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SRP068648
Mus musculus
6 Downloadable Samples
Illumina HiSeq 2500
Description
mRNA expression in Eµ-Myc lymphoma cells expressing or lacking miR-17~92 Overall design: Eµ-Myc B-cell lymphomas harboring conditional alleles of miR-17~92 were cultured with or without 4-OHT to generate isogenic tumour cells with homozygous deletion of miR-17~92. Wild type (fl) and miR-17~92-deleted (del) Eµ-Myc cells were cultured for 48 hours under regular growth conditions, and RNA isolated for sequencing analysis.
Publication Title
The miR-17∼92 microRNA Cluster Is a Global Regulator of Tumor Metabolism.
Sample Metadata Fields
Specimen part, Subject
View Samples- Mus musculus
- 18 Downloadable Samples
- Illumina HiSeq 2500
Description
Immune-Responsive Gene 1 (Irg1) is a mitochondrial enzyme that produces itaconate under inflammatory conditions principally in cells of myeloid lineage. Cell culture studies suggest that itaconate regulates inflammation through inhibitory effects on cytokine and reactive oxygen species production. To evaluate the functions of Irg1 in vivo, we challenged wild-type (WT) and Irg1 KO mice with Mycobacterium tuberculosis (Mtb) and monitored disease progression. Irg1 KO but not WT mice succumbed rapidly to Mtb, and mortality was associated with increased infection, inflammation, and pathology. Infection of LysM-Cre Irg1 flox, MPR8-Cre Irg1 flox, and CD11c-Cre Irg1 flox conditional knockout mice along with neutrophil depletion experiments revealed a role for Irg1 in alveolar macrophages and LysM+ myeloid cells in preventing neutrophil-mediated immunopathology and disease. RNA-seq analyses suggest that Irg1 and its production of itaconate temper Mtb-induced inflammatory responses in myeloid cells at the transcriptional level. Thus, Irg1 modulates inflammation to curtail Mtb-induced lung disease. Overall design: Neutrophils were purified from bone marrow of naïve mice by negative selection using magnetic-activated cell sorting beads (Miltenyi). Neutrophil purity (>95%) was assessed by flow cytometry as the percentage of Ly6G+ CD11b+ cells. Neutrophils were cultured in RPMI-1640 supplemented with 1% non-essential amino acids at 37°C, 5% CO2. GFP-Mtb was grown to mid-log phase, washed with PBS, sonicated to disperse clumps, and resuspended in neutrophil culture media. GFP-Mtb then was opsonized prior to infection by mixing with an equal volume of normal mouse sera (Sigma) and incubation at room temperature for 30 min. Neutrophils were mock-infected or infected with opsonized GFP-Mtb at MOI 1 and incubated at 37°C, 5% CO2.
Publication Title
<i>Irg1</i> expression in myeloid cells prevents immunopathology during <i>M. tuberculosis</i> infection.
