ABSTRACT
The co-inhibitory programmed death (PD)-1 signaling pathway plays a major role in the context of tumor-specific T cell responses. Conversely, it also contributes to the maintenance of peripheral tolerance, as patients receiving anti-PD-1 treatment are prone to developing immune-related adverse events. Yet, the physiological role of the PD-1/PDL-1 axis in T cell homeostasis is still poorly understood. Herein, we show that under steady-state conditions, the absence of PD-1 signaling led to a preferential expansion of CD8+ T cells in the liver. These cells exhibit an oligoclonal T cell receptor (TCR) repertoire and a terminally differentiated exhaustion profile. The transcription factor EOMES is required for the clonal expansion and acquisition of this differentiation program. Finally, single-cell transcriptomics coupled with TCR repertoire analysis support the notion that these cells arise locally from liver-resident memory CD8+ T cells. Overall, we show a role for PD-1 signaling in liver memory T cell homeostasis.
Subject(s)
CD8-Positive T-Lymphocytes , Gene Expression Regulation , Humans , CD8-Positive T-Lymphocytes/metabolism , Liver/metabolism , Signal Transduction , Receptors, Antigen, T-Cell/metabolism , T-Box Domain Proteins/genetics , T-Box Domain Proteins/metabolismABSTRACT
Climate change and human activities increasingly threaten lakes that store 87% of Earth's liquid surface fresh water. Yet, recent trends and drivers of lake volume change remain largely unknown globally. Here, we analyze the 1972 largest global lakes using three decades of satellite observations, climate data, and hydrologic models, finding statistically significant storage declines for 53% of these water bodies over the period 1992-2020. The net volume loss in natural lakes is largely attributable to climate warming, increasing evaporative demand, and human water consumption, whereas sedimentation dominates storage losses in reservoirs. We estimate that roughly one-quarter of the world's population resides in a basin of a drying lake, underscoring the necessity of incorporating climate change and sedimentation impacts into sustainable water resources management.
ABSTRACT
A consistent dataset of lake surface water temperature, ice cover, water-leaving reflectance, water level and extent is presented. The collection constitutes the Lakes Essential Climate Variable (ECV) for inland waters. The data span combined satellite observations from 1992 to 2020 inclusive and quantifies over 2000 relatively large lakes, which represent a small fraction of the number of lakes worldwide but a significant fraction of global freshwater surface. Visible and near-infrared optical imagery, thermal imagery and microwave radar data from satellites have been exploited. All observations are provided in a common grid at 1/120° latitude-longitude resolution, jointly in daily files. The data/algorithms have been validated against in situ measurements where possible. Consistency analysis between the variables has guided the development of the joint dataset. It is the most complete collection of consistent satellite observations of the Lakes ECV currently available. Lakes are of significant interest to scientific disciplines such as hydrology, limnology, climatology, biogeochemistry and geodesy. They are a vital resource for freshwater supply, and key sentinels for global environmental change.
ABSTRACT
Monitoring the evolution of the Sahelian environment is a major challenge because the great Sahelian droughts, marked by significant environmental consequences and social impacts, contributed, for example, to the drying up of Lake Chad. We combined remote sensing images with a water level database from the Hydroweb project to determine the response of Lake Chad vegetation cover and surface water variations to rainfall fluctuations in the Lake Chad watershed under recent climate conditions. The variance in lake surface water levels was determined by computing the monthly anomaly time series of surface water height and area from the Hydroweb datasets. The spatiotemporal variability of watershed rainfall and vegetation cover of Lake Chad was highlighted through multivariate statistical analysis. The spatial distribution of correlations between watershed rainfall and Lake Chad vegetation cover was investigated. The results show an increase in watershed rainfall, vegetation cover, and surface water area and height, as their slopes were all positive i.e., 5.1 10-4 (mm/day); 4.26 10-6 (ndvi unit/day); 1.2 10-3 (km2/day) and 6 10-5 (m/day), respectively. The rainfall variations in the watershed drive those of Lake Chad vegetation cover and surface water, as the rainfall trend was strongly and positively correlated with those of vegetation cover (0.79), surface water height (0.57), and area (0.53). The time lag between the watershed rainfall fluctuations and lake surface water variations corresponded to approximately â¼112 days. Between rainfall variations and vegetation cover changes, the spatial distribution of the time lag showed a response time of <16 days in the western shores of the lake and on both sides of the great barrier, about 16 days in the bare soils of the northern basin and the eastern part of the south basin, and >64 days in the marshlands of the southern basin. For the analysis of lakes around the world, this research provides a robust method that computes the spatiotemporal variances of their trends and seasonality and correlates these with the spatiotemporal variances of climate changes. The correlations obtained have strong potential for predicting future changes in lake surface water worldwide.
