ABSTRACT
Myeloid-derived suppressor cells (MDSCs) are a heterogeneous population of immature myeloid cells, which are characterized by their capability to suppress T-cell responses. While MDSCs have been traditionally associated with cancer diseases, their role as regulators of autoimmune diseases is emerging. Pemphigus is a chronic autoimmune blistering skin disease characterized by dysregulated T-cell responses and autoantibody production. The role of MDSCs in pemphigus disease has not been defined yet. The aim of this study was to characterize MDSCs in pemphigus patients and to dissect their relationship with CD4+ T-cell subsets and clinical disease assessments. For this purpose, we performed a cross-sectional analysis of 20 patients with pemphigus. Our results indicate that a population of CD66b+ CD11b+ polymorphonuclear-like MDSCs (PMN-MDSCs) is expanded in the peripheral blood mononuclear cell fraction of pemphigus patients compared to age-matched healthy donors. These PMN-MDSCs have the capability of suppressing allogeneic T-cell proliferation in vitro and show increased expression of characteristic effector molecules such as arginase I and interleukin-10. We further demonstrate that PMN-MDSCs are especially expanded in patients with active pemphigus, but not in patients in remission. Moreover, MDSC frequencies correlate with an increased Th2/Th1 cell ratio. In conclusion, the identification of a functional PMN-MDSC population suggests a possible role of these cells as regulators of Th cell responses in pemphigus.
Subject(s)
Arginase/metabolism , Myeloid-Derived Suppressor Cells/metabolism , Pemphigus/metabolism , Th1 Cells/metabolism , Th2 Cells/metabolism , Adult , Aged , Aged, 80 and over , Cross-Sectional Studies , Female , Humans , Male , Middle AgedABSTRACT
[18 F]FDG-PET/CT is a high sensitive functional diagnostic imaging modality to monitor tumor but also immune cell activation by determination of the glucose metabolism. Our results show that the anti-inflammatory effects of immunotherapeutics like DMF can be assessed non invasively in vivo during Th1/Th17 cell-mediated encephalomyelitis (EAE) by [18 F]FDG-PET/CT imaging of the draining lymph nodes.
Subject(s)
Dimethyl Fumarate/immunology , Drug Monitoring/methods , Encephalomyelitis, Autoimmune, Experimental/immunology , Glucose/metabolism , Lymph Nodes/immunology , Positron Emission Tomography Computed Tomography/methods , Animals , Dimethyl Fumarate/therapeutic use , Encephalomyelitis, Autoimmune, Experimental/diagnostic imaging , Encephalomyelitis, Autoimmune, Experimental/drug therapy , Fluorodeoxyglucose F18/metabolism , Humans , Lymph Nodes/drug effects , Lymph Nodes/metabolism , Mice , Th1 Cells/drug effects , Th1 Cells/immunology , Th1 Cells/metabolism , Th17 Cells/drug effects , Th17 Cells/immunology , Th17 Cells/metabolismABSTRACT
In a variety of diseases, from benign to life-threatening ones, inflammation plays a major role. Monitoring the intensity and extent of a multifaceted inflammatory process has become a cornerstone in diagnostics and therapy monitoring. However, the current tools lack the ability to provide insight into one of its most crucial aspects, namely, the alteration of the extracellular matrix (ECM). Using a radiolabeled platelet glycoprotein VI-based ECM-targeting fusion protein (GPVI-Fc), we investigated how binding of GPVI-Fc on fibrous tissue could uncover the progression of several inflammatory disease models at different stages (rheumatoid arthritis, cutaneous delayed-type hypersensitivity, lung inflammation and experimental autoimmune encephalomyelitis). Methods: The fusion protein GPVI-Fc was covalently linked to 1,4,7-triazacyclononane-1,4,7-triacetic acid (NOTA) and subsequently labeled with 64Cu. We analyzed noninvasively in vivo64Cu-GPVI-Fc accumulation in murine cutaneous delayed-type hypersensitivity, anti-glucose-6-phosphate isomerase serum-induced rheumatoid arthritis, lipopolysaccharide-induced lung inflammation and an experimental autoimmune encephalomyelitis model. Static and dynamic Positron Emission Tomography (PET) of the radiotracer distribution was performed in vivo, with ex vivo autoradiography confirmation, yielding quantitative accumulation and a distribution map of 64Cu-GPVI-Fc. Ex vivo tissue histological staining was performed on harvested samples to highlight the fusion protein binding to collagen I, II and III, fibronectin and fibrinogen as well as the morphology of excised tissue. Results:64Cu-GPVI-Fc showed a several-fold increased uptake in inflamed tissue compared to control tissue, particularly in the RA model, with a peak 24 h after radiotracer injection of up to half the injected dose. Blocking and isotype control experiments indicated a target-driven accumulation of the radiotracer in the case of chronic inflammation. Histological analysis confirmed a prolonged accumulation at the inflammation site, with a pronounced colocalization with the different components of the ECM (collagen III and fibronectin notably). Binding of the fusion protein appeared to be specific to the ECM but unspecific to particular components. Conclusion: Imaging of 64Cu-GPVI-Fc accumulation in the ECM matrix appears to be a promising candidate for monitoring chronic inflammation. By binding to exposed fibrous tissue (collagen, fibronectin, etc.) after extravasation, a new insight is provided into the fibrotic events resulting from a prolonged inflammatory state.
