Transcriptome and translatome profiling of Mtb-infected human DC identifies GSK-3beta as a molecular switch of rapamycin-driven immune stimulation
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PROVIDER: PRJNA686434 | ENA |
REPOSITORIES: ENA
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Project description:In human primary dendritic cells (DC) rapamycin - an autophagy inducer and protein synthesis inhibitor - overcomes the autophagy block induced by Mycobacterium tuberculosis (Mtb) and promotes Th1 response via IL-12 secretion. Here, the immunostimulatory activity of rapamycin in Mtb-infected DC was investigated by analyzing the transcriptome and the translatome profiles. Several differentially expressed genes (DEGs) were found in transcriptome and translatome of Mtb-infected DC, showing an additional increase in presence of rapamycin, which was instead ineffective in uninfected cells. The majority of DEGs identified in the translatome resulted also present in the transcriptome-modulated genes, suggesting that both RNAs are actively translated mRNAs. The DEG in silico analysis showed significant changes in intracellular cascades related to cytokine production, cytokine-induced signaling and immune response to pathogens. In particular, rapamycin treatment of Mtb-infected DC caused a significant enrichment of IFN-beta, IFN-lambda and IFN-stimulated genes in the polysome-associated RNA fraction. In addition, IL-12, IL-23, IL-1B, IL-6, and TNF-A were induced at RNA and protein level by rapamycin in Mtb-infected DC, while IL-10 was reduced. Interestingly, upon siRNA or pharmacologically GSK-3beta inhibition, rapamycin-driven modulation of the pro- and anti-inflammatory cytokine balance was lost, thus indicating that in Mtb-infected DC GSK-3beta functions as molecular switch for the regulation of cytokine milieu. In conclusion, our study sheds light on the molecular mechanism by which autophagy contributes to DC activation during Mtb infection and highlights that rapamycin and its analogs as well as GSK-3beta modulators are promising compounds for host-directed therapy in the control of Mtb infection.
2021-04-12 | GSE163531 | GEO
Project description:This project involves RNA-Seq analysis of samples obtained from the Phase IIA clinical trial TB-019 (NCT01669096) which evaluated kinetics of response, safety, and immunogenicity of the GSK Mycobacterium tuberculosis (MTB) vaccine M72/AS01E (“GSK M72”). GSK M72 consists of the M72 recombinant fusion of Mycobacterium tuberculosis (MTB) proteins Rv0125 and Rv1196 in combination with the liposome, TLR4 ligand (MPL), and QS21 saponin adjuvant AS01E (Leroux-Roels et al., 2013).
2023-12-30 | GSE102574 | GEO
Project description:In human primary dendritic cells (DC) rapamycin-an autophagy inducer and protein synthesis inhibitor-overcomes the autophagy block induced by Mycobacterium tuberculosis (Mtb) and promotes a Th1 response via IL-12 secretion. Here, the immunostimulatory activity of rapamycin in Mtb-infected DC was further investigated by analyzing both transcriptome and translatome gene profiles. Hundreds of differentially expressed genes (DEGs) were identified by transcriptome and translatome analyses of Mtb-infected DC, and some of these genes were found further modulated by rapamycin. The majority of transcriptome-associated DEGs overlapped with those present in the translatome, suggesting that transcriptionally stimulated mRNAs are also actively translated. In silico analysis of DEGs revealed significant changes in intracellular cascades related to cytokine production, cytokine-induced signaling and immune response to pathogens. In particular, rapamycin treatment of Mtb-infected DC caused an enrichment of IFN-β, IFN-λ and IFN-stimulated gene transcripts in the polysome-associated RNA fraction. In addition, rapamycin led to an increase of IL-12, IL-23, IL-1β, IL-6, and TNF-α but to a reduction of IL-10. Interestingly, upon silencing or pharmacological inhibition of GSK-3β, the rapamycin-driven modulation of the pro- and anti-inflammatory cytokine balance was lost, indicating that, in Mtb-infected DC, GSK-3β acts as molecular switch for the regulation of the cytokine milieu. In conclusion, our study sheds light on the molecular mechanism by which autophagy induction contributes to DC activation during Mtb infection and points to rapamycin and GSK-3β modulators as promising compounds for host-directed therapy in the control of Mtb infection.
