Unknown,Transcriptomics,Genomics,Proteomics

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Mechanisms Establishing TLR4-Responsive Activation States of Inflammatory Response Genes (Agilent expression data)

ABSTRACT: Precise control of the innate immune response is required for resistance to microbial infections and maintenance of normal tissue homeostasis. Because this response involves coordinate regulation of hundreds of genes, it provides a powerful biological system to elucidate the molecular strategies that underlie signal- and time-dependent transitions of gene expression. Using a combination of genome-wide and gene-specific approaches, we provide evidence that rather than representing off/on transitions, Toll-like receptor 4 (TLR4)-dependent activation of nearly all immediate/early (I/E) and late response genes results from a sequential process in which signal-independent factors, exemplified by Gabpa, initially establish basal levels of gene expression that are then amplified by signal-dependent transcription factors. Promoters of I/E genes are distinguished from those of late genes by the use of distinct sets of signal-dependent transcription factors, preferential binding of TBP and basal enrichment for RNA Pol II immediately downstream of transcriptional start sites. Global nuclear run-on (GRO) sequencing and total RNA sequencing further indicates that TLR4 signaling markedly increases efficiency of transcriptional elongation of nearly all I/E genes, while RNA splicing is largely unaffected. NCoR/SMRT co-repressor complexes are unexpectedly found to be associated with H3K4me3-positive promoters of TLR4-responsive genes that exhibit a broad range of basal expression levels, implying a dynamic, rather than static role in regulation of gene expression. Collectively, these findings reveal mechanisms underlying temporally distinct patterns of TLR4-dependent gene activation required for homeostasis and effective immune responses. ChIP-Seq, Total RNA-Seq, Gro-Seq, and gene expression profiling was performed in macrophages treated with Kdo2 Lipid A. Control samples for H3K4me3 and Input in Macrophages and B cells, in addition to control microarray data are included in GEO accession# GSE21512

ORGANISM(S): Mus musculus

SUBMITTER: Christopher Benner

PROVIDER: E-GEOD-23620 | biostudies-arrayexpress |

REPOSITORIES: biostudies-arrayexpress

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Project description:Precise control of the innate immune response is required for resistance to microbial infections and maintenance of normal tissue homeostasis. Because this response involves coordinate regulation of hundreds of genes, it provides a powerful biological system to elucidate the molecular strategies that underlie signal- and time-dependent transitions of gene expression. Using a combination of genome-wide and gene-specific approaches, we provide evidence that rather than representing off/on transitions, Toll-like receptor 4 (TLR4)-dependent activation of nearly all immediate/early (I/E) and late response genes results from a sequential process in which signal-independent factors, exemplified by Gabpa, initially establish basal levels of gene expression that are then amplified by signal-dependent transcription factors. Promoters of I/E genes are distinguished from those of late genes by the use of distinct sets of signal-dependent transcription factors, preferential binding of TBP and basal enrichment for RNA Pol II immediately downstream of transcriptional start sites. Global nuclear run-on (GRO) sequencing and total RNA sequencing further indicates that TLR4 signaling markedly increases efficiency of transcriptional elongation of nearly all I/E genes, while RNA splicing is largely unaffected. NCoR/SMRT co-repressor complexes are unexpectedly found to be associated with H3K4me3-positive promoters of TLR4-responsive genes that exhibit a broad range of basal expression levels, implying a dynamic, rather than static role in regulation of gene expression. Collectively, these findings reveal mechanisms underlying temporally distinct patterns of TLR4-dependent gene activation required for homeostasis and effective immune responses. ChIP-Seq, Total RNA-Seq, Gro-Seq, and gene expression profiling was performed in macrophages treated with Kdo2 Lipid A. Control samples for H3K4me3 in Macrophages and B cells, in addition to control microarray data are included in GEO accession# GSE21512. FASTA files are available for older experiments that lack quality read information (i.e. no FASTQ file)

Project description:Alternatively activated macrophages (AAMφs) play important roles in a number of Th2 driven pathologies including asthma and allergy, and a number of parasitic infections. Our studies, and those of others, investigating the pathologies associated with infection with the helminth Schistosoma japonicum implicate a role for AAMφs in fibrosis and immunomodulation.. In the present study we show that S. japonicum-secreted egg antigens are able to induce the alternative activation of macrophages as characterised by the induction of Chi3l3 and Arg1 expression. Retnla, another common marker of AAMφs, was not consistently induced in these macrophages suggesting that the specific function of these cells may differ to those induced by S. mansoni and other parasites. Closer examination of the gene expression profile and functionality of these cells identified pathways independent of Retnla expression that could be important for their immunomodulatory activity such as modulating expression of T-cell co-stimulatory molecules and chemokines. In vivo generated S. japonicum soluble egg antigen stimulated AAMφs also exhibited a reduction in their phagocytic ability likely related to the induction of IL4 and decreased expression of cell surface receptors. Additionally these macrophages exhibited reduced expression of Toll-like receptors (TLRs) and an associated reduction in responsiveness to stimulation with TLR ligands. We did not observe pathways that would suggest that AAMφs have a direct profibrotic activity. Taken together, these data describe a mechanism by which alternative activation of macrophages may be induced during S. japonicum infection and highlight the importance of the context of activation in directing AAMφ phenotype and function. The gene expression profile of Schistosoma japonicum soluble egg antigen (SEA)-stimulated macrophages was compared with that of PBS stimulated controls. Macrophages were isolated from the peritoneal cavity of BALB/c mice (n=6 per group) stimulated by intraperitoneal injection with SEA or PBS. The macrophages were pooled and RNA was extracted from these cells. Microarray analysis was performed on cRNA synthesised from total RNA derived from these macrophages. The experiment was performed twice creating two biological replicates. Fold-change (relative to the respective PBS control) reported in supplementary file linked below.

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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Mechanisms Establishing TLR4-Responsive Activation States of Inflammatory Response Genes (Agilent expression data)

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