Molecular Immunology 60 (2014) 129–134
Contents lists available at ScienceDirect
Molecular Immunology journal homepage: www.elsevier.com/locate/molimm
Effects of strenuous exercise on Th1/Th2 gene expression from human peripheral blood mononuclear cells of marathon participants Lianbin Xiang ∗ , Kristina E. Rehm, Gailen D. Marshall Jr. Laboratory of Behavioral Immunology Research, Division of Clinical Immunology and Allergy, Department of Medicine, University of Mississippi Medical Center, Jackson, MS, USA
a r t i c l e
i n f o
Article history: Received 28 August 2013 Received in revised form 5 March 2014 Accepted 18 March 2014 Available online 20 May 2014 Keywords: Strenuous exercise Cytokine Th1/Th2 balance Transcription factors PCR Array Gene expression
a b s t r a c t Physical stressors, such as strenuous exercise, can have numerous effects on the human body including the immune system. The aim of this study was to evaluate the gene expression profile of Th1/Th2 cytokines and related transcription factor genes in order to investigate possible immune imbalances before and after a marathon. Blood samples were collected from 16 normal volunteers 24–48 h before and one week after completing a marathon race. Gene expression of Th1 and Th2 related cytokines from human peripheral blood mononuclear cells (PBMC) was analyzed using Human Th1-Th2-Th3 RT2 Profiler PCR Array and qRT-PCR that measured the transcript levels of 84 genes related to T cell activation. We found that PBMC express a characteristic Th2-like gene profile one week post-marathon compared to pre-marathon. The majority of genes up-regulated one week post-marathon such as IL-4, GATA3, and CCR4 were Th2 associated. For Th1-related genes, CXCR3 and IRF1 were up-regulated one week post-marathon. There was a trend of down-regulation of two Th1 related genes, T-bet and STAT1. Th3-related gene expression patterns did not change in the study. The ratios of both IFN-␥/IL-4 and T-bet/GATA3 gene expressions were significantly lower one week after marathon. These findings suggest that a Th1/Th2 immune imbalance persisted at least 1 week after completion of a marathon which offers a mechanistic rationale for the increased risk of upper respiratory tract infections often reported after strenuous exercise. © 2014 Elsevier Ltd. All rights reserved.
1. Introduction Various studies have implied that regular and moderate exercise has a beneficial effect on immune function, which could protect against upper respiratory tract infections (URTI) (Barrett et al., 2012; Walsh et al., 2011). In contrast, extended and exhaustive exercise, such as a marathon race, may have negative effects on the immune system and increase the risk of URTI (Nieman et al., 1989; Shepard and Shek, 1996). After a strenuous bout of exercise, athletes are hypothetically more susceptible to URTI, although a causal relation has never been demonstrated. In a large study (2311 participants) of the Los Angeles Marathon, data suggested that runners
Abbreviations: PBMC, peripheral blood mononuclear cells; URTI, upper respiratory tract infections; Th cells, T helper cells; IFN-␥, interferon gamma; IL-4, interleukin 4; GATA3, GATA binding protein 3; T-bet, (TBX21): T-box 21; RT, reverse transcription. ∗ Corresponding author at: Laboratory of Behavioral Immunology Research, Division of Clinical Immunology and Allergy, Department of Medicine, University of Mississippi Medical Center, 2500 North State Street, Jackson, MS 39216, USA. Tel.: +1 601 815 3171. E-mail address: [email protected] (L. Xiang). http://dx.doi.org/10.1016/j.molimm.2014.03.004 0161-5890/© 2014 Elsevier Ltd. All rights reserved.
may experience increased odds for infectious episodes following a marathon race (Nieman et al., 1990). It has also been reported that prolonged training results in a significant decrease in both mucosal s-IgA and the secretion rate of s-IgA concomitant with an increase in the incidence of URTI (Fahlman and Engels, 2005). Gleeson et al. (2011) reported that high levels of intensive physical activity are associated with increased risk of URTI possibly due to an elevated anti-inflammatory cytokine response to antigen challenge. Animal studies have also demonstrated that intense exercise to exhaustion leads to increased susceptibility and severity of infections. For example, lymphocytic choriomeningitis virus (LCMV) infected C57BL/6 mice became to suppression of LCMV-specific CD8 and CD4 T-cell responses following exhaustive exercise (Kapasi et al., 2005). The kinetics of antigen-specific cytokine production may be also changed by exhaustive exercise (Kohut et al., 2001). We recently reported various changes to effector and regulatory CD4+ and CD8+ T cell components in recreational marathon runners 24–48 h before a marathon compared with 4 weeks before and one week after a marathon (Rehm et al., 2013). Our overall results were consistent with other studies that have reported that various exercise-induced immune changes typically return to baseline within 72 h post-race (Nieman and Pedersen, 1999;
130
L. Xiang et al. / Molecular Immunology 60 (2014) 129–134
Smith, 2003). Interestingly, both Th2 (CD3+ CD8− IFNg− IL4+ ) and Tc2 (CD3+ CD8− IFNg− IL4+ ) cells were significantly increased prerace compared to baseline but then continued to increase one week post-marathon. This resulted in a continued reduction of the T1/T2 ratios one week post-marathon, suggesting that some components of immunity may still be altered up to one week. In the current study we measured Th1, Th2 and Th3 related genes (cytokines, transcription factors, activation markers, cytokine/chemokine receptors, and accessory molecules) using a new PCR Array technology at the pre- and one week post-marathon time points with PBMC from a randomly selected subjects in our previous study with recreational marathon runners to determine if the continued decrease in the Th1/Th2 ratio one week post-race could be measured at the transcriptional level. 2. Materials and methods 2.1. Human subjects Sixteen healthy recreational marathoners aged 26–57 years (mean = 41 ± 2.6; 5 female, 11 male) were randomly selected from a larger cohort recruited for our marathon study (Rehm et al., 2013) in accordance with an approved University of Mississippi Medical Center Institutional Review Board protocol. All participants were from and trained in the Jackson, MS metropolitan area. Participants ran either the Mississippi Blues Marathon (Jackson, MS) in January 2012 or the Rock n’ Roll New Orleans Marathon (New Orleans, LA) in early March 2012. The average training mileage the week of the pre-race visit was 17.4 ± 9.1 miles/week and was 33 ± 8.8 miles/week the week of the post-race visit (this included the marathon). All participants finished the marathon in an average time of 249 ± 46.5 min. Subjects were excluded for any past medical history of smoking, psychiatric illnesses, cardiovascular diseases, or recent medical use of corticosteroids, catecholamines or adrenergic antagonists. 2.2. Blood collection and PBMC isolation Blood samples were collected 24–48 h before the marathon (pre-marathon) and 7–10 days after the marathon (postmarathon). All blood samples were obtained between 8 am and 10 am to control for diurnal hormone and immune variations. PBMC were isolated from venous blood by Ficoll-Hypaque gradient centrifugation as previously described (Salicru et al., 2007). 2.3. RNA preparation and PCR Array Total RNA was extracted from PBMC using RNAqueous RNA Isolation Kit (Applied Biosystems, Foster City, CA). RNA was treated with DNase to remove contaminated genomic DNA. One microgram of the total RNA was reverse transcribed to cDNA using the High Capacity cDNA Reverse Transcription Kit (Applied Biosystems, Foster City, CA). Gene expression was analyzed using RT2 Profiler PCR Array Human Th1-Th2-Th3 (SABiosciences, Frederick, MD). cDNA was amplified in the presence of 84 specific primers coated in 96well microtiter plates on an Applied Biosystems 7300 Real-Time PCR System. RT2 Real-Time SYBR Green/PCR Master Mix (SABiosciences, Frederick, MD) was used in qRT-PCR.