| S-EPMC8086600 | biostudies-literature
Project description:Gene expression data from AML cell lines, MOLM-14, U937, THP-1 and HL-60, that were infected with a scrambled control hairpin (shControl), two shRNAs directed against GSK-3B (shGSK3B_1 and shGSK3B_2), or two shRNAs directed against GSK-3A (shGSK3A_5 and shGSK3A_6). Acute myeloid leukemia (AML) is the most common form of acute leukemia in adults. Long-term survival of patients with AML has changed little over the past decade, necessitating the identification and validation of new AML targets. Integration of genomic approaches with small-molecule and genetic-based high-throughput screening holds the promise of improved discovery of candidate targets for cancer therapy. Here, we identified a role for glycogen synthase kinase 3A (GSK-3A) in AML by performing two independent small-molecule library screens and an shRNA screen for perturbations that induced a differentiation expression signature in AML cells. GSK-3 is a serine-threonine kinase involved in diverse cellular processes including differentiation, signal transduction, cell cycle regulation, and proliferation. We demonstrated that specific loss of GSK-3A induced differentiation in AML by multiple measurements, including induction of gene expression signatures, morphological changes, and cell surface markers consistent with myeloid maturation. GSK-3AM-bM-^@M-^Sspecific suppression also led to impaired growth and proliferation in vitro, induction of apoptosis, loss of colony formation in methylcellulose, and anti-AML activity in vivo. Although the role of GSK-3B has been well studied in cancer development, these studies support a role for GSK-3A in AML. The AML cell lines, MOLM-14, U937, THP-1 and HL-60, were infected with a scrambled control hairpin (shControl), two shRNAs directed against GSK-3B (shGSK3B_1 and shGSK3B_2), and two shRNAs directed against GSK-3A (shGSK3A_5 and shGSK3A_6).
2012-02-27 | E-GEOD-35200 | biostudies-arrayexpress
Project description:GSK-3b plays a critical role in regulating the Wnt/b-catenin signaling pathway, and manipulating GSK-3b in dendritic cells (DCs) has been shown to improve anti-tumor efficacy of DC vaccines. Since inhibition of GSK-3b leads to activation of b-catenin, we hypothesize that blocking GSK-3b in DCs negatively regulates DC-mediated CD8 T cell immunity and anti-tumor immunity. Using CD11c-GSK-3b-/- conditional knockout mice in which GSK-3b is genetically deleted in CD11c-expressing DCs, we surprisingly found that deletion of GSK-3b in DCs resulted in increased anti-tumor immunity, which contradicted our initial expectation of reduced anti-tumor immunity due to presumed upregulation of b-catenin in DCs. Indeed, we found by both western blot and flow cytometry that deletion of GSK-3b in DCs did not lead to augmented expression of b-catenin protein, suggesting that GSK-3b exerts its function independent of b-catenin. Supporting this notion, our single cell RNA sequencing (scRNA-seq) analysis revealed that GSK-3b-deficient DCs exhibited distinct gene expression patterns with minimally overlapping differentially expressed genes (DEGs) compared to DCs with activated b-catenin. This suggests that deletion of GSK-3b in DCs is unlikely to lead to upregulation of b-catenin at the transcriptional level. Consistent with enhanced anti-tumor immunity, we also found that CD11c-GSK-3b-/- mice exhibited significantly augmented cross-priming of antigen-specific CD8 T cells following DC-targeted vaccines. We further found that deletion of GSK-3b in DCs completely abrogated memory CD8 T cell responses, suggesting that GSK-3b in DCs also plays a negative role in regulating the differentiation and/or maintenance of memory CD8 T cells. scRNA-seq analysis further revealed that although deletion of GSK-3b in DCs positively regulated transcriptional programs for effector differentiation and function of primed antigen-specific CD8 T cells in CD11c-GSK-3b-/- mice during the priming phase, it resulted in significantly reduced antigen-specific memory CD8 T cells, consistent with diminished memory responses. Taken together, our data demonstrate that GSK-3b in DCs has opposite functions in regulating cross-priming and memory CD8 T cell responses, and GSK-3b exerts its functions independent of its regulation of b-catenin.
