Edited by: Sean X. Leng, Johns Hopkins University, United States
Reviewed by: Francesco Nicoli, University of Ferrara, Italy; Xiang Qiu, National Institutes of Health (NIH), United States; Lili Ji, Fudan University, China
*Correspondence: Gábor Papp,
This article was submitted to T Cell Biology, a section of the journal Frontiers in Immunology
†These authors have contributed equally to this work and share first authorship
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Age-related changes of the immune system lead to an increased morbidity and mortality due to enhanced vulnerability to infectious diseases and malignancies. Recent studies revealed the important effects of physical activity on immune functions, which may largely depend on the type of exercise, its intensity and duration. However, limited information is available regarding the immunological effects of sport activities in older ages. The aim of our study was to examine the changes in a wide spectrum of lymphocyte subtypes after regular workout among healthy elderly individuals. We enrolled 29 elderly women with sedentary lifestyle (mean age: 67.03 ± 3.74 years) to take part in a 6-week long functional conditioning gymnastic exercise program. The percentages of peripheral natural killer (NK), NKT cells, T and B lymphocyte subtypes (early-/late-activated T, naïve and memory T, cytotoxic T (Tc), T-helper (Th)1, Th2, Th17, T regulatory type 1 (Tr1), CD4+CD127lo/-CD25bright Treg, as well as naïve and memory B cells) were determined by flow cytometry. Evaluation of the changes in functional capability of Treg cells was based on
A properly functioning immune system is essential for the continuing survival of the host by maintaining a well-balanced defense against foreign organisms and protection from endogenous altered or virally transformed cells. However, old age inevitably leads to a number of changes that affect almost every element of the immune system and result in a progressive decline in immune functions. This age-associated process, called ‘immunosenescence’, disrupts the balance of immune homeostasis, consequently, elderly individuals become more susceptible to a wide range of infections, neoplasia and autoimmune diseases (
The changes associated with B lymphocytes are somewhat similar to the alterations observed for T cells. Naïve B cell proportion shows an age-related decrease presumably due to inadequate levels of B-cell activating factor (BAFF), which is a key maintenance factor for this lineage. On the contrary, memory B cells accumulate in older age and exhibit a more restricted repertoire of B cell antigen receptors (BCR) (
Interestingly, in older age, the intrinsic functional impairment of immune competence and the lack of proper immune defense are associated with a low-grade chronic systemic inflammation (
Twenty-nine healthy elderly volunteers (female/male ratio: 29/0; mean age: 67.03 ± 3.74 years; mean BMI: 26.52 ± 1.89 kg/m2) living independently in the community were enrolled in the present study. Each volunteer completed a diet and physical questionnaire in the beginning and at the end of the study in order to assess their health condition and determine whether they comply with the criteria. Participants enrolled in the study were non-smokers, and they were abstaining from any physical exercises or sport activities, special diet and vitamin supplements for at least 3 months prior to the study. Moreover, exclusion criteria included ongoing viral or bacterial infection, allergic or autoimmune disease, chronic disease treated with continuous drug therapy, cancer; alcohol or drug addiction, psychiatric illness, insufficient compliance and dietary changes or usage of dietary supplements during the study period, as well.
All participants attended 60-minute long functional exercise sessions twice a week, in the morning (between 8 and 9 a.m.) for 6 weeks under stable climatic conditions (18 - 20°C) at UniFit Fitness and Gym Centre in Debrecen. We designed the exercise protocol according to the latest American College of Sports Medicine (ACSM) position stand on “Quantity and Quality of Exercise for Developing and Maintaining Cardiorespiratory, Musculoskeletal, and Neuromotor Fitness in Apparently Healthy Adults: Guidance for Prescribing Exercise” (
Detailed training protocol.
