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. 2020 Nov 20;9(11):1155.
doi: 10.3390/antiox9111155.

The Effect of Physical Training on Peripheral Blood Mononuclear Cell Ex Vivo Proliferation, Differentiation, Activity, and Reactive Oxygen Species Production in Racehorses

Olga Witkowska-Piłaszewicz  1 , Rafał Pingwara  2 , Anna Winnicka  1
Affiliations

The Effect of Physical Training on Peripheral Blood Mononuclear Cell Ex Vivo Proliferation, Differentiation, Activity, and Reactive Oxygen Species Production in Racehorses

Olga Witkowska-Piłaszewicz et al. Antioxidants (Basel). .

Abstract

Physical activity has an influence on a variety of processes in an athlete's organism including the immune system. Unfortunately, there is a lack of studies regarding racehorse immune cells, especially when the horse model is compared to human exercise physiology. The aim of the study was to determine changes in immune cell proliferation, lymphocyte populations, and monocyte functionality in trained and untrained racehorses after exercise. In this study, field data were collected. The cells from 28 racehorses (14 untrained and 14 well-trained) were collected before and after exercise (800 m at a speed of about 800 m/min) and cultured for 4 days. The expression of CD4, CD8, FoxP3, CD14, MHCII, and CD5 in PBMC, and reactive oxygen species (ROS) production, as well as cell proliferation, were evaluated by flow cytometry. In addition, IL-1β, IL-4, IL-6, IL-10, IL-17, INF-γ, and TNF-α concentrations were evaluated by ELISA. The creation of an anti-inflammatory environment in well-trained horses was confirmed. In contrast, a pro-inflammatory reaction occurred in untrained horses after training. In conclusion, an anti-inflammatory state occurs in well-trained racehorses, which is an adaptational reaction to an increased workload during training.

Keywords: Tregs; anti-inflammatory state; cytokines; exercise; lymphocytes; monocytes; pro-inflammatory state; reactive oxygen species (ROS).

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Conflict of interest statement

The authors declare no conflict of interest.

Figures

Figure 1
Figure 1
The gating strategy. Doublets were removed from the analysis by setting the gate on single cells on the FSC-area (FSC-A) vs. FSC-high (FSC-H) dot plot. Cell proliferation was calculated from singlets. Next, the lymphocytes or monocytes were gated based on FSC and SSC dot plots. Then the gate included lymphocytes and analysis of CD4+, CD8+, and FoxP3 cells were made. The second sample included CD5+, CD14+, MHCII+ cells, and the median fluorescence intensity (MFI) of ROS was calculated on that cell population.
Figure 2
Figure 2
(A) Representative histogram showing T lymphocyte proliferation obtained before and after exercise from untrained and well-trained horses. The graph shows total (B), CD4+ (C), and CD8+ (D) lymphocyte proliferation. The results are presented as the mean ± SEM. Significance levels are: *** p < 0.001.
Figure 3
Figure 3
Graphic representation showing percentages of positive cells: CD4+ (A), CD8+ (B), CD4+FoxP3+ (C), and CD8+FoxP3+ (D) gated from total lymphocytes. The results are presented as the mean ± SEM. Significance levels are: * p < 0.05; *** p < 0.001.
Figure 4
Figure 4
Graphic representation showing percentages of positive cells: CD14-MHCII+ (A), CD14+MHCII− (B), and CD14+MHCII+ (C) gated from total monocytes. The results are presented as the mean ± SEM. Significance levels are: *** p < 0.001.
Figure 5
Figure 5
Graphic representation showing percentages of positive cells: CD14+CR+ (A), CD5+CR+ (B), and CD14+MHCII+ gated from total lymphocytes/monocytes and median fluorescent intensity of CR (CellRox) in CD14+ (C) and CD5+ (D) cells. The results are presented as the mean ± SEM. Significance levels are: ** p < 0.01.
Figure 6
Figure 6
Graphic representation showing the cytokine concentrations: IL-1β (A), IL-4 (B), IL-6 (C), IL-10 (D), IL-17 (E), TNF-α (F), and IFN-γ (G) presented in culture medium of PBMCs obtained from untrained and well-trained horses. The results are presented as the mean ± SEM. Significance levels are: * p < 0.05; *** p < 0.001.
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