Differential impacts of trail and ultra-trail running on cytokine profiles: An observational study

Abstract

BACKGROUND:

Endurance running events are known to cause inflammation and result in increased cytokine production. However, the effects of ultramarathons on cytokine profiles are not well characterized.

OBJECTIVE:

The aim of this study was to describe and compare the effects of a trail (40 km) race and an ultra-trail (171 km) race on leukocyte concentrations and cytokine profiles.

METHODS:

The study was conducted during the Ultra-Trail du Mont Blanc® ultra-marathon running event, and included 11 runners who completed the 40 km trail run and 12 runners who completed the 171 km ultra-trail. Blood samples were taken before and after the races.

RESULTS:

Leukocyte concentrations significantly increased after both races. Circulating levels of IL-6, IL-1β, MCP-1, and IFN-γ were significantly higher after the longer race compared to the shorter race. Furthermore, while both races resulted in significant increases in IL-6 and IL-8, only the longer race resulted in significant increases in MIP-1β, IL-7, IL-17a, and IL-4.

CONCLUSIONS:

These results illustrate that a 171 km ultra-trail race results in greater modulations in cytokine profiles than a traditional trail race.

Keywords

Running exercise ultra-trail inflammation cytokines profile leukocytes

1 Introduction

Many studies have demonstrated that acute strenuous exercise results in immune system activation, characterized by changes in leukocyte concentrations and cytokine profile [1 –5]. Cytokines are intracellular signaling molecules released by a wide variety of cells, including myocytes, adipocytes, immune cells, and endothelial cells, in response to stress [6]. A large body of evidence shows that acute bouts of endurance running can promote the production of both pro- and anti-inflammatory cytokines [1, 6]. Indeed, findings of multiple studies show that marathons consistently cause levels of IL-6, IL-8, and IL-10 to rise, with levels of IL-6 increasing over 100-fold in certain studies [7 –9]. This increased cytokine production is thought to have potentially positive and negative effects during and after exercise. For example, exercise-mediated changes in cytokine profile may help restrict muscle damage, improve muscle recovery, and play a role in exercise-related changes in metabolism [1, 6]. On the other hand, cytokines released during strenuous exercise may contribute to gastrointestinal distress during exercise bouts, and could acutely enhance susceptibility to infections [4 , 10].

Recently, the popularity of endurance races that exceed the length of a marathon (42.195 km), referred to as ultramarathons, has grown [11, 12]. These events, which often take place under extreme environmental conditions and over difficult terrain, have attracted scientific interest as participants’ bodies are pushed to their limits [11, 12]. Several studies have investigated the effect of these extreme races on cytokine profiles. For example, a study by Kasprowicz and colleagues showed that a 100 km ultramarathon resulted in increased IL-6, accompanied by elevated levels of white blood cells (WBC) and C-reactive protein (CRP) [13]. Additionally, findings from Gill et al showed that a 24-hour continuous ultramarathon (the total distance run ranged from 122–208 km) resulted in increased concentrations of CRP, IL-6, IL-10, IL-8, IL-1β, and TNF-α [14]. In accordance, results from a study by Neiman et al showed that IL-6, IL-10, IL-8, IL-1ra, MCP-1, MIP-1β, and G-CSF all increased after a 160 km race [15]. Previous research has demonstrated that intensity, duration of exercise, and ratio of eccentric to concentric exercise can affect cytokine profiles [2, 16]. However, to our knowledge, no studies have directly compared cytokine profiles following an ultra-trail race compared to a shorter trail race. Therefore, this study was conducted to evaluate differences in cytokine profiles after a 171 km ultra-trail and a 40 km trail race performed in similar environmental and technical conditions. Based on data from previous studies, we hypothesized that the ultra-trail race would result in significantly higher concentrations of IL-6, IL-8, IL-1β, MCP-1, and MIP-1β than the 40 km trail run due to the ultra-trail’s longer distance, greater duration, and larger elevation changes.

