Gene expression profiling of human IL-10+ B-cells
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ABSTRACT:
ORGANISM(S): Homo sapiens
PROVIDER: GSE50895 | GEO | 2014/06/25
SECONDARY ACCESSION(S): PRJNA219309
REPOSITORIES: GEO
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Project description:Lactobacilli are probiotics that, among other health promoting effects, have been ascribed immunostimulating and virus preventive properties. Certain lactobacilli species have been shown to possess strong IL-12 inducing properties. As IL-12 production depends on the up-regulation of type I interferons, we hypothesized that the strong IL-12 inducing capacity of L. acidophilus NCFM in murine bone marrow derived DC is caused by an up-regulation of IFN-β, which subsequently stimulates the induction of IL-12 and the dsRNA binding toll like receptor (TLR)-3. The expression of the genes encoding IFN-β, IL-12, IL-10 and TLR-3 in DC upon stimulation with L. acidophilus NCFM was measured. L. acidophilus NCFM induced a much stronger expression of ifn-β, il-12 and il-10 compared to the synthetic dsRNA ligand Poly I:C, whereas the levels of expressed tlr-3 were similar. By the use of whole genome microarray gene expression, we investigated whether other genes related to the viral defence were up-regulated in DC upon stimulation with L. acidophilus NCFM and found that various virus defence related genes, both early and late, were among the strongest up-regulated genes. The IFN-β stimulating capability was also detected in another L. acidophilus strain, but was not a property of other probiotic bacteria tested (B. bifidum and E. coli nissle).The IFN-β inducing capacity was markedly reduced in TLR-2 -/- DCs, dependent on endocytosis and the major cause of the induction of il-12 and tlr-3 in L. acidophilus NCFM stimulated cells. Collectively, our results reveal that certain lactobacilli trigger the expression of viral defence genes in DC in a TLR-2 manner through induction of IFN- β. Experiment Overall Design: In the experiment Lactobacillus NCFM were added to murine dendritic cells and stimulated for 4, 10 or 18 hours. These were compared to control experiment at the same timepoints. Experiments were run in triplicates except for control 10h and control 18h which were only in duplicate, giving a total of 16 arrays.
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Project description:Pro-inflammation triggered by microbial lipopolysaccharide (LPS) through Toll-like receptor (TLR) 4 in the presence of interferon (IFN)-g induces cytokine secretion in dendritic cells (DCs) tightly regulated by a defined differentiation program. This DC differentiation is characterized by a dynamic immune activating but also tolerance inducing phenotype associated with irreversible down-modulation of cytokines. CD40L on activated T cells further modifies DC differentiation. Using DNA micro arrays we showed down-regulated mRNA levels of TLR signaling molecules while CD40/CD40L signaling molecules were up-regulated at a time when LPS/IFN-g activated DCs have ceased cytokine expression. Accordingly we demonstrated that CD40/CD40L but not TLR4 or TLR3 signaling mediated by LPS or poly (cytidylic-inosinic) acid (poly I:C) and dsRNA re-established the capacity to secret interleukin (IL)-12 in LPS/IFN-g activated DCs, which have exhausted their potential for cytokine secretion. This resulting TH1 polarizing DC phenotype â which lacked accompanying secretion of the crucial immune suppressive IL-10 - enhanced activation of cytotoxic T lymphocytes (CTLs). We therefore conclude that immune modulation is restricted to a secondary T-cell mediated stimulus at an exhausted DC state which prevents an immune tolerant DC phenotype. These findings impacts on the rational design of TLR activated DC-based cancer vaccines for the induction of anti-tumoral CTL responses. Experiment Overall Design: Monocyte derived DCs were matured for 6 hours with 30 ng/ml lipopolysaccharide (LPS, E. coli strain O111:B4, Calbiochem, San Diego, CA) and 1.000 U/ml IFN-g (Imukin, Boehringer Ingelheim, Austria). RNA of DCs was isolated after 6, 12, 24, or 48 hours of maturation using the RNeasy kit (Qiagen, Hilden, Germany) and analyzed by the RZPD (German Resource Centre for Genome Research, Berlin) using the Affymetrix DNA micro array platform (U133 plus 2.0). DNA array data from different maturation-time experiments were compared to controls that were kept in culture for the same time without stimulation.
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