RESUMO
Sepsis is a life-threatening syndrome, and its associated mortality is increased when cardiac dysfunction and damage (septic cardiomyopathy [SCM]) occur. Although inflammation is involved in the pathophysiology of SCM, the mechanism of how inflammation induces SCM in vivo has remained obscure. NLRP3 inflammasome is a critical component of the innate immune system that activates caspase-1 (Casp1) and causes the maturation of IL-1ß and IL-18 as well as the processing of gasdermin D (GSDMD). Here, we investigated the role of the NLRP3 inflammasome in a murine model of lipopolysaccharide (LPS)-induced SCM. LPS injection induced cardiac dysfunction, damage, and lethality, which was significantly prevented in NLRP3-/- mice, compared to wild-type (WT) mice. LPS injection upregulated mRNA levels of inflammatory cytokines (Il6, Tnfa, and Ifng) in the heart, liver, and spleen of WT mice, and this upregulation was prevented in NLRP3-/- mice. LPS injection increased plasma levels of inflammatory cytokines (IL-1ß, IL-18, and TNF-α) in WT mice, and this increase was markedly inhibited in NLRP3-/- mice. LPS-induced SCM was also prevented in Casp1/11-/- mice, but not in Casp11mt, IL-1ß-/-, IL-1α-/-, or GSDMD-/- mice. Notably, LPS-induced SCM was apparently prevented in IL-1ß-/- mice transduced with adeno-associated virus vector expressing IL-18 binding protein (IL-18BP). Furthermore, splenectomy, irradiation, or macrophage depletion alleviated LPS-induced SCM. Our findings demonstrate that the cross-regulation of NLRP3 inflammasome-driven IL-1ß and IL-18 contributes to the pathophysiology of SCM and provide new insights into the mechanism underlying the pathogenesis of SCM.
Assuntos
Cardiomiopatias , Inflamassomos , Interleucina-18 , Interleucina-1beta , Proteína 3 que Contém Domínio de Pirina da Família NLR , Animais , Camundongos , Cardiomiopatias/genética , Caspase 1/genética , Caspase 1/metabolismo , Citocinas , Inflamassomos/metabolismo , Inflamação , Interleucina-18/genética , Interleucina-1beta/metabolismo , Lipopolissacarídeos/efeitos adversos , Camundongos Endogâmicos C57BL , Proteína 3 que Contém Domínio de Pirina da Família NLR/genética , Proteína 3 que Contém Domínio de Pirina da Família NLR/metabolismoRESUMO
Caspase-11 is an inflammatory caspase that triggers an inflammatory response by regulating non-canonical NLRP3 inflammasome activation. Although the deficiency of both caspase-11 and caspase-1, another inflammatory caspase that functions as an executor of the inflammasome, prevents the development of atherosclerosis, the effect of caspase-11 deficiency alone on the development of atherosclerosis has not been fully evaluated. In the present study, we found that caspase-11 deficiency prevented the formation of the necrotic core, whereas it did not affect the development of atherosclerosis in Apoe-deficient mice. Notably, the infiltration of neutrophils into atherosclerotic lesions was attenuated by caspase-11 deficiency. RNA-seq analysis of stage-dependent expression of atherosclerotic lesions revealed that both upregulations of caspase-11 and neutrophil migration are common features of advanced atherosclerotic lesions. Furthermore, similar expression profiles were observed in unstable human plaque. These data suggest that caspase-11 regulates neutrophil recruitment and plaque destabilization in advanced atherosclerotic lesions.
Assuntos
Aterosclerose , Placa Aterosclerótica , Animais , Humanos , Camundongos , Inflamassomos/metabolismo , Caspases , Infiltração de Neutrófilos , Camundongos Knockout , Aterosclerose/metabolismo , Placa Aterosclerótica/patologia , Apolipoproteínas E/genética , Apolipoproteínas/farmacologia , Camundongos Endogâmicos C57BLRESUMO
Hematopoietic stem cells (HSC) rarely divide, rest in quiescence, and proliferate only upon stress hematopoiesis. The cytokine thrombopoietin (Thpo) has been perplexingly described to induce quiescence and promote self-renewal divisions in HSCs. To clarify the contradictory effect of Thpo, we conducted a detailed analysis on conventional (Thpo-/-) and liver-specific (Thpofl/fl;AlbCre+/-) Thpo-deletion models. Thpo-/- HSCs exhibited profound loss of quiescence, impaired cell cycle progression, and increased apoptosis. Thpo-/- HSCs also exhibited diminished mitochondrial mass and impaired mitochondrial bioenergetics. Abnormal HSC phenotypes in Thpo-/- mice were reversible after HSC transplantation into wild-type recipients. Moreover, Thpo-/- HSCs acquired quiescence with extended administration of a Thpo receptor agonist, romiplostim, and were prone to subsequent stem cell exhaustion during competitive bone marrow transplantation. Thpofl/fl;AlbCre+/- HSCs exhibited similar stem cell phenotypes but to a lesser degree compared with Thpo-/- HSCs. HSCs that survive Thpo deficiency acquire quiescence in a dose-dependent manner through the modification of their metabolic state.