Subject(s)
Extracellular Matrix/metabolism , Fibrosis/diagnostic imaging , Glycoproteins/metabolism , Immunoglobulin Fc Fragments/metabolism , Positron-Emission Tomography/methods , Staining and Labeling/methods , Animals , Arthritis, Rheumatoid/complications , Copper Radioisotopes/metabolism , Dermatitis/complications , Disease Models, Animal , Encephalomyelitis, Autoimmune, Experimental/complications , Glycoproteins/genetics , Heterocyclic Compounds, 1-Ring/metabolism , Immunoglobulin Fc Fragments/genetics , Mice, Inbred C57BL , Pneumonia/complications , Sensitivity and SpecificityABSTRACT
Dimethyl fumarate (DMF) promotes an IL-17Alow IFN-γlow IL-4+ CD4+ T cell phenotype. Adoptive transfer of in vitro DMF-treated myelin peptide-reactive IL-17Alow IFN-γlow IL-4+ CD4+ T cells prior to immunization for EAE reduces the severity of encephalomyelitis. This beneficial effect of transferred DMF-treated CD4+ T cells requires an early in vivo recall.
Subject(s)
CD4-Positive T-Lymphocytes/immunology , Dimethyl Fumarate/pharmacology , Encephalomyelitis, Autoimmune, Experimental/immunology , Encephalomyelitis, Autoimmune, Experimental/prevention & control , Immunosuppressive Agents/pharmacology , Interferon-gamma/immunology , Interleukin-4/immunology , Th17 Cells/immunology , Adoptive Transfer , Animals , CD4-Positive T-Lymphocytes/transplantation , Interferon-gamma/biosynthesis , Interleukin-10/biosynthesis , Interleukin-10/immunology , Interleukin-17/biosynthesis , Interleukin-17/immunology , Interleukin-4/biosynthesis , Mice , Th1 Cells/immunology , Th2 Cells/immunologyABSTRACT
Fumaric acid esters (FAEs) such as dimethylfumarate (DMF) are used for the treatment of adults with moderate-to-severe psoriasis. The mode of action of FAEs is complex. Here, we provide a comprehensive review of the literature to describe the molecular mechanisms by which DMF and its active metabolite monomethylfumarate (MMF) exert their anti-inflammatory and immune modulatory effects. MMF can bind to the hydroxy-carboxylic acid receptor 2 (HCA2) on the cell surface and both DMF and MMF react with intracellular glutathione following cell penetration. DMF and to some extent also MMF modulate the activity of certain cellular signalling proteins such as the nuclear factor (erythroid-derived 2)-like 2 (Nrf2), nuclear factor kappa B (Nf-κB) and the cellular concentration of cyclic adenosine monophosphate. Some studies show that DMF can also affect the hypoxia-inducible factor 1-alpha (HIF-1α). These actions seem to be responsible for i) the downregulation of inflammatory cytokines and ii) an overall shift from a proinflammatory Th1/Th17 response to an anti-inflammatory/regulatory Th2 response. Both steps are necessary for the amelioration of psoriatic inflammation, although additional mechanisms have been proposed. There is a growing body of evidence to support the notion that DMF/MMF may also exert effects on granulocytes and non-immune cell lineages including keratinocytes and endothelial cells. A better understanding of the multiple molecular mechanisms involved in the cellular action of FAEs will help to adapt and further improve the use of such small molecules for the treatment of psoriasis and other chronic inflammatory diseases.
