Materials and Methods == == 2 . 1 . 1 . Introduction == The human intestinal microbial ecosystem includes at least 1, 000 distinct bacterial species that possess 100-fold more genes compared with their human hosts [1]. This complex microbial system modulates the pathogenesis of different diseases, including allergies, tumors, and autoimmune diseases such as T1D with either beneficial or detrimental effects [2]. In humans, T1D susceptibility has been linked to compositional changes in the gut microbiota and, specifically, with a significant increased representation of bacteria of the Bacteroidetes phylum and a decrease in the number ofBifidobacterium, Lactobacillus, andClostridiumstrains [35]. Studies in preclinical models of T1D have indicated that the gut microbiota plays a key role in controlling disease onset and severity. For example , the absence of MyD88, an adaptor molecule involved in TLR signaling, protects NOD mice from autoimmune T1D by inducing a protective microbiota profile characterized by a low Firmicutes/Bacteroidetes ratio and Lactobacillaceae strain enrichment [6]. The beneficial effect of Lactobacillaceae strains in T1D was also proven by the observation that treatment of NOD mice with VSL#3, a probiotic enriched in Lactobacillaceae largely used to modulate microbiota composition in humans, prevents T1D [7]. The mechanisms underlying the influence of the microbiota on T1D pathogenesis have not yet been clarified. Recent evidence indicates that T cells originating in the gut mucosa can modulate autoimmunity at sites distal from the intestines such as the central nervous system (CNS) in experimental allergic encephalomyelitis (EAE), the experimental model of multiple sclerosis [8]. Similarly, gut microbiota modifications can affect autoimmune T1D Lagociclovir by altering T cell immunity within the intestinal mucosa and in the pancreatic lymph nodes (PLN) and islets [9]. In Lagociclovir support of this hypothesis, there are some lines of evidence showing that gut immunity is altered in T1D. For example , alterations in immune homeostasis and signs of immune dysregulation such as increased expression of MHC II and inflammatory cytokines have been found in the intestines of both T1D patients and preclinical T1D models even before clinical onset of Lagociclovir the disease [1016]. In addition , intestinal immune regulation, specifically differentiation of FoxP3+Treg cells in the intestinal mucosa, is significantly impaired in T1D patients [17]. These observations have led to the hypothesis that intestinal immunity may be important for regulation of T1D. Lagociclovir That was also confirmed from finding that a large fraction of the T cells infiltrating the pancreatic islets of T1D patients and NOD mice originate in the intestine and express gut-homing receptors, such as CCR9 and47 [18, 19]. Thus, the microbiota composition may modulate T1D by altering intestinal immunity and immune regulation. Proinflammatory microbial species can elicit specific immune responses that trigger inflammation and promote T1D by destroying gut immune homeostasis. On the contrary, beneficial microbial species can confer Lagociclovir protection against T1D by inducing immune tolerance in the gut mucosa and a protective Teff/Treg cell ratio in the intestine, and in distal organs, such as the pancreatic islets. Dietary Rabbit Polyclonal to Smad1 (phospho-Ser465) factors, like vitamins, can similarly affect T1D pathogenesis by modulating gut immunity. A vitamin A-rich diet as well as the administration of vitamin A and its metabolite, all-trans-retinoic acid (RA), has been shown to prevent autoimmune T1D in preclinical models [20, 21]. Vitamin A and its metabolite RA are crucial for maintenance of immune tolerance in the intestine; thus their administration may counterregulate autoimmune T1D by regulating the reciprocal relationship between Treg and Teff cells in the intestine. Here, we demonstrate that administration of the Lactobacillaceae-enriched VSL#3 probiotic, alone or in combination with RA, prevents T1D in NOD mice by enriching the local microbiota with Lactobacillaceae strains and by inducing substantial modifications in the microbiota composition with increase in Clostridia and Rikenellaceae species and reduction in the Bacteroidetes strain S24-7. In addition , VSL#3 administration and the resulting microbiota modifications generate a protolerogenic intestinal microenvironment with high expression of IDO and low expression of inflammatory IL-1. The VSL#3-induced protolerogenic microenvironment promoted CD103+DC differentiation and reduced the Teff/Treg cell ratios within the gut mucosa, mesenteric lymph nodes (MLN), and PLN, thereby modulating T1D pathogenesis. == 2 . Materials and Methods == == 2 . 1 . Mice == Four-week-old NOD mice were purchased from Charles River Laboratories (Calco, Italy) and maintained under specific pathogen-free conditions in the animal facility of San Raffaele Scientific Institute..