Germ-free animals have been used to define the vital role of commensal bacteria on the maturation of the host immune system. However, the role of bacterial residues in diet in this setting is poorly understood. Here we investigated the effect of bacterial contamination in sterile diet on the level of allergic sensitization in germ-free mice. Sterile grain-based diets ST1 and R03 were tested for the level of bacterial contamination. ST1 contained higher amount of bacterial DNA, approximately ten times more endotoxin, and induced higher, TLR4-dependent, cytokine production in dendritic cells compared to R03. In a germ-free mouse model of sensitization to the major birch pollen allergen Bet v 1, feeding on ST1 for at least two generations was associated with decreased production of allergen-specific IgE and IgG1 antibodies in sera in comparison to R03. Furthermore, reduced levels of allergen-specific and ConA-induced cytokines IL-4, IL-5 and IL-13 accompanied by increased levels of IFN-γ were detected in splenocytes cultures of these mice. Our results show that contamination of experimental diet with bacterial residues, such as endotoxin, significantly affects the development of allergic sensitization in germ-free mice. Therefore, careful selection of sterile food is critical for the outcomes of germ-free or gnotobiotic experimental models of immune-deviated diseases.

• MeSH
• Hypersensitivity immunology   pathology   MeSH
• Antigens, Plant immunology   MeSH
• Cell Differentiation drug effects   MeSH
• Breeding MeSH
• Cytokines biosynthesis   MeSH
• Dendritic Cells drug effects   MeSH
• Diet * MeSH
• DNA, Bacterial analysis   MeSH
• Endotoxins toxicity   MeSH
• Epitopes immunology   MeSH
• Germ-Free Life MeSH
• HEK293 Cells MeSH
• Immunization * MeSH
• Immunoglobulin A immunology   MeSH
• Immunoglobulin G immunology   MeSH
• DNA Contamination MeSH
• Cells, Cultured MeSH
• Humans MeSH
• Ligands MeSH
• Mitogens pharmacology   MeSH
• Mice, Inbred BALB C MeSH
• Spleen pathology   MeSH
• Toll-Like Receptor 4 metabolism   MeSH
• Digestive System immunology   MeSH
• Animals MeSH
• Check Tag
• Humans MeSH
• Animals MeSH
• Publication type
• Journal Article MeSH
• Research Support, Non-U.S. Gov't MeSH
• Names of Substances
• Antigens, Plant MeSH
• Bet v 1 allergen, Betula MeSH Browser
• Cytokines MeSH
• DNA, Bacterial MeSH
• Endotoxins MeSH
• Epitopes MeSH
• Immunoglobulin A MeSH
• Immunoglobulin G MeSH
• Ligands MeSH
• Mitogens MeSH
• Toll-Like Receptor 4 MeSH

Increasing numbers of clinical trials and animal experiments have shown that probiotic bacteria are promising tools for allergy prevention. Here, we analyzed the immunomodulatory properties of three selected lactobacillus strains and the impact of their mixture on allergic sensitization to Bet v 1 using a gnotobiotic mouse model. We showed that Lactobacillus (L.) rhamnosus LOCK0900, L. rhamnosus LOCK0908 and L. casei LOCK0919 are recognized via Toll-like receptor 2 (TLR2) and nucleotide-binding oligomerization domain-containing protein 2 (NOD2) receptors and stimulate bone marrow-derived dendritic cells to produce cytokines in species- and strain-dependent manners. Colonization of germ-free (GF) mice with a mixture of all three strains (Lmix) improved the intestinal barrier by strengthening the apical junctional complexes of enterocytes and restoring the structures of microfilaments extending into the terminal web. Mice colonized with Lmix and sensitized to the Bet v 1 allergen showed significantly lower levels of allergen-specific IgE, IgG1 and IgG2a and an elevated total IgA level in the sera and intestinal lavages as well as an increased transforming growth factor (TGF)-β level compared with the sensitized GF mice. Splenocytes and mesenteric lymph node cells from the Lmix-colonized mice showed the significant upregulation of TGF-β after in vitro stimulation with Bet v 1. Our results show that Lmix colonization improved the gut epithelial barrier and reduced allergic sensitization to Bet v 1. Furthermore, these findings were accompanied by the increased production of circulating and secretory IgA and the regulatory cytokine TGF-β. Thus, this mixture of three lactobacillus strains shows potential for use in the prevention of increased gut permeability and the onset of allergies in humans.

