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1.
Nat Immunol ; 18(3): 293-302, 2017 03.
Article in English | MEDLINE | ID: mdl-28092373

ABSTRACT

The aggregation of hypertrophic macrophages constitutes the basis of all granulomatous diseases, such as tuberculosis or sarcoidosis, and is decisive for disease pathogenesis. However, macrophage-intrinsic pathways driving granuloma initiation and maintenance remain elusive. We found that activation of the metabolic checkpoint kinase mTORC1 in macrophages by deletion of the gene encoding tuberous sclerosis 2 (Tsc2) was sufficient to induce hypertrophy and proliferation, resulting in excessive granuloma formation in vivo. TSC2-deficient macrophages formed mTORC1-dependent granulomatous structures in vitro and showed constitutive proliferation that was mediated by the neo-expression of cyclin-dependent kinase 4 (CDK4). Moreover, mTORC1 promoted metabolic reprogramming via CDK4 toward increased glycolysis while simultaneously inhibiting NF-κB signaling and apoptosis. Inhibition of mTORC1 induced apoptosis and completely resolved granulomas in myeloid TSC2-deficient mice. In human sarcoidosis patients, mTORC1 activation, macrophage proliferation and glycolysis were identified as hallmarks that correlated with clinical disease progression. Collectively, TSC2 maintains macrophage quiescence and prevents mTORC1-dependent granulomatous disease with clinical implications for sarcoidosis.


Subject(s)
Granuloma/immunology , Macrophages/immunology , Multiprotein Complexes/metabolism , Sarcoidosis/immunology , TOR Serine-Threonine Kinases/metabolism , Tumor Suppressor Proteins/metabolism , Animals , Cell Line , Cyclin-Dependent Kinase 4/metabolism , Disease Progression , Granuloma/drug therapy , Humans , Macrophages/drug effects , Mechanistic Target of Rapamycin Complex 1 , Mice , Mice, Inbred C57BL , Mice, Knockout , RNA, Small Interfering/genetics , Sarcoidosis/drug therapy , Signal Transduction , Tuberous Sclerosis Complex 2 Protein , Tumor Suppressor Proteins/genetics
2.
J Immunol ; 190(4): 1519-27, 2013 Feb 15.
Article in English | MEDLINE | ID: mdl-23315073

ABSTRACT

The MAPK p38α senses environmental stressors and orchestrates inflammatory and immunomodulatory reactions. However, the molecular mechanism how p38α controls immunomodulatory responses in myeloid cells remains elusive. We found that in monocytes and macrophages, p38α activated the mechanistic target of rapamycin (mTOR) pathway in vitro and in vivo. p38α signaling in myeloid immune cells promoted IL-10 but inhibited IL-12 expression via mTOR and blocked the differentiation of proinflammatory CD4(+) Th1 cells. Cellular stress induced p38α-mediated mTOR activation that was independent of PI3K but dependent on the MAPK-activated protein kinase 2 and on the inhibition of tuberous sclerosis 1 and 2, a negative regulatory complex of mTOR signaling. Remarkably, p38α and PI3K concurrently modulated mTOR to balance IL-12 and IL-10 expression. Our data link p38α to mTOR signaling in myeloid immune cells that is decisive for tuning the immune response in dependence on the environmental milieu.


Subject(s)
Environmental Exposure , Immunity, Innate , Mitogen-Activated Protein Kinase 14/physiology , TOR Serine-Threonine Kinases/physiology , Animals , Cell Line, Transformed , Cells, Cultured , Dendritic Cells/immunology , Dendritic Cells/metabolism , Environmental Exposure/adverse effects , Humans , Immunity, Innate/genetics , Interleukin-10/biosynthesis , Interleukin-12 Subunit p40/biosynthesis , Macrophages/immunology , Macrophages/metabolism , Mice , Mice, Knockout , Mitogen-Activated Protein Kinase 14/genetics , Monocytes/immunology , Monocytes/metabolism , Signal Transduction/genetics , Signal Transduction/immunology
3.
Biochem Soc Trans ; 41(4): 927-33, 2013 Aug.
Article in English | MEDLINE | ID: mdl-23863158

ABSTRACT

The innate myeloid immune system is a complex network of cells that protect against disease by identifying and killing pathogens and tumour cells, but it is also implicated in homoeostatic mechanisms such as tissue remodelling and wound healing. Myeloid phagocytes such as monocytes, macrophages or dendritic cells are at the basis of controlling these immune responses in all tissues of the body. In the present review, we summarize recent studies demonstrating that mTOR [mammalian (or mechanistic) target of rapamycin] regulates innate immune reactions in macrophages and dendritic cells. The mTOR pathway serves as a decision maker to control the cellular response to pathogens and tumours by regulating the expression of inflammatory mediators such as cytokines, chemokines or interferons. In addition to various in vivo mouse models, kidney transplant patients under mTOR inhibitor therapy allowed the elucidation of important innate immune functions regulated by mTOR in humans. The role of the mTOR pathway in macrophages and dendritic cells enhances our understanding of the immune system and suggests new therapeutic avenues for the regulation of pro- versus anti-inflammatory mediators with potential relevance to cancer therapy, the design of novel adjuvants and the control of distinct infectious and autoimmune diseases.


