RESUMO
The aim of our study was to determine whether granzyme B-expressing regulatory B cells (GZMB+ B cells) are enriched in the blood of transplant patients with renal graft tolerance. To achieve this goal, we analysed two single-cell RNA sequencing (scRNAseq) datasets: (1) peripheral blood mononuclear cells (PBMCs), including GZMB+ B cells from renal transplant patients, i.e., patients with stable graft function on conventional immunosuppressive treatment (STA, n = 3), drug-free tolerant patients (TOL, n = 3), and patients with antibody-mediated rejection (ABMR, n = 3), and (2) ex-vivo-induced GZMB+ B cells from these groups. In the patient PBMCs, we first showed that natural GZMB+ B cells were enriched in genes specific to Natural Killer (NK) cells (such as NKG7 and KLRD1) and regulatory B cells (such as GZMB, IL10, and CCL4). We performed a pseudotemporal trajectory analysis of natural GZMB+ B cells and showed that they were highly differentiated B cells with a trajectory that is very different from that of conventional memory B cells and linked to the transcription factor KLF13. By specifically analysing GZMB+ natural B cells in TOLs, we found that these cells had a very specific transcriptomic profile associated with a reduction in the expression of HLA molecules, apoptosis, and the inflammatory response (in general) in the blood and that this signature was conserved after ex vivo induction, with the induction of genes associated with migration processes, such as CCR7, CCL3, or CCL4. An analysis of receptor/ligand interactions between these GZMB+/- natural B cells and all of the immune cells present in PBMCs also demonstrated that GZMB+ B cells were the B cells that carried the most ligands and had the most interactions with other immune cells, particularly in tolerant patients. Finally, we showed that these GZMB+ B cells were able to infiltrate the graft under inflammatory conditions, thus suggesting that they can act in locations where immune events occur.
Assuntos
Linfócitos B Reguladores , Granzimas , Transplante de Rim , Humanos , Granzimas/metabolismo , Granzimas/genética , Linfócitos B Reguladores/imunologia , Linfócitos B Reguladores/metabolismo , Diferenciação Celular , Feminino , Masculino , Sistema Imunitário/metabolismo , Pessoa de Meia-Idade , Rejeição de Enxerto/imunologia , Células Matadoras Naturais/imunologia , Células Matadoras Naturais/metabolismo , Leucócitos Mononucleares/metabolismo , Leucócitos Mononucleares/imunologiaRESUMO
Sepsis induces immune alterations, which last for months after the resolution of illness. The effect of this immunological reprogramming on the risk of developing cancer is unclear. Here we use a national claims database to show that sepsis survivors had a lower cumulative incidence of cancers than matched nonsevere infection survivors. We identify a chemokine network released from sepsis-trained resident macrophages that triggers tissue residency of T cells via CCR2 and CXCR6 stimulations as the immune mechanism responsible for this decreased risk of de novo tumor development after sepsis cure. While nonseptic inflammation does not provoke this network, laminarin injection could therapeutically reproduce the protective sepsis effect. This chemokine network and CXCR6 tissue-resident T cell accumulation were detected in humans with sepsis and were associated with prolonged survival in humans with cancer. These findings identify a therapeutically relevant antitumor consequence of sepsis-induced trained immunity.
Assuntos
Macrófagos , Neoplasias , Sepse , Humanos , Sepse/imunologia , Macrófagos/imunologia , Feminino , Neoplasias/imunologia , Neoplasias/terapia , Masculino , Receptores CXCR6/metabolismo , Animais , Linfócitos T/imunologia , Receptores CCR2/metabolismo , Pessoa de Meia-Idade , Camundongos , Idoso , Quimiocinas/metabolismo , AdultoRESUMO
Introduction: Human Granzyme B (GZMB) regulatory B cells (Bregs) have suppressive properties on CD4+ effector T cells by a mechanism partially dependent on GZMB. Moreover, these cells may be easily induced in vitro making them interesting for cell therapy. Methods: We characterized this population of in vitro induced GZMB+Bregs using single cell transcriptomics. To investigate their regulatory properties, Bregs or total B cells were also co-cultured with T cells and scRNAseq was used to identify receptor ligand interactions and to reveal gene expression changes in the T cells. Results: We find that Bregs exhibit a unique set of 149 genes differentially expressed and which are implicated in proliferation, apoptosis, metabolism, and altered antigen presentation capacity consistent with their differentiated B cells profile. Notably, Bregs induced a strong inhibition of T cell genes associated to proliferation, activation, inflammation and apoptosis compared to total B cells. We identified and validated 5 receptor/ligand interactions between Bregs and T cells. Functional analysis using specific inhibitors was used to test their suppressive properties and we identified Lymphotoxin alpha (LTA) as a new and potent Breg ligand implicated in Breg suppressive properties. Discussion: We report for the first time for a role of LTA in GZMB+Bregs as an enhancer of GZMB expression, and involved in the suppressive properties of GZMB+Bregs in human. The exact mechanism of LTA/GZMB function in this specific subset of Bregs remains to be determined.