• MeSH
• Hypersensitivity immunology   prevention & control   MeSH
• Antigens, Plant toxicity   MeSH
• Germ-Free Life immunology   MeSH
• HEK293 Cells MeSH
• Immunoglobulin A immunology   MeSH
• Immunoglobulin G immunology   MeSH
• Lacticaseibacillus rhamnosus immunology   MeSH
• Lacticaseibacillus casei immunology   MeSH
• Humans MeSH
• Mice, Inbred BALB C MeSH
• Mice MeSH
• Nod2 Signaling Adaptor Protein immunology   MeSH
• Intestinal Mucosa immunology   MeSH
• Toll-Like Receptor 2 immunology   MeSH
• Animals MeSH
• Check Tag
• Humans MeSH
• Mice MeSH
• Animals MeSH
• Publication type
• Journal Article MeSH
• Research Support, Non-U.S. Gov't MeSH
• Names of Substances
• Antigens, Plant MeSH
• Bet v 1 allergen, Betula MeSH Browser
• Immunoglobulin A MeSH
• Immunoglobulin G MeSH
• NOD2 protein, human MeSH Browser
• Nod2 protein, mouse MeSH Browser
• Nod2 Signaling Adaptor Protein MeSH
• TLR2 protein, human MeSH Browser
• Tlr2 protein, mouse MeSH Browser
• Toll-Like Receptor 2 MeSH

BACKGROUND: Reduced microbial diversity has been associated with inflammatory bowel disease (IBD) and probiotic bacteria have been proposed for its prevention and/or treatment. Nevertheless, comparative studies of strains of the same subspecies for specific health benefits are scarce. Here we compared two Bifidobacterium longum ssp. longum strains for their capacity to prevent experimental colitis. METHODS: Immunomodulatory properties of nine probiotic bifidobacteria were assessed by stimulation of murine splenocytes. The immune responses to B. longum ssp. longum CCM 7952 (Bl 7952) and CCDM 372 (Bl 372) were further characterized by stimulation of bone marrow-derived dendritic cell, HEK293/TLR2 or HEK293/NOD2 cells. A mouse model of dextran sulphate sodium (DSS)-induced colitis was used to compare their beneficial effects in vivo. RESULTS: The nine bifidobacteria exhibited strain-specific abilities to induce cytokine production. Bl 372 induced higher levels of both pro- and anti-inflammatory cytokines in spleen and dendritic cell cultures compared to Bl 7952. Both strains engaged TLR2 and contain ligands for NOD2. In a mouse model of DSS-induced colitis, Bl 7952, but not Bl 372, reduced clinical symptoms and preserved expression of tight junction proteins. Importantly, Bl 7952 improved intestinal barrier function as demonstrated by reduced FITC-dextran levels in serum. CONCLUSIONS: We have shown that Bl 7952, but not Bl 372, protected mice from the development of experimental colitis. Our data suggest that although some immunomodulatory properties might be widespread among the genus Bifidobacterium, others may be rare and characteristic only for a specific strain. Therefore, careful selection might be crucial in providing beneficial outcome in clinical trials with probiotics in IBD.

• MeSH
• Bifidobacterium classification   physiology   MeSH
• Dendritic Cells microbiology   pathology   MeSH
• HEK293 Cells MeSH
• Immunoenzyme Techniques MeSH
• Colitis chemically induced   prevention & control   MeSH
• Humans MeSH
• Disease Models, Animal * MeSH
• Mice, Inbred BALB C MeSH
• Mice MeSH
• Probiotics pharmacology   MeSH
• Nod2 Signaling Adaptor Protein genetics   metabolism   MeSH
• Dextran Sulfate toxicity   MeSH
• Intestines microbiology   physiopathology   MeSH
• Toll-Like Receptor 2 genetics   metabolism   MeSH
• Animals MeSH
• Check Tag
• Humans MeSH
• Mice MeSH
• Female MeSH
• Animals MeSH
• Publication type
• Journal Article MeSH
• Research Support, Non-U.S. Gov't MeSH
• Names of Substances
• Nod2 Signaling Adaptor Protein MeSH
• Dextran Sulfate MeSH
• Tlr2 protein, mouse MeSH Browser
• Toll-Like Receptor 2 MeSH