RESUMO
Trained innate immunity, induced via modulation of mature myeloid cells or their bone marrow progenitors, mediates sustained increased responsiveness to secondary challenges. Here, we investigated whether anti-tumor immunity can be enhanced through induction of trained immunity. Pre-treatment of mice with ß-glucan, a fungal-derived prototypical agonist of trained immunity, resulted in diminished tumor growth. The anti-tumor effect of ß-glucan-induced trained immunity was associated with transcriptomic and epigenetic rewiring of granulopoiesis and neutrophil reprogramming toward an anti-tumor phenotype; this process required type I interferon signaling irrespective of adaptive immunity in the host. Adoptive transfer of neutrophils from ß-glucan-trained mice to naive recipients suppressed tumor growth in the latter in a ROS-dependent manner. Moreover, the anti-tumor effect of ß-glucan-induced trained granulopoiesis was transmissible by bone marrow transplantation to recipient naive mice. Our findings identify a novel and therapeutically relevant anti-tumor facet of trained immunity involving appropriate rewiring of granulopoiesis.
Assuntos
Granulócitos/imunologia , Imunidade Inata , Neoplasias/imunologia , Imunidade Adaptativa , Transferência Adotiva , Animais , Epigênese Genética , Interferon Tipo I/metabolismo , Camundongos Endogâmicos C57BL , Monócitos/metabolismo , Neoplasias/patologia , Neutrófilos/metabolismo , Fenótipo , Receptor de Interferon alfa e beta/deficiência , Receptor de Interferon alfa e beta/metabolismo , Transcrição Gênica , Transcriptoma/genética , beta-Glucanas/metabolismoRESUMO
Interleukin-17A (IL-17A) is a key mediator of protective immunity to yeast and bacterial infections but also drives the pathogenesis of several autoimmune diseases, such as psoriasis or psoriatic arthritis. Here we show that the tetra-transmembrane protein CMTM4 is a subunit of the IL-17 receptor (IL-17R). CMTM4 constitutively associated with IL-17R subunit C to mediate its stability, glycosylation and plasma membrane localization. Both mouse and human cell lines deficient in CMTM4 were largely unresponsive to IL-17A, due to their inability to assemble the IL-17R signaling complex. Accordingly, CMTM4-deficient mice had a severe defect in the recruitment of immune cells following IL-17A administration and were largely resistant to experimental psoriasis, but not to experimental autoimmune encephalomyelitis. Collectively, our data identified CMTM4 as an essential component of IL-17R and a potential therapeutic target for treating IL-17-mediated autoimmune diseases.
Assuntos
Artrite Psoriásica , Encefalomielite Autoimune Experimental , Psoríase , Humanos , Camundongos , Animais , Receptores de Interleucina-17/genética , Receptores de Interleucina-17/metabolismo , Interleucina-17/metabolismo , Encefalomielite Autoimune Experimental/genética , Proteínas com Domínio MARVEL/genéticaRESUMO
T cells are pivotal in the adaptive immune defense, necessitating a delicate balance between robust response against infections and self-tolerance. Their activation involves intricate cross-talk among signaling pathways triggered by the T-cell antigen receptors (TCR) and co-stimulatory or inhibitory receptors. The molecular regulation of these complex signaling networks is still incompletely understood. Here, we identify the adaptor protein ABIN1 as a component of the signaling complexes of GITR and OX40 co-stimulation receptors. T cells lacking ABIN1 are hyper-responsive ex vivo, exhibit enhanced responses to cognate infections, and superior ability to induce experimental autoimmune diabetes in mice. ABIN1 negatively regulates p38 kinase activation and late NF-κB target genes. P38 is at least partially responsible for the upregulation of the key effector proteins IFNG and GZMB in ABIN1-deficient T cells after TCR stimulation. Our findings reveal the intricate role of ABIN1 in T-cell regulation.
