RESUMO
INTRODUCTION: Chronic hyperglycemia affects neutrophil functions, leading to reduced pathogen killing and increased morbidity. This impairment has been directly linked to increased glycemia, however, how this specifically affects neutrophils metabolism and their differentiation in the bone marrow is unclear and difficult to study. RESEARCH DESIGN AND METHODS: We used high-resolution respirometry to investigate the metabolism of resting and activated donor neutrophils, and flow cytometry to measure surface CD15 and CD11b expression. We then used HL-60 cells differentiated towards neutrophil-like cells in standard media and investigated the effect of doubling glucose concentration on differentiation metabolism. We measured the oxygen consumption rate (OCR), and the enzymatic activity of carnitine palmitoyl transferase 1 (CPT1) and citrate synthase during neutrophil-like differentiation. We compared the surface phenotype, functions, and OCR of neutrophil-like cells differentiated under both glucose concentrations. RESULTS: Donor neutrophils showed significant instability of CD11b and OCR after phorbol 12-myristate 13-acetate stimulation at 3 hours post-enrichment. During HL-60 neutrophil-like cell differentiation, there was a significant increase in surface CD15 and CD11b expression together with the loss of mitochondrial mass. Differentiated neutrophil-like cells also exhibited higher CD11b expression and were significantly more phagocytic. In higher glucose media, we measured a decrease in citrate synthase and CPT1 activities during neutrophil-like differentiation. CONCLUSIONS: HL-60 neutrophil-like differentiation recapitulated known molecular and metabolic features of human neutrophil differentiation. Increased glucose concentrations correlated with features described in hyperglycemic donor neutrophils including increased CD11b and phagocytosis. We used this model to describe metabolic features of neutrophil-like cell differentiation in hyperglycemia and show for the first time the downregulation of CPT1 and citrate synthase activity, independently of mitochondrial mass.
Assuntos
Diferenciação Celular , Hiperglicemia , Neutrófilos , Humanos , Neutrófilos/metabolismo , Células HL-60 , Hiperglicemia/metabolismo , Hiperglicemia/patologia , Antígeno CD11b/metabolismo , Glucose/metabolismo , Carnitina O-Palmitoiltransferase/metabolismo , Consumo de Oxigênio , Antígenos CD15/metabolismo , Citrato (si)-Sintase/metabolismoRESUMO
Neutrophils possess unique characteristics that render them indispensable to health, and patients with irregular neutrophil counts or functions suffer from increased morbidity and mortality. As neutrophils are short-lived postmitotic cells, genetic aberrations cannot be corrected directly in neutrophils and must be targeted in their progenitors. Neutrophils are increasingly being contemplated for a range of therapeutic applications, including restoration or modulation of immune function and targeting of solid tumors. This review addresses the state-of-the-art in neutrophil transfusions and their possible applications for infectious disease prevention and treatment. It offers a landscape of the most recent gene therapy approaches to address neutrophil-related genetic diseases. We also discuss how ongoing research could broaden the applicability of neutrophil-based therapies to solid cancer treatments and beyond.
Assuntos
Terapia Genética , Neoplasias , Neutrófilos , Humanos , Neutrófilos/imunologia , Neoplasias/terapia , Neoplasias/imunologia , Terapia Genética/métodos , AnimaisRESUMO
The gold-standard method to evaluate a functional antiviral immune response is to titer neutralizing antibodies (NAbs) against a viral pathogen. This is historically performed using an in vitro assay of virus-mediated infection, which requires BSL-3 facilities. As these are insufficient in Latin American countries, including Mexico, scant information is obtained locally about viral pathogens NAb, using a functional assay. An alternative solution to using a BSL-3 assay with live virus is to use a BSL-2-safe assay with a non-replicative pseudovirus. Pseudoviral particles can be engineered to display a selected pathogen's entry protein on their surface, and to deliver a reporter gene into target cells upon transduction. Here we comprehensively describe the first development of a BSL-2 safe NAbs-measuring functional assay in Mexico, based on the production of pseudotyped lentiviral particles. As proof-of-concept, the assay is based on Nanoluc luciferase-mediated luminescence measurements from target cells transduced with SARS-CoV-2 Spike-pseudotyped lentiviral particles. We applied the optimized assay in a BSL-2 facility to measure NAbs in 65 serum samples, which evidenced the assay with 100% sensitivity, 86.6% specificity and 96% accuracy. Overall, this is the first report of a BSL-2 safe pseudovirus-based functional assay developed in Mexico to measure NAbs, and a cornerstone methodology necessary to measure NAbs with a functional assay in limited resources settings.