RESUMO
Functionally diverse T cell populations interact to maintain homeostasis of the immune system. We found that human and mouse antigen-activated T cells with high expression of the lymphocyte surface marker CD52 suppressed other T cells. CD52(hi)CD4(+) T cells were distinct from CD4(+)CD25(+)Foxp3(+) regulatory T cells. Their suppression was mediated by soluble CD52 released by phospholipase C. Soluble CD52 bound to the inhibitory receptor Siglec-10 and impaired phosphorylation of the T cell receptor-associated kinases Lck and Zap70 and T cell activation. Humans with type 1 diabetes had a lower frequency and diminished function of CD52(hi)CD4(+) T cells responsive to the autoantigen GAD65. In diabetes-prone mice of the nonobese diabetic (NOD) strain, transfer of lymphocyte populations depleted of CD52(hi) cells resulted in a substantially accelerated onset of diabetes. Our studies identify a ligand-receptor mechanism of T cell regulation that may protect humans and mice from autoimmune disease.
Assuntos
Antígenos CD/imunologia , Antígenos de Neoplasias/imunologia , Linfócitos T CD4-Positivos/imunologia , Diabetes Mellitus Tipo 1/imunologia , Glicoproteínas/imunologia , Ativação Linfocitária/imunologia , Lectinas Semelhantes a Imunoglobulina de Ligação ao Ácido Siálico/imunologia , Proteínas Adaptadoras de Transdução de Sinal/imunologia , Animais , Antígenos CD/genética , Antígenos de Neoplasias/genética , Autoantígenos/imunologia , Antígeno CD52 , Feminino , Citometria de Fluxo , Glicoproteínas/genética , Homeostase/imunologia , Humanos , Masculino , Camundongos , Camundongos Endogâmicos NOD , Camundongos SCID , Fosforilação/imunologia , RNA Mensageiro/química , RNA Mensageiro/genética , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Proteína-Tirosina Quinase ZAP-70/imunologiaRESUMO
Blood pH is tightly maintained between 7.35 and 7.45, and acidosis (pH <7.3) indicates poor prognosis in sepsis, wherein lactic acid from anoxic tissues overwhelms the buffering capacity of blood. Poor sepsis prognosis is also associated with low zinc levels and the release of High mobility group box 1 (HMGB1) from activated and/or necrotic cells. HMGB1 added to whole blood at physiological pH did not bind leukocyte receptors, but lowering pH with lactic acid to mimic sepsis conditions allowed binding, implying the presence of natural inhibitor(s) preventing binding at normal pH. Testing micromolar concentrations of divalent cations showed that zinc supported the robust binding of sialylated glycoproteins with HMGB1. Further characterizing HMGB1 as a sialic acid-binding lectin, we found that optimal binding takes place at normal blood pH and is markedly reduced when pH is adjusted with lactic acid to levels found in sepsis. Glycan array studies confirmed the binding of HMGB1 to sialylated glycan sequences typically found on plasma glycoproteins, with binding again being dependent on zinc and normal blood pH. Thus, HMGB1-mediated hyperactivation of innate immunity in sepsis requires acidosis, and micromolar zinc concentrations are protective. We suggest that the potent inflammatory effects of HMGB1 are kept in check via sequestration by plasma sialoglycoproteins at physiological pH and triggered when pH and zinc levels fall in late stages of sepsis. Current clinical trials independently studying zinc supplementation, HMGB1 inhibition, or pH normalization may be more successful if these approaches are combined and perhaps supplemented by infusions of heavily sialylated molecules.