Table 1 Summary of gene expression from PBMC. Category
Significantly up-regulated genes
T helper cell 1 T helper cell 2
CXCR3, IRF1 CCR2, CCR3, CCR4, CEBPB, IL4, GATA3, GPR44, NFATC2, NFATC2IP, TMED1 LAG3, LAT, MAP2K7 CD28, CD86
CD4 TC marker T-cell activation
(CT ) generated from the identical reverse transcribed mRNA samples. Resulting CT values provided relative gene expression levels for mRNA transcripts between study groups. For calculation of fold changes between pre- and post-marathon, CT values were converted using the 2−CT method (Vandesompele et al., 2002). The data for quantitative gene expression from real-time RT-PCR were analyzed using a paired Student’s t-test. Data are presented as mean ± SEM. 3. Results Using an 84 gene Th1-Th2-Th3 cytokine profile, 17 genes were significantly up-regulated in PBMC one week post-marathon compared to pre-marathon samples (Table 1). Eight genes were down-regulated among various subjects but none consistently enough to achieve statistical significance (Table 2 and supplemental Table 1). It is noted that there was a trend of down-regulation of two Th1 related genes, T-bet and STAT1 (p = 0.089 and 0.093, respectively). Supplementary material related to this article can be found, in the online version, at http://dx.doi.org/10.1016/j.molimm. 2014.03.004. 3.1. Gene expression profile of Th1 and Th2 cytokine related genes after marathon Compared to pre-marathon, two Th1 related genes were upregulated one week post-marathon (Fig. 1): chemokine receptor 3 (CXCR3, p < 0.01) and interferon regulatory factor 1 (IRF1, p < 0.01). However, there was significant up-regulation post-marathon in ten Th2 related genes (Figs. 2 and 3): chemokine (C C motif) receptor 2 (CCR2, p < 0.05), CCR3 (p < 0.01), CCR4 (p < 0.05), CCAAT/enhancer binding protein beta (CEBPB, p < 0.05), prostaglandin D2 receptor 2 (GPR44, p < 0.01), nuclear factors of activated T-cells (NFATC2 and NFATC2IP, p < 0.01 and p < 0.05, respectively), transmembrane protein transport domain 1 (TMED1, p < 0.05), IL-4 (p < 0.01) and GATA binding protein 3 (GATA3, p < 0.01).
Table 2 Down-regulated genes from PBMC. Category
Gene
p-Value
Th1 related gene
T-bet TLR4 STAT1 SOCS5
0.089 0.431 0.093 0.259
Th2 related gene
IL10 IL13R PCGF2
0.327 0.975 0.278
CD4+TC marker
PTPRC
0.763
2.4. Statistical analysis The data from real-time PCR were calculated using the Ct method and analyzed by Analysis Template v3.0 (SABiosciences, Frederick, MD) as previously described (Xiang and Marshall, 2011; Hansel et al., 2008). The CT values of each target gene were normalized by subtraction of the CT value from the housekeeping gene
Bold denotes a borderline significant difference between pre- and post-marathon; T-bet: T-box 21; TLR4: toll-like receptor 4; STAT1: signal transducer and activator of transcription 1; SOCS5: suppressor of cytokine signaling 5; PCGF2: polycomb group ring finger 2; PTPRC: protein tyrosine phosphatase receptor C.
L. Xiang et al. / Molecular Immunology 60 (2014) 129–134
131
regulators for expression of IFN-␥ and IL-4, respectively, was also analyzed (Fig. 3, lower panel). One week after the marathon, Tbet expression was trending toward a decrease but GATA3 mRNA was significantly higher compared to pre-marathon (p < 0.01). This resulted in a significantly decreased (p < 0.001) T-bet/GATA3 mRNA ratio one week post-marathon compared to pre-marathon which is consistent with the observed decrease in the IFN␥/IL-4 mRNA ratio. 4. Discussion
Fig. 1. Expression of Th1 cytokine related genes from PBMC pre- and post-marathon. Values are means ± SEM. The asterisk denotes a significant difference between groups (**p < 0.01).