Assuntos
Células-Tronco Hematopoéticas/citologia , Trombopoetina/deficiência , Animais , Apoptose , Ciclo Celular , Autorrenovação Celular , Metabolismo Energético/efeitos dos fármacos , Células-Tronco Hematopoéticas/efeitos dos fármacos , Camundongos , Camundongos Knockout , Camundongos Transgênicos , Mitocôndrias/efeitos dos fármacos , Mitocôndrias/metabolismo , Estresse Oxidativo/efeitos dos fármacos , Receptores Fc , Receptores de Trombopoetina/agonistas , Proteínas Recombinantes de Fusão/farmacologia , Transdução de Sinais , Trombopoetina/genética , Trombopoetina/farmacologia , TranscriptomaRESUMO
Sphingosine-1-phosphate (S1P), a potent signalling lipid secreted by red blood cells and platelets, plays numerous biologically significant roles. However, the identity of its long-sought exporter is enigmatic. Here we show that the major facilitator superfamily transporter 2b (Mfsd2b), an orphan transporter, is essential for S1P export from red blood cells and platelets. Comprehensive lipidomic analysis indicates a dramatic and specific accumulation of S1P species in Mfsd2b knockout red blood cells and platelets compared with that of wild-type controls. Consistently, biochemical assays from knockout red blood cells, platelets, and cell lines overexpressing human and mouse Mfsd2b proteins demonstrate that Mfsd2b actively exports S1P. Plasma S1P level in knockout mice is significantly reduced by 42-54% of that of wild-type level, indicating that Mfsd2b pathway contributes approximately half of the plasma S1P pool. The reduction of plasma S1P in knockout mice is insufficient to cause blood vessel leakiness, but it does render the mice more sensitive to anaphylactic shock. Stress-induced erythropoiesis significantly increased plasma S1P levels and knockout mice were sensitive to these treatments. Surprisingly, knockout mice exhibited haemolysis associated with red blood cell stomatocytes, and the haemolytic phenotype was severely increased with signs of membrane fragility under stress erythropoiesis. We show that S1P secretion by Mfsd2b is critical for red blood cell morphology. Our data reveal an unexpected physiological role of red blood cells in sphingolipid metabolism in circulation. These findings open new avenues for investigating the signalling roles of S1P derived from red blood cells and platelets.
Assuntos
Plaquetas/metabolismo , Eritrócitos/metabolismo , Lisofosfolipídeos/metabolismo , Proteínas de Membrana/metabolismo , Esfingosina/análogos & derivados , Anemia/genética , Anemia/metabolismo , Animais , Transporte Biológico , Forma Celular , Contagem de Eritrócitos , Eritrócitos/citologia , Deleção de Genes , Células HEK293 , Humanos , Lisofosfolipídeos/sangue , Proteínas de Membrana/deficiência , Proteínas de Membrana/genética , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Transdução de Sinais , Esfingosina/sangue , Esfingosina/metabolismoRESUMO
RUNX1 is among the most frequently mutated genes in human leukemia, and the loss or dominant-negative suppression of RUNX1 function is found in myelodysplastic syndrome and acute myeloid leukemia (AML). How posttranslational modifications (PTMs) of RUNX1 affect its in vivo function, however, and whether PTM dysregulation of RUNX1 can cause leukemia are largely unknown. We performed targeted deep sequencing on a family with 3 occurrences of AML and identified a novel RUNX1 mutation, R237K. The mutated R237 residue is a methylation site by protein arginine methyltransferase 1, and loss of methylation reportedly impairs the transcriptional activity of RUNX1 in vitro. To explore the biologic significance of RUNX1 methylation in vivo, we used RUNX1 R233K/R237K double-mutant mice, in which 2 arginine-to-lysine mutations precluded RUNX1 methylation. Genetic ablation of RUNX1 methylation led to loss of quiescence and expansion of hematopoietic stem cells (HSCs), and it changed the genomic and epigenomic signatures of phenotypic HSCs to a poised progenitor state. Furthermore, loss of RUNX1 R233/R237 methylation suppressed endoplasmic reticulum stress-induced unfolded protein response genes, including Atf4, Ddit3, and Gadd34; the radiation-induced p53 downstream genes Bbc3, Pmaip1, and Cdkn1a; and subsequent apoptosis in HSCs. Mechanistically, activating transcription factor 4 was identified as a direct transcriptional target of RUNX1. Collectively, defects in RUNX1 methylation in HSCs confer resistance to apoptosis and survival advantage under stress conditions, a hallmark of a preleukemic clone that may predispose affected individuals to leukemia. Our study will lead to a better understanding of how dysregulation of PTMs can contribute to leukemogenesis.
