Cerebral palsy (CP) comprises a heterogenic group of neurodevelopmental disorders sharing common symptoms, mainly involving difficulties with movement and posture.¹ CP results from a non-progressive brain lesion occurring before or during early infancy. Individuals with CP present impairments such as muscle weakness, co-contraction, spasticity, and poor selective motor control as well as balance.¹ These features in turn are considered possible causes of various musculoskeletal pathologies that further develop into bone deformities and joint instability.² Moreover, previous studies showed that muscles of individuals with spastic CP are structurally and mechanically different compared to those of typically developing individuals.²

To date, it is well established that individuals with CP are less physically active compared to typically developing individuals.³ Individuals with CP with low motor function are the most sedentary group and usually use assistive mobility devices, which further negatively impact their activity level, cardiorespiratory fitness, and muscle health. Combining their reduced physical activity with other factors, such as nutrient deficiencies, individuals with CP have an increased risk for developing metabolic disorders such as obesity, type 2 diabetes, arthritis, and atherosclerosis.^{4, 5} All of these disorders have been associated with a chronic inflammatory status in typically developing individuals.^{6, 7} However, little is known about the existence of chronic inflammation and/or altered immune function in adults with CP.

Although studies in individuals with CP are scarce, evidence exists indicating the occurrence of systemic low-grade inflammation and immune dysregulation in young individuals with CP.^8-13 For instance, enhanced levels of specific cytokines (e.g. tumor necrosis factor alpha and interleukin-6)^{12, 13} as well as T-cell (e.g. CD3+, CD4+, and CD8+) and B-cell subsets (CD22+) were observed in young children with CP when compared to typically developing children.¹³ In addition, significantly increased levels of erythropoietin and a hyporesponsiveness to lipopolysaccharide for interleukin-1a, interleukin-1b, interleukin-2, and interleukin-6 levels were found in school-aged children with CP,⁹ an elevated gene expression of proinflammatory cytokines in skeletal muscles with fixed contractures¹¹ and significantly increased levels of transforming growth factor beta-1 and C-reactive protein were also found in children and adolescents with CP.¹⁰ The reported results indicate the existence of a systemic low-grade inflammation and altered immune function in children with CP. However, there is a lack of knowledge about the immune status of young adults with CP. Thus, more detailed investigations of immune cell subpopulations in young adults with CP are warranted.

Exercise influences the activation state of the immune system¹⁴ and also affects the inflammatory status and gene expression in its target tissues (e.g. muscle tissue).¹⁴ In response to physical activity, the immune cells infiltrate the tissues and mediate changes in physical performance capacity by expressing cytokines, producing antibodies, or modulating inflammation, defense, and regeneration processes.¹⁵ Importantly, the expression pattern of these responses and their extent depend on a number of factors such as work intensity, duration and frequency, and the amount of activated muscle mass. In typically developing populations, acute bouts of endurance exercise typically lead to characteristic biphasic changes in the numbers of circulating lymphocytes (i.e. T-cells and subpopulations, B-cells):^{7, 16} increase in cell numbers (i.e. lymphocytosis) during and immediately after exercise and decrease in numbers below pre-exercise levels during the early stages of recovery. Natural killer cells are rapidly mobilized into the circulation during exercise and decline within a few hours afterwards.⁷ Moreover, acute endurance exercise increased the number of circulating neutrophils.¹⁷ While these responses have been found in typically developed populations, there are currently no studies about the immune system response to acute exercise in young adults with CP. Information about the immune system response to exercise may uncover an altered behavior of the immune system in this group. This in turn could shift our focus on the evaluation of treatment strategies that may have the potential to better stimulate health-related immune cell responses in individuals with CP.

The main aims of this study were (1) to quantify and compare the abundance and proportions of circulating immune cell populations in young adults with CP and typically developing adults at rest, and (2) to examine the response of circulating immune cell populations after an acute bout of endurance exercise in individuals with CP and typically developing individuals. We hypothesized that the resting proportions would not differ between groups and that an endurance exercise bout would lead to a similar change in circulating immune cells in the individuals with CP and typically developing individuals.

METHOD

Immune cell analysis

After the complete collection from all participants, frozen samples were carefully thawed for analysis. Subtypes of PBMCs (see below) were quantified using fluorescence-activated cell sorting based on combinations of cell surface markers. For fluorescence-activated cell sorting analysis, about 500 000 cells were taken from each previously made cell stock to perform antibody staining in two separate panels (250 000 cells each). In panel A, PBMCs were stained for CD45 (BD #555482), CD177 (BD #564239), CD66b (BD #562254), CD19 (BD #560728), CD20 (BD #563067), CD14 (BD #555399), and CD16 (BD #562874). For panel B, PBMCs were stained for CD8 (BD #563919), CD4 (BD #561843), CD45 (BD #555482), CD3 (BD #561810), gamma delta (TCRγδ) T-cells (BioLegend #331221), CD16 (BD #562874), and CD56 (BD #560842). These panel markers were used in a gating strategy (Figure 1) to identify eight populations of PBMCs: B-cells, monocytes, macrophages, bright and dim natural killer cells, CD8+ T-cells, CD4+ T-cells, and TCRγδ+ T-cells.

