ABSTRACT
Statins are a well-established family of drugs that lower cholesterol levels via the competitive inhibition of the enzyme 3-hydroxy-3-methylglutaryl coenzyme A reductase (HMGCR). In addition, the pleiotropic anti-inflammatory effects of statins on T cells make them attractive as therapeutic drugs in T-cell-driven autoimmune disorders. Since statins do not exclusively target HMGCR and thus might have varying effects on different cell types, we generated a new mouse strain allowing for the tissue-specific deletion of HMGCR. Deletion of HMGCR expression in T cells led to a severe decrease in their numbers with the remaining cells displaying an activated phenotype, with an increased proportion of regulatory T cells (Tregs) in particular. However, deletion of HMGCR specifically in Tregs resulted in severe autoimmunity, suggesting that this enzyme is also essential for the maintenance of Tregs. We were able to prevent the death of HMGCR-deficient lymphocytes by the addition of either the direct metabolite of HMGCR, namely mevalonate, or the downstream metabolite geranylgeranyl pyrophosphate, which is essential for protein prenylation. However, the addition of cholesterol, which is the final product of the mevalonate pathway, did not inhibit cell death, indicating that protein prenylation rather than the cholesterol biosynthesis pathway is indispensible for T-cell survival.
Subject(s)
Hydroxymethylglutaryl CoA Reductases/metabolism , Protein Prenylation , T-Lymphocytes/cytology , T-Lymphocytes/enzymology , Animals , Cell Count , Cell Death/drug effects , Cell Survival/drug effects , Gene Deletion , Hydroxymethylglutaryl CoA Reductases/deficiency , Integrases/metabolism , Lymphocyte Activation/drug effects , Mevalonic Acid/analogs & derivatives , Mevalonic Acid/pharmacology , Mice, Inbred C57BL , Phenotype , Polyisoprenyl Phosphates/pharmacology , Protein Prenylation/drug effects , T-Lymphocytes/drug effects , T-Lymphocytes, Regulatory/cytology , T-Lymphocytes, Regulatory/drug effects , T-Lymphocytes, Regulatory/enzymologyABSTRACT
The link between the extensive usage of calcineurin (CN) inhibitors cyclosporin A and tacrolimus (FK506) in transplantation medicine and the increasing rate of opportunistic infections within this segment of patients is alarming. Currently, how peritoneal infections are favored by these drugs, which impair the activity of several signaling pathways including the Ca(++) /CN/NFAT, Ca(++) /CN/cofilin, Ca(++) /CN/BAD, and NF-κB networks, is unknown. Here, we show that Saccharomyces cerevisiae infection of peritoneal resident macrophages triggers the transient nuclear translocation of NFATc1ß isoforms, resulting in a coordinated, CN-dependent induction of the Ccl2, Ccl7, and Ccl12 genes, all encoding CCR2 agonists. CN inhibitors block the CCR2-dependent recruitment of inflammatory monocytes (IM) to the peritoneal cavities of S. cerevisiae infected mice. In myeloid cells, NFATc1/ß proteins represent the most prominent NFATc1 isoforms. NFATc1/ß ablation leads to a decrease of CCR2 chemokines, impaired mobilization of IMs, and delayed clearance of infection. We show that, upon binding to a composite NFAT/BCL6 regulatory element within the Ccl2 promoter, NFATc1/ß proteins release the BCL6-dependent repression of Ccl2 gene in macrophages. These findings suggest a novel CN-dependent cross-talk between NFAT and BCL6 transcription factors, which may affect the outcome of opportunistic fungal infections in immunocompromised patients.
