ABSTRACT
Regulatory T cells (Tregs) control adaptive immunity and restrain type 2 inflammation in allergic disease. Interleukin-33 promotes the expansion of tissue-resident Tregs and group 2 innate lymphoid cells (ILC2s); however, how Tregs locally coordinate their function within the inflammatory niche is not understood. Here, we show that ILC2s are critical orchestrators of Treg function. Using spatial, cellular, and molecular profiling of the type 2 inflamed niche, we found that ILC2s and Tregs engage in a direct (OX40L-OX40) and chemotaxis-dependent (CCL1-CCR8) cellular dialogue that enforces the local accumulation of Gata3high Tregs, which are transcriptionally and functionally adapted to the type 2 environment. Genetic interruption of ILC2-Treg communication resulted in uncontrolled type 2 lung inflammation after allergen exposure. Mechanistically, we found that Gata3high Tregs can modulate the local bioavailability of the costimulatory molecule OX40L, which subsequently controlled effector memory T helper 2 cell numbers. Hence, ILC2-Treg interactions represent a critical feedback mechanism to control adaptive type 2 immunity.
Subject(s)
Adaptive Immunity , GATA3 Transcription Factor , Mice, Inbred C57BL , T-Lymphocytes, Regulatory , Animals , T-Lymphocytes, Regulatory/immunology , GATA3 Transcription Factor/immunology , GATA3 Transcription Factor/metabolism , Mice , Adaptive Immunity/immunology , Lymphocytes/immunology , Immunity, Innate/immunology , Mice, Knockout , Th2 Cells/immunology , FemaleABSTRACT
T cell development is critically dependent on successful rearrangement of antigen-receptor chains. At the ß-selection checkpoint, only cells with a functional rearrangement continue in development. However, how nonselected T cells proceed in their dead-end fate is not clear. We identified low CD27 expression to mark pre-T cells that have failed to rearrange their ß-chain. Expression profiling and single-cell transcriptome clustering identified a developmental trajectory through ß-selection and revealed specific expression of the transcription factor Duxbl at a stage of high recombination activity before ß-selection. Conditional transgenic expression of Duxbl resulted in a developmental block at the DN3-to-DN4 transition due to reduced proliferation and enhanced apoptosis, whereas RNA silencing of Duxbl led to a decrease in apoptosis. Transcriptome analysis linked Duxbl to elevated expression of the apoptosis-inducing Oas/RNaseL pathway. RNaseL deficiency or sustained Bcl2 expression led to a partial rescue of cells in Duxbl transgenic mice. These findings identify Duxbl as a regulator of ß-selection by inducing apoptosis in cells with a nonfunctional rearrangement.
Subject(s)
Homeodomain Proteins/metabolism , T-Lymphocytes/physiology , Transcription Factors/metabolism , Animals , Apoptosis/genetics , Female , Gene Expression Regulation , Homeodomain Proteins/genetics , Male , Mice, Inbred C57BL , Mice, Transgenic , Receptors, Antigen, T-Cell, alpha-beta/metabolism , T-Lymphocytes/cytology , Thymus Gland/cytology , Transcription Factors/genetics , Tumor Necrosis Factor Receptor Superfamily, Member 7/metabolismABSTRACT
Hematopoietic stem cells require MLL1, which is one of six Set1/Trithorax-type histone 3 lysine 4 (H3K4) methyltransferases in mammals and clinically the most important leukemia gene. Here, we add to emerging evidence that all six H3K4 methyltransferases play essential roles in the hematopoietic system by showing that conditional mutagenesis of Setd1b in adult mice provoked aberrant homeostasis of hematopoietic stem and progenitor cells (HSPCs). Using both ubiquitous and hematopoietic-specific deletion strategies, the loss of Setd1b resulted in peripheral thrombo- and lymphocytopenia, multilineage dysplasia, myeloid-biased extramedullary hematopoiesis in the spleen, and lethality. By transplantation experiments and expression profiling, we determined that Setd1b is autonomously required in the hematopoietic lineages where it regulates key lineage specification components, including Cebpa, Gata1, and Klf1. Altogether, these data imply that the Set1/Trithorax-type epigenetic machinery sustains different aspects of hematopoiesis and constitutes a second framework additional to the transcription factor hierarchy of hematopoietic homeostasis.
