Quantitative analysis of miRNA changes in CD4+ T cells responding to myelin antigen
ABSTRACT: Mice were immunized with either MOG35-55 in CFA or with CFA alone and 14 days later PLN cells were isolated and stimulated in vitro with MOG35-55 for 3 days followed by CD4+ T cell sorting. Microarray analysis of miRNA expression profiles was performed comparing sorted CD4+ T cells from MOG35-55 in CFA immunized mice to CD4+ T cells from mice immunized with CFA alone. The data are from magnetic beads sorted CD4+ T cells from peripheral lymph nodes from 5 pooled animals per data point
Project description:Mice were immunized with either MOG35-55 in CFA or with CFA alone and 14 days later PLN cells were isolated and stimulated in vitro with MOG35-55 for 3 days followed by CD4+ T cell sorting. Microarray analysis of miRNA expression profiles was performed comparing sorted CD4+ T cells from MOG35-55 in CFA immunized mice to CD4+ T cells from mice immunized with CFA alone. Overall design: The data are from magnetic beads sorted CD4+ T cells from peripheral lymph nodes from 5 pooled animals per data point
Project description:C57BL/6 mice were immunized by subcutaneous injection of MOG35-55 peptide.DIM post-treatment was given at DAY 10 of the disease progression. CD4 T cells were isolated from the brain of the mice on day 15 of the MOG35-55 peptide immunization. Total RNA was extracted and subjected to microRNA high-throughput array with Affymetrix platform.
Project description:CD44 wild-type and knockout C57 BL/6 mice were immunized by subcutaneous injection of MOG35-55 peptide. CD4 T cells were isolated from spleens of the mice on day 15 of the MOG35-55 peptide immunization. Peripheral blood lymphocytes (PBLs) were prepared from multiple sclerosis patients and normal individuals. Total RNA was extracted and subjected to microRNA high-throughput array with Affymetrix platform.
Project description:The study assessed the efficacy of R-flurbiprofen in experimental autoimmune encephalomyelitis (EAE) models of multiple sclerosis in mice. R-flurbiprofen, also known as tarenflurbil, is the R-enantiomer of the cyclooxyygenase inhibitor S-flurbiprofen. It is ineffective in terms of cyclooxygenase inhibition and has no relevant toxicity in humans. Oral R-flurbiprofen prevented and attenuated primary progressive EAE in C57BL6/J mice and relapsing-remitting EAE in SJL mice, even if the treatment was initiated on or after the first flare of the disease. R-flurbiprofen reduced immune cell infiltration and microglia activation and inflammation in the spinal cord, brain and optic nerve and attenuated myelin destruction and EAE-evoked hyperalgesia. R-flurbiprofen treatment increased CD4+CD25+FoxP3+ regulatory T-cells, CTLA4+ inhibitory T-cells and interleukin-10, whereas the EAE-evoked upregulation of pro-inflammatory genes in the spinal cord was strongly reduced (Sentrix6 results). The effects were associated with an increase of plasma and cortical endocannabinoids but decreased spinal prostaglandins, the latter likely due to R- to S inversion. The promising results suggest potential efficacy of R-flurbiprofen in human MS. To assess effects of R-flurbiprofen on EAE evoked gene regulations in the spinal cord a genome wide expression analysis was performed using Illumina Sentrix 6 v2 BeadChips. For the microarray study female C57BL6/J mice were immunized according to a standard protocol using the Hooke Kit™ MOG35-55/CFA emulsion PTX (EK-2110, Hooke Labs, St Lawrence, MA), which contains 200 µg myelin oligodendrocyte glycoprotein (MOG) 35-55 emulsified in 200 µl Complete Freund’s Adjuvant (CFA). The emulsion was injected subcutaneously at two sites followed by two intraperitoneal (i.p.) injections of 200 ng pertussis toxin (PTX) in phosphate buffered saline (PBS), the first 1-2 h after MOG35-55, and the second 24 h after MOG35-55. Control mice received CFA without MOG35-55 (sham mice). Treatment with R-flurbiprofen or vehicle (n = 12 per group) was started 5 days after immunization and was administered continuously via the drinking water up to the end. Spinal cords were dissected out during the flare of the disease, day 16 after immunization. For microarray analysis, total RNA was extracted from homogenizedlumbar spinal cord tissue with Trizol reagent, followed by clean-up and DNase I treatment with QIAGEN RNeasy mini kit. RNA quality was checked (Nanodrop ND-1000, Agilent 2100 Bioanalyzer), and subsequently biotinylated and hybridized to Mouse Sentrix-6 V2 Expression BeadChips (Illumina). Each sample consisted of pooled lumbar spinal cord tissue from 3 animals and 3 replicate samples were analyzed per group, i.e. the analysis is based on 9 mice per group. Groups were CFA-control with vehicle treatment, CFA-control with R-flurbiprofen, EAE-vehicle and EAE-R-flurbiprofen treatment. Treatment was started 5 days after immunization. For dissection, pairs were matched according to the clinical scores. QC, labeling, hybridization and raw data evaluation and normalization were done according to standard protocols at the core facilities of the Deutsche Krebsforschungszentrum, Heidelberg, Germany.
