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Transcriptional profiling of macrophages derived from monocytes and iPS cells identifies a conserved response to LPS and novel alternative transcription.

ABSTRACT: Macrophages differentiated from human induced pluripotent stem cells (IPSDMs) are a potentially valuable new tool for linking genotype to phenotype in functional studies. However, at a genome-wide level these cells have remained largely uncharacterised. Here, we compared the transcriptomes of naïve and lipopolysaccharide (LPS) stimulated monocyte-derived macrophages (MDMs) and IPSDMs using RNA-Seq. The IPSDM and MDM transcriptomes were broadly similar and exhibited a highly conserved response to LPS. However, there were also significant differences in the expression of genes associated with antigen presentation and tissue remodelling. Furthermore, genes coding for multiple chemokines involved in neutrophil recruitment were more highly expressed in IPSDMs upon LPS stimulation. Additionally, analysing individual transcript expression identified hundreds of genes undergoing alternative promoter and 3' untranslated region usage following LPS treatment representing a previously under-appreciated level of regulation in the LPS response.

SUBMITTER: Alasoo K

PROVIDER: S-EPMC4519778 | biostudies-literature | 2015 Jul

REPOSITORIES: biostudies-literature

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Transcriptional profiling of macrophages derived from monocytes and iPS cells identifies a conserved response to LPS and novel alternative transcription.

Alasoo Kaur K Martinez Fernando O FO Hale Christine C Gordon Siamon S Powrie Fiona F Dougan Gordon G Mukhopadhyay Subhankar S Gaffney Daniel J DJ

Scientific reports 20150730

Macrophages differentiated from human induced pluripotent stem cells (IPSDMs) are a potentially valuable new tool for linking genotype to phenotype in functional studies. However, at a genome-wide level these cells have remained largely uncharacterised. Here, we compared the transcriptomes of naïve and lipopolysaccharide (LPS) stimulated monocyte-derived macrophages (MDMs) and IPSDMs using RNA-Seq. The IPSDM and MDM transcriptomes were broadly similar and exhibited a highly conserved response to ...[more]

PMID: 26224331

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Project description:BACKGROUND: Exposure of macrophages to bacterial products such as lipopolysaccharide (LPS) results in activation of the NF-kappaB transcription factor, which orchestrates a gene expression programme that underpins the macrophage-dependent immune response. These changes include the induction or repression of a wide range of genes that regulate inflammation, cell proliferation, migration and cell survival. This process is tightly regulated and loss of control is associated with conditions such as septic shock, inflammatory diseases and cancer. To study this response, it is important to have in vitro model systems that reflect the behaviour of cells in vivo. In addition, it is necessary to understand the natural differences that can occur between individuals. In this report, we have investigated and compared the LPS response in macrophage derived cell lines and peripheral blood mononuclear cell (PBMC) derived macrophages. RESULTS: Gene expression profiles were determined following LPS treatment of THP-1 cells for 1 and 4 hours. LPS significantly induced or repressed 72 out of 465 genes selected as being known or putative NF-kappaB target genes, which exhibited 4 temporal patterns of expression. Results for 34 of these genes, including several genes not previously identified as LPS target genes, were validated using real time PCR. A high correlation between microarray and real time PCR data was found. Significantly, the LPS induced expression profile of THP-1 cells, as determined using real time PCR, was found to be very similar to that of human PBMC derived macrophages. Interestingly, some differences were observed in the LPS response between the two donor PBMC macrophage populations. Surprisingly, we found that the LPS response in U937 cells was dramatically different to both THP-1 and PBMC derived macrophages. CONCLUSION: This study revealed a dynamic and diverse transcriptional response to LPS in macrophages, involving both the induction and repression of gene expression in a time dependent manner. Moreover, we demonstrated that the LPS induced transcriptional response in the THP-1 cell line is very similar to primary PBMC derived macrophages. Therefore, THP-1 cells represent a good model system for studying the mechanisms of LPS and NF-kappaB dependent gene expression.

Project description:Histone deacetylases (HDACs) are a group of enzymes that control histone deacetylation and bear potential to direct expression of large gene sets. We determined the effect of HDAC inhibitors (HDACi) on human monocytes and macrophages, with respect to their polarization, activation, and their capabilities of inducing endotoxin tolerance. To address the role for HDACs in macrophage polarization, we treated monocytes with HDAC3i, HDAC6i or pan-HDACi prior to polarization into M1 or M2 macrophages using IFNγ or IL-4 respectively. To study the HDAC inhibition effect on cytokine expression, macrophages were treated with HDACi prior to LPS-stimulation. TNFα, IL-6, and p40 were measured with ELISA, whereas modifications of Histone 3 and STAT1 were assessed using western blot. To address the role for HDAC3 in repeated LPS challenge induction, HDAC3i or HDAC3 siRNA was added to monocytes prior to incubation with IFNγ, which were then repeatedly challenged with LPS and analyzed by means of protein analyses and transcriptional profiling. Pan-HDACi and HDAC3i reduced cytokine secretion in monocytes and M1 macrophages, whereas HDAC6i yielded no such effect. Notably, neither pan-HDACi nor HDAC3i reduced cytokine secretion in M2 macrophages. In contrast to previous reports in mouse macrophages, HDAC3i did not affect macrophage polarization in human cells. Likewise, HDAC3 was not required for IFNγ signaling or IFNβ secretion. Cytokine and gene expression analyses confirmed that IFNγ-treated macrophages consistently develop a cytokine response after LPS repeated challenge, but pretreatment with HDAC3i or HDAC3 siRNA reinstates a state of tolerance reflected by general suppression of tolerizable genes, possibly through decreasing TLRs expression, and particularly TLR4/CD14. The development of endotoxin tolerance in macrophages is important to reduce exacerbated immune response and limit tissue damage. We conclude that HDAC3 is an attractive protein target to mediate macrophage reactivity and tolerance induction in inflammatory macrophages.

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Transcriptional profiling of macrophages derived from monocytes and iPS cells identifies a conserved response to LPS and novel alternative transcription.

Publications

Transcriptional profiling of macrophages derived from monocytes and iPS cells identifies a conserved response to LPS and novel alternative transcription.

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