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1.
Life Sci Alliance ; 4(6)2021 06.
Article in English | MEDLINE | ID: mdl-33771876

ABSTRACT

The mycobacterial cell wall glycolipid trehalose-6,6-dimycolate (TDM) activates macrophages through the C-type lectin receptor MINCLE. Regulation of innate immune cells relies on miRNAs, which may be exploited by mycobacteria to survive and replicate in macrophages. Here, we have used macrophages deficient in the microprocessor component DGCR8 to investigate the impact of miRNA on the response to TDM. Deletion of DGCR8 in bone marrow progenitors reduced macrophage yield, but did not block macrophage differentiation. DGCR8-deficient macrophages showed reduced constitutive and TDM-inducible miRNA expression. RNAseq analysis revealed that they accumulated primary miRNA transcripts and displayed a modest type I IFN signature at baseline. Stimulation with TDM in the absence of DGCR8 induced overshooting expression of IFNß and IFN-induced genes, which was blocked by antibodies to type I IFN. In contrast, signaling and transcriptional responses to recombinant IFNß were unaltered. Infection with live Mycobacterium bovis Bacille Calmette-Guerin replicated the enhanced IFN response. Together, our results reveal an essential role for DGCR8 in curbing IFNß expression macrophage reprogramming by mycobacteria.


Subject(s)
Macrophages/metabolism , Mycobacterium/metabolism , RNA-Binding Proteins/metabolism , Animals , Cytokines/metabolism , Female , Interferons/immunology , Interferons/metabolism , Lectins, C-Type/genetics , Lectins, C-Type/metabolism , Male , Membrane Proteins/metabolism , Mice , Mice, Inbred C57BL , MicroRNAs/genetics , Mycobacterium/genetics , Mycobacterium/pathogenicity , RNA-Binding Proteins/genetics , Sugar Phosphates/metabolism , Trehalose/analogs & derivatives , Trehalose/metabolism
2.
J Allergy Clin Immunol ; 139(6): 1873-1884.e10, 2017 Jun.
Article in English | MEDLINE | ID: mdl-27742396

ABSTRACT

BACKGROUND: Langerhans cell (LC) networks play key roles in immunity and tolerance at body surfaces. LCs are established prenatally and can be replenished from blood monocytes. Unlike skin-resident dermal DCs (dDCs)/interstitial-type DCs and inflammatory dendritic epidermal cells appearing in dermatitis/eczema lesions, LCs lack key monocyte-affiliated markers. Inversely, LCs express various epithelial genes critical for their long-term peripheral tissue residency. OBJECTIVE: Dendritic cells (DCs) are functionally involved in inflammatory diseases; however, the mechanisms remained poorly understood. METHODS: In vitro differentiation models of human DCs, gene profiling, gene transduction, and immunohistology were used to identify molecules involved in DC subset specification. RESULTS: Here we identified the monocyte/macrophage lineage identity transcription factor Kruppel-like factor 4 (KLF4) to be inhibited during LC differentiation from human blood monocytes. Conversely, KLF4 is maintained or induced during dermal DC and monocyte-derived dendritic cell/inflammatory dendritic epidermal cell differentiation. We showed that in monocytic cells KLF4 has to be repressed to allow their differentiation into LCs. Moreover, respective KLF4 levels in DC subsets positively correlate with proinflammatory characteristics. We identified epithelial Notch signaling to repress KLF4 in monocytes undergoing LC commitment. Loss of KLF4 in monocytes transcriptionally derepresses Runt-related transcription factor 3 in response to TGF-ß1, thereby allowing LC differentiation marked by a low cytokine expression profile. CONCLUSION: Monocyte differentiation into LCs depends on activation of Notch signaling and the concomitant loss of KLF4.


Subject(s)
Dendritic Cells/cytology , Kruppel-Like Transcription Factors/metabolism , Monocytes/cytology , Skin/cytology , Adult , Cell Differentiation/physiology , Cells, Cultured , Dendritic Cells/metabolism , Embryo, Mammalian , Fetal Blood/cytology , Humans , Inflammation/metabolism , Kruppel-Like Factor 4 , Monocytes/metabolism , Transforming Growth Factor beta1/pharmacology
3.
J Immunol ; 195(4): 1753-62, 2015 Aug 15.
Article in English | MEDLINE | ID: mdl-26170386

ABSTRACT

Plasmacytoid dendritic cells (pDCs) efficiently produce large amounts of type I IFN in response to TLR7 and TLR9 ligands, whereas conventional DCs (cDCs) predominantly secrete high levels of the cytokines IL-10 and IL-12. The molecular basis underlying this distinct phenotype is not well understood. In this study, we identified the MAPK phosphatase Dusp9/MKP-4 by transcriptome analysis as selectively expressed in pDCs, but not cDCs. We confirmed the constitutive expression of Dusp9 at the protein level in pDCs generated in vitro by culture with Flt3 ligand and ex vivo in sorted splenic pDCs. Dusp9 expression was low in B220(-) bone marrow precursors and was upregulated during pDC differentiation, concomitant with established pDC markers. Higher expression of Dusp9 in pDCs correlated with impaired phosphorylation of the MAPK ERK1/2 upon TLR9 stimulation. Notably, Dusp9 was not expressed at detectable levels in human pDCs, although these displayed similarly impaired activation of ERK1/2 MAPK compared with cDCs. Enforced retroviral expression of Dusp9 in mouse GM-CSF-induced cDCs increased the expression of TLR9-induced IL-12p40 and IFN-ß, but not of IL-10. Conditional deletion of Dusp9 in pDCs was effectively achieved in Dusp9(flox/flox); CD11c-Cre mice at the mRNA and protein levels. However, the lack of Dusp9 in pDC did not restore ERK1/2 activation after TLR9 stimulation and only weakly affected IFN-ß and IL-12p40 production. Taken together, our results suggest that expression of Dusp9 is sufficient to impair ERK1/2 activation and enhance IFN-ß expression. However, despite selective expression in pDCs, Dusp9 is not essential for high-level IFN-ß production by these cells.