Assuntos
Proteínas Adaptadoras de Transdução de Sinal , Transdução de Sinais , Linfócitos T Citotóxicos , Animais , Humanos , Camundongos , Proteínas Adaptadoras de Transdução de Sinal/metabolismo , Proteínas Adaptadoras de Transdução de Sinal/genética , Diabetes Mellitus Tipo 1/imunologia , Diabetes Mellitus Tipo 1/genética , Diabetes Mellitus Tipo 1/metabolismo , Proteína Relacionada a TNFR Induzida por Glucocorticoide , Interferon gama/metabolismo , Ativação Linfocitária/imunologia , Ativação Linfocitária/genética , Camundongos Endogâmicos C57BL , Camundongos Knockout , NF-kappa B/metabolismo , Proteínas Quinases p38 Ativadas por Mitógeno/metabolismo , Receptores de Antígenos de Linfócitos T/metabolismo , Receptores OX40/metabolismo , Receptores OX40/genética , Linfócitos T Citotóxicos/imunologia , Linfócitos T Citotóxicos/metabolismoRESUMO
Ag-inexperienced memory-like T (AIMT) cells are functionally unique T cells, representing one of the two largest subsets of murine CD8+ T cells. However, differences between laboratory inbred strains, insufficient data from germ-free mice, a complete lack of data from feral mice, and an unclear relationship between AIMT cells formation during aging represent major barriers for better understanding of their biology. We performed a thorough characterization of AIMT cells from mice of different genetic background, age, and hygienic status by flow cytometry and multiomics approaches, including analyses of gene expression, TCR repertoire, and microbial colonization. Our data showed that AIMT cells are steadily present in mice, independent of their genetic background and hygienic status. Despite differences in their gene expression profiles, young and aged AIMT cells originate from identical clones. We identified that CD122 discriminates two major subsets of AIMT cells in a strain-independent manner. Whereas thymic CD122LOW AIMT cells (innate memory) prevail only in young animals with high thymic IL-4 production, peripheral CD122HIGH AIMT cells (virtual memory) dominate in aged mice. Cohousing with feral mice changed the bacterial colonization of laboratory strains but had only minimal effects on the CD8+ T cell compartment, including AIMT cells.
Assuntos
Envelhecimento/genética , Antígenos/genética , Memória Imunológica/genética , Linfócitos T/imunologia , Envelhecimento/imunologia , Animais , Antígenos/imunologia , Evolução Clonal , Instabilidade Genômica , Memória Imunológica/imunologia , Camundongos , Camundongos Endogâmicos BALB C , Camundongos Endogâmicos C57BL , Camundongos Transgênicos , FenótipoRESUMO
Leukocytes are rapidly recruited to sites of inflammation via interactions with the vascular endothelium. The steroid hormone dehydroepiandrosterone (DHEA) exerts anti-inflammatory properties; however, the underlying mechanisms are poorly understood. In this study, we show that an anti-inflammatory mechanism of DHEA involves the regulation of developmental endothelial locus 1 (DEL-1) expression. DEL-1 is a secreted homeostatic factor that inhibits ß2-integrin-dependent leukocyte adhesion, and the subsequent leukocyte recruitment and its expression is downregulated upon inflammation. Similarly, DHEA inhibited leukocyte adhesion to the endothelium in venules of the inflamed mouse cremaster muscle. Importantly, in a model of lung inflammation, DHEA limited neutrophil recruitment in a DEL-1-dependent manner. Mechanistically, DHEA counteracted the inhibitory effect of inflammation on DEL-1 expression. Indeed, whereas TNF reduced DEL-1 expression and secretion in endothelial cells by diminishing C/EBPß binding to the DEL-1 gene promoter, DHEA counteracted the inhibitory effect of TNF via activation of tropomyosin receptor kinase A (TRKA) and downstream PI3K/AKT signaling that restored C/EBPß binding to the DEL-1 promoter. In conclusion, DHEA restrains neutrophil recruitment by reversing inflammation-induced downregulation of DEL-1 expression. Therefore, the anti-inflammatory DHEA/DEL-1 axis could be harnessed therapeutically in the context of inflammatory diseases.
Assuntos
Proteínas de Ligação ao Cálcio/imunologia , Moléculas de Adesão Celular/imunologia , Desidroepiandrosterona/farmacologia , Leucócitos/imunologia , Transdução de Sinais/imunologia , Animais , Proteína beta Intensificadora de Ligação a CCAAT/imunologia , Antígenos CD18/imunologia , Adesão Celular/imunologia , Endotélio Vascular/imunologia , Feminino , Regulação da Expressão Gênica/imunologia , Leucócitos/citologia , Camundongos , Fosfatidilinositol 3-Quinases/imunologia , Regiões Promotoras Genéticas/imunologia , Proteínas Proto-Oncogênicas c-akt/imunologia , Receptor trkA/imunologiaRESUMO
Endogenous steroid hormones, especially glucocorticoids and mineralocorticoids, derive from the adrenal cortex, and drastic or sustained changes in their circulatory levels affect multiple organ systems. Although hypoxia signaling in steroidogenesis has been suggested, knowledge on the true impact of the HIFs (Hypoxia-Inducible Factors) in the adrenocortical cells of vertebrates is scant. By creating a unique set of transgenic mouse lines, we reveal a prominent role for HIF1α in the synthesis of virtually all steroids in vivo. Specifically, mice deficient in HIF1α in adrenocortical cells displayed enhanced levels of enzymes responsible for steroidogenesis and a cognate increase in circulatory steroid levels. These changes resulted in cytokine alterations and changes in the profile of circulatory mature hematopoietic cells. Conversely, HIF1α overexpression resulted in the opposite phenotype of insufficient steroid production due to impaired transcription of necessary enzymes. Based on these results, we propose HIF1α to be a vital regulator of steroidogenesis as its modulation in adrenocortical cells dramatically impacts hormone synthesis with systemic consequences. In addition, these mice can have potential clinical significances as they may serve as essential tools to understand the pathophysiology of hormone modulations in a number of diseases associated with metabolic syndrome, auto-immunity or even cancer.
