RESUMEN
Metabolic dysfunction-associated steatotic liver disease (MASLD, formerly known as non-alcoholic fatty liver disease) is a leading cause of chronic liver disease worldwide. MASLD can progress to metabolic dysfunction-associated steatohepatitis (MASH, formerly known as non-alcoholic steatohepatitis) with subsequent liver cirrhosis and hepatocellular carcinoma formation. The advent of current technologies such as single-cell and single-nuclei RNA sequencing have transformed our understanding of the liver in homeostasis and disease. The next frontier is contextualizing this single-cell information in its native spatial orientation. This understanding will markedly accelerate discovery science in hepatology, resulting in a further step-change in our knowledge of liver biology and pathobiology. In this Review, we discuss up-to-date knowledge of MASLD development and progression and how the burgeoning field of spatial genomics is driving exciting new developments in our understanding of human liver disease pathogenesis and therapeutic target identification.
Asunto(s)
Genómica , Enfermedad del Hígado Graso no Alcohólico , Humanos , Enfermedad del Hígado Graso no Alcohólico/genética , Progresión de la Enfermedad , Análisis de la Célula Individual , Neoplasias Hepáticas/genética , Neoplasias Hepáticas/patologíaRESUMEN
The concept of hepatocyte functional zonation is well established, with differences in metabolism and xenobiotic processing determined by multiple factors including oxygen and nutrient levels across the hepatic lobule. However, recent advances in single-cell genomics technologies, including single-cell and nuclei RNA sequencing, and the rapidly evolving fields of spatial transcriptomic and proteomic profiling have greatly increased our understanding of liver zonation. Here we discuss how these transformative experimental strategies are being leveraged to dissect liver zonation at unprecedented resolution and how this new information should facilitate the emergence of novel precision medicine-based therapies for patients with liver disease.
Asunto(s)
Proteómica , Xenobióticos , Hepatocitos/metabolismo , Humanos , Hígado/metabolismo , Oxígeno/metabolismoRESUMEN
The mRNA N6-methyladenosine (m6A) modification has emerged as an essential regulator of normal and malignant hematopoiesis. Inactivation of the m6A mRNA reader YTHDF2, which recognizes m6A-modified transcripts to promote m6A-mRNA degradation, results in hematopoietic stem cell (HSC) expansion and compromises acute myeloid leukemia. Here we investigate the long-term impact of YTHDF2 deletion on HSC maintenance and multilineage hematopoiesis. We demonstrate that Ythdf2-deficient HSCs from young mice fail upon serial transplantation, display increased abundance of multiple m6A-modified inflammation-related transcripts, and chronically activate proinflammatory pathways. Consistent with the detrimental consequences of chronic activation of inflammatory pathways in HSCs, hematopoiesis-specific Ythdf2 deficiency results in a progressive myeloid bias, loss of lymphoid potential, HSC expansion, and failure of aged Ythdf2-deficient HSCs to reconstitute multilineage hematopoiesis. Experimentally induced inflammation increases YTHDF2 expression, and YTHDF2 is required to protect HSCs from this insult. Thus, our study positions YTHDF2 as a repressor of inflammatory pathways in HSCs and highlights the significance of m6A in long-term HSC maintenance.
Asunto(s)
Adenosina/análogos & derivados , Células Madre Hematopoyéticas/metabolismo , Inflamación/genética , Proteínas de Unión al ARN/metabolismo , Adenosina/metabolismo , Animales , Linaje de la Célula , Proliferación Celular , Senescencia Celular , Eliminación de Gen , Hematopoyesis , Trasplante de Células Madre Hematopoyéticas , Inflamación/patología , Linfocitos/metabolismo , Ratones Endogámicos C57BL , Células Mieloides/metabolismo , ARN Mensajero/genética , ARN Mensajero/metabolismoRESUMEN
Acute myeloid leukemia (AML) is an aggressive clonal disorder of hematopoietic stem cells (HSCs) and primitive progenitors that blocks their myeloid differentiation, generating self-renewing leukemic stem cells (LSCs). Here, we show that the mRNA m6A reader YTHDF2 is overexpressed in a broad spectrum of human AML and is required for disease initiation as well as propagation in mouse and human AML. YTHDF2 decreases the half-life of diverse m6A transcripts that contribute to the overall integrity of LSC function, including the tumor necrosis factor receptor Tnfrsf2, whose upregulation in Ythdf2-deficient LSCs primes cells for apoptosis. Intriguingly, YTHDF2 is not essential for normal HSC function, with YTHDF2 deficiency actually enhancing HSC activity. Thus, we identify YTHDF2 as a unique therapeutic target whose inhibition selectively targets LSCs while promoting HSC expansion.