Subject(s)
Lakes , Water , Chad , Climate Change , DroughtsABSTRACT
Severe COVID-19 disease is associated with dysregulation of the myeloid compartment during acute infection. Survivors frequently experience long-lasting sequelae, but little is known about the eventual persistence of this immune alteration. Herein, we evaluated TLR-induced cytokine responses in a cohort of mild to critical patients during acute or convalescent phases (n = 97). In the acute phase, we observed impaired cytokine production by monocytes in the patients with the most severe COVID-19. This capacity was globally restored in convalescent patients. However, we observed increased responsiveness to TLR1/2 ligation in patients who recovered from severe disease, indicating that these cells display distinct functional properties at the different stages of the disease. In patients with acute severe COVID-19, we identified a specific transcriptomic and epigenomic state in monocytes that can account for their functional refractoriness. The molecular profile of monocytes from recovering patients was distinct and characterized by increased chromatin accessibility at activating protein 1 (AP1) and MAF loci. These results demonstrate that severe COVID-19 infection has a profound impact on the differentiation status and function of circulating monocytes, during both the acute and the convalescent phases, in a completely distinct manner. This could have important implications for our understanding of short- and long-term COVID-19-related morbidity.
Subject(s)
COVID-19 , Cytokines/metabolism , Disease Progression , Humans , Monocytes/metabolism , SARS-CoV-2ABSTRACT
Cancer is caused primarily by genomic alterations resulting in deregulation of gene regulatory circuits in key growth, apoptosis, or DNA repair pathways. Multiple genes associated with the initiation and development of tumors are also regulated at the level of mRNA decay, through the recruitment of RNA-binding proteins to AU-rich elements (AREs) located in their 3'-untranslated regions. One of these ARE-binding proteins, tristetraprolin (TTP; encoded by Zfp36), is consistently dysregulated in many human malignancies. Herein, using regulated overexpression or conditional ablation in the context of cutaneous chemical carcinogenesis, we show that TTP represents a critical regulator of skin tumorigenesis. We provide evidence that TTP controlled both tumor-associated inflammation and key oncogenic pathways in neoplastic epidermal cells. We identify Areg as a direct target of TTP in keratinocytes and show that EGFR signaling potentially contributed to exacerbated tumor formation. Finally, single-cell RNA-Seq analysis indicated that ZFP36 was downregulated in human malignant keratinocytes. We conclude that TTP expression by epidermal cells played a major role in the control of skin tumorigenesis.
Subject(s)
Carcinogenesis/metabolism , Keratinocytes/metabolism , Skin Neoplasms/metabolism , Skin/metabolism , Tristetraprolin/metabolism , 3' Untranslated Regions , AU Rich Elements , Animals , Carcinogenesis/genetics , Disease Models, Animal , Down-Regulation , ErbB Receptors/metabolism , Gene Regulatory Networks , Humans , Inflammation/metabolism , Mice, Inbred C57BL , RNA Stability , RNA, Messenger/metabolism , RNA-Binding Proteins/metabolism , Signal Transduction , Skin Neoplasms/geneticsABSTRACT
AU-rich element (ARE)-mediated mRNA decay represents a key mechanism to avoid excessive production of inflammatory cytokines. Tristetraprolin (TTP, encoded by Zfp36) is a major ARE-binding protein, since Zfp36-/- mice develop a complex multiorgan inflammatory syndrome that shares many features with spondyloarthritis. The role of TTP in intestinal homeostasis is not known. Herein, we show that Zfp36-/- mice do not develop any histological signs of gut pathology. However, they display a clear increase in intestinal inflammatory markers and discrete alterations in microbiota composition. Importantly, oral antibiotic treatment reduced both local and systemic joint and skin inflammation. We further show that absence of overt intestinal pathology is associated with local expansion of regulatory T cells. We demonstrate that this is related to increased vitamin A metabolism by gut dendritic cells, and identify RALDH2 as a direct target of TTP. In conclusion, these data bring insights into the interplay between microbiota-dependent gut and systemic inflammation during immune-mediated disorders, such as spondyloarthritis.