Assuntos
Glândulas Suprarrenais/metabolismo , Regulação da Expressão Gênica , Subunidade alfa do Fator 1 Induzível por Hipóxia/metabolismo , Prolina Dioxigenases do Fator Induzível por Hipóxia/metabolismo , Esteroides/biossíntese , Animais , Feminino , Subunidade alfa do Fator 1 Induzível por Hipóxia/genética , Prolina Dioxigenases do Fator Induzível por Hipóxia/genética , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Transdução de SinaisRESUMO
A hallmark of proliferative retinopathies, such as retinopathy of prematurity (ROP), is a pathological neovascularization orchestrated by hypoxia and the resulting hypoxia-inducible factor (HIF)-dependent response. We studied the role of Hif2α in hematopoietic cells for pathological retina neovascularization in the murine model of ROP, the oxygen-induced retinopathy (OIR) model. Hematopoietic-specific deficiency of Hif2α ameliorated pathological neovascularization in the OIR model, which was accompanied by enhanced endothelial cell apoptosis. That latter finding was associated with up-regulation of the apoptosis-inducer FasL in Hif2α-deficient microglia. Consistently, pharmacological inhibition of the FasL reversed the reduced pathological neovascularization from hematopoietic-specific Hif2α deficiency. Our study found that the hematopoietic cell Hif2α contributes to pathological retina angiogenesis. Our findings not only provide novel insights regarding the complex interplay between immune cells and endothelial cells in hypoxia-driven retina neovascularization but also may have therapeutic implications for proliferative retinopathies.-Korovina, I., Neuwirth, A., Sprott, D., Weber, S., Sardar Pasha, S. P. B., Gercken, B., Breier, G., El-Armouche, A., Deussen, A., Karl, M. O., Wielockx, B., Chavakis, T., Klotzsche-von Ameln, A. Hematopoietic hypoxia-inducible factor 2α deficiency ameliorates pathological retinal neovascularization via modulation of endothelial cell apoptosis.
Assuntos
Apoptose/fisiologia , Fatores de Transcrição Hélice-Alça-Hélice Básicos/fisiologia , Células da Medula Óssea/metabolismo , Medula Óssea/metabolismo , Endotélio Vascular/patologia , Neovascularização Patológica , Vasos Retinianos/patologia , Proteína ADAM17/metabolismo , Animais , Fatores de Transcrição Hélice-Alça-Hélice Básicos/genética , Linhagem Celular Transformada , Modelos Animais de Doenças , Proteína Ligante Fas/metabolismo , Camundongos , Camundongos Knockout , Microglia/metabolismo , Retinopatia da Prematuridade/metabolismo , Retinopatia da Prematuridade/patologiaRESUMO
BACKGROUND & AIMS: Autoimmune polyendocrinopathy candidiasis ectodermal dystrophy (APECED) is an autoimmune disorder characterized by chronic mucocutaneous candidiasis, hypoparathyroidism, and adrenal insufficiency, but patients also develop intestinal disorders. APECED is an autosomal recessive disorder caused by mutations in the autoimmune regulator (AIRE, which regulates immune tolerance) that allow self-reactive T cells to enter the periphery. Enteric α-defensins are antimicrobial peptides secreted by Paneth cells. Patients with APECED frequently have gastrointestinal symptoms and seroreactivity against secretory granules of Paneth cells. We investigated whether enteric α-defensins are autoantigens in humans and mice with AIRE deficiency. METHODS: We analyzed clinical data, along with serum and stool samples and available duodenal biopsies from 50 patients with APECED collected from multiple centers in Europe. Samples were assessed for expression of defensins and other molecules by quantitative reverse transcription polymerase chain reaction and flow cytometry; levels of antibodies and other proteins were measured by immunohistochemical and immunoblot analyses. Histologic analyses were performed on biopsy samples. We used Aire(-/-) mice as a model of APECED, and studied the effects of transferring immune cells from these mice to athymic mice. RESULTS: Enteric defensins were detected in extraintestinal tissues of patients with APECED, especially in medullary thymic epithelial cells. Some patients with APECED lacked Paneth cells and were seropositive for defensin-specific autoantibodies; the presence of autoantibodies correlated with frequent diarrhea. Aire(-/-) mice developed defensin-specific T cells. Adoptive transfer of these T cells to athymic mice resulted in T-cell infiltration of the gut, loss of Paneth cells, microbial dysbiosis, and the induction of T-helper 17 cell-mediated autoimmune responses resembling those observed in patients with APECED. CONCLUSIONS: In patients with APECED, loss of AIRE appears to cause an autoimmune response against enteric defensins and loss of Paneth cells. Aire(-/-) mice developed defensin-specific T cells that cause intestinal defects similar to those observed in patients with APECED. These findings provide a mechanism by which loss of AIRE-mediated immune tolerance leads to intestinal disorders in patients with APECED.
