RESUMO
Introduction: Prior to pregnancy, hormonal changes lead to cellular adaptations in the endometrium allowing for embryo implantation. Critical for successful pregnancy establishment, innate immune cells constitute a significant proportion of uterine cells prior to arrival of the embryo and throughout the first trimester in humans and animal models. Abnormal uterine immune cell function during implantation is believed to play a role in multiple adverse pregnancy outcomes. Current work in humans has focused on uterine immune cells present after pregnancy establishment, and limited in vitro models exist to explore unique functions of these cells. Methods: With single-cell RNA-sequencing (scRNAseq), we comprehensively compared the human uterine immune landscape of the endometrium during the window of implantation and the decidua during the first trimester of pregnancy. Results: We uncovered global and cell-type-specific gene signatures for each timepoint. Immune cells in the endometrium prior to implantation expressed genes associated with immune metabolism, division, and activation. In contrast, we observed widespread interferon signaling during the first trimester of pregnancy. We also provide evidence of specific inflammatory pathways enriched in pre- and post-implantation macrophages and natural killer (NK) cells in the uterine lining. Using our novel implantation-on-a-chip (IOC) to model human implantation ex vivo, we demonstrate for the first time that uterine macrophages strongly promote invasion of extravillous trophoblasts (EVTs), a process essential for pregnancy establishment. Pre- and post-implantation uterine macrophages promoted EVT invasion to a similar degree as pre- and post-implantation NK cells on the IOC. Conclusions: This work provides a foundation for further investigation of the individual roles of uterine immune cell subtypes present prior to embryo implantation and during early pregnancy, which will be critical for our understanding of pregnancy complications associated with abnormal trophoblast invasion and placentation.
Assuntos
Decídua , Implantação do Embrião , Gravidez , Feminino , Animais , Humanos , Decídua/metabolismo , Útero , Células Matadoras Naturais , MacrófagosRESUMO
During infection, virus-specific CD8+ T cells undergo rapid bursts of proliferation and differentiate into effector cells that kill virus-infected cells and reduce viral load. This rapid clonal expansion can put T cells at significant risk for replication-induced DNA damage. Here, we find that c-Myc links CD8+ T cell expansion to DNA damage response pathways though the E3 ubiquitin ligase, Cullin 4b (Cul4b). Following activation, c-Myc increases the levels of Cul4b and other members of the Cullin RING Ligase 4 (CRL4) complex. Despite expressing c-Myc at high levels, Cul4b-deficient CD8+ T cells do not expand and clear the Armstrong strain of lymphocytic choriomeningitis virus (LCMV) in vivo. Cul4b-deficient CD8+ T cells accrue DNA damage and succumb to proliferative catastrophe early after antigen encounter. Mechanistically, Cul4b knockout induces an accumulation of p21 and Cyclin E2, resulting in replication stress. Our data show that c-Myc supports cell proliferation by maintaining genome stability via Cul4b, thereby directly coupling these two interdependent pathways. These data clarify how CD8+ T cells use c-Myc and Cul4b to sustain their potential for extraordinary population expansion, longevity and antiviral responses.
Assuntos
Linfócitos T CD8-Positivos , Proteínas Culina , Vírus da Coriomeningite Linfocítica , Proteínas Proto-Oncogênicas c-myc , Linfócitos T CD8-Positivos/imunologia , Ciclo Celular , Proteínas Culina/metabolismo , Ubiquitina-Proteína Ligases/metabolismo , Proteínas Proto-Oncogênicas c-myc/metabolismoRESUMO
BACKGROUNDCellular cholesterol efflux capacity (CEC) is a better predictor of cardiovascular disease (CVD) events than HDL-cholesterol (HDL-C) but is not suitable as a routine clinical assay.METHODSWe developed an HDL-specific phospholipid efflux (HDL-SPE) assay to assess HDL functionality based on whole plasma HDL apolipoprotein-mediated solubilization of fluorescent phosphatidylethanolamine from artificial lipid donor particles. We first assessed the association of HDL-SPE with prevalent coronary artery disease (CAD): study I included NIH severe-CAD (n = 50) and non-CAD (n = 50) participants, who were frequency matched for sex, BMI, type 2 diabetes mellitus, and smoking; study II included Japanese CAD (n = 70) and non-CAD (n = 154) participants. We also examined the association of HDL-SPE with incident CVD events in the Prevention of Renal and Vascular End-stage Disease (PREVEND) study comparing 340 patients with 340 controls individually matched for age, sex, smoking, and HDL-C levels.RESULTSReceiver operating characteristic curves revealed stronger associations of HDL-SPE with prevalent CAD. The AUCs in study I were as follows: HDL-SPE, 0.68; apolipoprotein A-I (apoA-I), 0.62; HDL-C, 0.63; and CEC, 0.52. The AUCs in study II were as follows: HDL-SPE, 0.83; apoA-I, 0.64; and HDL-C, 0.53. Also longitudinally, HDL-SPE was significantly associated with incident CVD events independent of traditional risk factors with ORs below 0.2 per SD increment in the PREVEND study (P < 0.001).CONCLUSIONHDL-SPE could serve as a routine clinical assay for improving CVD risk assessment and drug discovery.TRIAL REGISTRATIONClinicalTrials.gov NCT01621594.FUNDINGNHLBI Intramural Research Program, NIH (HL006095-06).