2 Materials and methods

2.1 Description of the trail running races

The study was performed during the Ultra-Trail du Mont Blanc® ultra-marathon running event, organized from August 26^th –September 1^st, 2019 in Chamonix, France. All participants were volunteers who completed either the MCC or the UTMB race. The protocol was approved by the Ethics Committee (CPP Ouest VI, ethics committee agreement 19.03.14.41740 received on 05/02/2019), and the study was conducted in accordance with the Declaration of Helsinki.

The MCC is a 40 km race with a 2,300 m positive altitude difference that must be completed in less than 10 hours. The race begins in Martigny-Combe, Switzerland, and finishes in Chamonix, France. The UTMB is a 171 km race with a positive altitude difference of 10,000 m that must be completed in less than 46.5 hours. The race begins in Chamonix, France, and passes through Italy and Switzerland before returning to Chamonix. To register for the UTMB, runners must have completed at least two qualifying ultra-marathon races in the year prior to the race. Both the MCC and UTMB races are completed in semi-autonomy. Beverage, food, and aid stations are positioned at intervals, ranging from 8 to 17 km, along the racecourse.

2.2 Participants and study design

Eleven (5 women, 6 men) runners who completed the MCC race volunteered to participate in the study. On average, participants in the MCC group were 37.5±9.0 years old, 174.3±7.4 cm, and weighed 65.4±10.2 kg. Twelve (4 women; 8 men) runners who completed the UTMB voluntarily participated in the study. The participants in the UTMB group were 38.3±6.9 years old, 171.4±9.7 cm, and weighed 63.5±9.3 kg.

All subjects attended a preliminary visit that occurred between 4–8 weeks prior to the race date. During this visit, participants performed an incremental maximal exercise test on a treadmill to determine his or her maximal oxygen consumption, as reported in detail in Roberts et al. [17]. The MCC race took place on August 26^th and the UTMB race began on August 30^th with the last participant finishing on September 1^st. Subjects were weighed pre- and post-race, and blood samples were drawn prior to the race and within 30 minutes after each subject finished the race.

2.3 Blood sample collection

Venous blood samples were drawn from the antecubital vein directly into EDTA tubes. Whole blood was used for hematological measurements. Plasma samples for cytokine assays were obtained by centrifuging blood from EDTA tubes at 2000 g for 10 minutes at ambient temperature. All samples were stored in multiple aliquots at –80°C until assayed.

2.4 Hematological parameters

White blood cell (WBC) count, red blood cell (RBC) count and hemoglobin, were measured using a hematological analyzer (XN-350, Sysmex, Japan). Hematocrit was measured using the micro-method following blood microcentrifugation. Plasma free hemoglobin was determined by measurement of hemoglobin absorbance at 576 nm using the Cripps method [18]. Creatine kinase concentrations were measured as described in our previous study [19].

2.5 Multiplex cytokine assay

The Bio-Plex Pro^TM Human Cyokine 17-plex Assay kit (Bio-Rad, Hercules, CA) was used to quantify plasma concentrations of human cytokines. The assays were performed according to the instructions from the kit. Cytokine concentrations were measured using the MAGPIX xPONENT 4.2 System (Luminex Corporation, Austin, TX).

2.6 Statistical analyses

All statistical analyses were conducted using SPSS Statistics software (version 24; IBM, Chicago, IL). The results are presented as mean and standard deviation. Main effects for within group differences over time (before and after the races), between group differences (comparing the MCC and UTMB races), and interactions were evaluated using two-way mixed ANOVAs. LSD post-hoc tests were used when necessary. Two-tailed Pearson’s correlations were conducted to analyze associations between cytokines and CK after the MCC and the UTMB. Statistical significance was defined as P < 0.05 for all analyses.

3 Results

3.1 Participants’ characteristics and race results

The subjects’ race results, maximal oxygen consumption (VO2max) values, and weight before and after the races are shown in Table 1. As expected, the race time was significantly longer, and the running speed was significantly slower for the UTMB race compared to the MCC. Both races resulted in significant weight loss, however weight loss during the race in the MCC group was significantly greater than in the UTMB group.

