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| Status |
Public on Nov 26, 2020 |
| Title |
AS_acute_3 |
| Sample type |
SRA |
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| Source name |
flow sorted spleen inflammatory monocytes
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| Organism |
Mus musculus |
| Characteristics |
strain: C57Bl/6J
cell type: flow sorted spleen inflammatory monocytes
malaria infection: acute blood-stage infection (day 7) with mosquito transmitted P. chabaudi AS
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| Treatment protocol |
We assessed the ex vivo transcriptome of 10,000 spleen inflammatory monocytes (Lineage- Ly6G- CD11b+ CD11c- Ly6C+) by flow sorting directly into 1.5 ml eppendorf tubes containing 1 ml Trizol Reagent (ThermoFisher, #15596026). Tubes were inverted ten times and incubated for 5 minutes at room temperature before snap freezing and storage at - 80°C.
To evaluate the transcriptional response of spleen inflammatory monocytes to LPS stimulation in vitro we flow-sorted 30,000 monocytes into a polypropylene tube with IMDM supplemented with 5 % FBS and 8 mM L-Glutamine. Following a gently spin (450 xg for 10 minutes, slow brake), monocytes were resuspended in 90 ul pre-warmed IMDM with 10 % FBS and 8 mM L-Glutamine and transferred to a ultra low attachment flat bottom 96 well cell culture plate (Corning). To stimulate cells 0.3 ng*µl-1 LPS (Lipopolysaccharides from Escherichia coli 0111:B4, Sigma) was added. Cell culture plates were incubated for 4 hours at 37 ̊C and 7 % CO2. RNA from both adherent and non-adherent cells was preserved in 1 ml Trizol Reagent and stored at - 80°C.
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| Growth protocol |
C57Bl/6J mice were bred and housed in individually ventilated cages under specific pathogen free conditions (22°C, 50% humidity) at the University of Edinburgh, UK. Since we have previously shown that mosquito transmission resets expression of the large sub-telomeric multi-gene families that control malaria parasite virulence, and in turn shapes the host immune response during the pathogenic blood-stage of infection (Spence, P.J., et al., Vector transmission regulates immune control of Plasmodium virulence. Nature, 2013. 498(7453): p. 228-31), we transmitted Plasmodium chabaudi chabaudi AJ (96AJ15) and AS (28AS11, both obtained from the European Malaria Reagent Repository (malariaresearch.eu)) through Anopheles stephensi mosquitoes (strain SD500, reared in house at the University of Edinburgh). For more information about mosquito transmission please see our published protocol: Spence, P.J., et al., Mosquito transmission of the rodent malaria parasite Plasmodium chabaudi. Malar J, 2012. 11: p. 407. Mice were injected intravenously with 200 sporozoites, which establish a blood-stage infection 52 hours later. To capture the acute response, we isolated inflammatory monocytes from the spleen for RNAseq on day 7 of blood-stage infection (n= 5 AJ, & AS). infection with P. chabaudi AJ causes severe symptoms (acute hyperparasitaemia, severe anaemia, hypothermia and prostration) while acute AS infection is mild (no hyperparasitaemia, no measurable clinical manifestations of disease other than low-grade anaemia). More than 50% of mice remain chronically infected with P. chabaudi AJ for 40 days (verified by quantitative PCR before sequencing, n = 5). the response to acute and chronic malaria infection was evaluated relative to uninfected age-matched control mice (n = 7).
To evaluate if the tolerant state of spleen inflammatory monocytes from chronically malaria-infected mice is reversible, we stimulated them in vitro with LPS (n = 5) and compared their transcriptional response to LPS stimulated spleen monocytes isolated from uninfected age-matched control mice (n = 4).
Chronic P. chabaudi AJ infection was drug treated using 100 mg/kg chloroquine diphosphate salt (Sigma, dissolved in water) administered by oral gavage daily for 10 days. Memory responses of once-malaria infected mice were assessed 30 days after the start of chloroquine treatment (n = 6) and compared to uninfected age-matched control mice that received an identical chloroquine regimen (n = 6).