Assuntos
Autoimunidade , Intestinos/imunologia , Poliendocrinopatias Autoimunes/imunologia , Fatores de Transcrição/genética , alfa-Defensinas/imunologia , Adolescente , Adulto , Idoso , Animais , Criança , Pré-Escolar , Feminino , Humanos , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Pessoa de Meia-Idade , Poliendocrinopatias Autoimunes/complicações , Linfócitos T/imunologia , Adulto Jovem , Proteína AIRERESUMO
Inflammation in the central nervous system (CNS) and disruption of its immune privilege are major contributors to the pathogenesis of multiple sclerosis (MS) and of its rodent counterpart, experimental autoimmune encephalomyelitis (EAE). We have previously identified developmental endothelial locus-1 (Del-1) as an endogenous anti-inflammatory factor, which inhibits integrin-dependent leukocyte adhesion. Here we show that Del-1 contributes to the immune privilege status of the CNS. Intriguingly, Del-1 expression decreased in chronic-active MS lesions and in the inflamed CNS in the course of EAE. Del-1-deficiency was associated with increased EAE severity, accompanied by increased demyelination and axonal loss. As compared with control mice, Del-1(-/-) mice displayed enhanced disruption of the blood-brain barrier and increased infiltration of neutrophil granulocytes in the spinal cord in the course of EAE, accompanied by elevated levels of inflammatory cytokines, including interleukin-17 (IL-17). The augmented levels of IL-17 in Del-1-deficiency derived predominantly from infiltrated CD8(+) T cells. Increased EAE severity and neutrophil infiltration because of Del-1-deficiency was reversed in mice lacking both Del-1 and IL-17 receptor, indicating a crucial role for the IL-17/neutrophil inflammatory axis in EAE pathogenesis in Del-1(-/-) mice. Strikingly, systemic administration of Del-1-Fc ameliorated clinical relapse in relapsing-remitting EAE. Therefore, Del-1 is an endogenous homeostatic factor in the CNS protecting from neuroinflammation and demyelination. Our findings provide mechanistic underpinnings for the previous implication of Del-1 as a candidate MS susceptibility gene and suggest that Del-1-centered therapeutic approaches may be beneficial in neuroinflammatory and demyelinating disorders.
Assuntos
Axônios/metabolismo , Barreira Hematoencefálica/metabolismo , Proteínas de Transporte/metabolismo , Bainha de Mielina/metabolismo , Neuroimunomodulação/fisiologia , Medula Espinal/metabolismo , Animais , Axônios/efeitos dos fármacos , Axônios/patologia , Barreira Hematoencefálica/efeitos dos fármacos , Barreira Hematoencefálica/patologia , Proteínas de Ligação ao Cálcio , Permeabilidade Capilar/efeitos dos fármacos , Permeabilidade Capilar/fisiologia , Proteínas de Transporte/genética , Moléculas de Adesão Celular , Encefalomielite Autoimune Experimental/tratamento farmacológico , Encefalomielite Autoimune Experimental/metabolismo , Encefalomielite Autoimune Experimental/patologia , Feminino , Granulócitos/efeitos dos fármacos , Granulócitos/metabolismo , Granulócitos/patologia , Homeostase/efeitos dos fármacos , Homeostase/fisiologia , Peptídeos e Proteínas de Sinalização Intercelular , Interleucina-17/metabolismo , Camundongos Endogâmicos C57BL , Camundongos Knockout , Bainha de Mielina/efeitos dos fármacos , Bainha de Mielina/patologia , Neuroimunomodulação/efeitos dos fármacos , Neutrófilos/efeitos dos fármacos , Neutrófilos/metabolismo , Neutrófilos/patologia , Receptores de Interleucina-17/genética , Receptores de Interleucina-17/metabolismo , Índice de Gravidade de Doença , Medula Espinal/efeitos dos fármacos , Medula Espinal/patologiaRESUMO