Assuntos
Linfócitos B Reguladores , Linfotoxina-alfa , Humanos , Granzimas , Ligantes , Linfócitos T CD4-Positivos , Proliferação de CélulasRESUMO
The BK polyomavirus (BKPyV) is an opportunistic pathogen, which is only pathogenic in immunosuppressed individuals, such as kidney transplant recipients, in whom BKPyV can cause significant morbidity. To identify broadly neutralizing antibodies against this virus, we used fluorescence-labeled BKPyV virus-like particles to sort BKPyV-specific B cells from the PBMC of KTx recipients, then single-cell RNAseq to obtain paired heavy- and light-chain antibody sequences from 2,106 sorted B cells. The BKPyV-specific repertoire was highly diverse in terms of both V-gene usage and clonotype diversity and included most of the IgM B cells, including many with extensive somatic hypermutation. In two patients where sufficient data were available, IgM B cells in the BKPyV-specific dataset had significant differences in V-gene usage compared with IgG B cells from the same patient. CDR3 sequence-based clustering allowed us to identify and characterize three broadly neutralizing "41F17-like" clonotypes that were predominantly IgG, suggesting that some specific BKPyV capsid epitopes are preferentially targeted by IgG.
Assuntos
Vírus BK , Transplante de Rim , Infecções por Polyomavirus , Humanos , Vírus BK/genética , Transplante de Rim/efeitos adversos , Leucócitos Mononucleares , Infecções por Polyomavirus/etiologia , Imunoglobulina G , Imunoglobulina MRESUMO
BACKGROUND: Regulatory T cells (Treg) in diverse species include CD4+ and CD8+ T cells. In all species, CD8+ Treg have been only partially characterized and there is no rat model in which CD4+ and CD8+ FOXP3+ Treg are genetically tagged. RESULTS: We generated a Foxp3-EGFP rat transgenic line in which FOXP3 gene was expressed and controlled EGFP. CD4+ and CD8+ T cells were the only cells that expressed EGFP, in similar proportion as observed with anti-FOXP3 antibodies and co-labeled in the same cells. CD4+EGFP+ Treg were 5-10 times more frequent than CD8+EGFP+ Treg. The suppressive activity of CD4+ and CD8+ Treg was largely confined to EGFP+ cells. RNAseq analyses showed similarities but also differences among CD4+ and CD8+ EGFP+ cells and provided the first description of the natural FOXP3+CD8+ Treg transcriptome. In vitro culture of CD4+ and CD8+ EGFP- cells with TGFbeta and IL-2 generated induced EGFP+ Treg. CD4+ and CD8+ EGFP+ Treg were expanded upon in vivo administration of a low dose of IL-2. CONCLUSIONS: This new and unique rat line constitutes a useful model to identify and isolate viable CD4+ and CD8+ FOXP3+ Treg. Additionally, it allows to identify molecules expressed in CD8+ Treg that may allow to better define their phenotype and function not only in rats but also in other species.
Assuntos
Linfócitos T CD8-Positivos , Linfócitos T Reguladores , Ratos , Animais , Linfócitos T Reguladores/metabolismo , Linfócitos T CD8-Positivos/metabolismo , Interleucina-2/genética , Interleucina-2/metabolismo , Fator de Crescimento Transformador beta/metabolismo , Fatores de Transcrição Forkhead/genética , Fatores de Transcrição Forkhead/metabolismoRESUMO
Tumors exploit numerous immune checkpoints, including those deployed by myeloid cells to curtail antitumor immunity. Here, we show that the C-type lectin receptor CLEC-1 expressed by myeloid cells senses dead cells killed by programmed necrosis. Moreover, we identified Tripartite Motif Containing 21 (TRIM21) as an endogenous ligand overexpressed in various cancers. We observed that the combination of CLEC-1 blockade with chemotherapy prolonged mouse survival in tumor models. Loss of CLEC-1 reduced the accumulation of immunosuppressive myeloid cells in tumors and invigorated the activation state of dendritic cells (DCs), thereby increasing T cell responses. Mechanistically, we found that the absence of CLEC-1 increased the cross-presentation of dead cell-associated antigens by conventional type-1 DCs. We identified antihuman CLEC-1 antagonist antibodies able to enhance antitumor immunity in CLEC-1 humanized mice. Together, our results demonstrate that CLEC-1 acts as an immune checkpoint in myeloid cells and support CLEC-1 as a novel target for cancer immunotherapy.