Assuntos
Acidose/sangue , Proteína HMGB1/sangue , Sepse/sangue , Sialoglicoproteínas/sangue , Zinco/sangue , Acidose/imunologia , Acidose/metabolismo , Acidose/patologia , Proteínas de Transporte , Proteína HMGB1/farmacologia , Humanos , Concentração de Íons de Hidrogênio , Imunidade Inata , Lipopolissacarídeos/farmacologia , Polissacarídeos/química , Sepse/imunologia , Sepse/patologia , Ácidos Siálicos/química , Sialoglicoproteínas/química , Zinco/metabolismoRESUMO
CD52, a glycophosphatidylinositol (GPI)-anchored glycoprotein, is released in a soluble form following T cell activation and binds to the Siglec (sialic acid-binding Ig-like lectin)-10 receptor on T cells to suppress their function. We show that binding of CD52-Fc to Siglec-10 and T cell suppression requires the damage-associated molecular pattern (DAMP) protein, high-mobility group box 1 (HMGB1). CD52-Fc bound specifically to the proinflammatory Box B domain of HMGB1, and this in turn promoted binding of the CD52 N-linked glycan, in α-2,3 sialic acid linkage with galactose, to Siglec-10. Suppression of T cell function was blocked by anti-HMGB1 antibody or the antiinflammatory Box A domain of HMGB1. CD52-Fc induced tyrosine phosphorylation of Siglec-10 and was recovered from T cells complexed with HMGB1 and Siglec-10 in association with SHP1 phosphatase and the T cell receptor (TCR). Thus, soluble CD52 exerts a concerted immunosuppressive effect by first sequestering HMGB1 to nullify its proinflammatory Box B, followed by binding to the inhibitory Siglec-10 receptor, triggering recruitment of SHP1 to the intracellular immunoreceptor tyrosine-based inhibitory motif of Siglec-10 and its interaction with the TCR. This mechanism may contribute to immune-inflammatory homeostasis in pathophysiologic states and underscores the potential of soluble CD52 as a therapeutic agent.
Assuntos
Antígeno CD52/imunologia , Proteína HMGB1/imunologia , Lectinas/imunologia , Receptores de Antígenos de Linfócitos T/imunologia , Linfócitos T/imunologia , Motivos de Aminoácidos , Anticorpos/farmacologia , Feminino , Proteína HMGB1/antagonistas & inibidores , Humanos , Masculino , Domínios Proteicos , Proteína Tirosina Fosfatase não Receptora Tipo 6/imunologiaRESUMO
Aim: To investigate the association between intravitreal ranibizumab therapy and serum cytokine concentrations in patients with diabetic macular edema (DME). Methods: Twenty-five patients with center-involved DME were recruited prospectively. Serum samples were collected from the patients before and 4 weeks after two ranibizumab injections. The levels of 32 cytokines at these two time points were assessed using a multiplex array assay. Results: Following two ranibizumab injections, there was a statistically significant decrease in the median [interquartile range] levels of Interleukin 1-1beta (IL-1ß) from 5.56 [3.6, 8.75] to 2.33 [1.51, 2.89], Interleukin 13 (IL-13) from 4.30 [1.84, 18.55] to 0.38 [0.38, 0.78], granulocyte-colony stimulating factor (G-CSF) from 64.65 [42.9, 108] to 37.8 [27.3, 46.37], Interferon gamma (IFN-γ) from 241 [103.33, 753.4] to 94.4626 [42.04, 118.58], Interferon gamma-induced protein 10 (IP-10) from 234.68 [144.16, 285.98] to 158.73 [94.71, 198.64], Macrophage Inflammatory Protein-1 alpha (MIP-1α) from 3.65 [2.62, 11.02] to 1.41 [0.94, 1.88], and Tumor necrosis factor- alpha (TNF-α) from 131.09 [100.68,28 240.27] to 45.19 [24.04, 68.55]. There was a statistically significant increase in the levels of Interleukin 9 (IL-9) from 0.76 [0.76, 7.03] to 19.67 [5.36 27.76], Macrophage Inflammatory Protein-1 beta (MIP-1ß) from 0.28 [0.28, 30 0.28] to 6.79 [I3.74, 14.16], Vascular endothelial growth factor (VEGF) from 2.55 [2.55, 2.55] to 25.24 [14.51, 41.73], and soluble vascular endothelial growth factor -1 (sVEGFR-1) from 333.92 [204.99, 440.43] to 500.12 [38.7, 786.91]. A Bonferroni-corrected p value of 0.00156 was considered statistically significant. Conclusions: In patients with DME, intravitreal ranibizumab therapy appears to influence the serum levels of a range of cytokines. After two injections, intravitreal ranibizumab therapy appears to be associated with a significant decrease in inflammatory mediators and a rise in VEGF and sVEGFR1.
Assuntos
Inibidores da Angiogênese/administração & dosagem , Citocinas/sangue , Retinopatia Diabética/sangue , Edema Macular/sangue , Ranibizumab/administração & dosagem , Fator A de Crescimento do Endotélio Vascular/metabolismo , Receptor 1 de Fatores de Crescimento do Endotélio Vascular/metabolismo , Idoso , Retinopatia Diabética/tratamento farmacológico , Feminino , Humanos , Injeções Intravítreas , Edema Macular/tratamento farmacológico , Masculino , Pessoa de Meia-IdadeRESUMO
BACKGROUND: Cord blood lipids are potential disease biomarkers. We aimed to determine if their concentrations were affected by delayed blood processing. METHOD: Refrigerated cord blood from six healthy newborns was centrifuged every 12 h for 4 days. Plasma lipids were analysed by liquid chromatography/mass spectroscopy. RESULTS: Of 262 lipids identified, only eight varied significantly over time. These comprised three dihexosylceramides, two phosphatidylserines and two phosphatidylethanolamines whose relative concentrations increased and one sphingomyelin that decreased. CONCLUSION: Delay in separation of plasma from refrigerated cord blood has minimal effect overall on the plasma lipidome.
