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1.
Sci Rep ; 11(1): 18721, 2021 09 21.
Article in English | MEDLINE | ID: mdl-34548527

ABSTRACT

Metformin is a first-line drug in the treatment of type-2 diabetes mellitus (T2DM). In addition to its antigluconeogenic and insulin-sensitizing properties, metformin has emerged as a potent inhibitor of the chronic inflammatory response of macrophages. In particular, metformin treatment has been shown to reduce expression of interleukin (IL-) 1ß during long-term exposure to the pro-inflammatory stimulus lipopolysaccharide (LPS) through a reduction in reactive oxygen species (ROS), which decreases the levels of the hypoxia-inducible factor (HIF) 1-α, and through enhanced expression of IL-10. However, the effect of metformin on the acute inflammatory response, before significant levels of ROS accumulate in the cell, has not been explored. Here, we show that metformin alters the acute inflammatory response through its activation of AMP-activated protein kinase (AMPK), but independently of HIF1-α and IL-10, in primary macrophages and two macrophage-like cell lines. Thus, metformin changes the acute and the chronic inflammatory response through fundamentally distinct mechanisms. Furthermore, RNA-seq analysis reveals that metformin pretreatment affects the levels of a large yet selective subset of inflammatory genes, dampening the response to short-term LPS exposure and affecting a wide range of pathways and biological functions. Taken together, these findings reveal an unexpected complexity in the anti-inflammatory properties of this widely used drug.


Subject(s)
Adenylate Kinase/metabolism , Hypoglycemic Agents/therapeutic use , Inflammation/prevention & control , Metformin/therapeutic use , Humans , Hypoglycemic Agents/pharmacology , Hypoxia-Inducible Factor 1, alpha Subunit/genetics , Interleukin-10/genetics , Interleukin-1beta/genetics , Lipopolysaccharides/pharmacology , Metformin/pharmacology , NF-kappa B/genetics , RNA, Messenger/genetics , Reactive Oxygen Species/metabolism
2.
Cell ; 170(6): 1096-1108.e13, 2017 Sep 07.
Article in English | MEDLINE | ID: mdl-28886380

ABSTRACT

Regulatory T cells (Tregs) play a pivotal role in the inhibition of anti-tumor immune responses. Understanding the mechanisms governing Treg homeostasis may therefore be important for development of effective tumor immunotherapy. We have recently demonstrated a key role for the canonical nuclear factor κB (NF-κB) subunits, p65 and c-Rel, in Treg identity and function. In this report, we show that NF-κB c-Rel ablation specifically impairs the generation and maintenance of the activated Treg (aTreg) subset, which is known to be enriched at sites of tumors. Using mouse models, we demonstrate that melanoma growth is drastically reduced in mice lacking c-Rel, but not p65, in Tregs. Moreover, chemical inhibition of c-Rel function delayed melanoma growth by impairing aTreg-mediated immunosuppression and potentiated the effects of anti-PD-1 immunotherapy. Our studies therefore establish inhibition of NF-κB c-Rel as a viable therapeutic approach for enhancing checkpoint-targeting immunotherapy protocols.


Subject(s)
Immunotherapy/methods , Melanoma/immunology , Melanoma/pathology , NF-kappa B/antagonists & inhibitors , Proto-Oncogene Proteins c-rel/antagonists & inhibitors , T-Lymphocytes, Regulatory/immunology , Animals , Disease Models, Animal , Female , Male , Melanoma/metabolism , Mice , Mice, Inbred C57BL , Mice, Knockout , NF-kappa B/metabolism
3.
Immunity ; 47(3): 450-465.e5, 2017 09 19.
Article in English | MEDLINE | ID: mdl-28889947

ABSTRACT

Both conventional T (Tconv) cells and regulatory T (Treg) cells are activated through ligation of the T cell receptor (TCR) complex, leading to the induction of the transcription factor NF-κB. In Tconv cells, NF-κB regulates expression of genes essential for T cell activation, proliferation, and function. However the role of NF-κB in Treg function remains unclear. We conditionally deleted canonical NF-κB members p65 and c-Rel in developing and mature Treg cells and found they have unique but partially redundant roles. c-Rel was critical for thymic Treg development while p65 was essential for mature Treg identity and maintenance of immune tolerance. Transcriptome and NF-κB p65 binding analyses demonstrated a lineage specific, NF-κB-dependent transcriptional program, enabled by enhanced chromatin accessibility. These dual roles of canonical NF-κB in Tconv and Treg cells highlight the functional plasticity of the NF-κB signaling pathway and underscores the need for more selective strategies to therapeutically target NF-κB.


Subject(s)
Cell Lineage/genetics , NF-kappa B/metabolism , T-Lymphocytes, Regulatory/immunology , T-Lymphocytes, Regulatory/metabolism , Transcription, Genetic , Animals , Autoimmunity/genetics , Autoimmunity/immunology , Binding Sites , Biomarkers , Cell Differentiation , Cell Survival/genetics , Cell Survival/immunology , Cluster Analysis , Cytokines/metabolism , Gene Deletion , Gene Expression Profiling , Gene Expression Regulation , Homeostasis/genetics , Homeostasis/immunology , Immune Tolerance , Immunophenotyping , Inflammation/genetics , Inflammation/immunology , Inflammation/metabolism , Lymphocyte Activation , Mice , Mice, Transgenic , NF-kappa B/genetics , Nucleotide Motifs , Phenotype , Protein Binding , Signal Transduction , T-Lymphocytes, Regulatory/cytology , Transcription Factor RelA/genetics , Transcription Factor RelA/metabolism , Transcriptome
4.
RNA ; 19(6): 811-27, 2013 Jun.
Article in English | MEDLINE | ID: mdl-23616639

ABSTRACT

The expression of eukaryotic mRNAs is achieved though an intricate series of molecular processes that provide many steps for regulating the production of a final gene product. However, the relationships between individual steps in mRNA biosynthesis and the rates at which they occur are poorly understood. By applying RNA-seq to chromatin-associated and soluble nucleoplasmic fractions of RNA from Lipid A-stimulated macrophages, we examined the timing of exon ligation and transcript release from chromatin relative to the induction of transcription. We find that for a subset of genes in the Lipid A response, the ligation of certain exon pairs is delayed relative to the synthesis of the complete transcript. In contrast, 3' end cleavage and polyadenylation occur rapidly once transcription extends through the cleavage site. Our data indicate that these transcripts with delayed splicing are not released from the chromatin fraction until all the introns have been excised. These unusual kinetics result in a chromatin-associated pool of completely transcribed and 3'-processed transcripts that are not yet fully spliced. We also find that long introns containing repressed exons that will be excluded from the final mRNA are excised particularly slowly relative to other introns in a transcript. These results indicate that the kinetics of splicing and transcript release contribute to the timing of expression for multiple genes of the inflammatory response.


Subject(s)
Alternative Splicing , Lipid A/pharmacology , Macrophages/drug effects , RNA, Messenger/metabolism , Animals , Cell Nucleus/genetics , Cell Nucleus/metabolism , Chromatin/genetics , Chromatin/metabolism , Exons , Gene Expression Regulation , Inflammation/genetics , Introns , Macrophages/cytology , Macrophages/metabolism , Mice , Mice, Inbred C57BL , Polyadenylation , RNA Cleavage , RNA Splice Sites , RNA, Messenger/genetics , Time Factors , Transcription, Genetic
5.
Article in English | MEDLINE | ID: mdl-24747344

ABSTRACT

Virtually all living organisms have evolved mechanisms to adapt to their environment by sensing environmental stresses and inducing the transcription of appropriate sets of response genes in a coordinated fashion. In the vertebrate immune system, the highly selective response to an environmental stimulus, often an invading microorganism, plays an especially important role in regulating the activities of, and interactions among, the many cell types involved in innate and adaptive immunity. It is now widely appreciated that the selective response to a stimulus requires the concerted action of signal transduction pathways, transcription factors, and chromatin structure. Many proteins and pathways that help to regulate a response have been characterized. However, our understanding of the gene-specific and global logic through which a highly selective response is elicited has only recently begun to emerge.


