Cross-sectional analysis of blood leukocyte responsiveness to interleukin-10 and interleukin-6 across age and physical activity level

Introduction

Inflammatory responses are critical for combatting illnesses, maintaining cellular homeostasis, and promoting tissue repair following injury, but unresolved sterile inflammation – commonly referred to as “chronic inflammation” – contributes to various chronic diseases [1, 2]. Regular exercise is a known powerful anti-inflammatory stimulus [3]. A major mediating mechanism is the release of anti-inflammatory cytokines from contracting muscle (e.g., interleukin [IL-6]), which stimulates the production of anti-inflammatory cytokines from circulating immune cells (e.g., IL-10), thereby counteracting the release of pro-inflammatory mediators that drive chronic inflammation [4, 5]. The purported existence of this “IL-6 cascade” has led to the widespread practice of quantifying the anti-inflammatory effects of exercise via measurement of circulating cytokine concentrations [6, 7].

Although reflective of changes in the crude systemic inflammatory milieu, circulating cytokine concentrations do not provide any insight into the cellular source/target of cytokine secretion/action nor do they capture exercise-induced changes in cellular inflammatory processes [8]. We have recently demonstrated that an acute bout of higher intensity exercise can induce a transient hyporesponsiveness to the anti-inflammatory actions of IL-10 (direct) and IL-6 (indirect) in blood leukocytes – as reflected by their ability to activate signal transducer and activator of transcription 3 (STAT3) and inhibit tumour necrosis factor-alpha (TNF-⍺) secretion in response to lipopolysaccharide (LPS) [9]. We have previously reported similar observations of attenuated anti-inflammatory cytokine action following 2 weeks of exercise training in individuals with obesity [10]. Importantly, in both these cases, circulating cytokine concentrations dissociated from changes in cellular cytokine action, accentuating the need to look beyond circulating cytokine concentrations when evaluating the immunomodulatory effects of exercise. Relatedly, whether the hyporesponsiveness to anti-inflammatory cytokine is a transient phenomenon observed after acute exercise and short-term training, or apparent under chronically high levels of physical activity is not known.

The primary objective of this exploratory cross-sectional study was to determine how blood leukocyte responsiveness to IL-10 and IL-6 – two cytokines most heavily implicated in the anti-inflammatory effects of exercise [8] – is altered across individuals at opposite ends of the physical activity spectrum. Given the known impacts of aging on the immune system [11], we also wanted to determine whether chronically high levels of physical activity were able to preserve IL-10/6 action in highly active older individuals compared to their younger counterparts. To test these objectives, we compared the ability of ex vivo IL-10/6 stimulation to induce STAT3 phosphorylation in circulating monocytes and lymphocytes from inactive younger individuals (18–30 years of age performing <150 min of weekly activity), younger highly active individuals (18–30-year-old varsity cross-country athletes performing >300 min of weekly activity), and older highly active individuals (>40-year-old adults performing >300 min of weekly activity). The ability of IL-10 and IL-6 to inhibit TNF-⍺ secretion in LPS-stimulated whole-blood was also determined in parallel. The summary of this article is presented in Figure 1.

Figure 1:

Graphical representation of this study. Key points: (1) A major mechanism by which exercise protects against chronic disease is by lowering chronic inflammation; (2) Much of what we know about the anti-inflammatory effects of exercise is derived from circulating cytokine concentrations, which may not reflect inflammatory processes at the cellular level; (3) Here we show that the ability of interleukin (IL)-10 and IL-6 to activate anti-inflammatory signaling in immune cells is enhanced in highly active individuals compared to those who are inactive – an effect that is apparent in both younger and older active individuals. These observations advance our understanding of the inflammation-lowering effects of physical activity. Figure created with BioRender.

Methods

Results

Basal IL-10/6 receptor expression

Basal (unstimulated) cytokine receptor expression in CD14 monocytes (panel A) and CD4 lymphocytes (D) are shown in Figure 2 (IL-10) and Figure 3 (IL-6). No between-group differences were apparent for unstimulated (basal) pSTAT3 MFI in either cell type (data not shown, p>0.48). IL-10 receptor expression was reduced in CD14 monocytes (p<0.01) and CD4 lymphocytes (p=0.05) in YA compared to YI (p<0.01). The OA group also had significantly lower CD4 IL-10 receptor expression compared to the YI group (p<0.05).

Figure 2:

IL-10 receptor expression and IL-10 mediated STAT3 phosphorylation in CD14+ monocytes (A–C) and CD4+ lymphocytes (D–F). Data are shown as means ± SD and were analyzed using a one-way ANOVA. p-values shown above bars correspond to Tukey’s post-hoc testing for pairwise comparisons.

