A systems biology approach to investigating the influence of exercise and fitness on the composition of leukocytes in peripheral blood

Abstract

Background: Exercise immunology has become a growing field in the past 20 years, with an emphasis on understanding how different forms of exercise affect immune function. Mechanistic studies are beginning to shed light on how exercise may impair the development of cancer or be used to augment cancer treatment. The beneficial effects of exercise on the immune system may be exploited to improve patient responses to cancer immunotherapy.

Methods: We investigated the effects of acute exercise on the composition of peripheral blood leukocytes over time in a male population of varying fitness. Subjects performed a brief maximal intensity cycling regimen and a longer less intense cycling regimen at separate visits. Leukocytes were measured by multi-parameter flow cytometry of more than 50 immunophenotypes for each collection sample.

Results: We found a differential induction of leukocytosis dependent on exercise intensity and duration. Cytotoxic natural killer cells demonstrated the greatest increase (average of 5.6 fold) immediately post-maximal exercise whereas CD15⁺ granulocytes demonstrated the largest increase at 3 h post-maximal exercise (1.6 fold). The longer, less intense endurance exercise resulted in an attenuated leukocytosis. Induction of leukocytosis did not differ in our limited study of active (n = 10) and sedentary (n = 5) subjects to exercise although we found that in baseline samples, sedentary individuals had elevated percentages of CD45RO⁺ memory CD4⁺ T cells and elevated proportions of CD4⁺ T cells expressing the negative immune regulator programmed death-1 (PD-1). Finally, we identified several leukocytes whose presence correlated with obesity related fitness parameters.

Conclusions: Our data suggests that leukocytes subsets are differentially mobilized into the peripheral blood and dependent on the intensity and duration of exercise. Pre-existing compositional differences of leukocytes were associated with various fitness parameters.

Keywords: Exercise immunology; Fitness; Monocytes; NK cells; Peripheral blood leukocytes; T cells.

Fig. 1

Peripheral blood leukocyte subsets are mobilized distinctly upon two different types of exercise. Blood samples were collected from 15 subjects at the indicated time points after maximal and endurance exercises. Leukocytes were enumerated by flow cytometry. Eight major subsets are shown: granulocytes, lymphocytes, monocytes, Natural Killer (NK) cells, B cells, T cells including CD4⁺ and CD8⁺ subsets. For each subset, the means were significantly different (p < 0.0001 by repeated measures ANOVA). ** = p <0.01 by the Bonferroni’s multiple comparison post-test of all pairs

Fig. 2

Natural killer cells show the greatest degree of induction upon exercise. a The values for each subset at the Post or 3HR time point were divided by the Pre sample value. Box and whisker plots show the 25th and 75th percentiles (box) with median (line in box) and the minimum and maximum values. b The changes from Pre to Post for CD56⁺⁺CD16⁻ NK cells and γδ T cells. c Changes in the distribution of Classical (CD14⁺CD16⁻), Intermediate (CD14⁺CD16⁺), and Non-classical monocytes (CD14^loCD16⁺). The means comparing Pre versus Post were significantly different (p < 0.0001 by repeated measures ANOVA). ** = p < 0.01 by the Bonferroni’s multiple comparison post-test of all pairs

Fig. 3

The quantity and composition of peripheral blood leukocytes change in response to different exercises. a The mean values of six subsets from 15 subjects were combined to visually recreate the entire peripheral blood compartment and plotted as pie graphs. The pie graphs are sized in relation to the Pre time point prior to maximal exercise. The means were significantly different (p < 0.0001 by repeated measures ANOVA). ** = p < 0.01 by the Bonferroni’s multiple comparison post-test of all pairs. b Principal component plot of the cumulative immune profiles of 15 subjects through each time point for maximal and endurance exercises. Each sphere represents the mean value of 16 immunophenotypes normalized to the baseline sample prior to the maximal exercise

Fig. 4

Immunophenotypic differences in sedentary versus active individuals at baseline samples. Immunophenotypes were measured by flow cytometry from baseline samples from 10 active subjects and 5 sedentary subjects. a CD45RO⁺ and PD-1⁺ cells were measured as a percent of parent CD4⁺ or CD8⁺ T cells in sedentary and active subjects. All subjects were included on the %CD45RO⁺ versus %PD-1⁺ plot. b Representative dot plots showing the distribution of positive and negative T cells for CD45RO and PD-1 (CD4 T cells on top, CD8 T cells on bottom.) The graph shows the percent positive PD-1 cells on memory T cells (CD45RO⁺) or naïve T cells (CD45RO⁻) for both CD4 and CD8. ** = p < 0.001 by a paired T test

Fig. 5

Immunophenotypic associations with fitness parameters. Selected baseline immunophenotypes measured as cell counts or as percentages of a parent population were correlated with three fitness parameters: Lean Body Mass (1st column), % Body Fat (2nd column), and Body Mass Index (BMI, 3rd column). Significant associations are shown with the corresponding p value. The last row of graphs show the percentages of CD4⁺CD45RO⁺CD62L⁺CCR7⁺ central memory T cells (closed circles) and CD4⁺CD45RO⁺CD62L⁻CCR7⁻ effector memory T cells (open circles) from total CD4⁺CD45RO⁺ memory T cells