Proteomic and Metabolic Analysis of Neutrophils from Septic Patients

• Grant number: 23/15909-6
• Support Opportunities: Scholarships in Brazil - Doctorate
• Start date: March 01, 2024
• End date: March 31, 2026
• Field of knowledge: Biological Sciences - Immunology - Cellular Immunology
• Principal Investigator: Reinaldo Salomão
• Grantee: Mônica Bragança Sousa
• Host Institution: Escola Paulista de Medicina (EPM). Universidade Federal de São Paulo (UNIFESP). Campus São Paulo. São Paulo , SP, Brazil
• Associated research grant: 17/21052-0 - Sepsis: mechanisms, therapeutic targets and epidemiology, AP.TEM

Abstract

AbstractSepsis is a public health problem that presents high morbidity and mortality, being one of the main causes of death in intensive care units. This condition, defined as a dysregulated host response to infection, has neutrophils as one of the first lines of defense against pathogens. Neutrophils are a priority in the lesion focus and even in adverse metabolic conditions they are capable of meeting large demands by storing energy through gluconeogenesis. There are several studies that demonstrate the glycolytic dependence of neutrophils in the immunomodulation of sepsis, however, this regulatory role is still little explored and studies suggest that dysfunctional neutrophils contribute to greater mortality. The objective of this project is to characterize the proteomic profile of neutrophils from patients with sepsis and use changes in metabolic pathways as a reference for carrying out functional studies, directed with the use of specific inhibitors to qualify the pathways related to cellular response and understand how the capacity metabolic influences the activation and response of these cells. For the proteomic analysis, we selected samples from 59 septic patients and 11 healthy controls from our biorepository. LC-MS/MS analyzes were performed on the Orbitrap Fusion Lumos system (Thermo), raw data were processed using Proteome Discoverer Suite and analyzed in R software. The assessment of the contribution of metabolic pathways to cellular function will be made by blocking the main supporting pathways, such as 2DG, using multiparameter flow cytometry. To carry out these experiments, we have a blood collection network from septic patients that are part of the thematic project FAPESP 2017/21052-0 and the use of the four-laser LSRFortessa (BD) cytometer, capable of reading up to twelve fluorochromes, where they will be Tests for the production of reactive oxygen species (ROS), extracellular traps (NETs), phagocytosis and glucose uptake were carried out. We will also evaluate oxygen consumption and extracellular acidification using the Seahorse XFe real-time metabolic analyzer (Agilent). Our preliminary results from proteomic analysis show that the most altered pathways are related to the immune system, neutrophil degranulation and metabolic pathways, indicating a regulation of these proteins that influence the cellular function of patient neutrophils. A better understanding of the immune response in sepsis may target mechanisms to reduce the exacerbated response that causes harm.