Subject(s)
Macrophages, Peritoneal/metabolism , NFATC Transcription Factors/immunology , NFATC Transcription Factors/physiology , Proto-Oncogene Proteins c-bcl-6/metabolism , Receptors, CCR2/agonists , Receptors, CCR2/immunology , Saccharomyces cerevisiae/immunology , Animals , Calcineurin/metabolism , Calcineurin Inhibitors , Chemokine CCL2/genetics , Chemokine CCL7/genetics , Macrophages, Peritoneal/microbiology , Mice , Monocyte Chemoattractant Proteins/genetics , Monocytes/immunology , NF-kappa B/metabolism , NFATC Transcription Factors/deficiency , NFATC Transcription Factors/genetics , Opportunistic Infections/immunology , Opportunistic Infections/virology , Promoter Regions, Genetic , Protein Isoforms , Protein Transport , Proto-Oncogene Proteins c-bcl-6/geneticsABSTRACT
During early embryogenesis, microglia arise from yolk sac progenitors that populate the developing central nervous system (CNS), but how the tissue-resident macrophages are maintained throughout the organism's lifespan still remains unclear. Here, we describe a system that allows specific, conditional ablation of microglia in adult mice. We found that the microglial compartment was reconstituted within 1 week of depletion. Microglia repopulation relied on CNS-resident cells, independent from bone-marrow-derived precursors. During repopulation, microglia formed clusters of highly proliferative cells that migrated apart once steady state was achieved. Proliferating microglia expressed high amounts of the interleukin-1 receptor (IL-1R), and treatment with an IL-1R antagonist during the repopulation phase impaired microglia proliferation. Hence, microglia have the potential for efficient self-renewal without the contribution of peripheral myeloid cells, and IL-1R signaling participates in this restorative proliferation process.
Subject(s)
Hematopoietic Stem Cells/cytology , Macrophages/cytology , Microglia/cytology , Receptors, Interleukin-1 Type I/biosynthesis , Animals , Base Sequence , Bone Marrow Cells/immunology , CX3C Chemokine Receptor 1 , Cell Differentiation , Cell Movement , Cell Proliferation , Central Nervous System/cytology , Interleukin-1beta/biosynthesis , Interleukin-1beta/genetics , Interleukin-1beta/metabolism , Mice , Mice, Inbred C57BL , Receptors, Chemokine/genetics , Receptors, Interleukin-1 Type I/antagonists & inhibitors , Sequence Analysis, DNA , Signal Transduction , Tumor Necrosis Factor-alpha/biosynthesis , Tumor Necrosis Factor-alpha/geneticsABSTRACT
Mechanisms underlying central neuropathic pain are poorly understood. Although glial dysfunction has been functionally linked with neuropathic pain, very little is known about modulation of pain by oligodendrocytes. Here we report that genetic ablation of oligodendrocytes rapidly triggers a pattern of sensory changes that closely resemble central neuropathic pain, which are manifest before overt demyelination. Primary oligodendrocyte loss is not associated with autoreactive T- and B-cell infiltration in the spinal cord and neither activation of microglia nor reactive astrogliosis contribute functionally to central pain evoked by ablation of oligodendrocytes. Instead, light and electron microscopic analyses reveal axonal pathology in the spinal dorsal horn and spinothalamic tract concurrent with the induction and maintenance of nociceptive hypersensitivity. These data reveal a role for oligodendrocytes in modulating pain and suggest that perturbation of oligodendrocyte functions that maintain axonal integrity can lead to central neuropathic pain independent of immune contributions.
Subject(s)
Adaptive Immunity/immunology , Axons/pathology , Immunity, Innate/immunology , Neuralgia/pathology , Oligodendroglia/physiology , Spinal Cord Dorsal Horn/pathology , Spinothalamic Tracts/pathology , Animals , Astrocytes , Axons/ultrastructure , B-Lymphocytes , Mice , Microglia , Microscopy, Electron , Neuralgia/immunology , Nociception , Oligodendroglia/metabolism , Spinal Cord/pathology , Spinal Cord/ultrastructure , Spinal Cord Dorsal Horn/immunology , Spinal Cord Dorsal Horn/ultrastructure , Spinothalamic Tracts/immunology , Spinothalamic Tracts/ultrastructure , T-LymphocytesABSTRACT
The lack of a generally accepted animal model for human psoriasis has hindered progress with respect to understanding the pathogenesis of the disease. Here we present a model in which transgenic IL-17A expression is targeted to the skin in mice, achievable after crossing our IL-17A(ind) allele to the K14-Cre strain. K14-IL-17A(ind/+) mice invariably develop an overt skin inflammation bearing many hallmark characteristics of human psoriasis including dermal infiltration of effector T cells, formation of neutrophil microabscesses, and hyperkeratosis. IL-17A expression in the skin results in upregulated granulopoiesis and migration of IL-6R-expressing neutrophils into the skin. Neutralization of IL-6 signaling efficiently reduces the observed pathogenesis in skin of IL-17A-overexpressing mice, with marked reductions in epidermal neutrophil abscess formation and epidermal thickening. Thus, IL-6 functions downstream of IL-17A to exacerbate neutrophil microabscess development in psoriasiform lesions.