Asunto(s)
Leucemia Mieloide Aguda/terapia , Células Madre Neoplásicas/fisiología , Proteínas de Unión al ARN/metabolismo , Animales , Autorrenovación de las Células , Hematopoyesis , Células Madre Hematopoyéticas , Humanos , Leucemia Mieloide Aguda/genética , Ratones , Ratones Endogámicos C57BL , Ratones Noqueados , ARN Interferente Pequeño/genética , Proteínas de Unión al ARN/genética , Células THP-1RESUMEN
There is a large amount of information in brightfield images that was previously inaccessible by using traditional microscopy techniques. This information can now be exploited by using machine-learning approaches for both image segmentation and the classification of objects. We have combined these approaches with a label-free assay for growth and differentiation of leukemic colonies, to generate a novel platform for phenotypic drug discovery. Initially, a supervised machine-learning algorithm was used to identify in-focus colonies growing in a three-dimensional (3D) methylcellulose gel. Once identified, unsupervised clustering and principle component analysis of texture-based phenotypic profiles were applied to group similar phenotypes. In a proof-of-concept study, we successfully identified a novel phenotype induced by a compound that is currently in clinical trials for the treatment of leukemia. We believe that our platform will be of great benefit for the utilization of patient-derived 3D cell culture systems for both drug discovery and diagnostic applications.
Asunto(s)
Descubrimiento de Drogas , Imagenología Tridimensional , Leucemia/diagnóstico por imagen , Leucemia/tratamiento farmacológico , Aprendizaje Automático , Fenotipo , Antineoplásicos/uso terapéutico , Diferenciación Celular/efectos de los fármacos , Proliferación Celular/efectos de los fármacos , Células Cultivadas , Humanos , Tamaño de la Partícula , Propiedades de Superficie , Células THP-1RESUMEN
Strict regulation of stem cell metabolism is essential for tissue functions and tumor suppression. In this study, we investigated the role of fumarate hydratase (Fh1), a key component of the mitochondrial tricarboxylic acid (TCA) cycle and cytosolic fumarate metabolism, in normal and leukemic hematopoiesis. Hematopoiesis-specific Fh1 deletion (resulting in endogenous fumarate accumulation and a genetic TCA cycle block reflected by decreased maximal mitochondrial respiration) caused lethal fetal liver hematopoietic defects and hematopoietic stem cell (HSC) failure. Reexpression of extramitochondrial Fh1 (which normalized fumarate levels but not maximal mitochondrial respiration) rescued these phenotypes, indicating the causal role of cellular fumarate accumulation. However, HSCs lacking mitochondrial Fh1 (which had normal fumarate levels but defective maximal mitochondrial respiration) failed to self-renew and displayed lymphoid differentiation defects. In contrast, leukemia-initiating cells lacking mitochondrial Fh1 efficiently propagated Meis1/Hoxa9-driven leukemia. Thus, we identify novel roles for fumarate metabolism in HSC maintenance and hematopoietic differentiation and reveal a differential requirement for mitochondrial Fh1 in normal hematopoiesis and leukemia propagation.
Asunto(s)
Fumarato Hidratasa/fisiología , Células Madre Hematopoyéticas/fisiología , Animales , Femenino , Fumaratos/metabolismo , Hematopoyesis , Histonas/metabolismo , Leucemia Mieloide Aguda/etiología , Ratones , Ratones Endogámicos C57BL , Mitocondrias/metabolismo , Factor 2 Relacionado con NF-E2/fisiología , Consumo de OxígenoRESUMEN
The hematopoietic stem cell (HSC) pool is maintained under hypoxic conditions within the bone marrow microenvironment. Cellular responses to hypoxia are largely mediated by the hypoxia-inducible factors, Hif-1 and Hif-2. The oxygen-regulated α subunits of Hif-1 and Hif-2 (namely, Hif-1α and Hif-2α) form dimers with their stably expressed ß subunits and control the transcription of downstream hypoxia-responsive genes to facilitate adaptation to low oxygen tension. An initial study concluded that Hif-1α is essential for HSC maintenance, whereby Hif-1α-deficient HSCs lost their ability to self-renew in serial transplantation assays. In another study, we demonstrated that Hif-2α is dispensable for cell-autonomous HSC maintenance, both under steady-state conditions and following transplantation. Given these unexpected findings, we set out to revisit the role of Hif-1α in cell-autonomous HSC functions. Here we demonstrate that inducible acute deletion of Hif-1α has no impact on HSC survival. Notably, unstressed HSCs lacking Hif-1α efficiently self-renew and sustain long-term multilineage hematopoiesis upon serial transplantation. Finally, Hif-1α-deficient HSCs recover normally after hematopoietic injury induced by serial administration of 5-fluorouracil. We therefore conclude that despite the hypoxic nature of the bone marrow microenvironment, Hif-1α is dispensable for cell-autonomous HSC maintenance.