Assuntos
Apresentação Cruzada , Neoplasias , Camundongos , Animais , Apresentação de Antígeno , Imunoterapia , Células Dendríticas , Neoplasias/terapiaRESUMO
INTRODUCTION: In kidney transplant recipients, belatacept is usually pursued indefinitely after it has been started. In the setting of the belatacept shortage and after having evaluated the benefit-risk ratio, we established a strategy consisting of time-limited belatacept therapy/transient calcineurin inhibitor withdrawal, whose results are analyzed in that study. METHODS: We considered all the kidney transplant recipients that had been switched from conventional immunosuppressive therapy to belatacept and then for whom belatacept has been withdrawn intentionally. Furthermore, in the first 8 patients, we assessed changes in peripheral blood mononuclear cells (PBMC) transcriptome using RNAseq before and 3 months after belatacept withdrawal. RESULTS: Over the study period, 28 out of 94 patients had belatacept intentionally withdrawn including 25 (89%) switched to low-dose CNI. One rejection due to poor compliance occurred. The eGFR after 12 months remained stable from 48 ± 19 mL.1.73 m-2 to 46 ± 17 mL.1.73 m-2 (p = 0.68). However, patients that resumed belatacept/withdrew CNIs (n = 10) had a trend towards a better eGFR comparing with the others (n = 15): 54 ± 20 mL.1.73 m-2 vs. eGFR 43 ± 16 mL.1.73 m-2, respectively (p = 0.15). The only factor associated with belatacept resumption was when the withdrawal took place during the COVID-19 outbreak. Transcriptome analysis of PBMCs, did not support rebound in alloimmune response. CONCLUSIONS: These findings underpin the use of belatacept as part of a time-limited therapy, in selected kidney transplant recipients, possibly as an approach to allow efficient vaccination against SARS-CoV-2.
RESUMO
Rationale: Brain injury induces systemic immunosuppression, increasing the risk of viral reactivations and altering neurological recovery. Objectives: To determine if systemic immune alterations and lung replication of herpesviridae are associated and can help predict outcomes after brain injury. Methods: We collected peripheral blood mononuclear cells in patients with severe brain injury requiring invasive mechanical ventilation. We systematically searched for respiratory herpes simplex virus (HSV) replications in tracheal aspirates. We also performed chromatin immunoprecipitation sequencing, RNA-sequencing, and in vitro functional assays of monocytes and CD4 T cells collected on Day 1 to characterize the immune response to severe acute brain injury. The primary outcome was the Glasgow Outcome Scale Extended at 6 months. Measurements and Main Results: In 344 patients with severe brain injury, lung HSV reactivations were observed in 39% of the 232 patients seropositive for HSV and independently associated with poor neurological recovery at 6 months (hazard ratio, 1.90; 95% confidence interval, 1.08-3.57). Weighted gene coexpression network analyses of the transcriptomic response of monocytes to brain injury defined a module of 721 genes, including PD-L1 and CD80, enriched for the binding DNA motif of the transcriptional factor Zeb2 and whose ontogenic analyses revealed decreased IFN-γ-mediated and antiviral response signaling pathways. This monocyte signature was preserved in a validation cohort and predicted the neurological outcome at 6 months with good accuracy (area under the curve, 0.786; 95% confidence interval, 0.593-0.978). Conclusions: A specific monocyte signature is associated with HSV reactivation and predicts poor recovery after brain injury. The alterations of the immune control of herpesviridae replication are understudied and represent a novel therapeutic target.