Assuntos
Doenças Cardiovasculares , Doença da Artéria Coronariana , Diabetes Mellitus Tipo 2 , Humanos , Lipoproteínas HDL , Doenças Cardiovasculares/diagnóstico , Doenças Cardiovasculares/epidemiologia , Apolipoproteína A-I , HDL-Colesterol , FosfolipídeosRESUMO
ApoB-100 is a member of a large lipid transfer protein superfamily and is one of the main apolipoproteins found on low-density lipoprotein (LDL) and very low-density lipoprotein (VLDL) particles. Despite its clinical significance for the development of cardiovascular disease, there is limited information on apoB-100 structure. We have developed a novel method based on the "divide and conquer" algorithm, using PSIPRED software, by dividing apoB-100 into five subunits and 11 domains. Models of each domain were prepared using I-TASSER, DEMO, RoseTTAFold, Phyre2, and MODELLER. Subsequently, we used disuccinimidyl sulfoxide (DSSO), a new mass spectrometry cleavable cross-linker, and the known position of disulfide bonds to experimentally validate each model. We obtained 65 unique DSSO cross-links, of which 87.5% were within a 26 Å threshold in the final model. We also evaluated the positions of cysteine residues involved in the eight known disulfide bonds in apoB-100, and each pair was measured within the expected 5.6 Å constraint. Finally, multiple domains were combined by applying constraints based on detected long-range DSSO cross-links to generate five subunits, which were subsequently merged to achieve an uninterrupted architecture for apoB-100 around a lipoprotein particle. Moreover, the dynamics of apoB-100 during particle size transitions was examined by comparing VLDL and LDL computational models and using experimental cross-linking data. In addition, the proposed model of receptor ligand binding of apoB-100 provides new insights into some of its functions.
Assuntos
Apolipoproteínas B , Cisteína , Apolipoproteína B-100 , Apolipoproteínas B/metabolismo , Simulação por Computador , Dissulfetos , Ligantes , Lipoproteínas LDL/química , Lipoproteínas VLDL , Modelos Estruturais , SulfóxidosRESUMO
Dennd5b plays a pivotal role in intestinal absorption of dietary lipids in mice and is associated with body mass index in humans. This study examined the impact of whole-body Dennd5b deletion on plasma lipid concentrations, atherosclerosis, and hepatic lipid metabolism in mice. Hypercholesterolemia was induced in Dennd5b-/- mice by infection with an adeno-associated virus expressing the proprotein convertase subtilisin/kexin type 9 serine protease (PCSK9) gain-of-function mutation (PCSK9D377Y) and feeding a Western diet for 12 weeks. Body weight and plasma lipid concentrations were monitored over 12 weeks, and then aortic atherosclerosis and hepatic lipid content were quantified. Compared to Dennd5b+/+ mice, Dennd5b-/- mice were resistant to diet-induced weight gain and PCSK9-induced hypercholesterolemia. Atherosclerosis quantified by en face analysis and in aortic root sections, revealed significantly smaller lesions in Dennd5b-/- compared to Dennd5b+/+ mice. Additionally, Dennd5b-/- mice had significantly less hepatic lipid content (triglyceride and cholesterol) compared to Dennd5b+/+ mice. To gain insight into the basis for reduced hepatic lipids, quantitative PCR was used to measure mRNA abundance of genes involved in hepatic lipid metabolism. Key genes involved in hepatic lipid metabolism and lipid storage were differentially expressed in Dennd5b-/- liver including Pparg, Cd36, and Pnpla3. These findings demonstrate a significant impact of Dennd5b on plasma and hepatic lipid concentrations and resistance to PCSK9-induced hypercholesterolemia in the absence of Dennd5b.
Assuntos
Aterosclerose , Fígado Gorduroso , Fatores de Troca do Nucleotídeo Guanina , Hipercolesterolemia , Animais , Humanos , Camundongos , Aciltransferases , Aterosclerose/patologia , Colesterol , Dieta , Fígado Gorduroso/genética , Hipercolesterolemia/genética , Fosfolipases A2 Independentes de Cálcio , Pró-Proteína Convertase 9/genética , Receptores de LDL/metabolismo , Fatores de Troca do Nucleotídeo Guanina/metabolismoRESUMO
Due to heightened awareness and advanced genetic tools, inborn errors of immunity (IEI) are increasingly recognized in children. However, diagnosing of IEI in premature infants is challenging and, subsequently, reports of IEI in premature infants remain rare. This review focuses on how common disorders of prematurity, such as sepsis, necrotizing enterocolitis, and bronchopulmonary dysplasia, can clinically overlap with presenting signs of IEI. We present four recent cases from a single neonatal intensive care unit that highlight diagnostic dilemmas facing neonatologists and clinical immunologists when considering IEI in preterm infants. Finally, we present a conceptual framework for when to consider IEI in premature infants and a guide to initial workup of premature infants suspected of having IEI.
