor does not affect the development of disease in SF mice, it plays a critical role in the immunomodulation by L. reuteri in Treg-deficiency disease. The adenosine A2A receptor and its activation may have a role in treating other Treg dysfunction-mediated autoimmune diseases.

GM-CSF Antibodies and Peripheral Treg Cells as Potential Markers of Disease Activity to Guide Therapeutic Decisions in Pediatric IBD

Objectives: 1. To measure and compare granulocytemacrophage colony-stimulating factor (GM-CSF) antibodies and peripheral regulatory T cells (Tregs) cells as non-invasive biomarkers in active inflammatory bowel disease (IBD) and remission in pediatric IBD. 2. To determine the ability of these biomarkers to predict severity/type and choice of therapy in pediatric IBD. Methods: We hypothesized that patients with severe IBD requiring biologics will have higher circulating GM-CSF antibodies and lower Foxp3+ Tregs at baseline, with subsequent improvement in response to therapy. 28 patients were prospectively analyzed. Blood collected preand post-treatment was analyzed for GM-CSF antibodies by ELISA and Tregs by flow cytometry. Clinically, PCDAI and PUCAI were scored. Wilcoxon Rank Sum tests were used to compare baseline values between groups, and paired signed-rank tests used to compare pre- and posttreatment values. Results: Median GM-CSF antibodies trended higher (0.50 µg/mL vs. 0.37) and Tregs lower (5.23% vs. 6.71%) in the biologic cohort with no significant difference between cohorts. In response to biologics, 37% showed decrease in GM-CSF antibodies and 75% showed increase in Tregs. GM-CSF antibodies trended higher in Crohn’s disease (CD) than ulcerative colitis (UC) (0.81 vs. 0.31, p=0.073). Activity indices showed significant improvement in the biologic cohort of UC patients (p=0.04). Conclusion: GM-CSF antibodies and peripheral Tregs may reflect disease activity and thereby guide IBD therapy. Although no individual biomarker could predict need for biologics, their trend favors their use in a predictive model to identify the most effective treatment strategy in pediatric IBD.