scholarly journals GCN2 drives macrophage and MDSC function and immunosuppression in the tumor microenvironment

2019 ◽  
Vol 4 (42) ◽  
pp. eaax8189 ◽  
Author(s):  
Marie Jo Halaby ◽  
Kebria Hezaveh ◽  
Sara Lamorte ◽  
M. Teresa Ciudad ◽  
Andreas Kloetgen ◽  
...  
Keyword(s):  
Transcription Factor ◽  
Macrophage Polarization ◽  
Suppressor Cells ◽  
Interferon Γ ◽  
Mass Cytometry ◽  
General Control ◽  
Immune Attack ◽  
Environmental Sensor ◽  
Rna Knockdown ◽  
Interfering Rna

General control nonderepressible 2 (GCN2) is an environmental sensor controlling transcription and translation in response to nutrient availability. Although GCN2 is a putative therapeutic target for immuno-oncology, its role in shaping the immune response to tumors is poorly understood. Here, we used mass cytometry, transcriptomics, and transcription factor–binding analysis to determine the functional impact of GCN2 on the myeloid phenotype and immune responses in melanoma. We found that myeloid-lineage deletion of GCN2 drives a shift in the phenotype of tumor-associated macrophages and myeloid-derived suppressor cells (MDSCs) that promotes antitumor immunity. Time-of-flight mass cytometry (CyTOF) and single-cell RNA sequencing showed that this was due to changes in the immune microenvironment with increased proinflammatory activation of macrophages and MDSCs and interferon-γ expression in intratumoral CD8+ T cells. Mechanistically, GCN2 altered myeloid function by promoting increased translation of the transcription factor CREB-2/ATF4, which was required for maturation and polarization of macrophages and MDSCs in both mice and humans, whereas targeting Atf4 by small interfering RNA knockdown reduced tumor growth. Last, analysis of patients with cutaneous melanoma showed that GCN2-dependent transcriptional signatures correlated with macrophage polarization, T cell infiltrates, and overall survival. Thus, these data reveal a previously unknown dependence of tumors on myeloid GCN2 signals for protection from immune attack.

scholarly journals Nuclear receptors RXRα:RARα are repressors for human MRP3 expression

2007 ◽  
Vol 292 (5) ◽  
pp. G1221-G1227 ◽  
Author(s):  
Wensheng Chen ◽  
Shi-Ying Cai ◽  
Shuhua Xu ◽  
Lee A. Denson ◽  
Carol J. Soroka ◽  
...  
Keyword(s):  
Transcription Factor ◽  
Promoter Activity ◽  
Regulatory Elements ◽  
Luciferase Reporter ◽  
Dependent Manner ◽  
Site Mutation ◽  
Transcription Factor Sp1 ◽  
Gc Box ◽  
Rna Knockdown ◽  
Interfering Rna

Multidrug resistance-associated protein MRP3/Mrp3 (ABCC3) is upregulated in cholestasis, an adaptive response that may protect the liver from accumulation of toxic compounds, such as bile salts and bilirubin conjugates. However, the mechanism of this upregulation is poorly understood. We and others have previously reported that fetoprotein transcription factor/liver receptor homolog-1 is an activator of MRP3/Mrp3 expression. In searching for additional regulatory elements in the human MRP3 promoter, we have now identified nuclear receptor retinoic X receptor-α:retinoic acid receptor-α (RXRα:RARα) as a repressor of MRP3 activation by transcription factor Sp1. A luciferase reporter assay demonstrated that cotransfection of transcription factor Sp1 stimulates the MRP3 promoter activity and that additions of RXRα:RARα abrogated this activation in a dose-dependent manner. Site mutations and gel shift assays have identified a Sp1 binding GC box motif at −113 to −108 nts upstream from the MRP3 translation start site, where RXRα:RARα specifically reduced Sp1 binding to this site. Mutation of the GC box also reduced MRP3 promoter activity. The functional role of RXRα:RARα as a repressor of MRP3 expression was further confirmed by RARα small-interfering RNA knockdown in HepG2 cells, which upregulated endogenous MRP3 expression. In summary, our results indicate that activator Sp1 and repressor RXRα:RARα act in concert to regulate MRP3 expression. Since RXRα:RARα expression is diminished by cholestatic liver injury, loss of RXRα:RARα may lead to upregulation of MRP3/Mrp3 expression in these disorders.