3.2. Comparison of CD4+ T-cell gene expression profile from preand post-marathon Expression of CD4+ T cell markers and activation genes in one week post-marathon PBMC was also compared to pre-marathon PBMC (Fig. 4). Results revealed that five genes were significantly up-regulated: lymphocyte-activation gene 3 (LAG3, p < 0.01), linker for activation of T cells (LAT, p < 0.01), mitogen-activated protein kinase 7 (MAP2K7, p < 0.05), CD28 (p < 0.05) and CD86 (p < 0.01). 3.3. Ratios of IFN-/IL-4 and T-bet/GATA3 We next determined the ratio of IFN-␥/IL-4 mRNA pre- and one week post-marathon (Fig. 3, upper panel). Results revealed that there was no significant difference in IFN-␥ mRNA levels before and one week after a marathon race, whereas IL-4 mRNA was significantly increased one week after the marathon (p < 0.05). It should be noted that the one week post-marathon IFN-␥/IL-4 mRNA ratio was significantly lower than the pre-marathon ratio (p < 0.01) indicating a shift toward a predominant Th2 response. In addition, gene expression of transcription factors T-bet and GATA3, which are key
Extreme exercise can potentially have adverse impact on host immunity (Shepard and Shek, 1996; Barrett et al., 2012). Previous studies have demonstrated an increased rate of URTI symptoms in marathon participants for various reported time periods following a marathon race (Nieman et al., 1990; Peters and Bateman, 1983). Robson-Ansley et al. (2012) reported that marathon runners were more likely to develop a URTI compared to nonparticipants living in the same household. Animal studies have demonstrated that antiviral-specific T cell responses are significantly inhibited by exhaustive exercise (Kohut et al., 2001; Kapasi et al., 2005). It has been hypothesized that even temporary altered immunity induced by exhaustive exercise can increase susceptibility to infection and that decreased performance in exercise is associated with URTI (Walsh et al., 2011). However, the precise immune mechanisms responsible for the increased susceptibility to URTI have not been well documented. In this study using PCR Array technology, our results demonstrated that a physical stressor, namely the completion of a marathon race, induced a shift in the Th1/Th2 balance to Th2 predominance. A decreased Th1/Th2 ratio has been associated with increased susceptibility to URTI following exhaustive physical exercise such as completing a marathon (Suzuki et al., 2002, 2003). The Th1/Th2 balance is important in maintaining host immune health (Kidd, 2003). Th1 cells promote resistance to intracellular pathogens, and secrete cytokines such as IFN-␥, a major cytokine associated with host resistance against viral infections. Th2 cells promote resistance to extracellular pathogens and, importantly to the current study, antagonize the production and activity of Th1 cytokines including IFN-␥ (Agnello et al., 2003; Romagnani, 2006). Thus, an altered Th1/Th2 balance can influence host susceptibility to a variety of immune-mediated diseases including allergy, autoimmunity and increased infections (Matsuzaki et al., 2005; Marshall, 2011). The Human Th1-Th2-Th3 RT2 Profiler PCR Array examines the expression of 84 genes which are related with Th cell immune responses. Our results showed that 17 of 84 genes were significantly altered in the study population one week
Fig. 2. Comparison of gene expression of Th2 cytokine related genes from PBMC of pre- and post-marathon. Values are means ± SEM. The asterisk denotes a significant difference between groups (*p < 0.05, **p < 0.01).
132
L. Xiang et al. / Molecular Immunology 60 (2014) 129–134
Fig. 3. Gene expression of IFN-␥, IL-4, T-bet, GATA3, IFN-␥/IL-4 ratio and T-bet/GATA3 ratio from PBMC of pre- and post-marathon. Values are means ± SEM. The asterisk denotes a significant difference between groups (**p < 0.01, ***p < 0.001).
post-marathon compared to pre-marathon samples. Among upregulated genes, only 2 (CXCR3 and IRF1) out of 21 genes were Th1 related, whereas 10 out of 29 genes were Th2 related genes including IL4 and GATA3. Another five up-regulated genes reflected CD4+ T cell marker or T cell activation genes (out of 32 genes) which can be involved in either Th1 or Th2 cell activation. In contrast, several genes were down-regulated but did not reach significant differences. Of importance, there was a trend of down-regulation of two Th1 related genes, T-bet and STAT1 (p = 0.089 and 0.093, respectively). These data should be verified by real-time PCR with a larger sample size in future studies. CCAAT/enhancer binding proteins (CEBPs) are transcription factors that involve in dimerization and DNA binding (Ramji and Foka, 2002). CEBP was first identified to regulate gene transcription in response to IL-1 and IL-6 (Akira et al., 1990; Poli et al., 1990). This regulatory protein is important in immune and inflammatory responses. Our study showed that CEBP expression was increased post-marathon compared to pre-marathon supporting the notion that altered CEBP may enhance Th2 cell response (Huber et al.,
Fig. 4. CD4+ T cell marker and T-cell activation gene expression profiles pre- vs. post-marathon were determined using PCR Arrays. Values are means ± SEM. The asterisk denotes a significant difference between groups (*p < 0.05, **p < 0.01).
2012). In addition, CD28 and CD86 were also up-regulated postmarathon. CD28 and CD86 are costimulatory molecules which are essential in the regulation of Th1 and Th2 cytokine production. CD28 interacts with two ligands on antigen presenting cells, CD80 and CD86, and plays a pivotal role in the costimulatory signal pathway for T cell activation and regulation (Sharpe and Freeman, 2002; Salomon and Bluestone, 2001; Alegre and Frauwirth, 2001). It has been reported that CD28 binding with CD80 preferentially activates Th1 cells while binding with CD86 activates Th2 cells (Bashian et al., 1997). The increased expression of CD28 and CD86 mRNA in post-marathon samples provide further evidence to explain the mechanisms for a preferential activation of Th2 cells by this physical stressor. The PCR Array data indicate more Th2 and Th2-related genes were activated after a marathon compared to Th1 genes. Although activation of specific Th genes is important in the immune response to specific antigens, the Th1/Th2 balance is more pivotal in the overall immunomodulatory process (Glaser and Kiecolt-Glaser, 2005; Marshall, 2011). We had measured Th gene expression profile one week after race to avoid immune changes that are known to happen as a result of inflammation from the race itself. One week after a marathon, there was little change in IFN-␥ mRNA level but IL-4 mRNA expression was significantly increased. Thus, the IFN-␥/IL4 mRNA balance was shifted toward a predominant Th2 response. Our previous study examined cytokine changes at the protein level of both IFNg and IL4 in PBMC gated on CD3+ CD8− and CD3− CD8+ cells (Rehm et al., 2013). Th2 (CD3+ CD8− IFNg− IL4+ ) cells were significantly increased pre-race compared to a 4 week prior baseline sample and continued to increase one week postmarathon resulting in a reduced Th1/Th2 ratio which is consistent with our more mechanistic-based gene array study results. Gene expression also revealed that transcription factors T-bet (TBX21) and GATA-3 expression were altered one week post-marathon. Differentiation to either Th1 or Th2 cells is significantly influenced by the relative expression of T-bet and GATA-3, respectively (Zhu et al., 2006; Jenner et al., 2009). T-bet acts as a key regulator of Th1 cell fate determination and directly activates IFN-␥ and suppresses IL-4 (Szabo et al., 2002; Djuretic et al., 2007), whereas GATA3 acts in the opposite manner to activate IL-4 and suppress IFN-␥ (Zheng and Flavell, 1997; Chang and Aune, 2007).
