Unknown,Transcriptomics,Genomics,Proteomics

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Transcription profiling of mouse dendritic cells compare the similarity of endogenous and exogenous antigens

ABSTRACT: Here we demonstrate by the use of extensive controls and stringent statistical analysis that dendritic cells differentially regulate hundreds of different genes based upon sequence similarity of endogenously- and exogenously-loaded antigens and in a T-cell independent fashion. When endogenously and exogenously-derived antigens are identical, dendritic cells upregulate many different components of the Th-1 response, favoring the priming of CD8+ effectors and promulgating cellular immunity. Experiment Overall Design: Dendritic cells were loaded by 1 of 5 different methods, then matured for 48 hours. Total cellular RNA was then extracted from 9 unloaded DC populations, 10 mRNA-loaded populations, 11 lysate-loaded populations, 7 doubly-loaded (matched) populations, and 7 doubly-loaded (mismatched) populations, a total of 44 different experiments. Each population was derived from a different normal human donor. Matched/mismatched refers to the source of the mRNA and lysate used to load the DCs. Matched signifies that the mRNA and lysate came from the same source. Mismatched signifies that the mRNA and lysate came from disparate sources. Gene expression profiles were then determined according to the method by which the DCs had been loaded. Genes were identified as differentially expressed by DCs doubly-loaded with matched antigens, only if they were significantly different from all of the other four controls. Significance was defined as Cohenâ??s |d| > 1.0 (large effect) and q-value (Benjamini-Hochberg false discovery) < 0.01.

ORGANISM(S): Homo sapiens

SUBMITTER: William Decker

PROVIDER: E-GEOD-7247 | 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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Transcription profiling of mouse dendritic cells compare the similarity of endogenous and exogenous antigens

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