2024-09-12 | GSE276602 | GEO
Project description:Gene expression data from AML cell lines, MOLM-14, U937, THP-1 and HL-60, that were infected with a scrambled control hairpin (shControl), two shRNAs directed against GSK-3B (shGSK3B_1 and shGSK3B_2), or two shRNAs directed against GSK-3A (shGSK3A_5 and shGSK3A_6). Acute myeloid leukemia (AML) is the most common form of acute leukemia in adults. Long-term survival of patients with AML has changed little over the past decade, necessitating the identification and validation of new AML targets. Integration of genomic approaches with small-molecule and genetic-based high-throughput screening holds the promise of improved discovery of candidate targets for cancer therapy. Here, we identified a role for glycogen synthase kinase 3A (GSK-3A) in AML by performing two independent small-molecule library screens and an shRNA screen for perturbations that induced a differentiation expression signature in AML cells. GSK-3 is a serine-threonine kinase involved in diverse cellular processes including differentiation, signal transduction, cell cycle regulation, and proliferation. We demonstrated that specific loss of GSK-3A induced differentiation in AML by multiple measurements, including induction of gene expression signatures, morphological changes, and cell surface markers consistent with myeloid maturation. GSK-3A–specific suppression also led to impaired growth and proliferation in vitro, induction of apoptosis, loss of colony formation in methylcellulose, and anti-AML activity in vivo. Although the role of GSK-3B has been well studied in cancer development, these studies support a role for GSK-3A in AML.
2012-02-27 | GSE35200 | GEO
Project description:Aeromonas sp. MTB isolate:MTB Genome sequencing and assembly
| PRJNA1083721 | ENA
Project description:Genome-wide expression data can provide important insights into normal and pathological cellular processes. However, the ability to use gene expression data to quantitatively assess the activation state of a given signaling pathway or transcriptional network in a sensitive and specific manner remains an important unmet goal. We now describe a computational algorithm, energy-paired scoring (EPS), that satisfies these criteria by predicting pathway activity using gene-gene interactions within a pathway signature in a manner analogous to the estimation of energy generated by two charged particles, as described by Coulomb’s law. We demonstrate the ability of EPS to: quantitatively assess pathway activation levels in vivo and in vitro; accurately estimate the extent of pathway inhibition achieved by gene knockdown; sensitively detect crosstalk between endogenous signaling pathways in vivo; and accurately identify compounds capable of inhibiting selected signaling pathways. Our findings indicate that EPS can accurately predict pathway activity over a wide dynamic range based upon gene expression data sets derived from multiple profiling platforms, as well as different species, tissues and cell types in both in vitro and in vivo contexts Four timepoints (0h, 24h, 48h and 96h) with 3 replicates per timepoint of doxycycline induction for MTB (Control), MTB/TAN, MTB/TOM and MTB/TWNT1
2013-09-18 | E-GEOD-50969 | biostudies-arrayexpress
Project description:GSK-3 is a serine-threonine protein kinase and consists of two isoforms, GSK-3alpha and GSK-3beta. GSK-3alpha and GSK-3beta are inactivated by phosphorylation at Ser21 and Ser9, respectively. GSK-3alpha Ser21Ala and GSK-3beta Ser9Ala phosphorylation-resistant knock-in mice have constitutively active form of GSK-3.
2019-03-11 | GSE112159 | GEO
Project description:GSK-3 is a serine-threonine protein kinase and consists of two isoforms, GSK-3alpha and GSK-3beta. GSK-3alpha and GSK-3beta are inactivated by phosphorylation at Ser21 and Ser9, respectively. GSK-3alpha Ser21Ala and GSK-3beta Ser9Ala phosphorylation-resistant knock-in mice have constitutively active form of GSK-3.
2019-03-11 | GSE112158 | GEO