L. Xiang et al. / Molecular Immunology 60 (2014) 129–134
Both factors exert their roles at gene loci that encode Th1 and Th2 cytokines, respectively. There was a trend toward downregulation on T-bet mRNA expression one week post-marathon compared to pre-marathon. The small sample size of this subset may have contributed to failure to detect a statistically significant decrease or it may reflect less effect of the stressor on T-bet expression. However, a significant increase of GATA3 expression was detected after the event. Thus, the T-bet/GATA3 mRNA ratio was significantly decreased post-marathon compared to pre-marathon. These results further contribute to understanding the molecular mechanisms associated with the reported stress-induced Th1/Th2 imbalance (Salicru et al., 2007; Xiang and Marshall, 2011; Xiang et al., 2012). Sixteen participants had been recruited in this study including 5 female and 11 male. It will be important to do future studies to search for effects of gender, race and age. This small sample size (n = 16) was predominantly male (69%), all Caucasians and a relatively young age. A larger sample is needed for a meaningful analysis in future investigation. We are only proposing that Th1/Th2 reduction lingers one week post-marathon and a potential mechanism to this is reduced mRNA expression as opposed to post transcriptional modifications. The current study provides intriguing data to suggest that strenuous exercise can cause a prolonged Th1/Th2 imbalance and that transcription factor gene expression associated with Th1 and Th2 cytokine production is affected. This may contribute to an increased susceptibility to post-race infections. Our study suggests that a mechanism for Th1/Th2 imbalance in this physical stress model (Rehm et al., 2013) is an altered cascade including IFN-␥/IL-4 mRNA ratio, T-bet/GATA3 mRNA ratio and the other Th2 related genes which were detected by PCR Array. Psychological stress can increase the production of stress hormones which are potential regulators in immune balance (Salicru et al., 2007). Previous studies by our group have shown the effects of stress level doses of dexamethasone (cortisol analog) on in vitro cultures of PBMC from normal individuals and demonstrated a shift in the secreted supernatant cytokine balance toward a Th2 predominant profile (Agarwal and Marshall, 1998, 2001). Using a focused PCR Array that allows screening of relevant genes, we have added additional data to further characterize the physiological basis of this physical stress-induced immune imbalance. Given that the ultimate clinical risks likely involve more pathways than just Th1/Th2 imbalances (such as altered regulatory T cell and other T cell subpopulations) in different individuals, the PCR Array technology may become a useful screening tool for categorizing and characterizing differing immune dysfunction profiles in specific patient populations as well as providing possible monitoring of effects that could help design and predict efficacy of therapeutic interventions. This would allow tailoring of these interventions for prophylaxis and/or early therapeutic interventions. Further definition of these mechanisms will require additional investigations in larger, more diverse populations. References Agarwal, S.K., Marshall, G.D., 1998. Glucocorticoid-induced type 1/type 2 cytokine alterations in humans: a model for stress-related immune dysfunction. J. Interferon Cytokine Res. 18, 1059–1068. Agarwal, S.K., Marshall, G.D., 2001. Dexamethasone promotes type 2 cytokine production primarily through inhibition of type 1 cytokines. J. Interferon Cytokine Res. 21, 147–155. Agnello, D., Lankford, C.S., Bream, J., Morinobu, A., Gadina, M., O’Shea, J.J., Frucht, D.M., 2003. Cytokines and transcription factors that regulate T helper cell differentiation: new players and new insights. J. Clin. Immunol. 23, 147–161. Akira, S., Isshiki, H., Sugita, H., Tanabe, O., Kinoshita, S., Nishio, Y., Nakajima, T., Hirano, T., Kishimoto, T., 1990. A nuclear factor for IL-6 expression (NF-IL6) is a member of a C/EBP family. EMBO J. 9, 1897–1906. Alegre, M.L., Frauwirth, K.A., 2001. Thompson CB: T-cell regulation by CD28 and CTLA-4. Nat. Rev. Immunol. 1, 220–228.
133
Barrett, B., Hayney, M.S., Muller, D., Rakel, D., Ward, A., Obasi, C.N., Brown, R., Zhang, Z., Zgierska, A., Gern, J., West, R., Ewers, T., Barlow, S., Gassman, M., Coe, C.L., 2012. Meditation or exercise for preventing acute respiratory infection: a randomized controlled trial. Ann. Fam. Med. 10, 337–346. Bashian, G.G., Braun, C.M., Huang, S.K., Kagey-Sobotka, A., Lichtenstein, L.M., Essayan, D.M., 1997. Differential regulation of human, antigen-specific Th1 and Th2 responses by the B-7 homologues, CD80 and CD86. Am. J. Respir. Cell Mol. Biol. 17, 235–242. Chang, S., Aune, T.M., 2007. Dynamic changes in histone-methylation ‘marks’ across the locus encoding interferon-gamma during the differentiation of T helper type 2 cells. Nat. Immunol. 8, 723–731. Djuretic, I.M., Levanon, D., Negreanu, V., Groner, Y., Rao, A., Ansel, K.M., 2007. Transcription factors T-bet and Runx3 cooperate to activate Ifng and silence II4 in T helper type 1 cells. Nat. Immunol. 8, 145–153. Fahlman, M.M., Engels, H.J., 2005. Mucosal IgA and URTI in American college football players: a year longitudinal study. Med. Sci. Sports Exerc. 37, 374–380. Glaser, R., Kiecolt-Glaser, J.K., 2005. Stress-induced immune dysfunction: implications for health. Nat. Rev. Immunol. 5, 243–251. Gleeson, M., Bishop, N., Oliveira, M., Tauler, P., 2013. Influence of training load on upper respiratory tract infection incidence and antigen-stimulated cytokine production.Scand. J. Med. Sci. Sports 23, 451–457. Hansel, N.N., Cheadle, C., Diette, G.B., Wright, J., Thompson, K.M., Barnes, K.C., Georas, S.N., 2008. Analysis of CD4+ T-cell gene expression in allergic subjects using two different microarray platforms. Allergy 63, 366–369. Huber, R., Pietsch, D., Panterodt, T., Brand, K., 2012. Regulation of C/EBP and resulting functions in cells of the monocytic lineage. Cell Signal. 24, 1287–1296. Jenner, R.G., Townsend, M.J., Jackson, I., Sun, K., Bouwman, R.D., Young, R.A., Glimcher, L.H., Lord, G.M., 2009. The transcription factors T-bet and GATA-3 control alternative pathways of T-cell differentiation through a shared set of target genes. Proc. Natl. Acad. Sci. U. S. A. 106, 17876–17881. Kapasi, Z.F., McRae, M.L., Ahmed, R., 2005. Suppression of viral specific primary Tcell response following intense physical exercise in young but not old mice. J. Appl. Physiol. 98, 663–671. Kidd, P., 2003. Th1/Th2 balance: the hypothesis, its limitations, and implications for health and disease. Altern. Med. Rev. 8, 223–246. Kohut, M.L., Boehm, G.W., Moynihan, J.A., 2001. Prolonged exercise suppresses antigen-specific cytokine response to upper respiratory infection. J. Appl. Physiol. 90, 678–684. Marshall, G.D., 2011. The adverse effects of psychological stress on immunoregulatory balance: applications to human inflammatory diseases. Immunol. Allergy Clin. North Am. 31, 133–140. Matsuzaki, J., Tsuji, T., Imazeki, I., Ikeda, H., Nishimura, T., 2005. Immunosteroid as a regulator for Th1/Th2 balance: its possible role in autoimmune diseases. Autoimmunity 38, 369–375. Nieman, D.C., Johanssen, L.M., Lee, J.W., 1989. Infectious episodes in runners before and after a roadrace. J. Sports Med. Phys. Fit. 29, 289–296. Nieman, D.C., Johanssen, L.M., Lee, J.W., Arabatzis, K., 1990. Infectious episodes in runners before and after the Los Angeles Marathon. J. Sports Med. Phys. Fit. 30, 316–328. Nieman, D.C., Pedersen, B.K., 1999. Exercise and immune function. Recent developments. Sports Med. 27, 73–80. Peters, E.M., Bateman, E.D., 1983. Ultramarathon running and upper respiratory tract infections. An epidemiological survey. S. Afr. Med. J. 64, 582–584. Poli, V., Mancini, F.P., Cortese, R., 1990. IL-6DBP, a nuclear protein involved in interleukin-6 signal transduction, defines a new family of leucine zipper proteins related to C/EBP. Cell 63, 643–653. Ramji, D.P., Foka, P., 2002. CCAAT/enhancer-binding proteins: structure, function and regulation. Biochem. J. 365, 561–575. Rehm, K.E., Elci, O.U., Hahn, K., Marshall, G.D., 2013. The impact of self-reported psychological stress levels on changes to peripheral blood immune biomarkers in recreational marathon runners during training and recovery. Neuroimmunomodulation 20, 164–176. Robson-Ansley, P., Howatson, G., Tallent, J., Mitcheson, K., Walshe, I., Toms, C., Du Toit, G., Smith, M., Ansley, L., 2012. Prevalence of allergy and upper respiratory tract symptoms in runners of the London marathon. Med. Sci. Sports Exerc. 44, 999–1004. Romagnani, S., 2006. Regulation of the T cell response. Clin. Exp. Allergy 36, 1357–1366. Salicru, A.N., Sams, C.F., Marshall, G.D., 2007. Cooperative effects of corticosteroids and catecholamines upon immune deviation of the type-1/type-2 cytokine balance in favor of type-2 expression in human peripheral blood mononuclear cells. Brain Behav. Immun. 21, 913–920. Salomon, B., Bluestone, J.A., 2001. Complexities of CD28/B7: CTLA-4 costimulatory pathways in autoimmunity and transplantation. Annu. Rev. Immunol. 19, 225–252. Sharpe, A., Freeman, G.J., 2002. The B7-CD28 superfamily. Nat. Rev. Immunol. 2, 116–126. Shepard, R.J., Shek, P.N., 1996. Impact of physical activity and sport on the immune system. Rev. Environ. Health 11, 133–147. Smith, L.L., 2003. Overtraining, excessive exercise, and altered immunity: is this a T helper-1 versus T helper-2 lymphocyte response? Sports Med. 33, 347–364. Suzuki, K., Nakaji, S., Kurakake, S., Totsuka, M., Sato, K., Kuriyama, T., Fujimoto, H., Shibusawa, K., Machida, K., Sugawara, K., 2003. Exhaustive exercise and type1/type-2 cytokine balance with special focus on interleukin-12 p40/p70. Exerc. Immunol. Rev. 9, 48–57.
134
L. Xiang et al. / Molecular Immunology 60 (2014) 129–134
Suzuki, K., Nakaji, S., Yamada, M., Totsuka, M., Sato, K., Sugawara, K., 2002. Systemic inflammatory response to exhaustive exercise. Cytokine kinetics. Exerc. Immunol. Rev. 8, 6–48. Szabo, S.J., Sullivan, B.M., Stemmann, C., Satoskar, A.R., Sleckman, B.P., Glimcher, L.H., 2002. Distinct effects of T-bet in TH1 lineage commitment and IFN-gamma production in CD4 and CD8 T cells. Science 295, 338–342. Vandesompele, J., De Preter, K., Pattyn, F., Poppe, B., Van Roy, N., De Paepe, A., Speleman, F., 2002. Accurate normalization of real-time quantitative RT-PCR data by geometric averaging of multiple internal control genes. Genome Biol. 3, 1–12. Walsh, N.P., Gleeson, M., Shephard, R.J., Gleeson, M., Woods, J.A., Bishop, N.C., Fleshner, M., Green, C., Pedersen, B.K., Hoffman-Goetz, L., Rogers, C.J., Northoff, H., Abbasi, A., Simon, P., 2011. Position statement. Part one: immune function and exercise. Exerc. Immunol. Rev. 17, 6–63.
Xiang, L., Marshall, G.D., 2011. Immunomodulatory effects of in vitro stress hormones on FoxP3, Th1/Th2 cytokine and costimulatory molecule mRNA expression in human PBMC. Neuroimmunomodulation 18, 1–10. Xiang, L., Del Ben, K., Rehm, K.E., Marshall, G.D., 2012. Effects of acute stress-induced immunomodulation on Th1/Th2 cytokine and catecholamine receptors expression in human peripheral blood cells. Psychoneurobiology 65, 12–19. Zheng, W., Flavell, R.A., 1997. The transcription factor GATA-3 is necessary and sufficient for Th2 cytokine gene expression in CD4 T cells. Cell 89, 587–596. Zhu, J., Yamane, H., Cote-Sierra, J., Guo, L., Paul, W.E., 2006. GATA-3 promotes Th2 responses through three different mechanisms: induction of Th2 cytokine production, selective growth of Th2 cells and inhibition of Th1 cell-specific factors. Cell Res. 16, 3–10.
