INTRODUCTION

It is well known that ageing is responsible for diverse physiological dysfunctions in different organs and systems. One of the physiological systems showing dramatic changes by ageing is the immune system (^{Franceschi et al., 2017}). This age-associated decline in immunity, known as immunosenescence, involves changes in both innate and adaptive immune components (Nikolich-Zugich, 2018). Regarding adaptive immune response, although affecting both humoral and cellular responses, changing in the T cell compartment is more evident and better documented. This is due mainly because of its role in orchestrate cellular responses and modulating of the immune system (^{Salam et al., 2013}). Thus, diminished numbers of naïve T cells, primarily due to thymic involution, that leads to the shrinking of the TCR repertoire, together with increased numbers of memory T cells are mayor hallmarks of T cells immunosenescence (^{Lazuardi et al., 2005}). Along with the reduction in the adaptive immune arm, increased innate immune response leads to a chronic, low-grade inflammation, characterized by increased production of proinflammatory cytokines such as IL-6 and Tumor necrosis factor (TNF)-α, termed “inflammaging”, another common factor during ageing (Franceschi & Campisi, 2014). A link between immunosenescence and inflammaging has been proposed by observation showing that memory CD8+ T cells during ageing can acquire a senescent phenotype that could promote inflammaging (^{Akbar et al., 2016}). Although this and other studies have described several factors capable of triggering inflammaging, it is less clear if negative signals or cells capable of modulating immune responses are compromised during ageing.

Physical activity (PA), described as any bodily movement resulting in energy expenditure, has been shown to improve the overall health, reducing the risk of suffering different disorders such as cardiovascular disease, diabetes and cancer among others (^{Hojman et al., 2018}). In advanced ages, PA reduces the risk of contracting the most relevant non-communicable chronic disorders. Although older adults are less active, with 40 to 80 % of older people not meeting PA guidelines of at least 150 minutes of PA per week, PA in older individuals have been shown to provide beneficial effects on muscular strength, aerobic capacity, cognitive function and overall reducing the risk for chronic diseases (^{Sáez de Asteasu et al., 2017}). However, how PA influences immunosenescence and inflammaging has received less attention. One recent study evaluating older people maintaining a high level of PA through cycling, showed increased numbers of circulating naïve T cells, and reduced numbers of memory T cells and IL-6 levels compared to healthy older controls, but PA did not show any effect over the frequency of senescent CD8+ T cells (^{Duggal et al., 2018}). However, a different study shows that aerobic fitness reduces senescent T cells (^{Spielmann et al., 2011}). The differences between studies could be explained because different ages between groups, different physical status between participants, differential analysis used, the type of PA developed, among other variables. Moreover, since functional dependency, autonomy and physical conditions are different between older adults, if inflammaging or immunosenescence are distinct between groups remains to be evaluated.

Here, we described increased immunosenescence, characterized by the reduction of circulating naïve T cell numbers, and inflammaging, with increased levels of IL-6, as age increases. We observed strong negative correlation between reduced naïve T cells and increased inflammaging in the older population. Older people that realize PA have reduced inflammaging compared to sedentary older controls. Finally, older people with reduced autonomy showed enhanced immunosenescence and inflammaging compared to the autonomous older adults, but this can be attributed to the increase of age in this group.

MATERIAL AND METHOD

The study was performed in the city of Talca (Chile), through a single cross-sectional measurement. The sample was selected for convenience visiting day clubs, senior clubs, associations and senior residencies in different locations in the city. A final sample of 30 older participants and 7 young adults was obtained. Subjects suffering autoimmune diseases, infections in the last month, or body mass index (BMI) >40 kg/m² were excluded. The study was approved by the bioethics committee of the Universidad Autónoma de Chile (Ethical Application number 142-18). Subjects agreed to the study terms signing an informed consent document.

The application of the questionnaire and blood extraction were carried out on the same day in the health centers or senior residences. The total application time of the test was approximately 45 minutes. All measures were made by technical team who were previously trained.

RESULTS

Reduced circulating naïve T cells in older adults. In order to evaluate the number of circulating naïve T cells and serum IL-6, 30 older volunteers ranging from 65 to 94 years of age (average 77±9 years) and 7 young volunteers ranging from 20 to 43 years of age (average 29±9 years), were recruited for this study. Table I lists the characteristics of older participants. 26 % of the participants were obese or overweight and 20 % of the older volunteers were categorized as active; 50 % of participants had low functionality. Health-related quality of life (HRQoL) measured by the EQ-5D-5L questionnaire, showed no significant differences between women versus men.

Table I Characteristics of the study sample, by sex. 

^{Abbreviations}: BMI: body mass index; CPF: composite physical function; IPAQ: International Physical Activity Questionnaire. Data are presented as mean ± Standard Deviation.

Dramatic reduction of circulating naïve CD4⁺ and CD8⁺ T cells were detected in the older population when compared to younger controls (Fig. 1). No differences in the number of naïve T cells were found when older volunteers where categorized by sex or body mass index (data not shown). These results suggest that ageing is one of the main contributors in reducing circulating naïve T cell numbers in the older population.