Assuntos
Lesões Encefálicas , Herpes Simples , Herpesvirus Humano 1 , Herpesvirus Humano 1/genética , Humanos , Leucócitos Mononucleares , MonócitosRESUMO
Nuclear deubiquitinase BAP1 (BRCA1-associated protein 1) is a core component of multiprotein complexes that promote transcription by reversing the ubiquitination of histone 2A (H2A). BAP1 is a tumor suppressor whose germline loss-of-function variants predispose to cancer. To our knowledge, there are very rare examples of different germline variants in the same gene causing either a neurodevelopmental disorder (NDD) or a tumor predisposition syndrome. Here, we report a series of 11 de novo germline heterozygous missense BAP1 variants associated with a rare syndromic NDD. Functional analysis showed that most of the variants cannot rescue the consequences of BAP1 inactivation, suggesting a loss-of-function mechanism. In T cells isolated from two affected children, H2A deubiquitination was impaired. In matching peripheral blood mononuclear cells, histone H3 K27 acetylation ChIP-seq indicated that these BAP1 variants induced genome-wide chromatin state alterations, with enrichment for regulatory regions surrounding genes of the ubiquitin-proteasome system (UPS). Altogether, these results define a clinical syndrome caused by rare germline missense BAP1 variants that alter chromatin remodeling through abnormal histone ubiquitination and lead to transcriptional dysregulation of developmental genes.
Assuntos
Proteína BRCA1/genética , Mutação em Linhagem Germinativa , Mutação com Perda de Função , Mutação de Sentido Incorreto , Transtornos do Neurodesenvolvimento/genética , Proteínas Supressoras de Tumor/genética , Ubiquitina Tiolesterase/genética , Adolescente , Proteína BRCA1/imunologia , Criança , Pré-Escolar , Cromatina/química , Cromatina/imunologia , Montagem e Desmontagem da Cromatina/genética , Montagem e Desmontagem da Cromatina/imunologia , Família , Feminino , Regulação da Expressão Gênica , Heterozigoto , Histonas/genética , Histonas/imunologia , Fator C1 de Célula Hospedeira/genética , Fator C1 de Célula Hospedeira/imunologia , Humanos , Lactente , Masculino , Transtornos do Neurodesenvolvimento/imunologia , Transtornos do Neurodesenvolvimento/patologia , Complexo de Endopeptidases do Proteassoma/genética , Complexo de Endopeptidases do Proteassoma/imunologia , Linfócitos T/imunologia , Linfócitos T/patologia , Proteínas Supressoras de Tumor/deficiência , Proteínas Supressoras de Tumor/imunologia , Ubiquitina/genética , Ubiquitina/imunologia , Ubiquitina Tiolesterase/deficiência , Ubiquitina Tiolesterase/imunologia , Ubiquitina-Proteína Ligases/genética , Ubiquitina-Proteína Ligases/imunologia , UbiquitinaçãoRESUMO
Intracellular ion fluxes emerge as critical actors of immunoregulation but still remain poorly explored. In this study, we investigated the role of the redundant cation channels TMEM176A and TMEM176B (TMEM176A/B) in retinoic acid-related orphan receptor γt+ cells and conventional dendritic cells (DCs) using germline and conditional double knockout mice. Although Tmem176a/b appeared surprisingly dispensable for the protective function of Th17 and group 3 innate lymphoid cells in the intestinal mucosa, we found that they were required in conventional DCs for optimal Ag processing and presentation to CD4+ T cells. Using a real-time imaging method, we show that TMEM176A/B accumulate in dynamic post-Golgi vesicles preferentially linked to the late endolysosomal system and strongly colocalize with HLA-DM. Taken together, our results suggest that TMEM176A/B ion channels play a direct role in the MHC class II compartment of DCs for the fine regulation of Ag presentation and naive CD4+ T cell priming.
