Impact of 12-Week Moderate-Intensity Aerobic Training on Inflammasome Complex Activation in Elderly Women

Abstract

Aging often associates with a chronic low-grade inflammatory status that can be consequent to the activation of Toll-like receptors (TLRs) and the downstream NLR family pyrin domain containing 3 (NLRP3) inflammasome and causes a chronic secretion of pro-inflammatory cytokines. Since exercise has known anti-inflammatory effects, we investigated the effect of Nordic walking training on inflammasome activation and downstream effectors in elderly women. A population of elderly women was divided into EXP (n = 29) that completed 12 weeks of the moderate-intensity aerobic training program and CTRL (n = 29), performing no activity. Blood samples were taken before and after the first (T1-pre and T1-post, respectively) and last (T2-pre and T2-post, respectively) exercise unit. Inflammasome activation status was assessed by whole blood NLRP3 and TLR4 expression by RT-qPCR. Serum levels of IL-1β, IL-6, TNFα, and IL-18 cytokines were assayed by multiplex fluorescent beads-based immunoassays or ELISA. NLRP3 and TLR4 levels were reduced 2 folds between T1-pre and T2-pre and induced at T2-post, compared to T2-pre, by 2.6- and 2.9-fold, respectively. A single exercise bout elicited a 1. 38-, 1. 5-, and 1.36-fold rise of IL-1β, TNFα, and IL-6 concentration, respectively, although not significant, at the beginning of the training (T1-pre vs. T1-post), a 1.4-fold decrease for IL-1β and TNFα at the end of the training (T1-pre vs. T2-pre), and a 2-, 1.8- and 1.26-fold increase after the last exercise session (T2-pre vs. T2-post) for the three cytokines. When stratifying the population based on BMI in normal weight (NW) and overweight (OW), NLRP3 and TLR4 expression was affected only in NW. As for inflammatory cytokines, IL-1β was modulated in NW at the beginning of the training, whereas in OW at the end of the training; for TNFα, this time-dependent modulation was significant only in OW. Applied aerobic training affected the resting expression of inflammasome constituents (NLRP3 and TLR4) and levels of downstream effectors (IL-1β, TNFα, and IL-6). However, at the end of the program, participants acquire an acute inflammatory response to exercise that was absent at baseline. Future studies would have to define the molecular mechanisms associated with, and how to potentiate, the exercise-associated inflammatory response.

Keywords: NLRP3 inflammasome; aerobic exercise; aging; physical activity; pro-inflammatory cytokines.

FIGURE 1

Expression profile of potential housekeeping genes, ACTB, PGK1, and PPIB, in whole blood. Expression profile of the three selected potential reference genes (ACTB, PGK1, and PPIB). Expression level of each potential reference gene was analyzed considering (A) samples from all groups (CTRL+EXP), (B) samples from the EXP (T1-pre, T1-post, T2-pre, and T2-post), and CTRL groups separated, and (C) samples from each time-point separated. (D) Heat-map of the expression profiles of ACTB, PGK1, and PPIB, from each group (CTRL, T1-pre, T1-post, T2-pre, and T2-post). Different colors represent differential gene expression.

FIGURE 2

Expression of inflammasome-related genes in whole blood samples. Fold change of NLRP3 and TLR4 gene expression, normalized on PPIB and PGK1, in whole blood from elderly women underwent a 12-week aerobic Nordic walking training program (EXP, n = 29) and untrained controls (CTRL, n = 35). (A) Expression of NLRP3 and TLR4 in the whole EXP group before and after the first (T1-pre and T1-post) and the last (T2-pre and T2-post) sessions of Nordic walking. (B) Expression of NLRP3 and TLR4 in the whole EXP group (orange dots), before the first and the last (T1-pre and T2-pre) Nordic walking session, and in the CTRL (blue squares) over the same observation period (T1, T2). (C) Expression of NLRP3 and TLR4 in the whole EXP cohort, grouped based on BMI in normal weight (NW, full dots) and overweight (OW, empty dots) subjects. (D) Expression of NLRP3 and TLR4 in the EXP (orange dots) and CTRL (blue squares) cohorts, grouped based on BMI in normal weight (NW, full symbols) and overweight (OW, empty symbols) subjects. Asterisks indicated significant differences according to the different statistic tests applied: *p < 0.05; **p < 0.01; ***p < 0.001.

FIGURE 3

Circulating levels of relevant cytokines associated to inflammasome pathway activation. Concentrations of IL-1β, TNFα, IL-6 and IL-18 in sera from elderly women underwent to a 12-week aerobic Nordic walking training program (EXP, n = 29) and untrained controls (CTRL, n = 35). (A) Circulating levels of the assayed cytokines in the whole EXP group before and after the first (T1-pre and T1-post) and the last (T2-pre and T2-post) sessions of Nordic walking. (B) Circulating levels of the assayed cytokines in the whole EXP group (orange dots), before the first and the last (T1-pre and T2-pre) Nordic walking session, and in the CTRL (blue squares) over the same observation period (T1, T2). (C) Circulating levels of the assayed cytokines in the EXP cohort, grouped based on BMI in normal weight (NW, full dots) and overweight (OW, empty dots) subjects. (D) Circulating levels of the assayed cytokines in the EXP (orange dots) and CTRL (blue squares) cohorts, grouped based on BMI in normal weight (NW, full symbols) and overweight (OW, empty symbols) subjects. Asterisks indicated significant differences according to the different statistic tests applied: *p < 0.05; **p < 0.01; ****p < 0.0001.

FIGURE 4

Schematic representation of the important findings of this study. A population of 29 elderly women (age 68 ± 8 years old) completed 12 weeks of Nordic walking moderate-intensity aerobic training program (EXP). Blood samples were taken before and after the first (T1-pre and T1-post, respectively) and last (T2-pre and T2-post, respectively) exercise unit, and inflammasome- and inflammation-related markers were assessed. Aerobic training mildly affected the inflammasome-related inflammatory response. Participants acquired an acute inflammatory response to exercise, that was absent at baseline, that resulted in the activation of NLRP3 and TLR4 gene expression and the consequent release in the blood stream of the inflammasome- and inflammation-related cytokines IL-1β, IL-6, TNFα, and IL-18. In particular, a single bout of exercise at the end of the training induced a significant upregulation of IL-1β (↑↑↑) and TNFα (↑↑), a non-significant increase of IL-6 (↑), and a non-significant down-modulation of IL-18 (↓). The arrows next to the cytokines refer to the entity of the effect of the upregulation or downregulation of these cytokines.