Subject(s)
Aldehyde Oxidoreductases/genetics , Dendritic Cells/immunology , Dendritic Cells/metabolism , Homeostasis , T-Lymphocytes, Regulatory/immunology , T-Lymphocytes, Regulatory/metabolism , Tristetraprolin/metabolism , Aldehyde Oxidoreductases/metabolism , Animals , Cytokines/metabolism , Disease Susceptibility , Gene Expression Regulation , Inflammation Mediators/metabolism , Mice , Mice, Knockout , RNA-Binding Proteins/metabolismABSTRACT
c-Jun N-terminal protein kinase 1 (JNK1) is involved in multiple biological processes but its implication in inflammatory skin diseases is still poorly defined. Herein, we studied the role of JNK1 in the context of Aldara®-induced skin inflammation. We observed that constitutive ablation of JNK1 reduced Aldara®-induced acanthosis and expression of inflammatory markers. Conditional deletion of JNK1 in myeloid cells led to reduced skin inflammation, a finding that was associated with impaired Aldara®-induced inflammasome activation in vitro. Next, we evaluated the specific role of JNK1 in epidermal cells. We observed reduced Aldara®-induced acanthosis despite similar levels of inflammatory markers. Transcriptomic and epigenomic analysis of keratinocytes revealed the potential involvement of JNK1 in the EGFR signaling pathway. Finally, we show that inhibition of the EGFR pathway reduced Aldara®-induced acanthosis. Taken together, these data indicate that JNK1 plays a dual role in the context of psoriasis by regulating the production of inflammatory cytokines by myeloid cells and the sensitivity of keratinocytes to EGFR ligands. These results suggest that JNK1 could represent a valuable therapeutic target in the context of psoriasis.
Subject(s)
ErbB Receptors/metabolism , Keratinocytes/enzymology , Mitogen-Activated Protein Kinase 8/metabolism , Myeloid Cells/enzymology , Psoriasis/enzymology , Skin/enzymology , Animals , Cytokines/genetics , Cytokines/metabolism , Disease Models, Animal , Epigenome , ErbB Receptors/genetics , Female , Imiquimod , Inflammation Mediators/metabolism , Keratinocytes/immunology , Keratinocytes/pathology , Mice, Inbred C57BL , Mice, Knockout , Mitogen-Activated Protein Kinase 8/genetics , Myeloid Cells/immunology , Psoriasis/chemically induced , Psoriasis/immunology , Psoriasis/pathology , Signal Transduction , Skin/immunology , Skin/pathology , TranscriptomeABSTRACT
Monocytes play a major role in the defense against pathogens. They are rapidly mobilized to inflamed sites where they exert both proinflammatory and regulatory effector functions. It is still poorly understood how this dynamic and exceptionally plastic system is controlled at the molecular level. Herein, we evaluated the differentiation process that occurs in Ly6Chi monocytes during oral infection by Toxoplasma gondii. Flow cytometry and single-cell analysis revealed distinct activation status and gene expression profiles in the bone marrow, the spleen and the lamina propria of infected mice. We provide further evidence that acquisition of effector functions, such as the capacity to produce interleukin-27, is accompanied by distinct waves of epigenetic programming, highlighting a role for STAT1/IRF1 in the bone marrow and AP-1/NF-κB in the periphery. This work broadens our understanding of the molecular events that occur in vivo during monocyte differentiation in response to inflammatory cues.