Assuntos
Apresentação de Antígeno/imunologia , Linfócitos T CD4-Positivos/imunologia , Células Dendríticas/imunologia , Antígenos de Histocompatibilidade Classe II/imunologia , Proteínas de Membrana/imunologia , Animais , Endossomos/imunologia , Feminino , Genes MHC da Classe II/imunologia , Complexo de Golgi/imunologia , Imunidade Inata/imunologia , Mucosa Intestinal/imunologia , Canais Iônicos/imunologia , Linfócitos/imunologia , Lisossomos/imunologia , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Células Th17/imunologia , Tretinoína/imunologiaRESUMO
Proteins involved in transcriptional regulation harbor a demonstrated enrichment of mutations in neurodevelopmental disorders. The Sin3 (Swi-independent 3)/histone deacetylase (HDAC) complex plays a central role in histone deacetylation and transcriptional repression. Among the two vertebrate paralogs encoding the Sin3 complex, SIN3A variants cause syndromic intellectual disability, but the clinical consequences of SIN3B haploinsufficiency in humans are uncharacterized. Here, we describe a syndrome hallmarked by intellectual disability, developmental delay, and dysmorphic facial features with variably penetrant autism spectrum disorder, congenital malformations, corpus callosum defects, and impaired growth caused by disruptive SIN3B variants. Using chromosomal microarray or exome sequencing, and through international data sharing efforts, we identified nine individuals with heterozygous SIN3B deletion or single-nucleotide variants. Five individuals harbor heterozygous deletions encompassing SIN3B that reside within a â¼230 kb minimal region of overlap on 19p13.11, two individuals have a rare nonsynonymous substitution, and two individuals have a single-nucleotide deletion that results in a frameshift and predicted premature termination codon. To test the relevance of SIN3B impairment to measurable aspects of the human phenotype, we disrupted the orthologous zebrafish locus by genome editing and transient suppression. The mutant and morphant larvae display altered craniofacial patterning, commissural axon defects, and reduced body length supportive of an essential role for Sin3 function in growth and patterning of anterior structures. To investigate further the molecular consequences of SIN3B variants, we quantified genome-wide enhancer and promoter activity states by using H3K27ac ChIP-seq. We show that, similar to SIN3A mutations, SIN3B disruption causes hyperacetylation of a subset of enhancers and promoters in peripheral blood mononuclear cells. Together, these data demonstrate that SIN3B haploinsufficiency leads to a hitherto unknown intellectual disability/autism syndrome, uncover a crucial role of SIN3B in the central nervous system, and define the epigenetic landscape associated with Sin3 complex impairment.
Assuntos
Transtorno do Espectro Autista/genética , Haploinsuficiência/genética , Histona Desacetilases/metabolismo , Deficiência Intelectual/genética , Proteínas Repressoras/genética , Acetilação , Adolescente , Animais , Criança , Pré-Escolar , Variações do Número de Cópias de DNA/genética , Feminino , Histonas/química , Histonas/metabolismo , Humanos , Lactente , Larva/genética , Imageamento por Ressonância Magnética , Masculino , Pessoa de Meia-Idade , Modelos Moleculares , Mutação , Proteínas Repressoras/deficiência , Proteínas Repressoras/metabolismo , Síndrome , Adulto Jovem , Peixe-Zebra/genética , Proteínas de Peixe-Zebra/deficiência , Proteínas de Peixe-Zebra/genéticaRESUMO
An amendment to this paper has been published and can be accessed via a link at the top of the paper.
RESUMO
Sepsis and trauma cause inflammation and elevated susceptibility to hospital-acquired pneumonia. As phagocytosis by macrophages plays a critical role in the control of bacteria, we investigated the phagocytic activity of macrophages after resolution of inflammation. After resolution of primary pneumonia, murine alveolar macrophages (AMs) exhibited poor phagocytic capacity for several weeks. These paralyzed AMs developed from resident AMs that underwent an epigenetic program of tolerogenic training. Such adaptation was not induced by direct encounter of the pathogen but by secondary immunosuppressive signals established locally upon resolution of primary infection. Signal-regulatory protein α (SIRPα) played a critical role in the establishment of the microenvironment that induced tolerogenic training. In humans with systemic inflammation, AMs and also circulating monocytes still displayed alterations consistent with reprogramming six months after resolution of inflammation. Antibody blockade of SIRPα restored phagocytosis in monocytes of critically ill patients in vitro, which suggests a potential strategy to prevent hospital-acquired pneumonia.
Assuntos
Epigênese Genética , Inflamação/etiologia , Pulmão/imunologia , Pulmão/metabolismo , Macrófagos Alveolares/metabolismo , Animais , Biomarcadores , Reprogramação Celular , Citocinas/metabolismo , Humanos , Tolerância Imunológica , Imunofenotipagem , Inflamação/metabolismo , Inflamação/patologia , Mediadores da Inflamação/metabolismo , Pulmão/patologia , Macrófagos/imunologia , Macrófagos/metabolismo , Macrófagos Alveolares/imunologia , Camundongos , Monócitos/imunologia , Monócitos/metabolismo , Fagocitose/imunologia , Pneumonia/etiologia , Pneumonia/metabolismo , Pneumonia/patologia , Subpopulações de Linfócitos T/imunologia , Subpopulações de Linfócitos T/metabolismoRESUMO
Innate lymphoid cells (ILCs) are tissue-resident lymphocytes that lack antigen-specific receptors and exhibit innate effector functions such as cytokine production that play an important role in immediate responses to pathogens especially at mucosal sites. Mouse and human ILC subsets have been extensively characterized in various tissues and in blood. In this study, we present the first characterization of ILCs and ILC subsets in rat gut and secondary lymphoid organs using flow cytometry and single cell RNA sequencing. Our results show that phenotype and function of rat ILC subsets are conserved as compared to human and mouse ILCs. However, and in contrast to human and mouse, our study unexpectedly revealed that ILC2 and not ILC3 was the dominant ILC subset in the rat intestinal lamina propria. ILC2 predominance in the gut was independent of rat strain, sex or housing facility. In contrast, ILC3 was the predominant ILC subset in mesenteric lymph nodes and Peyer patches. In conclusion, our study demonstrates that in spite of highly conserved phenotype and function between mice, rat and humans, the distribution of ILC subsets in the intestinal mucosa is dependent on the species likely in response to both genetic and environmental factors.
