Unknown,Transcriptomics,Genomics,Proteomics

Dataset Information

Exosomes isolated from M.tb infected cells modulate macrophage response to IFN-gamma

ABSTRACT: we studied the role of exosomes isolated from M.tb infected macrophages in modulating the macrophage response to IFN-γ. Nimblegen microarray gene expression studies were used to compare the suppression of IFN-γ inducible genes by exosomes relative to the virulent strain of M.tuberculosis. Overall our study suggest that exosomes, as carriers of M.tb pathogen associated molecular patterns (PAMPs), may provide a mechanistic link by which M.tb may exert its suppression of host immune response beyond the infected cell, and implies a physiological role for exosomes in immune surveillance of TB. Macrophages were treated with exosomes, infected with M.tb H37Rv or left untreated for 18 hours followed by +/- IFN-γ for an additional 18 hours. Cells were harvested and RNA was isolated and converted to double stranded cDNA and subsequently labeled and hybridized onto Mus musculus 4×72 Nimblegen microarray using Nimblegen Hybridization system 4 according to manufacturer’s instructions (Roche)

ORGANISM(S): Mus musculus

SUBMITTER: Erliang Zeng

PROVIDER: E-GEOD-29731 | biostudies-arrayexpress |

REPOSITORIES: biostudies-arrayexpress

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Publications

Exosomes released from M. tuberculosis infected cells can suppress IFN-γ mediated activation of naïve macrophages.

Singh Prachi P PP LeMaire Christopher C Tan John C JC Zeng Erliang E Zeng Erliang E Schorey Jeffery S JS

PloS one 20110414 4

<h4>Background</h4>Macrophages infected with Mycobacterium tuberculosis (M.tb) are known to be refractory to IFN-γ stimulation. Previous studies have shown that M.tb express components such as the 19-kDa lipoprotein and peptidoglycan that can bind to macrophage receptors including the Toll-like receptor 2 resulting in the loss in IFN-γ responsiveness. However, it is unclear whether this effect is limited to infected macrophages. We have previously shown that M.tb-infected macrophages release exo ...[more]

PMID: 21533172

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Project description:The woodchuck model of hepatitis B virus (HBV) infection displays many characteristics of human infection and has particular value for characterizing the host immune responses during the development of chronic infection. Using the newly developed custom woodchuck microarray platform, we compared the intrahepatic transcriptional responses of neonatal woodchucks with self-limiting and progressing persistent infection with woodchuck hepatitis virus (WHV). This revealed that WHV does not induce intrahepatic gene expression during the early acute stage of infection (8 weeks), suggesting it is a M-bM-^@M-^\stealthM-bM-^@M-^] virus. At the mid-acute phase of infection (14 weeks), resolution was associated with induction of a prominent cytotoxic T cell signature, with perforin and other markers of immune-mediated cytotolytic response being strongly expressed. Strikingly, this was accompanied by high level expression of PD-1 and various other inhibitory T cell receptors, which likely act to minimize liver damage by cytotoxic T cells during viral clearance. Conversely, self-limiting infection was not associated with a strong interferon-M-NM-1/M-NM-2 (IFN-M-NM-1/M-NM-2) transcriptional response, while the IFN-M-NM-3 signaling response (as measured by expression of CXCL9) in the mid-acute phase was comparable to that in chronically infected adult animals. Nevertheless, viperin and other antiviral genes were differentially expressed during resolving infection, suggesting that a subset of interferon-stimulated genes (ISGs) may play a role in the control of WHV replication. Conclusion: This study identifies new immune pathways associated with the clearance of hepadnavirus infection and reveals novel targets with potential for the treatment of chronic hepatitis B. Neonatal woodchucks of both genders were infected at 3 days of age with the same WHV7P1 inoculum containing 5 x 106 WID50 of WHV strain WHV7-11. Custom microarrays were generated from sequences obtained in transcriptome sequencing of woodchuck liver and PBMCs, and were used to examine liver gene expression in animals which eventually become chronically infected with WHV (8 weeks, n=5; 14 weeks, n=9), animals that eventually resolve WHV infection (8 weeks, n=10;14 weeks, n=7) and uninfected animals (8 weeks, n=5;14 weeks,n=5).