Subject(s)
Cell Differentiation/immunology , Monocytes/immunology , Toxoplasma/immunology , Toxoplasmosis/immunology , Toxoplasmosis/parasitology , Animals , Cellular Reprogramming/genetics , Computational Biology/methods , Epigenesis, Genetic , Gene Expression Profiling , Macrophages/immunology , Macrophages/metabolism , Mice , Monocytes/cytology , Monocytes/metabolism , Single-Cell Analysis , Toxoplasmosis/genetics , Toxoplasmosis/metabolismABSTRACT
Memory CD8+ T cells have the ability to provide lifelong immunity against pathogens. Although memory features generally arise after challenge with a foreign antigen, naïve CD8 single positive (SP) thymocytes may acquire phenotypic and functional characteristics of memory cells in response to cytokines such as interleukin-4. This process is associated with the induction of the T-box transcription factor Eomesodermin (EOMES). However, the underlying molecular mechanisms remain ill-defined. Using epigenomic profiling, we show that these innate memory CD8SP cells acquire only a portion of the active enhancer repertoire of conventional memory cells. This reprograming is secondary to EOMES recruitment, mostly to RUNX3-bound enhancers. Furthermore, EOMES is found within chromatin-associated complexes containing BRG1 and promotes the recruitment of this chromatin remodelling factor. Also, the in vivo acquisition of EOMES-dependent program is BRG1-dependent. In conclusion, our results support a strong epigenetic basis for the EOMES-driven establishment of CD8+ T cell innate memory program.
Subject(s)
CD8-Positive T-Lymphocytes/immunology , Core Binding Factor Alpha 3 Subunit/physiology , DNA Helicases/physiology , Epigenesis, Genetic , Immunologic Memory , Nuclear Proteins/physiology , T-Box Domain Proteins/metabolism , Transcription Factors/physiology , Animals , Core Binding Factor Alpha 3 Subunit/immunology , Core Binding Factor Alpha 3 Subunit/metabolism , DNA Helicases/immunology , DNA Helicases/metabolism , Female , Gene Expression Profiling , Male , Mice, Inbred BALB C , Mice, Inbred C57BL , Nuclear Proteins/immunology , Nuclear Proteins/metabolism , T-Box Domain Proteins/genetics , Transcription Factors/immunology , Transcription Factors/metabolismABSTRACT
CD8(+) T-cell memory phenotype and function are acquired after antigen-driven activation. Memory-like cells may also arise in absence of antigenic exposure in the thymus or in the periphery. Eomesodermin (Eomes) is a key transcription factor for the development of these unconventional memory cells. Herein, we show that type I interferon signalling in CD8(+) T cells directly activates Eomes gene expression. Consistent with this observation, the phenotype, function and age-dependent expansion of 'virtual memory' CD8(+) T cells are strongly affected in absence of type I interferon signalling. In addition, type I interferons induce a sustained expansion of 'virtual memory' CD8(+) T cells in an Eomes-dependent fashion. We further show that the development of 'innate thymic' CD8(+) T cells is dependent on the same pathway. In conclusion, we demonstrate that type I interferon signalling in CD8(+) T cells drives Eomes expression and thereby regulates the function and homeostasis of memory-like CD8(+) T cells.
Subject(s)
CD8-Positive T-Lymphocytes/metabolism , Immunologic Memory/genetics , Interferon Type I/metabolism , T-Box Domain Proteins/genetics , Animals , Antigens/metabolism , CD8-Positive T-Lymphocytes/drug effects , Gene Expression Regulation/drug effects , Homeostasis/drug effects , Immunity, Innate/drug effects , Immunologic Memory/drug effects , Interferon-Stimulated Gene Factor 3, gamma Subunit/metabolism , Interferon-gamma/biosynthesis , Mice, Inbred BALB C , Mice, Inbred C57BL , Poly I-C/pharmacology , Receptor, Interferon alpha-beta/deficiency , Receptor, Interferon alpha-beta/metabolism , Signal Transduction/drug effects , T-Box Domain Proteins/metabolism , Thymocytes/drug effects , Thymocytes/metabolismABSTRACT
IFN regulatory factor (IRF) 3 plays a key role in innate responses against viruses. Herein we assessed its contribution to T-cell activation. We observed that poly(I:C)-induced IRF3 activation in CD8 T cells represses IL-17 expression in a type I IFN-independent fashion. Even in the absence of poly(I:C), polyclonally activated naïve IRF3(-/-) CD8 T cells expressed high levels of IL-17 and IL-23R in comparison with wild-type cells. Furthermore, IRF3(-/-) OT1 cells adoptively transferred into wild-type hosts also produced higher IL-17 levels upon immunization than their wild-type counterparts. This phenotype could be reversed by ectopic expression of IRF3, confirming that this effect is intrinsic to T cells. We show that IRF3 directly interacts with RORγt in the cytoplasm through its IRF interaction domain and limits its ability to bind and transactivate the IL-17 promoter. These observations uncover an unexpected role of IRF3 in the control of CD8 T-cell polarization.