Subject(s)
Abscess/immunology , Interleukin-17/immunology , Interleukin-6/immunology , Neutrophils/immunology , Psoriasis/immunology , Abscess/drug therapy , Abscess/pathology , Animals , Disease Models, Animal , Epidermis/immunology , Epidermis/metabolism , Epidermis/pathology , Gene Expression/immunology , Granulocytes/immunology , Granulocytes/pathology , Interleukin-17/genetics , Interleukin-17/metabolism , Interleukin-6/metabolism , Interleukin-6/pharmacology , Macrophages/immunology , Macrophages/pathology , Mice , Mice, Knockout , Neutrophils/metabolism , Psoriasis/drug therapy , Psoriasis/pathology , Receptors, Interleukin-6/genetics , Receptors, Interleukin-6/immunology , Signal Transduction/drug effects , Signal Transduction/immunology , T-Lymphocytes/immunology , T-Lymphocytes/pathologyABSTRACT
The transcription factor Bcl-3 functions as a proto-oncogene via regulation of cell proliferation and apoptosis. Bcl-3 is an atypical member of the IκB family and plays a central role in the immune response through interactions with the NF-κB subunits p50 and p52. To investigate the impact of Bcl-3 on B-cell maturation and regulation, we generated mice that overexpress Bcl-3 specifically in B cells. Interestingly, these mice lack marginal zone B cells and exhibit a significant reduction in the number of B-1 B cells. Further, B cells from these mice are impaired in their proliferative capacity. Our data demonstrate that the overexpression of the transcription factor Bcl-3 inhibits germinal center formation, marginal zone B-cell development, and affects the B-1 B-cell compartment.
Subject(s)
B-Lymphocytes/metabolism , Gene Expression/genetics , Proto-Oncogene Proteins/genetics , Proto-Oncogene Proteins/metabolism , Transcription Factors/genetics , Transcription Factors/metabolism , Animals , B-Cell Lymphoma 3 Protein , Cell Proliferation , Germinal Center/metabolism , MiceABSTRACT
The second messengers cAMP and cGMP activate their target proteins by binding to a conserved cyclic nucleotide-binding domain (CNBD). Here, we identify and characterize an entirely novel CNBD-containing protein called CRIS (cyclic nucleotide receptor involved in sperm function) that is unrelated to any of the other members of this protein family. CRIS is exclusively expressed in sperm precursor cells. Cris-deficient male mice are either infertile due to a lack of sperm resulting from spermatogenic arrest, or subfertile due to impaired sperm motility. The motility defect is caused by altered Ca(2+) regulation of flagellar beat asymmetry, leading to a beating pattern that is reminiscent of sperm hyperactivation. Our results suggest that CRIS interacts during spermiogenesis with Ca(2+)-regulated proteins that--in mature sperm--are involved in flagellar bending.
Subject(s)
Carrier Proteins/genetics , Cyclic AMP/genetics , Flagella/genetics , Protein Binding/genetics , Spermatogenesis/genetics , Animals , Calcium/metabolism , Cyclic AMP/metabolism , Cyclic GMP/metabolism , Flagella/metabolism , Humans , Intracellular Signaling Peptides and Proteins , Male , Mice , Phosphorylation , Signal Transduction/genetics , Sperm Motility/genetics , Spermatozoa/metabolismABSTRACT
The t(8;21) chromosomal translocation activates aberrant expression of the AML1-ETO (AE) fusion protein and is commonly associated with core binding factor acute myeloid leukaemia (CBF AML). Combining a conditional mouse model that closely resembles the slow evolution and the mosaic AE expression pattern of human t(8;21) CBF AML with global transcriptome sequencing, we find that disease progression was characterized by two principal pathogenic mechanisms. Initially, AE expression modified the lineage potential of haematopoietic stem cells (HSCs), resulting in the selective expansion of the myeloid compartment at the expense of normal erythro- and lymphopoiesis. This lineage skewing was followed by a second substantial rewiring of transcriptional networks occurring in the trajectory to manifest leukaemia. We also find that both HSC and lineage-restricted granulocyte macrophage progenitors (GMPs) acquired leukaemic stem cell (LSC) potential being capable of initiating and maintaining the disease. Finally, our data demonstrate that long-term expression of AE induces an indolent myeloproliferative disease (MPD)-like myeloid leukaemia phenotype with complete penetrance and that acute inactivation of AE function is a potential novel therapeutic option.