Project description:Recent data from our group, demonstrate that infusion of myelin oligodendrocyte glycoprotein (MOG35-55) peptide, leads to induction of MOG35-55-specific Tregs and subsequent suppression of Experimental Autoimmune Encephalomyelitis (EAE), the mouse model of multiple sclerosis. Amelioration of EAE was accompanied by reduced MOG-specific Th1 and Th17 responses in the draining lymph nodes (dLNs). Phenotypic analysis of the dLNs of MOG-infused mice revealed a significant Treg-mediated reduction in the recruitment of 7AAD-CD3-CD19-CD11c+CD11bhighGr-1+ iDCs compared to non-infused control immunized mice. Focusing on the delineation of novel molecules/genes that are involved in the MOG-specific Treg-mediated suppression of autoimmune responses, we have isolated highly purified iDCs from MOG infused and non-infused control immunized mice. Gene expression profiles of iDCs isolated from tolerized or immunized mice. Affymetrix MG 430 2.0 whole genome arrays were performed in triplicates for tolerized iDCs and immunized iDCs (6 arrays in total, 5 of them were analyzed). To obtain genes significantly regulated in iDCs upon tolerization with MOG35-55, the expression profiles of iDCs isolated from tolerized or immunized mice were compared to each other. After total RNA extraction, reverse transcription, cDNA extraction, the biotinylated cRNA was transcribed, fragmented, and 15 µg cRNA hybridized to the 6 GeneChip arrays: Group1 iDCs isolated from tolerized mice, Group2 iDCs isolated from immunized mice.
Project description:Murine lymphocytes T helper cells were obtained from spleen of C57Bl/6 mice and miR-155 deficient mice strains that have been immunised with MOG peptide 35-55 in complete Freunds adiuvant 12 days prior. Subsequently cells were stimulated for 72 hours with MOG peptide 35-55 or left unstimulated followed by T helper cell sorting using magnetic beads and RNA isolation. qPCR gene expression profiling. Cells from three individual mice of the same genotype were pooled and treated separately as indicated in the summary. Equal amount total RNA from each mice was pooled prior to gene expression analysis.
Project description:The reassorted human 2009 pandemic H1N1 (H1N1pdm) virus infected millions of people and caused thousands of deaths with associated immunopathology. Traditional immunosuppressants have limited capabilities in controlling inflammatory responses associated with severe influenza infections. A new therapeutic strategy, therefore, must be developed. We evaluated several adjuvants including toll-like receptor (TLR) agonists, TLR independent adjuvants and Complete Freund’s adjuvant (CFA) for limiting severe H1N1pdm infection outcomes in a mouse model. In contrast to the TLR agonists and TLR independent adjuvants, CFA which contains multiple pattern recognition receptor (PRR) agonists significantly restrained pro-inflammatory responses and promoted survival in mice from lethal H1N1pdm infection. CFA reduced expression of the plamacytoid dendritic cell (pDC) markers and interferon alpha (IFN-α) after infection, whereas it elevated the T regulatory (Treg) cell suppressive molecules galectin-1 and CTLA-4 expression. Consequently, Th1 cell differentiation and CD8+ effector T cell responses were diminished via downregulated myeloid DC (mDC) costimulation. Furthermore, CTLA-4 expressing Treg cells were dramatically increased when the CFA primed splenocytes were restimulated with its stimuli-killed mycobacterium tuberculosis (M. TB). The elevated CTLA-4 on Treg cells led to reduced CD86 expressing pDCs/mDCs and less CD4+ effector T cells. Overall, our study highlights that the stimuli of innate immunity potentially controls overactive host immune responses in certain situations, and the uncovered mechanism(s) sheds light on the development of anti-influenza therapies. C57B6/J Mice were treated with CFA at day -2 and day 2 post H1N1pdm infection. Weight loss and lethality were monitored post infection. At day 3 and day 5 lung tissues were collected for RNA extraction and expression array analysis.
Project description:Normal SJL mice, 6 to 8 weeks old, were used for the isolation of bone marrow stem cells (BMSC). Bone marrow cells were obtained from the femurs and tibias of euthanized mice by flushing with PBS. Cells were subjected to negative magnetic sorting using the Lineage Cell Depletion Kit. Isolation of murine bone marrow Lin-Sca-1+ cells was performed using MACS Sca-1 MultiSort Kit (fBMSC). The differentiation of neural stem cells was induced by removing the bFGF-containing medium and resuspending cells in fresh bFGF-free medium. Microarray analysis of miRNA expression profiles was performed comparing non differentiated bone marrow stem cells (fBMSC) to bone marrow stem cells differentiated for 4 or 7 days. The data are from adult mouse stem cells isolated from bone marrow
Project description:We found that a number of Tfh cells downmodulated BCL6 protein after their development, and we sought to compare the gene expression between BCL6-hi Tfh cells and BCL6-low Tfh cells. Overall design: CD4+ T cells were sorted from immunized and non-immunized mice for RNA extraction and hybridization on Affymetrix microarrays. Bcl6yfp/+ OT-II cells were transferred to congenic recipient mice, and immunized with NP-OVA in CFA subcutaneously. Seven or ten days after immunization, cells were collected from draining lymph nodes, and sorted on FACSAria by the expression of CXCR5, PD-1 and BCL6-YFP. Naive CD4+ T cells were CD4+ CD44lo CD62Lhi cells from unimmunized mice.