Formyl peptide receptor 2 (FPR2) is a receptor for formylated peptides and specific pro-resolving mediators, and is involved in various inflammatory processes. Here, we aimed to elucidate the role of FPR2 in dendritic cell (DC) function and autoimmunity-related central nervous system (CNS) inflammation by using the experimental autoimmune encephalomyelitis (EAE) model. EAE induction was accompanied by increased Fpr2 mRNA expression in the spinal cord. FPR2-deficient (Fpr2 KO) mice displayed delayed onset of EAE compared to wild-type (WT) mice, associated with reduced frequencies of Th17 cells in the inflamed spinal cord at the early stage of the disease. However, FPR2 deficiency did not affect EAE severity after the disease reached its peak. FPR2 deficiency in mature DCs resulted in decreased expression of Th17 polarizing cytokines IL6, IL23p19, IL1ß, and thereby diminished the DC-mediated activation of Th17 cell differentiation. LPS-activated FPR2-deficient DCs showed upregulated Nos2 expression and nitric oxide (NO) production, as well as reduced oxygen consumption rate and impaired mitochondrial function, including decreased mitochondrial superoxide levels, lower mitochondrial membrane potential and diminished expression of genes related to the tricarboxylic acid cycle and genes related to the electron transport chain, as compared to WT DCs. Treatment with a NO inhibitor reversed the reduced Th17 cell differentiation in the presence of FPR2-deficient DCs. Together, by regulating DC metabolism, FPR2 enhances the production of DC-derived Th17-polarizing cytokines and hence Th17 cell differentiation in the context of neuroinflammation.
Assuntos
Diferenciação Celular , Células Dendríticas , Encefalomielite Autoimune Experimental , Camundongos Knockout , Receptores de Formil Peptídeo , Células Th17 , Animais , Células Dendríticas/imunologia , Células Dendríticas/metabolismo , Receptores de Formil Peptídeo/genética , Receptores de Formil Peptídeo/metabolismo , Células Th17/imunologia , Células Th17/metabolismo , Camundongos , Encefalomielite Autoimune Experimental/imunologia , Encefalomielite Autoimune Experimental/metabolismo , Camundongos Endogâmicos C57BL , Citocinas/metabolismo , Doenças Neuroinflamatórias/imunologia , Doenças Neuroinflamatórias/metabolismo , Feminino , Medula Espinal/imunologia , Medula Espinal/metabolismoRESUMO
Type 1 diabetes (T1D) is an autoimmune disease caused by T-cell mediated destruction of pancreatic beta cells. Recently, small cationic α-defensin molecules have been implicated in the pathogenesis of certain inflammatory and autoimmune diseases. The purpose of this study was to assess the α-defensin expression in patients with T1D and elucidate the cellular source of their production. Our results show that 30% of patients exhibit increased levels of α-defensin mRNAs in their capillary blood. Quantitative RT-PCR performed on FACS-sorted granulocytes identified CD15(dull)/CD14(weak) population as the cellular source of α-defensins. Surprisingly, this granulocyte subpopulation displayed augmentation of α-defensin expression in all T1D patients tested. The determination of cell surface markers, expression of cell-specific genes and confocal microscopy identified CD15(dull)/CD14(weak) cells as eosinophils. The presence of transcriptionally active eosinophils in diabetic patients suggests that eosinophils could be a part of an intricate innate immune cellular network involved in the development of diabetes.
