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SRP106053
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- Rattus norvegicus
- 6 Downloadable Samples
Affymetrix Rat Genome 230 2.0 Array (rat2302)
Description
Astrocyte-secreted proteins induce synapse formation between isolated retinal ganglion cell (RGC) neurons in culture. We asked whether 2 of these proteins, glypican 4 (Gpc4) or thrombospondin 1 (TSP1) induce synapse formation by regulating gene expression in RGCs.
Publication Title
Astrocyte-Secreted Glypican 4 Regulates Release of Neuronal Pentraxin 1 from Axons to Induce Functional Synapse Formation.
Sample Metadata Fields
Treatment
View Samples- Homo sapiens
- 507 Downloadable Samples
- Illumina HiSeq 2500
Description
Despite widespread interest in using human stem cells in neurological disease modeling, a suitable model system to study human neuronal connectivity is lacking. Here, we report a protocol for efficient differentiation of hippocampal pyramidal neurons and an in vitro model for hippocampal neuronal connectivity. We developed an embryonic stem cell (ESC)- and induced pluripotent stem cell (iPSC)-based protocol to differentiate human CA3 pyramidal neurons from patterned hippocampal neural progenitor cells (NPCs). This differentiation induces a comprehensive patterning and generates multiple CA3 neuronal subtypes. The differentiated CA3 neurons are functionally active and readily form neuronal connection with dentate granule (DG) neurons in vitro, recapitulating the synaptic connectivity within the hippocampus. When we applied this neuronal co-culture approach to study connectivity in schizophrenia, we found deficits in spontaneous activity in patient iPSC derived DG–CA3 co-culture by multi-electrode array recording. In addition, both multi-electrode array recording and whole cell patch clamp electrophysiology revealed a reduction in spontaneous and evoked neuronal activity in CA3 neurons derived from schizophrenia patients. Altogether these results underscore the relevance of this new model in studying diseases with hippocampal vulnerability. Overall design: 4 technical replicates were used and later pooled together for the bioinformatic analysis.
Publication Title
Efficient Generation of CA3 Neurons from Human Pluripotent Stem Cells Enables Modeling of Hippocampal Connectivity In Vitro.
Sample Metadata Fields
Specimen part, Subject
View Samples- Mus musculus
- 201 Downloadable Samples
- Illumina HiSeq 2500
Description
Single-cell sequencing methods have emerged as powerful tools for identification of heterogeneous cell types within defined brain regions. Application of single-cell techniques to study the transcriptome of activated neurons can offer insight into molecular dynamics associated with differential neuronal responses to a given experience. Through evaluation of common whole-cell and single-nuclei RNA-sequencing (snRNA-seq) methods, here we show that snRNA-seq faithfully re-capitulates transcriptional patterns associated with experience-driven induction of activity, including immediate early genes (IEGs) such as Fos, Arc, and Egr1. SnRNA-seq of mouse dentate granule cells reveals large-scale changes in the activated neuronal transcriptome after brief novel environment exposure, including induction of MAPK pathway genes . In addition, we observe a continuum of activation states, revealing a pseudo-temporal pattern of activation from gene expression alone. In summary, snRNA-seq of activated neurons enables the examination of gene expression beyond IEGs,allowing for novel insights into neuronal activation patterns in vivo. Overall design: Examination of 1) 82 whole-cell (WC) dentate granule cells from a PTZ- or saline-treated mouse, and 2) 23 single-nuclei (SN) from dentate granule cells of a homecage (HC) mouse or 96 nuclei from a mouse exposed to a novel environment (NE)
Publication Title
Nuclear RNA-seq of single neurons reveals molecular signatures of activation.
Sample Metadata Fields
Specimen part, Cell line, Treatment, Subject
View Samples- Homo sapiens
- 5 Downloadable Samples
- Affymetrix Human Genome U133 Plus 2.0 Array (hgu133plus2)
Description
C/EBPbeta-2 results in EMT and ErbB indpendence this project investigated the gene changes in related genes upon C/EBPbeta-2 overexpression in MCF10A cells.
Publication Title
Genomic profiling of C/EBPβ2 transformed mammary epithelial cells: a role for nuclear interleukin-1β.
