Optimization of the immune system is critical for maintaining health. Indeed, the decrease in immunity that occurs with aging, termed immunosenescence [14, 15], contributes to geriatric disorders such as dementia, Parkinson’s disease, arthritis, osteoporosis, heart disease, high cholesterol, and metabolic syndromes [17]. In particular, cellular senescence-induced inflammation has been linked to dysfunction of fat tissue, which causes an imbalance in metabolic homeostasis [17, 18]. Several studies have demonstrated that exercise has anti-aging effects and can ameliorate metabolic disease [9, 10, 35]. However, it remains unclear how exercise leads to metabolic benefits in aging. Here, we evaluated whether changes in immunosenescence induced by exercise were associated with metabolic abnormalities in aging.
Validation of anti-aging effect of exercise
It has been known that the expression of p16, as an in vivo marker of senescence, mediates cellular senescence [28, 36]. It was not surprising that exercise prevented the progression of senescence in the WAT of aged mice [37]. Consistent with the previous study [28, 37], p16 levels in WAT were increased in sedentary aged mice compared to sedentary young mice, and were decreased in aged mice after exercise. At the same time, the RER of exercised mice returned to the level of young mice. The RER was calculated by O2 consumption and CO2 production and is an indirect indicator of aerobic fitness. Additionally, body fat mass was reversed by exercise in aged mice. As shown in Supplementary Table 2, comparing differentially expressed genes related to biological processes in WAT of young and old mice, large metabolic changes are accompanying during aging. We found that exercise may affect metabolism in different directions, as the expression patterns of genes related to fatty acid biosynthesis such as acsl4 and olah, which were increased in the old mice, decreased after 4 weeks of exercise. Overall, we can conclude that 4 weeks of treadmill exercise had a profound effect on improving energy metabolism and body composition by reducing senescence in adipose tissue.
Exercise-induced immune cell alterations in the SVF of aged mice
The immune system, broadly consisting of the innate and adaptive immune systems, is a host defense system that responds specifically to external pathogens [38]. The innate immune response is the first line of defense, involving NK cells and phagocytic cells such as eosinophils, neutrophils, and macrophages. The adaptive immune response, represented by B and T cells, is mediated by antigen-specific defense mechanisms and takes several days for the complete immune response to develop. Immune cells also recognize and dispose of abnormal cells in our body. Both the innate and adaptive immune systems are strongly associated with aging and exercise [39]. To determine if the exercise-induced metabolic improvements in aged mice are related to the immune system, we investigated immune cells in the SVF of aged mice following exercise.
The most noticeable immunological changes associated with aging are defective function of T and B cells [40]. T-cell precursors are derived from the bone marrow and undergo lineage determination to become CD4+ (helper) or CD8+ (cytotoxic) T cells in the thymus. Many other age-associated changes in T-cell numbers and phenotypes have been reported [41]. Although not all T-cell compartments are equally affected by age, overall T-cell numbers decline with age as thymic involution leads to a decreased output of cells [41]. The decrease in naïve T-cell output from the thymus, increase in memory T cells from multiple antigenic encounters and homeostatic proliferation, and increase in regulatory T cells are profound qualitative and quantitative changes that occur with age [22, 42]. Lower numbers of hematopoietic stem cells and reduced output of antigen-specific naïve T cells from the thymus result in decreased numbers and proportions of naïve CD4+ and CD8+ T cells and increased numbers and proportions of late-stage differentiated EM CD4+ and CD8+ T cells during aging. In the elderly, however, an active lifestyle appears only to limit the accumulation of late-stage differentiated T cells and not to substantially affect the proportion of naïve T cells [43]. In accordance with a previous study, we did not observe a significant effect of aging or exercise on naïve CD4+ T cells, CM CD4+ T cells, or CD25+FoxP3+ regulatory T cells. However, we observed a marginal reduction in EM CD4+ T cells in the SVF of aged mice, indicating maladaptive immunity during senescence. Considering aged memory CD4+ T cells maintain a more catabolic state in lipid metabolism [44], these results provide evidence of disruption of fat metabolism in aged mice. Our data showed that exercise substantially increased CM CD8+ T cells only in the SVF of young mice; only a slight decrease was observed in older mice. The SVF of WAT is a reservoir for memory T cells, which protect against reinfection [45]. These findings support the idea that immunological memory, a critical step in the adaptive immune system, is boosted by exercise in young mice. Adipocytes directly interact with NKT cells by presenting lipid antigens and stimulate NK T cells to alleviate inflammation [46]. Dysfunction of NKT cells has been shown to lead to the development of autoimmune diseases such as diabetes, atherosclerosis, and cancer. However, in the present study, exercise and aging did not affect the proportion of NKT cells.
