The Prolonged Activation of the p65 Subunit of the NF-Kappa-B Nuclear Factor Sustains the Persistent Effect of Advanced Glycation End Products on Inflammatory Sensitization in Macrophages

Abstract

Advanced glycation end products (AGEs) prime macrophages for lipopolysaccharide (LPS)-induced inflammation. We investigated the persistence of cellular AGE-sensitization to LPS, considering the nuclear content of p50 and p65 nuclear factor kappa B (NFKB) subunits and the expression of inflammatory genes. Macrophages treated with control (C) or AGE-albumin were rested for varying intervals in medium alone before being incubated with LPS. Comparisons were made using one-way ANOVA or Student t-test (n = 6). AGE-albumin primed macrophages for increased responsiveness to LPS, resulting in elevated levels of TNF, IL-6, and IL-1beta (1.5%, 9.4%, and 5.6%, respectively), compared to C-albumin. TNF, IL-6, and IL-1 beta secretion persisted for up to 24 h even after the removal of AGE-albumin (area under the curve greater by 1.6, 16, and 5.2 times, respectively). The expressions of Il6 and RelA were higher 8 h after albumin removal, and Il6 and Abca1 were higher 24 h after albumin removal. The nuclear content of p50 remained similar, but p65 showed a sustained increase (2.9 times) for up to 24 h in AGE-albumin-treated cells. The prolonged activation of the p65 subunit of NFKB contributes to the persistent effect of AGEs on macrophage inflammatory priming, which could be targeted for therapies to prevent complications based on the AGE-RAGE-NFKB axis.

Keywords: NFKB; RAGE; advanced glycation end products; inflammation; lipopolysaccharide; toll-like receptor.

Figure 1

Lactate dehydrogenase (LDH) activity in RAW 264.7 macrophages treated with C- or AGE-albumin and challenged with LPS after resting for different intervals of time. RAW 264.7 macrophages were treated for 24 h with acetylated LDL and then with C- or AGE-albumin (2 mg/mL) for 48 h. After different resting intervals in RPMI/FAFA, cells were challenged with LPS for 24 h. Following the completion of treatments, the culture medium was collected for the LDH assay. Values were compared using Student’s t-test and represented as mean ± standard error of the mean (n = 6).

Figure 2

Persistence of the effect of AGE-albumin on the inflammatory response in RAW 264.7 macrophages. RAW 264.7 macrophages were treated for 24 h with acetylated LDL (50 µg/mL) and then with either C- or AGE-albumin (2 mg/mL) for 48 h. Following various resting intervals in RPMI/FAFA, cells were exposed to LPS (1 µg/mL) for 24 h. The concentrations of TNF, IL-6, and IL-1beta were determined by ELISA, with total concentrations presented in panels (A,C,E) and the area under the curve (AUC) depicted in panels (B,D,F). Normality was tested using the Shapiro–Wilk test, and comparisons were conducted using Student’s t-test. Results are expressed as mean ± standard error of the mean (n = 6 for each incubation time).

Figure 3

Persistence of the effect of AGE-albumin on the inflammatory response in RAW 264.7 macrophages. RAW 264.7 macrophages were treated for 24 h with acetylated LDL (50 µg/mL) and then with C- or AGE-albumin (2 mg/mL), alone or in the presence of HDL (50 µg/mL), for 48 h. Following various resting intervals in RPMI/FAFA, cells were exposed to LPS (1 µg/mL) for 24 h. The concentrations of TNF, IL-6, and IL-1beta were determined by ELISA, with total concentrations presented in panels (A,C) and the area under the curve (AUC) depicted in panels (B,D). Normality was tested using the Shapiro–Wilk test, and comparisons were conducted using Student’s t-test. Results are expressed as mean ± standard error of the mean (n = 6 for each incubation time).

Figure 4

Temporal profile of gene expression related to the inflammatory response in macrophages treated with C- or AGE-albumin and challenged with LPS. RAW264.7 macrophages were treated for 48 h with C- or AGE-albumin (2 mg/mL) and, after washing, maintained over time only in a culture medium containing fatty acid-free albumin. Subsequently, they were challenged with LPS for 24 h. Gene expressions of Il6 (A), Tnf (B), Nfkb1 (C), and RelA (D) were assessed by RT-qPCR. Comparisons were made using the Student’s t-test, with data normalization to the control condition for each time point; values are presented as mean ± standard error of the mean (n = 5).

Figure 5

Temporal profile of gene expression related to cholesterol efflux in macrophages treated with C- or AGE-albumin and challenged with LPS. RAW 264.7 macrophages were treated for 48 h with C- or AGE-albumin (2 mg/mL) and, after washing, maintained over time only in a culture medium containing fatty acid-free albumin. Subsequently, they were challenged with LPS for 24 h. Gene expressions of Ager (A), Tlr4 (B), Abca1 (C), Abcg1 (D), and Jak2 (E) were assessed by RT-qPCR. Comparisons were made using the Student’s t-test, with data normalization to the control condition for each time point; values are presented as mean ± standard error of the mean (n = 5).

Figure 6

Nuclear contents of p50 and p65 subunits of NFKB in macrophages treated with C- or AGE-albumin and challenged with LPS. RAW 264.7 macrophages were treated with C- or AGE-albumin (2 mg/mL) for 48 h, followed by washing and maintenance over time in a culture medium containing fatty acid-free albumin. Subsequently, they were challenged with LPS (1 µg/mL) for 24 h. The determination of the nuclear contents of p50 and p65 subunits of NFKB was performed by Western blot, and the quantification of band intensity was determined by optical densitometry, normalized to their respective controls (Ponceau staining). Panels (A,D): p50 and p65 proteins analyzed at each time point (0 h, 8 h, or 24 h) comparing the treatment with C- or AGE-albumin (Shapiro–Wilk normality test, followed by Student’s t-test, with values presented as mean ± SEM; n = 5). Panels (B,C,E,F): comparisons made among different time points (0 h, 8 h, or 24 h) under the same treatment (C or AGE-albumin) using the Shapiro–Wilk normality test, followed by one-way ANOVA (values presented as mean ± standard error of the mean (n = 5). AU = arbitrary unit.

Figure 6

Nuclear contents of p50 and p65 subunits of NFKB in macrophages treated with C- or AGE-albumin and challenged with LPS. RAW 264.7 macrophages were treated with C- or AGE-albumin (2 mg/mL) for 48 h, followed by washing and maintenance over time in a culture medium containing fatty acid-free albumin. Subsequently, they were challenged with LPS (1 µg/mL) for 24 h. The determination of the nuclear contents of p50 and p65 subunits of NFKB was performed by Western blot, and the quantification of band intensity was determined by optical densitometry, normalized to their respective controls (Ponceau staining). Panels (A,D): p50 and p65 proteins analyzed at each time point (0 h, 8 h, or 24 h) comparing the treatment with C- or AGE-albumin (Shapiro–Wilk normality test, followed by Student’s t-test, with values presented as mean ± SEM; n = 5). Panels (B,C,E,F): comparisons made among different time points (0 h, 8 h, or 24 h) under the same treatment (C or AGE-albumin) using the Shapiro–Wilk normality test, followed by one-way ANOVA (values presented as mean ± standard error of the mean (n = 5). AU = arbitrary unit.