Subject(s)
Hematopoiesis/genetics , Hematopoietic Stem Cells/metabolism , Histone-Lysine N-Methyltransferase/genetics , Homeostasis/genetics , Lymphopenia/genetics , Myeloid-Lymphoid Leukemia Protein/genetics , Thrombocytopenia/genetics , Animals , Bone Marrow Transplantation , CCAAT-Enhancer-Binding Proteins/genetics , CCAAT-Enhancer-Binding Proteins/metabolism , Cell Lineage/genetics , GATA1 Transcription Factor/genetics , GATA1 Transcription Factor/metabolism , Gene Expression Profiling , Gene Expression Regulation , Genes, Lethal , Hematopoietic Stem Cells/cytology , Histone-Lysine N-Methyltransferase/deficiency , Isoenzymes/deficiency , Isoenzymes/genetics , Kruppel-Like Transcription Factors/genetics , Kruppel-Like Transcription Factors/metabolism , Lymphopenia/metabolism , Lymphopenia/pathology , Mice , Mice, Knockout , Myeloid-Lymphoid Leukemia Protein/deficiency , Spleen/metabolism , Spleen/pathology , Thrombocytopenia/metabolism , Thrombocytopenia/pathology , Whole-Body IrradiationABSTRACT
BACKGROUND: Group 2 innate lymphoid cells (ILC2s) are major producers of the cytokines driving allergic asthma, and increased ILC2 numbers have been detected in blood and sputum of asthmatic patients. Asthma susceptibility has a strong genetic component, but the underlying mechanisms and whether asthma genetics affect ILC2 biology remain unclear. OBJECTIVE: We sought to study the ILC2 transcriptome and epigenome during airway inflammation (AI) to couple these to genes and genetic variants associated with asthma pathogenesis. METHODS: Mice harboring a reporter for the key ILC2 transcription factor GATA-3 were subjected to IL-33-driven AI, and ILC2s were isolated from bronchoalveolar lavage fluid and mediastinal lymph nodes. Human ILC2s were purified from peripheral blood and activated in vitro. We used RNA sequencing, genome-wide identification of histone-3 lysine-4 dimethylation-marked chromatin, and computational approaches to study the ILC2 transcriptome and epigenome. RESULTS: Activated ILC2s in mice displayed a tissue-specific gene expression signature that emerged from remarkably similar epigenomes. We identified superenhancers implicated in controlling ILC2 identity and asthma-associated genes. More than 300 asthma-associated genetic polymorphisms identified in genome-wide association studies localized to H3K4Me2+ gene regulatory elements in ILC2s. A refined set of candidate causal asthma-associated variants was uniquely enriched in ILC2, but not TH2 cell, regulatory regions. CONCLUSIONS: ILC2s in AI use a flexible epigenome that couples adaptation to new microenvironments with functional plasticity. Importantly, we reveal strong correlations between gene regulatory mechanisms in ILC2s and the genetic basis of asthma, supporting a pathogenic role for ILC2s in patients with allergic asthma.
Subject(s)
Asthma/genetics , Asthma/immunology , Genetic Predisposition to Disease , Lymphocytes/immunology , Animals , Epigenesis, Genetic , GATA3 Transcription Factor/genetics , Genome , Humans , Immunity, Innate , Mice , Regulatory Sequences, Nucleic Acid , TranscriptomeABSTRACT
Group 2 innate lymphoid cells (ILC2) are implicated in allergic asthma as an early innate source of the type 2 cytokines IL-5 and IL-13. However, their induction in house dust mite (HDM)-mediated airway inflammation additionally requires T cell activation. It is currently unknown whether phenotypic differences exist between ILC2s that are activated in a T cell-dependent or T cell-independent fashion. Here, we compared ILC2s in IL-33- and HDM-driven airway inflammation. Using flow cytometry, we found that surface expression levels of various markers frequently used to identify ILC2s were dependent on their mode of activation, highly variable over time, and differed between tissue compartments, including bronchoalveolar lavage (BAL) fluid, lung, draining lymph nodes, and spleen. Whereas in vivo IL-33-activated BAL fluid ILC2s exhibited an almost uniform CD25+CD127+T1/ST2+ICOS+KLRG1+ phenotype, at a comparable time point after HDM exposure BAL fluid ILC2s had a very heterogeneous surface marker phenotype. A major fraction of HDM-activated ILC2s were CD25lowCD127+T1/ST2low ICOSlowKLRG1low, but nevertheless had the capacity to produce large amounts of type 2 cytokines. HDM-activated CD25low ILC2s in BAL fluid and lung rapidly reverted to CD25high ILC2s upon in vivo stimulation with IL-33. Genome-wide transcriptional profiling of BAL ILC2s revealed ~1,600 differentially expressed genes: HDM-stimulated ILC2s specifically expressed genes involved in the regulation of adaptive immunity through B and T cell interactions, whereas IL-33-stimulated ILC2s expressed high levels of proliferation-related and cytokine genes. In both airway inflammation models ILC2s were present in the lung submucosa close to epithelial cells, as identified by confocal microscopy. In chronic HDM-driven airway inflammation ILC2s were also found inside organized cellular infiltrates near T cells. Collectively, our findings show that ILC2s are phenotypically more heterogeneous than previously thought, whereby their surface marker and gene expression profile are highly dynamic.