Fig. 1 Increased immunosenescence in older volunteers. A. Gating strategy used to define naïve CD4⁺ T cells and representative FACS profiles. B. Circulating numbers of naïve CD4⁺ (left) and naïve CD8⁺ (right) T cells, from younger and older volunteers. Error bars indicate SD. **** p < 0.0001 by two-tailed Student’s t test. 

Reduced immunosenescence and increased inflammaging as older adults get older. We next decided to analyze specifically the older population, evaluating the number of circulating naïve T cells and IL-6 levels from differently aged donors. Our results showed a negative correlation between naïve T cells and age, reducing both naïve CD4⁺ and CD8⁺ T cells as people get older (Fig. 2a). Moreover, inflammaging, measured by circulating levels of IL-6, positively correlated with age, suggesting that inflammaging also increased as older controls get older (Fig. 2b).

Fig. 2 Effect of ageing over immunosenescence and inflammaging in the older population. A. Correlation between circulating naïve CD4⁺ (left) and CD8⁺ (right) T cells versus age in the older population. B. Correlation between serum IL-6 levels versus age in the older population. Pearson correlation coefficient ranges are shown for each graph. 

Reduced naïve T cell number correlates with increased inflammaging in the older population. Although inflammaging has been well described, the factors promoting inflammaging are still matter of debate. In fact, Regulatory T cells (Tregs), one of the main immune cells involved in dampening immune responses by promoting peripheral tolerance, seems to work even more efficiently during elderly (^{Garg et al., 2014}). Thus, it is possible that other cell(s) type(s) with potential suppressive capabilities could be affected during ageing. To explore this point further, we evaluated the association between circulating naïve T cell numbers and inflammaging, measured by serum IL-6 levels. We observed a strong negative correlation particularly between naïve CD4⁺ T cells and IL-6 levels (Figs. 3a and 3b), suggesting that naïve T cell could possess an unexpected suppressive activity over innate immune cell components.

Fig. 3 Strong negative correlation between circulating naïve CD4⁺ T cell numbers and IL-6 levels in older volunteers. A. Correlation between circulating naïve CD4⁺ T cells versus serum IL-6 levels in the older population. B. Correlation between naïve CD8⁺ T cells versus serum IL- 6 levels in the older population. Pearson correlation coefficient ranges are shown for each graph. 

PA at youth did not affect immunosenescence or inflammaging during elderly. It has been previously demonstrated that PA has profound effect over both innate and adaptive immune system in healthy adults (^{Koelwyn et al., 2015}). However, how PA at youth modulates immune system during aging has not been yet evaluated. We decided to assess this point, by categorizing our older volunteers according the development of chronic PA during youth by using the widely used and validated International Physical Activity Questionnaire (IPAQ) and evaluating immunosenescence and inflammaging (^{Hagstromer et al., 2006}). We did not observe any significant differences between number of circulating naïve T cells or serum IL-6 levels between both, Active and Sedentary groups (Fig. 4), suggesting that the development of PA during youth has no significant effect over the immune system during aging.

Fig. 4 Ageing immune system is not affected by the realization of PA during youth. Older volunteers were categorized according to the development of chronic PA during youth and circulating naïve T cells and serum IL-6 levels were analyzed. A. Circulating numbers of naïve CD4⁺ (left) and naïve CD8⁺ (right) T cells, from older adults who declared to have performed intense PA during youth (Active) versus older controls (Sedentary) are shown. B. Serum IL-6 levels from both groups are shown. Error bars indicate SD. ns: not significant. 

PA during elderly increased naïve T cell numbers and reduced inflammaging. We next asked if PA during aging in the elderly has any effect over immunosenescence and inflammaging, by categorizing the older adults in Active (high PA) or Sedentary (low PA) by using IPAQ. Our results indicated that Active older adults showed increased circulating naïve T cells and lower serum IL-6 levels compared to Sedentary older volunteers (Fig. 5). We did not find significant difference between the two groups with respect to age, sex or body mass (not shown). These results suggest that PA during elderly could modulate immune system, reducing immunosenescence and inflammaging in older adults.

Finally, we categorized our older volunteers according to their functionality by using the Composite Physical Function scale (CPF) (Rikli & Jones). Although we did not observe differences between number of circulating naïve CD4⁺ or CD8⁺ T cells between both groups (not shown), we found increased IL-6 levels in the low functionality group (see in Additional file 1: Fig. S1a). However, this observation could in part be explained by the increased average age of this group compared to the high functionality group (Fig. S1b).

Collectively, these findings indicate that immunosenescence and inflammaging dramatically increase during aging, and there is a strong negative correlation between CD4⁺ naïve T cell reduction and higher serum IL- 6, suggesting a possible suppressive role of this population over innate immune cell components. Moreover, PA during aging improves the immune system, slowing naïve T cells decrease and reduced serum IL-6 levels.