Schedule | Exercises | Intensity level 1 (week 1-3) | Intensity level 2 (week 4-6) |
---|---|---|---|
10 mins | warm-up with treadmill/ellipse trainer/bicycle | 40-50% HRmax | 40-50% HRmax |
20 mins | cyclic aerobic exercises with treadmill/ellipse trainer/bicycle | 50-55% HRmax | 55-60% HRmax |
20 mins | TRX Squat | 3 sets of 12-15 reps | – |
TRX Single Leg Squat | – | 3 sets of 10 reps per foot | |
TRX Low Rows | 3 sets of 12-15 reps |
– | |
TRX Single Arm Low Rows | – | 3 sets of 10 reps per arm |
|
TRX Push Up | 3 sets of 12-15 reps |
– | |
TRX Push Up on one LEG | – | 3 sets of 6 reps per leg |
|
TRX Standing Hip Drop | 3 sets of 10 reps | 3 sets of 15 reps | |
10 mins | Balance with Fitball/Bosu | in pairs with help | alone - without help |
10 mins | Stretching | within normal range |
within maximum range |
HRmax, maximal heart rate.
Body composition analyses were carried out by an InBody 270 device (InBody, Seoul, South Korea). After the manual input of basic data such as height, sex and age, the measurements takes about 15 seconds. Besides the exact body weight, the analysis give a comprehensive picture on the mass of skeletal muscles and body fat regarding the whole body as well as its different parts including upper and lower extremities and trunk. Body Mass Indexes (BMI) were also calculated for each measurement.
Short Physical Performance Battery (SPPB) test was used for the evaluation of the severity of sarcopenia. SPPB evaluation includes a group of measures that combines the results of the gait speed, chair stand and balance tests (
For laboratory experiments, peripheral blood samples were taken baseline and 3 days after the 6-week long exercise program, in order to measure the immunological effects of the physical exercise program. All samples were collected between 8:00 and 9:00 a.m. to avoid circadian variation. Blood cell counts including total lymphocyte counts were analyzed from blood samples anticoagulated with ethylenediamine tetra‐acetic acid (EDTA) with ADVIA 2120i hematology system (Siemens, Munich, Germany).
For the comprehensive phenotypic analysis of peripheral lymphocyte populations, heparinized blood samples were collected from the healthy volunteers. The different cell populations were identified using fluorochrome-conjugated monoclonal antibodies against specific cell surface antigens. The basic characterization of lymphocytes performed by the combination of CD3-fluorescein isothiocyanate (FITC)/CD16+CD56-phycoerythrin (PE) (BD Biosciences, San Diego, CA, USA) and CD19-R-phycoerythrin-cyanine dye 5 (PE-Cy5) (Beckmann Coulter Inc., Brea, CA, USA) as well as CD4-FITC/CD8-R-PE/CD3-RPE-Cy5 (Dako Agilent Technologies, Santa Clara, CA, USA), monoclonal antibodies against cell surface markers. For the analysis of naïve and memory B cell subsets we used IgD-FITC, CD27-PE and CD19-PE-Cy5 (all from Beckman Coulter) antibodies, while the following monoclonal antibody combinations were used for phenotypic characterization of naïve and memory Th and Tc cells: CD45RA-FITC/CD4-PE (Beckman Coulter) and CD45RA-FITC/CD8-RPE (Bio-Rad Laboratories, Hercules, CA, USA), respectively, as well as CD62L-PE-Cy5 (Beckman Coulter). For the identification of early and late activated T cells we used CD69-PE-Cy5 (BD Biosciences) and human leukocyte antigen (HLA)-DR-PE (Bio-Rad) monoclonal antibodies, respectively, with the combination of CD3-FITC (Bio-Rad). We also investigated CD4+CD127lo/-CD25bright Treg cells with the following reagents: CD4-FITC (BD Biosciences), CD127-PE and CD25-PE-Cy5 (both from Beckman Coulter). NKT cells were identified according to the combination of 6B11-PE and CD3-PerCP (both from BD Biosciences) monoclonal antibodies. IgG1-FITC/PE/PE-Cy5 (Beckman Coulter) isotype-matched antibodies were used in all procedures. For the identification of lymphocyte subpopulations in peripheral blood, freshly drawn (<3 h) anti-coagulated whole blood was used. After the incubation (30 minutes, at room temperature) with monoclonal antibodies, the hemolysis of erythrocytes was performed with 0.2% solution of formic acid, then cells were washed and fixed with 1% solution of paraformaldehyde and stored at 4°C until further measurement. The stained cells were assessed with Coulter FC500 flow cytometer (Beckman Coulter) and data were analyzed using Kaluza 1.2a software (Beckman Coulter).
