Study on the role of T-bet in macrophages in the regulation of obesity-induced metabolic syndrome

• Grant number: 22/16083-1
• Support Opportunities: Scholarships in Brazil - Doctorate
• Effective date (Start): April 01, 2023
• Effective date (End): February 28, 2027
• Field of knowledge: Biological Sciences - Immunology
• Principal Investigator: Pedro Manoel Mendes de Moraes Vieira
• Grantee: Webster Leonardo Guimarães da Costa
• 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

Tbx21/T-bet is a transcription factor of the T-box binding domain protein family that was originally described in CD4 T cells and later in T CD8, NK, NKT and ILC1 cells, as being responsible for the transactivation of the Interferon (IFN)y gene, for Th1-type response maintenance. T-bet represses Gata3 and RoryT in helper T lymphocytes and induces IgG2c expression on B lymphocytes, all of which are highly pro-inflammatory functions. In dendritic cells, however, it represses TNFa, suggesting a regulatory role in inflammation in the myeloid lineage. However, whether macrophages are potentially regulated by this important transcription factor is still unknown. Macrophages are plastic phagocytes that adopt different phenotypes according to the stimulus received, such as classic or alternative. Our preliminary data with standard LPS or IL-4 stimuli do not induce Tbx21 expression in macrophages. However, treatment with IFNy is sufficient to induce Tbx21 expression in these cells after 3 hours. Stimulation of LPS together with IFNy potentiates this effect. These data indicate that Tbx21/T-bet expression is related to the regulation of a classic inflammatory profile (LPS+IFNy) and not the alternative resolution profile (IL-4). Deletion of Tbx21 in myeloid cells (LyzMcreTbx21f/f) resulted in higher Nos2 expression and Arg1 reduction in BMDM after LPS+IFNy treatment, with higher NO levels. We also observed that the deletion led to higher levels of IL-6, IL-12 and TNFa, and did not modulate IL-1b after treatment with LPS+IFNy, at the transcriptional level. T-bet deficiency in macrophages also led to a higher glycolytic rate and lactate secretion after treatment with LPS+IFNy. Taken together, our data suggest that T-bet regulates macrophage mediators and metabolism in order to prevent exacerbated levels of NO and pro-inflammatory cytokines, in addition to the accelerated flow of the glycolytic pathway related to classical polarization (LPS+IFNy). Thus, the same protein (T-bet) has antagonistic functions depending on the cell type (lymphoid or myeloid) to regulate the inflammatory scenario. The preliminary study with a classic model of activation by TLR4 agonist (LPS) aroused the interest of carrying out the obesity model, in which there is also activation of macrophages by high levels of IFNy, and by palmitate that binds to TLR4. Total deletion of T-bet in mice improves obesity-induced insulin resistance by decreasing a Th1 response in mice submitted to a high-fat diet. However, the role of T-bet in the myeloid lineage has not yet been evaluated. We observed that the MMe stimulus that mimics the obesogenic condition is able to induce Tbx21 in BMDM, which is intensified with the addition of IFNy. Analysis of scRNA-seq of resident adipose tissue macrophages (ATM) reveals that they express Tbx21, with an increasing trend in animals submitted to high-fat diet. Thus, obesity culminates in the induction of T-bet in ATMs, and its role will be explored by us. Thus, our aim is to determine the role of T-bet in modulating macrophage function and metabolism in the context of obesity-induced metabolic syndrome, as well as the mechanisms. We hypothesize that LyzMcreTbx21f/f mice will present a more exacerbated metabolic syndrome than animals without the conditional deletion, when submitted to a high-fat diet, due to the greater production of inflammatory cytokines, such as TNFa, which is a potent inhibitor of insulin receptor signaling in adipocytes, myocytes, and hepatocytes. We assume that T-bet mechanistically binds to the promoter of genes related to the inflammatory tone of macrophages, repressing them. Thus, a new perspective on the role of this transcription factor may suggest new strategies for the prevention and/or treatment of type 2 diabetes and other inflammatory diseases in which macrophages participate.