ABSTRACT
Maintaining immune tolerance requires the production of Foxp3-expressing regulatory T (Treg) cells in the thymus. Activation of NF-κB transcription factors is critically required for Treg cell development, partly via initiating Foxp3 expression. NF-κB activation is controlled by a negative feedback regulation through the ubiquitin editing enzyme A20, which reduces proinflammatory signaling in myeloid cells and B cells. In naive CD4+ T cells, A20 prevents kinase RIPK3-dependent necroptosis. Using mice deficient for A20 in T lineage cells, we show that thymic and peripheral Treg cell compartments are quantitatively enlarged because of a cell-intrinsic developmental advantage of A20-deficient thymic Treg differentiation. A20-deficient thymic Treg cells exhibit reduced dependence on IL-2 but unchanged rates of proliferation and apoptosis. Activation of the NF-κB transcription factor RelA was enhanced, whereas nuclear translocation of c-Rel was decreased in A20-deficient thymic Treg cells. Furthermore, we found that the increase in Treg cells in T cell-specific A20-deficient mice was already observed in CD4+ single-positive CD25+ GITR+ Foxp3- thymic Treg cell progenitors. Treg cell precursors expressed high levels of the tumor necrosis factor receptor superfamily molecule GITR, whose stimulation is closely linked to thymic Treg cell development. A20-deficient Treg cells efficiently suppressed effector T cell-mediated graft-versus-host disease after allogeneic hematopoietic stem cell transplantation, suggesting normal suppressive function. Holding thymic production of natural Treg cells in check, A20 thus integrates Treg cell activity and increased effector T cell survival into an efficient CD4+ T cell response.
Subject(s)
CD4-Positive T-Lymphocytes/immunology , T-Lymphocytes, Regulatory/physiology , Thymus Gland/cytology , Thymus Gland/physiology , Tumor Necrosis Factor alpha-Induced Protein 3/genetics , Tumor Necrosis Factor alpha-Induced Protein 3/metabolism , Animals , Apoptosis , Cell Differentiation , Flow Cytometry , Forkhead Transcription Factors/genetics , Forkhead Transcription Factors/metabolism , Gene Expression Regulation , Glucocorticoid-Induced TNFR-Related Protein/genetics , Graft vs Host Disease/prevention & control , Interleukin-2/immunology , Lymphocyte Activation , Mice , NF-kappa B/metabolism , Proto-Oncogene Proteins c-rel/genetics , Signal Transduction , Stem Cell Transplantation , Thymus Gland/immunology , Transcription Factor RelA/genetics , Tumor Necrosis Factor alpha-Induced Protein 3/deficiencyABSTRACT
Replication-deficient recombinant adenoviruses are potent vectors for the efficient transient expression of exogenous genes in resting immune cells. However, most leukocytes are refractory to efficient adenoviral transduction as they lack expression of the coxsackie/adenovirus receptor (CAR). To circumvent this obstacle, we generated the R26/CAG-CARΔ1(StopF) (where R26 is ROSA26 and CAG is CMV early enhancer/chicken ß actin promoter) knock-in mouse line. This strain allows monitoring of in situ Cre recombinase activity through expression of CARΔ1. Simultaneously, CARΔ1 expression permits selective and highly efficient adenoviral transduction of immune cell populations, such as mast cells or T cells, directly ex vivo in bulk cultures without prior cell purification or activation. Furthermore, we show that CARΔ1 expression dramatically improves adenoviral infection of in vitro differentiated conventional and plasmacytoid dendritic cells (DCs), basophils, mast cells, as well as Hoxb8-immortalized hematopoietic progenitor cells. This novel dual function mouse strain will hence be a valuable tool to rapidly dissect the function of specific genes in leukocyte physiology.
