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Enhanced immune response of BMDCs pulsed with H9N2 AIV and CpG

ABSTRACT: Dendritic cells (DCs), professional antigen presenting cells, have demonstrated effective in controllingthe initial of innate immune, while CpG could improve the performance of immune system. To explorethe mechanism of CpG enhancing the immune response, we compared different stimulated mouse DCswith systemic approach microarrays. Analysis revealed 1840 differentially expressed genes in H9N2stimulated group, more than 1728 altered genes in inactive H9N2 group. Investigation also proved thatCpG/inactive H9N2 co-stimulation changed 2140 genes, more than that in H9N2 group, strongly demon-strated that CpG improved the performance of inactive H9N2 vaccination. Pathways analysis founded thatDCs response rapid to shift in their maturation state, which involved Toll-like receptor (TLR) pathwaysignificantly. Microarrays results were also verified by qRT-PCR with 14 elected representative genes. Fur-ther analysis proved that co-stimulatory molecules (CD40, CD80, CD86 and MHC-II), regulatory protein(IRF-7 and TRAF-6) and pro-inflammatory cytokines (IL-1, IL-6 and IL-12) were all changed and involvedin DCs maturation. At last we demonstrated TLR signalling pathway in chicken bone marrow-deriveddendritic cells (chBM-DCs) stimulated with CpG. The distinct transcriptional profiles of DCs pulsed withvarious stimuli expanded our understanding of how DCs respond and recognize influenza.

ORGANISM(S): Mus musculus

PROVIDER: GSE41454 | GEO | 2012/10/11

SECONDARY ACCESSION(S): PRJNA177034

REPOSITORIES: GEO

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Project description:Toll-like receptor (TLR)-induced maturation of dendritic cells (DCs) leads to the production of proinflammatory cytokines as well as the upregulation of various molecules involved in T cell activation. These are believed to be the critical events that account for the induction of the adaptive immune response. Here, we have examined the role of micro-RNA-155 (miR-155) in DC function and the induction of immunity. Using a model where the transfer of self-antigen-pulsed, TLR-matured DCs can induce a functional CD8 T cell response and autoimmunity, we find that DCs lacking miR-155 have an impaired ability to break immune tolerance. Importantly, transfer of self- antigen-pulsed DCs overexpressing miR-155 was sufficient to break tolerance in the absence of TLR stimuli. Although these unstimulated DCs induced T cell function in vivo, there was no evidence for the upregulation of costimulatory ligands or cytokine secretion. Further analysis showed that miR-155 influenced the level of the phosphatase SHIP1 in DCs, and that the lack of SHIP1 in DCs was sufficient to break T cell tolerance in vivo, again in the absence of TLR induced DC maturation. Our study demonstrates that the overexpression of miR-155 in DCs is a critical event that is alone sufficient to break self tolerance and promote a CD8-mediated autoimmune response in vivo. This process is independent of the induction of conventional DC maturation markers, indicating that miR-155 regulation of SHIP represents a unique axis that regulates DC function in vivo. Dendritic cell culture: Bone marrow was flushed from tibias and femurs of mice with HBSS. Bone marrow cells were cultured at 2x106 cells/ml in 10 ml non-tissue culture treated dishes in RPMI 1640 containing 10% LPS-free FBS, Penicillin:streptomycin glutamine 2-mercaptoethanol (cRPMI), with 40ng/ml murine GM-CSF (Peprotech). On day 3 of cultures 10ml of fresh media was added also containing 40ng/ml GM-CSF. On days 6 and 8, 10ml of media was removed and centrifuged to collect cells, which were resuspended in 10ml fresh media containing GM-CSF and were added back to dishes. Non-adherent cells were isolated on day 9. DCs were plated in 24-well plates at 2x106 cells/ml in 1ml of cRPMI with or without the class B CpG ODN1826 (ACGT DNA Technologies corporation, Toronto, ON, Canada) at 10?M final concentration overnight. Array analysis of mi-RNA expression: BMDCs from WT mice were either stimulated with CpG or left in media alone overnight. Whole RNA was isolated using mirVana miRNA isolation kit following the manufacturers instructions. RNA samples were labeled and hybridized to Agilent mouse miRNA 8x15K arrays (Agilent). Data was generated from 8 individual samples (4 unstimulated and 4 CpG-treated).

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Enhanced immune response of BMDCs pulsed with H9N2 AIV and CpG

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