Sample Metadata Fields
Specimen part, Cell line, Subject, Time
View Samples- Mus musculus
- 15 Downloadable Samples
- Illumina HiSeq 2500
Description
Immune-Responsive Gene 1 (Irg1) is a mitochondrial enzyme that produces itaconate under inflammatory conditions principally in cells of myeloid lineage. Cell culture studies suggest that itaconate regulates inflammation through inhibitory effects on cytokine and reactive oxygen species production. To evaluate the functions of Irg1 in vivo, we challenged wild-type (WT) and Irg1-/- mice with Mycobacterium tuberculosis (Mtb) and monitored disease progression. Irg1-/- but not WT mice succumbed rapidly to Mtb, and mortality was associated with increased infection, inflammation, and pathology. Infection of LysM-Cre Irg1fl/fl, MPR8-Cre Irg1fl/fl, and CD11c-Cre Irg1fl/fl conditional knockout mice along with neutrophil depletion experiments revealed a role for Irg1 in alveolar macrophages and LysM+ myeloid cells in preventing neutrophil-mediated immunopathology and disease. RNA-seq analyses suggest that Irg1 and its production of itaconate temper Mtb-induced inflammatory responses in myeloid cells at the transcriptional level. Thus, Irg1 modulates inflammation to curtail Mtb-induced lung disease. Overall design: Macrophages were obtained by culturing bone marrow cells in RPMI-1640 (Invitrogen) supplemented with 10% heat inactivated fetal bovine serum, 2 mM L-glutamine, 1% non-essential amino acids, 100 U penicillin per mL, 100 µg streptomycin per mL, and 22 ng M-CSF (Peprotech) per ml for 6 days at 37°C, 5% CO2. Fresh media was added on day 3 of culture. After 6 days of culture, non-adherent cells were discarded. Adherent macrophages were switched into antibiotic-free media and seeded at 105 cells per well and 9 x 105 cells per well in tissue culture-treated 96 and 6 well plates respectively. In some cases, macrophages were treated with 0.25 mM itaconic acid (Sigma) for 12 h prior to inoculation with Mtb. Mtb was grown to mid-log phase, washed with PBS, sonicated to disperse clumps, and resuspended in antibiotic-free macrophage culture media. Macrophage cultures were inoculated by adding Mtb-containing media at a multiplicity of infection (MOI) of 1 and centrifuging for 10 min at 200 x g. Cells were washed twice with PBS to remove unbound Mtb, fresh culture media was added, and cells were incubated at 37°C, 5% CO2. In some cases culture media was supplemented with 0.25 mM itaconic acid.
Publication Title
<i>Irg1</i> expression in myeloid cells prevents immunopathology during <i>M. tuberculosis</i> infection.
Sample Metadata Fields
Specimen part, Treatment, Subject
View Samples- Mus musculus
- 11 Downloadable Samples
- Illumina HiSeq 2500
Description
Immune-Responsive Gene 1 (Irg1) is a mitochondrial enzyme that produces itaconate under inflammatory conditions principally in cells of myeloid lineage. Cell culture studies suggest that itaconate regulates inflammation through inhibitory effects on cytokine and reactive oxygen species production. To evaluate the functions of Irg1 in vivo, we challenged wild-type (WT) and Irg1 KO mice with Mycobacterium tuberculosis (Mtb) and monitored disease progression. Irg1 KO but not WT mice succumbed rapidly to Mtb, and mortality was associated with increased infection, inflammation, and pathology. Infection of LysM-Cre Irg1 flox, MPR8-Cre Irg1 flox, and CD11c-Cre Irg1 flox conditional knockout mice along with neutrophil depletion experiments revealed a role for Irg1 in alveolar macrophages and LysM+ myeloid cells in preventing neutrophil-mediated immunopathology and disease. RNA-seq analyses suggest that Irg1 and its production of itaconate temper Mtb-induced inflammatory responses in myeloid cells at the transcriptional level. Thus, Irg1 modulates inflammation to curtail Mtb-induced lung disease. Overall design: C57BL/6N (WT) mice were purchased from Charles River. B6.SJL (CD45.1) mice were obtained from Jackson Laboratories. Irg1-/- mice (embryonic stem cells obtained from KOMP (C57BL/6N background), MGI: 103206) were generated at Washington University. Adult mice (6-13 weeks of age) of both sexes were used, and sex was randomized between experiments. Neutrophils were purified by magnetic-activated cell sorting from the bone marrow of naïve mice (negative selection) or the lungs of Mtb-infected mice at 16 dpi (selection for Ly6G+ cells) (Miltenyi).
Publication Title
<i>Irg1</i> expression in myeloid cells prevents immunopathology during <i>M. tuberculosis</i> infection.