Assuntos
Sangue Fetal/química , Lipídeos/sangue , Lipídeos/isolamento & purificação , Cromatografia Líquida de Alta Pressão , Humanos , Recém-Nascido , Espectrometria de Massas por Ionização por Electrospray , Espectrometria de Massas em TandemRESUMO
Differentiation of naïve CD4(+) T cells into effector (Th1, Th2, and Th17) and induced regulatory (iTreg) T cells requires lineage-specifying transcription factors and epigenetic modifications that allow appropriate repression or activation of gene transcription. The epigenetic silencing of cytokine genes is associated with the repressive H3K27 trimethylation mark, mediated by the Ezh2 or Ezh1 methyltransferase components of the polycomb repressive complex 2 (PRC2). Here we show that silencing of the Ifng, Gata3, and Il10 loci in naïve CD4(+) T cells is dependent on Ezh2. Naïve CD4(+) T cells lacking Ezh2 were epigenetically primed for overproduction of IFN-γ in Th2 and iTreg and IL-10 in Th2 cells. In addition, deficiency of Ezh2 accelerated effector Th cell death via death receptor-mediated extrinsic and intrinsic apoptotic pathways, confirmed in vivo for Ezh2-null IFN-γ-producing CD4(+) and CD8(+) T cells responding to Listeria monocytogenes infection. These findings demonstrate the key role of PRC2/Ezh2 in differentiation and survival of peripheral T cells and reveal potential immunotherapeutic targets.
Assuntos
Apoptose/imunologia , Diferenciação Celular/imunologia , Inativação Gênica/imunologia , Complexo Repressor Polycomb 2/imunologia , Linfócitos T Auxiliares-Indutores/imunologia , Animais , Sobrevivência Celular/imunologia , Proteína Potenciadora do Homólogo 2 de Zeste , Feminino , Humanos , Interferon gama/imunologia , Interleucina-10/imunologia , Listeria monocytogenes/imunologia , Listeriose/imunologia , Listeriose/patologia , Masculino , Camundongos , Linfócitos T Auxiliares-Indutores/citologiaRESUMO
BACKGROUND: Biochemical changes induced in red blood cells (RBCs) during storage may impair their function upon transfusion. Transfusion-associated stresses may further amplify storage lesion effects including increased phosphatidylserine (PS) exposure at the RBC membrane, microparticle (MP) release, and adhesion to endothelial cells (ECs). RBC stress susceptibility in vitro was investigated in relation to storage time and additive solution. STUDY DESIGN AND METHODS: Leukoreduced whole blood donations (n = 18) were paired, mixed, and resplit before separating the RBCs for storage in saline-adenine-glucose-mannitol (SAGM) or AS-1. Samples were taken after 3, 21, or 35 days. For oxidative stress treatment, RBCs were exposed to 0.5 mmol/L tert-butylhydroperoxide. Transfusion-associated stress was simulated by overnight culture at 37 °C with plasma containing inflammatory mediators. PS exposure and MPs were measured by flow cytometry and adhesion to ECs was tested under flow conditions. PS specificity of adhesion was tested by blocking with PS-containing lipid vesicles. RESULTS: Oxidative stress induced significantly higher PS exposure and adhesion to ECs in RBCs stored for 35 days compared to 3 days (p < 0.04). PS-containing vesicles blocked RBC-EC adhesion. After overnight culture with or without plasma, PS exposure and EC adhesion were significantly increased (p < 0.05). MP numbers increased with longer RBC storage and after RBC culture with plasma. Culture conditions influenced MP numbers from Day 35 RBCs. RBCs stored in SAGM had significantly higher PS exposure after stress treatment than AS-1 RBCs (p < 0.02). CONCLUSION: Storage for 35 days significantly increased RBC susceptibility to oxidative and in vitro transfusion-associated stresses and was higher for RBCs stored in SAGM compared to AS-1.