For the identification of CD4+ Th cell subsets and CD8+ Tc cells, we used cytoplasmic cytokine staining method. Briefly, whole blood were diluted to 1:1 with saline solution and incubated with phorbol-12-myristate 13-acetate (PMA) (25 ng/ml), ionomycin (1 μg/ml) and Golgi Stop brefeldin A (10 μg/ml) (all from Sigma Aldrich, St. Louis, Missouri, USA) for 5 h at 37°C in 5% CO2 milieu. The following monoclonal antibodies were used for cell surface staining: CD4-PE-Cy5 or CD8-PE-Cy5 (both from Beckman Coulter). The cells were then fixed and permeabilized with Intraprep™ permeabilization reagent (Beckman Coulter) according to the manufacturer’s instructions, and intracellular cytokines were stained with the combination of interferon (IFN)-γ-FITC, interleukin (IL)-4-PE, IL-10-PE (all from BD Biosciences) and IL-17-PE (R&D Systems, Minneapolis, MN, USA) monoclonal antibodies. Measurements were performed and data were analyzed on Coulter FC500 flow cytometer (Beckman Coulter) equipped with Kaluza 1.2a software. IgG1-FITC (BD Biosciences) and IgG1-PE (R&D Systems) isotype-matched antibodies were used during the identification. Cells were quantified as their percentage in the CD4+ or CD8+ lymphocyte population.
The CD4+CD25+CD127lo/– Regulatory T Cell Isolation Kit II (Miltenyi Biotech GmbH, Bergisch Gladbach, Germany) was used to obtain Treg cells, while with the CD4+ T Cell Isolation Kit (Miltenyi) we managed to gain effector Th cells from peripheral blood mononuclear cells (PBMCs) after Ficoll (Sigma-Aldrich, St Louis, MO, USA) gradients centrifugation. In case of Treg cells, the isolation was performed in a two-step procedure first using an LD, then an MS column according to the manufacturer’s instructions. The purity of isolated cell populations was above 98% in case of CD4+CD25- effector Th cells and 95–98% regarding CD4+CD127lo/-CD25+ Treg cells. To carry out the suppressor activity assay, 8x104 Treg cells were co-cultured with 8x104 effector Th cells in the presence of human T cell activator CD3/CD28 microbeads (Thermo Fisher Scientific, Waltham, Massachusetts, USA) in 200 µl RPMI-1640 in a 96-well flat-bottom plate for 72 hours at 37°C in 5% CO2 condition. For controls and background measurements, Treg and effector Th cells were cultured separately as well. All measurements were performed in duplicate. Cell proliferation was measured by EZ4U colorimetric cell proliferation assay (Biomedica, Vienna, Austria) and the suppressor activity was detected by LabSystems 352 Multiskan MS Microplate Reader (Thermo Fisher Scientific) at 450/620 nm for optical density (OD) values, as described previously (
Data were analyzed with GraphPad Prism 8 software (Graphpad Software, San Diego, USA). To assess the distribution of the data, Kolmogorov–Smirnov and Shapiro-Wilk normality tests were used. In case of Gaussian distribution, two-tail paired t test was used, on the other hand, if the data set differed from normal distribution, Wilcoxon test was performed. Differences were considered statistically significant at p < 0.05.
The number of enrolled individuals was estimated with
In order to determine the effects of regular exercise on the fitness level of participants, measurements on body composition and physical performance were performed at baseline and repeated after the last exercise. A mild significant increase was observed between the before and after values of BMI (27.51 ± 4.132 vs. 27.66 ± 4.335; p = 0.018) (
Measurement of physical activity in elderly women before and after the exercise training program.
Flow cytometric analyses were performed to evaluate the percentages of a wide-spectrum of immune-competent cell subsets in the peripheral blood of the participants. According to different cell surface antigens, listed in detail in the previous section, certain lymphocyte subpopulations, including NKT cells, Treg cells as well as activated, naïve and memory cell subsets were identified. Regarding the proportions of adaptive immune cells, flow cytometric evaluation included CD3+ T cells, CD3-CD56+CD16+ NK cells, CD19+ B cells, CD3+CD4+ Th cells and CD3+CD8+ Tc cells. However, considering the pronounced but transient exercise-induced changes in lymphocyte counts (
The distribution of peripheral lymphocyte subsets in elderly women before and after the exercise program. Whole blood of 29 participants were stained with fluorochrome-conjugated monoclonal antibodies as described previously. CD56+ NK, CD19+ B and CD3+ T cells were quantified as their percentage in the lymphocyte population, while CD4+ Th and CD8+ Tc cells were quantified as their frequencies in CD3+ cells.