Contents lists available at ScienceDirect
Molecular Immunology journal homepage: www.elsevier.com/locate/molimm
Effects of strenuous exercise on Th1/Th2 gene expression from human peripheral blood mononuclear cells of marathon participants Lianbin Xiang ∗ , Kristina E. Rehm, Gailen D. Marshall Jr. Laboratory of Behavioral Immunology Research, Division of Clinical Immunology and Allergy, Department of Medicine, University of Mississippi Medical Center, Jackson, MS, USA
a r t i c l e
i n f o
Article history: Received 28 August 2013 Received in revised form 5 March 2014 Accepted 18 March 2014 Available online 20 May 2014 Keywords: Strenuous exercise Cytokine Th1/Th2 balance Transcription factors PCR Array Gene expression
a b s t r a c t Physical stressors, such as strenuous exercise, can have numerous effects on the human body including the immune system. The aim of this study was to evaluate the gene expression profile of Th1/Th2 cytokines and related transcription factor genes in order to investigate possible immune imbalances before and after a marathon. Blood samples were collected from 16 normal volunteers 24–48 h before and one week after completing a marathon race. Gene expression of Th1 and Th2 related cytokines from human peripheral blood mononuclear cells (PBMC) was analyzed using Human Th1-Th2-Th3 RT2 Profiler PCR Array and qRT-PCR that measured the transcript levels of 84 genes related to T cell activation. We found that PBMC express a characteristic Th2-like gene profile one week post-marathon compared to pre-marathon. The majority of genes up-regulated one week post-marathon such as IL-4, GATA3, and CCR4 were Th2 associated. For Th1-related genes, CXCR3 and IRF1 were up-regulated one week post-marathon. There was a trend of down-regulation of two Th1 related genes, T-bet and STAT1. Th3-related gene expression patterns did not change in the study. The ratios of both IFN-␥/IL-4 and T-bet/GATA3 gene expressions were significantly lower one week after marathon. These findings suggest that a Th1/Th2 immune imbalance persisted at least 1 week after completion of a marathon which offers a mechanistic rationale for the increased risk of upper respiratory tract infections often reported after strenuous exercise. © 2014 Elsevier Ltd. All rights reserved.
1. Introduction Various studies have implied that regular and moderate exercise has a beneficial effect on immune function, which could protect against upper respiratory tract infections (URTI) (Barrett et al., 2012; Walsh et al., 2011). In contrast, extended and exhaustive exercise, such as a marathon race, may have negative effects on the immune system and increase the risk of URTI (Nieman et al., 1989; Shepard and Shek, 1996). After a strenuous bout of exercise, athletes are hypothetically more susceptible to URTI, although a causal relation has never been demonstrated. In a large study (2311 participants) of the Los Angeles Marathon, data suggested that runners
Abbreviations: PBMC, peripheral blood mononuclear cells; URTI, upper respiratory tract infections; Th cells, T helper cells; IFN-␥, interferon gamma; IL-4, interleukin 4; GATA3, GATA binding protein 3; T-bet, (TBX21): T-box 21; RT, reverse transcription. ∗ Corresponding author at: Laboratory of Behavioral Immunology Research, Division of Clinical Immunology and Allergy, Department of Medicine, University of Mississippi Medical Center, 2500 North State Street, Jackson, MS 39216, USA. Tel.: +1 601 815 3171. E-mail address: [email protected] (L. Xiang). http://dx.doi.org/10.1016/j.molimm.2014.03.004 0161-5890/© 2014 Elsevier Ltd. All rights reserved.
may experience increased odds for infectious episodes following a marathon race (Nieman et al., 1990). It has also been reported that prolonged training results in a significant decrease in both mucosal s-IgA and the secretion rate of s-IgA concomitant with an increase in the incidence of URTI (Fahlman and Engels, 2005). Gleeson et al. (2011) reported that high levels of intensive physical activity are associated with increased risk of URTI possibly due to an elevated anti-inflammatory cytokine response to antigen challenge. Animal studies have also demonstrated that intense exercise to exhaustion leads to increased susceptibility and severity of infections. For example, lymphocytic choriomeningitis virus (LCMV) infected C57BL/6 mice became to suppression of LCMV-specific CD8 and CD4 T-cell responses following exhaustive exercise (Kapasi et al., 2005). The kinetics of antigen-specific cytokine production may be also changed by exhaustive exercise (Kohut et al., 2001). We recently reported various changes to effector and regulatory CD4+ and CD8+ T cell components in recreational marathon runners 24–48 h before a marathon compared with 4 weeks before and one week after a marathon (Rehm et al., 2013). Our overall results were consistent with other studies that have reported that various exercise-induced immune changes typically return to baseline within 72 h post-race (Nieman and Pedersen, 1999;
130
L. Xiang et al. / Molecular Immunology 60 (2014) 129–134
Smith, 2003). Interestingly, both Th2 (CD3+ CD8− IFNg− IL4+ ) and Tc2 (CD3+ CD8− IFNg− IL4+ ) cells were significantly increased prerace compared to baseline but then continued to increase one week post-marathon. This resulted in a continued reduction of the T1/T2 ratios one week post-marathon, suggesting that some components of immunity may still be altered up to one week. In the current study we measured Th1, Th2 and Th3 related genes (cytokines, transcription factors, activation markers, cytokine/chemokine receptors, and accessory molecules) using a new PCR Array technology at the pre- and one week post-marathon time points with PBMC from a randomly selected subjects in our previous study with recreational marathon runners to determine if the continued decrease in the Th1/Th2 ratio one week post-race could be measured at the transcriptional level. 2. Materials and methods 2.1. Human subjects Sixteen healthy recreational marathoners aged 26–57 years (mean = 41 ± 2.6; 5 female, 11 male) were randomly selected from a larger cohort recruited for our marathon study (Rehm et al., 2013) in accordance with an approved University of Mississippi Medical Center Institutional Review Board protocol. All participants were from and trained in the Jackson, MS metropolitan area. Participants ran either the Mississippi Blues Marathon (Jackson, MS) in January 2012 or the Rock n’ Roll New Orleans Marathon (New Orleans, LA) in early March 2012. The average training mileage the week of the pre-race visit was 17.4 ± 9.1 miles/week and was 33 ± 8.8 miles/week the week of the post-race visit (this included the marathon). All participants finished the marathon in an average time of 249 ± 46.5 min. Subjects were excluded for any past medical history of smoking, psychiatric illnesses, cardiovascular diseases, or recent medical use of corticosteroids, catecholamines or adrenergic antagonists. 2.2. Blood collection and PBMC isolation Blood samples were collected 24–48 h before the marathon (pre-marathon) and 7–10 days after the marathon (postmarathon). All blood samples were obtained between 8 am and 10 am to control for diurnal hormone and immune variations. PBMC were isolated from venous blood by Ficoll-Hypaque gradient centrifugation as previously described (Salicru et al., 2007). 2.3. RNA preparation and PCR Array Total RNA was extracted from PBMC using RNAqueous RNA Isolation Kit (Applied Biosystems, Foster City, CA). RNA was treated with DNase to remove contaminated genomic DNA. One microgram of the total RNA was reverse transcribed to cDNA using the High Capacity cDNA Reverse Transcription Kit (Applied Biosystems, Foster City, CA). Gene expression was analyzed using RT2 Profiler PCR Array Human Th1-Th2-Th3 (SABiosciences, Frederick, MD). cDNA was amplified in the presence of 84 specific primers coated in 96well microtiter plates on an Applied Biosystems 7300 Real-Time PCR System. RT2 Real-Time SYBR Green/PCR Master Mix (SABiosciences, Frederick, MD) was used in qRT-PCR.