Assuntos
Subunidade alfa 2 de Fator de Ligação ao Core/genética , Subunidade alfa 2 de Fator de Ligação ao Core/metabolismo , Células-Tronco Hematopoéticas/fisiologia , Leucemia/genética , Metiltransferases/metabolismo , Processamento de Proteína Pós-Traducional/genética , Animais , Apoptose/genética , Sobrevivência Celular/genética , Família , Feminino , Predisposição Genética para Doença , Genótipo , Células-Tronco Hematopoéticas/metabolismo , Humanos , Leucemia/metabolismo , Leucemia/patologia , Leucemia Mieloide Aguda/sangue , Leucemia Mieloide Aguda/genética , Leucemia Mieloide Aguda/metabolismo , Masculino , Metilação , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Transgênicos , Pessoa de Meia-Idade , Mutação de Sentido Incorreto , Síndromes Mielodisplásicas/sangue , Síndromes Mielodisplásicas/genética , Síndromes Mielodisplásicas/metabolismo , LinhagemRESUMO
Adult erythropoiesis entails a series of well-coordinated events that produce mature red blood cells. One of such events is the mitochondria clearance that occurs cell-autonomously via autophagy-dependent mechanisms. Interestingly, recent studies have shown mitochondria transfer activities between various cell types. In the context of erythropoiesis, macrophages are known to interact closely with the early stages of erythroblasts to provide a specialized niche, termed erythroblastic islands (EBI). However, whether mitochondria transfer can occur in the EBI niche has not been explored. Here, we report that mitochondria transfer in the EBI niche occurs in vivo. We observed mitochondria transfer activities from the early stages of erythroblasts to macrophages in the reconstituted in vitro murine EBI via different modes, including tunnelling nanotubes (TNT). Moreover, we demonstrated that Wiskott-Aldrich syndrome protein (WASp) in macrophages mediates TNT formation and mitochondria transfer via the modulation of F-actin filamentation, thus promoting mitochondria clearance from erythroid cells, to potentially enhance their differentiation. Taken together, our findings provide novel insight into the mitochondria clearance machineries that mediate erythroid maturation.
Assuntos
Diferenciação Celular , Eritroblastos/metabolismo , Macrófagos/metabolismo , Mitocôndrias/transplante , Nanotubos/química , Nicho de Células-Tronco , Animais , Camundongos , Camundongos Transgênicos , Mitocôndrias/metabolismoRESUMO
Takayasu arteritis (TAK) is a systemic vasculitis with severe complications that affects the aorta and its large branches. HLA-B*52 is an established susceptibility locus to TAK. To date, there are still only a limited number of reports concerning non-HLA susceptibility loci to TAK. We conducted a genome-wide association study (GWAS) and a follow-up study in a total of 633 TAK cases and 5,928 controls. A total of 510,879 SNPs were genotyped, and 5,875,450 SNPs were imputed together with HLA-B*52. Functional annotation of significant loci, enhancer enrichment, and pathway analyses were conducted. We identified four unreported significant loci, namely rs2322599, rs103294, rs17133698, and rs1713450, in PTK2B, LILRA3/LILRB2, DUSP22, and KLHL33, respectively. Two additional significant loci unreported in non-European GWAS were identified, namely HSPA6/FCGR3A and chr21q.22. We found that a single variant associated with the expression of MICB, a ligand for natural killer (NK) cell receptor, could explain the entire association with the HLA-B region. Rs2322599 is strongly associated with the expression of PTK2B Rs103294 risk allele in LILRA3/LILRB2 is known to be a tagging SNP for the deletion of LILRA3, a soluble receptor of HLA class I molecules. We found a significant epistasis effect between HLA-B*52 and rs103294 (P = 1.2 × 10-3). Enhancer enrichment analysis and pathway analysis suggested the involvement of NK cells (P = 8.8 × 10-5, enhancer enrichment). In conclusion, four unreported TAK susceptibility loci and an epistasis effect between LILRA3 and HLA-B*52 were identified. HLA and non-HLA regions suggested a critical role for NK cells in TAK.
