Metabolomics,Unknown,Transcriptomics,Genomics,Proteomics

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LPS hypersensitivity of gp130 mutant mice is independent of elevated hematopoietic TLR4 signaling

ABSTRACT: The inflammatory mediator IL6 induced by LPS, which signals via TLR4, has been shown to feedback and augment TLR4 signaling when over-produced in LPS hypersensitive gp130F/F mice. The identity of the LPS/TLR4 responsive inflammatory signaling pathways and gene networks which are modulated by IL6 are unknown. Therefore, to understand the molecular consequences of gp130 hyperactivity in non-haemopoietic tissue on LPS-induced systemic inflammation, global gene expression profiling of livers was performed. The mouse 22K compugen oligo array from the Adelaide Microarray Facility was used to investigate the effect of LPS treatment on the livers of wildtype and gp130F/F mutant mice. Livers of both wildtype mice and gp130F/F mice at 6hr post LPS injection and also control livers wer collected. Each experimental group had 3 independant mice. In total 12 arrays were performed with a total of three biological replicates. A common reference design was used for this experiment. RNA extracted from eight pooled 17.5dpc C57BL/6 mouse embryos was used as the reference sample. 6 wildytpe mice and 6 gp130F/F mice were used in this study, with 3 mice of each genotype treated with LPS.

ORGANISM(S): Mus musculus

SUBMITTER: Jodee Gould

PROVIDER: E-GEOD-29311 | biostudies-arrayexpress |

REPOSITORIES: biostudies-arrayexpress

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Project description:Siglec-E is a murine CD33-related siglec that functions as an inhibitory receptor and is expressed mainly on neutrophils and macrophage populations. Recent studies have suggested that siglec-E is an important negative regulator of LPS-TLR4 signalling and one report (Wu et al 2016) claimed that siglec-E is required for TLR4 endocytosis following uptake of Escherichia coli by macrophages and dendritic cells (DCs). Our attempts to reproduce these observations using cells from wildtype (WT) and siglec E deficient mice were unsuccessful. We used a variety of assays to determine if siglec-E expressed by different macrophage populations can regulate TLR-4 signalling in response to LPS, but found no consistent differences in cytokine secretion in vitro and in vivo, comparing 3 different strains of siglec-E-deficient mice with matched WT controls. No evidence was found that the siglec-E deficiency was compensated by expression of siglecs-F and –G, the other murine inhibitory CD33-related siglecs. Quantitative proteomics was used as an unbiased approach and provided additional evidence that siglec-E does not suppress inflammatory TLR4 signaling. Interestingly, proteomics revealed a siglec E dependent alteration in macrophage phenotype that could be relevant to functional responses in host defence. In support of this, siglec-E-deficient mice exhibited enhanced growth of Salmonella enterica serovar Typhimurium in the liver following intravenous infection, but macrophages lacking siglec-E did not show altered uptake or killing of bacteria in vitro. Using various cell types including bone marrow derived DCs (BMDC), splenic DCs and macrophages from WT and siglec-E-deficient mice, we showed that siglec-E is not required for TLR4 endocytosis following E.coli uptake or LPS challenge. We failed to see expression of siglec-E by BMDC even after LPS-induced maturation, but confirmed previous studies that splenic DCs express low levels of siglec-E. Taken together, our findings do not support a major role of siglec-E in regulation of TLR4 signalling functions or TLR4 endocytosis in macrophages or DCs. Instead, they reveal that induction of siglec-E by LPS can modulate the phenotype of macrophages, the functional significance of which is currently unclear.

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LPS hypersensitivity of gp130 mutant mice is independent of elevated hematopoietic TLR4 signaling

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