Subject(s)
Dendritic Cells/immunology , Macrophages/immunology , Signal Transduction , TOR Serine-Threonine Kinases/metabolism , Cytokines/metabolism , Dendritic Cells/metabolism , Humans , Immunity, Innate , Inflammation Mediators/metabolism , Macrophages/metabolism , Neoplasms/therapy , TOR Serine-Threonine Kinases/antagonists & inhibitors
4.
Blood ; 117(16): 4273-83, 2011 Apr 21.
Article in English | MEDLINE | ID: mdl-21368289

ABSTRACT

A central role for the mammalian target of rapamycin (mTOR) in innate immunity has been recently defined by its ability to limit proinflammatory mediators. Although glucocorticoids (GCs) exert potent anti-inflammatory effects in innate immune cells, it is currently unknown whether the mTOR pathway interferes with GC signaling. Here we show that inhibition of mTOR with rapamycin or Torin1 prevented the anti-inflammatory potency of GC both in human monocytes and myeloid dendritic cells. GCs could not suppress nuclear factor-κB and JNK activation, the expression of proinflammatory cytokines, and the promotion of Th1 responses when mTOR was inhibited. Interestingly, long-term activation of monocytes with lipopolysaccharide enhanced the expression of TSC2, the principle negative regulator of mTOR, whereas dexamethasone blocked TSC2 expression and reestablished mTOR activation. Renal transplant patients receiving rapamycin but not those receiving calcineurin inhibitors displayed a state of innate immune cell hyper-responsiveness despite the concurrent use of GC. Finally, mTOR inhibition was able to override the healing phenotype of dexamethasone in a murine lipopolysaccharide shock model. Collectively, these data identify a novel link between the glucocorticoid receptor and mTOR in innate immune cells, which is of considerable clinical importance in a variety of disorders, including allogeneic transplantation, autoimmune diseases, and cancer.


Subject(s)
Anti-Inflammatory Agents/pharmacology , Glucocorticoids/pharmacology , Immunosuppressive Agents/pharmacology , Myeloid Cells/drug effects , Sirolimus/pharmacology , TOR Serine-Threonine Kinases/antagonists & inhibitors , Animals , Anti-Inflammatory Agents/therapeutic use , Cells, Cultured , Dendritic Cells/drug effects , Dendritic Cells/immunology , Dexamethasone/pharmacology , Dexamethasone/therapeutic use , Glucocorticoids/therapeutic use , Humans , Immunosuppressive Agents/therapeutic use , Kidney Transplantation , Leukocytes, Mononuclear/drug effects , Leukocytes, Mononuclear/immunology , Mice , Mice, Inbred C57BL , Monocytes/drug effects , Monocytes/immunology , Myeloid Cells/immunology , NF-kappa B/immunology , Naphthyridines/pharmacology , Naphthyridines/therapeutic use , Sirolimus/therapeutic use , TOR Serine-Threonine Kinases/immunology , Tuberous Sclerosis Complex 2 Protein , Tumor Suppressor Proteins/immunology
5.
J Am Soc Nephrol ; 23(5): 934-47, 2012 May.
Article in English | MEDLINE | ID: mdl-22282592

ABSTRACT

Uremia impairs the atheroprotective properties of HDL, but the mechanisms underlying why this occurs are unknown. Here, we observed that HDL isolated from healthy individuals inhibited the production of inflammatory cytokines by peripheral monocytes stimulated with a Toll-like receptor 2 agonist. In contrast, HDL isolated from the majority of patients with ESRD did not show this anti-inflammatory property; many HDL samples even promoted the production of inflammatory cytokines. To investigate this difference, we used shotgun proteomics to identify 49 HDL-associated proteins in a uremia-specific pattern. Proteins enriched in HDL from patients with ESRD (ESRD-HDL) included surfactant protein B (SP-B), apolipoprotein C-II, serum amyloid A (SAA), and α-1-microglobulin/bikunin precursor. In addition, we detected some ESRD-enriched proteins in earlier stages of CKD. We did not detect a difference in oxidation status between HDL isolated from uremic and healthy patients. Regarding function of these uremia-specific proteins, only SAA mimicked ESRD-HDL by promoting inflammatory cytokine production. Furthermore, SAA levels in ESRD-HDL inversely correlated with its anti-inflammatory potency. In conclusion, HDL has anti-inflammatory activities that are defective in uremic patients as a result of specific changes in its molecular composition. These data suggest a potential link between the high levels of inflammation and cardiovascular mortality in uremia.