Assuntos
Diagnóstico Tardio , Síndromes de Imunodeficiência/diagnóstico , Doenças do Prematuro/diagnóstico , Infecções/etiologia , Infecções Bacterianas/etiologia , Displasia Broncopulmonar/complicações , Candida parapsilosis , Candidíase Invasiva/complicações , Criança , Diagnóstico Diferencial , Enterocolite Necrosante/complicações , Evolução Fatal , Feminino , Estudos de Associação Genética , Humanos , Síndromes de Imunodeficiência/complicações , Síndromes de Imunodeficiência/genética , Recém-Nascido , Recém-Nascido Prematuro , Doenças do Prematuro/genética , Inflamação , Unidades de Terapia Intensiva Neonatal , Linfo-Histiocitose Hemofagocítica/diagnóstico , Masculino , Meningites Bacterianas/etiologia , Recidiva , Sepse/etiologiaRESUMO
Disruption in homeostasis of IL-15 is linked to poor maternal and fetal outcomes during pregnancy. The only cells described to respond to IL-15 at the early maternal-fetal interface have been NK cells. We now show a novel population of macrophages, evident in several organs but enriched in the uterus of mice and humans, expressing the ß-chain of the IL-15R complex (CD122) and responding to IL-15. CD122+ macrophages (CD122+Macs) are morphologic, phenotypic, and transcriptomic macrophages that can derive from bone marrow monocytes. CD122+Macs develop in the uterus and placenta with kinetics that mirror IFN activity at the maternal-fetal interface. M-CSF permits macrophages to express CD122, and IFNs are sufficient to drive expression of CD122 on macrophages. Neither type I nor type II IFNs are required to generate CD122+Macs, however. In response to IL-15, CD122+Macs activate the ERK signaling cascade and enhance production of proinflammatory cytokines after stimulation with the TLR9 agonist CpG. Finally, we provide evidence of human cells that phenocopy murine CD122+Macs in secretory phase endometrium during the implantation window and in first-trimester uterine decidua. Our data support a model wherein IFNs local to the maternal-fetal interface direct novel IL-15-responsive macrophages with the potential to mediate IL-15 signals critical for optimal outcomes of pregnancy.
Assuntos
Interferons/metabolismo , Interleucina-15/metabolismo , Macrófagos/metabolismo , Adolescente , Adulto , Animais , Ilhas de CpG/fisiologia , Citocinas/metabolismo , Decídua/metabolismo , Feminino , Humanos , Inflamação/metabolismo , Subunidade beta de Receptor de Interleucina-2/metabolismo , Células Matadoras Naturais/metabolismo , Sistema de Sinalização das MAP Quinases/fisiologia , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Monócitos/metabolismo , Placenta/metabolismo , Gravidez , Primeiro Trimestre da Gravidez/metabolismo , Transdução de Sinais/fisiologia , Receptor Toll-Like 9/metabolismo , Transcriptoma/fisiologia , Adulto JovemRESUMO
BACKGROUND: Eicosapentaenoic acid (EPA) and docosahexaenoic acid (DHA) have both shared and different cardiovascular effects, and commonly used fish oil supplements have considerably varied EPA/DHA ratios. AIMS: We compared the effects of fish oil supplements with different EPA/DHA ratios on lipoprotein metabolism. METHODS: In a double-blind, randomized cross-over study, normolipidemic adults (n = 30) consumed 12 g/day of EPA-rich (EPA/DHA: 2.3) or DHA-rich (EPA/DHA: 0.3) fish oil for 8-weeks, separated by an 8-week washout period. RESULTS: Both fish oil supplements similarly lowered plasma TG levels and TG-related NMR parameters versus baseline (p < 0.05). There were no changes in plasma cholesterol-related parameters due to either fish oil, although on-treatment levels for LDL particle number were slightly higher for DHA-rich oil compared with EPA-rich oil (p < 0.05). Both fish oil supplements similarly altered HDL subclass profile and proteome, and down regulated HDL proteins related to inflammation, with EPA-rich oil to a greater extent. Furthermore, EPA-rich oil increased apoM abundance versus DHA-rich oil (p < 0.05). CONCLUSIONS: Overall, fish oil supplements with varied EPA/DHA ratios had similar effects on total lipids/lipoproteins, but differences were observed in lipoprotein subfraction composition and distribution, which could impact on the use of EPA versus DHA for improving cardiovascular health.