Figure 3:

IL-6 receptor expression and IL-6 mediated STAT3 phosphorylation in CD14+ monocytes (A–C) and CD4+ lymphocytes (D–F). Data are shown as means ± SD and were analyzed using a one-way ANOVA. p-values shown above bars correspond to Tukey’s post-hoc testing for pairwise comparisons.

LPS stimulated TNF-⍺ secretion and its inhibition by IL-10/6

No significant between-group differences (p>0.71) were observed for LPS-stimulated TNF-⍺ secretion (YI: 314 ± 163 pg/mL; YA: 328 ± 101; OA: 365 ± 127). The ability of various concentrations of IL-10 and IL-6 to inhibit LPS-stimulated TNF-⍺ are shown in Figure 4A and B , respectively. Both cytokines inhibited TNF-⍺ secretion (main effect of concentration, p<0.01) though IL-10 was overall more anti-inflammatory eliciting a stronger dose-response inhibition of TNF-⍺ secretion. A significant group*concentration interaction effect (p=0.02) was observed with maximal IL-6 (10 ng/mL) co-treatment and appeared to be driven by a tendency for less TNF-⍺ inhibition in the OA vs. the YI group (p<0.10). No other differences for IL-10/6 mediated inhibition of TNF-⍺ secretion were apparent (p>0.05).

Figure 4:

IL-10 (A) and IL-6 (B) mediated inhibition of TNF-⍺ secretion from LPS-stimulated whole-blood. Data are shown as means ± SD and were analyzed using mixed effects analysis. p-values shown above bars correspond to Tukey’s post-hoc testing for pairwise comparisons.

Discussion

The purpose of this cross-sectional study was to compare blood leukocyte responsiveness to the anti-inflammatory cytokines IL-10 and IL-6 between individuals of varying activity levels and ages. The major finding was that the ability of IL-10 and IL-6 to activate STAT3 appeared to be enhanced in younger and older highly active individuals – despite lower leukocyte IL-10 receptor expression – when compared to younger inactive adults. These differences in leukocyte signaling were not associated with alterations in the ability of IL-10 or IL-6 to elicit decreases in the level of the pro-inflammatory cytokine TNF-⍺, which was similar across activity levels and preserved in older active individuals. These findings provide new insights into cellular cytokine action across age and physical activity status and support the anti-inflammatory potential of engaging in high levels of exercise/activity.

The impacts of exercise and training on systemic cytokines have been extensively studied but comparatively little is known about the associated impact on cytokine action at the cellular level [8]. This is important because cytokines typically function locally to impact cellular function after engaging with specific receptors and activating distinct signaling pathways [13]. We recently demonstrated that an acute bout of higher intensity exercise blunts the overall anti-inflammatory effects of IL-10/6 in active young adults [9] – a finding that appeared contradictory to the known anti-inflammatory benefits of regular physical activity [3, 4]. We speculated that this acute hyporesponsiveness to IL-10/6 action is likely transient in nature and confined to the immediate post-exercise period to enable inflammatory processes that facilitate cellular adaptations [14]. In this scenario, anti-inflammatory cytokine action would then return to or exceed pre-exercise levels, supporting an overall anti-inflammatory profile. Our current work partly supports this supposition by demonstrating that blood leukocytes from highly active individuals are indeed more responsive to IL-10/6 when studied under non-exercised (rested) conditions. Thus, despite the apparent transient hyporesponsiveness to anti-inflammatory cytokine action following each exercise bout [9], the ability of IL-10/6 to activate STAT3 signaling appears to be enhanced in highly active individuals compared to those who are inactive. The functional consequence of this greater STAT3 responsiveness remains to be determined, as the inhibition of TNF-⍺ secretion in response to LPS stimulation was similar between groups. Future work should therefore investigate addition events downstream of STAT3 signaling and/or cytokine secretion in response to other immune-activating stimuli to fully elucidate how anti-inflammatory cytokine action may be altered with chronically high levels of physical activity.

Intriguingly, the enhanced responsiveness to IL-10/6 in the highly active groups was apparent despite lower leukocyte IL-10 receptor expression (with a similar albeit non-significant pattern also observed for IL-6 receptor expression). Downregulation of the IL-10 receptor is likely an adaptive response to the high levels of physical activity in the YA/OA groups and the repeated stimulation of anti-inflammatory cytokines that is often reported following individual exercise bouts [4, 8]. The enhanced responsiveness of blood leukocytes to IL-10/6 is likely needed to allow activation of intracellular signaling pathways with fewer cytokine receptors – particularly since cytokine responses to acute exercise can sometimes be attenuated with training [15]. The enhanced sensitivity to IL-10 action could also be a contributor to the frequently purported immunosuppressive effects of very high levels of activity [16, 17]. Others have also reported associations between antigen stimulated IL-10 production and training load in athletes [18, 19]. The present data suggest a role of altered IL-10 action – in addition to production – in the potential immunosuppressive effects of high exercise volumes.