Assuntos
Mucosa Intestinal/imunologia , Subpopulações de Linfócitos/imunologia , Linfócitos/imunologia , Animais , Contagem de Células , Células Cultivadas , Citocinas/metabolismo , Citometria de Fluxo , Humanos , Imunidade Inata , Masculino , Camundongos , Ratos , Ratos Sprague-Dawley , Células Th2/imunologiaRESUMO
The lack of plasticity of neurons to respond to dietary changes, such as high fat and high fructose diets, by modulating gene and protein expression has been associated with functional and behavioral impairments that can have detrimental consequences. The inhibition of high fat-induced rewiring of hypothalamic neurons induced obesity. Feeding rodents with high fructose is a recognized and widely used model to trigger obesity and metabolic syndrome. However the adaptive response of the retina to short term feeding with high fructose is poorly documented. We therefore aimed to characterize both the functional and gene expression changes in the neurosensory retina of Brown Norway rats fed during 3 and 8 days with a 60%-rich fructose diet (n = 16 per diet and per time point). Glucose, insulin, leptin, triacylglycerols, total cholesterol, HDL-cholesterol, LDL-cholesterol and fructosamine were quantified in plasma (n = 8 in each group). Functionality of the inner retina was studied using scotopic single flash electroretinography (n = 8 in each group) and the individual response of rod and cone photoreceptors was determined using 8.02 Hz Flicker electroretinography (n = 8 in each group). Analysis of gene expression in the neurosensory retina was performed by Affymetrix genechips, and confirmed by RT-qPCR (n = 6 in each group). Elevated glycemia (+13%), insulinemia (+83%), and leptinemia (+172%) was observed after 8 days of fructose feeding. The cone photoreceptor response was altered at day 8 in high fructose fed rats (Δ = 0.5 log unit of light stimulus intensity). Affymetrix analysis of gene expression highlighted significant modulation of the pathways of eIF2 signaling and endoplasmic reticulum stress, regulation of eIF4 and p70S6K signaling, as well as mTOR signaling and mitochondrial dysfunction. RT-qPCR analysis confirmed the down regulation of Crystallins, Npy, Nid1 and Optc genes after 3 days of fructose feeding, and up regulation of End2. Meanwhile, a trend towards an increased expression of αA- and αB-crystallin proteins was observed at day 8. Our results are consistent with early alterations of the functioning and gene expression in the retina in a pro diabetogenic environment.
Assuntos
Diabetes Mellitus Experimental , Dieta , Carboidratos da Dieta/administração & dosagem , Frutose/administração & dosagem , Retina/fisiologia , Células Fotorreceptoras Retinianas Cones/fisiologia , Animais , Glicemia/análise , Colesterol/sangue , Cristalinas/metabolismo , Diabetes Mellitus Experimental/metabolismo , Diabetes Mellitus Experimental/fisiopatologia , Eletrorretinografia , Estresse do Retículo Endoplasmático/fisiologia , Fator de Iniciação 2 em Eucariotos/fisiologia , Frutosamina/sangue , Perfilação da Expressão Gênica , Regulação da Expressão Gênica , Insulina/sangue , Leptina/sangue , Masculino , RatosRESUMO
Oxidation of cholesterol into oxysterols is a major way of elimination of cholesterol from the liver and extrahepatic tissues, including the brain and the retina. Oxysterols are involved in various cellular processes. Numerous links have been established between oxysterols and several disorders such as neurodegenerative pathologies, retinopathies and atherosclerosis. Different components of the lipid layer such as sphingolipids, sterols and proteins participate to membrane fluidity and forme lipid rafts microdomains. Few data are available on the links between lipids rafts and oxysterols. The purpose of this review is to suggest the potential role of lipid rafts microdomains in the development of retinopathies with special emphasis and opening perspectives of their interactions with oxysterols. Actually cholesterol oxidation mechanism may have deleterious effect on its ability to support rafts formation .This review suggest that the effect of oxysterols of lipid rafts would probably depend on the oxysterol molecule and cell type.