To answer if memory of first malaria infection alters the spleen inflammatory monocyte response to second malaria exposure we developed a novel reinfection model: mice were first infected with the avirulent P. chabaudi clone AS to induce chronic recrudescing parasitaemia and then chloroquine treated after 40 days of blood-stage infection to clear circulating and sequestered parasites. 30 days later, mice were infected for a second time - but now with the virulent parasite clone AJ. Critically, parasite density and the dynamics of red cell loss between first infection with P. chabaudi AS and second infection are identical. We therefore completely remove pathogen load as confounding factor when analysing the host response to reinfection. The transcriptional response of inflammatory spleen monocytes to second infection was assessed at day 7 of blood-stage infection (n = 5) relative to uninfected age-matched control mice, which were also chloroquine treated (n = 5).
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| Extracted molecule |
total RNA |
| Extraction protocol |
Total RNA was extracted using 1 - Bromo 3 - chloropropane (Sigma, #B9673) and Isopropanol (VWR, #437423R). total RNA was quantified and assessed for quality and integrity by Bioanalyser (RNA Pico 6000 Chips, Agilent). cDNA was generated from > 2 ng of RNA using the SMART-Seq v4 Ultra Low Input RNA Kit (Clontech Laboratories) and amplified using 11 cycles of PCR. Amplified cDNA was purified using Agencourt AMPure XP beads (Beckman Coulter), quantified by Qubit (Qubit 2.0 Fluorometer - dsDNA HS assay, ThermoFisher) and quality assessed by Bioanalyser (DNA HS Kit, Agilent).
Sequencing libraries were constructed from 150 pg of cDNA using the Nextera XT DNA Library Preparation Kit (Illumina) according to manufacturers instruction. Library DNA was purified and short fragments were removed with AMPure XP beads. libraries were quantified by Qubit (dsDNA HS assay) and library fragment quality and size distribution assessed by Bioanalyser (DNA HS Kit). Equimolar library pools were sequenced at the Wellcome Trust Clinical Research Facility, Western General Hospital (Edinburgh, UK) using the NextSeq 550 platform (Illumina) generating 75 bp paired end reads.
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| Library strategy |
RNA-Seq |
| Library source |
transcriptomic |
| Library selection |
cDNA |
| Instrument model |
NextSeq 550 |
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| Description |
Phil_13
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| Data processing |
Raw sequence data were assessed for quality and content using FastQC
Paired end sequences were aligned to the Ensembl release 96 murine transcripts set, using bowtie2 v2.2.7, applying parameters --very-sensitive -p 30 --no-mixed --no-discordant --no-unal.
The bowtie2 alignment outputs were stored as sorted, indexed bam files, and counts for each transcript obtained from the bam files using samtools idxstats.
Raw counts were imported into the R/Bioconductor environment using the DESeq2 package.
Genome_build: Murine transcript sequences (coding and non-coding) were obtained from Ensembl release 96 in fasta format and made into a bowtie2-searchable database using bowtie2-build; annotation information was obtained for the transcripts using Ensembl BioMart.
Supplementary_files_format_and_content: Processed files contain the outputs from samtools idxstats, namely three tab-separated columns comprising transcript name, transcript length, and read counts for the given sample. A combined file, with the data from all the samples, is also available as NWRmatrix.xls.gz
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| Submission date |
May 07, 2020 |
| Last update date |
Nov 27, 2020 |
| Contact name |
Alasdair Ivens |
| E-mail(s) |
al.ivens@ed.ac.uk
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| Phone |
44 131 6513605
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| Organization name |
Centre for Immunity, Infection and Evolution
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| Street address |
Kings Buildings
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| City |
Edinburgh |
| ZIP/Postal code |
EH9 3FL |
| Country |
United Kingdom |
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| Platform ID |
GPL21626 |
| Series (2) |
| GSE150047 |
A single malaria episode induces mechanisms that minimise inflammation and promote tolerance in spleen inflammatory monocytes. |
| GSE150479 |
Inducible mechanisms of disease tolerance provide an alternative strategy of acquired immunity to malaria. |
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| Relations |
| BioSample |
SAMN14848722 |
| SRA |
SRX8288011 |
| Supplementary file |
Size |
Download |
File type/resource |
| GSM4521294_Phil_13.REL96.counts.txt.gz |
961.8 Kb |
(ftp)(http) |
TXT |
SRA Run Selector |
| Raw data are available in SRA |
| Processed data provided as supplementary file |
| Processed data are available on Series record |
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