Project description:Shifts in the gut microbiota composition, called dysbiosis, have been directly associated with acute and chronic diseases. However, the underlying biological systems connecting gut dysbiosis to systemic inflammatory pathologies are not well understood. Phospholipids (PLs) act as precursors of both, bioactive inflammatory and resolving mediators. Their dysregulation is associated with chronic diseases including cancer. Gut microbial-derived lipids are structurally unique and capable of modulating host’s immunity. Lactobacillus johnsonii N6.2 is a Gram-positive gut symbiont with probiotic characteristics. L. johnsonii N6.2 reduces the incidence of autoimmunity in animal models of Type 1 Diabetes and improves general wellness in healthy volunteers by promoting, in part, local and systemic anti-inflammatory responses. By utilizing bioassay-guided fractionation methods with bone marrow-derived dendritic cells (BMDCs), we report here that L. johnsonii N6.2 purified lipids induce a transcriptional signature that resembles that of migratory (mig)DCs. RNAseq-based analysis showed that BMDCs stimulated with L. johnsonii N6.2 total lipids upregulate maturation-mig related genes Cd86, Cd40, Ccr7, Icam1 along with immunoregulatory genes including Itgb8, Nfkbiz, Jag1, Adora2a, IL2ra, Arg1, and Cd274. Quantitative reverse transcription (qRT)-PCR analysis indicated that PLs are the bioactive lipids triggering the BMDCs response. Antibody-blocking of surface Toll-like receptor (TLR)2 resulted in boosted PL-mediated upregulation of pro-inflammatory Il6. Chemical inhibition of the IKKα kinase from the non-canonical NF-κB pathway specifically restricted upregulation of Il6 and Tnf. Phenotypically, PL-stimulated BMDCs display an immature like-phenotype with significantly increased surface ICAM-1. This study provides insight into the immunoregulatory capacity of Gram-positive, gut microbial-derived phospholipids on innate immune responses.
Project description:Oxidative stress due to endogenous hydrogen peroxide production by Lactobacillus species is a well-known issue in the food industry. In this study, the transcriptional response to oxygen of Lactobacillus johnsonii, one of the H2O2-producing strains used in the food industry, was analyzed. It was found that aerobic growth conditions led to a more than two-fold downregulation of 45 genes as compared to anaerobic growth, whereas 6 genes were more than twofold upregulated. Among the upregulated genes were two genes that displayed significant homology to NADH-dependent oxidoreductase (NOX). The postulated transcriptional regulation of the nox promoter by oxygen was studied using a GUS-reporter construct, confirming a 2.1-fold upregulated GUS-expression upon aerobic growth. Exposure to sublethal levels of hydrogen peroxide did not result in significant regulation of the nox promoter. In a previous study of hydrogen peroxide production by L. johnsonii, a NADH flavin reductase (NFR) was identified to be involved in hydrogen peroxide production. An NFR-deficient derivative was strongly impaired in H2O2 production, but regained a partial H2O2 producing capacity upon prolonged oxygen exposure. The nox-promoter appeared to be 3.6-fold upregulated under aerobic conditions in the NFR-deficient background, which may imply a role of this gene in the regained H2O2 production. Indeed, deletion of the nox-gene in the NFR-deletion background, resulted in a strain that no longer produced H2O2, also during prolonged exposure to oxygen. The double-mutant (nfr, nox) displayed strongly impaired aerobic growth and oxygenation induced rapid growth stagnation that is not caused by H2O2. We conclude that H2O2 production in L. johnsonii is primarily dependent on NFR but can also involve an oxygen-inducible NADH oxidase under aerobic conditions. Moreover, our results imply that H2O2 production plays a prominent role in oxygen tolerance of L. johnsonii. loop design of the samples including two shortcuts