RESULTS

Exercise performance and baseline immune cell populations

Significantly lower mean heart rates (p < 0.05) during the 45-minute running exercise were observed in the young adults with CP compared to typically developing adults (Figure 2a). In contrast, no difference was observed in perceived exertion (mean [SD]: typically developing: 15 [1]; CP: 15 [1]; p = 0.920, Figure 2b).

Regarding the baseline composition of the immune cell populations (Figure 3a and Figure 4a), a difference between groups was found in the TCRγδ+ T-cells (Figure 4c). More information on the baseline proportions can be found in Table S4.

Acute responses of circulating immune cell populations

In Figure 3, a significant main effect of time (p < 0.05) was found for the B-cells, monocytes, and macrophages, indicating a general effect of exercise on these cell subpopulations in both individuals with CP and typically developing participants (see also Table S5).

In Figure 4b, a significant effect of time (p < 0.05) was found in CD56+ bright natural killer cells mainly due to an overall increase immediately after the acute exercise bout. Moreover, a significant time×group interaction (p < 0.05) was observed in the CD8+ T-cell population. The post hoc comparison revealed that CD8+ T-cells increased after exercise in the typically developing participants but not in the individuals with CP (Figure 4e). To evaluate the effect of exercise intensity on the response of the CD8+ T-cells, the response of these cells was additionally correlated to heart rate using a Pearson correlation. The analysis revealed a positive correlation between the two parameters (Figure 5; r = 0.537, p = 0.003).

DISCUSSION

In this study, we assessed circulating immune cell populations in young adults with CP compared to their typically developing peers and examined the response of the cells to acute endurance exercise. The most important findings of the present study were that (1) at rest, the TCRγδ+ T-cell population was significantly different between the individuals with CP and typically developing individuals, and (2) the CD8+ T-cells of the two groups responded differently to the acute endurance exercise bout, with significantly higher levels observed immediately after the exercise bout in the typically developing participants.

To date, little is known about the extent to which the immune system is involved in individuals with CP. For diseases of the central nervous system, studies emphasize the occurrence of chronic low-grade inflammation and immunodeficiency in patients with, for instance, spinal cord injury.²⁰ In agreement, recent studies found markers of chronic low-grade inflammation also in young patients with CP. For instance, increased levels of tumor necrosis factor alpha^{12, 13} and interleukin-6 levels^{13, 21} were observed in children with CP aged 5 years or under. Moreover, these studies indicate that the highest levels of proinflammatory cytokines are present in the youngest children (1–3 years).^{12, 21} This finding is in line with the results presented by Pingel et al.¹⁰ and Zareen et al.⁹, who found no alterations in older children as well as adults with CP, when compared to typically developing controls. Taken together, the mentioned studies support the relevance of age regarding the existence of proinflammatory cytokines/chronic inflammation in individuals with CP. Sharova et al.¹³ observed increased levels of CD3+ T-cells (+22%) and both CD4+ (+20%) and CD8+ T-cell (+21%) subsets along with increased levels of CD22+ B-cells (+28%) in young children with CP compared to typically developing children. In contrast, significant differences in most circulating immune cell populations could not be found between young adults with CP and typically developing young adults in the current study. This result indicates a similar immune system state in individuals with CP and typically developing individuals in adulthood. However, we found an increased baseline of TCRγδ T-cells in individuals with CP compared to typically developing individuals. These rare T-cells connect innate and adaptive immune system, can exert strong antitumor cytotoxicity,²² and have an extensive cytokine production capacity. Interestingly, they have previously been implicated in the immune systems response to periventricular leukomalacia in infants, a major cause of CP.²³ In this context, TCRγδ T-cells appear to contribute to the injury of the brain by inducing amongst others the production of interleukin-17, subsequently leading to central nervous system inflammation and recruitment of other immune cell types that result in demyelinating lesions.^{24, 25} While increased frequency and absolute numbers of γδ T-cells (i.e. Vδ2 T-cells) were also found in newborn infants with neonatal encephalopathy, school-aged children post neonatal encephalopathy, and children with CP,²⁵ retained high levels of TCRγδ T-cells in adults have not been reported before. This may emphasize the continued relevance of this cell subtype into adulthood for its inflammatory and immune role in central nervous system disease. However, more information about the mechanisms by which these cells contribute to CP in general and assessments of the cytokine profiles of young adults with CP are needed to clarify and to support this finding.

