RESUMO
Macrophages are integral to the pathogenesis of atherosclerosis, but the contribution of distinct macrophage subsets to disease remains poorly defined. Using single cell technologies and conditional ablation via a LysMCre+ Clec4a2flox/DTR mouse strain, we demonstrate that the expression of the C-type lectin receptor CLEC4A2 is a distinguishing feature of vascular resident macrophages endowed with athero-protective properties. Through genetic deletion and competitive bone marrow chimera experiments, we identify CLEC4A2 as an intrinsic regulator of macrophage tissue adaptation by promoting a bias in monocyte-to-macrophage in situ differentiation towards colony stimulating factor 1 (CSF1) in vascular health and disease. During atherogenesis, CLEC4A2 deficiency results in loss of resident vascular macrophages and their homeostatic properties causing dysfunctional cholesterol metabolism and enhanced toll-like receptor triggering, exacerbating disease. Our study demonstrates that CLEC4A2 licenses monocytes to join the vascular resident macrophage pool, and that CLEC4A2-mediated macrophage homeostasis is critical to combat cardiovascular disease.
Assuntos
Apolipoproteínas E/genética , Aterosclerose/genética , Vasos Sanguíneos/metabolismo , Lectinas Tipo C/genética , Macrófagos/metabolismo , Animais , Apolipoproteínas E/deficiência , Aterosclerose/metabolismo , Aterosclerose/patologia , Vasos Sanguíneos/patologia , Células da Medula Óssea/metabolismo , Células da Medula Óssea/patologia , Morte Celular/genética , Diferenciação Celular , Linhagem da Célula/genética , Colesterol/metabolismo , Modelos Animais de Doenças , Regulação da Expressão Gênica , Homeostase/genética , Humanos , Lectinas Tipo C/deficiência , Fator Estimulador de Colônias de Macrófagos/genética , Fator Estimulador de Colônias de Macrófagos/metabolismo , Macrófagos/patologia , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Monócitos/metabolismo , Monócitos/patologia , Transdução de Sinais , Análise de Célula ÚnicaRESUMO
Recent studies indicate that Toll-like receptors (TLRs) and C-type lectin receptors (CLRs) can function as the signal of pattern recognition receptors, which play a pivotal role in the pathogenesis of the autoimmune disease. Systemic lupus erythematosus (SLE) is a classic autoimmune disease. Previous reports mainly focused on the potential role of TLRs in regulating the development of SLE, but little is known about the role of CLRs in the progression of SLE. Our previous studies showed that the inflammation-mediated accumulation of myeloid-derived suppressor cells (MDSCs) including granulocytic (G-MDSCs) and monocytic (M-MDSCs) participated in the pathogenesis of lupus. Mice deficient in Card9 (the downstream molecule of CLRs) were more susceptible to colitis-associated cancer via promoting the expansion of MDSCs. Whether the abnormal activation of CLRs regulates the expansion of MDSCs to participate in the pathogenesis of lupus remains unknown. In the present study, the expressions of CLRs were examined in both SLE patients and mouse models, revealing the expression of Dectin3 was positively correlated with SLEDAI. Dectin3 deficiency retarded the lupus-like disease by regulating the expansion and function of MDSCs. The mechanistic analysis revealed that Dectin3 deficiency promoted FoxO1-mediated apoptosis of MDSCs. Syk-Akt1-mediated nuclear transfer of FoxO1 increased in Dectin3-deficient MDSCs. Notedly, the accumulation of M-MDSCs mainly decreased in Dectin3-/- lupus mice, and the nuclear transfer of FoxO1 negatively correlated with the expression of LOX-1 on M-MDSCs. The silencing of FoxO1 expression in Dectin3-/- mice promoted the expansion of LOX-1+ M-MDSCs in vivo, and LOX-1+ M-MDSCs increased the differentiation of Th17 cells. Both LOX-1 expression on M-MDSCs and Dectin3 expression on MDSCs increased in patients with SLE. These data indicated that increased LOX-1+ M-MDSCs were related to the exacerbation of SLE development and might be potential target cells for the treatment of SLE.
Assuntos
Proteína Forkhead Box O1/metabolismo , Lectinas Tipo C/deficiência , Lúpus Eritematoso Sistêmico/patologia , Monócitos/patologia , Células Supressoras Mieloides/metabolismo , Receptores Imunológicos/deficiência , Receptores Depuradores Classe E/metabolismo , Transferência Adotiva , Adulto , Animais , Apoptose , Diferenciação Celular/genética , Núcleo Celular/metabolismo , Modelos Animais de Doenças , Feminino , Regulação da Expressão Gênica , Inativação Gênica , Humanos , Imiquimode , Lectinas Tipo C/metabolismo , Lúpus Eritematoso Sistêmico/genética , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Pessoa de Meia-Idade , Proteínas Proto-Oncogênicas c-akt/metabolismo , Células RAW 264.7 , Receptores Imunológicos/metabolismo , Receptores Depuradores Classe E/deficiência , Quinase Syk/metabolismo , TerpenosRESUMO
We previously described a new osteogenic growth factor, osteolectin/Clec11a, which is required for the maintenance of skeletal bone mass during adulthood. Osteolectin binds to Integrin α11 (Itga11), promoting Wnt pathway activation and osteogenic differentiation by leptin receptor+ (LepR+) stromal cells in the bone marrow. Parathyroid hormone (PTH) and sclerostin inhibitor (SOSTi) are bone anabolic agents that are administered to patients with osteoporosis. Here we tested whether osteolectin mediates the effects of PTH or SOSTi on bone formation. We discovered that PTH promoted Osteolectin expression by bone marrow stromal cells within hours of administration and that PTH treatment increased serum osteolectin levels in mice and humans. Osteolectin deficiency in mice attenuated Wnt pathway activation by PTH in bone marrow stromal cells and reduced the osteogenic response to PTH in vitro and in vivo. In contrast, SOSTi did not affect serum osteolectin levels and osteolectin was not required for SOSTi-induced bone formation. Combined administration of osteolectin and PTH, but not osteolectin and SOSTi, additively increased bone volume. PTH thus promotes osteolectin expression and osteolectin mediates part of the effect of PTH on bone formation.
