Endurance Exercise Diverts the Balance Between Th17 Cells and Regulatory T Cells
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Endurance Exercise Diverts the Balance between Th17 Cells and Regulatory T Cells Chava Perry1*., Marjorie Pick2., Nir Bdolach3, Inbal Hazan-Halevi1, Sigi Kay1, Idit Berr1, Adi Reches3, Yair Harishanu1, Dan Grisaru3 1 Department of Hematology and Bone Marrow Transplantation, Tel Aviv Sourasky Medical Center, Tel Aviv, Israel, 2 Department of Hematology, Hadassah University Hospital, The Hebrew University, Jerusalem, Israel, 3 Gynecologic Oncology Unit, Department of Obstetrics and Gynecology, Lis Maternity Hospital, Tel Aviv Sourasky Medical Center, Tel Aviv, Israel Abstract Endurance, marathon-type exertion is known to induce adverse changes in the immune system. Increased airway hyper- responsiveness and airway inflammation are well documented in endurance athletes and endurance exercise is considered a major risk factor for asthma in elite athletes. Yet, the mechanisms underlying this phenomenon are still to be deduced. We studied the effect of strenuous endurance exercise (marathon and half-ironman triathlon) on CD4+ lymphocyte sub- populations and on the balance between effector and regulatory CD4+ lymphocytes in the peripheral blood of trained athletes, Endurance exercise induced a significant increase in Th17 cells and a sustained decrease in peripheral blood regulatory T cells (Tregs). While interleukin (IL)-2 levels remained undetectable, post-race serum IL-6 and transforming growth factor (TGF) b levels were significantly elevated. Treg levels in sedentary controls’ decreased in vitro after incubation with athletes’ post-exercise serum, an effect that was attenuated by supplements of IL-2 or anti IL-6 neutralizing antibodies. Our data suggest that exercise-induced changes in serum cytokine levels promote alterations in Tregs and Th17 cell populations, which may divert the subtle balance in the immune system towards inflammation. This may explain allergic and autoimmune phenomena previously reported in endurance athletes and contribute to our understanding of exercise- related asthma. Citation: Perry C, Pick M, Bdolach N, Hazan-Halevi I, Kay S, et al. (2013) Endurance Exercise Diverts the Balance between Th17 Cells and Regulatory T Cells. PLoS ONE 8(10): e74722. doi:10.1371/journal.pone.0074722 Editor: Derya Unutmaz, New York University, United States of America Received May 28, 2013; Accepted August 4, 2013; Published October 9, 2013 Copyright: ß 2013 Perry et al. This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. Funding: The work was funded by Department of Hematology, Tel Aviv Medical Center. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript. Competing Interests: The authors have declared that no competing interests exist. * E-mail: [email protected] . These authors contributed equally to this work. Introduction regulatory T cells (Tregs) are differentiated T lymphocytes actively involved in control of peripheral immunity. The identification of Accumulating clinical data support the notion that the immune these cells has led to new insights into mechanisms of tolerance system responds to increased physical activity. The reported effects breakdown in human diseases, including those resulting from of endurance exercise on the immune system differ from that of allergic, autoimmune, or infectious causes [11,12]. moderate physical activity. Moderate exercise is associated with The transcription factor Forkhead box P (FoxP)3 plays a key reduced incidence of upper respiratory tract (URT) infections role in Tregs cell function and is a useful characteristic that allows while endurance, elite athletes report of susceptibility to upper identification for these cells. Transforming growth factor (TGF)b respiratory infections [1–4]. Endurance sport is considered a triggers FoxP3 expression in CD4+CD25- precursors and together major risk factor for asthma. In fact, self-reported and physician- with IL-2, is a key regulator of the signaling pathways that diagnosed asthma are twice as common in elite Norwegian and maintain FoxP3 expression and suppressive function in Tregs Finnish athletes than in randomly selected age-matched and sex- [13,14]. However, a combined signal of TGFb and the pro- matched control populations [5,6]. It was recently reported that inflammatory cytokine IL-6 may down regulate FoxP3 function, the prevalence of allergy in a large cohort of runners competing in promote the induction of the transcription factor RAR-related the 2010 London Marathon was 40% and almost half of the orphan protein gamma (RORc) and the differentiation of Tregs runners experienced upper respiratory tract (URT) symptoms [7]. into Th17 cells [15,16]. The balance between Th17 and Tregs Ceasing high-level training could lead to attenuation or even CD4+ lymphocytes is tightly controlled as perturbed equilibrium disappearance of bronchial hyper responsiveness and asthma [8]. might lead to decreased immune reactivity or, in contrast, to The effector T-cell lineage shows great plasticity. T helper (h)17 pathological inflammation [17]. cells are CD4+ lymphocytes that produce Interleukin (IL)-17, a We hypothesized that strenuous endurance exercise affects the cytokine that play a crucial role in allergic inflammation and are balance between effector and regulatory CD4+ lymphocytes. As a known as powerful pro-inflammatory cells that promote autoim- result, we evaluated the peripheral blood CD4+ T cell sub- munity [9,10]. On the other end of the spectrum CD4+CD25+ populations’ profile in trained athletes throughout extreme PLOS ONE | www.plosone.org 1 October 2013 | Volume 8 | Issue 10 | e74722 Endurance Exercise May Promote Inflammation exertion (marathon and half Ironman triathlon). Our data shows ciences). Using four-color flow cytometry a lymphocyte population that endurance exercise induced a significant increase in Th17 was first gated from the mononuclear cell population according to cells and a sustained decline in peripheral blood Tregs population. their low forward versus side low scatter variables. Of the total Athletes’ serum collected post-exercise induced, in vitro, a decrease lymphocyte gate only CD4+ cells were identified, gated and in controls’ peripheral blood Treg levels. These alterations in analyzed for their expression of IL-17. Isotype control IgG PerCp- CD4+ T cell sub-populations may be attributed to changes in Cy5 was used to detect background staining. TGFb, IL-6 and IL-2 serum levels, and perhaps, play a pivotal role in the mechanism(s) underlying allergic and autoimmune Cytokines phenomena previously reported in endurance athletes. TGF- b, IL-2, IL-6 and IL-10 serum levels were evaluated by ELISA, according to the manufacturer’s instructions (Quantikines Materials and Methods kits, R&D Systems). Tumor necrosis factor (TNF) a, interferon (INF) c and IL-4 serum levels were evaluated by flow cytometry, Athletes using a particle-based immunoassay (CBA kit; BD Biosciences) Forty two athletes who participated in the 2007 Emek- according to the manufacturer instructions. Hayarden Half Ironman triathlon (consisting of 1.9 kilometer Serum cortisol levels were measured by electrochemilumines- (km) of swimming, 90 km of cycling and 21 km of running), or in cent (ECL) method (Elecsys 2010 Immunoassay Analyzer, the 2009 Tiberia marathon (a 42.2 km run), were included in this Boehringer, Mannheim, Germany). study. Athletes were included if they were over the age of 18 years old and up to 60 years old, with no previous known medical history of ischemic heart disease, hypertension or diabetes. In vitro studies Athletes with previous known medical history of ischemic heart PBMNC were separated from whole blood of healthy sedentary disease, hypertension or diabetes were excluded from the study. volunteers (males, median age 36 years, range 30–43), who were This research was approved by the Tel Aviv Sourasky Medical not regularly engaged in any sportive activity, on Ficoll-Hypaque 6 Center Ethics Committee and all subjects participating in this gradients. 10610 cells were plated in 6 well plates and incubated research signed an informed consent form, approved by the Tel for 4 hours in RPMI (Biological Industries, Beit Haemek, Israel), Aviv Sourasky Medical Center Ethics Committee. supplemented with 10% serum collected either pre-race or post- All athletes filled a questionnaire documenting their age, race from 4 of the participating athletes. One of the athletes’ sera gender, medical history, use of medications and any physical was used in 2 different sets of the in vitro experiments, with two phenomena that occurred in the seven days following the race. different controls’ PBMNC. Each serum was tested individually on Blood samples were collected 4 days prior to the race (pre-race), individual control PBMNC. at the end of the race (post-race) and 10 days after the race Either IL-2 (50 U/ml), anti human IL-6 neutralizing antibodies (recovery). Blood was drawn into EDTA tubes for complete blood (5 mg/ml) or mouse IgG isotype control antibodies (R&D Systems) count and flow cytometry immunophenotyping. Serum was were added to the post-race serum. Cells were then analyzed by separated from whole blood by centrifugation for 10 minutes at flow cytometry for Treg cells, as described above. 2000 rpm and stored at 280uC until use. The fraction of dead cells was evaluated by 7-amino-actinomy-