Subject(s)
Hematopoietic Stem Cells/metabolism , Neoplastic Stem Cells/metabolism , Oncogene Proteins, Fusion/metabolism , Animals , Antibiotics, Antineoplastic/pharmacology , Antibiotics, Antineoplastic/therapeutic use , Cell Lineage , Disease Models, Animal , Doxorubicin/pharmacology , Doxorubicin/therapeutic use , Gene Expression Regulation/drug effects , Granulocyte-Macrophage Progenitor Cells/cytology , Granulocyte-Macrophage Progenitor Cells/metabolism , Hematopoietic Stem Cells/cytology , Immunophenotyping , Leukemia, Myeloid, Acute/drug therapy , Leukemia, Myeloid, Acute/metabolism , Leukemia, Myeloid, Acute/pathology , Mice , Mice, Inbred C57BL , Neoplastic Stem Cells/cytology , Phenotype , Sequence Analysis, RNA , Translocation, Genetic/drug effectsABSTRACT
A major goal in current neuroscience is to understand the causal links connecting protein functions, neural activity, and behavior. The cannabinoid CB1 receptor is expressed in different neuronal subpopulations, and is engaged in fine-tuning excitatory and inhibitory neurotransmission. Studies using conditional knock-out mice revealed necessary roles of CB1 receptor expressed in dorsal telencephalic glutamatergic neurons in synaptic plasticity and behavior, but whether this expression is also sufficient for brain functions is still to be determined. We applied a genetic strategy to reconstitute full wild-type CB1 receptor functions exclusively in dorsal telencephalic glutamatergic neurons and investigated endocannabinoid-dependent synaptic processes and behavior. Using this approach, we partly restored the phenotype of global CB1 receptor deletion in anxiety-like behaviors and fully restored hippocampus-dependent neuroprotection from chemically induced epileptiform seizures. These features coincided with a rescued hippocampal depolarization-induced suppression of excitation (DSE), a CB1 receptor-dependent form of synaptic plasticity at glutamatergic neurons. By comparison, the rescue of the CB1 receptor on dorsal telencephalic glutamatergic neurons prolonged the time course of DSE in the amygdala, and impaired fear extinction in auditory fear conditioning. These data reveal that CB1 receptor in dorsal telencephalic glutamatergic neurons plays a sufficient role to control neuronal functions that are in large part hippocampus-dependent, while it is insufficient for proper amygdala functions, suggesting an unexpectedly complex circuit regulation by endocannabinoid signaling in the amygdala. Our data pave the way to a better understanding of neuronal networks in the context of behavior, by fine-tuned interference with synaptic transmission processes.