Assuntos
Fatores de Crescimento de Células Hematopoéticas/metabolismo , Lectinas Tipo C/metabolismo , Osteogênese/efeitos dos fármacos , Hormônio Paratireóideo/farmacologia , Proteínas Adaptadoras de Transdução de Sinal/antagonistas & inibidores , Proteínas Adaptadoras de Transdução de Sinal/metabolismo , Animais , Osso Esponjoso/efeitos dos fármacos , Osso Esponjoso/patologia , Feminino , Fatores de Crescimento de Células Hematopoéticas/sangue , Fatores de Crescimento de Células Hematopoéticas/deficiência , Humanos , Lectinas Tipo C/sangue , Lectinas Tipo C/deficiência , Camundongos Endogâmicos C57BL , Tamanho do Órgão/efeitos dos fármacos , Osteoporose/sangue , Pré-Menopausa/sangue , Via de Sinalização Wnt/efeitos dos fármacosRESUMO
Increasing evidence has shown that the NOD-like receptor protein 3 (NLRP3) inflammasome and pyroptotic cell death play vital roles in the pathophysiology of myocardial infarction (MI), a common cardiovascular disease characterized by cardiac dysfunction. C-type lectin member 5A (CLEC5A) has been reported to be strongly associated with activation of the NLRP3 inflammasome and pyroptosis. In this study, an in vivo MI model was established by ligation of the left anterior descending coronary artery in male C57BL/6 mice, and CLEC5A knockdown was further achieved by intra-myocardial injection of adenovirus delivering shRNA-CLEC5A. CLEC5A was found to be highly expressed in the left ventricle of MI mice, while CLEC5A knockdown alleviated cardiac dysfunction in MI mice. In addition, MI-induced classical activation of macrophages was significantly inhibited after CLEC5A silencing. Additionally, CLEC5A knockdown dramatically inhibited MI-triggered activation of NLRP3 inflammasome, pyroptosis, and NF-κB signaling in the left ventricle of mice. In vitro experiments further validated that CLEC5A knockdown suppressed M1 polarization in LPS/IFNγ-stimulated RAW264.7 cells and inhibited the polarized RAW264.7-induced activation of NLRP3 inflammasome/pyroptosis signaling in co-cultured cardiomyocytes. In conclusion, CLEC5A knockdown protects against MI-induced cardiac dysfunction by regulating macrophage polarization, NLRP3 inflammasome, and cell pyroptosis.
Assuntos
Cardiomiopatias/metabolismo , Inflamassomos/metabolismo , Lectinas Tipo C/metabolismo , Macrófagos/metabolismo , Infarto do Miocárdio/metabolismo , Proteína 3 que Contém Domínio de Pirina da Família NLR/metabolismo , Receptores de Superfície Celular/metabolismo , Animais , Cardiomiopatias/patologia , Células Cultivadas , Lectinas Tipo C/deficiência , Macrófagos/patologia , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Infarto do Miocárdio/patologia , Piroptose , Células RAW 264.7 , Receptores de Superfície Celular/deficiênciaRESUMO
Asthma is an allergic chronic respiratory disease that affects more than 300 million people around the world. Dysbiosis of intestinal commensal microbiota influences the development of asthma. Dectin-1 (gene symbol: Clec7a), a C-type lectin receptor, plays an important role in the intestinal immune homeostasis by controlling regulatory T (Treg) cell differentiation through regulation of intestinal microbiota. However, it is not clear whether intestinal immune conditions affect immune responses in other organs. In this study, we examined the effects of Dectin-1 deficiency on allergic airway inflammation (AAI). OVA-induced AAI was attenuated in Clec7a -/- mice. Treg cells were more abundant in colonic lamina propria, mesenteric lymph nodes, and bronchoalveolar lavage fluid of Clec7a -/- mice after AAI induction. Treatment with antibiotics, but not an antifungal agent, decreased the abundance of intestinal Treg cells and aggravated the symptoms of AAI in Clec7a -/- mice. Transplantation of gut microbiota from Clec7a -/- mice into antibiotic-treated hosts increased the abundance of intestinal Treg cells and ameliorated AAI. Overcolonization by Lactobacillus murinus, a Dectin-1 signaling-regulated commensal bacterium, also promoted expansion of Treg cells in the colon and suppressed lung inflammation. Depletion of Treg cells with anti-CD25 Ab eliminated the phenotypic differences between wild-type and Clec7a -/- mice in OVA-induced AAI. These observations suggest that inhibition of Dectin-1 signaling ameliorates AAI by increasing the abundance of Treg cells in lungs through modification of intestinal commensal bacteria, suggesting a role for commensal microbiota in regulating inflammation in organs other than the intestine.
Assuntos
Intestinos/imunologia , Lectinas Tipo C/deficiência , Pulmão/imunologia , Ovalbumina/imunologia , Pneumonia/imunologia , Linfócitos T Reguladores/imunologia , Animais , Bactérias/classificação , Bactérias/genética , Bactérias/imunologia , Líquido da Lavagem Broncoalveolar/imunologia , Microbioma Gastrointestinal/imunologia , Hipersensibilidade/genética , Hipersensibilidade/imunologia , Intestinos/microbiologia , Lactobacillus/imunologia , Lactobacillus/fisiologia , Lectinas Tipo C/genética , Pulmão/metabolismo , Camundongos Endogâmicos BALB C , Camundongos Knockout , Pneumonia/genética , RNA Ribossômico 16S/genética , Linfócitos T Reguladores/metabolismoRESUMO
During early embryonic development in mammals, including humans and mice, megakaryocytes (Mks) first originate from primitive hematopoiesis in the yolk sac. These embryonic Mks (eMks) circulate in the vasculature with unclear function. Herein, we report that podoplanin (PDPN), the ligand of C-type lectin-like receptor (CLEC-2) on Mks/platelets, is temporarily expressed in neural tissue during midgestation in mice. Loss of PDPN or CLEC-2 resulted in aneurysms and spontaneous hemorrhage, specifically in the lower diencephalon during midgestation. Surprisingly, more eMks/platelets had enhanced granule release and localized to the lower diencephalon in mutant mouse embryos than in wild-type littermates before hemorrhage. We found that PDPN counteracted the collagen-1-induced secretion of angiopoietin-1 from fetal Mks, which coincided with enhanced TIE-2 activation in aneurysm-like sprouts of PDPN-deficient embryos. Blocking platelet activation prevented the PDPN-deficient embryo from developing vascular defects. Our data reveal a new role for PDPN in regulating eMk function during midgestation.