Subject(s)
Adaptive Immunity/immunology , CD8-Positive T-Lymphocytes/metabolism , Gene Expression Regulation/immunology , Immunity, Innate/immunology , Interferon Regulatory Factor-3/metabolism , Interleukin-17/metabolism , Animals , Blotting, Western , Chromatin Immunoprecipitation , Enzyme-Linked Immunosorbent Assay , HEK293 Cells , Humans , Luciferases , Mice , Mice, Inbred C57BL , Mice, Knockout , Microscopy, Fluorescence , Real-Time Polymerase Chain Reaction , Reverse Transcriptase Polymerase Chain ReactionABSTRACT
The generation of high-affinity antibodies and the development of B cell memory are dependent on the help provided by CD4 T cells. Mouse studies indicate that STAT3 signaling in CD4 T cells promotes the acquisition of the B cell help function. However, the role of STAT3 in humans has been controversial. In this study, we show that IL-6 and other STAT3 activating cytokines (IL-21 and IL-27) induce the differentiation of CD4 T cells promoting antibody production by B cells. The acquisition of B cell stimulating properties by naive cord blood CD4 T cells required the STAT3-dependent expression of ICOS and IL-21. Gene reporter and ChIP experiments unambiguously demonstrated that upon IL-6 stimulation, STAT3 induces the transcription of the ICOS gene through direct recruitment to the proximal promoter region indicating that STAT3 acts in part through the direct activation of the ICOS gene.
Subject(s)
B-Lymphocytes/immunology , CD4-Positive T-Lymphocytes/immunology , Inducible T-Cell Co-Stimulator Protein/immunology , Interleukin-6/immunology , STAT3 Transcription Factor/immunology , Animals , Antibody Formation , B-Lymphocytes/cytology , CD4-Positive T-Lymphocytes/cytology , CD4-Positive T-Lymphocytes/metabolism , Cell Differentiation , Cells, Cultured , Humans , Inducible T-Cell Co-Stimulator Protein/genetics , Interleukin-12/immunology , Interleukin-27/immunology , Lymphocyte Activation , Mice , Signal Transduction , Transcriptional ActivationABSTRACT
BACKGROUND: Characteristics of the human neonatal immune system are thought to be responsible for heightened susceptibility to infectious pathogens and poor responses to vaccine antigens. Using cord blood as a source of immune cells, many reports indicate that the response of neonatal monocytes and dendritic cells (DC) to Toll-like receptor (TLR) agonists differs significantly from that of adult cells. Herein, we analyzed the evolution of these responses within the first year of life. METHODOLOGY/PRINCIPAL FINDINGS: Blood samples from children (0, 3, 6, 9, 12 month old) and healthy adults were stimulated ex vivo with bacterial lipopolysaccharide (LPS, TLR4 agonist) or CpG oligonucleotides (TLR9 agonist). We determined phenotypic maturation of monocytes, myeloid (m) and plasmacytoid (p) DC and production of cytokines in the culture supernatants. We observed that surface expression of CD80 and HLA-DR reaches adult levels within the first 3 months of life for mDCs and 6-9 months of life for monocytes and pDCs. In response to LPS, production of TNF-alpha, IP-10 and IL-12p70 reached adult levels between 6-9 months of life. In response to CpG stimulation, production of type I IFN-dependent chemokines (IP-10 and CXCL9) gradually increased with age but was still limited in 1-year old infants as compared to adult controls. Finally, cord blood samples stimulated with CpG ODN produced large amounts of IL-6, IL-8, IL-1beta and IL-10, a situation that was not observed for 3 month-old infants. CONCLUSIONS: The first year of life represents a critical period during which adult-like levels of TLR responses are reached for most but not all cytokine responses.