Subject(s)
Adenoviridae/genetics , Gene Targeting , Genes, Reporter , Genetic Vectors/genetics , Homologous Recombination , Integrases/metabolism , Transduction, Genetic , Animals , Coxsackie and Adenovirus Receptor-Like Membrane Protein/genetics , Gene Expression , Gene Targeting/methods , Humans , Integrases/genetics , Leukocytes/immunology , Leukocytes/metabolism , Mice , Mice, Transgenic , Myeloid Cells/cytology , Myeloid Cells/immunology , Myeloid Cells/metabolism , Organ SpecificityABSTRACT
Caspase-11 is a highly inducible caspase that controls both inflammatory responses and cell death. Caspase-11 controls interleukin 1ß (IL-1ß) secretion by potentiating caspase-1 activation and induces caspase-1-independent pyroptosis downstream of noncanonical NLRP3 inflammasome activators such as lipopolysaccharide (LPS) and Gram-negative bacteria. However, we still know very little about the downstream mechanism of caspase-11 in regulating inflammation because the known substrates of caspase-11 are only other caspases. Here, we identify the cationic channel subunit transient receptor potential channel 1 (TRPC1) as a substrate of caspase-11. TRPC1 deficiency increases the secretion of IL-1ß without modulating caspase-1 cleavage or cell death in cultured macrophages. Consistently, trpc1(-/-) mice show higher IL-1ß secretion in the sepsis model of intraperitoneal LPS injection. Altogether, our data suggest that caspase-11 modulates the cationic channel composition of the cell and thus regulates the unconventional secretion pathway in a manner independent of caspase-1.
Subject(s)
Caspases/metabolism , Interleukin-1beta/metabolism , TRPC Cation Channels/physiology , Animals , Caspases, Initiator , HEK293 Cells , Humans , Mice , Mice, Inbred C57BL , Mice, Knockout , TRPC Cation Channels/deficiency , TRPC Cation Channels/metabolism , TransfectionABSTRACT
Mast cells are abundantly situated at contact sites between the body and its environment, such as the skin and, especially during certain immune responses, at mucosal surfaces. They mediate allergic reactions and degrade toxins as well as venoms. However, their roles during innate and adaptive immune responses remain controversial and it is likely that major functions remain to be discovered. Recent developments in mast cell-specific conditional gene targeting in the mouse promise to enhance our understanding of these fascinating cells. To complete the genetic toolbox to study mast cell development, homeostasis and function, it is imperative to inducibly manipulate their gene expression. Here, we report the generation of a novel knock-in mouse line expressing a tamoxifen-inducible version of the Cre recombinase from within the endogenous c-Kit locus. We demonstrate highly efficient and specific inducible expression of a fluorescent reporter protein in mast cells both in vivo and in vitro. Furthermore, induction of diphtheria toxin A expression allowed selective and efficient ablation of mast cells at various anatomical locations, while other hematopoietic cells remain unaffected. This novel mouse strain will hence be very valuable to study mast cell homeostasis and how specific genes influence their functions in physiology and pathology.
Subject(s)
Diphtheria Toxin/metabolism , Gene Targeting/methods , Integrases/metabolism , Mast Cells/immunology , Mice, Transgenic/immunology , Peptide Fragments/metabolism , Animals , Cell Death/drug effects , Cell Death/genetics , Diphtheria Toxin/genetics , Gene Expression Regulation/drug effects , Gene Knock-In Techniques , Genetic Loci/genetics , Integrases/genetics , Mast Cells/drug effects , Mast Cells/pathology , Mice , Organ Specificity , Peptide Fragments/genetics , Proto-Oncogene Proteins c-kit/genetics , Tamoxifen/administration & dosage , Transgenes/geneticsABSTRACT
The phytopharmaceutical agent St. John's wort (SJW) is currently under intense investigation. Studies of drug interactions resulting from concomitant use of SJW and conventional medication are of fundamental importance, since the use of SJW as a complementary and alternative medicine is highly popular. Intake of SJW often remains unrecognized by physicians resulting in clinical relevant alterations of treatment, as this phytopharmaceutical agent is available without prescription. This review elicits molecular explanations for clinical observations in terms of concomitant use of SJW and conventional drugs. Since patients suffering from severe diseases such as cancer are especially at risk, we focus on chemotherapeutic agents. There is strong evidence that SJW extract lowers drug plasma levels of various anti-cancer agents by pregnane X receptor activation resulting in induction of cytochrome P450 isotype 3A4, P glycoprotein and several other enzymes. New methods such as photophysical diagnosis (PPD) and photodynamic therapy (PDT) seem to be highly promising with respect to their clinical application. Due to its fluorescent activity and an intense accumulation in cancer cells, hypericin could be applied to locate tumorous tissues. Upon excitation by light, hypericin generates cytotoxic products rendering its use attractive as photosensitizing agent. In this review both PPD and PDT are explained in detail, with a particular focus on molecular mechanisms.