Assuntos
Epistasia Genética , Antígeno HLA-B52/genética , Polimorfismo de Nucleotídeo Único , Receptores Imunológicos/genética , Arterite de Takayasu/genética , Estudos de Casos e Controles , Células Cultivadas , Predisposição Genética para Doença , Estudo de Associação Genômica Ampla , Genótipo , Humanos , Células Matadoras Naturais/metabolismo , Células Matadoras Naturais/patologia , Arterite de Takayasu/patologiaRESUMO
Inherited thrombocytopenia is a genetically heterogeneous disease characterized by varying degrees of thrombocytopenia and risk of haematological malignancy, and the genetic cause of many cases remains unknown. We performed whole-exome sequencing of a family with thrombocytopenia and myeloid malignancy and identified a novel TUBB1 variant, T149P. Screening of other thrombocytopenia pedigrees identified another TUBB1 variant, R251H. TUBB1 encodes the tubulin ß-1 chain, a major component of microtubules abundant in megakaryocytes. Variant TUBB1 disrupted the normal assembly of microtubules and impaired proplatelet formation in vitro. In addition, DNA damage response was severely attenuated by loss of TUBB1. We found that the nuclear accumulation of p53 (also termed TP53) and the expression of pro-apoptotic genes triggered by genotoxic stress were blocked in TUBB1-deficient cells and, accordingly, apoptosis after DNA damage was diminished by knockdown of TUBB1. Thus, we have demonstrated that microtubule dysfunction confers resistance to apoptosis, even in DNA damage-accumulated cells, which explains genome instability in the affected individuals. These studies will lead us to a better understanding of how microtubule dysfunction can contribute to the accumulation of DNA damage, genetic instability and leukaemogenesis.
Assuntos
Trombocitopenia/genética , Tubulina (Proteína)/genética , Idoso , Sequência de Aminoácidos , Animais , Apoptose/fisiologia , Linhagem Celular Tumoral , Proliferação de Células/fisiologia , Feminino , Instabilidade Genômica , Mutação em Linhagem Germinativa , Humanos , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Pessoa de Meia-Idade , Linhagem , Alinhamento de Sequência , Trombocitopenia/patologia , Proteína Supressora de Tumor p53/genética , Sequenciamento do ExomaRESUMO
Takayasu arteritis (TAK) is an autoimmune systemic vasculitis of unknown etiology. Although previous studies have revealed that HLA-B*52:01 has an effect on TAK susceptibility, no other genetic determinants have been established so far. Here, we performed genome scanning of 167 TAK cases and 663 healthy controls via Illumina Infinium Human Exome BeadChip arrays, followed by a replication study consisting of 212 TAK cases and 1,322 controls. As a result, we found that the IL12B region on chromosome 5 (rs6871626, overall p = 1.7 × 10(-13), OR = 1.75, 95% CI 1.42-2.16) and the MLX region on chromosome 17 (rs665268, overall p = 5.2 × 10(-7), OR = 1.50, 95% CI 1.28-1.76) as well as the HLA-B region (rs9263739, a proxy of HLA-B*52:01, overall p = 2.8 × 10(-21), OR = 2.44, 95% CI 2.03-2.93) exhibited significant associations. A significant synergistic effect of rs6871626 and rs9263739 was found with a relative excess risk of 3.45, attributable proportion of 0.58, and synergy index of 3.24 (p ≤ 0.00028) in addition to a suggestive synergistic effect between rs665268 and rs926379 (p ≤ 0.027). We also found that rs6871626 showed a significant association with clinical manifestations of TAK, including increased risk and severity of aortic regurgitation, a representative severe complication of TAK. Detection of these susceptibility loci will provide new insights to the basic mechanisms of TAK pathogenesis. Our findings indicate that IL12B plays a fundamental role on the pathophysiology of TAK in combination with HLA-B(∗)52:01 and that common autoimmune mechanisms underlie the pathology of TAK and other autoimmune disorders such as psoriasis and inflammatory bowel diseases in which IL12B is involved as a genetic predisposing factor.