Assuntos
Oftalmopatias/metabolismo , Microdomínios da Membrana/metabolismo , Esteróis/metabolismo , Animais , Humanos , Modelos Biológicos , Esteróis/químicaRESUMO
Cholesterol 24S-hydroxylase (CYP46A1) converts cholesterol into 24S-hydroxycholesterol in neurons and participates in cholesterol homeostasis in the central nervous system, including the retina. We aimed to evaluate the consequences of CYP46A1 inhibition by voriconazole on cholesterol homeostasis and function in the retina. Rats received daily intraperitoneal injections of voriconazole (60mg/kg), minocycline (22mg/kg), voriconazole plus minocycline, or vehicle during five consecutive days. The rats were submitted to electroretinography to monitor retinal functionality. Cholesterol and 24S-hydroxycholesterol were measured in plasma, brain and retina by gas chromatography-mass spectrometry. The expression of CYP46A1, and GFAP as a marker for glial activation was analyzed in the retina and brain. Cytokines and chemokines were measured in plasma, vitreous, retina and brain. Voriconazole significantly impaired the functioning of the retina as exemplified by the reduced amplitude and increased latency of the b-wave of the electroretinogram, and altered oscillary potentials. Voriconazole decreased 24S-hydroxycholesterol levels in the retina. Unexpectedly, CYP46A1 and GFAP expression was increased in the retina of voriconazole-treated rats. ICAM-1 and MCP-1 showed significant increases in the retina and vitreous body. Minocycline did not reverse the effects of voriconazole. Our data highlighted the cross talk between retinal ganglion cells and glial cells in the retina, suggesting that reduced 24S-hydroxycholesterol concentration in the retina may be detected by glial cells, which were consequently activated.
Assuntos
Colesterol/metabolismo , Pirimidinas/farmacologia , Retina/efeitos dos fármacos , Esteroide Hidroxilases/antagonistas & inibidores , Triazóis/farmacologia , Animais , Colesterol/sangue , Colesterol 24-Hidroxilase , Citocinas/sangue , Citocinas/metabolismo , Eletrorretinografia , Inibidores Enzimáticos/farmacologia , Homeostase/efeitos dos fármacos , Hidroxicolesteróis/metabolismo , Masculino , Microglia/efeitos dos fármacos , Proteínas do Tecido Nervoso/metabolismo , Neuroglia/efeitos dos fármacos , Neuroglia/metabolismo , Ratos , Ratos Wistar , Retina/citologia , Retina/metabolismo , Esteroide Hidroxilases/metabolismo , Regulação para Cima/efeitos dos fármacos , VoriconazolRESUMO
PURPOSE: We investigated the association of single nucleotide polymorphism (SNP) in the cholesterol-24S-hydroxylase (CYP46A1) gene, according to CFH and LOC387715 SNPs, with age-related macular degeneration (AMD). METHODS: We enrolled 1388 AMD patients with neovascular AMD or geographic atrophy and 487 unrelated control subjects. SNPs were genotyped in the CYP46A1 (rs754203), LOC387715 (rs10490924), and CFH (rs1061170) genes. Plasma 24S-hydroxycholesterol, the metabolic product of CYP46A1, was quantified by gas chromatography-mass spectrometry using an authentic deuterated internal standard in subgroups of patients and controls. The χ(2) test was used to compare categoric allelic and genotype distributions between cases and controls. The odds ratio (OR) with a 95% confidence interval (95% CI) was calculated for AMD risk, and adjusted for age and gender. Significance levels were set at P < 0.05. RESULTS: The rs754203 SNP in the CYP46A1 gene was not associated with AMD (crude OR = 1.2, 95% CI = 0.9-1.4, P = 0.2). The crude OR for risk of AMD was 2.9 (95% CI = 2.4-3.4, P < 0.0001) according to the number of rs10490924 T alleles in the LOC387715 gene, and 2.0 (95% CI = 1.7-2.3, P < 0.0001) according to the number of rs1061170 C alleles in the CFH gene. After adjustment for age and gender, an OR of 2.2 (95% CI = 1.1-4.1, P = 0.04) was obtained for AMD cases with the C allele in the CYP46A1 gene, and carrying no risk alleles in the CFH and LOC387715 genes. CONCLUSIONS: The rs754203 C allele in the CYP46A1 gene may confer a higher risk for exudative AMD in patients who carry no risk alleles in the CFH and LOC387715 genes. Additional studies with larger sample sizes are needed in AMD subjects at no risk in CFH and LOC387715.
