Study of the mitophagic pathway regulation in macrophages by HIF-1a

• Grant number: 23/01856-8
• Support Opportunities: Scholarships in Brazil - Doctorate (Direct)
• Start date: March 01, 2023
• End date: February 28, 2027
• Field of knowledge: Biological Sciences - Immunology
• Principal Investigator: Pedro Manoel Mendes de Moraes Vieira
• Grantee: Thaís Pirola dos Santos
• Host Institution: Instituto de Biologia (IB). Universidade Estadual de Campinas (UNICAMP). Campinas , SP, Brazil
• Associated research grant: 20/16030-0 - Immunometabolic adaptation of tissue resident macrophages in health and disease, AP.TEM

Abstract

Mitophagy is the process of eliminating defective mitochondria, which acts as a mitochondrial quality control - reducing inflammation and signs of cell death, in addition to preventing the development of chronic diseases. The process occurs through the PINK1/Parkin pathway or also through the receptor-mediated pathway, in which both induce the formation of autolysosomes, where damaged mitochondria is degraded. The transcription factor HIF-1±, described as fundamental in the regulation of the immune response, demonstrated in our preliminary studies, to be involved in the expression of lysosomal proteins - Cathepsin C and LAMP1 - and mitophagic- BNIP3. Thus, the project aims to elucidate the influence of the transcription factor HIF-1± on the regulation of mitophagy in macrophages, verifying its impact in the presence and functionality of lysosomes and its influence in mitophagy, as well as in the functionality of macrophages with HIF-1± modulation. Lysosomal proteins will be evaluated by qPCR, Western Blot and enzymatic assay to verify the abundance and activity of lysosomes, being further characterized early and late autophagolysosomes by immunofluorescence and electron microscopy. To assess how HIF-1± affects mitophagy in macrophages, LysMcreHif1±flox, LysMcreVHLflox mice will be used with Phamflox animals, and the mitophagic proteins will also be verified by Western Blot and confocal microscopy. The metabolism of macrophages will be evaluated using expression of glycolytic pathway genes by qPCR and cytokines will be evaluated by intracellular cytometry and ELISA. Finally, the phagocytic capacity of macrophages will be analyzed by flow cytometry and CFU, and the response of in vivo macrophages to infection with E. coli will be evaluated. With this, it is expected to determine how the HIF-1± modulates the functionality of lysosomes and the process of mitophagy in macrophages. (AU)