Sample Metadata Fields
Cell line
View Samples- Homo sapiens
- 52 Downloadable Samples
- Illumina HiSeq 2500, Illumina HiSeq 2000, NextSeq 500
Description
Background: NK cells during chronic viral infection have been well studied over the last decade. We performed an unbiased next-generation RNA-sequencing approach to identify commonalities or differences of the effect of HIV, HCV and HBV viremia on NK cell transcriptomes. Methods: Using cell sorting, we obtained CD3-CD56+ NK cells from blood of 6 HIV, 11 HCV, and 32 HBV infected and untreated patients. Library preparation and sequencing were done using Illumina mRNA-Seq Sample Prep Kit and the HiSeq 2000, HiSeq2500 or NextSeq 500, and further analysis by an in-house analytic pipeline. Results: In NK cells from HIV, HCV and HBV patients, transcriptome analysis identified 272, 53, and 56 differentially expressed genes, respectively (fold change >1.5, q-value 0.2). Interferon stimulated genes were induced in NK cells from HIV/HCV patients, but not during HBV infection. HIV viremia downregulated ribosome assembly genes in NK cells. In HBV, viral load and ALT variation had little effect on genes related to NK effector function. Conclusion: We compare, for the first time, NK cell transcripts of viremic HIV, HCV and HBV patients. We clearly demonstrate distinctive NK cell gene signatures in 3 different populations, suggestive for a different degree of functional alterations of the NK cell compartment as compared to healthy individuals. Overall design: We analyzed NK cell transcripts collected from the blood of well-characterized chronic HBV patients (n=32), chronic HCV patients (n=8), and HIV patients (n=6). Differential gene expression analysis, global module analysis, and unsupervised clustering analysis were performed by employing RNA-sequencing on blood NK cell transcriptomes.
Publication Title
Persistent Replication of HIV, Hepatitis C Virus (HCV), and HBV Results in Distinct Gene Expression Profiles by Human NK Cells.
Sample Metadata Fields
Sex, Specimen part, Disease, Subject
View Samples- Homo sapiens
- 18 Downloadable Samples
- Affymetrix Human Genome U133 Plus 2.0 Array (hgu133plus2)
Description
Keloids are benign tumors of the dermis that form during a protracted wound healing process. Susceptibility to keloid formation occurs predominantly in people of African and Asian descent. The key alteration(s) responsible for keloid formation has not been identified and there is no satisfactory treatment for this disorder. The altered regulatory mechanism is limited to dermal wound healing, although several diseases characterized by an exaggerated response to injury are prevalent in individuals of African ancestry. We have observed a complex pattern of phenotypic differences in keloid fibroblasts grown in standard culture medium or induced by hydrocortisone. In this study Affymetrix-based microarray was performed on RNA obtained from fibroblasts cultured from normal scars and keloids grown in the absence and presence of hydrocortisone. We observed differential regulation of approximately 500 genes of the 38,000 represented on the Affymetrix chip. Of particular interest was increased expression of several IGF-binding and IGF-binding related proteins and decreased expression of a subset of Wnt pathway inhibitors and multiple IL-1-inducible genes. Increased expression of CTGF and IGFBP-3 was observed in keloid fibroblasts only in the presence of hydrocortisone. These findings support a role for multiple fibrosis-related pathways in the pathogenesis of keloids
Publication Title
Gene profiling of keloid fibroblasts shows altered expression in multiple fibrosis-associated pathways.
Sample Metadata Fields
No sample metadata fields
View Samples- Homo sapiens
- 12 Downloadable Samples
- Affymetrix Human Genome U133 Plus 2.0 Array (hgu133plus2)
Description
Thermal injury incites inflammatory responses that often transcend the local environment and lead to structural deficiencies in skin that give way to scar formation. We hypothesized that extensive perturbations within burned skin following thermal insult and during subsequent events of wound repair induce vast alterations in gene expression that likely serve as a wound and systemic healing deterrent. A high-throughput microarray experiment was designed to analyze genetic expression patterns and identify potential genes to target for therapeutic augmentation or silencing. The study compares gene expression from burn wound margins at various times following thermal injury to expression observed in normal skin. Utilizing this design, we report that the totality of gene expression alterations is indeed enormous. Further, we observed that the differential expression of many inflammatory and immune response genes appear to be continually up-regulated in burn wound margins seven days or more after initial thermal insult. As it is well established that the inflammatory process must abate for wound healing to proceed, the finding of ongoing local inflammation is cause for further investigation. To our knowledge, this is the first report of the gene expression alterations induced by thermal injury of human skin. As such, it provides a wealth of data to mine with the ultimate goal of better understanding the local pathophysiologic changes at the site of thermal injury that not only affect wound healing capacity, but may also contribute to systemic derangements within the burn patient.
Publication Title
A microarray analysis of temporal gene expression profiles in thermally injured human skin.
Sample Metadata Fields
Sex
View Samples