The prolonged activation of the p65 subunit of the nuclear factor NF-kappa-B sustains the persistent effect of advanced glycation end products on inflammatory sensitization in macrophages

Author(s)

Sayonara Ivana Santos de Assis

• Document type: Master's Dissertation
• Press: São Paulo.
• Institution: Universidade de São Paulo (USP). Faculdade de Medicina (FM/SBD)
• Defense date: 2024-03-18

Examining board members

Marisa Passarelli; William Tadeu Lara Festuccia; Helena Coutinho Franco de Oliveira

• Advisor: Marisa Passarelli

Abstract

Advanced glycation end products (AGEs) elicit inflammation by triggering the activation of nuclear factor NF-kappa-B (NFKB) upon binding to the receptor for AGE (RAGE). Moreover, RAGE signaling converges with toll-like receptor 4 (TLR4) signaling, and AGEs prime macrophage to the lipopolysaccharide (LPS)-induced inflammation. This effect is sustained in these cells, persisting even after a long time in an AGE-free environment, and depends on glycemic control in individuals with diabetes mellitus (DM). The persistence ofsensitization to inflammatory stimulation by LPS was determined in macrophages treated with control albumin (C) and AGE-albumin, inferred by 1) secretion of inflammatory cytokines; 2) nuclear content of NFKB subunits, p50, and p65; 3) expression of inflammatory genes. AGE-albumin was prepared by incubation with 10mM glycolaldehyde for 4 days at 37ºC in the dark, and C-albumin was incubated only with a phosphate-buffered solution. RAW-264.7 macrophages overloaded for 24h with acetylated low-density lipoprotein were treated with C or AGE-albumin (2 mg/mL, 48 h), in the presence or absence of HDL. They were then kept in a medium free of these albumins for different intervals and subsequently incubated for 24 h with LPS. Gene expression of RelA, Nfkb1, Ager, Tlr4, IL6, TNF, Abca1, and Abcg1 was determined by RT-qPCR, nuclear content of p50 and p65 by western blot, and secretion of inflammatory cytokines TNF, IL-6, and IL-1beta by ELISA. Comparisons were done by one-way ANOVA or Student t-test (n=6). AGE-albumin sensitized macrophages to LPS stimulation, favoring increased secretion of TNF, IL-6, and IL-1 beta by 1.5%, 9.4%, and 5.6%, respectively, compared to C-albumin (zero time). The increase in TNF, IL-6, and IL-1beta secretion persisted, respectively, up to 24 h, 24 h, and 12 h, even after the removal of AGE-albumin from the medium, with an area under the curve greater by 1.6, 16, and 5.2 times; incubation in the presence of HDL did not alter the persistent inflammatory profile. The expression of Il6 and RelA genes (after 8h of albumin removal from the culture medium) and Il6 and Abca1 (after 24 h of albumin removal from the culture medium) was higher in cells treated with AGE-albumin compared to C-albumin. The nuclear content of p50 was similar between groups, but p65 remained increased 2.9 times for up to 24 h in cells treated with AGE-albumin. The results demonstrate that prolonged activation of the p65 subunit of nuclear factor NFKB sustains the persistent effect of AGE on gene expression alterations and the secretion of inflammatory cytokines in LPS-challenged macrophages, contributing to cellular metabolic memory (AU)

• FAPESP's process: 21/04989-3 - Molecular mediators involved in the persistent effect of advanced glycation products on the inflammatory response and cholesterol efflux disruption in macrophages
• Grantee: Sayonara Ivana Santos de Assis
• Support Opportunities: Scholarships in Brazil - Master