Assuntos
Adenina/farmacologia , Preservação de Sangue , Membrana Eritrocítica/metabolismo , Transfusão de Eritrócitos , Glucose/farmacologia , Manitol/farmacologia , Estresse Oxidativo/efeitos dos fármacos , Cloreto de Sódio/farmacologia , Adesão Celular/efeitos dos fármacos , Técnicas de Cocultura , Células Endoteliais/metabolismo , Feminino , Humanos , Masculino , Soluções Farmacêuticas/farmacologia , Fosfatidilserinas/metabolismo , Fatores de TempoRESUMO
Skin inflammation is a complex process implicated in various dermatological disorders. The chronic proliferative dermatitis (cpd) phenotype driven by the cpd mutation (cpdm) in the Sharpin gene is characterized by dermal inflammation and epidermal abnormalities. Tumour necrosis factor (TNF) and caspase-8-driven cell death causes the pathogenesis of Sharpincpdm mice; however, the role of mind bomb 2 (MIB2), a pro-survival E3 ubiquitin ligase involved in TNF signaling, in skin inflammation remains unknown. Here, we demonstrate that MIB2 antagonizes inflammatory dermatitis in the context of the cpd mutation. Surprisingly, the role of MIB2 in limiting skin inflammation is independent of its known pro-survival function and E3 ligase activity. Instead, MIB2 enhances the production of wound-healing molecules, granulocyte colony-stimulating factor, and Eotaxin, within the skin. This discovery advances our comprehension of inflammatory cytokines and chemokines associated with cpdm pathogenesis and highlights the significance of MIB2 in inflammatory skin disease that is independent of its ability to regulate TNF-induced cell death.
RESUMO
Insulin-dependent or type 1 diabetes (T1D) is a paradigm for prevention of autoimmune disease: Pancreatic ß-cell autoantigens are defined, at-risk individuals can be identified before the onset of symptoms, and autoimmune diabetes is preventable in rodent models. Intervention in asymptomatic individuals before or after the onset of subclinical islet autoimmunity places a premium on safety, a requirement met only by lifestyle-dietary approaches or autoantigen-based vaccination to induce protective immune tolerance. Insulin is the key driver of autoimmune ß-cell destruction in the nonobese diabetic (NOD) mouse model of T1D and is an early autoimmune target in children at risk for T1D. In the NOD mouse, mucosal administration of insulin induces regulatory T cells that protect against diabetes. The promise of autoantigen-specific vaccination in humans has yet to be realized, but recent trials of oral and nasal insulin vaccination in at-risk humans provide grounds for cautious optimism.
Assuntos
Autoantígenos/imunologia , Diabetes Mellitus Tipo 1/imunologia , Diabetes Mellitus Tipo 1/prevenção & controle , Vacinação , Animais , Glutamato Descarboxilase/imunologia , Humanos , Insulina/imunologia , Ensaios Clínicos Controlados Aleatórios como AssuntoRESUMO
AIMS/INTRODUCTION: Autoantibodies to pancreatic islet antigens identify young children at high risk of type 1 diabetes. On a background of genetic susceptibility, islet autoimmunity is thought to be driven by environmental factors, of which enteric viruses are prime candidates. We sought evidence for enteric pathology in children genetically at-risk for type 1 diabetes followed from birth who had developed islet autoantibodies ("seroconverted"), by measuring mucosa-associated cytokines in their sera. MATERIALS AND METHODS: Sera were collected 3 monthly from birth from children with a first-degree type 1 diabetes relative, in the Environmental Determinants of Islet Autoimmunity (ENDIA) study. Children who seroconverted were matched for sex, age, and sample availability with seronegative children. Luminex xMap technology was used to measure serum cytokines. RESULTS: Of eight children who seroconverted, for whom serum samples were available at least 6 months before and after seroconversion, the serum concentrations of mucosa-associated cytokines IL-21, IL-22, IL-25, and IL-10, the Th17-related cytokines IL-17F and IL-23, as well as IL-33, IFN-γ, and IL-4, peaked from a low baseline in seven around the time of seroconversion and in one preceding seroconversion. These changes were not detected in eight sex- and age-matched seronegative controls, or in a separate cohort of 11 unmatched seronegative children. CONCLUSIONS: In a cohort of children at risk for type 1 diabetes followed from birth, a transient, systemic increase in mucosa-associated cytokines around the time of seroconversion lends support to the view that mucosal infection, e.g., by an enteric virus, may drive the development of islet autoimmunity.
