Moderate-vigorous physical activity attenuates premature senescence of immune cells in sedentary adults with obesity: a pilot randomized controlled trial

Introduction

A sedentary or inactive lifestyle is one of the leading causes of mortality, accelerated aging, and a wide array of age-related diseases, such as metabolic diseases, cardiovascular diseases, and cancer. In contrast, being physically active has long been recognized as a safe and effective “medicine” for the aging population [1]. Nevertheless, according to the World Health Organization (WHO), approximately one in five men and one in three women globally fail to meet the recommendations for weekly minimal moderate–vigorous physical activity (MVPA) [2]. The world is aging rapidly, with around 13% of the world population (one billion) aged 60 years and above, as reported by WHO in 2020 [3]. This has led to a dramatic increase in the burden of aging and aging-related diseases. Many studies have revealed that physical inactivity is a critical determinant of life and health spans [4, 5]. Additionally, due to the effectiveness and cost-effectiveness of physical activity (PA), being physically active during aging, known as “active aging,” is a promising strategy to reduce the burden of aging and age-related diseases [6, 7]. In this context, a sedentary or inactive lifestyle serves as a therapeutic target for accelerated aging and multiple age-related diseases; however, its adverse effects on the health of the aging population and the relevant underlying mechanisms are not yet fully understood.

Cellular senescence, an irreversible state of cell cycle arrest that occurs under conditions of stress, is a key mechanism underlying aging. The aberrant accumulation of senescent cells that express atypical levels of p16^INK4a and p21^Cip1, vital senescent markers, contributes to premature aging and age-related diseases [8]. Thus, senescent cells are therapeutic targets for cellular senescence in aging and age-related diseases and premature senescence in diseases that occur at a younger age, such as obesity [9]. Notably, a recent study revealed that senescent immune cells could trigger systemic and premature aging of organs and tissues throughout the body; in contrast, alleviation of immune senescence slows down whole-body aging [10]. Therefore, the removal or attenuation of senescent immune cells, also known as senolytics or senostatics, is a potential therapy for premature aging and age-related diseases at not only the cellular but also the whole-organism level. Senolytics are a novel class of medicine that targets cellular senescence in various conditions, which have shown great potential in treating many diseases, such as diabetic kidney disease and idiopathic pulmonary fibrosis [11–13]. However, no senolytic targeting immune senescence is currently available, though many senolytic candidates have been discovered and are currently being tested in clinical trials [12, 13].

Previous studies have shown that self-reported exercise frequency is negatively correlated with p16^INK4a levels in T lymphocytes, independent of age [14, 15]. This implies that premature senescence of immune cells may account for sedentary lifestyle-related accelerated aging and age-related diseases. However, studies on objectively measured physical behaviors, such as PA, standing, sedentary behaviors, and sleep, have not yet been reported. Nonetheless, our systematic review and other studies have uncovered the senolytic effect of physical exercise or activity interventions on the immune system in older individuals, and potentially in individuals with obesity [16, 17]. Undoubtedly, physical exercise is an effective senolytic against aging and age-related diseases in older adults. However, the senolytic effects of habitual PA and PA interventions on premature senescence of immune cells, particularly in sedentary adults with obesity, remain unknown. Compared with administering treatments against severe diseases at a later stage, alleviating accelerated aging at a younger age is a better strategy for prolonging the health span and reducing the risk of diseases in the aging population. Therefore, this pilot study aimed to investigate the role of objectively measured physical behaviors and PA intervention in premature senescence of immune cells in sedentary adults with obesity. The findings generated by this study may contribute to understanding the associations between PA and premature senescence and provide a novel approach against accelerated aging in the aging population.

Results

The demographic, anthropometric, and behavioral characteristics of the included 45 participants (age, 31.71 ± 7.30 years) are shown in Table 1. The cross-sectional analysis indicated that chronological age, maximal oxygen consumption (VO₂max), body fat, and blood pressures (diastolic blood pressure and systolic blood pressure) were not associated with the log₂-transformed p16^INK4a and p21^Cip1 levels in peripheral blood mononuclear cell (PBMCs) in sedentary adults with obesity (p > 0.05) (Figure 1A–1C, Tables 2, 3; Supplementary Figure 1). Notably, MVPA was independently and reversely correlated with log₂-transformed p16^INK4a (B = −8.83, 95% CI = −15.98 to −1.68, p = 0.02) and p21^Cip1 (B = −8.30, 95% CI = −14.74 to −1.85, p = 0.01) levels in PBMCs; however, no significant correlation was found in the other physical behaviors, including light PA (LPA), vigorous PA, standing, sedentary behaviors, and sleep duration (p > 0.05) (Figure 1D and Table 2; Supplementary Figure 1). Although the body mass index (BMI) and daily steps were also significantly correlated with log₂-transformed p16^INK4a and p21^Cip1 levels in PBMCs (p < 0.05) (Figure 1C; Supplementary Figure 1), the correlation became non-significant after adjusting for other factors (p > 0.05) (Table 2). These findings suggested that insufficient MVPA is a major driver of cellular senescence of immune cells in sedentary adults with obesity.