Subject(s)
Immune System , Toll-Like Receptor 4/immunology , Toll-Like Receptor 9/immunology , Adult , Cells, Cultured , Child , Cytokines/biosynthesis , Dendritic Cells , Fetal Blood/cytology , Human Development , Humans , Infant , Lipopolysaccharides/pharmacology , Monocytes , Oligodeoxyribonucleotides/pharmacologyABSTRACT
IL-27 is a heterodimeric cytokine composed of EBV-induced gene 3 and p28. Produced by dendritic cells (DCs) in response to TLR ligands, IL-27 recently emerged as a key regulator of inflammatory responses. In this study, we first demonstrate that Toll/IL-1R-containing adaptor inducing IFN-beta and its associated IFN regulatory factor (IRF) 3 transcription factor are critically involved in IL-27p28 expression in mouse DCs stimulated by TLR ligands. We then show that IL-27 serum levels are dramatically reduced in IRF3(-/-) upon LPS injection, indicating a critical role for IRF3 in TLR4-mediated IL-27 production in vivo. We identified an IRF3-binding site within the IL-27p28 promoter region which is required for IL-27p28 gene activation in reporter gene assays. In human DCs, IL-27p28 mRNA was preferentially induced by Toll/IL-1R-containing adaptor inducing IFN-beta-coupled TLR ligands and following CMV infection. Furthermore, chromatin immunoprecipitation studies demonstrate that IRF3 is recruited to the endogenous p28 promoter in TLR4-stimulated human DCs. We conclude that IRF3 activation is a master switch for IL-27 synthesis.
Subject(s)
Adaptor Proteins, Vesicular Transport/metabolism , Gene Expression Regulation , Interferon Regulatory Factor-3/metabolism , Interleukins/genetics , Amino Acid Sequence , Animals , Binding Sites , Chromatin Immunoprecipitation , Dendritic Cells/immunology , Humans , Interferon Regulatory Factor-3/genetics , Interferon-beta/metabolism , Interleukins/biosynthesis , Interleukins/blood , Lipopolysaccharides/immunology , Mice , Minor Histocompatibility Antigens , Molecular Sequence Data , Myeloid Differentiation Factor 88/metabolism , Promoter Regions, Genetic , Receptors, Cytokine/genetics , Receptors, Interleukin-1/metabolism , Toll-Like Receptor 4/agonists , Toll-Like Receptors/metabolism , Transcriptional ActivationABSTRACT
Protein kinase C (PKC) isoforms are critically involved in the regulation of innate immune responses. Herein, we investigated the role of conventional PKCalpha in the regulation of IFN-beta gene expression mediated by the Toll-like receptor 3 (TLR3) signaling pathway. Inhibition of conventional PKC (cPKC) activity in monocyte-derived dendritic cells or TLR3-expressing cells by an isoform-specific inhibitor, Gö6976, selectively inhibited IFN-beta synthesis induced by double-stranded RNA polyinosine-polycytidylic acid. Furthermore, reporter gene assays confirmed that PKCalpha regulates IFN-beta promoter activity, since overexpression of dominant negative PKCalpha but not PKCbeta(I) repressed interferon regulatory factor 3 (IRF-3)-dependent but not NF-kappaB-mediated promoter activity upon TLR3 engagement in HEK 293 cells. Dominant negative PKCalpha inhibited IRF-3 transcriptional activity mediated by overexpression of TIR domain-containing adapter inducing IFN-beta and Tank-binding kinase-1. Additional biochemical analysis demonstrated that Gö6976-treated dendritic cells exhibited IRF-3 phosphorylation, dimerization, nuclear translocation, and DNA binding activity analogous to their control counterparts in response to polyinosine-polycytidylic acid. In contrast, co-immunoprecipitation experiments revealed that TLR3-induced cPKC activity is essential for mediating the interaction of IRF-3 but not p65/RelA with the co-activator CREB-binding protein. Furthermore, PKCalpha knock-down with specific small interfering RNA inhibited IFN-beta expression and down-regulated IRF-3-dependent promoter activity, establishing PKCalpha as a component of TLR3 signaling that regulates IFN-beta gene expression by targeting IRF-3-CREB-binding protein interaction. Finally, we analyzed the involvement of cPKCs in other signaling pathways leading to IFN-beta synthesis. These experiments revealed that cPKCs play a role in the synthesis of IFN-beta induced via both TLR-dependent and -independent pathways.
Subject(s)
Down-Regulation/physiology , Interferon Regulatory Factor-3/metabolism , Interferon-beta/biosynthesis , Protein Kinase C-alpha/metabolism , Signal Transduction/physiology , Toll-Like Receptor 3/metabolism , Active Transport, Cell Nucleus/drug effects , Active Transport, Cell Nucleus/physiology , Carbazoles/pharmacology , Cell Line , Cell Nucleus/metabolism , Down-Regulation/drug effects , Enzyme Inhibitors/pharmacology , Humans , Indoles/pharmacology , Interferon Regulatory Factor-3/genetics , Interferon-beta/genetics , NF-kappa B/metabolism , Phosphorylation/drug effects , Poly I-C/pharmacology , Promoter Regions, Genetic/physiology , Protein Kinase C/metabolism , Protein Kinase C beta , Protein Kinase C-alpha/antagonists & inhibitors , Protein Kinase C-alpha/genetics , Protein Serine-Threonine Kinases/metabolism , RNA, Double-Stranded/pharmacology , RNA, Small Interfering/genetics , Signal Transduction/drug effects , Transcription Factor RelA/metabolism , Transcription, Genetic/drug effects , Transcription, Genetic/physiologyABSTRACT
The synthesis of interferon-beta (IFNbeta) and IFN-inducible factors elicited by lipopolysaccharide (LPS) depends on the transcriptional activity of interferon regulatory factor 3 (IRF-3) downstream of Toll-like receptor-4 (TLR4). To examine the ability of human newborns to mount TLR4-mediated IRF-3-dependent responses, we analyzed the pattern of genes expressed on the addition of LPS to cord blood or cord blood monocyte-derived dendritic cells (moDCs). Expression of IFNbeta and IFN-inducible genes was selectively impaired in neonatal blood and moDCs as compared with their adult counterparts. This selective defect was confirmed by microarray experiments on moDCs. Altered expression of IFN-inducible genes was related to impaired IFNbeta synthesis because IFNbeta signaling was functional in neonatal moDCs. However, addition of exogenous IFNbeta failed to restore LPS-induced IL-12p70 synthesis which was previously shown to be defective in neonatal moDCs. Although LPS-induced IRF-3 nuclear translocation was observed both in adult and neonatal moDCs, IRF-3 DNA-binding activity and association with the coactivator CREB-binding protein (CBP) were decreased in neonatal as compared with adult moDCs. We conclude that impaired IRF-3/CBP interaction in neonatal blood cells exposed to LPS is associated with impaired expression of IFNbeta and IFN-inducible genes. Because IRF-3 activity is also required for IL-12p70 synthesis, our findings provide a molecular basis for the decreased ability of LPS-stimulated neonatal moDCs to elicit Th1-type responses.
Subject(s)
Fetal Blood/drug effects , Fetal Blood/metabolism , Interferon Regulatory Factor-3/blood , Lipopolysaccharides/pharmacology , Active Transport, Cell Nucleus/drug effects , Adult , CREB-Binding Protein/blood , Fetal Blood/cytology , Gene Expression/drug effects , Humans , In Vitro Techniques , Infant, Newborn , Interferon-beta/blood , Interferon-beta/genetics , RNA, Messenger/blood , RNA, Messenger/geneticsABSTRACT
Interleukin-12 (IL-12) is a heterodimeric cytokine produced by dendritic cells (DCs) in response to Toll-like receptor (TLR) ligation. While the mechanisms regulating IL-12p40 chain gene expression are well characterized, molecular events involved in IL-12p35 chain gene activation remain to be clarified. Since IL-12p35 mRNA was induced in human DCs activated through TLR3 or TLR4 but not TLR2, we investigated the potential role of interferon regulatory factor 3 (IRF-3) in IL-12p35 gene transactivation. First, a binding site for IRF-3 named interferon-stimulated response element-1 (ISRE-1) was identified in the human IL-12p35 promoter region between nucleotides -251 and -240. The ISRE-1 site was required for IL-12p35 gene activation in RAW 264.7 cells stimulated by lipopolysaccharide (LPS) or PolyI:C. Ectopic expression of IRF-3 was found to up-regulate IL-12p35 gene activation in the same system. Furthermore, chromatin immunoprecipitation (ChIP) studies demonstrated that IRF-3 is recruited to ISRE-1 site in TLR4- or TLR3-stimulated human DCs. Finally, experiments on DCs from IRF-3-deficient mice established that TLR4-induced IL-12p35 mRNA and IL-12p70 synthesis are impaired in absence of IRF-3. We conclude that IRF-3 binds to a critical cis-acting element in the IL-12p35 gene promoter and thereby represents a key factor for the induction of IL-12p70 synthesis in DCs.