Table 1 Summary of gene expression from PBMC. Category
Significantly up-regulated genes
T helper cell 1 T helper cell 2
CXCR3, IRF1 CCR2, CCR3, CCR4, CEBPB, IL4, GATA3, GPR44, NFATC2, NFATC2IP, TMED1 LAG3, LAT, MAP2K7 CD28, CD86
CD4 TC marker T-cell activation
(CT ) generated from the identical reverse transcribed mRNA samples. Resulting CT values provided relative gene expression levels for mRNA transcripts between study groups. For calculation of fold changes between pre- and post-marathon, CT values were converted using the 2−CT method (Vandesompele et al., 2002). The data for quantitative gene expression from real-time RT-PCR were analyzed using a paired Student’s t-test. Data are presented as mean ± SEM. 3. Results Using an 84 gene Th1-Th2-Th3 cytokine profile, 17 genes were significantly up-regulated in PBMC one week post-marathon compared to pre-marathon samples (Table 1). Eight genes were down-regulated among various subjects but none consistently enough to achieve statistical significance (Table 2 and supplemental Table 1). It is noted that there was a trend of down-regulation of two Th1 related genes, T-bet and STAT1 (p = 0.089 and 0.093, respectively). Supplementary material related to this article can be found, in the online version, at http://dx.doi.org/10.1016/j.molimm. 2014.03.004. 3.1. Gene expression profile of Th1 and Th2 cytokine related genes after marathon Compared to pre-marathon, two Th1 related genes were upregulated one week post-marathon (Fig. 1): chemokine receptor 3 (CXCR3, p < 0.01) and interferon regulatory factor 1 (IRF1, p < 0.01). However, there was significant up-regulation post-marathon in ten Th2 related genes (Figs. 2 and 3): chemokine (C C motif) receptor 2 (CCR2, p < 0.05), CCR3 (p < 0.01), CCR4 (p < 0.05), CCAAT/enhancer binding protein beta (CEBPB, p < 0.05), prostaglandin D2 receptor 2 (GPR44, p < 0.01), nuclear factors of activated T-cells (NFATC2 and NFATC2IP, p < 0.01 and p < 0.05, respectively), transmembrane protein transport domain 1 (TMED1, p < 0.05), IL-4 (p < 0.01) and GATA binding protein 3 (GATA3, p < 0.01).
Table 2 Down-regulated genes from PBMC. Category
Gene
p-Value
Th1 related gene
T-bet TLR4 STAT1 SOCS5
0.089 0.431 0.093 0.259
Th2 related gene
IL10 IL13R PCGF2
0.327 0.975 0.278
CD4+TC marker
PTPRC
0.763
2.4. Statistical analysis The data from real-time PCR were calculated using the Ct method and analyzed by Analysis Template v3.0 (SABiosciences, Frederick, MD) as previously described (Xiang and Marshall, 2011; Hansel et al., 2008). The CT values of each target gene were normalized by subtraction of the CT value from the housekeeping gene
Bold denotes a borderline significant difference between pre- and post-marathon; T-bet: T-box 21; TLR4: toll-like receptor 4; STAT1: signal transducer and activator of transcription 1; SOCS5: suppressor of cytokine signaling 5; PCGF2: polycomb group ring finger 2; PTPRC: protein tyrosine phosphatase receptor C.
L. Xiang et al. / Molecular Immunology 60 (2014) 129–134
131
regulators for expression of IFN-␥ and IL-4, respectively, was also analyzed (Fig. 3, lower panel). One week after the marathon, Tbet expression was trending toward a decrease but GATA3 mRNA was significantly higher compared to pre-marathon (p < 0.01). This resulted in a significantly decreased (p < 0.001) T-bet/GATA3 mRNA ratio one week post-marathon compared to pre-marathon which is consistent with the observed decrease in the IFN␥/IL-4 mRNA ratio. 4. Discussion
Fig. 1. Expression of Th1 cytokine related genes from PBMC pre- and post-marathon. Values are means ± SEM. The asterisk denotes a significant difference between groups (**p < 0.01).