Asunto(s)
Células Madre Adultas/fisiología , Proliferación Celular/genética , Células Madre Hematopoyéticas/fisiología , Subunidad alfa del Factor 1 Inducible por Hipoxia/genética , Células Madre Adultas/metabolismo , Animales , División Celular/genética , Células Cultivadas , Femenino , Hematopoyesis/genética , Células Madre Hematopoyéticas/metabolismo , Subunidad alfa del Factor 1 Inducible por Hipoxia/metabolismo , Masculino , Ratones , Ratones Endogámicos C57BL , Ratones TransgénicosRESUMEN
OBJECTIVE: To evaluate the sensitivity and specificity of an enzyme immunoassay (EIA) for antibodies to a recombinant Blastomyces adhesin-1 repeat antigen (rBAD-1) to aid in the diagnosis of blastomycosis in dogs and compare the findings with results from other tests used for this purpose. DESIGN: Prospective analytic study. SAMPLE: Serum and urine from 70 dogs with and without blastomycosis. PROCEDURES: Serum and urine samples were collected from dogs with blastomycosis (n = 21), histoplasmosis (8), or nonfungal pulmonary disease (21) and from healthy control dogs living in a blastomycosis-endemic area (20). Serum was tested for antibodies against Blastomyces dermatitidis with the rBAD-1 antibody EIA and an A-antigen antibody agar gel immunodiffusion (AGID) assay. Serum and urine were tested for B dermatitidis antigen with a quantitative EIA. RESULTS: Sensitivity of the quantitative antigen EIA was 100% in serum and urine samples from dogs with blastomycosis, with specificity of 95% in urine samples from dogs with nonfungal pulmonary disease and 100% in urine samples from healthy dogs. Sensitivity of the rBAD-1 antibody EIA (95%) was significantly greater than that of the A-antigen antibody AGID assay (65%). Specificity of the antibody EIA was 88% in dogs with histoplasmosis, 95% in healthy dogs, and 100% in dogs with nonfungal pulmonary disease. CONCLUSIONS AND CLINICAL RELEVANCE: The rBAD-1 antibody EIA had greater sensitivity than the A-antigen antibody AGID assay in dogs with blastomycosis. This antibody EIA may assist in distinguishing histoplasmosis from blastomycosis. Further evaluation in a larger prospective study is needed to verify these results.
Asunto(s)
Anticuerpos Antifúngicos/inmunología , Antígenos Fúngicos/inmunología , Blastomyces/metabolismo , Blastomicosis/veterinaria , Enfermedades de los Perros/microbiología , Técnicas para Inmunoenzimas/veterinaria , Animales , Anticuerpos Antifúngicos/sangre , Anticuerpos Antifúngicos/orina , Blastomicosis/sangre , Blastomicosis/diagnóstico , Blastomicosis/orina , Enfermedades de los Perros/diagnóstico , Perros , Femenino , Técnicas para Inmunoenzimas/métodos , Masculino , Sensibilidad y EspecificidadRESUMEN
BACKGROUND: Individual enteropathogen infections in healthy and clinically ill cats are well described, but prevalence and patterns of enteropathogen co-infection have only been reported on a limited basis. We studied enteropathogen co-infection in diarrhoeic UK cats using results of a real time PCR assay for 8 enteropathogenic species; feline coronavirus (Co), feline panleukopenia virus (Pa), Clostridium perfringens (Cl), Salmonella enterica (Sa), Giardia spp. (Gi), Tritrichomonas foetus (Tr), Cryptosporidium spp. (Cr), and Toxoplasma gondii (To). Age, gender, breed and history were recorded. PCR panels from 1088 diarrhoeic cats were available for analysis. RESULTS: Overall enteropathogen prevalence was 56.9% (Co), 22.1% (Pa), 56.6% (Cl), 0.8% (Sa), 20.6% (Gi), 18.8% (Tr), 24.4% (Cr) and 1.0% (To). Prevalence of Co, Gi and Tr was higher in pedigree cats compared to non-pedigree cats (DSH) and prevalence decreased with increasing age for Co, Pa, Gi, Cr and Tr. Co-infection was common: ≥2 enteropathogens were detected in 62.5% of cats, and 13.3% of cats had ≥4 enteropathogens. Mean ( x¯) enteropathogen co-infection 2.01 (±1.3 SD), was significantly higher in pedigree cats ( x¯ =2.51) compared to DSH ( x¯ =1.68) and decreased with age ( x¯ =2.64 <6 months, x¯ =1.68 for >1 yr). More cats were negative for all 8 enteropathogens tested (12.7%) than expected. When exact combinations of co-infection were examined, Tr tended to be found in combinations with Co, Cl, and Gi. CONCLUSIONS: Multiple infections should be considered the most likely result of faecal testing in cats, and case management needs to take this into account. In contrast, the relatively high percentage of cats negative for all 8 enteropathogens tested could indicate an innate resistance to infection. Alternatively it could indicate a lack of exposure to these 8 enteropathogens or the presence of other enteropathogens not assessed by this assay.