Reprogramming of Macrophage Metabolism and Function by Crotoxin

• Grant number: 18/09326-0
• Support Opportunities: Scholarships in Brazil - Doctorate
• Start date: March 01, 2019
• End date: August 31, 2022
• Field of knowledge: Health Sciences - Medicine
• Principal Investigator: Sandra Coccuzzo Sampaio Vessoni
• Grantee: Patricia Pereira Nunes Traldi
• Host Institution: Instituto Butantan. Secretaria da Saúde (São Paulo - Estado). São Paulo , SP, Brazil

Abstract

Macrophages (MØ) are cells with high plasticity that can be reprogrammed into two distinct phenotypes - MØ M1 (pro-inflammatory) and MØ M2 (anti-inflammatory) - according to the nature of the disorder detected in the organism. Experimentally, it is possible to induce reprogramming of macrophages to M1 and M2 and the functional and metabolic status of both phenotypes have already been characterized. Previous studies of our group have demonstrated that Crotoxin (CTX) exerts on macrophages two actions that make them relevant for cellular therapy: 1) it changes its metabolic and functional profile and 2) these effects are long lasting. Such effects are observed after a single and short exposure to the toxin. In this context, this research seeks to characterize the metabolism and functional status of MØ treated with different concentrations of CTX, in order to verify whether in each concentration is possible to identify intermediates phenotypes other than those already described as M1 and M2 profiles, then to seek to reprogram these cells according to specific pathophysiological processes. The CTX-induced phenotypic reprogramming ability of monocytes will be evaluated in in vitro assays, when cells of the human monocytic lineage (THP-1) will be incubated in absence (control) or in the presence of CTX at different concentrations (2 nM; 10 nM; 50 nM and 100 nM) over 1 hour and then maintained in culture for 24 hours. After the different protocols of incubation with the toxin, the phenotypes markers will be characterized by qPCR technique, using as markers of M1 profile the toll type receptor (TLR-4), CD25 and CD80 and for M2 profile, the mannose receptor, SR-A, CD163 and CD209. As a method of evaluation of cellular function, it will be analyzed the capacity of spreading, phagocytosis and cytokines secretion (IL-1², IL-6, TNF-± and IL-10), besides the generation of reactive oxygen species (H2O2) and nitrogen (NO) by MØ. Considering that the main substrates of MØ metabolism are glucose and glutamine, to evaluate the metabolic function, the gene expression and the activity of key enzymes of the glycolytic pathway (hexoquinase, G6PDH e fosfofrutoquinase) and glyutaminolytic pathway (glutaminase) will be analyzed. The key enzyme of the citric acid cycle (citrate synthase) in addition to the enzymes fatty acid synthase, ATP-citrate lyase and malic enzyme, involved in fatty acid metabolism and the key enzyme of beta-oxidation - carnitine palmitoyl-CoA transferase will also be evaluated. Finally, the concentration of citrate and succinate substrates will be measured. This study, through the use of CTX as an important scientific tool capable of modulating metabolism of MØ, will provide evidence of key points of cellular metabolism involved in the capacity of the immune system to control and/or reverse the progression of different inflammatory diseases, generating important impact in the studies of immunometabolism and immunomodulation.