Project description:Oxidative stress due to endogenous hydrogen peroxide production by Lactobacillus species is a well-known issue in the food industry. In this study, the transcriptional response to oxygen of Lactobacillus johnsonii, one of the H2O2-producing strains used in the food industry, was analyzed. It was found that aerobic growth conditions led to a more than two-fold downregulation of 45 genes as compared to anaerobic growth, whereas 6 genes were more than twofold upregulated. Among the upregulated genes were two genes that displayed significant homology to NADH-dependent oxidoreductase (NOX). The postulated transcriptional regulation of the nox promoter by oxygen was studied using a GUS-reporter construct, confirming a 2.1-fold upregulated GUS-expression upon aerobic growth. Exposure to sublethal levels of hydrogen peroxide did not result in significant regulation of the nox promoter. In a previous study of hydrogen peroxide production by L. johnsonii, a NADH flavin reductase (NFR) was identified to be involved in hydrogen peroxide production. An NFR-deficient derivative was strongly impaired in H2O2 production, but regained a partial H2O2 producing capacity upon prolonged oxygen exposure. The nox-promoter appeared to be 3.6-fold upregulated under aerobic conditions in the NFR-deficient background, which may imply a role of this gene in the regained H2O2 production. Indeed, deletion of the nox-gene in the NFR-deletion background, resulted in a strain that no longer produced H2O2, also during prolonged exposure to oxygen. The double-mutant (nfr, nox) displayed strongly impaired aerobic growth and oxygenation induced rapid growth stagnation that is not caused by H2O2. We conclude that H2O2 production in L. johnsonii is primarily dependent on NFR but can also involve an oxygen-inducible NADH oxidase under aerobic conditions. Moreover, our results imply that H2O2 production plays a prominent role in oxygen tolerance of L. johnsonii.
2014-07-01 | GSE52876 | GEO
Project description:Internalization of extracellular vesicles from Lactobacillus johnsonii N6.2 elicit an RNA sensory response in human pancreatic cell lines.
Project description:Background: Lactobacillus plantarum is found in a variety of fermented foods and as such, consumed for centuries. Some strains are natural inhabitants of the human gastro-intestinal tract and like other Lactobacillus species, L. plantarum has been extensively studied for its immunomodulatory properties and its putative health-promoting effects (probiotic). Being the first line of host defense intestinal epithelial cells (IEC) are key players in the recognition and initiation of responses to gut microorganisms. Results: Using high-density oligonucleotide microarrays we examined the gene expression profiles of differentiated Caco-2 cells exposed to various doses of L. plantarum. In addition, the effects were correlated to monolayer permeability studies and measurement of lactic acid production. A transcriptional dose-dependent IEC response to L. plantarum was found. Incubation of Caco-2 with a low bacterial dose induced a specific response, not due to cytotoxicity or production of lactic acid, including modulation of cell cycle and cell signaling functions. Exposure of Caco-2 cells to larger amounts of bacteria, accompanied by the production of lactic acid and glucose depletion, provoked increased permeability and supposed non-specific defense responses. Conclusions: These results suggest that IEC are able to sense and react to the presence of gut bacteria. This study provides the first description of global transcriptional response of human IEC to a commensal lactic acid bacterium, and it shows the importance of choosing physiological bacterial doses to prevent the observation of non-specific host reactions. Caco-2 cells were exposed for 10h to Lactobacillus. Fourteen samples are analyzed: 4 control Caco-2, 4 Caco-2 exposed to a low dose (10) of Lactobacillus, 4 Caco-2 exposed to a medium dose (100) of Lactobacillus, 2 Caco-2 exposed to a high dose (1000) of Lactobacillus. All 14 RNA samples are labeled with Cy5 and hybridized to a common reference (undifferentiated Caco-2, untreated) RNA labeled with Cy3
Project description:Whole genome DNA microarray designed for the probiotic L. johnsonii strain NCC533 was used for comparative genomic hybridization (CGH) of L. johnsonii ATCC 33200T, L. johnsonii BL261, L. gasseri ATCC 33323T and L. iatae BL263 (CECT 7394T). In these experiments, the fluorescence ratio distributions obtained with L. iatae and L. gasseri showed characteristic inter-species profiles. The percentage of conserved L. johnsonii NCC533 genes was about 83% in the L. johnsonii strains comparisons and decreased to 51% and 47% for L. iatae and L. gasseri, respectively. These results confirmed the separate status of L. iatae from L. johnsonii at the level of species, and also that it is closer to L. johnsonii than L. gasseri. L. johnsonii, L. gasseri, and L. iatae strains were hybridized versus L. johnsonii NCC533, some with replicates