Subject(s)
Dermatologic Agents/metabolism , Dermatologic Agents/pharmacology , Dimethyl Fumarate/metabolism , Dimethyl Fumarate/pharmacology , Psoriasis/drug therapy , Animals , Cytokines/metabolism , Dermatologic Agents/pharmacokinetics , Dimethyl Fumarate/pharmacokinetics , Down-Regulation/drug effects , Endothelial Cells/drug effects , Glutathione/metabolism , Humans , Keratinocytes/drug effects , Lymphocytes/drug effects , NF-E2-Related Factor 2/metabolism , NF-kappa B/metabolism , Neutrophils/drug effects , Psoriasis/etiology , Psoriasis/metabolism , Receptors, G-Protein-Coupled/metabolism , Signal Transduction/drug effectsABSTRACT
BACKGROUND: Psoriasis is an inflammatory skin disease with aberrant keratinocyte proliferation, presumably as a result of immune cell activation. Th17 cytokines like IL-17A and IL-22 are critically implicated in epidermal thickening, altered keratinocyte differentiation and production of innate factors such as antimicrobial peptides. Psoriasis treatment options include modern targeted therapies using anti-cytokine antibodies and traditional non-targeted treatments like anthralin (dithranol). While the mode of action of anti-cytokine antibodies is defined, the effects of topical anthralin on psoriatic skin are not fully understood. OBJECTIVE: This study aims to unravel the direct effects of anthralin on keratinocyte proliferation, differentiation and production of psoriasis-associated factors. METHODS: We tested the effects of anthralin on cell proliferation, cytokeratin expression and changes in the expression of antimicrobial peptides using primary keratinocytes and 3D psoriasis tissue models with and without stimulation of the psoriasis-promoting cytokines IL-17A and IL-22. Moreover, we compared the findings derived from monolayer and multilayer cultures to data derived from lesional skin of patients with psoriasis before and under treatment with anthralin. RESULTS: Our study shows that anthralin directly induces cell apoptosis in vitro in monolayer cultures but not in 3D psoriasis tissue models treated with IL-17A and IL-22. Yet, keratinocyte proliferation as determined by Ki-67 staining is impaired by anthralin in vivo. In lesional skin but not in 3D psoriasis tissue models anthralin rapidly normalizes cytokeratin (CK)16 expression. Furthermore, anthralin directly inhibits DEFB4 expression in vitro and in vivo, while other antimicrobial peptides and cytokines studied like IL-6 and IL-8 are regulated differently in vitro and in vivo. CONCLUSIONS: Our results show that anthralin directly regulates DEFB4A expression. However, its beneficial effects on psoriasis cannot be explained by direct effects on keratinocyte differentiation or cytokine expression.
Subject(s)
Anthralin/pharmacology , Dermatologic Agents/pharmacology , Keratin-16/metabolism , Keratinocytes/drug effects , Psoriasis/drug therapy , beta-Defensins/metabolism , Administration, Cutaneous , Anthralin/therapeutic use , Apoptosis/drug effects , Biopsy , Cell Differentiation/drug effects , Cell Proliferation/drug effects , Cells, Cultured , Dermatologic Agents/therapeutic use , Fluorescent Antibody Technique , Humans , Interleukin-17/metabolism , Interleukin-6/metabolism , Interleukin-8/metabolism , Interleukins/metabolism , Keratin-10/metabolism , Keratin-5/metabolism , Keratinocytes/metabolism , Ki-67 Antigen/metabolism , Psoriasis/pathology , Skin/cytology , Skin/drug effects , Skin/pathology , Tissue Culture Techniques/methods , Interleukin-22ABSTRACT
BACKGROUND: Atopic dermatitis (AD) is a common inflammatory skin disease with dysfunction of the skin barrier, an abnormal immune response and frequent allergies to environmental antigens like food antigens. Clinical observations suggest that certain diets can influence the course of AD. OBJECTIVE: Here we compared the phenotype of food allergen-specific T cells activated through skin or gut allergen exposure to transfer skin inflammation into naïve recipients upon epicutaenous allergen challenge. METHODS: Ovalbumin (OVA) TCR-transgenic mice were treated epicutaneously with OVA or were fed OVA. CD4+ T cells from skin lymph nodes or mesenteric lymph nodes were transferred into naïve BALB/c mice, which were challenged with OVA epicutaneously. Skin inflammation was determined by histological parameters. In addition, we analyzed the phenotype of the immune response in lymphoid tissues and in skin tissue. RESULTS: TCR-transgenic T cells activated through epicutaneous or oral OVA exposure both migrate to skin lymph nodes after adoptive transfer and epicutaenous OVA exposure. AD-like skin inflammation could only be induced by the transfer of epicutaneously primed OVA T cells. Analysis of the immune phenotype demonstrated an IL-22/IL-17A-dominated immune phenotype of skin-pathogenic T cells. CONCLUSION: IL-22 seems to be the critical cytokine for the development of AD and is induced in this model by epicutaneous sensitization with OVA.