Assuntos
Diabetes Mellitus Tipo 1 , Ilhotas Pancreáticas , Criança , Humanos , Lactente , Pré-Escolar , Citocinas , Soroconversão , Autoimunidade , AutoanticorposRESUMO
Type 1 diabetes in children is heralded by a preclinical phase defined by circulating autoantibodies to pancreatic islet antigens. How islet autoimmunity is initiated and then progresses to clinical diabetes remains poorly understood. Only one study has reported gene expression in specific immune cells of children at risk associated with progression to islet autoimmunity. We analyzed gene expression with RNA sequencing in CD4+ and CD8+ T cells, natural killer (NK) cells, and B cells, and chromatin accessibility by assay for transposase-accessible chromatin sequencing (ATAC-seq) in CD4+ T cells, in five genetically at risk children with islet autoantibodies who progressed to diabetes over a median of 3 years ("progressors") compared with five children matched for sex, age, and HLA-DR who had not progressed ("nonprogressors"). In progressors, differentially expressed genes (DEGs) were largely confined to CD4+ T cells and enriched for cytotoxicity-related genes/pathways. Several top-ranked DEGs were validated in a semi-independent cohort of 13 progressors and 11 nonprogressors. Flow cytometry confirmed that progression was associated with expansion of CD4+ cells with a cytotoxic phenotype. By ATAC-seq, progression was associated with reconfiguration of regulatory chromatin regions in CD4+ cells, some linked to differentially expressed cytotoxicity-related genes. Our findings suggest that cytotoxic CD4+ T cells play a role in promoting progression to type 1 diabetes.
Assuntos
Linfócitos T CD4-Positivos/metabolismo , Cromatina/química , Citotoxicidade Imunológica/genética , Diabetes Mellitus Tipo 1/imunologia , Progressão da Doença , Regulação da Expressão Gênica , Adolescente , Autoimunidade/genética , Linfócitos B/metabolismo , Linfócitos T CD4-Positivos/imunologia , Linfócitos T CD4-Positivos/ultraestrutura , Linfócitos T CD8-Positivos/metabolismo , Criança , Pré-Escolar , Diabetes Mellitus Tipo 1/genética , Predisposição Genética para Doença , Humanos , Ilhotas Pancreáticas/imunologia , Células Matadoras Naturais/metabolismo , Análise de Sequência de RNARESUMO
AIMS: Studies of the gut microbiome have focused on its bacterial composition. We aimed to characterize the gut fungal microbiome (mycobiome) across pregnancy in women with and without type 1 diabetes. METHODS: Faecal samples (n = 162) were collected from 70 pregnant women (45 with and 25 without type 1 diabetes) across all trimesters. Fungi were analysed by internal transcribed spacer 1 amplicon sequencing. Markers of intestinal inflammation (faecal calprotectin) and intestinal epithelial integrity (serum intestinal fatty acid binding protein; I-FABP), and serum antibodies to Saccharomyces cerevisiae (ASCA) were measured. RESULTS: Women with type 1 diabetes had decreased fungal alpha diversity by the third trimester, associated with an increased abundance of Saccharomyces cerevisiae that was inversely related to the abundance of the anti-inflammatory butyrate-producing bacterium Faecalibacterium prausnitzii. Women with type 1 diabetes had higher concentrations of calprotectin, I-FABP and ASCA. CONCLUSIONS: Women with type 1 diabetes exhibit a shift in the gut mycobiome across pregnancy associated with evidence of gut inflammation and impaired intestinal barrier function. The relevance of these findings to the higher rate of pregnancy complications in type 1 diabetes warrants further study.
Assuntos
Diabetes Mellitus Tipo 1 , Microbioma Gastrointestinal , Micobioma , Fezes/microbiologia , Feminino , Microbioma Gastrointestinal/genética , Humanos , Inflamação , Gravidez , Saccharomyces cerevisiaeRESUMO
Following activation by antigen, T cells enter the cell cycle in stages that can be defined with flow cytometric markers. We show that these markers include the increase of forward light scatter width (FSC-W) signal and the ratio of FSC area/peak. This change in light scatter precedes the first cell division and may reflect blast transformation. We show that the FSC-W parameter can be used, alone or in combination with other activation markers, to monitor the relative and absolute numbers of T cells responding to a proliferative stimulus. In contrast to dye dilution assays, the FSC-W method does not allow discrimination between consecutive cell divisions, but it has several advantages and could complement the dye dilution assay. Our findings also show that the routine elimination of doublets based on FSC signals may exclude proliferating T cells from the analysis.