Table 1
Participants’ characteristics and race results

MCC UTMB

Mass before race (kg) 65.4±10.2 63.5±9.3

Mass weight after race (kg) 63.6±9.8^††† 62.8±9.1^†

VO₂max (ml/min/kg) 59.0±11.6 59.0±7.5

Race time (hr:min:sec) 6:50:12±0:39:12 39:53:00±3:27:55^*

Race speed (km/h) 6.1±1.6 4.5±0.9^**

	MCC	UTMB
Mass before race (kg)	65.4±10.2	63.5±9.3
Mass weight after race (kg)	63.6±9.8^†††	62.8±9.1^†
VO₂max (ml/min/kg)	59.0±11.6	59.0±7.5
Race time (hr:min:sec)	6:50:12±0:39:12	39:53:00±3:27:55^*
Race speed (km/h)	6.1±1.6	4.5±0.9^**

^*p < 0.05; ^**p < 0.01 compared to MCC. ^†p < 0.05; ^†††p < 0.001 compared to weight before race.

3.2 Hematological parameters

Hematological parameters measured before and after each race are reported in Table 2. WBC count significantly increased in both groups after the race. Hematocrit and hemoglobin significantly decreased in the UTMB group only, and were significantly lower in the UTMB group compared to the MCC group after the race as previously reported by our team [17]. RBC counts were significantly lower in the UTMB group compared to the MCC group both before and after the race. There were no significant differences in plasma free hemoglobin concentrations within or between the groups. Creatine kinase significantly increased following both the MCC and the UTMB, but was significantly higher in the UTMB group compared to the MCC group post-race.

Table 2
Hematological parameters

MCC UTMB

Before After Before After

WBC (10^∧3/uL) 6.9±1.4 16.8±5.1^††† 6.1±1.5 13.1±3.1^†††

RBC (10^∧6/ul) 5.0±0.3 5.0±0.3 4.6±0.2^ 4.2±0.2^†††

HGB (g/dL) 145.7±9.4 144.9±9.0 140.2±9.7 127.9±7.7^†††

HCT % 43.4±1.9 44.1±1.7 43.3±2.6 38.8±2.0^†††

Plasma free Hb (mg/dL) 16.8±5.7 16.8±16.9 13.5±3.5 12.7±6.6

CK (U/L) 212.0±304.1 575.3±491.7^††† 126.8±53.0 8323.5±7277.8^**††

	MCC	UTMB
WBC (10^∧3/uL)	6.9±1.4	16.8±5.1^†††	6.1±1.5	13.1±3.1^*†††
RBC (10^∧6/ul)	5.0±0.3	5.0±0.3	4.6±0.2^**	4.2±0.2^***†††
HGB (g/dL)	145.7±9.4	144.9±9.0	140.2±9.7	127.9±7.7^***†††
HCT %	43.4±1.9	44.1±1.7	43.3±2.6	38.8±2.0^***†††
Plasma free Hb (mg/dL)	16.8±5.7	16.8±16.9	13.5±3.5	12.7±6.6
CK (U/L)	212.0±304.1	575.3±491.7^†††	126.8±53.0	8323.5±7277.8^**††

^*p < 0.05; ^**p < 0.01; ^***p < 0.001 compared to after MCC. ^†p < 0.05; ^††p < 0.01 ^††† p < 0.001 compared to before race. White blood cells –WBC; Red Blood Cells –RBC; Hemoglobin –HGB; Hematocrit –HCT; Plasma hemoglobin –Plasma Hb; Creatine Kinase –CK.

3.3 Cytokines

Cytokine concentrations from before and after both races are shown in Fig. 1. Significant interactions between time (before and after the races) and group (MCC or UTMB races) were observed for MIP-1β, MCP-1, IFN-γ, IL-8, IL-7, IL-6, IL-4, IL-2, and IL-1β. Post-hoc analyses showed that plasma levels of IL-6, IL-1β, MCP-1, and INF-γ were significantly higher in the UTMB group compared to the MCC group following the races, and IL-8 tended to be higher in the UTMB group compared to the MCC group post-race. Furthermore, levels of IL-6 and IL-8 significantly increased following both the UTMB and MCC races, while levels of MIP-1β, IL-7, and IL-4 only increased significantly in the UTMB group following the race. In addition, main effects of time were observed for TNF-alpha and IL-17a. There were no significant interactions, main within group differences, or main between group differences for IL-2, GM-CSF, IL-12, or IL-5.