Fig. 5 PA during elderly slow down immunosenescence and inflammaging. Older volunteers were categorized according to the development of PA during elderly and circulating naïve T cells and serum IL-6 levels were analyzed. A. Circulating numbers of naïve CD4⁺ (left) and naïve CD8⁺ (right) T cells, from Active older adults versus Sedentary older controls are shown. B. Serum IL-6 levels from both groups are shown. Error bars indicate SD. * p < 0.05; **p<0.01 by two-tailed Student’s t test. 

Supplementary information Additional File 1: Figure S1. Reduced functionality increased inflammaging and immunosenescence, but it can be explained by the differences in age between groups. Older volunteers were categorized according to their functional status and serum IL-6 levels were analyzed. A. Serum IL-6 levels from both groups is shown. B. Age average between both groups. Error bars indicate SD. ** p < 0.01; ***p<0.001 by two-tailed Student’s t test. 

DISCUSSION

Inflammaging and immunosenescence are two key events occurring during aging in the elderly that affect immune system. Although these processes have been generally accepted to occur independently, the relationship between immunosenescence and inflammaging has begun to be elucidated (^{Fulop et al., 2018}). Here, by evaluating young and older volunteers from the Maule Region, Chile, we provide evidence showing that reducing numbers of naïve T cells, especially CD4⁺ T cells negatively correlates with increasing levels of serum IL-6. Our results indicate that these processes could be directly connected, suggesting that performing PA during aging has a beneficial effect on the immune system.

Diminished naïve T cells activation and proliferation in aged mice and humans have been previously demonstrated (^{Goronzy et al., 2015}). The main determinant of these changes seems to be explained in part by the age-related regression of the thymus (Lazuardi et al.). In fact, patients thymectomized during early childhood showed premature immunosenescence with dramatic reduction of naïve T cells and accumulation of oligoclonal memory T cells (^{Sauce et al., 2009}). Our results showed a strong negative correlation between circulating naïve CD4⁺ T cell and serum IL-6 levels, suggesting a suppressive capability of this population over innate immune cells. Moreover, we observed increased levels of CD25⁺ Tregs in the aged volunteers (not shown), similar to what has been previously published (Garg et al.). Interestingly, human immunodeficiency virus infection (HIV), is characterized by CD4⁺ T cell depletion and also by elevated IL-6 levels, mimicking some aspects observed during ageing (^{Breen et al., 1990}). A negative correlation between peripheral CD4⁺ T cell numbers but not Tregs and IL-6 levels has been observed in HIV patients, closely resembling what we observed in our study regarding older adults (^{González-Hernández et al., 2012}). Although the possible suppressive activity of naïve CD4⁺ T cells could have implications beyond ageing, further studies are necessary to confirm these findings.

PA during lifetime reduces the risk cancer, cardiovascular disease, and other inflammatory disorders. How PA during youth could affects the immune system while aging remains largely unknown. Here, by using a validated socio- demographic and lifestyle characteristics questionnaire, we evaluated this question and found no differences regarding immunosenescence or inflammaging parameters between older adults that performed ongoing PA during youth versus the control group. Since we did not categorize the type of PA developed during youth, our results could in part be explained by different routines of PA (type of exercise, dose, among others), that could affect in different ways the immunosenescence parameters in individuals (^{Turner, 2016}). Further studies specifically focusing on the different types of exercise, dose, time per week among other parameters are necessary to determine the contribution of specific PA during youth to immunosenescence and inflammaging during aging.

We have also evaluated the effect of PA in the elderly. Our results showed increased number of circulating naïve T cells and reduced IL-6 levels in active older adults (i.e., accumulating 150 minutes of moderate intensity activity on a weekly basis), compared to non-active older controls. These results are in line with a recent study showing that active older cyclist people have increased number of naïve T cells and reduced levels of IL-6 compared to sedentary older controls (Duggal et al.). Although in that study they include adults below 60 years old and they did not take into account the differences of age between both groups, our results showed that age is the main factor in triggering immunosenescence and inflammaging in the older population and also highlights the importance of PA during aging to slow these processes.

How is the immune system status in functional versus dependent older adults is still unknown. By using the CPF scale, we observed increased inflammaging and immunosenescence in the dependent group compared to the functional adults. However, our observations can in part, be explained by the different mean of age between both groups. Thus, and although interesting, more research focusing in functional versus dependent older people of similar age is necessary to validate our observation.

Some limitations of our work should be considered. First, the number of participants was relatively low, and the differences in the age between the older volunteers was significant, although the sample size allowed statistical comparisons that showed significant changes. Second, we evaluated PA in older adults by using validated questionnaires but did not directly assay PA in our volunteers. However, we consider that this study provides enough evidence to correlate naïve T cell number reduction and inflammaging, to explore possible suppressive capabilities of naïve T cells over innate immune components. Furthermore, to evaluate different PA interventions in older people and their differential impact over immunosenescence and inflammaging.

In summary, our observations indicate that immunosenescence and inflammaging dramatically increase as people get older. There is a strong negative correlation between circulating naïve CD4⁺ T cells and serum IL-6 levels in older adults, suggesting that naïve T cells can have a direct suppressive activity over innate immune components. Furthermore, PA in the elderly may reduce immunosenescence and inflammaging in older subjects.