ORGAN-SPECIFICITY OF METABOLIC REPROGRAMMING AND EXTRACELLULAR VESICLE-BASED COMMUNICATION IN RESIDENT MACROPHAGES

• Grant number: 23/14912-3
• Support Opportunities: Scholarships abroad - Research Internship - Post-doctor
• Start date: April 01, 2024
• End date: March 31, 2025
• Field of knowledge: Biological Sciences - Physiology - Physiology of Organs and Systems
• Principal Investigator: Alexandre Alarcon Steiner
• Grantee: Eduardo Hermogenes Moretti
• Supervisor: Stefano Pluchino
• Host Institution: Instituto de Ciências Biomédicas (ICB). Universidade de São Paulo (USP). São Paulo , SP, Brazil
• Institution abroad: University of Cambridge, England
• Associated to the scholarship: 20/09399-7 - Development of hypothermia in systemic inflammation: the brain hypoxia hypothesis, BP.PD

Abstract

Resident macrophages are sentinel immune cells that sense microbes and the tissue microenvironment, being essential for host defense. They are highly heterogeneous, with each subpopulation playing tissue-specific roles. However, the cellular mechanisms underlying such heterogeneity are not understood, and neither are the mechanisms by which macrophages from distinct organs communicate with each other. Here, we propose to probe for mechanistic differences in the responses elicited by microbial products in macrophages that reside in different organs, and for the potential relevance of these differences to inter-organ communication. Metabolic reprogramming and its relationship to extracellular membrane vesicles will be investigated under three specific aims. Specific Aim 1 (metabolic reprogramming): During the transition to an inflammation-promoting phenotype, macrophages switch energy metabolism from oxidative phosphorylation to high-rate aerobic glycolysis, with a "breakdown" of the Krebs cycle. This results in accumulation of certain Krebs cycle intermediates, such as succinate, citrate, isocitrate and itaconate. We propose that macrophages that reside in different organs will exhibit distinct metabolite profiles (ratios), both intracellularly and extracellularly, during activation by microbial products. This notion will be tested in macrophages harvested from the spleen, liver, lungs and brain of naïve C57/BL male mice. The macrophages from each organ will be stimulated with lipopolysaccharide (LPS), poly(I:C) or CpG oligodeoxynucleotide, and Krebs cycle intermediates from both macrophages (intracellular) and cell culture media (extracellular) will be assessed by LC-MS/MS-based metabolomics. The metabolic profiles will be analyzed in relation to cytokine profiles, assessed by next-generation RNA sequencing (RNAseq). Specific Aim 2 (metabolic reprogramming ’ EVs): We propose that organ-specific metabolic reprogramming in macrophages will be linked not only to specific cytokine expression profiles, but also to specific profiles of EVs release. Macrophages from the spleen, liver, lungs and brain will be harvested and stimulated as in Specific Aim 1. EV size and quantity in culture media will be determined by tunable resistive pulse sensing analysis, nanoparticle tracking analysis, and transmission electron microscopy. EV cargo will be profiled by untargeted proteomic and lipidomic approaches. Specific Aim 3 (EVs ’ metabolic reprogramming): Here, we will investigate the potential of EVs to interorgan communication among macrophage subpopulations. EVs released by LPS-stimulated macrophages from one organ will be purified and added to the culture of macrophages from a different organ prior to their stimulation. As a starting point, and based on our previous study involving splenectomy, this will be done by incubating EVs released by splenic macrophages with macrophages harvested from the liver, lungs and brain. Metabolic reprogramming and cytokine profiles will be assessed as in Specific Aim 1. This proof-of-concept project may provide a novel approach to investigate infectious diseases from an integrative, systems-oriented perspective.