Subject(s)
Inflammation/etiology , Lipoproteins, HDL/physiology , Serum Amyloid A Protein/physiology , Uremia/blood , Adult , Aged , Female , Humans , Iron/metabolism , Kidney Failure, Chronic/blood , Male , Middle Aged , Oxidation-Reduction , Proteomics , Pulmonary Surfactant-Associated Protein B/blood
6.
Cell Stress ; 5(12): 176-182, 2021 Dec.
Article in English | MEDLINE | ID: mdl-34917890

ABSTRACT

Programmed cell death protein 4 (PDCD4) exerts critical functions as tumor suppressor and in immune cells to regulate inflammatory processes. The phosphoinositide 3-kinase (PI3K) promotes degradation of PDCD4 via mammalian target of rapamycin complex 1 (mTORC1). However, additional pathways that may regulate PDCD4 expression are largely ill-defined. In this study, we have found that activation of the mitogen-activated protein kinase p38 promoted degradation of PDCD4 in macrophages and fibroblasts. Mechanistically, we identified a pathway from p38 and its substrate MAP kinase-activated protein kinase 2 (MK2) to the tuberous sclerosis complex (TSC) to regulate mTORC1-dependent degradation of PDCD4. Moreover, we provide evidence that TSC1 and TSC2 regulate PDCD4 expression via an additional mechanism independent of mTORC1. These novel data extend our knowledge of how PDCD4 expression is regulated by stress- and nutrient-sensing pathways.

7.
Cell Rep ; 30(5): 1542-1552.e7, 2020 02 04.
Article in English | MEDLINE | ID: mdl-32023468

ABSTRACT

Mechanistic or mammalian target of rapamycin complex 1 (mTORC1) is an important regulator of effector functions, proliferation, and cellular metabolism in macrophages. The biochemical processes that are controlled by mTORC1 are still being defined. Here, we demonstrate that integrative multiomics in conjunction with a data-driven inverse modeling approach, termed COVRECON, identifies a biochemical node that influences overall metabolic profiles and reactions of mTORC1-dependent macrophage metabolism. Using a combined approach of metabolomics, proteomics, mRNA expression analysis, and enzymatic activity measurements, we demonstrate that Tsc2, a negative regulator of mTORC1 signaling, critically influences the cellular activity of macrophages by regulating the enzyme phosphoglycerate dehydrogenase (Phgdh) in an mTORC1-dependent manner. More generally, while lipopolysaccharide (LPS)-stimulated macrophages repress Phgdh activity, IL-4-stimulated macrophages increase the activity of the enzyme required for the expression of key anti-inflammatory molecules and macrophage proliferation. Thus, we identify Phgdh as a metabolic checkpoint of M2 macrophages.


Subject(s)
Cell Polarity , Genomics , Macrophages/cytology , Macrophages/metabolism , Models, Biological , Phosphoglycerate Dehydrogenase/metabolism , Animals , Cell Polarity/drug effects , Cell Proliferation/drug effects , Gene Expression Regulation, Enzymologic/drug effects , Glutamic Acid/metabolism , Glycine/metabolism , Interleukin-4/pharmacology , Ketoglutaric Acids/metabolism , Kinetics , Macrophages/drug effects , Macrophages/enzymology , Mechanistic Target of Rapamycin Complex 1/metabolism , Mice, Inbred C57BL , Phosphoglycerate Dehydrogenase/genetics , Principal Component Analysis , Serine/metabolism , Tuberous Sclerosis Complex 2 Protein/metabolism
8.
JCI Insight ; 4(20)2019 10 17.
Article in English | MEDLINE | ID: mdl-31619583

ABSTRACT

The mechanistic target of rapamycin complex 2 (mTORC2) is a potentially novel and promising anticancer target due to its critical roles in proliferation, apoptosis, and metabolic reprogramming of cancer cells. However, the activity and function of mTORC2 in distinct cells within malignant tissue in vivo is insufficiently explored. Surprisingly, in primary human and mouse colorectal cancer (CRC) samples, mTORC2 signaling could not be detected in tumor cells. In contrast, only macrophages in tumor-adjacent areas showed mTORC2 activity, which was downregulated in stromal macrophages residing within human and mouse tumor tissues. Functionally, inhibition of mTORC2 by specific deletion of Rictor in macrophages stimulated tumorigenesis in a colitis-associated CRC mouse model. This phenotype was driven by a proinflammatory reprogramming of mTORC2-deficient macrophages that promoted colitis via the cytokine SPP1/osteopontin to stimulate tumor growth. In human CRC patients, high SPP1 levels and low mTORC2 activity in tumor-associated macrophages correlated with a worsened clinical prognosis. Treatment of mice with a second-generation mTOR inhibitor that inhibits mTORC2 and mTORC1 exacerbated experimental colorectal tumorigenesis in vivo. In conclusion, mTORC2 activity is confined to macrophages in CRC and limits tumorigenesis. These results suggest activation but not inhibition of mTORC2 as a therapeutic strategy for colitis-associated CRC.