scholarly journals Expression of Interferon-γ by a Coronavirus Defective-Interfering RNA Vector and Its Effect on Viral Replication, Spread, and Pathogenicity

Virology ◽  
1997 ◽  
Vol 233 (2) ◽  
pp. 327-338 ◽  
Author(s):  
Xuming Zhang ◽  
David R. Hinton ◽  
Daniel J. Cua ◽  
Stephen A. Stohlman ◽  
Michael M.C. Lai
Keyword(s):  
Viral Replication ◽  
Interferon Γ ◽  
Defective Interfering Rna ◽  
Interfering Rna

Potentiation of flagellin responses in gut epithelial cells by interferon-γ is associated with STAT-independent regulation of MyD88 expression

2009 ◽  
Vol 423 (1) ◽  
pp. 119-128 ◽  
Author(s):  
Ciara Bannon ◽  
Pam J. Davies ◽  
Andrew Collett ◽  
Geoffrey Warhurst
Keyword(s):  
Epithelial Cells ◽  
Enteric Bacteria ◽  
Interferon Γ ◽  
Janus Kinase ◽  
Cxcl8 Secretion ◽  
Health And Disease ◽  
Toll Like Receptor 5 ◽  
Myeloid Differentiation Factor ◽  
Interfering Rna ◽  
Dose Dependent

Flagellin acting via TLR5 (Toll-like receptor 5) is a key regulator of the host response to the gut microbial flora in both health and disease. The present study has investigated regulation of flagellin–TLR5 signalling in human colonocytes (HT29-19A) by IFNγ (interferon-γ), a cytokine released early in the inflammatory process which has multiple effects on gut epithelial function that may facilitate abnormal responses to enteric bacteria. Flagellin induced a dose-dependent secretion of chemokines CXCL8 and CCL2 in the human colonocyte line, HT29-19A. Exposure to IFNγ did not induce chemokine secretion, but markedly potentiated responses to flagellin, increasing CXL8 gene expression and protein secretion by approx. 4-fold. Potentiation by IFNγ was independent of changes in TLR5 and was associated with a rapid, sustained increase in expression of the downstream adaptor molecule MyD88 (myeloid differentiation factor 88). Knockdown of MyD88 expression using siRNA (small interfering RNA) abolished flagellin-dependent CXCL8 secretion and the potentiating effect of IFNγ. Exposure of non-transformed mouse and human colonocytes to IFNγ also increased MyD88 expression. STAT (signal transducer and activator of transcription) 1 knockdown and use of the broad-spectrum JAK (Janus kinase)-STAT inhibitor AG490 had no effect on IFNγ-mediated up-regulation of MyD88. The findings of the present study suggest that IFNγ sensitizes colonic epithelial cells to bacterial flagellin via a largely STAT-independent up-regulation of MyD88 expression leading to increased secretion of immunomodulatory factors. These results indicate that epithelial responses to flagellin are potentiated by IFNγ, most likely mediated by increased MyD88 expression. The present study adds to our understanding of the spectrum of effects of this cytokine on gut epithelium that may contribute to bacterial-driven inflammation in the gut.

scholarly journals Identification of human immune cell subtypes most responsive to IL-1β–induced inflammatory signaling using mass cytometry

2021 ◽  
Vol 14 (673) ◽  
pp. eabc5763 ◽  
Author(s):  
Hema Kothari ◽  
Corey M. Williams ◽  
Chantel McSkimming ◽  
Fabrizio Drago ◽  
Melissa A. Marshall ◽  
...  
Keyword(s):  
T Cells ◽  
Transcription Factor ◽  
Mononuclear Cells ◽  
Immune Cell ◽  
Human Peripheral Blood ◽  
Effector Memory ◽  
High Morbidity ◽  
Cytokine Storm ◽  
Mass Cytometry ◽  
Myeloid Dendritic Cells