Subject(s)
Dendritic Cells/metabolism , Dual-Specificity Phosphatases/genetics , Gene Expression , Interferon-beta/biosynthesis , Animals , Cell Differentiation/genetics , Cluster Analysis , Computational Biology/methods , Dendritic Cells/cytology , Dendritic Cells/immunology , Extracellular Signal-Regulated MAP Kinases/metabolism , Gene Expression Profiling , Humans , Interferon-beta/genetics , Interleukin-12 , Mice , Mice, Knockout , Organ Specificity/genetics , Phosphorylation , Reproducibility of Results , Toll-Like Receptor 7/metabolism , Toll-Like Receptor 9/metabolism , Transcriptome
4.
Am J Respir Cell Mol Biol ; 47(5): 575-82, 2012 Nov.
Article in English | MEDLINE | ID: mdl-22904196

ABSTRACT

High concentrations of particulate matter (PM(10)) were measured in classrooms. This study addresses the hazard of indoor particles in comparison to the better-studied outdoor particles. Samples were taken from six schools during teaching hours. Genome-wide gene expression in human BEAS-2B lung epithelial cells was analyzed and verified by quantitative PCR. Polycyclic aromatic hydrocarbons, endotoxin, and cat allergen (Fel d 1) were analyzed by standard methods. Enhancement of allergic reactivity by PM(10) was confirmed in human primary basophils. Acceleration of human blood coagulation was determined with supernatants of PM(10)-exposed human peripheral blood monocytes. Indoor PM(10) induced serine protease inhibitor B2 (involved in blood coagulation) and inflammatory genes (such as CXCL6, CXCL1, IL6, IL8; all P < 0.001). Outdoor PM(10) induced xenobiotic metabolizing enzymes (cytochrome P450 [CYP] 1A1, CYP1B1, TIPARP; all P < 0.001). The induction of inflammatory genes by indoor PM(10) was explained by endotoxin (indoor 128.5 ± 42.2 EU/mg versus outdoor 13.4 ± 21.5 EU/mg; P < 0.001), the induction of CYP by outdoor polycyclic aromatic hydrocarbons (indoor 8.3 ± 4.9 ng/mg versus outdoor 16.7 ± 15.2 ng/mg; P < 0.01). The induction of serine protease inhibitor B2 was confirmed by a more rapid human blood coagulation (P < 0.05). Indoor PM(10) only affected allergic reactivity from human primary basophils from cat-allergic individuals. This was explained by varying Fel d 1 concentrations in indoor PM(10) (P < 0.001). Indoor PM(10), compared with outdoor PM(10), was six times higher and, on an equal weight basis, induced more inflammatory and allergenic reactions, and accelerated blood coagulation. Outdoor PM(10) had significantly lower effects, but induced detoxifying enzymes. Therefore, preliminary interventions for the reduction of classroom PM(10) seem reasonable, perhaps through intensified ventilation.


Subject(s)
Air Pollutants/toxicity , Air Pollution, Indoor , Particulate Matter/toxicity , Schools , Air Pollutants/analysis , Air Pollutants/immunology , Allergens/analysis , Analysis of Variance , Animals , Basophils/drug effects , Basophils/immunology , Basophils/physiology , Blood Coagulation Tests , Cats , Cell Line , Endotoxins/analysis , Gene Expression Regulation/drug effects , Humans , Hypersensitivity , Monocytes/drug effects , Oligonucleotide Array Sequence Analysis , Particulate Matter/analysis , Particulate Matter/immunology , Polycyclic Aromatic Hydrocarbons/analysis , Transcriptome
5.
Mol Syst Biol ; 6: 371, 2010 Jun 08.
Article in English | MEDLINE | ID: mdl-20531401

ABSTRACT

Recognition of microbial danger signals by toll-like receptors (TLR) causes re-programming of macrophages. To investigate kinase cascades triggered by the TLR4 ligand lipopolysaccharide (LPS) on systems level, we performed a global, quantitative and kinetic analysis of the phosphoproteome of primary macrophages using stable isotope labelling with amino acids in cell culture, phosphopeptide enrichment and high-resolution mass spectrometry. In parallel, nascent RNA was profiled to link transcription factor (TF) phosphorylation to TLR4-induced transcriptional activation. We reproducibly identified 1850 phosphoproteins with 6956 phosphorylation sites, two thirds of which were not reported earlier. LPS caused major dynamic changes in the phosphoproteome (24% up-regulation and 9% down-regulation). Functional bioinformatic analyses confirmed canonical players of the TLR pathway and highlighted other signalling modules (e.g. mTOR, ATM/ATR kinases) and the cytoskeleton as hotspots of LPS-regulated phosphorylation. Finally, weaving together phosphoproteome and nascent transcriptome data by in silico promoter analysis, we implicated several phosphorylated TFs in primary LPS-controlled gene expression.