Fig. 1

Cytokine concentrations in pg·mL^-1 before and after the MCC and UTMB races. Statistical analyses revealed significant interactions between time (before and after the races) and group (MCC or UTMB races) for MIP-1β, MCP-1, IFN-γ, IL-8, IL-7, IL-6, IL-4, IL-2, and IL-1β. Main effects of time with no interactions were observed for IL-17 and TNF-α. Simple within groups effects (^*p < 0.05; ^**p < 0.01; ^***p < 0.001) and simple between group effects (^†p < 0.05; ^††p < 0.01) are shown.

3.4 Correlation analyses

Pearson’s correlation analyses revealed significant correlations between CK and IL-1β (R = 0.65, p = 0.03), MCP-1 (R = 0.66, p = 0.02), IL-8 (R = 0.60, p = 0.04), and IL-4 (R = 0.59, p = 0.04) in the UTMB group only (p < 0.05).

4 Discussion

Past research has shown that strenuous exercise can cause circulating levels of cytokines, such as IL-6, IL-8, and IL-10, to increase significantly, with relatively smaller and inconsistent changes in other cytokines, including MCP-1 and IL-1β [20]. However, our study is the first to show that, when comparing the effects of a “classic” 40 km trail running race (the MCC) versus a 171 km ultra-trail (the UTMB), post-race levels of IL-6, IL-1β, MCP-1, IFN-γ, and IL-8 were higher after the longer race compared to the shorter race. Furthermore, both races resulted in significant increases in IL-6 and IL-8, whereas only the longer race resulted in significant increases in MIP-1β, IL-7, and IL-4. Past research has shown that although the intensity, type, and duration of exercise can all affect cytokine profiles, the degree of increase of cytokine concentration is most closely linked to exercise duration [2, 21]. Furthermore, a study by Donnikov et al measured IL-6 before and after an ultramarathon race, during which subjects ran as far as they could in six hours. The results showed that the magnitude of IL-6 increase correlated with the distance each participant ran [16]. The UTMB is 4.3 times longer than the MCC, and on average subjects who completed the UTMB ran significantly slower for a much longer duration than those who completed the MCC. Therefore, our results suggest that, like IL-6, increases in plasma concentrations of IL-1β, IL-8, MCP-1, MIP-1β, IFN-γ, IL-4, and IL-7 may be more closely related to exercise duration and running distance than exercise intensity. However, our understanding of these results is limited because we were unable to measure cytokine production over the course of the race. Thus, we could not evaluate the time-course of cytokine appearance or removal, or confirm that cytokine production truly peaked at the end of each race.

IL-6 was the first myokine to be identified because plasma levels have been shown to increase consistently, and in larger amounts than any other cytokine, following strenuous aerobic exercise [22]. Contracting muscles appear to be the largest generators of IL-6 during exercise. Although release of this myokine was originally thought to be a result of muscle damage, evidence now indicates that neither eccentric exercise, nor markers of muscle damage are associated with increased plasma IL-6 [3, 6]. Our results showing no significant correlations between IL-6 and CK following the MCC or the UTMB are in agreement with these past findings. Research over the last three decades has revealed that IL-6 modulates lipolysis and glucose metabolism in multiple ways, and carbohydrate availability appears to regulate IL-6 release during exercise [6]. In our study, circulating IL-6 was over two times greater in the UTMB group than in the MCC group post-race. This significantly larger increase in IL-6 could potentially be related to greater alterations in carbohydrate utilization and availability as well as increased lipolysis during the ultramarathon compared to the shorter trail race.