Subject(s)
CD4-Positive T-Lymphocytes/immunology , Dermatitis, Atopic/immunology , Food Hypersensitivity/immunology , Interleukins/immunology , Skin/immunology , Allergens/immunology , Animals , CD4-Positive T-Lymphocytes/metabolism , Disease Models, Animal , Female , Humans , Interleukin-17/immunology , Interleukin-17/metabolism , Interleukins/metabolism , Intestines/immunology , Mice , Mice, Inbred BALB C , Mice, Transgenic , Ovalbumin/genetics , Ovalbumin/immunology , Vaccination/methods , Interleukin-22ABSTRACT
The nutritional curcumin (CUR) is beneficial in cell-mediated autoimmune diseases. The molecular mechanisms underlying this food-mediated silencing of inflammatory immune responses are poorly understood. By investigating antigen-specific immune responses we found that dietary CUR impairs the differentiation of Th1/Th17 cells in vivo during encephalomyelitis and instead promoted Th2 cells. In contrast, feeding CUR had no inhibitory effect on ovalbumin-induced airway inflammation. Mechanistically, we found that CUR induces an anti-inflammatory phenotype in dendritic cells (DC) with enhanced STAT3 phosphorylation and suppressed expression of Il12b and Il23a. On the molecular level CUR readily induced NRF2-sensitive heme oxygenase 1 (HO-1) mRNA and protein in LPS-activated DC. HO-1 enhanced STAT3 phosphorylation, which enriched to Il12b and Il23a loci and negatively regulated their transcription. These findings demonstrate the underlying mechanism through which a nutritional can interfere with the immune response. CUR silences IL-23/Th17-mediated pathology by enhancing HO-1/STAT3 interaction in DC.
Subject(s)
Autoimmune Diseases/drug therapy , Curcumin/administration & dosage , Heme Oxygenase-1/genetics , Inflammation/drug therapy , Interleukin-23/genetics , Membrane Proteins/genetics , STAT3 Transcription Factor/genetics , Animals , Autoimmune Diseases/genetics , Autoimmune Diseases/pathology , Dendritic Cells/drug effects , Encephalomyelitis, Autoimmune, Experimental , Immunity, Cellular/drug effects , Inflammation/chemically induced , Inflammation/genetics , Inflammation/pathology , Mice , Ovalbumin/toxicity , Phosphorylation , Th17 Cells/drug effects , Th2 Cells/drug effects , Th2 Cells/immunologyABSTRACT
T helper (Th) cells producing interleukin (IL)-17, IL-22, and tumor necrosis factor (TNF) form the key T cell population driving psoriasis pathogenesis. They orchestrate the inflammation in the skin that results in the proliferation of keratinocytes and endothelial cells. Besides Th17 cells, other immune cells that are capable of producing IL-17-associated cytokines participate in psoriatic inflammation. Recent advances in psoriasis research improved our understanding of the cellular and molecular players that are involved in Th17 pathology and inflammatory pathways in the skin. The inflammation-driving actions of TNF in psoriasis are already well known and antibodies against TNF are successful in the treatment of Th17-mediated psoriatic skin inflammation. A further key cytokine with potent IL-17-/IL-22-promoting properties is IL-23. Therapeutics directly neutralizing IL-23 or IL-17 itself are now extending the therapeutic spectrum of antipsoriatic agents and further developments are on the way. The enormous progress in psoriasis research allows us to control this Th17-mediated inflammatory skin disease in many patients.
ABSTRACT
Small interfering RNA (siRNA)-based therapies allow targeted correction of molecular defects in distinct cell populations. Although efficient in multiple cell populations, dendritic cells (DCs) seem to resist siRNA delivery. Using fluorescence labeling and radiolabeling, we show that cholesterol modification enables siRNA uptake by DCs in vitro and in vivo. Delivery of cholesterol-modified p40 siRNA selectively abolished p40 transcription and suppressed TLR-triggered p40 production by DCs. During immunization with peptide in CFA, cholesterol-modified p40 siRNA generated p40-deficient, IL-10-producing DCs that prevented IL-17/Th17 and IFN-γ/Th1 responses. Only cholesterol-modified p40-siRNA established protective immunity against experimental autoimmune encephalomyelitis and suppressed IFN-γ and IL-17 expression by CNS-infiltrating mononuclear cells without inducing regulatory T cells. Because cholesterol-modified siRNA can thus modify selected DC functions in vivo, it is intriguing for targeted immune therapy of allergic, autoimmune, or neoplastic diseases.