Project description:Signaling pathways controlling vasculogenesis and angiogenesis are still poorly understood. Zebrafish Ets1-related protein (Etsrp) which encodes an ETS domain transcription factor, evolutionary related to the mammalian ER71 protein subfamily, has been identified as a major regulator of vasculogenesis and myelopoiesis and functions at the hemangioblast stage affecting the formation of both lineages. In the absence of Etsrp, angioblasts do not migrate or differentiate while overexpression of Etsrp results in the expansion of vascular endothelial and myeloid lineages. To identify genes functioning downstream of Etsrp we performed microarray analysis of etsrp-overexpressing embryos. Etsrp RNA injected embryos and control uninjected embryos were frozen at the tailbud stage and analyzed for expression of more than 30,000 genes using Nimblegen expression arrays. Approximately 300 genes showed greater than two-fold induction in etsrp-overexpressing embryos. Scl, crl, egfl7, aqp8, fli1a, fli1b, lmo2, cdh5 were among the previously known hemangioblast or vasculature-specific genes which were strongly upregulated in Etsrp-overexpressing embryos. We isolated and characterized a number of genes that were novel or previously unassociated with the zebrafish vasculature formation. Eight of them which include angiotensin II type 2 receptor (agtr2), src homology 2 domain containing E (she), similar to mannose receptor C1 (mrc1), endothelial cell-specific adhesion molecule (esam), cdc42 guanine nucleotide exchange factor 9 (arhgef9), yes-related kinase (yrk), zinc finger protein, multitype 2b (zfpm2b/fog2b) and stabilin 2 (stab2) were specifically expressed in vascular endothelial cells during early embryonic development while keratin18 expression was localized to the myeloid cells among others. Identification of novel vasculature and myeloid-specific genes that are regulated by Etsrp will be important for dissecting molecular mechanisms that regulate vasculogenesis / angiogenesis and myelopoiesis. Approximately 50 pg of etsrp RNA was microinjected into zebrafish embryos at the 1-4 cell stages. Three separate batches of approximately 150 microinjected embryos and uninjected controls were frozen on dry ice at the tailbud stage. Total RNA was purified using the RNAquous-4PCR kit (Ambion). Approximately 10-20 Âµg RNA from each batch was sent to the NimbleGen facility, were it was processed, labeled and hybridized to standard NimbleGen 385K gene expression array. Briefly, RNA was converted into cDNA using the SuperScript II cDNA Conversion Kit (Invitrogen, Carlsbad, CA). Double-stranded cDNA was random-prime labeled with Cy3-nonamers and hybridized to the microarrays for 16 hours at 42Â°C. The zebrafish microarray was based on Zv6 genome assembly and contained approximately 32,000 genes with approximately 12 probes of 60mer oligonucleotides per each gene. The arrays were washed, dried, and scanned at 5Î¼m resolution using a GenePix 4000B microarray scanner (Molecular Devices, Sunnyvale, CA). Data were extracted from scanned images using NimbleScan software (Roche NimbleGen). Quantile normalization was performed across replicate arrays, and RMA (Robust Multichip Average) analysis was performed to generate gene expression values. Average ratios of expression values of etsrp-injected vs. control were calculated from three experiments. Results were sorted by the fold change from the highest to the lowest. Genes that displayed values of p>0.05 or showed expression levels less than 2-fold above the background level in both control and experimental samples were eliminated from further analysis.

Dataset Information

Exosomes isolated from M.tb infected cells modulate macrophage response to IFN-gamma

Publications

Exosomes released from M. tuberculosis infected cells can suppress IFN-γ mediated activation of naïve macrophages.

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