Although our study focused on the anti-inflammatory potential of IL-6 in the context of physical activity, this cytokine has known pro-inflammatory effects in other contexts that should be considered when interpreting the biological significance of IL-6 action [20, 21]. In our study, the IL-6 mediated activation of STAT3 was accompanied by inhibition of TNF-⍺ secretion from LPS-stimulated whole blood co-treated with IL-6, supporting an overall anti-inflammatory effect that is consistent with previous work 22], [23], [24. However, the inhibition of TNF-⍺ secretion by IL-6 was less pronounced than IL-10 – an observation that we have reported previously [10] – confirming that IL-6 is less anti-inflammatory than IL-10. Moreover, it is unclear if the inhibition of TNF-⍺ secretion in IL-6 treated whole-blood cultures was due to direct effects of IL-6 per se or via downstream stimulation of IL-10 secretion [23, 25]. Additional work is needed to discern the direct and indirect anti-inflammatory actions of IL-6 in blood leukocytes and how these may be impacted by physical activity.

Age-related alternations in immune function – often termed immunosenescence or inflammaging – are a proposed contributor to poor health and heightened chronic disease risk in older individuals [11, 26]. Although the accompanying reductions in physical activity make it difficult to disentangle the direct effects of aging per se from physical inactivity, exercise has known immunomodulatory benefits in older adults [11]. Our observation of greater STAT3 activation in IL-6 – stimulated blood leukocytes from older active individuals compared to their younger inactive counterparts provides an additional mechanism by which high levels of activity may help maintain an anti-inflammatory profile through the lifespan. Importantly, the ability of both IL-10 and IL-6 to inhibit the secretion of TNF-⍺ secretion was preserved in the older active adults in our study, suggesting that the ability of these cytokines to combat inflammation does not deteriorate through the middle age with high levels of physical activity. These observations are in line with studies examining plasma cytokine concentrations in older adults, which are also supportive of a more anti-inflammatory profile in those of higher physical activity levels [27].

The enhanced ability of IL-10/6 to activate intracellular signaling in monocytes and lymphocytes from active individuals may have implications for broader changes in immune function with physical activity. For instance, it is well-established that chronically high levels of physical activity lower the risk of mortality and morbidity via reduced chronic inflammation (e.g., via lowered expression and/or activation of toll-like receptors on innate immune cells) [3]. The ability of physical activity to modulate responsiveness of innate immune cells such as monocytes to IL-10 stimulation could be a contributor to the inflammation-lowering effects of physical activity. On the other hand, a heightened responsiveness of monocyte signaling to IL-10 could also contribute to tempered innate immune responses in highly active individuals, making them more susceptible to immunosuppression and potentially contributing to the elevated rates of respiratory infections that are sometime reported in athletes [16, 17]. Given the role of IL-6 in regulation of T cell differentiation and effector functions, the greater responsiveness of CD4 cells to IL-6 in more active individuals could also be associated with altered innate immunity [28]. Future research including additional immune cell subtypes, immune activating stimuli, and/or the assessment of immune cell effector responses is needed to further elucidate how physical activity alters the regulation of innate and adaptions immune functions by IL-10/6.

The current study had some noteworthy strength and limitations. First, we directly measured cytokine action by quantifying the ability of various concentrations of IL-10/6 to activate STAT3 signaling in distinct blood leukocyte populations and inhibit TNF-⍺ secretion ex vivo. Although we recognize the small sample size of the current study, we matched the number of males and females within each group to equalize the potential impact of biological sex, which we have previously shown to be a modulator of anti-inflammatory cytokine action [12]. We also recognize that due to the lack of an inactive older group, the direct effects of aging per se on cytokine action could not be disentangled from physical activity providing an important next step for future work in the area. Although comorbidities and medications can often make age-related comparisons difficult, future studies should also seek to explore anti-inflammatory cytokine action in older age groups.

Conclusions

Blood leukocytes from active individuals exhibit more responsiveness to IL-10/6 stimulation – as reflected by greater cytokine stimulated STAT3 phosphorylation ex vivo – compared to individuals who are physically inactive. The heightened ability of these anti-inflammatory cytokines to activate STAT3 was apparent despite lower cytokine receptor expression, pointing to enhanced responsiveness of intracellular signaling with high levels of activity. The ability of IL-10/6 to inhibit pro-inflammatory cytokine secretion was not impacted by physical activity level, but was preserved in older active individuals compared to their younger counterparts.