Assuntos
Ácidos Docosa-Hexaenoicos/administração & dosagem , Ácido Eicosapentaenoico/administração & dosagem , Lipoproteínas HDL/sangue , Lipoproteínas LDL/sangue , Triglicerídeos/sangue , Adulto , Estudos Cross-Over , Método Duplo-Cego , Feminino , Humanos , MasculinoRESUMO
BACKGROUND: Patients with type 1 diabetes (T1DM) typically have normal or even elevated plasma high density lipoprotein (HDL) cholesterol concentrations; however, HDL protein composition can be altered without a change in cholesterol content. Alteration of the HDL proteome can result in dysfunctional HDL particles with reduced ability to protect against cardiovascular disease (CVD). The objective of this study was to compare the HDL proteomes of youth with T1DM and healthy controls (HC) and to evaluate the influence of glycemic control on HDL protein composition. METHODS: This was a cross-sectional case-control study. Blood samples were obtained from patients with T1DM and HC. HDL was isolated from plasma by size-exclusion chromatography and further purified using a lipid binding resin. The HDL proteome was analyzed by mass spectrometry using label-free SWATH peptide quantification. RESULTS: Samples from 26 patients with T1DM and 13 HC were analyzed and 78 HDL-bound proteins were measured. Youth with T1DM had significantly increased amounts of complement factor H related protein 2 (FHR2; adjusted P < 0.05), compared to HC. When patients were analyzed based on glucose control, several trends emerged. Some proteins were altered in T1DM and not influenced by glycemic control (e.g. FHR2) while others were partially or completely corrected with optimal glucose control (e.g. alpha-1-beta glycoprotein, A1BG). In a subgroup of poorly controlled T1DM patients, inter alpha trypsin inhibitor 4 (ITIH4) was dramatically elevated (P < 0.0001) and this was partially reversed in patients with optimal glucose control. Some proteins including complement component C3 (CO3) and albumin (ALB) were significantly different only in T1DM patients with optimal glucose control, suggesting a possible effect of exogenous insulin. CONCLUSIONS: Youth with T1DM have proteomic alterations of their HDL compared to HC, despite similar concentration of HDL cholesterol. The influence of these compositional changes on HDL function are not yet known. Future efforts should focus on investigating the role of these HDL associated proteins in regard to HDL function and their role in CVD risk in patients with T1DM. Trial registration NCT02275091.
Assuntos
Glicemia/efeitos dos fármacos , Diabetes Mellitus Tipo 1/sangue , Diabetes Mellitus Tipo 1/tratamento farmacológico , Hipoglicemiantes/uso terapêutico , Lipoproteínas HDL/sangue , Proteômica/métodos , Adolescente , Fatores Etários , Animais , Biomarcadores/sangue , Glicemia/metabolismo , Estudos de Casos e Controles , Linhagem Celular , Criança , HDL-Colesterol/sangue , Cromatografia Líquida , Diabetes Mellitus Tipo 1/diagnóstico , Feminino , Humanos , Macrófagos/metabolismo , Masculino , Camundongos , Espectrometria de Massas em Tandem , Resultado do Tratamento , Adulto JovemRESUMO
Lecithin:cholesterol acyltransferase (LCAT) catalyzes plasma cholesteryl ester formation and is defective in familial lecithin:cholesterol acyltransferase deficiency (FLD), an autosomal recessive disorder characterized by low high-density lipoprotein, anemia, and renal disease. This study aimed to investigate the mechanism by which compound A [3-(5-(ethylthio)-1,3,4-thiadiazol-2-ylthio)pyrazine-2-carbonitrile], a small heterocyclic amine, activates LCAT. The effect of compound A on LCAT was tested in human plasma and with recombinant LCAT. Mass spectrometry and nuclear magnetic resonance were used to determine compound A adduct formation with LCAT. Molecular modeling was performed to gain insight into the effects of compound A on LCAT structure and activity. Compound A increased LCAT activity in a subset (three of nine) of LCAT mutations to levels comparable to FLD heterozygotes. The site-directed mutation LCAT-Cys31Gly prevented activation by compound A. Substitution of Cys31 with charged residues (Glu, Arg, and Lys) decreased LCAT activity, whereas bulky hydrophobic groups (Trp, Leu, Phe, and Met) increased activity up to 3-fold (P < 0.005). Mass spectrometry of a tryptic digestion of LCAT incubated with compound A revealed a +103.017 m/z adduct on Cys31, consistent with the addition of a single hydrophobic cyanopyrazine ring. Molecular modeling identified potential interactions of compound A near Cys31 and structural changes correlating with enhanced activity. Functional groups important for LCAT activation by compound A were identified by testing compound A derivatives. Finally, sulfhydryl-reactive ß-lactams were developed as a new class of LCAT activators. In conclusion, compound A activates LCAT, including some FLD mutations, by forming a hydrophobic adduct with Cys31, thus providing a mechanistic rationale for the design of future LCAT activators.
Assuntos
Cisteína/fisiologia , Fosfatidilcolina-Esterol O-Aciltransferase/metabolismo , Compostos de Sulfidrila/farmacologia , Relação Dose-Resposta a Droga , Ativação Enzimática/efeitos dos fármacos , Ativação Enzimática/fisiologia , Ativadores de Enzimas/química , Ativadores de Enzimas/metabolismo , Ativadores de Enzimas/farmacologia , Células HEK293 , Humanos , Deficiência da Lecitina Colesterol Aciltransferase/metabolismo , Modelos Moleculares , Fosfatidilcolina-Esterol O-Aciltransferase/química , Compostos de Sulfidrila/químicaRESUMO
BACKGROUND AND AIMS: Concentrated fish oils, containing a mixture of long-chain monounsaturated fatty acids (LCMUFA) with aliphatic chains longer than 18 C atoms (i.e., C20:1 and C22:1), have been shown to attenuate atherosclerosis development in mouse models. It is not clear, however, how individual LCMUFA isomers may act on atherosclerosis. METHODS: In the present study, we used saury fish oil-derived concentrates enriched in either C20:1 or C22:1 isomer fractions to investigate their individual effect on atherosclerosis and lipoprotein metabolism. LDLR-deficient (LDLr-/-) mice were fed a Western diet supplemented with 5% (w/w) of either C20:1 or C22:1 concentrate for 12 wk. RESULTS: Compared to the control Western diet with no supplement, both LCMUFA isomers increased hepatic levels of LCMUFA by 2â¼3-fold (p < 0.05), and decreased atherosclerotic lesion areas by more than 40% (p < 0.05), although there were no major differences in plasma lipoproteins or hepatic lipid content. Both LCMUFA isomers significantly decreased plasma CRP levels, improved Abca1-dependent cholesterol efflux capacity of apoB-depleted plasma, and enhanced Ppar transcriptional activities in HepG2 cells. LC-MS/MS proteomic analysis of lipoproteins (HDL, LDL and VLDL) revealed that both LCMUFA isomer diets resulted in similar potentially beneficial alterations in proteins involved in complement activation, blood coagulation, and lipid metabolism. Several lipoprotein proteome changes were significantly correlated with atherosclerotic plaque reduction. CONCLUSIONS: Dietary supplementation with the LCMUFA isomers C20:1 or C22:1 was equally effective in reducing atherosclerosis in LDLr-/-mice and this may partly occur through activation of the Ppar signaling pathways and favorable alterations in the proteome of lipoproteins.
Assuntos
Doenças da Aorta/prevenção & controle , Aterosclerose/prevenção & controle , Suplementos Nutricionais , Ácidos Graxos Monoinsaturados/farmacologia , Óleos de Peixe/farmacologia , Hiperlipidemias/tratamento farmacológico , Lipoproteínas/sangue , Proteoma , Receptores de LDL/deficiência , Animais , Doenças da Aorta/sangue , Doenças da Aorta/genética , Aterosclerose/sangue , Aterosclerose/genética , Aterosclerose/patologia , Biomarcadores/sangue , Proteína C-Reativa/metabolismo , Cromatografia Líquida , Dieta Ocidental , Modelos Animais de Doenças , Predisposição Genética para Doença , Células Hep G2 , Humanos , Hiperlipidemias/sangue , Hiperlipidemias/genética , Hiperlipidemias/patologia , Fígado/efeitos dos fármacos , Fígado/metabolismo , Camundongos Knockout , Receptores Ativados por Proliferador de Peroxissomo/genética , Receptores Ativados por Proliferador de Peroxissomo/metabolismo , Fenótipo , Placa Aterosclerótica , Proteômica/métodos , Receptores de LDL/genética , Espectrometria de Massas em TandemRESUMO
Apolipoprotein B (apoB) is a large amphipathic protein that is the structural scaffold for the formation of several classes of lipoproteins involved in lipid transport throughout the body. The goal of the present study was to identify specific domains in the apoB sequence that contribute to its lipid binding properties. A sequence analysis algorithm was developed to identify stretches of hydrophobic amino acids devoid of charged amino acids, which are referred to as hydrophobic cluster domains (HCDs). This analysis identified 78 HCDs in apoB with hydrophobic stretches ranging from 6 to 26 residues. Each HCD was analyzed in silico for secondary structure and lipid binding properties, and a subset was synthesized for experimental evaluation. One HCD peptide, B38, showed high affinity binding to both isolated HDL and LDL, and could exchange between lipoproteins. All-atom molecular dynamics simulations indicate that B38 inserts 3.7Å below the phosphate plane of the bilayer. B38 forms an unusual α-helix with a broad hydrophobic face and polar serine and threonine residues on the opposite face. Based on this structure, we hypothesized that B38 could efflux cholesterol from cells. B38 showed a 12-fold greater activity than the 5A peptide, a bihelical Class A amphipathic helix (EC50 of 0.2658 vs. 3.188µM; p<0.0001), in promoting cholesterol efflux from ABCA1 expressing BHK-1 cells. In conclusion, we have identified novel domains within apoB that contribute to its lipid biding properties. Additionally, we have discovered a unique amphipathic helix design for efficient ABCA1-specific cholesterol efflux.