Sample Metadata Fields
Specimen part, Cell line, Subject
View Samples- Mus musculus
- 32 Downloadable Samples
- Illumina HiSeq 2500
Description
The most common form of senile dementia, Alzheimer’s disease (AD), is characterized by Aß plaques and neurofibrillary tangles in the CNS. AD genetic studies have identified high-risk hypomorphic variants in TREM2, a myeloid cell surface receptor that enables concerted microglial responses to Aß plaques and neuronal cell death, including proliferation, survival, clustering and phagocytosis. How TREM2 promotes these responses is not known. Here, we demonstrate that TREM2 drives mTOR signaling, which maintains high ATP levels, supports biosynthetic pathways and suppresses AMPK phosphorylation and autophagy. In vitro, TREM2-deficient macrophages undergo dramatically increased autophagy and die in response to growth factor limitation or ER stress. Excessive autophagy is also evident in microglia from Trem2-/- 5XFAD mice and in post-mortem specimens from AD patients carrying TREM2 risk variants. Metabolic derailment, autophagy and cell death can be circumvented by engaging alternative energy production pathways. Thus, restoring microglial energetic and anabolic levels may be a future therapeutic avenue for TREM2-associated neurological disease. Overall design: Bone marrow-derived macrophages (BMDMs) from WT and Trem2–/– mice were cultured in either 0.5% or 10% LCCM overnight in complete RPMI. Some samples cells were stimulated with 10 ng/ml LPS for 4 hours.
Publication Title
TREM2 Maintains Microglial Metabolic Fitness in Alzheimer's Disease.
Sample Metadata Fields
Specimen part, Cell line, Treatment, Subject
View Samples- Mus musculus
- 14 Downloadable Samples
- Illumina HiSeq 2500
Description
The emergence of multidrug resistant (MDR) Mycobacterium tuberculosis (Mtb) strains, resistant to the frontline anti-tubercular drugs rifampicin and isoniazid, forces treatment with less effective and toxic second-line drugs and stands to derail TB control efforts. However, the immune response to MDR Mtb infection remains poorly understood. Here, we determined the RNA transcriptional profile of in vitro generated macrophages to infection with either drug susceptible Mtb HN878 or MDR Mtb W_7642 infection. Overall design: Bone marrow-derived macrophages (BMDMs) from WT and Il1r1–/– mice were derived in 7 days in GM-CSF supplemented complete DMEM. Cells were infected with either Mtb HN878 or Mtb W_7642 (multiplicity of infection = 1) and RNA samples collected after 6 days.
Publication Title
Mycobacterium tuberculosis carrying a rifampicin drug resistance mutation reprograms macrophage metabolism through cell wall lipid changes.
Sample Metadata Fields
Cell line, Subject
View Samples- Mus musculus
- 8 Downloadable Samples
- Illumina HiSeq 2500
Description
Interplay between metabolic state of the cell and its ability to undergo immunological activation has been recently recognized as a treasure chest of novel fundamental regulatory principles. Itaconate, and its membrane permeable derivative dimethyl itaconate (DI) were recently shown to selectively inhibit subset of cytokines during macrophage activation (e.g. Il1b, il6, Il12b but not TNF), yet the precise mechanism of this effect remained unclear. We find that selectivity of DI action stems from the inhibitory effects of electrophilic stress exerted by DI on IkB-zeta protein translation, leading to selective control of the secondary wave of Nfkb-signaling. Mechanistically, DI leads to glutathione depletion and subsequent activation of both Nrf2-dependent and Nrf2-independent stress responses. We find that IkB-zeta regulation is carried out in Nrf2-independent manner, and identify Atf3 as a key mediator of DI effects on IkB-zeta/IL6. This inhibitory effect is conserved across species and cell types, as evident from inhibition of IkB-zeta production in activating human monocytes and IL-17A stimulated keratinocytes of both human and mice. Finally, DI administration in vivo ameliorated IL17/IkB-zeta-driven skin pathology in the mouse model of psoriasis, highlighting therapeutic potential of this regulatory pathway. Overall design: Bone marrow-derived macrophages (BMDMs) from WT and Nrf2–/– mice were derived in 7 days in MCSF supplemented complete RPMI. Some samples cells were stimulated with 250 uM DimethylItaconate(DI) for 12 hours prior to collection for RNA-seq.