Assuntos
Proliferação de Células , Citometria de Fluxo/métodos , Ativação Linfocitária , Linfócitos T/imunologia , Animais , Citocinas/análise , Fluoresceínas/química , Corantes Fluorescentes/química , Humanos , Luz , Camundongos , Espalhamento de Radiação , Succinimidas/químicaRESUMO
Remodelling of chromatin architecture is known to regulate gene expression and has been well characterized in cell lineage development but less so in response to cell perturbation. Activation of T cells, which triggers extensive changes in transcriptional programs, serves as an instructive model to elucidate how changes in chromatin architecture orchestrate gene expression in response to cell perturbation. To characterize coordinate changes at different levels of chromatin architecture, we analyzed chromatin accessibility, chromosome conformation and gene expression in activated human T cells. T cell activation was characterized by widespread changes in chromatin accessibility and interactions that were shared between activated CD4+ and CD8+ T cells, and with the formation of active regulatory regions associated with transcription factors relevant to T cell biology. Chromatin interactions that increased and decreased were coupled, respectively, with up- and down-regulation of corresponding target genes. Furthermore, activation was associated with disruption of long-range chromatin interactions and with partitioning of topologically associating domains (TADs) and remodelling of their TAD boundaries. Newly formed/strengthened TAD boundaries were associated with higher nucleosome occupancy and lower accessibility, linking changes in lower and higher order chromatin architecture. T cell activation exemplifies coordinate multi-level remodelling of chromatin underlying gene transcription.
Assuntos
Montagem e Desmontagem da Cromatina/genética , Montagem e Desmontagem da Cromatina/fisiologia , Cromatina/química , Cromatina/genética , Regulação da Expressão Gênica no Desenvolvimento/genética , Ativação Linfocitária/genética , Linfócitos T/imunologia , Linfócitos T CD4-Positivos , Linfócitos T CD8-Positivos , Células Cultivadas , Humanos , Masculino , Nucleossomos/genética , Fatores de Transcrição , Transcrição Gênica/genéticaRESUMO
BACKGROUND: The gut microbiome changes in response to a range of environmental conditions, life events and disease states. Pregnancy is a natural life event that involves major physiological adaptation yet studies of the microbiome in pregnancy are limited and their findings inconsistent. Pregnancy with type 1 diabetes (T1D) is associated with increased maternal and fetal risks but the gut microbiome in this context has not been characterized. By whole metagenome sequencing (WMS), we defined the taxonomic composition and function of the gut bacterial microbiome across 70 pregnancies, 36 in women with T1D. RESULTS: Women with and without T1D exhibited compositional and functional changes in the gut microbiome across pregnancy. Profiles in women with T1D were distinct, with an increase in bacteria that produce lipopolysaccharides and a decrease in those that produce short-chain fatty acids, especially in the third trimester. In addition, women with T1D had elevated concentrations of fecal calprotectin, a marker of intestinal inflammation, and serum intestinal fatty acid-binding protein (I-FABP), a marker of intestinal epithelial damage. CONCLUSIONS: Women with T1D exhibit a shift towards a more pro-inflammatory gut microbiome during pregnancy, associated with evidence of intestinal inflammation. These changes could contribute to the increased risk of pregnancy complications in women with T1D and are potentially modifiable by dietary means. Video abstract.
Assuntos
Diabetes Mellitus Tipo 1 , Microbioma Gastrointestinal , Gravidez em Diabéticas/microbiologia , Diabetes Mellitus Tipo 1/microbiologia , Fezes , Feminino , Microbioma Gastrointestinal/genética , Humanos , Intestinos , Metagenoma , GravidezRESUMO
The complex cytopathology of mitochondrial diseases is usually attributed to insufficient ATP. AMP-activated protein kinase (AMPK) is a highly sensitive cellular energy sensor that is stimulated by ATP-depleting stresses. By antisense-inhibiting chaperonin 60 expression, we produced mitochondrially diseased strains with gene dose-dependent defects in phototaxis, growth, and multicellular morphogenesis. Mitochondrial disease was phenocopied in a gene dose-dependent manner by overexpressing a constitutively active AMPK alpha subunit (AMPKalphaT). The aberrant phenotypes in mitochondrially diseased strains were suppressed completely by antisense-inhibiting AMPKalpha expression. Phagocytosis and macropinocytosis, although energy consuming, were unaffected by mitochondrial disease and AMPKalpha expression levels. Consistent with the role of AMPK in energy homeostasis, mitochondrial "mass" and ATP levels were reduced by AMPKalpha antisense inhibition and increased by AMPKalphaT overexpression, but they were near normal in mitochondrially diseased cells. We also found that 5-aminoimidazole-4-carboxamide-1-beta-D-ribofuranoside, a pharmacological AMPK activator in mammalian cells, mimics mitochondrial disease in impairing Dictyostelium phototaxis and that AMPKalpha antisense-inhibited cells were resistant to this effect. The results show that diverse cytopathologies in Dictyostelium mitochondrial disease are caused by chronic AMPK signaling not by insufficient ATP.