Subject(s)
Cholesterol/immunology , Dendritic Cells/immunology , Encephalomyelitis, Autoimmune, Experimental/immunology , Interleukin-12 Subunit p40/immunology , RNA, Small Interfering/immunology , Animals , Cholesterol/chemistry , Dendritic Cells/metabolism , Encephalomyelitis, Autoimmune, Experimental/genetics , Encephalomyelitis, Autoimmune, Experimental/therapy , Female , Flow Cytometry , Gene Expression/immunology , Immunization/methods , Interleukin-10/genetics , Interleukin-10/immunology , Interleukin-12 Subunit p40/blood , Interleukin-12 Subunit p40/genetics , Mice, Inbred Strains , Molecular Structure , RNA, Small Interfering/genetics , RNAi Therapeutics/methods , Reverse Transcriptase Polymerase Chain Reaction , Th1 Cells/immunology , Th1 Cells/metabolism , Th17 Cells/immunology , Th17 Cells/metabolism , Treatment OutcomeABSTRACT
T cells are key players in inflammation, autoimmune diseases, and immunotherapy. Thus, holistic and noninvasive in vivo characterizations of the temporal distribution and homing dynamics of lymphocytes in mammals are of special interest. Herein, we show that PET-based T-cell labeling facilitates quantitative, highly sensitive, and holistic monitoring of T-cell homing patterns in vivo. We developed a new T-cell receptor (TCR)-specific labeling approach for the intracellular labeling of mouse T cells. We found that continuous TCR plasma membrane turnover and the endocytosis of the specific (64)Cu-monoclonal antibody (mAb)-TCR complex enables a stable labeling of T cells. The TCR-mAb complex was internalized within 24 h, whereas antigen recognition was not impaired. Harmful effects of the label on the viability, DNA-damage and apoptosis-necrosis induction, could be minimized while yielding a high contrast in in vivo PET images. We were able to follow and quantify the specific homing of systemically applied (64)Cu-labeled chicken ovalbumin (cOVA)-TCR transgenic T cells into the pulmonary and perithymic lymph nodes (LNs) of mice with cOVA-induced airway delayed-type hypersensitivity reaction (DTHR) but not into pulmonary and perithymic LNs of naïve control mice or mice diseased from turkey or pheasant OVA-induced DTHR. Our protocol provides consequent advancements in the detection of small accumulations of immune cells in single LNs and specific homing to the sites of inflammation by PET using the internalization of TCR-specific mAbs as a specific label of T cells. Thus, our labeling approach is applicable to other cells with constant membrane receptor turnover.
Subject(s)
Antibodies, Monoclonal/pharmacology , Positron-Emission Tomography/methods , Receptors, Antigen/antagonists & inhibitors , T-Lymphocytes/diagnostic imaging , Animals , Apoptosis/immunology , Copper Radioisotopes , DNA Damage/immunology , Mice , Radioactive Tracers , Radiography , Receptors, Antigen/immunology , T-Lymphocytes/immunologyABSTRACT
Psoriasis is a T helper (Th)17/Th1-mediated autoimmune disease affecting the skin and joints. So far, distinct traditional oral compounds and modern biologics have been approved in most countries for the treatment of patients with moderate to severe psoriasis or psoriatic arthritis. Yet, the anti-psoriatic therapeutic spectrum is to be extended by a number of novel targeted therapies including biologics and modern oral compounds. The next set of anti-psoriatic biologics targets mainly Th17-associated cytokines such as IL-17 or IL-23. In contrast, modern oral anti-psoriatics, such as dimethyl fumarate (DMF), apremilast or Janus kinase (JAK) inhibitors interfere with intracellular proteins and affect signaling pathways. Here we summarize the current systemic therapies for psoriasis and their immunological mechanism. The recent advances in psoriasis therapy will help treat our patients efficiently and complete our understanding of disease pathogenesis.
ABSTRACT
Sulforaphane (SFN), an isothiocyanate, is part of an important group of naturally occurring small molecules with anti-inflammatory properties. The published reports are best conceivable with an inhibition of T cell function, but the mode of action remains unknown. We therefore analyzed the effect of SFN on T cell-mediated autoimmune disease. Feeding mice with SFN protected from severe experimental autoimmune encephalomyelitis. Disease amelioration was associated with reduced IL-17 and IFN-γ expression in draining lymph nodes. In vitro, SFN treatment of T cells did not directly alter T cell cytokine secretion. In contrast, SFN treatment of dendritic cells (DCs) inhibited TLR4-induced IL-12 and IL-23 production, and severely suppressed Th1 and Th17 development of T cells primed by SFN-treated DCs. SFN regulated the activity of the TLR4-induced transcription factor NF-κB, without affecting the degradation of its inhibitor IκB-α. Instead, SFN treatment of DCs resulted in strong expression of the stress response protein heme oxygenase-1 (HO-1), which interacts with and thereby inhibits NF-κB p65. Consistent with these findings, HO-1 bound to p65 and subsequently inhibited the p65 activity at the IL23a and IL12b promoters. Importantly, SFN suppressed Il23a and Il12b expression in vivo and silenced Th17/Th1 responses within the CNS. Thus, our data show that SFN improves Th17/Th1-mediated autoimmune disease by inducing HO-1 and inhibiting NF-κB p65-regulated IL-23 and IL-12 expression.
Subject(s)
Autoimmune Diseases/immunology , Autoimmune Diseases/metabolism , Interleukin-12/metabolism , Interleukin-23/metabolism , Isothiocyanates/pharmacology , T-Lymphocyte Subsets/immunology , T-Lymphocyte Subsets/metabolism , Animals , Autoimmune Diseases/prevention & control , Cell Differentiation/drug effects , Cluster Analysis , Cytokines/biosynthesis , DNA/metabolism , Dendritic Cells/drug effects , Dendritic Cells/immunology , Dendritic Cells/metabolism , Encephalomyelitis, Autoimmune, Experimental/immunology , Encephalomyelitis, Autoimmune, Experimental/metabolism , Encephalomyelitis, Autoimmune, Experimental/prevention & control , Female , Gene Expression Profiling , Gene Expression Regulation/drug effects , Heme Oxygenase-1/metabolism , Interleukin-12/genetics , Interleukin-23/genetics , Isothiocyanates/administration & dosage , Lymphocyte Activation/drug effects , Lymphocyte Activation/immunology , Mice , NF-kappa B/metabolism , Phenotype , Protein Binding/drug effects , Sulfoxides , T-Lymphocyte Subsets/drug effects , Th1 Cells/cytology , Th1 Cells/drug effects , Th1 Cells/immunology , Th1 Cells/metabolism , Th17 Cells/cytology , Th17 Cells/drug effects , Th17 Cells/immunology , Th17 Cells/metabolismABSTRACT
Sepsis, sepsis-induced hyperinflammation and subsequent sepsis-associated immunosuppression (SAIS) are important causes of death. Here we show in humans that the loss of the major reactive oxygen species (ROS) scavenger, glutathione (GSH), during SAIS directly correlates with an increase in the expression of activating transcription factor 3 (ATF3). In endotoxin-stimulated monocytes, ROS stress strongly superinduced NF-E2-related factor 2 (NRF2)-dependent ATF3. In vivo, this ROS-mediated superinduction of ATF3 protected against endotoxic shock by inhibiting innate cytokines, as Atf3(-/-) mice remained susceptible to endotoxic shock even under conditions of ROS stress. Although it protected against endotoxic shock, this ROS-mediated superinduction of ATF3 caused high susceptibility to bacterial and fungal infections through the suppression of interleukin 6 (IL-6). As a result, Atf3(-/-) mice were protected against bacterial and fungal infections, even under conditions of ROS stress, whereas Atf3(-/-)Il6(-/-) mice were highly susceptible to these infections. Moreover, in a model of SAIS, secondary infections caused considerably less mortality in Atf3(-/-) mice than in wild-type mice, indicating that ROS-induced ATF3 crucially determines susceptibility to secondary infections during SAIS.
Subject(s)
Activating Transcription Factor 3/metabolism , Coinfection/immunology , Glutathione/metabolism , Immune Tolerance , Interleukin-6/metabolism , Reactive Oxygen Species/metabolism , Shock, Septic/immunology , Activating Transcription Factor 3/genetics , Animals , Coinfection/microbiology , Female , Gene Expression Regulation , Glutathione/blood , Humans , Interleukin-6/genetics , Macrophages, Peritoneal/immunology , Mice , Mice, Inbred C57BL , Monocytes/metabolism , NF-E2-Related Factor 2/metabolism , Oxidative Stress , Reactive Oxygen Species/blood , Shock, Septic/metabolism , Signal TransductionABSTRACT
Fumarates improve multiple sclerosis (MS) and psoriasis, two diseases in which both IL-12 and IL-23 promote pathogenic T helper (Th) cell differentiation. However, both diseases show opposing responses to most established therapies. First, we show in humans that fumarate treatment induces IL-4-producing Th2 cells in vivo and generates type II dendritic cells (DCs) that produce IL-10 instead of IL-12 and IL-23. In mice, fumarates also generate type II DCs that induce IL-4-producing Th2 cells in vitro and in vivo and protect mice from experimental autoimmune encephalomyelitis. Type II DCs result from fumarate-induced glutathione (GSH) depletion, followed by increased hemoxygenase-1 (HO-1) expression and impaired STAT1 phosphorylation. Induced HO-1 is cleaved, whereupon the N-terminal fragment of HO-1 translocates into the nucleus and interacts with AP-1 and NF-κB sites of the IL-23p19 promoter. This interaction prevents IL-23p19 transcription without affecting IL-12p35, whereas STAT1 inactivation prevents IL-12p35 transcription without affecting IL-23p19. As a consequence, GSH depletion by small molecules such as fumarates induces type II DCs in mice and in humans that ameliorate inflammatory autoimmune diseases. This therapeutic approach improves Th1- and Th17-mediated autoimmune diseases such as psoriasis and MS by interfering with IL-12 and IL-23 production.