Assuntos
Diabetes Mellitus Tipo 1/imunologia , Eosinófilos/imunologia , Peroxidase/imunologia , alfa-Defensinas/imunologia , Adolescente , Adulto , Autoimunidade , Criança , Pré-Escolar , Diabetes Mellitus Tipo 1/sangue , Diabetes Mellitus Tipo 1/genética , Eosinófilos/metabolismo , Eosinófilos/patologia , Citometria de Fluxo , Expressão Gênica , Humanos , Tolerância Imunológica , Imunidade Inata , Células Secretoras de Insulina/imunologia , Células Secretoras de Insulina/metabolismo , Células Secretoras de Insulina/patologia , Antígenos CD15/imunologia , Receptores de Lipopolissacarídeos/imunologia , Peroxidase/sangue , Peroxidase/genética , RNA Mensageiro/biossíntese , Reação em Cadeia da Polimerase Via Transcriptase Reversa , alfa-Defensinas/sangue , alfa-Defensinas/genéticaRESUMO
The aim of this study was to find out whether protease inhibitor 9 (PI-9) and granzyme B (GrB) molecules that contribute to immune response and the immunological privilege of various tissues are expressed in healthy and pathological human corneas. Using cryosections, cell imprints of control corneoscleral discs, we showed that PI-9 was expressed particularly in the endothelium, the superficial and suprabasal epithelium of healthy corneas, limbus, and conjunctiva. GrB was localized in healthy corneal and conjunctival epithelium, while the endothelium showed weak immunostaining. The expression of PI-6 and GrB was confirmed by qRT-PCR. Increased expression levels of the PI-9 and GrB genes were determined when the corneas were cultured with proinflammatory cytokines. Fluorescent and enzymatic immunohistochemistry of pathological corneal explants (corneal melting and herpes virus keratitis) showed pronounced PI-9, GrB, human leucocyte antigen (HLA)-DR, and leukocyte-common antigen (CD45) signals localized in multicellular stromal infiltrates and inflammatory cells scattered in the corneal stroma. We conclude that increased expression of the PI-9 and GrB proteins under pathological conditions and their upregulation in an inflammatory environment indicate their participation in immune response of the cornea during the inflammatory process.
RESUMO
Mature T cells are selected for recognizing self-antigens with low to intermediate affinity in the thymus. Recently, the relative differences in self-reactivity among individual T-cell clones were appreciated as important factors regulating their fate and immune response, but the role of self-reactivity in T-cell biology is incompletely understood. We addressed the role of self-reactivity in T-cell diversity by generating an atlas of mouse peripheral CD8+ T cells, which revealed two unconventional populations of antigen-inexperienced T cells. In the next step, we examined the steady-state phenotype of monoclonal T cells with various levels of self-reactivity. Highly self-reactive clones preferentially differentiate into antigen-inexperienced memory-like cells, but do not form a population expressing type I interferon-induced genes, showing that these two subsets have unrelated origins. The functional comparison of naïve monoclonal CD8+ T cells specific to the identical model antigen did not show any correlation between the level of self-reactivity and the magnitude of the immune response.
Assuntos
Linfócitos T CD8-Positivos , Interferon Tipo I , Camundongos , Animais , Timo , Células Clonais , AutoantígenosRESUMO
The causative role of inflammation in hypertension-related cardiovascular diseases is evident and calls for development of specific immunomodulatory therapies. We tested the therapeutic efficacy and mechanisms of action of developmental endothelial locus-1 (DEL-1), an endogenous antiinflammatory factor, in angiotensin II- (ANGII-) and deoxycorticosterone acetate-salt-induced (DOCA-salt-induced) cardiovascular organ damage and hypertension. By using mice with endothelial overexpression of DEL-1 (EC-Del1 mice) and performing preventive and interventional studies by injecting recombinant DEL-1 in mice, we showed that DEL-1 improved endothelial function and abrogated aortic adventitial fibrosis, medial thickening, and loss of elastin. DEL-1 also protected the mice from cardiac concentric hypertrophy and interstitial and perivascular coronary fibrosis and improved left ventricular function and myocardial coronary perfusion. DEL-1 prevented aortic stiffness and abolished the progression of hypertension. Mechanistically, DEL-1 acted by inhibiting αvß3 integrin-dependent activation of pro-MMP2 in mice and in human isolated aorta. Moreover, DEL-1 stabilized αvß3 integrin-dependent CD25+FoxP3+ Treg numbers and IL-10 levels, which were associated with decreased recruitment of inflammatory cells and reduced production of proinflammatory cytokines in cardiovascular organs. The demonstrated effects and immune-modulating mechanisms of DEL-1 in abrogation of cardiovascular remodeling and progression of hypertension identify DEL-1 as a potential therapeutic factor.
Assuntos
Proteínas de Ligação ao Cálcio/metabolismo , Moléculas de Adesão Celular/metabolismo , Hipertensão , Remodelação Ventricular , Animais , Cardiomegalia , Fibrose , Hipertensão/complicações , Imunomodulação/genética , Integrinas , Camundongos , Remodelação Ventricular/genéticaRESUMO
Experimental autoimmune encephalomyelitis (EAE) represents the mouse model of multiple sclerosis, a devastating neurological disorder. EAE development and progression involves the infiltration of different immune cells into the brain and spinal cord. However, less is known about a potential role of eosinophil granulocytes for EAE disease pathogenesis. In the present study, we found enhanced eosinophil abundance accompanied by increased concentration of the eosinophil chemoattractant eotaxin-1 in the spinal cord in the course of EAE induced in C57BL/6 mice by immunization with MOG35-55 peptide. However, the absence of eosinophils did not affect neuroinflammation, demyelination and clinical development or severity of EAE, as assessed in ∆dblGATA1 eosinophil-deficient mice. Taken together, despite their enhanced abundance in the inflamed spinal cord during disease progression, eosinophils were dispensable for EAE development.