IL-1β is a key mediator of the cytokine storm linked to high morbidity and mortality from COVID-19, and IL-1β blockade with anakinra and canakinumab during COVID-19 infection has entered clinical trials. Using mass cytometry of human peripheral blood mononuclear cells, we identified effector memory CD4+ T cells and CD4−CD8low/−CD161+ T cells, specifically those positive for the chemokine receptor CCR6, as the circulating immune subtypes with the greatest response to IL-1β. This response manifested as increased phosphorylation and, thus, activation of the proinflammatory transcription factor NF-κB and was also seen in other subsets, including CD11c+ myeloid dendritic cells, classical monocytes, two subsets of natural killer cells (CD16−CD56brightCD161− and CD16−CD56dimCD161+), and lineage− (Lin−) cells expressing CD161 and CD25. IL-1β also induced a rapid but less robust increase in the phosphorylation of the kinase p38 as compared to that of NF-κB in most of these immune cell subsets. Prolonged IL-1β stimulation increased the phosphorylation of the transcription factor STAT3 and to a lesser extent that of STAT1 and STAT5 across various immune cell types. IL-1β–induced production of IL-6 likely led to the activation of STAT1 and STAT3 at later time points. Interindividual heterogeneity and inhibition of STAT activation by anakinra raise the possibility that assays measuring NF-κB phosphorylation in response to IL-1β in CCR6+ T cell subtypes could identify those patients at higher risk of cytokine storm and most likely to benefit from IL-1β–neutralizing therapies.

scholarly journals The role of the Th1 transcription factor T-bet in a mouse model of immune-mediated bone-marrow failure

Blood ◽  
2010 ◽  
Vol 115 (3) ◽  
pp. 541-548 ◽  
Author(s):  
Yong Tang ◽  
Marie J. Desierto ◽  
Jichun Chen ◽  
Neal S. Young
Keyword(s):  
Bone Marrow ◽  
T Cells ◽  
Transcription Factor ◽  
Transforming Growth Factor ◽  
Bone Marrow Failure ◽  
Critical Role ◽  
Interferon Γ ◽  
Interleukin 17 ◽  
Immune Mediated

Abstract The transcription factor T-bet is a key regulator of type 1 immune responses. We examined the role of T-bet in an animal model of immune-mediated bone marrow (BM) failure using mice carrying a germline T-bet gene deletion (T-bet−/−). In comparison with normal C57BL6 (B6) control mice, T-bet−/− mice had normal cellular composition in lymphohematopoietic tissues, but T-bet−/− lymphocytes were functionally defective. Infusion of 5 × 106 T-bet−/− lymph node (LN) cells into sublethally irradiated, major histocompatibility complex–mismatched CByB6F1 (F1) recipients failed to induce the severe marrow hypoplasia and fatal pancytopenia that is produced by injection of similar numbers of B6 LN cells. Increasing T-bet−/− LN-cell dose to 10 to 23 × 106 per recipient led to only mild hematopoietic deficiency. Recipients of T-bet−/− LN cells had no expansion in T cells or interferon-γ–producing T cells but showed a significant increase in Lin−Sca1+CD117+CD34− BM cells. Plasma transforming growth factor-β and interleukin-17 concentrations were increased in T-bet−/− LN-cell recipients, possibly a compensatory up-regulation of the Th17 immune response. Continuous infusion of interferon-γ resulted in hematopoietic suppression but did not cause T-bet−/− LN-cell expansion or BM destruction. Our data provided fresh evidence demonstrating a critical role of T-bet in immune-mediated BM failure.

scholarly journals The DDX23 Negatively Regulates Translation and Replication of Foot-and-Mouth Disease Virus and Is Degraded by 3C Proteinase

Viruses ◽  
2020 ◽  
Vol 12 (12) ◽  
pp. 1348
Author(s):  
Sahibzada Waheed Abdullah ◽  
Shichong Han ◽  
Jin’en Wu ◽  
Yun Zhang ◽  
Manyuan Bai ◽  
...  
Keyword(s):  
Disease Virus ◽  
Small Interfering Rna ◽  
Foot And Mouth Disease ◽  
Mouth Disease ◽  
Dependent Manner ◽  
Mouth Disease Virus ◽  
Foot And Mouth ◽  
Rna Knockdown ◽  
Interfering Rna ◽  
Fmdv Replication

DEAD-box helicase 23 (DDX23) is a host nuclear helicase, which is a part of the spliceosomal complex and involved in pre-mRNA splicing. To investigate whether DDX23, an internal ribosomal entry sites transacting factor (ITAF) affects foot-and-mouth disease virus (FMDV) replication and translation through internal ribosome entry site (IRES)-dependent manner. For this, we utilized a pull-down assay, Western blotting, quantitative real-time PCR, confocal microscopy, overexpression and small interfering RNA knockdown, as well as the median tissue culture infective dose. Our findings showed that FMDV infection inhibited DDX23 expression and the overexpression of DDX23 reduced viral replication, however, CRISPR Cas9 knockout/small interfering RNA knockdown increased FMDV replication. FMDV IRES domain III and IV interacted with DDX23, whereas DDX23 interacted with FMDV 3C proteinase and significantly degraded. The enzymatic activity of FMDV 3C proteinase degraded DDX23, whereas FMDV degraded DDX23 via the lysosomal pathway. Additionally, IRES-driven translation was suppressed in DDX23-overexpressing cells, and was enhanced in DDX23 knocked down. Collectively, our results demonstrated that DDX23 negatively affects FMDV IRES-dependent translation, which could be a useful target for the design of antiviral drugs.

