Role of PKM2 in neutrophil metabolism and function

• Grant number: 19/25298-9
• Support Opportunities: Scholarships abroad - Research Internship - Post-doctor
• Start date: January 04, 2021
• End date: January 03, 2022
• Field of knowledge: Biological Sciences - Immunology - Cellular Immunology
• Principal Investigator: José Carlos Farias Alves Filho
• Grantee: Juliana Escher Toller
• Supervisor: Luke Anthony John O'Neill
• Host Institution: Faculdade de Medicina de Ribeirão Preto (FMRP). Universidade de São Paulo (USP). Ribeirão Preto , SP, Brazil
• Institution abroad: Trinity College Dublin, Ireland
• Associated to the scholarship: 17/01714-8 - PKM2 contribution to neutrophils' activation in experimental systemic lupus erythematosus onset, BP.PD

Abstract

Pyruvate kinase type M2 (PKM2) is an enzyme that regulates the final step of glycolysis - converting phosphoenolpyruvate (PEP) in pyruvate - that has a critical role in regulating metabolism and function of immune cells. However, the role of PKM2 in the regulation of neutrophils' functions remains poorly understood. We proposed to investigate the contribution of PKM2 to neutrophil activation in which has been funded by FAPESP. We already found that both human and mouse neutrophils show constitutive expression of PKM2. The genetic deficiency of PKM2 in neutrophils decreased lactate production induced by zymosan, indicating that PKM2 plays an important role in the glycolytic flux of these cells. Moreover, pharmacological inhibition with oxalate or genetic deficiency of PKM2 decreased reactive oxygen species (ROS) production, but not phagocytic capacity, in zymosan-activated neutrophils. On the other hand, pharmacological activation of PKM2 with TEPP-46 increased ROS production. Consequently, the killing capacity was decreased in both oxalate-treated or PKM2-deficient neutrophils and increased in TEPP-46-treated neutrophils. Moreover, we found that neutrophils produce less ROS under glucose-free medium condition, indicating that glycolysis may play a role in ROS production. It is known that ROS production by NADPH oxidase involves the activation of protein kinase C (PKC) by a membrane-bound second messenger diacylglycerol (DAG), that is generated from the cleavage of phosphatidylinositol (4,5)-bisphosphate (PIP2) by phospholipase C (PLC). Interestingly, DAG can also be produced via de novo synthesis from the glycolytic intermediate dihydroxyacetone phosphate (DHAP). In parallel, it has been shown that low activity of PK results in accumulation of its substrate PEP which can inhibit triosephosphate isomerase (TPI), an enzyme that catalyzes the reversible interconversion of DHAP into glyceraldehyde 3-phosphate (G3P). Altogether, we hypothesized that the inhibition or deficiency of PKM2 in neutrophils leads to PEP accumulation, which in turn inhibits TPI, resulting in reduction of DHAP concentration. This would compromise the de novo synthesis of DAG from glucose, impairing ROS production in neutrophils. Indeed, we found that TPI activity was decreased in zymosan-activated neutrophils when PKM2 was pharmacologically inhibited. Moreover, inhibition or deficiency of PKM2 decreased DHAP concentration in zymosan-activated neutrophils. Accordingly, ROS production was restored by pre-treating PKM2-deficient neutrophils with 1-oleoyl-2-acetyl-sn-glycerol (OAG), a functional analog of DAG. Therefore, we intend to further investigate the role of PKM2 in neutrophil metabolism and function, by performing metabolomic and lipidomic analysis. Moreover, another aim is to investigate the role of PKM2 in the flux of pentose phosphate pathway (PPP) and in the glutathione antioxidant system in neutrophils. In order to reach these aims, we have set up a collaborative study with Prof. Luke A.J. O'Neill from Trinity College Dublin in Ireland, which is expert in the field of immunometabolism and metabolic reprogramming in immunity. To this end, we are applying for the BEPE fellowship program to spend 12 months in his laboratory to develop this project. (AU)

Scientific publications

(References retrieved automatically from Web of Science and SciELO through information on FAPESP grants and their corresponding numbers as mentioned in the publications by the authors)

TOLLER-KAWAHISA, JULIANA ESCHER; HIROKI, CARLOS HIROJI; SILVA, CAMILA MEIRELLES DE SOUZA; NASCIMENTO, DANIELE CARVALHO; PUBLIO, GABRIEL AZEVEDO; MARTINS, TIMNA VARELA; DAMASCENO, LUIS EDUARDO ALVES; VERAS, FLAVIO PROTASIO; VIACAVA, PAULA RAMOS; SUKESADA, FABIO YUJI; et al. The metabolic function of pyruvate kinase M2 regulates reactive oxygen species production and microbial killing by neutrophils. NATURE COMMUNICATIONS, v. 14, n. 1, p. 16-pg., 2023-07-17. (13/08216-2, 19/25298-9, 17/01714-8, 20/02207-5)