Assuntos
Encéfalo/irrigação sanguínea , Aneurisma Intracraniano/etiologia , Megacariócitos/patologia , Glicoproteínas de Membrana/deficiência , Aneurisma Roto/embriologia , Aneurisma Roto/etiologia , Angiopoietina-1/metabolismo , Animais , Encéfalo/embriologia , Células Cultivadas , Hemorragia Cerebral/embriologia , Hemorragia Cerebral/etiologia , Colágeno/farmacologia , Diencéfalo/irrigação sanguínea , Diencéfalo/embriologia , Regulação da Expressão Gênica no Desenvolvimento , Idade Gestacional , Aneurisma Intracraniano/embriologia , Aneurisma Intracraniano/genética , Aneurisma Intracraniano/patologia , Lectinas Tipo C/deficiência , Lectinas Tipo C/genética , Lectinas Tipo C/fisiologia , Megacariócitos/metabolismo , Glicoproteínas de Membrana/genética , Glicoproteínas de Membrana/fisiologia , Camundongos , Camundongos Knockout , Neovascularização Patológica/genética , Neovascularização Patológica/fisiopatologia , Neovascularização Fisiológica/fisiologia , Ativação Plaquetária , Agregação Plaquetária/efeitos dos fármacos , Inibidores da Agregação Plaquetária/farmacologia , Receptor TIE-2/metabolismoRESUMO
BACKGROUND: Ischemic heart disease is a leading cause of heart failure and despite advanced therapeutic options, morbidity and mortality rates remain high. Although acute inflammation in response to myocardial cell death has been extensively studied, subsequent adaptive immune activity and anti-heart autoimmunity may also contribute to the development of heart failure. After ischemic injury to the myocardium, dendritic cells (DC) respond to cardiomyocyte necrosis, present cardiac antigen to T cells, and potentially initiate a persistent autoimmune response against the heart. Cross-priming DC have the ability to activate both CD4+ helper and CD8+ cytotoxic T cells in response to necrotic cells and may thus be crucial players in exacerbating autoimmunity targeting the heart. This study investigates a role for cross-priming DC in post-myocardial infarction immunopathology through presentation of self-antigen from necrotic cardiac cells to cytotoxic CD8+ T cells. METHODS: We induced type 2 myocardial infarction-like ischemic injury in the heart by treatment with a single high dose of the ß-adrenergic agonist isoproterenol. We characterized the DC population in the heart and mediastinal lymph nodes and analyzed long-term cardiac immunopathology and functional decline in wild type and Clec9a-depleted mice lacking DC cross-priming function. RESULTS: A diverse DC population, including cross-priming DC, is present in the heart and activated after ischemic injury. Clec9a-/- mice deficient in DC cross-priming are protected from persistent immune-mediated myocardial damage and decline of cardiac function, likely because of dampened activation of cytotoxic CD8+ T cells. CONCLUSION: Activation of cytotoxic CD8+ T cells by cross-priming DC contributes to exacerbation of postischemic inflammatory damage of the myocardium and corresponding decline in cardiac function. Importantly, this provides novel therapeutic targets to prevent postischemic immunopathology and heart failure.
Assuntos
Apresentação Cruzada , Células Dendríticas/imunologia , Miocárdio/patologia , Animais , Apresentação de Antígeno , Linfócitos T CD8-Positivos/imunologia , Linfócitos T CD8-Positivos/metabolismo , Células Dendríticas/metabolismo , Modelos Animais de Doenças , Feminino , Insuficiência Cardíaca/patologia , Humanos , Lectinas Tipo C/deficiência , Lectinas Tipo C/genética , Linfonodos/imunologia , Linfonodos/metabolismo , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Pessoa de Meia-Idade , Infarto do Miocárdio/imunologia , Infarto do Miocárdio/metabolismo , Infarto do Miocárdio/patologia , Miocárdio/imunologia , Miocárdio/metabolismo , Receptores de Quimiocinas/metabolismo , Receptores Imunológicos/deficiência , Receptores Imunológicos/genéticaRESUMO
S-layer (glyco)-proteins (SLPs) form a nanostructured envelope that covers the surface of different prokaryotes and show immunomodulatory activity. Previously, we have demonstrated that the S-layer glycoprotein from probiotic Lactobacillus kefiri CIDCA 8348 (SLP-8348) is recognized by Mincle (macrophage inducible C-type lectin receptor), and its adjuvanticity depends on the integrity of its glycans. However, the glycan's structure has not been described so far. Herein, we analyze the glycosylation pattern of three SLPs, SLP-8348, SLP-8321, and SLP-5818, and explore how these patterns impact their recognition by C-type lectin receptors and the immunomodulatory effect of the L. kefiri SLPs on antigen-presenting cells. High-performance anion-exchange chromatography-pulse amperometric detector performed after ß-elimination showed glucose as the major component in the O-glycans of the three SLPs; however, some differences in the length of hexose chains were observed. No N-glycosylation signals were detected in SLP-8348 and SLP-8321, but SLP-5818 was observed to have two sites carrying complex N-glycans based on a site-specific analysis and a glycomic workflow of the permethylated glycans. SLP-8348 was previously shown to enhance LPS-induced activation on both RAW264.7 macrophages and murine bone marrow-derived dendritic cells; we now show that SLP-8321 and SLP-5818 have a similar effect regardless of the differences in their glycosylation patterns. Studies performed with bone marrow-derived dendritic cells from C-type lectin receptor-deficient mice revealed that the immunostimulatory activity of SLP-8321 depends on its recognition by Mincle, whereas SLP-5818's effects are dependent on SignR3 (murine ortholog of human DC-SIGN). These findings encourage further investigation of both the potential application of these SLPs as new adjuvants and the protein glycosylation mechanisms in these bacteria.
