Analysis of LPS-induced gene expression of OTULIN-deficient murine macrophages by RNA sequencing
Ontology highlight
ABSTRACT:
PROVIDER: PRJNA975500 | ENA |
REPOSITORIES: ENA
Dataset's files
| Action | DRS | |||
|---|---|---|---|---|
| SRR24718200.fastq.gz | Fastqsanger.gz | |||
| SRR24718201.fastq.gz | Fastqsanger.gz | |||
| SRR24718202.fastq.gz | Fastqsanger.gz | |||
| SRR24718203.fastq.gz | Fastqsanger.gz | |||
| SRR24718204.fastq.gz | Fastqsanger.gz |
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Project description:In order to unravel the role of the linear deubiquitinase OTULIN in LPS-induced gene expression in macrophages, we performed RNA-sequencing on primary bone marrow-derived macrophages of mice lacking OTULIN selectively in myeloid cells. Myeloid cell-specific OTULIN knock-out mice develop an auto-inflammatory phenotype and macrophages derived from these mice display increased LPS-induced inflammasome activation and cell death. Through analysing LPS-induced gene expression analyses on OTULIN-deficient macrophages we aimed to identify signalling pathways and genes that regulate inflammasome activation and cell death in these cells, in order to get new insights on the molecular events that regulate the development of autoinflammation in myeloid cell specific OTULIN-deficient mice.
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Project description:Lipid A (a hexaacylated 1,4 bis-phosphate) is a potent immune stimulant for TLR4/MD-2. Upon lipid A ligation, the TLR4/MD-2 complex dimerizes and initiates signal transduction. Historically, studies also suggested the existence of TLR4/MD-2-independent LPS signaling. Here we define the role of TLR4 and MD-2 in LPS signaling by using genome wide expression profiling in TLR4- and MD-2-deficient macrophages after stimulations with peptidoglycan-free LPS and synthetic E.coli lipid A. Of the 1,396 genes found significantly induced or repressed by any one of the treatments in the wildtype macrophages, none was present in the TLR4- or MD-2-deficient macrophages, confirming that the TLR4/MD-2 complex is the only receptor for endotoxin, and are both absolutely required for responses to LPS. Using a molecular genetics approach, we investigated the mechanism of TLR4/MD-2 activation by combining the known crystal structure of TLR4/MD-2 with computer modeling. We used lipid IVa, a defined lipid A mimetic to model the activation of mouse TLR4/MD2. The two phosphates on lipid A were predicted to interact extensively with the two positively charged patches mouse TLR4 according to our dimeric murine TLR4/MD-2/lipid IVa model. These two patches are composed of K263, R337, and K360 (Positive Patch 1), and K367 and R434 (Positive Patch 2). When either Positive Patch was abolished by mutagenesis into Ala, the responses to LPS and lipid A were almost abrogated. Thus, ionic interactions between the two phosphates on lipid A and the two positively charged patches on murine TLR4 appear to be essential for LPS receptor activation. Bone marrow-derived macrophages were pooled from four individual WT or TLR4-deficient mice and stimulated with either 10 ng LPS /mL, 100 ng lipid A/mL or 10 nM Pam2 for 2 hours and compared to PBS-stimulated control cells. We also compared PBS-stimulated WT cells directly to PBS-stimulated TLR4-deficient cells to compare the basal expression of genes in the two genotypes. This experiment was repeated once in its entirety.
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