Furthermore, a randomized controlled trial (RCT) was conducted to investigate the 12-week PA intervention on the anthropometric, behavioral, and senescent markers in adults with obesity. Significant increases in MVPA (group effect, p = 0.004) and steps (group effect, p = 0.04 [activPAL™]; group effect, p < 0.01 [Fitbit Watch]) were observed in the PA intervention group compared with those in the control group (Table 3; Supplementary Figures 2, 3). In contrast, the other physical behaviors, including sedentary behaviors, LPA, and sleep duration, remained unchanged after the 12-week PA intervention (p > 0.05) (Table 3). However, the change in habitual MVPA showed no effect on the anthropometric measures, including BMI, body weight, body fat, and VO₂max (p > 0.05). More importantly, the 12-week PA intervention significantly attenuated the elevated log₂-transformed p16^INK4a levels in PBMCs (interaction effect, p = 0.04) (Table 3; Supplementary Figure 4A). However, the intervention had no impact on the expression of p21^Cip1, interleukin (IL)-1β, IL-6, and tumor necrosis factor (TNF)-α in PBMCs and serum IL-1β, IL-6, IL-8, C-C motif chemokine ligand 2 (CCL2), intercellular adhesion molecule 1 (ICAM-I), vascular endothelial growth factor (VEGF), and plasminogen activator inhibitor-1 (PAI-I) (p > 0.05) levels, which are also known as senescence-associated secretory phenotypes (SASPs) (Table 3; Supplementary Figure 4B). Intriguingly, while MVPA was inversely correlated with both p16^INK4a and p21^Cip1 levels in PBMCs, the 12-week PA intervention showed a sole effect on p16^INK4a but not on p21^Cip1 and other SASPs. No adverse event was reported during the 12-week PA intervention.

Discussion

Premature senescence accelerates aging and elevates the risk of many diseases in the aging population, including cancer, metabolic diseases, and neurological diseases, in an age-independent manner [18, 19]. Thus, effective senolytics or senostatics against premature senescence will contribute to a lower burden of aging and age-related diseases worldwide. Although chronological age has long been identified as the main driver of senescence, the determinants of premature senescence in adults with obesity are less studied [20]. This pilot cross-sectional analysis and RCT demonstrated that a sedentary lifestyle, especially the lack of MVPA, was a major contributing factor to premature senescence of immune cells in adults with obesity in age-, VO₂max-, and BMI-independent manners. Furthermore, the 12-week PA intervention significantly attenuated the elevated p16^INK4a levels in the immune cells of sedentary adults with obesity. These findings highlight the importance of an active lifestyle in maintaining a youthful immune system and the senolytic or senostatic effects of PA on premature senescent immune cells in sedentary adults with obesity.

A strong correlation between log₂-transformed p16^INK4a mRNA level in immune cells and chronological age was previously reported in an aging population [14]. However, our findings suggested that chronological age and cardiorespiratory fitness (VO₂max) were not the major drivers of cellular senescence of immune cells in sedentary adults with obesity. Instead, physical inactivity, especially lacking MVPA, was a determinant of premature senescence of immune cells in sedentary adults with obesity. However, sedentary behaviors, LPA, standing, and sleep duration were not determinants of premature senescence. Physical inactivity is a potential mechanism underlying the adverse effects of a sedentary lifestyle on healthy aging via accelerating immune system senescence [10]. Expectedly, chronological age was not a driver of premature senescence in a relatively younger population. Premature senescence is commonly triggered by other stressful conditions, such as an unhealthy lifestyle [21]. Besides, cardiorespiratory fitness (VO₂max) has long been associated with a higher proportion of senescent immune cells, which are distinguished by the surface markers of immune cells in middle-aged but not in young adults [22]. While the surface markers of immune cells can also classify senescent cells by the stage of the cell cycle, such as the CD28^-CD57⁺KLRG1⁺ terminally-differentiated memory T cell subset, more reliable markers involved in the pathway of cellular senescence are needed [23]. By using golden standard markers of cellular senescence, including p16^INK4a and p21^Cip1, our findings showed that VO₂max is not a determinant of cellular senescence in the immune cells of adults with obesity. Previous cohort studies have reported that self-reported exercise frequency is independently and negatively correlated with p16^INK4a levels in immune cells. However, the evidence is too preliminary since only a simple question about exercise was used in the questionnaire [14, 15]. To the best of our knowledge, this is the first study to report that insufficient objectively measured MVPA is a major factor contributing to the cellular senescence of immune cells in sedentary adults with obesity. Compared with previous studies, we identified MVPA, a specific type of PA, as a key determinant of cellular senescence in immune cells. We used activPAL™ to objectively measure physical behaviors, which provided a more precise therapeutic target against immune aging for the aging population, with more solid evidence than that of previous studies [14, 15]. In addition, sedentary behavior is another vital physical behavior-based risk factor for unhealthy aging, independent of PA [24]. Surprisingly, LPA and sedentary behaviors were not major contributing factors to the premature senescence of immune cells in sedentary adults with obesity. Collectively, inadequate MVPA was a vital driver of premature senescence of immune cells in sedentary adults with obesity; in contrast, chronological age, cardiorespiratory fitness, LPA, and sedentary behavior showed subtle effects.