ABSTRACT
Retinoid-related orphan receptor gamma t (RORγt) is a master transcription factor central to type 17 immunity involving cells such as T helper 17, group 3 innate lymphoid cells or IL-17-producing γδ T cells. Here we show that the intracellular ion channel TMEM176B and its homologue TMEM176A are strongly expressed in these RORγt(+) cells. We demonstrate that TMEM176A and B exhibit a similar cation channel activity and mainly colocalise in close proximity to the trans-Golgi network. Strikingly, in the mouse, the loss of Tmem176b is systematically associated with a strong upregulation of Tmem176a. While Tmem176b single-deficiency has no effect on the course of experimental autoimmune encephalomyelitis, T cell or DSS-induced colitis, it significantly reduces imiquimod-induced psoriasis-like skin inflammation. These findings shed light on a potentially novel specific process linked to post-Golgi trafficking for modulating the function of RORγt(+) cells and indicate that both homologues should be simultaneously targeted to clearly elucidate the role of this intracellular ion flow.
Subject(s)
Membrane Proteins/genetics , Nuclear Receptor Subfamily 1, Group F, Member 3/metabolism , T-Lymphocytes, Helper-Inducer/cytology , Animals , Cells, Cultured , Colitis/chemically induced , Colitis/genetics , Colitis/immunology , Disease Models, Animal , Encephalomyelitis, Autoimmune, Experimental/chemically induced , Encephalomyelitis, Autoimmune, Experimental/genetics , Humans , Membrane Proteins/metabolism , Mice , Psoriasis/chemically induced , Psoriasis/genetics , T-Lymphocytes, Helper-Inducer/metabolism , trans-Golgi Network/genetics , trans-Golgi Network/metabolismABSTRACT
Multiple sclerosis (MS) is caused by T cells that are reactive for brain antigens. In experimental autoimmune encephalomyelitis, the animal model for MS, myelin-reactive T cells initiate the autoimmune process when entering the nervous tissue and become reactivated upon local encounter of their cognate CNS antigen. Thereby, the strength of the T-cellular reactivation process within the CNS tissue is crucial for the manifestation and the severity of the clinical disease. Recently, B cells were found to participate in the pathogenesis of CNS autoimmunity, with several diverse underlying mechanisms being under discussion. We here report that B cells play an important role in promoting the initiation process of CNS autoimmunity. Myelin-specific antibodies produced by autoreactive B cells after activation in the periphery diffused into the CNS together with the first invading pathogenic T cells. The antibodies accumulated in resident antigen-presenting phagocytes and significantly enhanced the activation of the incoming effector T cells. The ensuing strong blood-brain barrier disruption and immune cell recruitment resulted in rapid manifestation of clinical disease. Therefore, myelin oligodendrocyte glycoprotein (MOG)-specific autoantibodies can initiate disease bouts by cooperating with the autoreactive T cells in helping them to recognize their autoantigen and become efficiently reactivated within the immune-deprived nervous tissue.
Subject(s)
Autoantibodies/immunology , Autoimmune Diseases/immunology , Central Nervous System Diseases/immunology , Lymphocyte Activation/immunology , T-Lymphocytes/immunology , Cell Differentiation , Humans , T-Lymphocytes/pathologyABSTRACT
T cell progenitors are known to arise from the foetal liver in embryos and the bone marrow in adults; however different studies have shown that a pool of T cell progenitors may also exist in the periphery. Here, we identified a lymphoid population resembling peripheral T cell progenitors which transiently seed the epidermis during late embryogenesis in both wild-type and T cell-deficient mice. We named these cells ELCs (Epidermal Lymphoid Cells). ELCs expressed Thy1 and CD2, but lacked CD3 and TCRαß/γδ at their surface, reminiscent of the phenotype of extra- or intra- thymic T cell progenitors. Similarly to Dendritic Epidermal T Cells (DETCs), ELCs were radioresistant and capable of self-renewal. However, despite their progenitor-like phenotype and expression of T cell lineage markers within the population, ELCs did not differentiate into conventional T cells or DETCs in in vitro, ex vivo or in vivo differentiation assays. Finally, we show that ELC expressed NK markers and secreted IFN-γ upon stimulation. Therefore we report the discovery of a unique population of lymphoid cells within the murine epidermis that appears related to NK cells with as-yet-unidentified functions.
Subject(s)
Epidermis/metabolism , Animals , CD2 Antigens/metabolism , CD3 Complex/metabolism , Cell Differentiation , Cells, Cultured , Coculture Techniques , Epidermal Cells , Homeodomain Proteins/genetics , Homeodomain Proteins/metabolism , Interferon-gamma/metabolism , Interleukin-2/metabolism , Mice , Mice, Inbred C57BL , Mice, Knockout , Mice, Nude , Microscopy, Fluorescence, Multiphoton , Phenotype , Receptors, Antigen, T-Cell, gamma-delta/metabolism , T-Lymphocytes/cytology , T-Lymphocytes/immunology , Thy-1 Antigens/metabolismABSTRACT
To study gene functions specifically in NKp46+ cells we developed novel Cre mice allowing for conditional gene targeting in cells expressing Ncr1 (encoding NKp46). We generated transgenic Ncr1(greenCre) mice carrying an EGFPcre fusion under the control of a proximal Ncr1 promoter that faithfully directed EGFPcre expression to NKp46+ cells from lymphoid and nonlymphoid tissues. This approach allowed for direct detection of Cre-expressing NKp46+ cells via their GFP signature by flow cytometry and histology. Cre was functional as evidenced by the NKp46+ cell-specific expression of RFP in Ncr1(greenCre) Rosa-dtRFP reporter mice. We generated Ncr1(greenCre) Il2rg(fl/fl) mice that lack NKp46+ cells in an otherwise intact hematopoietic environment. Il2rg encodes the common gamma chain (γc ), which is an essential receptor subunit for cytokines (IL-2, -4, -7, -9, -15, and -21) that stimulate lymphocyte development and function. In Ncr1(greenCre) Il2rg(fl/fl) mice, NK cells are severely reduced and the few remaining NKp46+ cells escaping γc deletion failed to express GFP. Using this new NK-cell-deficient model, we demonstrate that the homeostasis of NKp46+ cells from all tissues (including the recently described intraepithelial ILC1 subset) requires Il2rg. Finally, Ncr1(greenCre) Il2rg(fl/fl) mice are unable to reject B16 lung metastases demonstrating the essential role of NKp46+ cells in antimelanoma immune responses.
Subject(s)
Antigens, Ly/genetics , Killer Cells, Natural/immunology , Lung Neoplasms/secondary , Melanoma, Experimental/pathology , Natural Cytotoxicity Triggering Receptor 1/genetics , Animals , Antigens, Ly/biosynthesis , B-Lymphocytes/immunology , CD4-Positive T-Lymphocytes/immunology , CD8-Positive T-Lymphocytes/immunology , Green Fluorescent Proteins/genetics , Interleukin Receptor Common gamma Subunit/genetics , Lung Neoplasms/immunology , Melanoma, Experimental/immunology , Mice , Mice, Inbred C57BL , Mice, Knockout , Natural Cytotoxicity Triggering Receptor 1/biosynthesisABSTRACT
Staining of transcription factors (TFs) together with retention of fluorescent reporter proteins is hindered by loss of fluorescence using current available methods. In this study, it is shown that current TF staining protocols do not destroy fluorescent proteins (FPs) but rather that fixation is not sufficient to retain FPs in the cytosol of the permeabilized cells. In this article, a simple and reliable protocol is elaborated, which allows efficient TF and cytokine staining while retaining FPs inside fixed cells.
Subject(s)
Cytokines/analysis , Flow Cytometry/methods , Nuclear Proteins/analysis , Transcription Factors/analysis , Animals , Cytoplasm/metabolism , Fixatives , Fluorescent Dyes , Forkhead Transcription Factors , Mice , Mice, Inbred C57BL , Mice, Transgenic , Mycobacterium tuberculosis/immunology , Nuclear Receptor Subfamily 1, Group F, Member 3 , Staining and Labeling , T-Box Domain Proteins , T-Lymphocytes/cytology , Tissue Fixation/methodsABSTRACT
The clinical relevance of gene therapy using the recombinant adeno-associated virus (rAAV) vectors often requires widespread distribution of the vector, and in this case, systemic delivery is the optimal route of administration. Humoral blood factors, such as antibodies or complement, are the first barriers met by the vectors administered systemically. We have found that other blood proteins, galectin 3 binding protein (G3BP) and C-reactive protein (CRP), can interact with different AAV serotypes in a species-specific manner. While interactions of rAAV vectors with G3BP, antibodies, or complement lead to a decrease in vector efficacy, systemic transduction of the CRP-deficient mouse and its respective control clearly established that binding to mouse CRP (mCRP) boosts rAAV vector 1 (rAAV-1) and rAAV-6 transduction efficiency in skeletal muscles over 10 times. Notably, the high efficacy of rAAV-6 in CRP-deficient mice can be restored by reconstitution of the CRP-deficient mouse with mCRP. Human CRP (hCRP) does not interact with either rAAV-1 or rAAV-6, and, consequently, the high efficiency of mCRP-mediated muscle transduction by these serotypes in mice cannot be translated to humans. No interaction of mCRP or hCRP was observed with rAAV-8 and rAAV-9. We show, for the first time, that serum components can significantly enhance rAAV-mediated tissue transduction in a serotype- and species-specific manner. Bioprocessing in body fluids should be considered when transfer of a preclinical proof of concept for AAV-based gene therapy to humans is planned.
Subject(s)
C-Reactive Protein/physiology , Dependovirus/genetics , Genetic Vectors/administration & dosage , Muscle, Skeletal/metabolism , Transduction, Genetic , Animals , Blotting, Western , Dependovirus/classification , Humans , Immunoprecipitation , Luciferases/metabolism , Male , Mice , Mice, Inbred C57BL , Mice, Knockout , Muscle, Skeletal/cytology , RNA, Messenger/genetics , Real-Time Polymerase Chain Reaction , Reverse Transcriptase Polymerase Chain ReactionABSTRACT
Expression of the pre-T cell receptor α (pTα) gene has been exploited in previous studies as a molecular marker to identify tiny cell populations in bone marrow (BM) and blood that were suggested to contain physiologically relevant thymus settling progenitors (TSPs). But to what extent these cells genuinely contribute to thymopoiesis has remained obscure. We have generated a novel pTα(iCre) knockin mouse line and performed lineage-tracing experiments to precisely quantitate the contribution of pTα-expressing progenitors to distinct differentiation pathways and to the genealogy of mature hematopoietic cells under physiological in vivo conditions. Using these mice in combination with fluorescent reporter strains, we observe highly consistent labeling patterns that identify pTα expression as a faithful molecular marker of T lineage commitment. Specifically, the fate of pTα-expressing progenitors was found to include all αß and most γδ T cells but, in contrast to previous assumptions, to exclude B, NK, and thymic dendritic cells. Although we could detect small numbers of T cell progenitors with a history of pTα expression in BM and blood, our data clearly exclude these populations as physiologically important precursors of thymopoiesis and indicate that they instead belong to a pathway of T cell maturation previously defined as extrathymic.
Subject(s)
Cell Differentiation/immunology , Gene Expression Regulation/immunology , Lymphopoiesis/physiology , Membrane Glycoproteins/immunology , Receptors, Antigen, T-Cell, alpha-beta/immunology , Thymocytes/immunology , Thymus Gland/immunology , Animals , Bone Marrow/immunology , Cell Differentiation/genetics , Gene Expression Regulation/genetics , Membrane Glycoproteins/genetics , Mice , Mice, Knockout , Receptors, Antigen, T-Cell, alpha-beta/genetics , Receptors, Antigen, T-Cell, gamma-delta/genetics , Receptors, Antigen, T-Cell, gamma-delta/immunology , Thymocytes/cytology , Thymus Gland/cytologyABSTRACT
Dendritic cell (DC) migration via lymphatic vessels to draining lymph nodes (dLNs) is crucial for the initiation of adaptive immunity. We imaged this process by intravital microscopy (IVM) in the ear skin of transgenic mice bearing red-fluorescent vasculature and yellow-fluorescent DCs. DCs within lymphatic capillaries were rarely transported by flow, but actively migrated within lymphatics and were significantly faster than in the interstitium. Pharmacologic blockade of the Rho-associated protein kinase (ROCK), which mediates nuclear contraction and de-adhesion from integrin ligands, significantly reduced DC migration from skin to dLNs in steady-state. IVM revealed that ROCK blockade strongly reduced the velocity of interstitial DC migration, but only marginally affected intralymphatic DC migration. By contrast, during tissue inflammation, ROCK blockade profoundly decreased both interstitial and intralymphatic DC migration. Inhibition of intralymphatic migration was paralleled by a strong up-regulation of ICAM-1 in lymphatic endothelium, suggesting that during inflammation ROCK mediates de-adhesion of DC-expressed integrins from lymphatic-expressed ICAM-1. Flow chamber assays confirmed an involvement of lymphatic-expressed ICAM-1 and DC-expressed ROCK in DC crawling on lymphatic endothelium. Overall, our findings further define the role of ROCK in DC migration to dLNs and reveal a differential requirement for ROCK in intralymphatic DC crawling during steady-state and inflammation.
Subject(s)
Cell Movement , Dendritic Cells/metabolism , Dermatitis, Contact/metabolism , Endothelium, Lymphatic/immunology , rho-Associated Kinases/metabolism , Animals , Cell Adhesion Molecules/genetics , Cell Adhesion Molecules/metabolism , Cell Movement/drug effects , Crosses, Genetic , Dendritic Cells/drug effects , Dendritic Cells/immunology , Dendritic Cells/pathology , Dermatitis, Contact/drug therapy , Dermatitis, Contact/immunology , Dermatitis, Contact/pathology , Endothelium, Lymphatic/drug effects , Endothelium, Lymphatic/metabolism , Endothelium, Lymphatic/pathology , Intercellular Adhesion Molecule-1/metabolism , Luminescent Proteins/genetics , Luminescent Proteins/metabolism , Mice , Mice, Inbred C57BL , Mice, Transgenic , Microscopy, Fluorescence , Microscopy, Video , Protein Kinase Inhibitors/pharmacology , Radiation Chimera , Recombinant Fusion Proteins/metabolism , Skin/drug effects , Skin/immunology , Skin/metabolism , Skin/pathology , Up-Regulation/drug effects , rho-Associated Kinases/antagonists & inhibitors , rho-Associated Kinases/geneticsABSTRACT
Brain circuits are assembled from a large variety of morphologically and functionally diverse cell types. It is not known how the intermingled cell types of an individual adult brain region differ in their expressed genomes. Here we describe an atlas of cell type transcriptomes in one brain region, the mouse retina. We found that each adult cell type expressed a specific set of genes, including a unique set of transcription factors, forming a 'barcode' for cell identity. Cell type transcriptomes carried enough information to categorize cells into morphological classes and types. Several genes that were specifically expressed in particular retinal circuit elements, such as inhibitory neuron types, are associated with eye diseases. The resource described here allows gene expression to be compared across adult retinal cell types, experimenting with specific transcription factors to differentiate stem or somatic cells to retinal cell types, and predicting cellular targets of newly discovered disease-associated genes.
Subject(s)
Nerve Tissue Proteins/metabolism , Neurons/classification , Neurons/physiology , Retina/cytology , Retinal Diseases/genetics , Transcription Factors/metabolism , Animals , Cluster Analysis , Connexins/genetics , Eye Proteins/genetics , Eye Proteins/metabolism , Flow Cytometry , Gene Expression/genetics , Gene Library , Membrane Potentials/genetics , Mice , Mice, Inbred C57BL , Mice, Transgenic , Microarray Analysis/methods , Mutation/genetics , Nerve Tissue Proteins/genetics , Patch-Clamp Techniques , Potassium Channels, Voltage-Gated/genetics , Receptors, Kainic Acid/genetics , Transcription Factors/genetics , Visual Pathways/metabolism , GluK2 Kainate ReceptorABSTRACT
Salmonella Typhimurium causes diarrhea by infecting the epithelium and lamina propria of the intestinal mucosa and by secreting various effector proteins through type III secretion systems (TTSSs). However, the mechanisms by which Salmonella transverses the epithelium and is subsequently released into the lamina propria are poorly understood. Using a murine Salmonella-diarrhea model and in vivo microscopy, we show that epithelial traversal requires TTSS-1-mediated invasion and TTSS-2-dependent trafficking to the basolateral side. After being released into the lamina propria, the bacterium is transiently extracellular before being taken up by phagocytes, including CD11c(+)CX(3)CR1(high) monocytic phagocytes (MPs), which were found to constitutively sample cellular material shed from the basolateral side of the epithelium. Thus, Salmonella infects the cecal mucsa through a step-wise process wherein the bacterium transverses the epithelium through TTSS-2-dependent trafficking and then likely exploits lamina propria MPs, which are sampling the epithelium, to enter and replicate within the host.
Subject(s)
Epithelium/microbiology , Gastrointestinal Tract/microbiology , Membrane Transport Proteins/metabolism , Mucous Membrane/microbiology , Phagocytes/microbiology , Salmonella typhimurium/pathogenicity , Virulence Factors/metabolism , Animals , Disease Models, Animal , Mice , Microscopy , Mucous Membrane/cytology , Salmonella Infections, AnimalABSTRACT
Immunological functions of mast cells remain poorly understood. Studies in Kit mutant mice suggest key roles for mast cells in certain antibody- and T cell-mediated autoimmune diseases. However, Kit mutations affect multiple cell types of both immune and nonimmune origin. Here, we show that targeted insertion of Cre-recombinase into the mast cell carboxypeptidase A3 locus deleted mast cells in connective and mucosal tissues by a genotoxic Trp53-dependent mechanism. Cre-mediated mast cell eradication (Cre-Master) mice had, with the exception of a lack of mast cells and reduced basophils, a normal immune system. Cre-Master mice were refractory to IgE-mediated anaphylaxis, and this defect was rescued by mast cell reconstitution. This mast cell-deficient strain was fully susceptible to antibody-induced autoimmune arthritis and to experimental autoimmune encephalomyelitis. Differences comparing Kit mutant mast cell deficiency models to selectively mast cell-deficient mice call for a systematic re-evaluation of immunological functions of mast cells beyond allergy.
Subject(s)
Autoantibodies/immunology , Autoimmunity/immunology , Integrases/metabolism , Mast Cells/immunology , T-Lymphocytes/immunology , Anaphylaxis/genetics , Anaphylaxis/immunology , Animals , Arthritis, Experimental/genetics , Arthritis, Experimental/immunology , Carboxypeptidases A/genetics , Carboxypeptidases A/metabolism , Disease Models, Animal , Encephalomyelitis, Autoimmune, Experimental/genetics , Encephalomyelitis, Autoimmune, Experimental/immunology , Gene Expression Profiling , Gene Targeting , Genetic Predisposition to Disease , Immunoglobulin E/immunology , Intestinal Mucosa/immunology , Intestinal Mucosa/metabolism , Intestinal Mucosa/pathology , Mast Cells/metabolism , Mice , Mice, Inbred C57BL , Mice, Knockout , Stem Cell Factor/deficiency , T-Lymphocytes/metabolism , Th2 Cells/immunology , Tumor Suppressor Protein p53/metabolismABSTRACT
C-reactive protein (CRP), a phylogenetically highly conserved plasma protein, is the classical acute phase reactant in humans. Upon infection, inflammation, or tissue damage, its plasma level can rise within hours >1000-fold, providing an early, nonspecific disease indicator of prime clinical importance. In recent years, another aspect of CRP expression has attracted much scientific and public attention. Apart from transient, acute phase-associated spikes in plasma concentration, highly sensitive measurements have revealed stable interindividual differences of baseline CRP values in healthy persons. Strikingly, even modest elevations in stable baseline CRP plasma levels have been found to correlate with a significantly increased risk of future cardiovascular disease. These observations have triggered intense controversies about potential atherosclerosis-promoting properties of CRP. To directly assess potential effects of CRP on atherogenesis, we have generated CRP-deficient mice via gene targeting and introduced the inactivated allele into atherosclerosis-susceptible ApoE(-/-) and LDLR(-/-) mice, two well established mouse models of atherogenesis. Morphometric analyses of atherosclerotic plaques in CRP-deficient animals revealed equivalent or increased atherosclerotic lesions compared with controls, an experimental result, which does not support a proatherogenic role of CRP. In fact, our data suggest that mouse CRP may even mediate atheroprotective effects, adding a cautionary note to the idea of targeting CRP as therapeutic intervention against progressive cardiovascular disease.
Subject(s)
Atherosclerosis/metabolism , C-Reactive Protein/metabolism , Animals , Apolipoproteins E/genetics , Apolipoproteins E/metabolism , Atherosclerosis/genetics , C-Reactive Protein/genetics , Disease Models, Animal , Humans , Mice , Mice, Knockout , Receptors, LDL/genetics , Receptors, LDL/metabolismABSTRACT
Lymphoid tissue-inducer (LTi) cells initiate the development of lymphoid tissues through the activation of local stromal cells in a process similar to inflammation. LTi cells express the nuclear hormone receptor RORγt, which also directs the expression of the proinflammatory cytokine interleukin-17 in T cells. We show here that LTi cells are part of a larger family of proinflammatory RORγt(+) innate lymphoid cells (ILCs) that differentiate from distinct fetal liver RORγt(+) precursors. The fate of RORγt(+) ILCs is determined by mouse age, and after birth, favors the generation of cells involved in intestinal homeostasis and defense. Contrary to RORγt(+) T cells, however, RORγt(+) ILCs develop in the absence of microbiota. Our study indicates that RORγt(+) ILCs evolve to preempt intestinal colonization by microbial symbionts.
Subject(s)
Intestinal Mucosa/immunology , Intestine, Small/immunology , Lymphocyte Subsets/immunology , Lymphocytes/immunology , Lymphoid Progenitor Cells/cytology , Nuclear Receptor Subfamily 1, Group F, Member 3/metabolism , Adaptive Immunity , Adoptive Transfer , Animals , Bacterial Physiological Phenomena , Cell Differentiation , Cell Lineage , Fetus/cytology , Fetus/immunology , Fetus/metabolism , Homeostasis , Immunity, Mucosal , Intestine, Small/embryology , Intestine, Small/microbiology , Liver/cytology , Liver/embryology , Liver/immunology , Lymphocyte Subsets/cytology , Lymphocyte Subsets/metabolism , Lymphocytes/cytology , Lymphocytes/metabolism , Lymphoid Tissue/cytology , Lymphoid Tissue/immunology , Lymphopoiesis , Mice , Mice, Transgenic , Receptors, CCR6/metabolism , Symbiosis , T-Lymphocyte Subsets/cytology , T-Lymphocyte Subsets/immunology , T-Lymphocyte Subsets/metabolismABSTRACT
Dendritic cells (DCs) are central for the induction of T cell immunity and tolerance. Fundamental for DCs to control the immune system is their differentiation from precursors into various DC subsets with distinct functions and locations in lymphoid organs and tissues. In contrast to the differentiation of epidermal Langerhans cells (LCs) and their seeding into the epidermis, LC maturation, turnover, and MHC class II Ag presentation capacities are strictly dependent on the presence of Dicer, which generates mature microRNAs (miRNAs). Absence of miRNAs caused a strongly disturbed steady-state homeostasis of LCs by increasing their turnover and apoptosis rate, leading to progressive ablation of LCs with age. The failure to maintain LCs populating the epidermis was accompanied by a proapoptotic gene expression signature. Dicer-deficient LCs showed largely increased cell sizes and reduced expression levels of the C-type lectin receptor Langerin, resulting in the lack of Birbeck granules. In addition, LCs failed to properly upregulate MHC class II, CD40, and CD86 surface molecules upon stimulation, which are critical hallmarks of functional DC maturation. This resulted in inefficient induction of CD4 T cell proliferation, whereas Dicer-deficient LCs could properly stimulate CD8 T cells. Taken together, Dicer-dependent generation of miRNAs affects homeostasis and function of epidermal LCs.
Subject(s)
Cell Differentiation/genetics , Cell Differentiation/immunology , DEAD-box RNA Helicases/genetics , Endoribonucleases/genetics , Langerhans Cells/cytology , Langerhans Cells/immunology , MicroRNAs/physiology , Animals , Apoptosis/genetics , Apoptosis/immunology , CD4-Positive T-Lymphocytes/immunology , CD4-Positive T-Lymphocytes/metabolism , CD8-Positive T-Lymphocytes/immunology , CD8-Positive T-Lymphocytes/metabolism , Cell Movement/genetics , Cell Movement/immunology , Cell Survival/genetics , Cell Survival/immunology , DEAD-box RNA Helicases/deficiency , DEAD-box RNA Helicases/physiology , Dendritic Cells/immunology , Dendritic Cells/metabolism , Dermatitis, Contact/genetics , Dermatitis, Contact/immunology , Dermatitis, Contact/pathology , Endoribonucleases/deficiency , Endoribonucleases/physiology , Gene Expression Regulation/immunology , Homeostasis/genetics , Homeostasis/immunology , Langerhans Cells/ultrastructure , Mice , Mice, Inbred C57BL , Mice, Transgenic , MicroRNAs/genetics , Ribonuclease IIIABSTRACT
It has long been presumed that after leaving the germinal centers (GCs), memory B cells colonize the marginal zone or join the recirculating pool. Here we demonstrate the preferential localization of nitrophenol-chicken gamma-globulin-induced CD38(+)IgG1(+) memory B cells adjacent to contracted GCs in the spleen. The memory B cells in this region proliferated after secondary immunization, a response that was abolished by depletion of CD4(+) T cells. We also found that these IgG1(+) memory B cells could present antigen on their surface, and that this activity was required for their activation. These results implicate this peri-GC region as an important site for survival and reactivation of memory B cells.