Assuntos
Antígenos CD/metabolismo , Lactobacillus/metabolismo , Lectinas Tipo C/metabolismo , Glicoproteínas de Membrana/metabolismo , Sequência de Aminoácidos , Animais , Antígenos CD/genética , Cromatografia Líquida de Alta Pressão , Células Dendríticas/citologia , Células Dendríticas/efeitos dos fármacos , Células Dendríticas/metabolismo , Glicopeptídeos/análise , Glicopeptídeos/química , Glicosilação , Imunização , Interferon gama/metabolismo , Lectinas Tipo C/deficiência , Lectinas Tipo C/genética , Lipopolissacarídeos/farmacologia , Glicoproteínas de Membrana/química , Proteínas de Membrana/deficiência , Proteínas de Membrana/genética , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Polissacarídeos/análise , Polissacarídeos/química , Células RAW 264.7 , Espectrometria de Massas por Ionização e Dessorção a Laser Assistida por MatrizRESUMO
Accumulating evidence indicates that cell death triggers sterile inflammation and that impaired clearance of dead cells causes nonresolving inflammation; however, the underlying mechanisms are still unclear. Here, we show that macrophage-inducible C-type lectin (Mincle) senses renal tubular cell death to induce sustained inflammation after acute kidney injury in mice. Mincle-deficient mice were protected against tissue damage and subsequent atrophy of the kidney after ischemia-reperfusion injury. Using lipophilic extract from the injured kidney, we identified ß-glucosylceramide as an endogenous Mincle ligand. Notably, free cholesterol markedly enhanced the agonistic effect of ß-glucosylceramide on Mincle. Moreover, ß-glucosylceramide and free cholesterol accumulated in dead renal tubules in proximity to Mincle-expressing macrophages, where Mincle was supposed to inhibit clearance of dead cells and increase proinflammatory cytokine production. This study demonstrates that ß-glucosylceramide in combination with free cholesterol acts on Mincle as an endogenous ligand to induce cell death-triggered, sustained inflammation after acute kidney injury.
Assuntos
Injúria Renal Aguda/metabolismo , Morte Celular/genética , Lectinas Tipo C/metabolismo , Proteínas de Membrana/metabolismo , Injúria Renal Aguda/imunologia , Animais , Citocinas/metabolismo , Modelos Animais de Doenças , Feminino , Glucosilceramidase/metabolismo , Proteínas de Fluorescência Verde/genética , Inflamação/metabolismo , Lectinas Tipo C/deficiência , Lectinas Tipo C/genética , Ligantes , Macrófagos/imunologia , Macrófagos/metabolismo , Masculino , Proteínas de Membrana/deficiência , Proteínas de Membrana/genética , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Transdução de Sinais/genética , Transdução de Sinais/imunologiaRESUMO
The ability of cancer cells to invade and disseminate can be affected by components of the surrounding microenvironment. To identify dermal components that regulate the growth of epidermal carcinomas, we studied the genetic disease called xeroderma pigmentosum that bears mutations in genes involved in the nucleotide excision repair of DNA. Patients with xeroderma pigmentosum are more prone to develop cutaneous tumors than the general population and their dermal fibroblasts display the features of dermal cancer-associated fibroblasts, which promote the invasion of keratinocytes. Here, we report that 3-dimensional dermal cultures of fibroblasts from healthy donors but not from patients with xeroderma pigmentosum complementation group C express CLEC2A, which is the ligand of the activating NK cell receptor NKp65. A similar loss of CLEC2A was observed in sporadic dermal cancer-associated fibroblasts and upon the culture of fibroblasts with cutaneous squamous cell carcinoma-conditioned medium. Using an innovative 3-dimensional organotypic skin culture model that contain NK cells in addition to fibroblasts and squamous cell carcinoma cells, we unveiled a key role of CLEC2A that orchestrates a crosstalk between fibroblasts and NK cells, thereby leading to the control of squamous cell carcinoma invasion. These findings indicate that CLEC2A-expressing dermal fibroblasts play a major role in immune surveillance of the skin.
Assuntos
Fibroblastos Associados a Câncer/patologia , Carcinoma de Células Escamosas/imunologia , Lectinas Tipo C/deficiência , Neoplasias Cutâneas/imunologia , Xeroderma Pigmentoso/patologia , Adolescente , Adulto , Idoso , Idoso de 80 Anos ou mais , Biópsia , Fibroblastos Associados a Câncer/imunologia , Carcinoma de Células Escamosas/patologia , Comunicação Celular/imunologia , Células Cultivadas , Criança , Pré-Escolar , Técnicas de Cocultura , Proteínas de Ligação a DNA/genética , Feminino , Perfilação da Expressão Gênica , Humanos , Vigilância Imunológica , Lactente , Recém-Nascido , Células Matadoras Naturais/imunologia , Masculino , Invasividade Neoplásica/imunologia , Invasividade Neoplásica/patologia , Cultura Primária de Células , Receptores Semelhantes a Lectina de Células NK/metabolismo , Pele/imunologia , Pele/patologia , Neoplasias Cutâneas/patologia , Microambiente Tumoral/imunologia , Xeroderma Pigmentoso/genética , Xeroderma Pigmentoso/imunologia , Adulto JovemRESUMO
BACKGROUND: C-type lectin receptors, including Dectin-2, are pattern recognition receptors on monocytes and macrophages that mainly recognize sugars and sugar-like structures present on fungi. Activation of C-type lectin receptors induces downstream CARD9 signalling, leading to the production of cytokines. We hypothesized that under hyperglycaemic conditions, as is the case in diabetes mellitus, glycosylated protein (sugar-like) structures activate C-type lectin receptors, leading to immune cell activation and increased atherosclerosis development. METHODS: Low-density lipoprotein receptor-deficient mice were lethally irradiated and transplanted with bone marrow from control wild-type, Dectin-2-/- or Card9-/- mice. After 6 weeks of recovery, mice received streptozotocin injections (50 mg/g BW; 5 days) to induce hyperglycaemia. After an additional 2 weeks, mice were fed a Western-type diet (0.1% cholesterol) for 10 weeks. RESULTS AND CONCLUSION: Deletion of haematopoietic Dectin-2 reduced the number of circulating Ly6Chi monocytes, increased pro-inflammatory cytokine production, but did not affect atherosclerosis development. Deletion of haematopoietic CARD9 tended to reduce macrophage and collagen content in atherosclerotic lesions, again without influencing the lesion size. Deletion of haematopoietic Dectin-2 did not influence atherosclerosis development under hyperglycaemic conditions, despite some minor effects on inflammation. Deletion of haematopoietic CARD9 induced minor alterations in plaque composition under hyperglycaemic conditions, without affecting lesion size.