Subject(s)
Amygdala/physiology , Behavior, Animal/physiology , Glutamic Acid/physiology , Hippocampus/physiology , Neurons/physiology , Receptor, Cannabinoid, CB1/physiology , Synapses/physiology , Telencephalon/physiology , Animals , Anxiety/psychology , Blotting, Western , Electrophysiological Phenomena , Excitatory Amino Acid Agonists/toxicity , Immunohistochemistry , Kainic Acid/toxicity , Light , Mice , Mice, Inbred C57BL , Mice, Knockout , Neuronal Plasticity/physiology , RNA/biosynthesis , RNA/isolation & purification , Real-Time Polymerase Chain Reaction , Telencephalon/cytologyABSTRACT
Topical application of imiquimod (IMQ) on the skin of mice induces inflammation with common features found in psoriatic skin. Recently, it was postulated that IL-17 has an important role both in psoriasis and in the IMQ model. To further investigate the impact of IL-17RA signaling in psoriasis, we generated IL-17 receptor A (IL-17RA)-deficient mice (IL-17RA(del)) and challenged these mice with IMQ. Interestingly, the disease was only partially reduced and delayed but not abolished when compared with controls. In the absence of IL-17RA, we found persisting signs of inflammation such as neutrophil and macrophage infiltration within the skin. Surprisingly, already in the naive state, the skin of IL-17RA(del) mice contained significantly elevated numbers of Th17- and IL-17-producing γδ T cells, assuming that IL-17RA signaling regulates the population size of Th17 and γδ T cells. Upon IMQ treatment of IL-17RA(del) mice, these cells secreted elevated amounts of tumor necrosis factor-α, IL-6, and IL-22, accompanied by increased levels of the chemokine CXCL2, suggesting an alternative pathway of neutrophil and macrophage skin infiltration. Hence, our findings have major implications in the potential long-term treatment of psoriasis by IL-17-targeting drugs.
Subject(s)
Aminoquinolines/pharmacology , Interleukin-17/immunology , Psoriasis/chemically induced , Psoriasis/immunology , Receptors, Interleukin-17/immunology , Signal Transduction/immunology , Adjuvants, Immunologic/pharmacology , Animals , Disease Models, Animal , Female , Imiquimod , Interleukin-17/metabolism , Interleukin-6/immunology , Interleukin-6/metabolism , Interleukins/immunology , Interleukins/metabolism , Macrophages/drug effects , Macrophages/immunology , Mice , Mice, Knockout , Neutrophil Infiltration/drug effects , Neutrophil Infiltration/immunology , Psoriasis/genetics , Receptors, Interleukin-17/genetics , Receptors, Interleukin-17/metabolism , Signal Transduction/drug effects , Skin/immunology , Skin/metabolism , Skin/pathology , Interleukin-22ABSTRACT
Anti-myelin immunity is commonly thought to drive multiple sclerosis, yet the initial trigger of this autoreactivity remains elusive. One of the proposed factors for initiating this disease is the primary death of oligodendrocytes. To specifically test such oligodendrocyte death as a trigger for anti-CNS immunity, we inducibly killed oligodendrocytes in an in vivo mouse model. Strong microglia-macrophage activation followed oligodendrocyte death, and myelin components in draining lymph nodes made CNS antigens available to lymphocytes. However, even conditions favoring autoimmunity-bystander activation, removal of regulatory T cells, presence of myelin-reactive T cells and application of demyelinating antibodies-did not result in the development of CNS inflammation after oligodendrocyte death. In addition, this lack of reactivity was not mediated by enhanced myelin-specific tolerance. Thus, in contrast with previously reported impairments of oligodendrocyte physiology, diffuse oligodendrocyte death alone or in conjunction with immune activation does not trigger anti-CNS immunity.
Subject(s)
Encephalomyelitis, Autoimmune, Experimental/immunology , Encephalomyelitis, Autoimmune, Experimental/pathology , Oligodendroglia/immunology , Oligodendroglia/pathology , Animals , Cell Death/immunology , Cells, Cultured , Gene Knock-In Techniques , Mice , Mice, Transgenic , Multiple Sclerosis/immunology , Multiple Sclerosis/pathologyABSTRACT
The recent decades have shown that multiple sclerosis (MS) is not a uniform disease entity with common etiology, but rather a disease or syndrome characterized by a heterogeneous pattern of manifestations and pathological principles. Apart from the older distinctions of the Devic's disease from the standard Western form of relapsing remitting MS or the more Asian form of opticospinal MS, specific pathological patterns indicating distinct etiologies have been established by analyses of biopsies and autopsies. Further, the distinct responses of patients to drugs targeting either specific cell types or immunoregulatory mechanisms such as Rituximab or IFNß clearly demonstrate the heterogeneity of the disease and their driving mechanisms. Finally, the late neurodegenerative phase, which severe cases of MS patients experience, is now in the focus of research. Here, a mechanism independent of or with low participation of the adaptive immune system takes place, which is therefore not treatable by current immunotargeting drugs. In this review, we will summarize previous and latest efforts to establish new mouse models mirroring these distinct disease patterns and pathways.
Subject(s)
Disease Models, Animal , Multiple Sclerosis , Animals , Encephalomyelitis, Autoimmune, Experimental/immunology , Encephalomyelitis, Autoimmune, Experimental/pathology , Encephalomyelitis, Autoimmune, Experimental/physiopathology , Humans , Mice , Mice, Inbred C57BL , Multiple Sclerosis/immunology , Multiple Sclerosis/pathology , Multiple Sclerosis/physiopathology , Nerve Degeneration/immunology , Nerve Degeneration/pathology , Nerve Degeneration/physiopathology , Rats , Rats, Inbred LewABSTRACT
IL-17-producing CD4(+) T cells (Th17) have been classified as a new T helper cell subset. Using an IL-17 fate mapping mouse strain, which genetically fixes the memory of IL-17 expression, we demonstrate that IL-17A/F-expressing T helper cells generated either in vitro or in vivo are not a stable T-cell subset. Upon adoptive transfer of IL-17F-reporter-positive Th17 cells to RAG-deficient or WT animals, encephalitogenic Th17 cells partially lose IL-17 expression and upregulate IFN-γ. Additionally, we show that Th1 cells can convert in vivo to IL-17A/IFN-γ-coexpressing cells in the mesenteric lymph nodes (mLN). Our data classify IL-17A and IL-17F as cytokines produced transiently in response to the local microenvironment, thus showing that IL-17 expression does not define an end-stage T helper cell subset.
Subject(s)
Encephalomyelitis, Autoimmune, Experimental/immunology , Gene Expression Regulation/immunology , T-Lymphocyte Subsets/metabolism , Th1 Cells/metabolism , Th17 Cells/metabolism , Adoptive Transfer , Animals , Cell Differentiation/immunology , Gene Expression Regulation/genetics , Genes, RAG-1/genetics , Genes, Reporter/genetics , Integrases/genetics , Interferon-gamma/metabolism , Lymphocyte Activation/genetics , Lymphocyte Activation/immunology , Mice , Mice, Inbred C57BL , Mice, Knockout , Mice, Transgenic , Receptors, Antigen, T-Cell/genetics , T-Lymphocyte Subsets/immunology , T-Lymphocyte Subsets/pathology , Th1 Cells/immunology , Th1 Cells/pathology , Th17 Cells/immunology , Th17 Cells/pathologyABSTRACT
BACKGROUND: Conditional gene activation is an efficient strategy for studying gene function in genetically modified animals. Among the presently available gene switches, the tetracycline-regulated system has attracted considerable interest because of its unique potential for reversible and adjustable gene regulation. RESULTS: To investigate whether the ubiquitously expressed Gt(ROSA)26Sor locus enables uniform DOX-controlled gene expression, we inserted the improved tetracycline-regulated transcription activator iM2 together with an iM2 dependent GFP gene into the Gt(ROSA)26Sor locus, using gene targeting to generate ROSA26-iM2-GFP (R26t1Δ) mice. Despite the presence of ROSA26 promoter driven iM2, R26t1Δ mice showed very sparse DOX-activated expression of different iM2-responsive reporter genes in the brain, mosaic expression in peripheral tissues and more prominent expression in erythroid, myeloid and lymphoid lineages, in hematopoietic stem and progenitor cells and in olfactory neurons. CONCLUSIONS: The finding that gene regulation by the DOX-activated transcriptional factor iM2 in the Gt(ROSA)26Sor locus has its limitations is of importance for future experimental strategies involving transgene activation from the endogenous ROSA26 promoter. Furthermore, our ROSA26-iM2 knock-in mouse model (R26t1Δ) represents a useful tool for implementing gene function in vivo especially under circumstances requiring the side-by-side comparison of gene manipulated and wild type cells. Since the ROSA26-iM2 mouse allows mosaic gene activation in peripheral tissues and haematopoietic cells, this model will be very useful for uncovering previously unknown or unsuspected phenotypes.