Assuntos
Atrofia Geográfica/genética , Polimorfismo de Nucleotídeo Único/genética , Proteínas/genética , Esteroide Hidroxilases/genética , Degeneração Macular Exsudativa/genética , Idoso , Alelos , Colesterol/sangue , Colesterol 24-Hidroxilase , Fator H do Complemento/genética , Feminino , Cromatografia Gasosa-Espectrometria de Massas , Técnicas de Genotipagem , Atrofia Geográfica/sangue , Humanos , Hidroxicolesteróis/sangue , Masculino , Razão de Chances , Fatores de Risco , Degeneração Macular Exsudativa/sangueRESUMO
PURPOSE: Our previous studies suggested that CYP46A1 and 24S-hydroxycholesterol (24SOH) may be associated with glaucoma. Loss of CYP46A1-expressing retinal ganglion cells is involved in the activation of glia, and therefore possibly in the disbalance of cholesterol. In this context, the purpose of our present work was to emphasize the glial and longitudinal CYP46A1 expression after an interventional glaucoma-related stress triggered by elevated intraocular pressure (IOP). METHODS: Sprague-Dawley rats were submitted to laser photocoagulation of the trabecular meshwork, limbus and episcleral veins in one eye to induce elevated IOP. Rats were euthanized at days 3, 14, 30 and 60 (n = 10 per time-point), and 24SOH was measured in plasma and retina by gas chromatography-mass spectrometry. CYP46A1 was quantified by Western blotting. Glial activation was assessed by glial fibrillary acidic protein immunoreactivity in Western blots and retinal cryosections. RESULTS: Sustained high IOP was observed in experimental eyes from day 1 to day 21. Plasma MCP-1 and ICAM-1, quantified using multiplex assay kits, were increased at day 3. Glial activation was observed bilaterally at all time-points, at lower levels in contralateral eyes than in experimental eyes. In experimental retinas, CYP46A1 expression showed a transient increase at day 3 and then returned to baseline. Plasma and retinal 24SOH peaked at day 14 and 30, respectively. CONCLUSIONS: These data show that CYP46A1 expression was induced early in response to retinal stress but remained constant at late time-points, reinforcing the constitutive role of CYP46A1 in maintaining cholesterol balance in neuronal tissues.
Assuntos
Gliose/sangue , Hidroxicolesteróis/sangue , Pressão Intraocular , Neuroglia/metabolismo , Hipertensão Ocular/sangue , Animais , Western Blotting , Colesterol 24-Hidroxilase , Citocinas/sangue , Modelos Animais de Doenças , Cromatografia Gasosa-Espectrometria de Massas , Proteína Glial Fibrilar Ácida/metabolismo , Gliose/enzimologia , Homeostase , Peptídeos e Proteínas de Sinalização Intercelular/sangue , Hipertensão Ocular/enzimologia , Ratos , Ratos Sprague-Dawley , Retina/metabolismo , Esteroide Hidroxilases/metabolismoRESUMO
Free cholesterol is the predominant form of cholesterol in the neural retina. The vertebrate neural retina exhibits its own capacity to synthesize cholesterol and meets its demand also by taking it from the circulation. Defects in cholesterol synthesis and trafficking in the neural retina has detrimental consequences on its structure and function, highlighting the crucial importance of maintaining cholesterol homeostasis in the retina. Our purpose was to give a review on the functioning of the retina, the role of cholesterol and cholesterol metabolism therein, with special emphasis on cholesterol-24S-hydroxylase (CYP46A1). Similar to the brain, the retina expresses cholesterol-24S-hydroxylase (CYP46A1) and is enriched in its metabolic product, 24S-hydroxycholesterol. We recently published that one single nucleotide polymorphism in CYP46A1 gene, designated as rs754203, was a risk factor for glaucoma. Glaucoma is the second leading cause of blindness worldwide, affecting more than 60 million people. Glaucoma is characterized by the loss of retinal ganglion cells, which show high CYP46A1 expression. These data suggest the potential involvement of CYP46A1 and 24S-hydroxycholesterol in the pathophysiology of glaucoma.