Metabolomics,Unknown,Transcriptomics,Genomics,Proteomics

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Whole transcriptome profiling of the steady state and Hh-treated (8 hrs) Alpk1+/- (heterozygous) and Alpk1-/- (knockout) BMDMs using RNA-Sequencing approach

ABSTRACT: Alpk1-deficient mice demonstrate exacerbated colitis and increased IL-12/Th1 response upon challenge with an intestinal pathobiont, Helicobacter hepaticus (Hh). Hematopoietic compartment is driving the pathogenic phenotype in this animal model, and Alpk1 is highly expressed in myeloid cells (macrophages and dendritic cells). Alpk1 deficiency has a recessive phenotype, since Alpk1+/- (heterozygous) mice show the same phenotype as the wild type mice. Mouse bone-marrow derived macrophages (BMDMs) show elevated IL-12 production in Alpk1-/- mice in response to stimulation with Hh. Since the molecular mechanism of how Alpk1 deficiency affects macrophage response to Hh is unknown, we aimed to characterise global changes in gene expression in Alpk1+/- vs Alpk1-/- bone-marrow differentiated cells (BMDMs). Cells were isolated from bone marrow of Alpk1+/- and Alpk1-/- (mixture of bone marrows from three mice per genotype) and plated in BMDM differentiation medium (RPMI, 10% FCS, penicillin and streptomycin, 50 micro beta-mercapthoethanol, 20 ng/ml recombinant mouse GM-CSF(Peprotech)), 7 million cells in per 10 sm uncoated TC dish in 10 ml of medium for eight days, extra 10 ml of medium was added to plates at day 4, before collection and replating in 96-well plates, 150 thousand cells/200 microliters of differentiation medium per well in technical triplicates per genotype/stimulation condition (R1-R3 labels of the samples). The following day BMDMs were stimulated with MOI of 10 of Hh and 10ng/ml of mouse IFNg (Peprotech) (Alpk1+/- BMDMs – het _Hh_8h vs Alpk1-/- BMDMs – alpk1_Hh_8h) or IFNg only (het _nonstim_8h vs alpk1_nonstim_8h) before lysis for RNA extraction using Quick-RNA kit from Zymo Research. Purified RNA was submitted to the Welcome Trust Centre for Human Genetics (Oxford) for RNA-Sequencing

INSTRUMENT(S): Illumina HiSeq 4000

ORGANISM(S): Mus musculus

SUBMITTER: Stephen Sansom

PROVIDER: E-MTAB-7076 | biostudies-arrayexpress |

REPOSITORIES: biostudies-arrayexpress

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Project description:To recruit phagocytes, apoptotic cells characteristically release ATP, which functions as a “danger” signal. Here, we found that the culture supernatant of apoptotic cells activated the macrophages to express anti-inflammatory genes such as NR4A and Thbs1. A high level of AMP accumulated in the apoptotic cell supernatant in a Pannexin1-dependent manner. A nucleotidase inhibitor and A2a adenosine receptor antagonist inhibited the apoptotic supernatant-induced gene expression, suggesting AMP was metabolized to adenosine by an ecto-5’-nucleotidase expressed on macrophages, to activate the macrophage A2a adenosine receptor. Intraperitoneal injection of zymosan into AdoR A2a- or Panx1-deficient mice produced high, sustained levels of inflammatory mediators in the peritoneal lavage. These results indicated that AMP from apoptotic cells suppresses inflammation as a “calm down” signal. If apoptotic cells produce “danger” or “anti-danger” signal(s), we rationalized that such signals would activate gene expression in macrophages. To investigate this possibility, we examined the effect of the culture supernatant from apoptotic cells on macrophage gene expression by using microarrays. For mouse BMDMs, bone marrow cells from female C57BL/6J mice at 8 weeks of age were cultured for more than 7 days with DMEM containing 10% FCS supplemented with mouse M-CSF. We used adherent cells as BMDMs in the study. W3 cells, mouse T cell line expressing Fas, were treated with human Fas ligand at 37°C for 30 min to induce apoptosis. The cells were then washed and re-suspended at a concentration of 1 × 107 cells/ml with RPMI containing 1% FCS, and further incubated for 60 min at 37°C. Following Fas ligand treatment, more than 90% of the W3 cells were Annexin V positive, and only small percentage were positive for both Annexin V and propidium iodide (PI). The culture supernatant was collected from apoptotic W3 cells. Next, BMDMs were incubated with medium (BMDMs-Medium) or apoptotic W3 cell supernatant (BMDMs-Apoptotic cell supernatant) for 1 h. Total RNA was extracted from the cells and hybridized on Affymetrix microarrays.

Dataset Information

Whole transcriptome profiling of the steady state and Hh-treated (8 hrs) Alpk1+/- (heterozygous) and Alpk1-/- (knockout) BMDMs using RNA-Sequencing approach

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