Subject(s)
Dendritic Cells/immunology , Fumarates/immunology , Fumarates/therapeutic use , Multiple Sclerosis/drug therapy , Multiple Sclerosis/immunology , Psoriasis/drug therapy , Psoriasis/immunology , Animals , Cell Differentiation/immunology , Dendritic Cells/cytology , Dendritic Cells/drug effects , Encephalomyelitis, Autoimmune, Experimental/immunology , Female , Heme Oxygenase-1/metabolism , Humans , Interleukin-12/immunology , Interleukin-23/immunology , Macrophages/immunology , Mice , NIH 3T3 Cells , Promoter Regions, Genetic , Reactive Oxygen Species/metabolism , Signal Transduction/immunology , T-Lymphocytes/cytology , T-Lymphocytes/immunology , Transcription Factor RelA/metabolismABSTRACT
The balance between GSH-levels and oxidative stress is critical for cell survival. The GSH-levels of erythrocytes are dramatically decreased during infection with Plasmodium spp. We therefore investigated the consequences of targeting GSH for erythrocyte and Plasmodium survival in vitro and in vivo using dimethylfumarate (DMF) at therapeutically established dosage. We first show that noninfected red blood cells (RBC) exposed to DMF undergo changes typical of apoptosis or eryptosis, such as cell shrinkage and cell membrane scrambling with subsequent phosphatidylserine (PS) exposure. DMF did not induce appreciable hemolysis. DMF-triggered PS exposure was mediated by intracellular GSH depletion and reversed by the antioxidative N-acetyl-l-cysteine. DMF treatment controlled intraerythrocyte DNA amplification and in vitro parasitemia of Plasmodium falciparum-infected RBC. In vivo, DMF treatment had no effect on RBC count or GSH levels in noninfected mice. Consistent with its effects on infected RBC, DMF treatment abrogated parasitemia and enhanced the survival of mice infected with Plasmodium berghei from 0% to 60%. In conclusion, DMF sensitizes the erythrocytes to the effect of Plasmodium infection on PS exposure, thus accelerating the clearance of infected erythrocytes. Accordingly, DMF treatment favorably influences the clinical course of malaria. As DMF targets mechanisms within the host cell, it is not likely to generate resistance of the pathogen.
Subject(s)
Erythrocyte Membrane/drug effects , Fumarates , Glutathione/metabolism , Immunosuppressive Agents , Malaria , Animals , Antioxidants/metabolism , Dimethyl Fumarate , Erythrocyte Membrane/chemistry , Erythrocyte Membrane/metabolism , Erythrocytes/cytology , Erythrocytes/drug effects , Erythrocytes/metabolism , Erythrocytes/parasitology , Fumarates/pharmacology , Fumarates/therapeutic use , Humans , Immunosuppressive Agents/pharmacology , Immunosuppressive Agents/therapeutic use , Malaria/blood , Malaria/drug therapy , Mice , Phosphatidylserines/metabolism , Plasmodium/drug effects , Plasmodium/metabolism , Plasmodium/parasitologyABSTRACT
Experimental evidence has suggested a role for CD8+ cytotoxic T lymphocytes (CTL) in the pathophysiology of vitiligo, a pigmentation disorder with focal loss of melanocytes in the skin. The discovery of tyrosinase-related protein 2 (TRP2) as a model melanocytic self-antigen recognized by CD8+ CTL in C57BL/6 mice allowed us to analyze the requirements for CD8+ T cell-mediated autoimmune destruction of melanocytes in an experimental model. Using two different genetic methods for the induction of cellular immunity in vivo, gene gun bombardment of the skin and injection of recombinant adenovirus, we show that peripheral tolerance of CD8+ T cells recognizing a single TRP2-derived H2-Kb-binding peptide is regulated in two steps. In the induction phase, stimulation and expansion of TRP2-specific CD8+ T cells in vivo depend on CD4+ T cell help. In the effector phase, autoimmune destruction of melanocytes in the skin depends on local inflammation. Our results suggest that accidental stimulation of CD8+ CTL recognizing major histocompatibility complex class I-binding peptides derived from melanocytic proteins in the context of an inflammatory skin disease may play an important role in the pathophysiology of vitiligo.