Assuntos
Doenças da Aorta/etiologia , Aterosclerose/etiologia , Glicemia/metabolismo , Proteínas Adaptadoras de Sinalização CARD/genética , Diabetes Mellitus Experimental/complicações , Deleção de Genes , Células-Tronco Hematopoéticas/metabolismo , Lectinas Tipo C/genética , Animais , Antígenos Ly/metabolismo , Aorta/metabolismo , Aorta/patologia , Doenças da Aorta/genética , Doenças da Aorta/metabolismo , Doenças da Aorta/patologia , Aterosclerose/genética , Aterosclerose/metabolismo , Aterosclerose/patologia , Biomarcadores/sangue , Transplante de Medula Óssea , Proteínas Adaptadoras de Sinalização CARD/deficiência , Células Cultivadas , Colágeno/metabolismo , Citocinas/metabolismo , Diabetes Mellitus Experimental/sangue , Dieta Ocidental , Predisposição Genética para Doença , Lectinas Tipo C/deficiência , Macrófagos Peritoneais/metabolismo , Macrófagos Peritoneais/patologia , Masculino , Camundongos Endogâmicos C57BL , Camundongos Knockout , Monócitos/metabolismo , Monócitos/patologia , Placa Aterosclerótica , Receptores de LDL/deficiência , Receptores de LDL/genéticaRESUMO
BACKGROUND & AIMS: Alcohol-associated liver disease is a leading indication for liver transplantation and a leading cause of mortality. Alterations to the gut microbiota contribute to the pathogenesis of alcohol-associated liver disease. Patients with alcohol-associated liver disease have increased proportions of Candida spp. in the fecal mycobiome, yet little is known about the effect of intestinal Candida on the disease. Herein, we evaluated the contributions of Candida albicans and its exotoxin candidalysin in alcohol-associated liver disease. METHODS: C. albicans and the extent of cell elongation 1 (ECE1) were analyzed in fecal samples from controls, patients with alcohol use disorder and those with alcoholic hepatitis. Mice colonized with different and genetically manipulated C. albicans strains were subjected to the chronic-plus-binge ethanol diet model. Primary hepatocytes were isolated and incubated with candidalysin. RESULTS: The percentages of individuals carrying ECE1 were 0%, 4.76% and 30.77% in non-alcoholic controls, patients with alcohol use disorder and patients with alcoholic hepatitis, respectively. Candidalysin exacerbates ethanol-induced liver disease and is associated with increased mortality in mice. Candidalysin enhances ethanol-induced liver disease independently of the ß-glucan receptor C-type lectin domain family 7 member A (CLEC7A) on bone marrow-derived cells, and candidalysin does not alter gut barrier function. Candidalysin can damage primary hepatocytes in a dose-dependent manner in vitro and is associated with liver disease severity and mortality in patients with alcoholic hepatitis. CONCLUSIONS: Candidalysin is associated with the progression of ethanol-induced liver disease in preclinical models and worse clinical outcomes in patients with alcoholic hepatitis. LAY SUMMARY: Candidalysin is a peptide toxin secreted by the commensal gut fungus Candida albicans. Candidalysin enhances alcohol-associated liver disease independently of the ß-glucan receptor CLEC7A on bone marrow-derived cells in mice without affecting intestinal permeability. Candidalysin is cytotoxic to primary hepatocytes, indicating a direct role of candidalysin on ethanol-induced liver disease. Candidalysin might be an effective target for therapy in patients with alcohol-associated liver disease.
Assuntos
Candida albicans/metabolismo , Exotoxinas/metabolismo , Proteínas Fúngicas/metabolismo , Hepatite Alcoólica/metabolismo , Hepatite Alcoólica/microbiologia , Hepatopatias Alcoólicas/metabolismo , Hepatopatias Alcoólicas/microbiologia , Adulto , Idoso , Animais , Estudos de Casos e Controles , Células Cultivadas , Modelos Animais de Doenças , Exotoxinas/análise , Exotoxinas/farmacologia , Fezes/microbiologia , Feminino , Proteínas Fúngicas/análise , Proteínas Fúngicas/farmacologia , Microbioma Gastrointestinal , Hepatite Alcoólica/mortalidade , Hepatócitos/efeitos dos fármacos , Humanos , Lectinas Tipo C/deficiência , Lectinas Tipo C/genética , Hepatopatias Alcoólicas/mortalidade , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Pessoa de Meia-Idade , Índice de Gravidade de DoençaRESUMO
BACKGROUNDInnate immune activation impacts lung transplant outcomes. Dectin-1 is an innate receptor important for pathogen recognition. We hypothesized that genotypes reducing dectin-1 activity would be associated with infection, graft dysfunction, and death in lung transplant recipients.METHODSWe assessed the rs16910526 CLEC7A gene polymorphism Y238X, which results in dectin-1 truncation, in 321 lung allograft recipients at a single institution and in 1,129 lung allograft recipients in the multicenter Lung Transplant Outcomes Group (LTOG) cohort. Differences in dectin-1 mRNA, cytokines, protein levels, immunophenotypes, and clinical factors were assessed.RESULTSY238X carriers had decreased dectin-1 mRNA expression (P = 0.0001), decreased soluble dectin-1 protein concentrations in bronchoalveolar lavage (P = 0.008) and plasma (P = 0.04), and decreased monocyte surface dectin-1 (P = 0.01) compared with wild-type subjects. Y238X carriers had an increased risk of fungal pathogens (HR 1.17, CI 1.0-1.4), an increased risk of graft dysfunction or death (HR 1.6, CI 1.0-2.6), as well increased mortality in the UCSF cohort (HR 1.8, CI 1.1-3.8) and in the LTOG cohort (HR 1.3, CI 1.1-1.6), compared with wild-type CLEC7A subjects.CONCLUSIONIncreased rates of graft dysfunction and death associated with this dectin-1 polymorphism may be amplified by immunosuppression that drives higher fungal burden from compromised pathogen recognition.FUNDINGThe UCSF Nina Ireland Program for Lung Health Innovative Grant program, the Clinical Sciences Research & Development Service of the VA Office of Research and Development, and the Joel D. Cooper Career Development Award from the International Society for Heart and Lung Transplantation.