scholarly journals Vimentin filaments drive migratory persistence in polyploidal cancer cells

2020 ◽  
Vol 117 (43) ◽  
pp. 26756-26765
Author(s):  
Botai Xuan ◽  
Deepraj Ghosh ◽  
Joy Jiang ◽  
Rachelle Shao ◽  
Michelle R. Dawson
Keyword(s):  
Cancer Cells ◽  
Cancer Progression ◽  
Structural Integrity ◽  
Single Cell Analysis ◽  
Critical Role ◽  
Critical Function ◽  
Rna Knockdown ◽  
Interfering Rna ◽  
The Impact

Polyploidal giant cancer cells (PGCCs) are multinucleated chemoresistant cancer cells found in heterogeneous solid tumors. Due in part to their apparent dormancy, the effect of PGCCs on cancer progression has remained largely unstudied. Recent studies have highlighted the critical role of PGCCs as aggressive and chemoresistant cancer cells, as well as their ability to undergo amitotic budding to escape dormancy. Our recent study demonstrated the unique biophysical properties of PGCCs, as well as their unusual migratory persistence. Here we unveil the critical function of vimentin intermediate filaments (VIFs) in maintaining the structural integrity of PGCCs and enhancing their migratory persistence. We performed in-depth single-cell analysis to examine the distribution of VIFs and their role in migratory persistence. We found that PGCCs rely heavily on their uniquely distributed and polarized VIF network to enhance their transition from a jammed to an unjammed state to allow for directional migration. Both the inhibition of VIFs with acrylamide and small interfering RNA knockdown of vimentin significantly decreased PGCC migration and resulted in a loss of PGCC volume. Because PGCCs rely on their VIF network to direct migration and to maintain their enlarged morphology, targeting vimentin or vimentin cross-linking proteins could provide a therapeutic approach to mitigate the impact of these chemoresistant cells in cancer progression and to improve patient outcomes with chemotherapy.

scholarly journals Targeted Deletion of the Murine apobec-1 Complementation Factor (acf) Gene Results in Embryonic Lethality

2005 ◽  
Vol 25 (16) ◽  
pp. 7260-7269 ◽  
Author(s):  
Valerie Blanc ◽  
Jeffrey O. Henderson ◽  
Elizabeth P. Newberry ◽  
Susan Kennedy ◽  
Jianyang Luo ◽  
...  
Keyword(s):  
Rna Binding ◽  
Cytidine Deaminase ◽  
Mrna Editing ◽  
Targeted Deletion ◽  
Apob Mrna ◽  
Rna Knockdown ◽  
Interfering Rna ◽  
Binding Subunit ◽  
Apparently Healthy

ABSTRACT apobec-1 complementation factor (ACF) is an hnRNP family member which functions as the obligate RNA binding subunit of the core enzyme mediating C-to-U editing of the nuclear apolipoprotein B (apoB) transcript. ACF binds to both apoB RNA and apobec-1, the catalytic cytidine deaminase, which then results in site-specific posttranscriptional editing of apoB mRNA. Targeted deletion of apobec1 eliminates C-to-U editing of apoB mRNA but is otherwise well tolerated. However, the functions and potential targets of ACF beyond apoB mRNA editing are unknown. Here we report the results of generating acf knockout mice using homologous recombination. While heterozygous acf +/ − mice were apparently healthy and fertile, no viable acf − / − mice were identified. Mutant acf − / − embryos were detectable only until the blastocyst (embryonic day 3.5 [E3.5]) stage. No acf − / − blastocysts were detectable following implantation at E4.5, and isolated acf − / − blastocysts failed to proliferate in vitro. Small interfering RNA knockdown of ACF in either rat (apobec-1-expressing) or human (apobec-1-deficient) hepatoma cells decreased ACF protein expression and induced a commensurate increase in apoptosis. Taken together, these data suggest that ACF plays a crucial role, which is independent of apobec-1 expression, in cell survival, particularly during early embryonic development.

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