B cells play a critical role in immunosenescence via antibody production [47]. Although previous studies have demonstrated that B cells in the blood are notably impacted by exercise [48], the frequency was stable in the SVF of both young and aged mice after exercise or no exercise, suggesting that B cells might play a role in immune homeostasis in the SVF of WAT.
Low-grade inflammation in adipose tissue is closely associated with obesity and insulin resistance [49]. M1 macrophage inhibits proliferation of adipocytes and causes tissue damage, whereas M2 macrophage promotes proliferation of adipocytes and tissue repair [50]. A recent study suggested exercise-mediated switching of M1 to M2 macrophages reduced inflammation in obese mice [50], while other studies suggested that improvements of inflammation may involve an attenuation of both M1 and M2 macrophages in adipose tissue [51, 52]. Although we did not observe any differences in the proportion of M1 or M2 macrophages in the aged or exercised groups, experimental conditions such as duration and intensity of exercise, timing of dissection, sample type (SVF, WAT, and blood), and senescence in adipose tissue may have affected our results.
Neutrophils are abundant myeloid cells in the blood. Neutrophils, which serve as a link between phagocytosis and immunomodulatory responses, are increased by catecholamines during exercise [53]. Studies have shown that the number of neutrophils is not altered in healthy elderly [54, 55]. Consistent with previous studies, we observed that aging did not influence the number of neutrophils, while exercise marginally increased the number of neutrophils in aged mice.
The effects of aging and exercise on eosinophils, another circulating myeloid cell, are still unclear [56, 57]. In this study, exercise increased the proportion of eosinophils in aged mice. Because eosinophils play a critical role in the regulation of fat homeostasis and systemic metabolism, the exercise-mediated increase in eosinophils may affect the metabolism of WAT in aged mice.
Our finding that was most consistent with previous studies was the change in the proportion of NK cells. NK cells not only play a major role in virally infected cells but also eliminate abnormal cells, such as cancer and senescent cells. In the line with previous studies [16, 58], we observed a decreased proportion of NK cells in the SVF of aged mice. By contrast, an exercise-induced increase in NK cells was observed in young and aged mice. Because metabolic disorders prevent the ability of NK cells to regulate the local immune system [59], NK cells in the SVF of aged mice may be decreased because of fat accumulation in adipose tissue during aging. Jahn and colleagues [60] showed that weight loss in obese individuals via diet and exercise resulted in increased interferon-γ production by NK cells. Barra and colleagues [61] showed that high-intensity interval training increased the proportion and function of NK cells in obese women and mice. NK cells are also systemic innate immune cells defensing against viral infection as well as tumor surveillance [62]. In preparation for the recent corona virus (COVID-19) pandemic, the exercise to boost NK cells of vulnerable groups should be emphasized. Collectively, these studies suggest a critical role of exercise in restoring immune-cell profiles, especially that of NK cells, in the SVF of WAT, as well as a therapeutic potential of exercise in metabolic rejuvenation in aging.
Exercise triggered an immune response in the adipose tissue of aged mice
The immune system works with other tissues to defend the body. Many studies have reported that exercise increases the number of NK cells with various anti-aging phenotypes [63]; however, little is known about the underlying physiological and genetic changes. To understand the effect of exercise-induced immune system changes in the SVF of adipose tissue and the improvement of energy metabolism in aged mice, we examined gene expression patterns in adipose tissue using microarray analysis. As expected, a high prevalence of functional annotation clusters, with greater enrichment scores, associated with cell projection assembly, wound healing, anion transport, cytokine production, and immune system process in the exercise group of aged mice. It should be noted that the expression levels of exercise-, immune-, and scavenging receptor-related genes were altered in the adipose tissue of aged mice. It has been suggested that exercise can affect the removal of aging-related problematic cells by regulating immune cells. KEGG pathway analysis confirmed the changes in various genes related to the function of NK cells. We confirmed an increase in genes related to NK cell function, such as Cd48, Ncr1, and Fcer1g; we also found that the expression of NK-cell-induced phagocytosis-related genes such as Gzmb and Cd244 was decreased in adipose tissue by exercise. These complex changes may be due to dynamic changes such as proliferation, differentiation, and apoptosis in aged adipose tissue according to environmental metabolic requirements [64].
Recent studies have supported the role of immune cells in the removal of dead, senescent, or damaged cells [11, 14]. Although there is still no direct evidence that the changes in immune cells triggered by exercise provide metabolic benefits via the removal of senescent adipocytes, the increases in NK cells and neutrophils as well as the changes in NK cell-mediated cytotoxicity-related genes in adipose tissue suggest that immune cells can mediate local and systemic metabolic rejuvenation via exercise.