Naïve and memory B cells were identified as CD19+IgD+CD27- naïve B cells, CD19+IgD+CD27+ un-switched memory B cells, CD19+IgD-CD27+ switched memory B cells, CD19+IgD-CD27- double-negative (DN) B cells. We found that the ratio of DN B cells was significantly decreased (4.747 ± 2.042 vs. 4.020 ± 1.763; p = 0.0298) and the percentages of un-switched memory B cells were significantly heightened (11.619 ± 6.591 vs. 12.299 ± 6.524; p = 0.0246) compared to baseline values (
Changes in the division of naïve and memory lymphocyte subsets in elderly women after the workout program. Whole blood of 29 elderly individuals were stained with labeled monoclonal antibodies as described previously. B cell subsets were quantified as their proportions in CD19+ lymphocytes, Th subpopulations were assessed as their ratio in the CD4+ cells, while Tc cell subsets were quantified as their percentages in CD8+ cells.
Naïve and memory Th and Tc cells were referred to as CD4+CD45RA+CD62L+ naïve Th, CD4+CD45RA-CD62L+ central memory (CM) Th, CD4+CD45RA-CD62L- effector memory (EM) Th, CD4+CD45RA+CD62L- effector memory RA+ (EMRA) Th cells as well as CD8+CD45RA+CD62L+ naïve Tc, CD8+CD45RA-CD62L+ CM Tc, CD8+CD45RA-CD62L- EM Tc, CD8+CD45RA+CD62L- EMRA Tc cells. Although there was no significant difference in case of naïve and memory Th cell subsets (
Additionally, CD3+CD69+ early-activated T lymphocytes, CD3+HLADR+ late-activated T lymphocytes, CD3+6B11+ NKT cells and CD4+CD127lo/-CD25bright Treg cells were also determined. When we analyzed the ratio of activated T cells, we found that late-activated T cells differed significantly (13.189 ± 5.396 vs. 14.144 ± 6.547; p = 0.0412) from the baseline values (
Assessment of the changes of activated T cells, Treg cells and NKT cells in elderly women after exercise program. Whole blood of 29 elderly women were stained with labeled monoclonal antibodies as described previously. Activated T cells were quantified as their percentages in the lymphocyte population, Treg cells were assessed as their frequencies in the CD4+ cells and NKT cells were quantified as their percentages in CD3+ cells.
The phenotypes within CD4+ cells were determined as CD4+IFN-γ+IL-4- Th1 cells, CD4+IFN-γ-IL-4+ Th2 cells, CD4+IFN-γ-IL17+ Th17 cells and CD4+IL-10+ type-1 regulatory T (Tr1) cells. Cytotoxic T cells were identified as CD8+IFN-γ+ T cells. All cell subsets were quantified as their percentage in the CD4+ or CD8+ lymphocyte population. We found no significant differences in peripheral blood Th1, Th2, Th17 and Tc cells (
Determination of T helper and T cytotoxic cells with intracellular cytokine analysis in elderly women before and after the exercise program. Whole blood of 29 healthy participants were stimulated for 5h and dyed with fluorochrome-conjugated monoclonal antibodies with intracellular staining method as described previously. All cell subsets were quantified as their percentage in the CD4+ or CD8+ lymphocyte population.
We measured the functional activity of Treg cells obtained from 10 healthy elderly volunteers at the baseline and at the end of the training program. To investigate the suppressor activity of Treg cells, magnetically isolated CD4+CD25- Th and CD4+CD127lo/-CD25+ Treg cells were cultured alone and together in the presence of human T cell activator anti-CD3/CD28 beads (
Suppressor activity of regulatory T cells in elderly women before and after the functional exercise program.