Assuntos
Aloenxertos/metabolismo , Lectinas Tipo C , Transplante de Pulmão , Pulmão/metabolismo , Disfunção Primária do Enxerto , Adulto , Idoso , Aloenxertos/fisiopatologia , Feminino , Humanos , Lectinas Tipo C/deficiência , Lectinas Tipo C/genética , Lectinas Tipo C/metabolismo , Pulmão/fisiopatologia , Transplante de Pulmão/efeitos adversos , Transplante de Pulmão/mortalidade , Masculino , Pessoa de Meia-Idade , Disfunção Primária do Enxerto/genética , Disfunção Primária do Enxerto/mortalidadeRESUMO
The lung is a specialized barrier organ that must tightly regulate interstitial fluid clearance and prevent infection in order to maintain effective gas exchange. Lymphatic vessels are important for these functions in other organs, but their roles in the lung have not been fully defined. In the present study, we addressed how the lymphatic vasculature participates in lung homeostasis. Studies using mice carrying a lymphatic reporter allele revealeded that, in contrast to other organs, lung lymphatic collecting vessels lack smooth muscle cells entirely, suggesting that forward lymph flow is highly dependent on movement and changes in pressure associated with respiration. Functional studies using CLEC2-deficient mice in which lymph flow is impaired due to loss of lympho-venous hemostasis or using inducible lung-specific ablation of lymphatic endothelial cells in a lung transplant model revealeded that loss of lymphatic function leads to an inflammatory state characterized by the formation of tertiary lymphoid organs (TLOs). In addition, impaired lymphatic flow in mice resulteds in hypoxia and features of lung injury that resemble emphysema. These findings reveal both a lung-specific mechanism of lymphatic physiology and a lung-specific consequence of lymphatic dysfunction that may contribute to chronic lung diseases that arise in association with TLO formation.
Assuntos
Células Endoteliais , Lectinas Tipo C/deficiência , Alvéolos Pulmonares , Enfisema Pulmonar , Estruturas Linfoides Terciárias , Animais , Células Endoteliais/imunologia , Células Endoteliais/patologia , Lectinas Tipo C/imunologia , Camundongos , Alvéolos Pulmonares/imunologia , Alvéolos Pulmonares/lesões , Alvéolos Pulmonares/patologia , Enfisema Pulmonar/genética , Enfisema Pulmonar/imunologia , Enfisema Pulmonar/patologia , Estruturas Linfoides Terciárias/imunologia , Estruturas Linfoides Terciárias/patologiaRESUMO
BACKGROUND: Epicutaneous sensitization is an important route of immunization for allergens in atopic diseases; however, studies have also shown that application with protein on the intact skin induces antigen-specific tolerance. Langerhans cells (LCs) play an immunosuppressive role in several inflammatory skin diseases and mouse models, and the role of LCs in the skin-induced tolerance is not fully understood. METHODS: Langerin-DTA mice that were deficient in LCs were utilized to produce the model of skin-induced tolerance to ovalbumin (OVA). Binding of Langerin to OVA was analyzed by enzyme-linked immunosorbent assay, flow cytometry, and immunofluorescence. Homozygous Langerin-DTR mice that were deficient in Langerin were introduced to assess the role of Langerin in the skin-induced tolerance. RESULTS: Application with OVA onto the intact, but not tape-stripped, skin attenuated the production of OVA-specific IgE, IgG1, and IgG2a induced by subsequent subcutaneous immunization with OVA, and the inhibitory effects were abolished in Langerin-DTA mice. In contrast to the tape-stripped skin, the intact skin induced the production of IL-10 by LCs in draining lymph node after application with OVA. Langerin could bind OVA, and homozygous Langerin-DTR mice demonstrated similar humoral and cellular immune responses in the model of skin-induced tolerance compared to wide-type mice. CONCLUSION: Our data suggested that LCs were critical in the intact skin-induced tolerance to protein antigen via Langerin, and LCs might be targeted via Langerin to regulate the immune responses in systemic and (or) skin inflammatory diseases.
Assuntos
Alérgenos/imunologia , Tolerância Imunológica/genética , Células de Langerhans/imunologia , Células de Langerhans/metabolismo , Ovalbumina/imunologia , Pele/imunologia , Pele/metabolismo , Animais , Antígenos de Superfície , Linhagem Celular , Células Cultivadas , Citocinas/biossíntese , Modelos Animais de Doenças , Ensaio de Imunoadsorção Enzimática , Humanos , Imunização , Lectinas Tipo C/deficiência , Lectinas de Ligação a Manose/deficiência , Camundongos , Pele/patologiaRESUMO
Microglia are resident brain macrophages with key roles in development and brain homeostasis. Cytomegalovirus (CMV) readily infects microglia cells, even as a possible primary target of infection in development. Effects of CMV infection on a cellular level in microglia are still unclear; therefore, the aim of this research was to assess the immunometabolic changes of BV-2 microglia cells following the murine cytomegalovirus (MCMV) infection. In light of that aim, we established an in vitro model of ramified BV-2 microglia (BV-2∅FCS, inducible nitric oxide synthase (iNOSlow), arginase-1 (Arg-1high), mannose receptor CD206high, and hypoxia-inducible factor 1α (HIF-1αlow)) to better replicate the in vivo conditions by removing FCS from the cultivation media, while the cells cultivated in 10% FCS DMEM displayed an ameboid morphology (BV-2FCS high, iNOShigh, Arg-1low, CD206low, and HIF-1αhigh). Experiments were performed using both ramified and ameboid microglia, and both of them were permissive to productive viral infection. Our results indicate that MCMV significantly alters the immunometabolic phenotypic properties of BV-2 microglia cells through the manipulation of iNOS and Arg-1 expression patterns, along with an induction of a glycolytic shift in the infected cell cultures.
Assuntos
Arginase/imunologia , Infecções por Herpesviridae/imunologia , Interações Hospedeiro-Patógeno/imunologia , Microglia/virologia , Muromegalovirus/genética , Óxido Nítrico Sintase Tipo II/imunologia , Animais , Arginase/genética , Linhagem Celular , Meios de Cultura Livres de Soro/farmacologia , Embrião de Mamíferos , Fibroblastos/imunologia , Fibroblastos/virologia , Regulação da Expressão Gênica , Infecções por Herpesviridae/genética , Infecções por Herpesviridae/virologia , Interações Hospedeiro-Patógeno/genética , Subunidade alfa do Fator 1 Induzível por Hipóxia/deficiência , Subunidade alfa do Fator 1 Induzível por Hipóxia/genética , Subunidade alfa do Fator 1 Induzível por Hipóxia/imunologia , Lectinas Tipo C/deficiência , Lectinas Tipo C/genética , Lectinas Tipo C/imunologia , Receptor de Manose , Lectinas de Ligação a Manose/deficiência , Lectinas de Ligação a Manose/genética , Lectinas de Ligação a Manose/imunologia , Camundongos , Camundongos Endogâmicos BALB C , Microglia/imunologia , Modelos Biológicos , Muromegalovirus/crescimento & desenvolvimento , Muromegalovirus/metabolismo , Óxido Nítrico Sintase Tipo II/deficiência , Óxido Nítrico Sintase Tipo II/genética , Cultura Primária de Células , Receptores de Superfície Celular/deficiência , Receptores de Superfície Celular/genética , Receptores de Superfície Celular/imunologia , Transdução de SinaisRESUMO
Macrophages have been suggested to contribute to constructing a cancer stem cell (CSC) niche. However, whether and how macrophages regulate the activity of CSCs through juxtacrine signaling are poorly understood. Here we report LSECtin, a transmembrane protein highly expressed on tumor-associated macrophages (TAMs), enhances stemness of breast cancer cells (BCCs). We identified BTN3A3, a B7 family member with previously unknown functions as the receptor for LSECtin on BCCs responsible for stemness-promoting effect of LSECtin. In mice bearing human tumor xenografts, either macrophage-specific ablation of LSECtin or silencing of BTN3A3 in BCCs decreased CSC frequency and tumor growth. Admixture of LSECtin-positive macrophages increased the tumorigenic activity of BCCs dependent on BTN3A3. Disruption of the LSECtin-BTN3A3 axis with BTN3A3-Fc or anti-BTN3A3 mAb has a therapeutic effect on breast cancer. These findings define a juxtacrine signaling mechanism by which TAMs promote cancer stemness. Targeting this axis in the CSC niche may provide potential therapies to breast cancer.
Assuntos
Neoplasias da Mama/patologia , Butirofilinas/metabolismo , Lectinas Tipo C/metabolismo , Macrófagos/metabolismo , Animais , Antígenos CD/metabolismo , Antígenos de Diferenciação Mielomonocítica/metabolismo , Neoplasias da Mama/metabolismo , Neoplasias da Mama/mortalidade , Butirofilinas/antagonistas & inibidores , Butirofilinas/genética , Linhagem Celular Tumoral , Proliferação de Células , Feminino , Humanos , Estimativa de Kaplan-Meier , Lectinas Tipo C/deficiência , Lectinas Tipo C/genética , Macrófagos/citologia , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Camundongos Nus , Células-Tronco Neoplásicas/metabolismo , Ligação Proteica , Interferência de RNA , RNA Interferente Pequeno/metabolismo , Transdução de SinaisRESUMO
Platelets promote wound healing by forming a vascular plug and by secreting growth factors and cytokines. Glycoprotein (GP)VI and C-type lectin-like receptor (CLEC)-2 signal through a (hem)-immunoreceptor tyrosine-based activation motif, which induces platelet activation. GPVI and CLEC-2 support vascular integrity during inflammation in the skin through regulation of leukocyte migration and function, and by sealing sites of vascular damage. In this study, we investigated the role of impaired vascular integrity due to GPVI and/or CLEC-2 deficiency in wound repair using a full-thickness excisional skin wound model in mice. Transgenic mice deficient in both GPVI and CLEC-2 exhibited accelerated skin wound healing, despite a marked impairment in vascular integrity. The local and temporal bleeding in the skin led to greater plasma protein entry, including fibrinogen and clotting factors, was associated with increased fibrin generation, reduction in wound neutrophils and M1 macrophages, decreased level of tumor necrosis factor (TNF)-α, and enhanced angiogenesis at day 3 after injury. Accelerated wound healing was not due to developmental defects in CLEC-2 and GPVI double-deficient mice as similar results were observed in GPVI-deficient mice treated with a podoplanin-blocking antibody. The rate of wound healing was not altered in mice deficient in either GPVI or CLEC-2. Our results show that, contrary to defects in coagulation, bleeding following a loss of vascular integrity caused by platelet CLEC-2 and GPVI deficiency facilitates wound repair by increasing fibrin(ogen) deposition, reducing inflammation, and promoting angiogenesis.