Our previous systematic review and meta-analysis confirmed that chronic physical exercise is a senolytic for cellular senescence in immune cells [16]. Moreover, a novel study also reported that a 12-week constructed exercise training program effectively reduced senescent markers of immune cells and SASPs in older adults [17]. However, the effect of free-living PA on cellular senescence of immune cells in the aging population, especially premature senescence in adults with obesity, remains largely unknown. The current study was the first to demonstrate the senolytic effects of PA intervention on senescent immune cells in sedentary adults with obesity. Unlike the previous interventional studies using structured exercise programs [16, 17], the current study provides evidence that increasing daily MVPA is an effective strategy against premature senescence of immune cells in sedentary adults with obesity. More importantly, the habitual PA of participants was improved by our intervention program, which will bring more prolonged benefits than previous physical exercise programs. Alleviation of cellular senescence in immune cells is potentially a key molecular mechanism underlying “active aging,” and the “exercise as medicine” [7] approach can go a long way in alleviating premature immune senescence in adults with obesity. Additionally, this study focused on the premature senescence of immune cells in sedentary adults with obesity, who are neglected in currently available senolytic studies. Premature senescence may account for accelerated senescence and the elevated burden of aging and age-related diseases. A previous interventional study reported that exercise lowered elevated p16^INK4a and p21^Cip1 levels in immune cells and circulating SASPs in older individuals [17]. However, elevated p16^INK4a and declined p21^Cip1 levels were observed in sedentary adults with obesity, which probably indicated early-stage accumulation of senescence immune cells during aging. This is because p16^INK4a maintains the senescent phenotypes while p21^Cip1 initiates cellular senescence [25]. Moreover, consistent with the findings of previous animal studies, our results demonstrated that PA intervention only exerted a senolytic effect on p16^INK4a in the immune cells but not on p21^Cip1 and SASPs of sedentary adults with obesity [26, 27]. This suggested distinct senescent phenotypes and senolytic effects of exercise between older adults and adults with obesity. Specifically, the senolytic effects of PA in older adults involved both the inhibition of the production of senescent cells (p21^Cip1) and the removal of excess senescent cells (p16^INK4a), whereas it only reduced excess senescent cells in sedentary adults with obesity. It should be noted that cellular senescence is not only a key molecular mechanism behind aging but also a vital biological process that is essential for tissue repair, cancer suppression, and health maintenance [28]. Our results show that PA intervention is a relatively safe senolytic for sedentary adults with obesity during aging. This senolytic only targets p16^INK4a+ excess senescent cells without affecting the p21^Cip1-regulated initiation of cellular senescence [25].

This study has several limitations. The main limitation is that this small-scale pilot study only included 45 participants with a narrow age range, which limits our ability to investigate the diversity of key senescent determinants in various age groups. In addition, the levels of p16^INK4a and p21^Cip1 in PBMCs measured in this study remained at the mRNA level. While it is a conventional and reliable method in the determination of cellular senescence in immune cells [14, 15], findings based on the level of p16^INK4a and p21^Cip1 at the protein level are more convincing. In addition, our primary protocol deviations from the planned trial are as follows: we included senescent markers as outcomes for studying immune senescence in sedentary adults with obesity and removed the PA + middle-to-high-intensity exercise group due to the coronavirus disease 2019 pandemic. Nevertheless, our study provides a feasible anti-aging strategy involving increasing daily steps, which will contribute to healthy aging and a reduced burden of aging and age-related diseases for individuals and society, respectively. Nevertheless, large-scale RCTs on the senolytic effect of PA intervention targeting premature senescence in different populations at various age stages, such as healthy sedentary adults, middle-aged adults, and adults with type-2 diabetes, are still needed. These studies may contribute to developing exercise prescriptions for promoting healthy aging and reducing the burden of age-related diseases in the accelerated aging populations worldwide.

Conclusion

Physical inactivity at a younger age is an independent determinant of premature senescence in immune cells, potentially leading to accelerated aging of the whole body. A 12-week free-living PA intervention targeting MVPA is a senolytic for premature immune senescence in sedentary adults with obesity. Being physically active by increasing daily steps is an effective and cost-effective strategy to slow the aging process and reduce the burden of aging and age-related diseases.

Materials and Methods

Supplementary Materials

Supplementary Figures

Supplementary Table 1