3.2. Comparison of CD4+ T-cell gene expression profile from preand post-marathon Expression of CD4+ T cell markers and activation genes in one week post-marathon PBMC was also compared to pre-marathon PBMC (Fig. 4). Results revealed that five genes were significantly up-regulated: lymphocyte-activation gene 3 (LAG3, p < 0.01), linker for activation of T cells (LAT, p < 0.01), mitogen-activated protein kinase 7 (MAP2K7, p < 0.05), CD28 (p < 0.05) and CD86 (p < 0.01). 3.3. Ratios of IFN-/IL-4 and T-bet/GATA3 We next determined the ratio of IFN-␥/IL-4 mRNA pre- and one week post-marathon (Fig. 3, upper panel). Results revealed that there was no significant difference in IFN-␥ mRNA levels before and one week after a marathon race, whereas IL-4 mRNA was significantly increased one week after the marathon (p < 0.05). It should be noted that the one week post-marathon IFN-␥/IL-4 mRNA ratio was significantly lower than the pre-marathon ratio (p < 0.01) indicating a shift toward a predominant Th2 response. In addition, gene expression of transcription factors T-bet and GATA3, which are key
Extreme exercise can potentially have adverse impact on host immunity (Shepard and Shek, 1996; Barrett et al., 2012). Previous studies have demonstrated an increased rate of URTI symptoms in marathon participants for various reported time periods following a marathon race (Nieman et al., 1990; Peters and Bateman, 1983). Robson-Ansley et al. (2012) reported that marathon runners were more likely to develop a URTI compared to nonparticipants living in the same household. Animal studies have demonstrated that antiviral-specific T cell responses are significantly inhibited by exhaustive exercise (Kohut et al., 2001; Kapasi et al., 2005). It has been hypothesized that even temporary altered immunity induced by exhaustive exercise can increase susceptibility to infection and that decreased performance in exercise is associated with URTI (Walsh et al., 2011). However, the precise immune mechanisms responsible for the increased susceptibility to URTI have not been well documented. In this study using PCR Array technology, our results demonstrated that a physical stressor, namely the completion of a marathon race, induced a shift in the Th1/Th2 balance to Th2 predominance. A decreased Th1/Th2 ratio has been associated with increased susceptibility to URTI following exhaustive physical exercise such as completing a marathon (Suzuki et al., 2002, 2003). The Th1/Th2 balance is important in maintaining host immune health (Kidd, 2003). Th1 cells promote resistance to intracellular pathogens, and secrete cytokines such as IFN-␥, a major cytokine associated with host resistance against viral infections. Th2 cells promote resistance to extracellular pathogens and, importantly to the current study, antagonize the production and activity of Th1 cytokines including IFN-␥ (Agnello et al., 2003; Romagnani, 2006). Thus, an altered Th1/Th2 balance can influence host susceptibility to a variety of immune-mediated diseases including allergy, autoimmunity and increased infections (Matsuzaki et al., 2005; Marshall, 2011). The Human Th1-Th2-Th3 RT2 Profiler PCR Array examines the expression of 84 genes which are related with Th cell immune responses. Our results showed that 17 of 84 genes were significantly altered in the study population one week
Fig. 2. Comparison of gene expression of Th2 cytokine related genes from PBMC of pre- and post-marathon. Values are means ± SEM. The asterisk denotes a significant difference between groups (*p < 0.05, **p < 0.01).
132
L. Xiang et al. / Molecular Immunology 60 (2014) 129–134
Fig. 3. Gene expression of IFN-␥, IL-4, T-bet, GATA3, IFN-␥/IL-4 ratio and T-bet/GATA3 ratio from PBMC of pre- and post-marathon. Values are means ± SEM. The asterisk denotes a significant difference between groups (**p < 0.01, ***p < 0.001).
post-marathon compared to pre-marathon samples. Among upregulated genes, only 2 (CXCR3 and IRF1) out of 21 genes were Th1 related, whereas 10 out of 29 genes were Th2 related genes including IL4 and GATA3. Another five up-regulated genes reflected CD4+ T cell marker or T cell activation genes (out of 32 genes) which can be involved in either Th1 or Th2 cell activation. In contrast, several genes were down-regulated but did not reach significant differences. Of importance, there was a trend of down-regulation of two Th1 related genes, T-bet and STAT1 (p = 0.089 and 0.093, respectively). These data should be verified by real-time PCR with a larger sample size in future studies. CCAAT/enhancer binding proteins (CEBPs) are transcription factors that involve in dimerization and DNA binding (Ramji and Foka, 2002). CEBP was first identified to regulate gene transcription in response to IL-1 and IL-6 (Akira et al., 1990; Poli et al., 1990). This regulatory protein is important in immune and inflammatory responses. Our study showed that CEBP expression was increased post-marathon compared to pre-marathon supporting the notion that altered CEBP may enhance Th2 cell response (Huber et al.,
Fig. 4. CD4+ T cell marker and T-cell activation gene expression profiles pre- vs. post-marathon were determined using PCR Arrays. Values are means ± SEM. The asterisk denotes a significant difference between groups (*p < 0.05, **p < 0.01).
2012). In addition, CD28 and CD86 were also up-regulated postmarathon. CD28 and CD86 are costimulatory molecules which are essential in the regulation of Th1 and Th2 cytokine production. CD28 interacts with two ligands on antigen presenting cells, CD80 and CD86, and plays a pivotal role in the costimulatory signal pathway for T cell activation and regulation (Sharpe and Freeman, 2002; Salomon and Bluestone, 2001; Alegre and Frauwirth, 2001). It has been reported that CD28 binding with CD80 preferentially activates Th1 cells while binding with CD86 activates Th2 cells (Bashian et al., 1997). The increased expression of CD28 and CD86 mRNA in post-marathon samples provide further evidence to explain the mechanisms for a preferential activation of Th2 cells by this physical stressor. The PCR Array data indicate more Th2 and Th2-related genes were activated after a marathon compared to Th1 genes. Although activation of specific Th genes is important in the immune response to specific antigens, the Th1/Th2 balance is more pivotal in the overall immunomodulatory process (Glaser and Kiecolt-Glaser, 2005; Marshall, 2011). We had measured Th gene expression profile one week after race to avoid immune changes that are known to happen as a result of inflammation from the race itself. One week after a marathon, there was little change in IFN-␥ mRNA level but IL-4 mRNA expression was significantly increased. Thus, the IFN-␥/IL4 mRNA balance was shifted toward a predominant Th2 response. Our previous study examined cytokine changes at the protein level of both IFNg and IL4 in PBMC gated on CD3+ CD8− and CD3− CD8+ cells (Rehm et al., 2013). Th2 (CD3+ CD8− IFNg− IL4+ ) cells were significantly increased pre-race compared to a 4 week prior baseline sample and continued to increase one week postmarathon resulting in a reduced Th1/Th2 ratio which is consistent with our more mechanistic-based gene array study results. Gene expression also revealed that transcription factors T-bet (TBX21) and GATA-3 expression were altered one week post-marathon. