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Single-cell RNA-sequencing of mouse ChP tissue and CSF infiltrate following intracerebroventricular injection of LPS

ABSTRACT: scRNA-seq of mouse ChP and CSF following ICV administration of LPS.

ORGANISM(S): Mus musculus

PROVIDER: GSE239318 | GEO | 2024/07/31

REPOSITORIES: GEO

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Single-cell RNA-sequencing of mouse ChP tissue and CSF infiltrate following intracerebroventricular injection of LPS

Project description:Single-cell RNA-sequencing of mouse ChP tissue and CSF infiltrate following intracerebroventricular injection of LPS

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Analysis of differential gene expression in the choroid plexus (ChP) of P60 mice at 9am and 9pm

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Analysis of differential gene expression in the choroid plexus (ChP) of E16.5 and P60 mice

Project description:Age-appropriate regulation of cerebrospinal fluid (CSF) dynamics including production, flow, and clearance (e.g. via glymphatics and dural lymphatics) is essential for brain health. The choroid plexus (ChP) is an important compartment in CSF dynamics but its contribution is poorly understood. To identify temporally regulated ChP epithelial cell functions that may relate to increased ATP demand, we investigated transcripts that are prioritized for translation, therefore reflecting the functional preference of the ChP tissue. We employed Translating Ribosomal Affinity Purification. We identified ChP Na-K-Cl co-transporter [NKCC1] as developmentally regulated, in both expression and function, by mechanisms ranging from epigenetic (NuRD) to post-translational, coupled with increased ATP production. Premature NKCC1 overexpression in ChP accelerated CSF [K+] reduction, reduced ventricle size, and increased intracranial compliance by adulthood as measured by a murine intracranial pressu re monitor paired serially with MRI. Our data point to a critical developmental period when ChP transiently absorbs CSF through NKCC1-mediated K+ transport, which bears implications on lifelong brain fluid homeostasis and health.

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GM-CSF Production by Non-Classical Monocytes Controls Antagonistic LPS-driven Functions In Allergic Inflammation

Project description:Lipopolysaccharide (LPS) can either promote or prevent T helper-2 (Th2) cell-allergic responses. However, the underlying mechanism remains unknown. We show here that LPS activity switches from pro-pathogenic to protective depending on the production of GM-CSF by non-classical monocytes. In the absence of GM-CSF, LPS can favor pathogenic Th2 cell responses by supporting the trafficking of lung migratory dendritic cells (mDC2s) into the lung-draining lymph node. However, when non-classical monocytes produce GM-CSF, LPS and GM-CSF synergize to differentiate monocyte-derived DCs (moDCs) that instruct mDC2s for Th2 cell suppression. Importantly, only allergens with cysteine protease activity trigger GM-CSF production by non-classical monocytes. Hence, the therapeutic effect of LPS is restricted to allergens with this enzymatic activity. Treatment with GM-CSF, however, restores the protective effects of LPS. Thus, GM-CSF produced by non-classical monocytes acts as a rheostat that fine-tunes the pathogenic and therapeutic functions of LPS.

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Project description:Purpose: The goals of this study are to compare NGS-derived GM-CSF-cultured bone marrow derived cells transcriptome profiling (RNA-seq) normalized counts and differential expression of genes between different stimulations (untreated, Beauvericin, LPS or Beauvericin with LPS). Methods: mRNA profiles of GM-CSF-cultured bone marrow derived FACS purified MHCII high CD11c+ cells from wild-type C57BL/6N mice that were left untreated or stimulated with Beauvericin, LPS or Beauvericin with LPS for 4h were generated by deep sequencing, in quadruplicate, using the Illumina NextSeq550 system. DNase digested total RNA samples used for transcriptome analyses were quantified (Qubit RNA HS Assay, Thermo Fisher Scientific) and quality measured by capillary electrophoresis using the Fragment Analyzer and the ‘Total RNA Standard Sensitivity Assay’ Results: The reads of all probes were adapter trimmed (Illumina TruSeq). Mapping was done against the Mus musculus (mm39; GRCm39) (June 24, 2020) genome sequence. After grouping of samples (four biological replicates each) according to their respective experimental condition, multi-group comparisons were made and statistically determined using DESeq2. The Resulting P values were corrected for multiple testing by FDR. A P value of <0.05 was considered significant. Conclusions: Our study represents the first detailed analysis of untreated and Beauvericin stimulated GM-CSF-cultured bone marrow derived cells transcriptomes.Our results show that NGS offers a comprehensive and more accurate quantitative and qualitative evaluation of mRNA content within GM-CSF-cultured bone marrow derived cells. We conclude that Beauvericin activates GM-CSF-cultured bone marrow derived cells inducing inflammatory cytokine,chemokine and Type I IFN production via a TLR4 dependent signaling pathway, but induces a gene expression profile different from LPS.

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Expression data of LPS-stimulated bone marrow macrophages induced by M-CSF from wild-type and Jmjd3-/- mice.

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Transcriptional effects of IL-10 neutralization on LPS-activated M-CSF-derived macrophages

Project description:Analysis of the role of IL-10 on the transcriptional signature of LPS-activated human M-CSF-dependent monocyte-derived macrophages. Methods: Human Peripheral Blood Mononuclear Cells (PBMC) were isolated from buffy coats from donors over a Lymphoprep gradient according to standard procedures. Monocytes were purified from PBMC by magnetic cell sorting using anti-CD14 microbeads (>95% CD14+ cells). Monocytes from three independent donors (0.5 x 106 cells/ml, >95% CD14+ cells) were cultured in RPMI 1640 supplemented with 10% fetal bovine serum (FBS) at 37°C in a humidified atmosphere with 5% CO2 and 21% O2 for 7 days in the presence of 1000 U/ml M-CSF, with cytokine addition every two days. Then, monocyte-derived macrophages were treated with an anti-IL-10 blocking antibody (anti-IL10) or an isotype-matched antibody (IgG2b) at 2.5 micrograms/ml for 1 hour, and exposed to LPS (10 ng/ml). After 4 hours of LPS treatment, cells were lysed and RNA isolated for transcriptional analysis.

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Transcription profiling of effect of icv administration of LPS (4 + 16 hr) on mouse hippocampal and microgial samples

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