Publication Title
Electrophilic properties of itaconate and derivatives regulate the IκBζ-ATF3 inflammatory axis.
Sample Metadata Fields
Specimen part, Cell line, Treatment, Subject
View Samples- Homo sapiens
- 9 Downloadable Samples
Affymetrix Human Genome U133 Plus 2.0 Array (hgu133plus2)
Description
ILLUMINATE (Investigation of Lipid Level Management to Understand its Impact in Atherosclerotic Events), the phase 3 morbidity and mortality trial of torcetrapib, a cholesteryl ester transfer protein (CETP) inhibitor, identified previously undescribed changes in plasma levels of potassium, sodium, bicarbonate, and aldosterone. A key question after this trial is whether the failure of torcetrapib was a result of CETP inhibition or of some other pharmacology of the molecule. The direct effects of torcetrapib and related molecules on adrenal steroid production were assessed in cell culture using the H295R as well as the newly developed HAC15 human adrenal carcinoma cell lines. Torcetrapib induced the synthesis of both aldosterone and cortisol in these two in vitro cell systems. Analysis of steroidogenic gene expression indicated that torcetrapib significantly induced the expression of CYP11B2 and CYP11B1, two enzymes in the last step of aldosterone and cortisol biosynthesis pathway, respectively. Transcription profiling indicated that torcetrapib and angiotensin II share overlapping pathways in regulating adrenal steroid biosynthesis. Hormone-induced steroid production is mainly mediated by two messengers, calcium and cAMP. An increase of intracellular calcium was observed after torcetrapib treatment, whereas cAMP was unchanged. Consistent with intracellular calcium being the key mediator of torcetrapibs effect in adrenal cells, calcium channel blockers completely blocked torcetrapib-induced corticoid release and calcium increase. A series of compounds structurally related to torcetrapib as well as structurally distinct compounds were profiled. The results indicate that the pressor and adrenal effects observed with torcetrapib and related molecules are independent of CETP inhibition.
Publication Title
Torcetrapib induces aldosterone and cortisol production by an intracellular calcium-mediated mechanism independently of cholesteryl ester transfer protein inhibition.
Sample Metadata Fields
Specimen part, Cell line, Time
View Samples- Homo sapiens
- 12 Downloadable Samples
- NextSeq 500
Description
Glucocorticoids (GCs) are essential steroid hormones that regulate the immune system. GCs have been widely used to treat various inflammation disorders and auto-immune diseases, due to their potent immune repression properties. Overall design: HeLa cells were cultured with DMEM plus 10% charcoal-stripped FBS. HeLa cells were treated in the presence of 100 nM triamcinolone acetonide (TA) for 4 hours. Cells were then collected for RNA-seq.
Publication Title
Extensive epigenomic integration of the glucocorticoid response in primary human monocytes and in vitro derived macrophages.
Sample Metadata Fields
Cell line, Treatment, Subject
View Samples- Homo sapiens
- 8 Downloadable Samples
- NextSeq 500
Description
Glucocorticoids (GCs) are essential steroid hormones that regulate the immune system. GCs have been widely used to treat various inflammation disorders and auto-immune diseases, due to their potent immune repression properties. Overall design: Monocytes from healthy donors were cultured in the presence of 100 nM triamcinolone acetonide (TA), 100 nM Dexamethasone (Dex) or 100 nM Prednisolone (Pred) for 4 hours. Cells were then collected for RNA-seq.
Publication Title
Extensive epigenomic integration of the glucocorticoid response in primary human monocytes and in vitro derived macrophages.
Sample Metadata Fields
Specimen part, Disease, Treatment, Subject
View Samples