Assuntos
Encefalomielite Autoimune Experimental/imunologia , Eosinófilos/imunologia , Esclerose Múltipla/imunologia , Medula Espinal/patologia , Animais , Quimiocina CCL11/metabolismo , Encefalomielite Autoimune Experimental/sangue , Encefalomielite Autoimune Experimental/diagnóstico , Encefalomielite Autoimune Experimental/patologia , Eosinófilos/metabolismo , Feminino , Humanos , Camundongos , Camundongos Transgênicos , Esclerose Múltipla/sangue , Esclerose Múltipla/diagnóstico , Esclerose Múltipla/patologia , Glicoproteína Mielina-Oligodendrócito/administração & dosagem , Glicoproteína Mielina-Oligodendrócito/imunologia , Fragmentos de Peptídeos/administração & dosagem , Fragmentos de Peptídeos/imunologia , Índice de Gravidade de Doença , Medula Espinal/imunologiaRESUMO
The goal of our study was to determine whether proteins typical of the human mesothelial cell phenotype, such as mesothelin, HBME-1 (Hector Battifora mesothelial cell-1) protein and calbindin 2, are expressed in the human cornea, especially in endothelial cells. Cryosections and endothelial and epithelial imprints of sixteen human cadaverous corneoscleral discs were used. The presence of proteins was examined using immunohistochemistry and Western blotting, while mRNA levels were determined by qRT-PCR. A strong signal for mesothelin was present in the corneal epithelium, while less intense staining was visible in the endothelium. Similarly, higher and lower mRNA levels were detected using qRT-PCR in the corneal epithelium and endothelium, respectively. HBME-1 antibody strongly stained the corneal endothelium and stromal keratocytes. Marked positivity was present in the corneal stromal extracellular matrix, while no staining was present in the sclera. Calbindin 2 was detected using immunohistochemistry and Western blotting in the corneal epithelium, endothelium and stroma. qRT-PCR confirmed its expression in epithelial and endothelial cells. Three proteins expressed constitutively in mesothelial cells were detected in the human cornea. The possible function of mesothelin in cell-cell contact on the ocular surface is discussed. The presence of HBME-1 protein in the endothelial layer may indicate a still unknown function that could be shared with mesothelial cells of the pleura and peritoneum. The much more pronounced occurrence of calbindin 2 in the corneal epithelium compared to fewer positive endothelial cells explains the higher turnover of epithelial cells compared to the proliferatively inactive endothelium.
Assuntos
Biomarcadores Tumorais/metabolismo , Substância Própria/metabolismo , Endotélio Corneano/metabolismo , Epitélio Corneano/metabolismo , Glicoproteínas de Membrana/metabolismo , Proteína G de Ligação ao Cálcio S100/metabolismo , Biomarcadores Tumorais/genética , Western Blotting , Calbindina 2 , Proteínas do Olho/metabolismo , Feminino , Técnica Indireta de Fluorescência para Anticorpo , Proteínas Ligadas por GPI , Humanos , Técnicas Imunoenzimáticas , Masculino , Glicoproteínas de Membrana/genética , Mesotelina , Pessoa de Meia-Idade , RNA Mensageiro/genética , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Proteína G de Ligação ao Cálcio S100/genéticaRESUMO
Mononuclear phagocytes, such as macrophages and microglia, are key regulators of organ homeostasis including vascularization processes. Here, we investigated the role of the suppressor of cytokine signaling 3 (SOCS3) in myeloid cells as a regulator of mononuclear phagocyte function and their interaction with endothelial cells in the context of sprouting angiogenesis. As compared to SOCS3-sufficient counterparts, SOCS3-deficient microglia and macrophages displayed an increased phagocytic activity toward primary apoptotic endothelial cells, which was associated with an enhanced expression of the opsonin growth arrest-specific 6 (Gas6), a major prophagocytic molecule. Furthermore, we found that myeloid SOCS3 deficiency significantly reduced angiogenesis in an ex vivo mouse aortic ring assay, which could be reversed by the inhibition of the Gas6 receptor Mer. Together, SOCS3 in myeloid cells regulates the Gas6/Mer-dependent phagocytosis of endothelial cells, and thereby angiogenesis-related processes. Our findings provide novel insights into the complex crosstalk between mononuclear phagocytes and endothelial cells, and may therefore provide a new platform for the development of new antiangiogenic therapies.