Our findings are in accordance with previous research showing that strenuous endurance exercise causes concentrations of the proinflammatory chemokines IL-8, MIP-1β, and MCP-1 to increase [3 , 23]. IL-8, MCP-1, and MIP-1β play major roles in the recruitment of neutrophils, monocytes, and lymphocytes to sites of inflammation. Following muscle damage, neutrophils infiltrate the tissue, promote inflammation, and attract macrophages, which contribute to both the pro-inflammatory and anti-inflammatory responses in the muscle [24]. We observed significantly higher post-race levels of CK in the UTMB group compared to the MCC group, indicating that there was a greater degree of muscle damage following the longer race. Furthermore, correlational analyses revealed significant associations between CK and several cytokines, including MCP-1 and IL-8, following the UTMB but not the MCC. Thus, during the UTMB, the enhanced production of these chemokines could contribute to the promotion or resolution of inflammation through neutrophil and macrophage recruitment. Furthermore, significant increases in muscle-derived production of IL-8 are mainly seen following exercise with an eccentric component [6, 9]. The IL-8 generated during exercise is thought to act locally to stimulate angiogenesis, and some evidence suggests that IL-8 may help inhibit post-exercise muscle soreness following strenuous exercise in this way [25]. The UTMB includes large elevation changes that caused subjects to run many more kilometers downhill than those who completed the 40-km trail race. Therefore, it is possible that the increased IL-8 production during the ultra-trail could be related to greater overall physiological strain caused by the UTMB’s extreme distance and downhill running.

IL-1β and TNF-α are traditionally considered the main cytokines that stimulate acute phase reactions. However, although local gene expression of these cytokines increases in skeletal muscle during exercise, the majority of studies show that circulating concentrations of these cytokines either do not change, or increase following endurance exercise [5 , 26]. In our study, both the MCC and UTMB appeared to be associated with a rise in TNF-α, and post-race levels did not differ between the two groups. One potential reason why concentrations of TNF-α do not differ significantly between groups after the races is that IL-6 was significantly elevated following the UTMB compared to the MCC. Muscle-derived IL-6 has been shown to exert a number of anti-inflammatory effects, including inhibition of TNF-α production. Therefore, the elevated levels of IL-6 following the UTMB may have suppressed TNF-α production in this group. On the other hand, levels of IL-1β were significantly higher post-UTMB compared to post-MCC, and only increased significantly after the 171 km race. IL-1β can stimulate production of IL-6, IL-8, and MCP-1 [20], which were all elevated following the UTMB. Therefore, our findings suggest that running an ultramarathon, but not a 40 km race, caused levels of IL-1β to increase significantly, and this could be linked to elevations in other cytokines like IL-6, IL-8, and MCP-1. Moreover, IL-1β release can be triggered by inflammatory cell death, which can be caused by the activation of the NLRP3 inflammasome pathway [27]. A previous study showed that high-intensity exercise, but not medium or low-intensity exercise, results in increased plasma concentrations of IL-1β accompanied by increased peripheral-blood mononuclear cell expression of NLRP3 [28]. Although the UTMB is generally run at a medium or low-intensity, the race is far more strenuous than the MCC because of its extremely long distance and greater vertical climbs. Thus, the increased plasma concentrations of IL-1β after the 171 km race could reflect exercise-mediated inflammasome NLRP3 pathway activation during the UTMB, but not the MCC, caused by the extreme effort. Oxidized hemoglobin, a hemolysis related product, has been identified as a potent trigger of NLRP3 activation and IL-1β production in-vivo. In the present study, hematocrit significantly decreased post-race in the UTMB group only, however levels of plasma free hemoglobin did not change. Therefore, our results indicate that a trigger other than hemolysis could be at the source of any potential NLRP3 activation that would contribute to the increase in IL-1β.

The results of the present study showed that levels of IL-3, IL-2, GM-CSF, IL-12, and IL-5 did not change significantly after either race. The cytokines GM-CSF, IL-3, and IL-5 are members of the β cytokine family, and are considered pleiotropic regulators of inflammation in response to pathogens, but also contribute to pathology in chronic inflammation [30]. IL-12 is a pro-inflammatory cytokine produced by dendritic cells and macrophages in response to antigenic stimulation [31]. IL-2 promotes the generation, survival, and function of regulatory T cells, and thus plays an essential role in the immune response [32]. Few studies have measured the effects of endurance exercise on these various cytokines. However, a study by Nielson and colleagues reported that levels of IL-2, IL-4, IL-5, IL-12, TNF-α, GM-CSF, and IFN-γ did not change after a marathon [5]. In accordance with these past findings, our results indicate that endurance exercise likely does not cause concentrations of IL-3, IL-2, GM-CSF, or IL-5 to increase. However, our results suggest that an exercise that is much longer in duration and distance than a marathon may cause IL-4 and IFN-γ to increase.

Leukocytosis is a well-described physiological response to stress, and a large number of studies have shown that levels of WBC increase following an ultra-marathon [1 , 34]. Therefore, it is unsurprising that our results show significant elevations in WBC after both races. Interestingly, the increase in WBC was greater after the 40 km race (2.45-fold increase) than after the 171 km race (2.13-fold increase). A previous study by Shin et al showed that the leukocytosis following a 308 km ultramarathon was mainly due to increases in neutrophils and monocytes, which peaked at 100 km, and were accompanied by a relative decrease in lymphocytes [23]. Therefore, it is possible that total leukocyte count peaked before the end of the 171 km race, and then decreased by the time the participants finished. However, additional studies would need to be conducted to confirm this, and to determine how concentrations of different kinds of leukocytes evolved over the course of the two races. In addition, ultra-endurance exercise is known to cause plasma volume expansion, resulting in hemodilution [35 –37]. Past studies have suggested that this phenomenon could be linked to inflammation and IL-6 release [35 , 38–40]. Thus, plasma volume expansion, related to increased inflammation, could contribute to the lower relative WBC concentration in the UTMB group after the race.

In conclusion, the results of this study demonstrate that both a 40 km and 171 km trail running race result in increased levels of WBC, however levels of IL-6, IL-1β, MCP-1, and INF-γ were significantly higher in the UTMB group than in the MCC group post-race. Furthermore, only the 171 km race caused levels of IL-1β, MCP-1, MIP-1β, IL-7, and IL-4 to increase significantly. These cytokines can be produced by muscle cells and white blood cells. Future research should be conducted to determine whether the same types of cells contribute to the production of these cytokines during ultra-trails and more “classic” trail running races. Furthermore, increased cytokine production following acute strenuous exercise is thought to modulate exercise-mediated metabolic and immune system changes that could help prevent or repair muscle damage and improve overall health. Conversely, these alterations could cause immune system suppression and increase risk of infection in the period after exercise. As the popularity of ultra-trails continues to rise, future research should focus on understanding both the positive and negative effects of the large changes in cytokine profiles caused by these extreme events.

Funding

This research did not receive any specific grant from funding agencies in the public, commercial, or not-for-profit sectors.

Footnotes

Acknowledgments

We would like to thank the organizers of ‘The Ultra-Trail du Mont-blanc ®’ and the ‘Ecole Nationale de Ski et d’Alpinisme’ of Chamonix for their help in data collection.

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	MCC		UTMB
	Before	After	Before	After
WBC (10^∧3/uL)	6.9±1.4	16.8±5.1^†††	6.1±1.5	13.1±3.1^*†††
RBC (10^∧6/ul)	5.0±0.3	5.0±0.3	4.6±0.2^**	4.2±0.2^***†††
HGB (g/dL)	145.7±9.4	144.9±9.0	140.2±9.7	127.9±7.7^***†††
HCT %	43.4±1.9	44.1±1.7	43.3±2.6	38.8±2.0^***†††
Plasma free Hb (mg/dL)	16.8±5.7	16.8±16.9	13.5±3.5	12.7±6.6
CK (U/L)	212.0±304.1	575.3±491.7^†††	126.8±53.0	8323.5±7277.8^**††