Subject(s)
Autoantigens/immunology , CD4-Positive T-Lymphocytes/immunology , CD8-Positive T-Lymphocytes/immunology , Melanocytes/immunology , Vitiligo/immunology , Vitiligo/physiopathology , Animals , Binding, Competitive/immunology , Biolistics , Dermatitis/immunology , Dermatitis/physiopathology , Epitopes/immunology , Epitopes/metabolism , Green Fluorescent Proteins/genetics , Immune Tolerance/immunology , Intramolecular Oxidoreductases/genetics , Intramolecular Oxidoreductases/immunology , Mice , Mice, Inbred C57BL , Mice, Mutant Strains , Plasmids , Recombinant Fusion Proteins/geneticsABSTRACT
BACKGROUND: A genetic melanoma vaccine consisting of cDNA encoding the model self-antigen tyrosinase-related protein 2 (TRP2) fused in-frame to the immunogenic enhanced green fluorescent protein (EGFP) was able to break immune tolerance and stimulate CD8+ T cells in vivo. In the present study we investigated whether alteration of the intracellular antigen localization as a result of the linkage with immune-enhancing helper proteins affects the resulting immune response. METHODS: Expression plasmids and recombinant adenoviruses were constructed encoding various fusion proteins with different intracellular sorting signals which direct the antigen to the cytosol, the endoplasmic reticulum or the endosomal compartments. Genetic immunization of C57BL/6 mice was performed with all constructs using particle-bombardment of the skin and injection of recombinant adenoviruses. The resulting immune response was analyzed using ELISPOT and tumor rejection assays. RESULTS: Induction of TRP2-specific CD8+ T cells in vivo and autoimmune-mediated destruction of melanocytes in the bombarded area of the skin were observed with all constructs expressing fusion proteins between TRP2 and EGFP. Importantly, injections of the different recombinant adenoviruses all mediated protective immunity against transplanted B16 melanoma cells. CONCLUSIONS: Altered intracellular sorting signals do not significantly influence the efficacy of genetic melanoma vaccines incorporating helper determinants in our model system. These results further support the concept that linkage of immunogenic helper sequences can be successfully applied for antigen-specific immunotherapy of melanoma and provide a scientific basis for the translation of this strategy in future clinical applications.
Subject(s)
Cancer Vaccines/administration & dosage , Genetic Therapy/methods , Intramolecular Oxidoreductases/immunology , Melanoma, Experimental/therapy , Recombinant Fusion Proteins/pharmacokinetics , T-Lymphocytes, Helper-Inducer/immunology , Animals , Autoimmunity , CD4 Antigens/genetics , Genetic Vectors/administration & dosage , Green Fluorescent Proteins/genetics , Green Fluorescent Proteins/immunology , Immunization , Intramolecular Oxidoreductases/genetics , Melanoma, Experimental/immunology , Melanoma, Experimental/prevention & control , Mice , Mice, Inbred C57BL , Protein Transport , Recombinant Fusion Proteins/genetics , Recombinant Fusion Proteins/immunology , Skin Neoplasms/immunology , Skin Neoplasms/prevention & control , Skin Neoplasms/therapyABSTRACT
Long-lasting, high-level gene expression in the absence of a toxic or inflammatory response to viral Ags is necessary for the successful application of genetically modified dendritic cell (DC). We previously demonstrated that efficient transduction of mature DC using DeltaE1DeltaE3 adenoviruses suppressed their stimulatory capacity for T cells. The current study was designed to investigate in more detail the suppressive effect of Ad-DC. We demonstrate that immunosuppression is not mediated by alterations in the T cell phenotype or cytokine profiles released by stimulated T cells. Also DC phenotypes are not affected. However, we demonstrate a cell cycle arrest of the T cell population stimulated by adenovirally transduced DC. Surprisingly, only freshly transduced DC are perturbed in their stimulatory capacity. Experiments using cycloheximide to block early intracellular viral gene expression showed that viral genes expressed in DC are responsible for this transient immunosuppression. In agreement with these findings, high-capacity (gutless) Ad-vectors that differ in viral gene expression from conventional DeltaE1DeltaE3 adenovirus are suitable for an efficient transduction of human DC. DC transduced with gutless Ad-vectors showed a high allostimulatory capacity for CD4(+) and CD8(+) T cells. Thus, the immunosuppressive effect of DeltaE1DeltaE3 Ad-transduced mature DC seems to be the result of early viral gene expression in DC that can be prevented using gutless Ad-vectors for transduction. These results have important implications for the use of genetically modified DC for therapeutic application.