Subject(s)
Carcinogenesis/immunology , Colitis, Ulcerative/pathology , Colorectal Neoplasms/immunology , Macrophages/immunology , Mechanistic Target of Rapamycin Complex 2/metabolism , Animals , Carcinogenesis/drug effects , Cell Line, Tumor , Cell Proliferation/drug effects , Cells, Cultured , Colitis, Ulcerative/blood , Colitis, Ulcerative/chemically induced , Colitis, Ulcerative/immunology , Colon/cytology , Colon/drug effects , Colon/immunology , Colon/pathology , Colorectal Neoplasms/mortality , Colorectal Neoplasms/pathology , Colorectal Neoplasms/prevention & control , Dextran Sulfate/toxicity , Disease Models, Animal , Female , Humans , Intestinal Mucosa/cytology , Intestinal Mucosa/drug effects , Intestinal Mucosa/immunology , Intestinal Mucosa/pathology , Kaplan-Meier Estimate , Macrophages/metabolism , Male , Mechanistic Target of Rapamycin Complex 2/antagonists & inhibitors , Mice , Mice, Transgenic , Morpholines/pharmacology , Osteopontin/blood , Osteopontin/metabolism , Primary Cell Culture , Prognosis , Survival Rate
9.
Methods Mol Biol ; 1228: 15-23, 2015.
Article in English | MEDLINE | ID: mdl-25311118

ABSTRACT

The isolation of nuclei from eukaryotic cells is essential for studying the composition and the dynamic changes of the nuclear proteome to gain insight into the mechanisms of gene expression and cell signalling. Primary cells are particularly challenging for standard nuclear isolation protocols due to low protein content, sample degradation, or nuclear clumping. Here, we describe a rapid and flexible protocol for the isolation of clean and intact nuclei, which results in the recovery of 90-95 % highly pure nuclei. The method, called lysis gradient centrifugation (LGC), is based on an iso-osmolar discontinuous iodixanol-based density gradient including a detergent-containing lysis layer. A single low g-force centrifugation step enables mild cell lysis and prevents extensive contact of the nuclei with the cytoplasmic environment. This fast method shows high reproducibility due to the relatively little cell manipulation required by the investigator. Further advantages are the low amount of starting material required, easy parallel processing of multiple samples, and isolation of nuclei and cytoplasm at the same time from the same sample.


Subject(s)
Cell Fractionation/methods , Cell Nucleus , Centrifugation/methods , Animals , Blotting, Western , Cell Death , Cell Nucleus/metabolism , Cells, Cultured , Humans , Mice
10.
J Immunol Methods ; 373(1-2): 167-73, 2011 Oct 28.
Article in English | MEDLINE | ID: mdl-21889513

ABSTRACT

Due to their low protein content and limited nuclear detergent stability, primary human immune cells such as monocytes or T lymphocytes represent a great challenge for standard nuclear isolation protocols. Nuclei clumping during the multiple centrifugation steps or contamination of isolated nuclei with cytoplasmic proteins due to membrane lysis is a frequently observed problem. Here we describe a versatile and novel method for the isolation of clean and intact nuclei from primary human monocytes, which can be applied for virtually any cell type. Living cells were applied on an iso-osmolar discontinuous iodixanol-based density gradient including a detergent-containing lysis layer. Mild cell lysis as well as efficient washing of the nuclei was performed during the course of one single low g-force centrifugation step. The isolation procedure, which we call lysis gradient centrifugation (LGC), results in the recovery of 90-95% of highly pure nuclei. This easy and highly reproducible procedure allows an effective preparation of nuclei and the cytoplasm in only 15 min with the ability to handle as little as one million cells per sample and easy parallel processing of multiple samples.


Subject(s)
Cell Fractionation/methods , Cell Nucleus/metabolism , Centrifugation, Density Gradient/methods , Monocytes/metabolism , Blotting, Western , Cell Line, Tumor , Cell Nucleus/drug effects , Cells, Cultured , Cytoplasm/metabolism , Humans , Jurkat Cells , Lipopolysaccharides/pharmacology , Macrophages/metabolism , Microscopy, Fluorescence , Reproducibility of Results , Subcellular Fractions/metabolism , T-Lymphocytes/metabolism , Time Factors , Transcription Factors/metabolism , Triiodobenzoic Acids/chemistry
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