Assuntos
Lectinas Tipo C/deficiência , Glicoproteínas de Membrana/deficiência , Neovascularização Fisiológica/genética , Glicoproteínas da Membrana de Plaquetas/deficiência , Cicatrização/genética , Animais , Biomarcadores , Feminino , Imunofluorescência , Imuno-Histoquímica , Lectinas Tipo C/genética , Lectinas Tipo C/metabolismo , Macrófagos/imunologia , Macrófagos/metabolismo , Macrófagos/patologia , Masculino , Glicoproteínas de Membrana/genética , Glicoproteínas de Membrana/metabolismo , Camundongos , Camundongos Knockout , Neutrófilos/imunologia , Neutrófilos/metabolismo , Neutrófilos/patologia , Glicoproteínas da Membrana de Plaquetas/genética , Glicoproteínas da Membrana de Plaquetas/metabolismo , Pele/metabolismo , Pele/patologiaRESUMO
RATIONALE: CD69 is an immunomodulatory molecule induced during lymphocyte activation. Following stroke, T-lymphocytes upregulate CD69 but its function is unknown. OBJECTIVE: We investigated whether CD69 was involved in brain damage following an ischemic stroke. METHODS AND RESULTS: We used adult male mice on the C57BL/6 or BALB/c backgrounds, including wild-type mice and CD69-/- mice, and CD69+/+ and CD69-/- lymphocyte-deficient Rag2-/- mice, and generated chimeric mice. We induced ischemia by transient or permanent middle cerebral artery occlusion. We measured infarct volume, assessed neurological function, and studied CD69 expression, as well as platelet function, fibrin(ogen) deposition, and VWF (von Willebrand factor) expression in brain vessels and VWF content and activity in plasma, and performed the tail-vein bleeding test and the carotid artery ferric chloride-induced thrombosis model. We also performed primary glial cell cultures and sorted brain CD45-CD11b-CD31+ endothelial cells for mRNA expression studies. We blocked VWF by intravenous administration of anti-VWF antibodies. CD69-/- mice showed larger infarct volumes and worse neurological deficits than the wild-type mice after ischemia. This worsening effect was not attributable to lymphocytes or other hematopoietic cells. CD69 deficiency lowered the time to thrombosis in the carotid artery despite platelet function not being affected. Ischemia upregulated Cd69 mRNA expression in brain endothelial cells. CD69-deficiency increased fibrin(ogen) accumulation in the ischemic tissue, and plasma VWF content and activity, and VWF expression in brain vessels. Blocking VWF reduced infarct volume and reverted the detrimental effect of CD69-/- deficiency. CONCLUSIONS: CD69 deficiency promotes a prothrombotic phenotype characterized by increased VWF and worse brain damage after ischemic stroke. The results suggest that CD69 acts as a downregulator of endothelial activation.
Assuntos
Antígenos CD/metabolismo , Antígenos de Diferenciação de Linfócitos T/metabolismo , Encéfalo/irrigação sanguínea , Encéfalo/metabolismo , Células Endoteliais/metabolismo , Infarto da Artéria Cerebral Média/metabolismo , Lectinas Tipo C/metabolismo , Ativação Linfocitária , Linfócitos T/metabolismo , Animais , Antígenos CD/genética , Antígenos de Diferenciação de Linfócitos T/genética , Coagulação Sanguínea , Plaquetas/metabolismo , Encéfalo/patologia , Células Cultivadas , Proteínas de Ligação a DNA/deficiência , Proteínas de Ligação a DNA/genética , Modelos Animais de Doenças , Células Endoteliais/patologia , Infarto da Artéria Cerebral Média/genética , Infarto da Artéria Cerebral Média/patologia , Lectinas Tipo C/deficiência , Lectinas Tipo C/genética , Masculino , Camundongos Endogâmicos BALB C , Camundongos Endogâmicos C57BL , Transdução de Sinais , Linfócitos T/patologia , Fator de von Willebrand/metabolismoRESUMO
BACKGROUND: Although the role of Th17 and regulatory T cells in the progression of atherosclerosis has been highlighted in recent years, their molecular mediators remain elusive. We aimed to evaluate the association between the CD69 receptor, a regulator of Th17/regulatory T cell immunity, and atherosclerosis development in animal models and in patients with subclinical disease. METHODS: Low-density lipoprotein receptor-deficient chimeric mice expressing or not expressing CD69 on either myeloid or lymphoid cells were subjected to a high fat diet. In vitro functional assays with human T cells were performed to decipher the mechanism of the observed phenotypes. Expression of CD69 and NR4A nuclear receptors was evaluated by reverse transcription-polymerase chain reaction in 305 male participants of the PESA study (Progression of Early Subclinical Atherosclerosis) with extensive (n=128) or focal (n=55) subclinical atherosclerosis and without disease (n=122). RESULTS: After a high fat diet, mice lacking CD69 on lymphoid cells developed large atheroma plaque along with an increased Th17/regulatory T cell ratio in blood. Oxidized low-density lipoprotein was shown to bind specifically and functionally to CD69 on human T lymphocytes, inhibiting the development of Th17 cells through the activation of NR4A nuclear receptors. Participants of the PESA study with evidence of subclinical atherosclerosis displayed a significant CD69 and NR4A1 mRNA downregulation in peripheral blood leukocytes compared with participants without disease. The expression of CD69 remained associated with the risk of subclinical atherosclerosis in an adjusted multivariable logistic regression model (odds ratio, 0.62; 95% CI, 0.40-0.94; P=0.006) after adjustment for traditional risk factors, the expression of NR4A1, the level of oxidized low-density lipoprotein, and the counts of different leucocyte subsets. CONCLUSIONS: CD69 depletion from the lymphoid compartment promotes a Th17/regulatory T cell imbalance and exacerbates the development of atherosclerosis. CD69 binding to oxidized low-density lipoprotein on T cells induces the expression of anti-inflammatory transcription factors. Data from a cohort of the PESA study with subclinical atherosclerosis indicate that CD69 expression in PBLs inversely correlates with the presence of disease. The expression of CD69 remained an independent predictor of subclinical atherosclerosis after adjustment for traditional risk factors.