Assuntos
Apoptose/imunologia , Células Endoteliais/imunologia , Células Mieloides/imunologia , Neovascularização Fisiológica/imunologia , Proteína 3 Supressora da Sinalização de Citocinas/deficiência , Animais , Apoptose/genética , Camundongos , Camundongos Transgênicos , Neovascularização Fisiológica/genética , Fagocitose , Proteína 3 Supressora da Sinalização de Citocinas/imunologiaRESUMO
The development and function of CD4(+) CD25(+) Foxp3(+) regulatory T cells (Tregs) are strictly regulated by cytokines. Here we show that transforming growth factor-beta (TGF-beta) and interleukin-4 (IL-4) play a crucial and antagonistic role in the development of Tregs. Additionally, these cytokines also have distinct effects on the maintenance of natural (nTregs) and antigen-induced (iTregs) Tregs. Using double-staining and tracking of proliferation of purified and carboxyflourescein succinimidyl ester (CFSE)-labelled mouse T-cell subpopulations we demonstrated that CD4(+) CD25(+) Foxp3(+) iTregs develop upon alloantigenic stimulation in the presence of TGF-beta exclusively from CD4(+) CD25(-) Foxp3(-) precursors. Both the induction of Foxp3 expression and Treg proliferation were prevented when the cells were stimulated in the presence of IL-4. By contrast, nTregs did not proliferate in the presence of the antigen and TGF-beta, and partially lost their Foxp3 expression. IL-4 not only prevented the development of iTregs, but also down-regulated the level of Foxp3 mRNA and decreased the number of Foxp3(+) cells in a population of iTregs. Further analyses proved that IL-4 decreased the expression of Foxp3 only in a population of iTregs, whereas it substantially supported the survival of nTregs. Functional experiments showed that Tregs induced in the presence of alloantigen and TGF-beta inhibited, on a per-cell basis, cell proliferation comparably to nTregs, and their suppressive capacity was not modulated by IL-4. These data suggest that TGF-beta and IL-4 differentially regulate the development of Tregs and distinctly sustain Foxp3 expression and the number of nTregs and iTregs, but have no influence on the suppressive activity of Tregs on a per-cell basis.
Assuntos
Linfócitos T CD4-Positivos/imunologia , Fatores de Transcrição Forkhead/biossíntese , Interleucina-4/fisiologia , Subpopulações de Linfócitos T/imunologia , Linfócitos T Reguladores/imunologia , Fator de Crescimento Transformador beta/fisiologia , Animais , Linfócitos T CD4-Positivos/efeitos dos fármacos , Linfócitos T CD4-Positivos/metabolismo , Diferenciação Celular/efeitos dos fármacos , Diferenciação Celular/imunologia , Feminino , Fatores de Transcrição Forkhead/antagonistas & inibidores , Interleucina-4/farmacologia , Masculino , Camundongos , Camundongos Endogâmicos BALB C , Camundongos Endogâmicos C57BL , RNA Mensageiro/imunologia , RNA Mensageiro/metabolismo , Subpopulações de Linfócitos T/efeitos dos fármacos , Subpopulações de Linfócitos T/metabolismo , Linfócitos T Reguladores/efeitos dos fármacos , Fator de Crescimento Transformador beta/farmacologiaRESUMO
The aim of this study was to determine if cytokeratins (CKs) 8 and 18--typical epithelial cell markers--are constitutively expressed in adult human corneal endothelium. Cryosections, paraffin-embedded sections and corneal endothelial imprints obtained from eleven adult human corneal discs not suitable for transplantation were used. Different fixative solutions were applied before indirect immunofluorescent or enzymatic staining was performed with antibodies against CK8 (Chemicon), CK18 (Dako and Sigma) and CK8/18 (Novocastra). Semi-quantitative RT-PCR and Western blotting (mRNA or proteins were isolated from Millicell membranes) were used to determine cytokeratin mRNA and protein levels. Approximately 50% of the corneal endothelial cells were positive for CK8 (Chemicon), CK18 (Sigma) and the CK pair 8/18 (Novocastra) in the endothelium when acetone was used for fixation. Four and 52% CK18-positive cells were observed using immunofluorescent and enzymatic immunohistochemistry, respectively, when the CK18 antibody from Dako was used. No signal was detected when 4% formalin or 10% paraformaldehyde was used as a fixative, irrespective of the antibody used. CK8 and CK18 proteins and mRNAs were detected in the endothelium of all tested corneas by Western blotting or semi-quantitative RT-PCR, respectively. We detected both CK8 and CK18 in the endothelium of all specimens at both the protein and mRNA levels. These results clearly demonstrate that cells of the corneal endothelium express CKs 8 and 18 and share some features with simple epithelia.