Subject(s)
Gene Expression Regulation , Transcription, Genetic , Animals , Chromatin/chemistry , Chromatin/metabolism , Enhancer Elements, Genetic , Gene Regulatory Networks , Humans , Interleukin-12 Subunit p40/metabolism , Mice , Nucleosomes/metabolism , Sequence Analysis, RNA , Signal Transduction , Transcription Factors/metabolism
6.
Cell ; 150(2): 279-90, 2012 Jul 20.
Article in English | MEDLINE | ID: mdl-22817891

ABSTRACT

Macrophages respond to inflammatory stimuli by modulating the expression of hundreds of genes in a defined temporal cascade, with diverse transcriptional and posttranscriptional mechanisms contributing to the regulatory network. We examined proinflammatory gene regulation in activated macrophages by performing RNA-seq with fractionated chromatin-associated, nucleoplasmic, and cytoplasmic transcripts. This methodological approach allowed us to separate the synthesis of nascent transcripts from transcript processing and the accumulation of mature mRNAs. In addition to documenting the subcellular locations of coding and noncoding transcripts, the results provide a high-resolution view of the relationship between defined promoter and chromatin properties and the temporal regulation of diverse classes of coexpressed genes. The data also reveal a striking accumulation of full-length yet incompletely spliced transcripts in the chromatin fraction, suggesting that splicing often occurs after transcription has been completed, with transcripts retained on the chromatin until fully spliced.


Subject(s)
Chromatin/genetics , Gene Expression Profiling , Inflammation/genetics , Macrophages/metabolism , RNA Splicing , Animals , Gene Expression Regulation , Lipid A/immunology , Macrophages/immunology , Mice , Mice, Inbred C57BL , Promoter Regions, Genetic , Receptor, Interferon alpha-beta/genetics , Receptors, Interferon/genetics , Sequence Analysis, RNA , Transcription, Genetic
7.
Cell ; 138(1): 114-28, 2009 Jul 10.
Article in English | MEDLINE | ID: mdl-19596239

ABSTRACT

We describe a broad mechanistic framework for the transcriptional induction of mammalian primary response genes by Toll-like receptors and other stimuli. One major class of primary response genes is characterized by CpG-island promoters, which facilitate promiscuous induction from constitutively active chromatin without a requirement for SWI/SNF nucleosome remodeling complexes. The low nucleosome occupancy at promoters in this class can be attributed to the assembly of CpG islands into unstable nucleosomes, which may lead to SWI/SNF independence. Another major class consists of non-CpG-island promoters that assemble into stable nucleosomes, resulting in SWI/SNF dependence and a requirement for transcription factors that promote selective nucleosome remodeling. Some stimuli, including serum and tumor necrosis factor-alpha, exhibit a strong bias toward activation of SWI/SNF-independent CpG-island genes. In contrast, interferon-beta is strongly biased toward SWI/SNF-dependent non-CpG-island genes. By activating a diverse set of transcription factors, Toll-like receptors induce both classes and others for an optimal response to microbial pathogens.


Subject(s)
Chromatin Assembly and Disassembly , CpG Islands , Transcriptional Activation , Animals , CD4-Positive T-Lymphocytes/metabolism , Chromosomal Proteins, Non-Histone/metabolism , Humans , Interferon Regulatory Factor-3/metabolism , Lipopolysaccharides/immunology , Mice , Nucleosomes/metabolism , Promoter Regions, Genetic , Transcription Factors/metabolism , Transcription, Genetic
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