In addition to the quantification of the circulating immune cell populations at rest, we investigated the response of the circulating immune cell populations to an acute endurance exercise bout in both groups. While the cell responses observed in individuals with CP and typically developing individuals are mostly in line with the behavior of the immune cells reported in previous studies,^{7, 16} a different response was observed for the CD8+ T-cells. It is well established that endurance exercise leads to an increase in T-cells and their subsets, such as the cytotoxic CD8+ T-cells.^{26, 27} In the present study, the participants with CP demonstrated a significantly lower number of circulating CD8+ T-cells directly after the acute bout of exercise compared with the typically developing participants. We assume that exercise intensity might have been the main influencing factor. The influence of exercise intensity on lymphocytosis has been previously described.²⁸ Moreover, studies emphasize the intensity-related response of the CD8+ T-cells to (endurance and resistance) exercise in typically developed people,^{29, 30} with higher intensities causing elevated responses. In the present study, perceived exertion was rated as 15 in both groups, characterizing the endurance exercise bout as mostly ‘hard (heavy)’. Although no differences in perceived exertion were detected, the individuals with CP reached lower heart rates (−11.1%) in comparison to their typically developing peers. Since ratings of perceived exertion are subjective,³¹ heart rate data may be seen as more objective criteria to determine exercise intensity. Therefore, considering heart rate as an objective representation of the exercise-imposed strain on the cardiovascular system, this finding may suggest a lower strain evoked during the 45 minutes of frame running in the participants with CP. Since cardiovascular strain affects blood pressure, shear pressure in the blood vessels, and cardiac output, the lower intensity may have resulted in a lower level of immune cell mobilization and thus a lower number of CD8+ T-cells was measured from peripheral venous blood in this study. Supporting this assumption, we found a positive correlation between the response of the CD8+ T-cells to heart rate. The discrepancy between groups might have been caused by the performance of the endurance bout (i.e. frame running [cycling] vs running) or the lower physical fitness level of the individuals with CP. Both aspects have to be further elucidated in future studies. Moreover, the use of intervals with higher intensities and shorter duration could potentially be considered as a strategy to positively affect immune cell mobilization, while exercise intensities should also be standardized and equalized between groups. Although we investigated the immune cell responses to an acute bout of endurance exercise, the results may indicate positive effects on the immune system of individuals with CP when performing frame running for a prolonged period. However, this is yet to be confirmed.

Notably, other factors such as poor motor control and motivation might also have had an influence on exercise performance and intensity, therefore also influencing the results of this study. Furthermore, the idea that the responsiveness of the CD8+ T-cells is reduced in general in individuals with CP cannot be verified. However, the responses of the other measured immune cells did not show significant alterations in comparison to those of the typically developing participants, which may indicate an altered function of CD8+ T-cells.

There are certain aspects that need to be considered when interpreting the results of the current study. First, many factors might have contributed to the immune profiles observed before and after the endurance exercise bout as, for instance, vaccination status, nutrition status, medications, and vitamin D status. Although we asked the participants about other diagnoses, medications, and comorbidities, we did not ask for permission to look further into their medical records and to investigate other related factors such as vitamin D status. We suggest these factors are considered in future studies on this topic to gain more information about potential associations. Second, we note that our groups did significantly differ in their anthropometric characteristics (e.g. body height, body mass, body mass index), which might have influenced the results. Moreover, the participating individuals with CP were already familiar with frame running and were mainly moderately physically active. Therefore, the findings might not be transferable to a less active population. Furthermore, the employed flow cytometric method of immune system analysis can only be a peripheral snapshot of immune system activity and should be interpreted as such. As we performed broad panel analysis rather than investigating individual subpopulations at a high resolution, there might also be additional shifts at the subpopulation level that are not detectable with our analysis. Finally, investigation of cytokine profiles may be needed to further support our findings.

To the best of our knowledge, this is the first study investigating the immune status and immune cell responses in young adults with CP. In summary, no evidence for an impaired immune function could be found in this group when compared to their typically developing peers. The findings largely suggest a ‘recovery’ of immune disturbances with age/maturation likely present throughout childhood. However, this should be further elucidated in future longitudinal studies. We further emphasize the importance of exercise intensity to evoke immune cell responses in individuals with CP. Thus, our data underline the importance of treatment and training strategies of adequate exercise intensities that cause positive responses of the immune system.

CONFLICT OF INTEREST STATEMENT

The authors have stated that they had no interests which might be perceived as posing a conflict or bias.