Subject(s)
Macrophage Activation/immunology , Macrophages/immunology , Phosphoproteins/metabolism , Proteome/metabolism , Toll-Like Receptor 4/immunology , Animals , Cells, Cultured , Enzyme Activation/drug effects , Lipopolysaccharides/pharmacology , Macrophage Activation/drug effects , Macrophages/drug effects , Macrophages/enzymology , Mice , Phosphorylation/drug effects , Protein Kinases/metabolism , Signal Transduction/drug effects , Transcription Factors/metabolism , Transcriptional Activation/drug effects , Transcriptional Activation/genetics
6.
J Immunol ; 184(2): 713-24, 2010 Jan 15.
Article in English | MEDLINE | ID: mdl-20007528

ABSTRACT

Although several markers have been associated with the characterization of regulatory T cells (Tregs) and their function, no studies have investigated the dynamics of their phenotype during infection. Since the necessity of Tregs to control immunopathology has been demonstrated, we used the chronic helminth infection model Schistosoma mansoni to address the impact on the Treg gene repertoire. Before gene expression profiling, we first studied the localization and Ag-specific suppressive nature of classically defined Tregs during infection. The presence of Foxp3+ cells was predominantly found in the periphery of granulomas and isolated CD4+CD25(hi)Foxp3+ Tregs from infected mice and blocked IFN-gamma and IL-10 cytokine secretion from infected CD4+CD25- effector T cells. Furthermore, the gene expression patterns of Tregs and effector T cells showed that 474 genes were significantly regulated during schistosomiasis. After k-means clustering, we identified genes exclusively regulated in all four populations, including Foxp3, CD103, GITR, OX40, and CTLA-4--classic Treg markers. During infection, however, several nonclassical genes were upregulated solely within the Treg population, such as Slpi, Gzmb, Mt1, Fabp5, Nfil3, Socs2, Gpr177, and Klrg1. Using RT-PCR, we confirmed aspects of the microarray data and also showed that the expression profile of Tregs from S. mansoni-infected mice is simultaneously unique and comparable with Tregs derived from other infections.


Subject(s)
Gene Expression Profiling , Gene Expression Regulation , Helminthiasis, Animal/genetics , T-Lymphocytes, Regulatory/metabolism , Animals , Chronic Disease , Cluster Analysis , Lymphocyte Activation , Mice , Phenotype , Schistosoma mansoni , T-Lymphocytes, Regulatory/parasitology
7.
Am J Physiol Cell Physiol ; 297(5): C1146-56, 2009 Nov.
Article in English | MEDLINE | ID: mdl-19726747

ABSTRACT

Neuronal nitric oxide synthase (nNOS)-derived nitric oxide (NO) acts as a neurotransmitter and intracellular signaling molecule in the central and peripheral nervous system. NO regulates multiple processes like neuronal development, plasticity, and differentiation and is a mediator of neurotoxicity. The nNOS gene is highly complex with 12 alternative first exons, exon 1a-1l, transcribed from distinct promoters, leading to nNOS variants with different 5'-untranslated regions. Transcriptional control of the nNOS gene is not understood in detail. To investigate regulation of nNOS gene expression by retinoic acid (RA), we used the human neuroblastoma cell line TGW-nu-I as a model system. We show that RA induces nNOS transcription in a protein synthesis-dependent fashion. We identify the phosphatidylinositol 3-kinase (PI3K)/Akt signaling pathway and the atypical orphan nuclear receptor DAX1 (NR0B1) as critical mediators involved in RA-induced nNOS gene transcription. RA treatment increases DAX1 expression via PI3K/Akt signaling. Upregulation of DAX1 expression in turn induces nNOS transcription in response to RA. These results identify nNOS as a target gene of a novel RA/PI3K/Akt/DAX1-dependent pathway in human neuroblastoma cells and stress the functional importance of the transcriptional regulator DAX1 for nNOS gene expression in response to RA treatment.


Subject(s)
Antineoplastic Agents/pharmacology , Neuroblastoma/metabolism , Nitric Oxide Synthase Type I/drug effects , Signal Transduction/drug effects , Tretinoin/pharmacology , Blotting, Western , Cell Line, Tumor , DAX-1 Orphan Nuclear Receptor , DNA-Binding Proteins/drug effects , DNA-Binding Proteins/metabolism , Gene Expression , Gene Expression Regulation/drug effects , Gene Expression Regulation/physiology , Humans , Immunoprecipitation , Nitric Oxide Synthase Type I/biosynthesis , Phosphatidylinositol 3-Kinases/drug effects , Phosphatidylinositol 3-Kinases/metabolism , Proto-Oncogene Proteins c-akt/drug effects , Proto-Oncogene Proteins c-akt/metabolism , Receptors, Retinoic Acid/drug effects , Receptors, Retinoic Acid/metabolism , Repressor Proteins/drug effects , Repressor Proteins/metabolism , Reverse Transcriptase Polymerase Chain Reaction , Signal Transduction/physiology , Transfection
8.
Cell Stem Cell ; 5(2): 157-67, 2009 Aug 07.
Article in English | MEDLINE | ID: mdl-19664990

ABSTRACT

Secreted frizzled-related protein 1 (Sfrp1) is highly expressed by stromal cells maintaining hematopoietic stem cells (HSCs). Sfrp1 loss in stromal cells increases production of hematopoietic progenitors, and in knockout mice, dysregulates hemostasis and increases Flk2- Cd34- Lin- Sca1+ Kit+ (LSK) cell numbers in bone marrow. Also, LSK and multipotent progenitors (MPPs) resided mainly in the G0/G1 phase of cell cycle, with an accompanying decrease in intracellular beta-catenin levels. Gene-expression studies showed a concomitant decrease Ccnd1 and Dkk1 in Cd34- LSK cells and increased expression of Pparg, Hes1, and Runx1 in MPP. Transplantation experiments showed no intrinsic effect of Sfrp1 loss on the number of HSCs or their ability to engraft irradiated recipients. In contrast, serial transplantations of wild-type HSCs into Sfrp1(-/-) mice show a progressive decrease of wild-type LSK and MPP numbers. Our results demonstrate that Sfrp1 is required to maintain HSC homeostasis through extrinsic regulation of beta-catenin.


Subject(s)
Hematopoiesis , Hematopoietic Stem Cells/metabolism , Proteins/metabolism , Animals , Basic Helix-Loop-Helix Transcription Factors/metabolism , Cell Cycle/physiology , Core Binding Factor Alpha 2 Subunit/metabolism , Cyclin D1/metabolism , Homeodomain Proteins/metabolism , Intercellular Signaling Peptides and Proteins/metabolism , Intracellular Signaling Peptides and Proteins , Mice , Mice, Knockout , PPAR gamma/metabolism , Proteins/genetics , Transcription Factor HES-1 , beta Catenin/metabolism
9.
PLoS Pathog ; 5(7): e1000506, 2009 Jul.
Article in English | MEDLINE | ID: mdl-19578441

ABSTRACT

The genome of Epstein-Barr virus (EBV) encodes 86 proteins, but only a limited set is expressed in EBV-growth transformed B cells, termed lymphoblastoid cell lines (LCLs). These cells proliferate via the concerted action of EBV nuclear antigens (EBNAs) and latent membrane proteins (LMPs), some of which are rate limiting to establish a stable homeostasis of growth promoting and anti-apoptotic activities. We show here that EBV mutants, which lack the EBNA-3A gene, are impaired but can still initiate cell cycle entry and proliferation of primary human B cells in contrast to an EBNA-2 deficient mutant virus. Surprisingly, and in contrast to previous reports, these viral mutants are attenuated in growth transformation assays but give rise to permanently growing EBNA-3A negative B cell lines which exhibit reduced proliferation rates and elevated levels of apoptosis. Expression profiles of EBNA-3A deficient LCLs are characterized by 129 down-regulated and 167 up-regulated genes, which are significantly enriched for genes involved in apoptotic processes or cell cycle progression like the tumor suppressor gene p16/INK4A, or might contribute to essential steps of the viral life cycle in the infected host. In addition, EBNA-3A cellular target genes remarkably overlap with previously identified targets of EBNA-2. This study comprises the first genome wide expression profiles of EBNA-3A target genes generated within the complex network of viral proteins of the growth transformed B cell and permits a more detailed understanding of EBNA-3A's function and contribution to viral pathogenesis.


Subject(s)
B-Lymphocytes/physiology , B-Lymphocytes/virology , Epstein-Barr Virus Nuclear Antigens/biosynthesis , Herpesvirus 4, Human/genetics , B-Lymphocytes/immunology , B-Lymphocytes/metabolism , Cell Line , Cell Proliferation , Cluster Analysis , Epstein-Barr Virus Nuclear Antigens/genetics , Gene Expression Profiling/methods , Herpesvirus 4, Human/growth & development , Herpesvirus 4, Human/metabolism , Humans , Lymphocyte Activation , Oligonucleotide Array Sequence Analysis , Reproducibility of Results , Reverse Transcriptase Polymerase Chain Reaction
10.
J Immunol ; 183(2): 1301-12, 2009 Jul 15.
Article in English | MEDLINE | ID: mdl-19561100

ABSTRACT

Elevated IL-10 has been implicated in reactivation tuberculosis (TB). Since macrophages rather than T cells were reported to be the major source of IL-10 in TB, we analyzed the consequences of a macrophage-specific overexpression of IL-10 in transgenic mice (macIL-10-transgenic) after aerosol infection with Mycobacterium tuberculosis (Mtb). MacIL-10 transgenic mice were more susceptible to chronic Mtb infection than nontransgenic littermates, exhibiting higher bacterial loads in the lung after 12 wk of infection and dying significantly earlier than controls. The differentiation, recruitment, and activation of Th1 cells as well as the induction of IFN-gamma-dependent effector genes against Mtb were not affected by macrophage-derived IL-10. However, microarray analysis of pulmonary gene expression revealed patterns characteristic of alternative macrophage activation that were overrepresented in Mtb-infected macIL-10 transgenic mice. Importantly, arginase-1 gene expression and activity were strikingly enhanced in transgenic mice accompanied by a reduced production of reactive nitrogen intermediates. Moreover, IL-10-dependent arginase-1 induction diminished antimycobacterial effector mechanisms in macrophages. Taken together, macrophage-derived IL-10 triggers aspects of alternative macrophage activation and promotes Mtb recrudescence independent of overt effects on anti-TB T cell immunity.


Subject(s)
Autocrine Communication/immunology , Interleukin-10/physiology , Macrophage Activation/immunology , T-Lymphocytes/immunology , Tuberculosis/immunology , Animals , Arginase/genetics , Gene Expression Profiling , Lung/metabolism , Lung/microbiology , Macrophages/metabolism , Mice , Mice, Transgenic , Mycobacterium tuberculosis
11.
Gastroenterology ; 137(1): 361-71, 371.e1-5, 2009 Jul.
Article in English | MEDLINE | ID: mdl-19362090

ABSTRACT

BACKGROUND & AIMS: Early metastasis is a hallmark of pancreatic ductal adenocarcinoma and responsible for >90% of pancreatic cancer death. Because little is known about the biology and genetics of the metastatic process, we desired to elucidate molecular pathways mediating pancreatic cancer metastasis in vivo by an unbiased forward genetic approach. METHODS: Highly metastatic pancreatic cancer cell populations were selected by serial in vivo passaging of parental cells with low metastatic potential and characterized by global gene expression profiling, chromatin immunoprecipitation, and in vivo metastatic assay. RESULTS: In vivo selection of highly metastatic pancreatic cancer cells induced epithelial-mesenchymal transition (EMT), loss of E-cadherin expression, and up-regulation of mesenchymal genes such as Snail. Genetic inactivation of E-cadherin in parental cells induced EMT and increased metastasis in vivo. Silencing of E-cadherin in highly metastatic cells is mediated by a transcriptional repressor complex containing Snail and histone deacetylase 1 (HDAC1) and HDAC2. In line, mesenchymal pancreatic cancer specimens and primary cell lines from genetically engineered Kras(G12D) mice showed HDAC-dependent down-regulation of E-cadherin and high metastatic potential. Finally, transforming growth factor beta-driven E-cadherin silencing and EMT of human pancreatic cancer cells depends on HDAC activity. CONCLUSIONS: We provide the first in vivo evidence that HDACs and Snail play an essential role in silencing E-cadherin during the metastatic process of pancreatic cancer cells. These data link the epigenetic HDAC machinery to EMT and metastasis and provide preclinical evidence that HDACs are promising targets for antimetastatic therapy.


Subject(s)
Cadherins/metabolism , Histone Deacetylases/metabolism , Lung Neoplasms/enzymology , Pancreatic Neoplasms/enzymology , Transcription Factors/metabolism , Animals , Antigens, CD , Antineoplastic Agents/pharmacology , Cadherins/genetics , Cell Line, Tumor , Cell Transdifferentiation , Gene Expression Profiling , Gene Expression Regulation, Neoplastic , Gene Silencing , Histone Deacetylase 1 , Histone Deacetylase 2 , Histone Deacetylase Inhibitors , Histone Deacetylases/genetics , Humans , Hydroxamic Acids/pharmacology , Lung Neoplasms/drug therapy , Lung Neoplasms/genetics , Lung Neoplasms/secondary , Mice , Mice, Nude , Pancreatic Neoplasms/drug therapy , Pancreatic Neoplasms/genetics , Pancreatic Neoplasms/pathology , Promoter Regions, Genetic , RNA Interference , Repressor Proteins/metabolism , Snail Family Transcription Factors , Transcription Factors/genetics , Transfection
12.
Gastroenterology ; 136(7): 2247-57, 2009 Jun.
Article in English | MEDLINE | ID: mdl-19272387

ABSTRACT

BACKGROUND & AIMS: Helicobacter pylori infects half of the world's population, thereby causing significant human morbidity and mortality. The mechanisms by which professional antigen-presenting cells recognize the microbe are poorly understood. METHODS: Using dendritic cells (DCs) from TRIF, MyD88, TLR 2/4/7/9(-/-), and multiple double/triple/quadruple mutant mice, we characterized receptors and pathways mediating innate immune recognition of H pylori. RESULTS: We identified a MyD88-dependent component of the DC activation program, which was induced by surface TLRs, with TLR2 and to a minor extent also TLR4 being the exclusive surface receptors recognizing H pylori. A second MyD88-dependent component could be blocked in TLR2/4(-/-) DCs by inhibitors of endosomal acidification and depended on intracellular TLRs. We identified TLR9-mediated recognition of H pylori DNA as a principal H pylori-induced intracellular TLR pathway and further showed that H pylori RNA induces proinflammatory cytokines in a TLR-dependent manner. Microarray analysis showed complementary, redundant, and synergistic interactions between TLRs and additionally revealed gene expression patterns specific for individual TLRs, including a TLR2-dependent anti-inflammatory signature. A third component of the DC activation program was primarily composed of type I interferon-stimulated genes. This response was MyD88 and TRIF independent but was inducible by RIG-I-dependent recognition of H pylori RNA. CONCLUSIONS: These results provide novel comprehensive insights into the mechanisms of H pylori recognition by DCs. Understanding these processes provides a basis for the rational design of new vaccination strategies.


Subject(s)
Helicobacter pylori/immunology , Immunity, Innate/physiology , Receptors, Pattern Recognition/immunology , Signal Transduction/physiology , Animals , Antigen-Presenting Cells/immunology , Antigen-Presenting Cells/metabolism , Cells, Cultured , DNA, Bacterial/metabolism , Dendritic Cells/immunology , Dendritic Cells/metabolism , Enzyme-Linked Immunosorbent Assay , Flow Cytometry , Gene Expression Profiling , Gene Expression Regulation , Helicobacter Infections/diagnosis , Helicobacter pylori/genetics , Immunity, Innate/genetics , Mice , Mice, Inbred C57BL , Mice, Knockout , Models, Animal , RNA, Bacterial/metabolism , Receptors, Pattern Recognition/genetics , Receptors, Pattern Recognition/metabolism , Sensitivity and Specificity , Signal Transduction/genetics , Toll-Like Receptors/deficiency
13.
J Exp Med ; 206(1): 89-97, 2009 Jan 16.
Article in English | MEDLINE | ID: mdl-19139169

ABSTRACT

Novel vaccination strategies against Mycobacterium tuberculosis (MTB) are urgently needed. The use of recombinant MTB antigens as subunit vaccines is a promising approach, but requires adjuvants that activate antigen-presenting cells (APCs) for elicitation of protective immunity. The mycobacterial cord factor Trehalose-6,6-dimycolate (TDM) and its synthetic analogue Trehalose-6,6-dibehenate (TDB) are effective adjuvants in combination with MTB subunit vaccine candidates in mice. However, it is unknown which signaling pathways they engage in APCs and how these pathways are coupled to the adaptive immune response. Here, we demonstrate that these glycolipids activate macrophages and dendritic cells (DCs) via Syk-Card9-Bcl10-Malt1 signaling to induce a specific innate activation program distinct from the response to Toll-like receptor (TLR) ligands. APC activation by TDB and TDM was independent of the C-type lectin receptor Dectin-1, but required the immunoreceptor tyrosine-based activation motif-bearing adaptor protein Fc receptor gamma chain (FcRgamma). In vivo, TDB and TDM adjuvant activity induced robust combined T helper (Th)-1 and Th-17 T cell responses to a MTB subunit vaccine and partial protection against MTB challenge in a Card9-dependent manner. These data provide a molecular basis for the immunostimulatory activity of TDB and TDM and identify the Syk-Card9 pathway as a rational target for vaccine development against tuberculosis.


Subject(s)
Adaptor Proteins, Signal Transducing/genetics , Immunity, Innate/immunology , Intracellular Signaling Peptides and Proteins/genetics , Protein-Tyrosine Kinases/genetics , Receptors, IgE/genetics , Tuberculosis Vaccines/immunology , Adjuvants, Immunologic/pharmacology , Animals , B-Cell CLL-Lymphoma 10 Protein , CARD Signaling Adaptor Proteins , CD4-Positive T-Lymphocytes/immunology , CD4-Positive T-Lymphocytes/metabolism , Caspases/genetics , Cytokines/genetics , Cytokines/metabolism , Dendritic Cells/drug effects , Dendritic Cells/immunology , Dendritic Cells/metabolism , Glycolipids/immunology , Glycolipids/pharmacology , Immunoglobulin G/blood , Immunoglobulin G/immunology , Lung/immunology , Lung/microbiology , Lymph Nodes/immunology , Lymph Nodes/microbiology , Macrophage Activation/drug effects , Macrophage Activation/immunology , Macrophages/drug effects , Macrophages/immunology , Macrophages/metabolism , Mice , Mice, Inbred C57BL , Mice, Knockout , Mucosa-Associated Lymphoid Tissue Lymphoma Translocation 1 Protein , Neoplasm Proteins/genetics , Signal Transduction/immunology , Syk Kinase , T-Lymphocytes, Helper-Inducer/drug effects , T-Lymphocytes, Helper-Inducer/immunology , T-Lymphocytes, Helper-Inducer/metabolism , Tuberculosis Vaccines/administration & dosage , Tuberculosis Vaccines/genetics , Tuberculosis, Pulmonary/immunology , Tuberculosis, Pulmonary/microbiology , Tuberculosis, Pulmonary/prevention & control , Vaccines, Subunit/administration & dosage , Vaccines, Subunit/immunology
14.
J Cell Mol Med ; 13(8B): 2069-2082, 2009 Aug.
Article in English | MEDLINE | ID: mdl-18705699

ABSTRACT

In a mouse model of mycobacteria-induced immunopathology, wild-type C57BL/6 (WT), IL-18-knockout (KO) and IFN-alphabeta receptor-KO mice developed circumscript, centrally necrotizing granulomatous lesions in response to aerosol infection with M. avium, whereas mice deficient in the IFN-gamma receptor, STAT-1 or IRF-1 did not exhibit granuloma necrosis. Comparative, microarray-based gene expression analysis in the lungs of infected WT and IRF-1-KO mice identified a set of genes whose differential regulation was closely associated with granuloma necrosis, among them cathepsin K, cystatin F and matrix metalloprotease 10. Further microarray-based comparison of gene expression in the lungs of infected WT, IFN-gamma-KO and IRF-1-KO mice revealed four distinct clusters of genes with variable dependence on the presence of IFN-gamma, IRF-1 or both. In particular, IRF-1 appeared to be directly involved in the differentiation of a type I immune response to mycobacterial infection. In summary, IRF-1, rather than being a mere transcription factor downstream of IFN-gamma, may be a master regulator of mycobacteria-induced immunopathology.


Subject(s)
Granuloma/microbiology , Interferon Regulatory Factor-1/physiology , Mycobacterium avium/pathogenicity , Animals , Base Sequence , DNA Primers , Enzyme-Linked Immunosorbent Assay , Interferon Regulatory Factor-1/genetics , Interleukin-18/genetics , Mice , Mice, Inbred C57BL , Mice, Knockout , Necrosis , Reverse Transcriptase Polymerase Chain Reaction
15.
Eur J Immunol ; 38(11): 2981-92, 2008 Nov.
Article in English | MEDLINE | ID: mdl-18924132

ABSTRACT

The mammalian target of rapamycin (mTOR) can be viewed as cellular master complex scoring cellular vitality and stress. Whether mTOR controls also innate immune-defenses is currently unknown. Here we demonstrate that TLR activate mTOR via phosphoinositide 3-kinase/Akt. mTOR physically associates with the MyD88 scaffold protein to allow activation of interferon regulatory factor-5 and interferon regulatory factor-7, known as master transcription factors for pro-inflammatory cytokine- and type I IFN-genes. Unexpectedly, inactivation of mTOR did not prevent but increased lethality of endotoxin-mediated shock, which correlated with increased levels of IL-1beta. Mechanistically, mTOR suppresses caspase-1 activation, thus inhibits release of bioactive IL-1beta. We have identified mTOR as indispensable component of PRR signal pathways, which orchestrates the defense program of innate immune cells.


Subject(s)
Immunity, Innate , Protein Kinases/physiology , Animals , Caspase 1/physiology , Cells, Cultured , Cytokines/biosynthesis , Female , Humans , Interferon Regulatory Factor-7/physiology , Interleukin-1beta/biosynthesis , Lipopolysaccharides/toxicity , Mice , Mice, Inbred C57BL , Signal Transduction , TOR Serine-Threonine Kinases , Toll-Like Receptors/physiology , Transcription, Genetic
16.
Proc Natl Acad Sci U S A ; 105(39): 15058-63, 2008 Sep 30.
Article in English | MEDLINE | ID: mdl-18815378

ABSTRACT

NF-kappaB is a key transcriptional regulator of inflammatory responses, but also controls expression of prosurvival genes, whose products protect tissues from damage and may thus act indirectly in an antiinflammatory fashion. The variable importance of these two distinct NF-kappaB-controlled responses impacts the potential utility of NF-kappaB inhibition as a treatment strategy for intractable inflammatory conditions, such as inflammatory bowel disease. Here, we show in murine models that inhibition of IKKbeta-dependent NF-kappaB activation exacerbates acute inflammation, but attenuates chronic inflammatory disease in the intestinal tract. Acute ulcerating inflammation is aggravated because of diminished NF-kappaB-mediated protection against epithelial cell apoptosis and delayed mucosal regeneration secondary to reduced NF-kappaB-dependent recruitment of inflammatory cells that secrete cytoprotective factors. In contrast, in IL-10-deficient mice, which serve as a model of chronic T cell-dependent colitis, ablation of IKKbeta in the intestinal epithelium has no impact, yet IKKbeta deficiency in myeloid cells attenuates inflammation and prolongs survival. These results highlight the striking context and tissue dependence of the proinflammatory and antiapoptotic functions of NF-kappaB. Our findings caution against the therapeutic use of IKKbeta/NF-kappaB inhibitors in acute inflammatory settings dominated by cell loss and ulceration.


Subject(s)
Colitis, Ulcerative/metabolism , I-kappa B Kinase/metabolism , Acute Disease , Animals , Chronic Disease , Colitis, Ulcerative/chemically induced , Colitis, Ulcerative/drug therapy , Dextran Sulfate/toxicity , Disease Models, Animal , Gene Expression , I-kappa B Kinase/antagonists & inhibitors , Interleukin-1/genetics , Intestinal Mucosa , Mice , Mice, Mutant Strains , NF-kappa B/metabolism , STAT3 Transcription Factor/metabolism
17.
Int J Cancer ; 123(9): 2138-47, 2008 Nov 01.
Article in English | MEDLINE | ID: mdl-18709639

ABSTRACT

Pancreatic cancer is a serious disease with poor patient outcome, often as a consequence of late diagnosis in advanced stages. This is in large part due to the lack of diagnostic tools for early detection. To address this deficiency, we have investigated novel molecular near-infrared fluorescent (NIRF) in vivo imaging techniques in clinically relevant mouse models of pancreatic cancer. Genome wide gene expression profiling was used to identify cathepsin cystein proteases and matrix metalloproteinases (MMP) as targets for NIRF imaging. Appropriate protease activatable probes were evaluated for detection of early-stage pancreatic cancer in mice with orthotopically implanted pancreatic cancer cell lines. Mice with pancreatitis served as controls. Whole body in vivo NIRF imaging using activatable cathepsin sensitive probes specifically detected pancreatic tumors as small as 1-2 mm diameter. Imaging of MMP activity demonstrated high specificity for MMP positive tumors. Intravital flexible confocal fluorescence lasermicroscopy of protease activity enabled specific detection of pancreatic tumors at the cellular level. Importantly, topical application of NIRF-probes markedly reduced background without altering signal intensity. Taken together, macroscopic and confocal lasermicroscopic molecular in vivo imaging of protease activity is highly sensitive, specific and allows discrimination between normal pancreatic tissue, inflammation and pancreatic cancer. Translation of this approach to the clinic could significantly improve endoscopic and laparoscopic detection of early-stage pancreatic cancer.


Subject(s)
Matrix Metalloproteinases/analysis , Pancreatic Neoplasms/diagnosis , Spectroscopy, Near-Infrared/methods , Animals , Cathepsins/analysis , Cathepsins/genetics , Cell Line, Tumor , Early Diagnosis , Fluorescence , Gene Expression Profiling , Humans , Matrix Metalloproteinases/genetics , Mice , Neoplasm Staging , Pancreatic Neoplasms/enzymology , Pancreatic Neoplasms/pathology , Sensitivity and Specificity
18.
J Immunol ; 181(4): 2694-704, 2008 Aug 15.
Article in English | MEDLINE | ID: mdl-18684960

ABSTRACT

Emerging evidence suggests an important role for human epidermal keratinocytes in innate immune mechanisms against bacterial and viral skin infections. The proinflammatory effect of viral infections can be mimicked by double-stranded RNA (dsRNA). Herein, we demonstrate that keratinocytes express all known dsRNA sensing receptors at a constitutive and inducible level, and that they use several downstream signaling pathways leading to a broad pattern of gene expression, not only proinflammatory and immune response genes under the control of NF-kappaB, but also genes under transcriptional control of IRF3. As a consequence, dsRNA, a stimulus for TLR3, protein kinase R (PKR), and the RNA helicases retinoic acid-inducible gene I (RIG-I) and MDA5, induces a status of antiviral defense in keratinocytes. Using inhibitors for the various dsRNA signaling pathways and specific small interfering RNA for TLR3, RIG-I, and MDA5, we demonstrated that in human keratinocytes, TLR3 seems to be necessary for NF-kappaB but not for IRF3 activation, whereas RIG-I and MDA5 are crucial for IRF3 activation. PKR is essential for the dsRNA response in both signaling pathways and thus represents the central antiviral receptor for dsRNA stimulation. Moreover, human keratinocytes up-regulate TLR7, the receptor for single-stranded RNA, in response to stimulation with dsRNA, which renders keratinocytes functionally responsive to the TLR7 agonist gardiquimod, a member of the imidazoquinoline antiviral immune response modifier family. Thus, in addition to building a physical barrier against infectious pathogens, keratinocytes are specially equipped with a full antiviral defense program that enables them to efficiently target viral infections of the skin.


Subject(s)
Antiviral Agents/metabolism , DEAD-box RNA Helicases/physiology , Keratinocytes/virology , RNA, Double-Stranded/physiology , Receptors, Retinoic Acid/physiology , Signal Transduction/immunology , Toll-Like Receptor 3/physiology , eIF-2 Kinase/physiology , Cells, Cultured , Gene Expression Regulation/immunology , Humans , Interferon Regulatory Factor-3/metabolism , Interferon Regulatory Factor-3/physiology , Interferon-Induced Helicase, IFIH1 , Keratinocytes/enzymology , Keratinocytes/immunology , Keratinocytes/metabolism , Papillomaviridae/immunology , Poly I-C/biosynthesis , Poly I-C/pharmacology , Receptors, Pattern Recognition/biosynthesis , Signal Transduction/genetics , Toll-Like Receptor 3/deficiency , Toll-Like Receptor 3/genetics
19.
RNA ; 14(9): 1959-72, 2008 Sep.
Article in English | MEDLINE | ID: mdl-18658122

ABSTRACT

RNA levels in a cell are determined by the relative rates of RNA synthesis and decay. State-of-the-art transcriptional analyses only employ total cellular RNA. Therefore, changes in RNA levels cannot be attributed to RNA synthesis or decay, and temporal resolution is poor. Recently, it was reported that newly transcribed RNA can be biosynthetically labeled for 1-2 h using thiolated nucleosides, purified from total cellular RNA and subjected to microarray analysis. However, in order to study signaling events at molecular level, analysis of changes occurring within minutes is required. We developed an improved approach to separate total cellular RNA into newly transcribed and preexisting RNA following 10-15 min of metabolic labeling. Employing new computational tools for array normalization and half-life determination we simultaneously study short-term RNA synthesis and decay as well as their impact on cellular transcript levels. As an example we studied the response of fibroblasts to type I and II interferons (IFN). Analysis of RNA transcribed within 15-30 min at different times during the first three hours of interferon-receptor activation resulted in a >10-fold increase in microarray sensitivity and provided a comprehensive profile of the kinetics of IFN-mediated changes in gene expression. We identify a previously undisclosed highly connected network of short-lived transcripts selectively down-regulated by IFNgamma in between 30 and 60 min after IFN treatment showing strong associations with cell cycle and apoptosis, indicating novel mechanisms by which IFNgamma affects these pathways.


Subject(s)
Gene Expression Profiling/methods , Gene Expression Regulation , RNA Stability/genetics , RNA, Messenger/biosynthesis , Animals , Fibroblasts/drug effects , Fibroblasts/metabolism , Interferon Type I/pharmacology , Interferon-gamma/pharmacology , Mice , NIH 3T3 Cells , RNA, Messenger/analysis , Transcription, Genetic
20.
Immunity ; 28(5): 675-86, 2008 May.
Article in English | MEDLINE | ID: mdl-18424188

ABSTRACT

The action of type I interferons in the central nervous system (CNS) during autoimmunity is largely unknown. Here, we demonstrate elevated interferon beta concentrations in the CNS, but not blood, of mice with experimental autoimmune encephalomyelitis (EAE), a model for CNS autoimmunity. Furthermore, mice devoid of the broadly expressed type I IFN receptor (IFNAR) developed exacerbated clinical disease accompanied by a markedly higher inflammation, demyelination, and lethality without shifting the T helper 17 (Th17) or Th1 cell immune response. Whereas adoptive transfer of encephalitogenic T cells led to enhanced disease in Ifnar1(-/-) mice, newly created conditional mice with B or T lymphocyte-specific IFNAR ablation showed normal EAE. The engagement of IFNAR on neuroectodermal CNS cells had no protective effect. In contrast, absence of IFNAR on myeloid cells led to severe disease with an enhanced effector phase and increased lethality, indicating a distinct protective function of type I IFNs during autoimmune inflammation of the CNS.


Subject(s)
Autoimmunity , Central Nervous System/immunology , Encephalomyelitis, Autoimmune, Experimental/immunology , Interferon-beta/metabolism , Myeloid Cells/immunology , Receptor, Interferon alpha-beta/metabolism , T-Lymphocyte Subsets/immunology , Adoptive Transfer , Animals , B-Lymphocytes/immunology , Brain/immunology , Brain/metabolism , Central Nervous System/metabolism , Disease Progression , Encephalomyelitis, Autoimmune, Experimental/metabolism , Female , Histocompatibility Antigens Class II/metabolism , Interferon-beta/immunology , Mice , Mice, Mutant Strains , Microglia/metabolism , Myeloid Cells/metabolism , Receptor, Interferon alpha-beta/immunology , Signal Transduction , Spinal Cord/immunology , Spinal Cord/metabolism , T-Lymphocyte Subsets/metabolism , Transcription, Genetic
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