Assuntos
Fatores de Transcrição de Zíper de Leucina e Hélice-Alça-Hélix Básicos/genética , Predisposição Genética para Doença , Antígeno HLA-B52/genética , Subunidade p40 da Interleucina-12/genética , Arterite de Takayasu/genética , Adulto , Idoso , Povo Asiático , Estudos de Casos e Controles , Cromossomos Humanos Par 17 , Cromossomos Humanos Par 5 , Feminino , Ligação Genética , Humanos , Masculino , Pessoa de Meia-Idade , Mutação , Fatores de Risco , Arterite de Takayasu/etnologiaRESUMO
Takayasu arteritis (TAK) is an acute and chronic vasculitis of unknown etiology. Recently, our group reported that SNP rs6871626 in the IL12B region had significant association with disease susceptibility to TAK. However, association of the SNP with clinical characteristics of TAK has yet to be determined. Therefore, we assessed whether this SNP was associated with TAK disease severity as represented by early onset and/or refractoriness to medical therapy. A total of 90 patients were genotyped for rs6871626 and their clinical charts were reviewed retrospectively. By examining the relationship between genotype and clinical profiles of patients, we found a strong association between the number of risk alleles and the frequency of severe cases as defined by (1) age at onset <20 years old, (2) steroid resistance, and/or (3) a relapse of disease [p = 0.03; odds ratio 3.75 (95 % confidence interval 1.13-13.5)]. Thus, our study points to potential diagnostic use of SNP rs6871626 for predicting disease severity of TAK, with the goal of genotyping-oriented therapy in the near future.
Assuntos
Subunidade p40 da Interleucina-12/genética , Polimorfismo de Nucleotídeo Único , Arterite de Takayasu/genética , Adulto , Idade de Início , Idoso , Resistência a Medicamentos/genética , Feminino , Frequência do Gene , Estudos de Associação Genética , Predisposição Genética para Doença , Humanos , Modelos Logísticos , Masculino , Pessoa de Meia-Idade , Razão de Chances , Fenótipo , Valor Preditivo dos Testes , Recidiva , Estudos Retrospectivos , Fatores de Risco , Índice de Gravidade de Doença , Esteroides/uso terapêutico , Arterite de Takayasu/diagnóstico , Arterite de Takayasu/tratamento farmacológico , Resultado do TratamentoRESUMO
Importance: Vasospastic angina (VSA) is vasospasm of the coronary artery and is particularly prevalent in East Asian populations. However, the specific genetic architecture for VSA at genome-wide levels is not fully understood. Objective: To identify genetic factors associated with VSA. Design, Setting, and Participants: This was a case-control genome-wide association study of VSA. Data from Biobank Japan (BBJ; enrolled patients from 2002-2008 and 2013-2018) were used, and controls without coronary artery disease (CAD) were enrolled. Patients from the BBJ were genotyped using arrays or a set of arrays. Patients recruited between 2002 and 2005 were classified within the first dataset, and those recruited between 2006 and 2008 were classified within the second dataset. To replicate the genome-wide association study in the first and second datasets, VSA cases and control samples from the latest patients in the BBJ recruited between 2013 and 2018 were analyzed in a third dataset. Exposures: Single-nucleotide variants associated with VSA. Main Outcomes and Measures: Cases with VSA and controls without CAD. Results: A total of 5720 cases (mean [SD] age, 67 [10] years; 3672 male [64.2%]) and 153â¯864 controls (mean [SD] age, 62 [15] years; 77â¯362 male [50.3%]) in 3 datasets were included in this study. The variants at the RNF213 locus showed the strongest association with VSA across the 3 datasets (odds ratio [OR], 2.34; 95% CI, 1.99-2.74; P = 4.4 × 10-25). Additionally, rs112735431, an Asian-specific rare deleterious variant (p.Arg4810Lys) experimentally shown to be associated with reduced angiogenesis and a well-known causal risk for Moyamoya disease was the most promising candidate for a causal variant explaining the association. The effect size of rs112735431 on VSA was distinct from that of other CADs. Furthermore, homozygous carriers of rs112735431 showed an association with VSA characterized by a large effect estimate (OR, 18.34; 95% CI, 5.15-65.22; P = 7.0 × 10-6), deviating from the additive model (OR, 4.35; 95% CI, 1.18-16.05; P = .03). Stratified analyses revealed that rs112735431 exhibited a stronger association in males (χ21 = 7.24; P = .007) and a younger age group (OR, 3.06; 95% CI, 2.24-4.19), corresponding to the epidemiologic features of VSA. In the registry, carriers without CAD of the risk allele rs112735431 had a strikingly high mortality rate due to acute myocardial infarction during the follow-up period (hazard ratio, 2.71; 95% CI, 1.57-4.65; P = 3.3 × 10-4). As previously reported, a possible overlap between VSA and Moyamoya disease was not found. Conclusions and Relevance: Results of this study suggest that vascular cell dysfunction mediated by variants in the RNF213 locus may promote coronary vasospasm, and the presence of the risk allele could serve as a predictive factor for the prognosis.
Assuntos
Adenosina Trifosfatases , Estudo de Associação Genômica Ampla , Infarto do Miocárdio , Polimorfismo de Nucleotídeo Único , Ubiquitina-Proteína Ligases , Humanos , Masculino , Feminino , Idoso , Estudos de Casos e Controles , Infarto do Miocárdio/genética , Infarto do Miocárdio/epidemiologia , Ubiquitina-Proteína Ligases/genética , Adenosina Trifosfatases/genética , Pessoa de Meia-Idade , Japão/epidemiologia , Vasoespasmo Coronário/genética , Predisposição Genética para Doença , Angina Pectoris Variante/genética , Fatores de RiscoRESUMO
Menkes disease is an X-linked disorder of copper metabolism caused by mutations in the ATP7A gene, and female carriers are usually asymptomatic. We describe a 7-month-old female patient with severe intellectual disability, epilepsy, and low levels of serum copper and ceruloplasmin. While heterozygous deletion of exons 16 and 17 of the ATP7A gene was detected in the proband, her mother, and her grandmother, only the proband suffered from Menkes disease clinically. Intriguingly, X chromosome inactivation (XCI) analysis demonstrated that the grandmother and the mother showed skewing of XCI toward the allele with the ATP7A deletion and that the proband had extremely skewed XCI toward the normal allele, resulting in exclusive expression of the pathogenic ATP7A mRNA transcripts. Expression bias analysis and recombination mapping of the X chromosome by the combination of whole genome and RNA sequencing demonstrated that meiotic recombination occurred at Xp21-p22 and Xq26-q28. Assuming that a genetic factor on the X chromosome enhanced or suppressed XCI of its allele, the factor must be on either of the two distal regions derived from her grandfather. Although we were unable to fully uncover the molecular mechanism, we concluded that unfavorable switching of skewed XCI caused Menkes disease in the proband.
Assuntos
Síndrome dos Cabelos Torcidos , Humanos , Lactente , Feminino , Síndrome dos Cabelos Torcidos/genética , Inativação do Cromossomo X/genética , Cobre/metabolismo , Cromossomos Humanos X/genética , MutaçãoRESUMO
Recent evidence indicates ferroptosis is implicated in the pathophysiology of various liver diseases; however, the organ-specific regulation mechanism is poorly understood. Here, we demonstrate 7-dehydrocholesterol reductase (DHCR7), the terminal enzyme of cholesterol biosynthesis, as a regulator of ferroptosis in hepatocytes. Genetic and pharmacological inhibition (with AY9944) of DHCR7 suppress ferroptosis in human hepatocellular carcinoma Huh-7 cells. DHCR7 inhibition increases its substrate, 7-dehydrocholesterol (7-DHC). Furthermore, exogenous 7-DHC supplementation using hydroxypropyl ß-cyclodextrin suppresses ferroptosis. A 7-DHC-derived oxysterol metabolite, 3ß,5α-dihydroxycholest-7-en-6-one (DHCEO), is increased by the ferroptosis-inducer RSL-3 in DHCR7-deficient cells, suggesting that the ferroptosis-suppressive effect of DHCR7 inhibition is associated with the oxidation of 7-DHC. Electron spin resonance analysis reveals that 7-DHC functions as a radical trapping agent, thus protecting cells from ferroptosis. We further show that AY9944 inhibits hepatic ischemia-reperfusion injury, and genetic ablation of Dhcr7 prevents acetaminophen-induced acute liver failure in mice. These findings provide new insights into the regulatory mechanism of liver ferroptosis and suggest a potential therapeutic option for ferroptosis-related liver diseases.
Assuntos
Ferroptose , Hepatopatias , Oxirredutases atuantes sobre Doadores de Grupo CH-CH , Camundongos , Animais , Humanos , Dicloridrato de trans-1,4-Bis(2-clorobenzaminometil)ciclo-hexano , Oxirredutases atuantes sobre Doadores de Grupo CH-CH/metabolismoRESUMO
The genetic lesions that drive acute megakaryoblastic leukemia (AMKL) have not been fully elucidated. To search for genetic alterations in AMKL, we performed targeted deep sequencing in 34 AMKL patient samples and 8 AMKL cell lines and detected frequent genetic mutations in the NOTCH pathway in addition to previously reported alterations in GATA-1 and the JAK-STAT pathway. Pharmacological and genetic NOTCH activation, but not inhibition, significantly suppressed AMKL cell proliferation in both in vitro and in vivo assays employing a patient-derived xenograft model. These results suggest that NOTCH inactivation underlies AMKL leukemogenesis. and NOTCH activation holds the potential for therapeutic application in AMKL.
RESUMO
Significance: Photothrombosis is a widely used model of ischemic stroke in rodent experiments. In the photothrombosis model, the photosensitizer rose bengal (RB) is systemically introduced into the blood stream and activated by green light to induce aggregation of platelets that eventually cause vessel occlusion. Since the activation of RB is a one-photon phenomenon and the molecules in the illuminated area (light path) are subject to excitation, targeting of thrombosis is unspecific, especially in the depth dimension. We developed a photothrombosis protocol that can target a single vessel in the cortical parenchyma by two-photon excitation. Aim: We aim to induce a thrombotic stroke in the cortical parenchyma by two-photon activation of RB to confine photothrombosis within a vessel of a target depth. Approach: FITC-dextran is injected into the blood stream to visualize the cerebral blood flow in anesthetized adult mice with a cranial window. After a target vessel is chosen by two-photon imaging (950 nm), RB is injected into the blood stream. The scanning wavelength is changed to 720 nm, and photothrombosis is induced by scanning the target vessel. Results: Two-photon depth-targeted single-vessel photothrombosis was achieved with a success rate of 84.9 % ± 1.7 % and an irradiation duration of < 80 s . Attempts without RB (i.e., only with FITC) did not result in photothrombosis at the excitation wavelength of 720 nm. Conclusions: We described a protocol that achieves depth-targeted single-vessel photothrombosis by two-photon excitation. Simultaneous imaging of blood flow in the targeted vessel using FITC dextran enabled the confirmation of vessel occlusion and prevention of excess irradiation that possibly induces unintended photodamage.
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Rhabdomyolysis is a severe condition that commonly leads to acute kidney injury (AKI). While double-stranded DNA (dsDNA) released from injured muscle can be involved in its pathogenesis, the exact mechanism of how dsDNA contributes to rhabdomyolysis-induced AKI (RIAKI) remains obscure. A dsDNA sensor, absent in melanoma 2 (AIM2), forms an inflammasome and induces gasdermin D (GSDMD) cleavage resulting in inflammatory cell death known as pyroptosis. In this study using a mouse model of RIAKI, we found that Aim2-deficiency led to massive macrophage accumulation resulting in delayed functional recovery and perpetuating fibrosis in the kidney. While Aim2-deficiency compromised RIAKI-induced kidney macrophage pyroptosis, it unexpectedly accelerated aberrant inflammation as demonstrated by CXCR3+CD206+ macrophage accumulation and activation of TBK1-IRF3/NF-κB. Kidney macrophages with intact AIM2 underwent swift pyroptosis without IL-1ß release in response to dsDNA. On the other hand, dsDNA-induced Aim2-deficient macrophages escaped from swift pyroptotic elimination and instead engaged STING-TBK1-IRF3/NF-κB signalling, leading to aggravated inflammatory phenotypes. Collectively, these findings shed light on a hitherto unknown immunoregulatory function of macrophage pyroptosis. dsDNA-induced rapid macrophage cell death potentially serves as an anti-inflammatory program and determines the healing process of RIAKI.
Assuntos
Injúria Renal Aguda , Proteínas de Ligação a DNA , Rabdomiólise , Humanos , Injúria Renal Aguda/etiologia , Injúria Renal Aguda/metabolismo , DNA , Proteínas de Ligação a DNA/metabolismo , Inflamassomos/metabolismo , Inflamação , NF-kappa B , Piroptose/genética , Rabdomiólise/complicaçõesRESUMO
Cryopyrin-associated periodic syndrome (CAPS) is an autoinflammatory syndrome caused by mutations of NLRP3 gene encoding cryopyrin. Familial cold autoinflammatory syndrome, the mildest form of CAPS, is characterized by cold-induced inflammation induced by the overproduction of IL-1ß. However, the molecular mechanism of how mutated NLRP3 causes inflammasome activation in CAPS remains unclear. Here, we found that CAPS-associated NLRP3 mutants form cryo-sensitive aggregates that function as a scaffold for inflammasome activation. Cold exposure promoted inflammasome assembly and subsequent IL-1ß release triggered by mutated NLRP3. While K+ efflux was dispensable, Ca2+ was necessary for mutated NLRP3-mediated inflammasome assembly. Notably, Ca2+ influx was induced during mutated NLRP3-mediated inflammasome assembly. Furthermore, caspase-1 inhibition prevented Ca2+ influx and inflammasome assembly induced by the mutated NLRP3, suggesting a feed-forward Ca2+ influx loop triggered by mutated NLRP3. Thus, the mutated NLRP3 forms cryo-sensitive aggregates to promote inflammasome assembly distinct from canonical NLRP3 inflammasome activation.
Assuntos
Síndromes Periódicas Associadas à Criopirina , Proteínas de Transporte/genética , Caspase 1/genética , Síndromes Periódicas Associadas à Criopirina/genética , Humanos , Inflamassomos/metabolismo , Interleucina-1beta/metabolismo , Proteína 3 que Contém Domínio de Pirina da Família NLR/genéticaRESUMO
The transcription factor MYB is a crucial regulator of hematopoietic stem and progenitor cells. However, the nature of lineage-specific enhancer usage of the Myb gene is largely unknown. We identify the Myb -68 enhancer, a regulatory element which marks basophils and mast cells. Using the Myb -68 enhancer activity, we show a population of granulocyte-macrophage progenitors with higher potential to differentiate into basophils and mast cells. Single cell RNA-seq demonstrates the differentiation trajectory is continuous from progenitors to mature basophils in vivo, characterizes bone marrow cells with a gene signature of mast cells, and identifies LILRB4 as a surface marker of basophil maturation. Together, our study leads to a better understanding of how MYB expression is regulated in a lineage-associated manner, and also shows how a combination of lineage-related reporter mice and single-cell transcriptomics can overcome the rarity of target cells and enhance our understanding of gene expression programs that control cell differentiation in vivo.
Assuntos
Basófilos , Hematopoese , Camundongos , Animais , Contagem de Leucócitos , Diferenciação Celular/genética , Células-Tronco/metabolismoRESUMO
Immunoglobulin G4 (IgG4)-related systemic disease was first recognized as a clinicopathological entity about 10 years ago, and since then, it has attracted growing attention. It is an autoimmune disease which affects multiple organs including the pancreas, bile duct, salivary glands and retroperitoneum. Further, it was recently reported that it can be manifested as periarteritis, often as inflammatory abdominal aortic aneurysm. We describe the case of a 75-year-old man with autoimmune pancreatitis and parotitis who presented with angina. The serum concentration of IgG4 was significantly increased at 2,510 mg/dl. Coronary angiography showed multiple stenotic lesions and pronounced dilatation of the right coronary artery. Cardiac computed tomography disclosed increased wall thickness of the coronary arteries and focal tumorous lesions surrounding the right coronary artery. Treatment with steroids proved only marginally effective and he underwent surgical resection of the aneurysm and coronary artery bypass grafting. The diagnosis of IgG4-related systemic disease was confirmed by histological examination of the resected mass, which showed a massive infiltration of IgG4-positive plasma cells. This case emphasizes the importance of considering the diagnosis in any patient with abnormally increased wall thickness or ectatic lesions in the coronary arteries.
Assuntos
Anti-Inflamatórios/uso terapêutico , Doenças Autoimunes/tratamento farmacológico , Doença da Artéria Coronariana/tratamento farmacológico , Hipergamaglobulinemia/tratamento farmacológico , Imunoglobulina G , Idoso , Angina Pectoris/etiologia , Aneurisma Coronário/diagnóstico por imagem , Aneurisma Coronário/etiologia , Angiografia Coronária , Doença da Artéria Coronariana/complicações , Doença da Artéria Coronariana/diagnóstico por imagem , Estenose Coronária/diagnóstico por imagem , Estenose Coronária/etiologia , Humanos , Hipergamaglobulinemia/complicações , Masculino , Pancreatite/complicações , Parotidite/complicações , Prednisolona/uso terapêuticoRESUMO
Interrupted inferior vena cava (IVC) with azygos continuation is a rare congenital anomaly, and is frequently associated with other cardiovascular malformations and situs anomalies, such as left isomerism. These patients usually develop deep vein thrombosis (DVT), and asymptomatic patients above 60 years of age are very rare. Here we report a case of interrupted IVC which we diagnosed in a 72-year-old woman. She was admitted to our hospital suffering from heart failure and supraventricular tachycardia. Echocardiography detected secundum atrial septal defect (ASD). An abnormal paravertebral pleural line on the chest X-rays indicated the existence of venous anomaly. Anatomical images obtained by Multidetector Computed Tomography (MDCT) helped us to successfully perform right heart catheterization procedures through azygos continuation including blood sampling from pulmonary veins. Even in elderly patients, a careful examination of chest X-rays can indicate undiagnosed venous anomalies; thus, it is critically important before planning surgical or interventional procedures.