Assuntos
Doenças Mitocondriais/enzimologia , Doenças Mitocondriais/patologia , Complexos Multienzimáticos/metabolismo , Proteínas Serina-Treonina Quinases/metabolismo , Proteínas Quinases Ativadas por AMP , Trifosfato de Adenosina/biossíntese , Sequência de Aminoácidos , Animais , Sequência de Bases , DNA de Protozoário/genética , Dictyostelium/enzimologia , Dictyostelium/genética , Dictyostelium/crescimento & desenvolvimento , Dosagem de Genes , Genes de Protozoários , Humanos , Doenças Mitocondriais/genética , Modelos Biológicos , Dados de Sequência Molecular , Complexos Multienzimáticos/antagonistas & inibidores , Complexos Multienzimáticos/química , Complexos Multienzimáticos/genética , Fagocitose , Fotobiologia , Pinocitose , Proteínas Serina-Treonina Quinases/antagonistas & inibidores , Proteínas Serina-Treonina Quinases/química , Proteínas Serina-Treonina Quinases/genética , Estrutura Terciária de Proteína , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Transdução de SinaisRESUMO
Human CD52 is a small glycopeptide (12 amino acid residues) with one N-linked glycosylation site at asparagine 3 (Asn3) and several potential O-glycosylation serine/threonine sites. Soluble CD52 is released from the surface of activated T cells and mediates immune suppression via its glycan moiety. In suppressing activated T cells, it first sequesters the pro-inflammatory high mobility group Box 1 (HMGB1) protein, which facilitates its binding to the inhibitory sialic acid-binding immunoglobulin-like lectin-10 (Siglec-10) receptor. We aimed to identify the features of CD52 glycan that underlie its bioactivity. Analysis of native CD52 purified from human spleen revealed extensive heterogeneity in N-glycosylation and multi-antennary sialylated N-glycans with abundant polyLacNAc extensions, together with mainly di-sialylated O-glycosylation type structures. Glycomic (porous graphitized carbon-ESI-MS/MS) and glycopeptide (C8-LC-ESI-MS) analysis of recombinant soluble human CD52-immunoglobulin Fc fusion proteins revealed that CD52 bioactivity was correlated with a high abundance of tetra-antennary α-2,3/6 sialylated N-glycans. Removal of α-2,3 sialylation abolished bioactivity, which was restored by re-sialylation with α-2,3 sialyltransferases. When glycoforms of CD52-Fc were fractionated by anion exchange MonoQ-GL chromatography, bioactive fractions displayed mainly tetra-antennary, α-2,3 sialylated N-glycan structures and a lower relative abundance of bisecting GlcNAc structures compared to non-bioactive fractions. In addition, O-glycan core type-2 di-sialylated structures at Ser12 were more abundant in bioactive CD52 fractions. Understanding the structural features of CD52 glycan required for its bioactivity will aid its development as an immunotherapeutic agent.
Assuntos
Antígeno CD52/imunologia , Antígeno CD52/metabolismo , Imunomodulação , Antígeno CD52/sangue , Antígeno CD52/isolamento & purificação , Cromatografia por Troca Iônica , Ensaio de Imunoadsorção Enzimática , Glicosilação , Humanos , Polissacarídeos/metabolismo , Proteínas Recombinantes , Baço/imunologia , Baço/metabolismoRESUMO
RIPK1 is an essential downstream component of many pattern recognition and death receptors. RIPK1 can promote the activation of caspase-8 induced apoptosis and RIPK3-MLKL-mediated necroptosis, however, during development RIPK1 limits both forms of cell death. Accordingly, Ripk1-/- mice present with systemic cell death and consequent multi-organ inflammation, which is driven through the activation of both FADD-caspase-8 and RIPK3-MLKL signaling pathways causing perinatal lethality. TRADD is a death domain (DD) containing molecule that mediates signaling downstream of TNFR1 and the TLRs. Following the disassembly of the upstream receptor complexes either RIPK1 or TRADD can form a complex with FADD-caspase-8-cFLIP, via DD-DD interactions with FADD, facilitating the activation of caspase-8. We show that genetic deletion of Ripk1 licenses TRADD to complex with FADD-caspase-8 and activates caspase-8 during development. Deletion of Tradd provided no survival advantage to Ripk1-/- animals and yet was sufficient to reduce the systemic cell death and inflammation, rescue the intestinal and thymic histopathologies, reduce cleaved caspases in most tissues and rescue the anemia observed in Ripk1-/- neonates. Furthermore, deletion of Ripk3 is sufficient to rescue the neonatal lethality of Ripk1-/-Tradd-/- animals and delays but does not completely prevent early mortality. Although Ripk3 deletion provides a significant survival advantage, Ripk1-/-Tradd-/-Ripk3-/- animals die between 22 and 49 days, are runty compared to littermate controls and present with splenomegaly. These findings reveal a new mechanism by which RIPK1 limits apoptosis through blocking TRADD recruitment to FADD and preventing aberrant activation of caspase-8.
Assuntos
Desenvolvimento Embrionário/genética , Proteína Serina-Treonina Quinases de Interação com Receptores/genética , Proteína de Domínio de Morte Associada a Receptor de TNF/genética , Animais , Animais Recém-Nascidos , Apoptose/genética , Caspase 8/genética , Morte Celular/genética , Proteína de Domínio de Morte Associada a Fas/genética , Inflamação/genética , Inflamação/patologia , Camundongos , Camundongos Knockout , Receptores Tipo I de Fatores de Necrose Tumoral/genética , Transdução de Sinais/genética , Fator de Necrose Tumoral alfa/genéticaRESUMO
Purpose: To evaluate the effect of intravitreal ranibizumab injections on aqueous concentrations of angiogenic or inflammatory cytokines in patients with diabetic macular edema (DME). Methods: Thirty eyes of 25 patients with center-involved DME were recruited to the study. All had a central macular thickness (CMT) of >300 µm and best-corrected visual acuity (BCVA) between 28 and 70 logMAR letters (Snellen equivalent 20/320-20/40). At baseline, all eyes had 0.1 mL of aqueous collected before ranibizumab treatment. At week 4, a second ranibizumab injection was administered and at week 8, aqueous sampling was repeated before a third ranibizumab injection. From week 12, all eyes were followed at 4-weekly intervals and the need for ranibizumab treatment was determined by BCVA and CMT measurements. Levels of 32 cytokines were assessed at baseline and at week 8 using a multiplex array assay. Results: Following two consecutive ranibizumab injections, there was a statistically significant reduction in VEGF (P < 0.00001), as well as IL-1ß (P = 0.00006), IL-7 (P = 0.00002), IL-8 (P = 0.00023), IL-10 (P < 0.00001), IL-12 (P < 0.00001), IL-17 (P = 0.00024), MCP-1 (P = 0.00023), and TNF-α (P < 0.00001). There was also an upregulation of soluble VEGF receptor-2 (P = 0.00004). A P < 0.0015 was considered significant in this study. Conclusions: Ranibizumab treatment influences various inflammatory cytokine concentrations in addition to reducing aqueous VEGF concentrations in patients with DME. This may contribute to its therapeutic effect in patients with DME.
Assuntos
Inibidores da Angiogênese/uso terapêutico , Humor Aquoso/metabolismo , Citocinas/metabolismo , Retinopatia Diabética/tratamento farmacológico , Inflamação/metabolismo , Edema Macular/tratamento farmacológico , Ranibizumab/uso terapêutico , Retinopatia Diabética/metabolismo , Feminino , Humanos , Injeções Intravítreas , Edema Macular/metabolismo , Masculino , Pessoa de Meia-Idade , Tomografia de Coerência Óptica , Fator A de Crescimento do Endotélio Vascular/antagonistas & inibidores , Acuidade Visual/fisiologiaRESUMO
Soluble CD52 is a small glycoprotein that suppresses T-cell activation, but its effect on innate immune cell function is unknown. Here we demonstrate that soluble CD52 inhibits Toll-like receptor and tumor necrosis factor receptor signaling to limit activation of NF-κB and thereby suppress the production of inflammatory cytokines by macrophages, monocytes and dendritic cells. At higher concentrations, soluble CD52 depletes the short-lived pro-survival protein MCL-1, contributing to activation of the BH3-only proteins BAX and BAK to cause intrinsic apoptotic cell death. In vivo, administration of soluble CD52 suppresses lipopolysaccharide (LPS)-induced cytokine secretion and other features of endotoxic shock, whereas genetic deletion of CD52 exacerbates LPS responses. Thus, soluble CD52 exhibits broad immune suppressive effects that signify its potential as an immunotherapeutic agent.