Assuntos
Apolipoproteínas B/química , Apolipoproteínas B/metabolismo , Lipídeos/química , Estrutura Secundária de Proteína/fisiologia , Transportador 1 de Cassete de Ligação de ATP/química , Transportador 1 de Cassete de Ligação de ATP/metabolismo , Aminoácidos/química , Aminoácidos/metabolismo , Sítios de Ligação/fisiologia , Células Cultivadas , HDL-Colesterol/química , HDL-Colesterol/metabolismo , LDL-Colesterol/química , LDL-Colesterol/metabolismo , Humanos , Interações Hidrofóbicas e Hidrofílicas , Peptídeos/química , Peptídeos/metabolismo , Ligação Proteica/fisiologiaRESUMO
Statins lower plasma cholesterol by as much as 50%, thus reducing future cardiovascular events. However, the physiological effects of statins are diverse and not all are related to low density lipoprotein cholesterol (LDL-C) lowering. We performed a small clinical pilot study to assess the impact of statins on lipoprotein-associated proteins in healthy individuals (n = 10) with normal LDL-C (<130 mg/dL), who were treated with rosuvastatin (20 mg/day) for 28 days. Proteomic analysis of size-exclusion chromatography isolated LDL, large high density lipoprotein (HDL-L), and small HDL (HDL-S) fractions and spectral counting was used to compare relative protein detection before and after statin therapy. Significant protein changes were found in each lipoprotein pool and included both increases and decreases in several proteins involved in lipoprotein metabolism, complement regulation and acute phase response. The most dramatic effect of the rosuvastatin treatment was an increase in α-1-antirypsin (A1AT) spectral counts associated with HDL-L particles. Quantitative measurement by ELISA confirmed an average 5.7-fold increase in HDL-L associated A1AT. Molecular modeling predictions indicated that the hydrophobic reactive center loop of A1AT, the functional domain responsible for its protease inhibitor activity, is likely involved in lipid binding and association with HDL was found to protect A1AT against oxidative inactivation. Cell culture experiments, using J774 macrophages, demonstrated that the association of A1AT with HDL enhances its antiprotease activity, preventing elastase induced production of tumor necrosis factor α. In conclusion, we show that statins can significantly alter the protein composition of both LDL and HDL and our studies reveal a novel functional relationship between A1AT and HDL. The up-regulation of A1AT on HDL enhances its anti-inflammatory functionality, which may contribute to the non-lipid lowering beneficial effects of statins.
Assuntos
Lipoproteínas HDL/metabolismo , Proteoma/efeitos dos fármacos , Rosuvastatina Cálcica/administração & dosagem , alfa 1-Antitripsina/metabolismo , Adulto , Animais , Sítios de Ligação/efeitos dos fármacos , Linhagem Celular , Feminino , Voluntários Saudáveis , Humanos , Lipoproteínas LDL/metabolismo , Macrófagos/citologia , Macrófagos/metabolismo , Masculino , Camundongos , Pessoa de Meia-Idade , Modelos Moleculares , Projetos Piloto , Proteômica , Rosuvastatina Cálcica/farmacologia , Regulação para Cima/efeitos dos fármacos , alfa 1-Antitripsina/químicaRESUMO
The cAMP/PKA signaling system constitutes an inhibitory pathway in T cells and, although its biochemistry has been thoroughly investigated, its possible effects on ion channels are still not fully understood. K(V)1.3 channels play an important role in T-cell activation, and their inhibition suppresses T-cell function. It has been reported that PKA modulates K(V)1.3 activity. Two PKA isoforms are expressed in human T cells: PKAI and PKAII. PKAI has been shown to inhibit T-cell activation via suppression of the tyrosine kinase Lck. The aim of this study was to determine the PKA isoform modulating K(V)1.3 and the signaling pathway underneath. 8-Bromoadenosine 3',5'-cyclic monophosphate (8-BrcAMP), a nonselective activator of PKA, inhibited K(V)1.3 currents both in primary human T and in Jurkat cells. This inhibition was prevented by the PKA blocker PKI(6-22). Selective knockdown of PKAI, but not PKAII, with siRNAs abolished the response to 8-BrcAMP. Additional studies were performed to determine the signaling pathway mediating PKAI effect on K(V)1.3. Overexpression of a constitutively active mutant of Lck reduced the response of K(V)1.3 to 8-Br-cAMP. Moreover, knockdown of the scaffolding protein disc large 1 (Dlg1), which binds K(V)1.3 to Lck, abolished PKA modulation of K(V)1.3 channels. Immunohistochemistry studies showed that PKAI, but not PKAII, colocalizes with K(V)1.3 and Dlg1 indicating a close proximity between these proteins. These results indicate that PKAI selectively regulates K(V)1.3 channels in human T lymphocytes. This effect is mediated by Lck and Dlg1. We thus propose that the K(V)1.3/Dlg1/Lck complex is part of the membrane pathway that cAMP utilizes to regulate T-cell function.
Assuntos
Proteínas Adaptadoras de Transdução de Sinal/fisiologia , Proteína Quinase Tipo I Dependente de AMP Cíclico/fisiologia , Canal de Potássio Kv1.3/metabolismo , Proteína Tirosina Quinase p56(lck) Linfócito-Específica/fisiologia , Proteínas de Membrana/fisiologia , Linfócitos T/enzimologia , 8-Bromo Monofosfato de Adenosina Cíclica/farmacologia , Células Cultivadas , Proteína 1 Homóloga a Discs-Large , Humanos , Imunossupressores/farmacologia , Células Jurkat , Proteína Tirosina Quinase p56(lck) Linfócito-Específica/antagonistas & inibidores , Transdução de Sinais/efeitos dos fármacos , Transdução de Sinais/fisiologia , Linfócitos T/efeitos dos fármacos , Linfócitos T/metabolismoRESUMO
Natural killer (NK) cells play critical roles defending against tumors and pathogens. We show that mice lacking both transcription factors Eomesodermin (Eomes) and T-bet failed to develop NK cells. Developmental stability of immature NK cells constitutively expressing the death ligand TRAIL depended on T-bet. Conversely, maturation characterized by loss of constitutive TRAIL expression and induction of Ly49 receptor diversity and integrin CD49b (DX5(+)) required Eomes. Mature NK cells from which Eomes was deleted reverted to phenotypic immaturity if T-bet was present or downregulated NK lineage antigens if T-bet was absent, despite retaining expression of Ly49 receptors. Fetal and adult hepatic hematopoiesis restricted Eomes expression and limited NK development to the T-bet-dependent, immature stage, whereas medullary hematopoiesis permitted Eomes-dependent NK maturation in adult mice. These findings reveal two sequential, genetically separable checkpoints of NK cell maturation, the progression of which is metered largely by the anatomic localization of hematopoiesis.
Assuntos
Ciclo Celular/genética , Diferenciação Celular , Células Matadoras Naturais/citologia , Células Matadoras Naturais/imunologia , Proteínas com Domínio T/metabolismo , Animais , Linhagem da Célula , Citometria de Fluxo , Deleção de Genes , Camundongos , Camundongos Knockout , Modelos Imunológicos , Reação em Cadeia da Polimerase em Tempo Real , Proteínas com Domínio T/genéticaRESUMO
Cytotoxic CD4 Th1 cells are emerging as a therapeutically useful T cell lineage that can effectively target tumors, but until now the pathways that govern their differentiation have been poorly understood. We demonstrate that CD134 (OX40) costimulation programs naive self- and virus-reactive CD4 T cells to undergo in vivo differentiation into cytotoxic Th1 effectors. CD137 (4-1BB) costimulation maximized clonal expansion, and IL-2 was necessary for cytotoxic Th1 differentiation. Importantly, the T-box transcription factor Eomesodermin was critical for inducing the cytotoxic marker granzyme B. CD134 plus CD137 dual costimulation also imprinted a cytotoxic phenotype on bystanding CD4 T cells. Thus, to our knowledge, the current study identifies for the first time a specific costimulatory pathway and an intracellular mechanism relying on Eomesodermin that induces both Ag-specific and bystander cytotoxic CD4 Th1 cells. This mechanism might be therapeutically useful because CD134 plus CD137 dual costimulation induced CD4 T cell-dependent tumoricidal function in a mouse melanoma model.
Assuntos
Diferenciação Celular/imunologia , Receptores OX40/imunologia , Proteínas com Domínio T/imunologia , Células Th1/citologia , Membro 9 da Superfamília de Receptores de Fatores de Necrose Tumoral/imunologia , Animais , Linfócitos T CD4-Positivos/citologia , Linfócitos T CD4-Positivos/imunologia , Linfócitos T CD4-Positivos/metabolismo , Separação Celular , Imunoprecipitação da Cromatina , Citometria de Fluxo , Melanoma Experimental/imunologia , Camundongos , Camundongos Transgênicos , Receptores OX40/metabolismo , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Transdução de Sinais/imunologia , Proteínas com Domínio T/metabolismo , Células Th1/imunologia , Células Th1/metabolismo , Membro 9 da Superfamília de Receptores de Fatores de Necrose Tumoral/metabolismoRESUMO
Conventional and nonconventional T cell development occur in the thymus. Nonconventional thymocytes that bear characteristics typically associated with innate immune cells are termed innate-like lymphocytes (ILLs). Mice harboring a tyrosine to phenylalanine mutation in the adaptor protein Src homology 2 domain-containing leukocyte protein of 76 kDa at residue 145 (Y145F mice) develop an expanded population of CD8(+)CD122(+)CD44(+) ILLs, typified by expression of the T-box transcription factor eomesodermin. Y145F mice also have an expanded population of γδ T cells that produce copious amounts of IL-4 via a mechanism that is dependent on the BTB-ZF transcription factor promyelocytic leukemia zinc finger. Using mice with T cell-specific deletion of Eomes, we demonstrate that this transcription factor is required for CD8(+) ILL development in Y145F as well as wild-type mice. Moreover, we show that promyelocytic leukemia zinc finger and IL-4 are also required for the generation of this ILL population. Taken together, these data shed light on the cell-intrinsic and cell-extrinsic factors that drive CD8(+) ILL differentiation.
Assuntos
Proteínas Adaptadoras de Transdução de Sinal/imunologia , Linfócitos T CD8-Positivos/imunologia , Fatores de Transcrição Kruppel-Like/imunologia , Fosfoproteínas/imunologia , Proteínas com Domínio T/imunologia , Proteínas Adaptadoras de Transdução de Sinal/genética , Substituição de Aminoácidos , Animais , Linfócitos T CD4-Positivos/imunologia , Linfócitos T CD4-Positivos/metabolismo , Linfócitos T CD8-Positivos/metabolismo , Células Cultivadas , Citometria de Fluxo , Interferon gama/imunologia , Interferon gama/metabolismo , Interleucina-4/imunologia , Interleucina-4/metabolismo , Fatores de Transcrição Kruppel-Like/genética , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Mutação , Fosfoproteínas/genética , Proteína com Dedos de Zinco da Leucemia Promielocítica , Proteínas com Domínio T/genética , Timo/citologia , Timo/imunologia , Timo/metabolismo , Dedos de Zinco/genética , Dedos de Zinco/imunologiaRESUMO
A hallmark of autoimmune lymphoproliferative syndrome (ALPS), caused by mutation of the Fas death receptor, is massive lymphadenopathy from aberrant expansion of CD4(-)CD8(-) (double-negative [DN]) T cells. Eomesodermin (Eomes) is a member of the T-box family of transcription factors and plays critical roles in effector cell function and memory cell fitness of CD8(+) T lymphocytes. We provide evidence in this study that DN T cells exhibit dysregulated expression of Eomes in humans and mice with ALPS. We also find that T cell-specific deletion of Eomes prevents lymphoid hypertrophy and accumulation of DN T cells in Fas-mutant mice. Although Eomes has critical physiological roles in the function and homeostasis of CD8(+) T cells, overexpression of Eomes appears to enable pathological induction or expansion of unusual CD8-related T cell subsets. Thus, antagonism of Eomes emerges as a therapeutic target for DN T cell ablation in ALPS.
Assuntos
Síndrome Linfoproliferativa Autoimune/imunologia , Linfócitos T CD8-Positivos/imunologia , Proteínas com Domínio T/imunologia , Subpopulações de Linfócitos T/imunologia , Receptor fas , Animais , Síndrome Linfoproliferativa Autoimune/genética , Síndrome Linfoproliferativa Autoimune/patologia , Síndrome Linfoproliferativa Autoimune/terapia , Linfócitos T CD8-Positivos/patologia , Feminino , Regulação da Expressão Gênica/genética , Regulação da Expressão Gênica/imunologia , Humanos , Memória Imunológica/genética , Memória Imunológica/imunologia , Masculino , Camundongos , Camundongos Knockout , Proteínas com Domínio T/genética , Subpopulações de Linfócitos T/patologiaRESUMO
When intracellular pathogens invade mammalian hosts, naïve CD8+ T cells differentiate into cytotoxic killers, which lyse infected target cells and secrete cytokines that activate intracellular microbicides. We show that CD8+ T cells deficient in the transcription factors T-bet and eomesodermin (Eomes) fail to differentiate into functional killers required for defense against lymphocytic choriomeningitis virus. Instead, virus-specific CD8+ T cells lacking both T-bet and Eomes differentiate into an interleukin-17-secreting lineage, reminiscent of the helper T cell fate that has been implicated in autoimmunity and extracellular microbial defense. Upon viral infection, mice with T cells lacking both T-bet and Eomes develop a CD8+ T cell-dependent, progressive inflammatory and wasting syndrome characterized by multi-organ infiltration of neutrophils. T-bet and Eomes, thus, ensure that CD8+ T cells adopt an appropriate course of intracellular rather than extracellular destruction.