Age-related changes in the immune system and their consequences became crucial issues lately, when human life expectancy keeps increasing. In many countries, the proportions of individuals aged 65 years or older are close or even above 20%; moreover, based on the present trends, by 2050, older people will represent approximately 25% of the population worldwide (
In our present study, besides examining the improvements in physical conditions, we focused on the immunological changes in healthy elderly women performing a 6-week training program with moderate-intensity. Due to the regular physical exercises the characteristics of body composition improved, the fat mass decreased, while skeletal muscle mass increased significantly, and the latter led mild increase in BMI value. In parallel with alteration in skeletal muscle mass, muscular strength also increased which reflects a significant improvement in the physical performance of the elderly individuals.
Importantly, changes induced by intense physical exercise may last at least 24 hours, and even moderate acute exercise induces significant immune alterations for several hours (
Focusing on the distribution of naïve and memory subpopulations of B cells, we revealed that the ratio of DN B cells decreased, while percentages of un-switched memory B cells increased at the end of workout program. Of note, the generation of DN B cells seems to be a typical phenomenon due to the aging of immune system. These cells may be exhausted memory B cells that have reverted their expression of CD27 as they have switched the heavy chain of immunoglobulin. Former studies suggest that these cells do not express HLA-DR, CD80 and CD40 which are important molecules for antigen presentation and T-B cooperation (
As for T cell subtypes, we revealed an increase in HLA-DR+ late-activated T cell percentages, which may indicate a more activated immunological state of the elderly individuals at the end of the 6-week long training program. It has already confirmed that the ratio of HLA-DR+ cells in CD4 and CD8 lymphocytes are increased in elderly individuals compared to young adults, however it is more pronounced in CD8 than in CD4 T cells. In vitro stimulation of both subset revealed that activated CD8 T cells gained more HLA-DR expression during culture (
In our study, total CD8+ Tc cell levels decreased and the distribution of CD8+ Tc cell subsets showed significant changes at the end of workout program. These results are in accordance with previous observations. Age-associated changes has been described to occur more profoundly in peripheral blood CD8+ Tc cell population than CD4+ Th cells (
We also observed an increase in total CD4+ Th cell proportions; nevertheless, the regular exercises did not lead to a change in Th1:Th2 ratio in the elderly population. Importantly, it is known that aging is associated with a predominance of Th2 cells, whereas there is a decline in Th1 cell proportions among T-helper cell population (
Regarding T cell subsets with immunoregulatory functions, it was reported that acute, high intensity exercises may cause a significant elevation in Treg cells (
Our findings suggest that exercise-induced changes in the distribution of certain naïve and memory B and T cell subsets as well as in the proportions of regulatory T cells presumably indicate a retuned immune regulation and a restored responsiveness of the immune system. Thereby, regular exercise, besides improving physical condition and age-related sarcopenia, may also delay or even reverse immunosenescence therefore can be particularly beneficial in maintaining appropriate immune functions in older ages.
The original contributions presented in the study are included in the article/
The studies involving human participants were reviewed and approved by the Ethics Committee of the University of Debrecen (protocol number: 4839-2017) and the Policy Administration Services of Public Health of the Government Office (registration number: 25040-4/2017/EÜIG). All experiments carried out were in compliance with the Declaration of Helsinki. The patients/participants provided their written informed consent to participate in this study.
GP designed the study, supervised the research program, analyzed data, and wrote the main manuscript text. KS performed laboratory experiments, analyses data, prepared figures, and contributed to manuscript writing. IJ performed laboratory experiments. AB and AA contributed to enrolling participants and data collection. GM instructed exercise sessions. MM designed and supervised the workout program. ZC and PS contributed to the interpretation of findings and edited the manuscript. LB provided conceptual advices and contributed to the final version of the manuscript. All authors contributed to the article and approved the submitted version.
The research was supported by the GINOP-2.3.2-15-2016-00062 project, which was co-financed by the European Union from the European Regional Development Fund. The work of GP was supported by the János Bolyai Research Scholarship of the Hungarian Academy of Sciences and the ÚNKP-20-5 New National Excellence Program of the Ministry for Innovation and Technology. AA received scholarship from the talent development program of EFOP-3.6.1-16-2016-00022 project co-financed by the European Union and the European Social Fund.
The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.
The Supplementary Material for this article can be found online at: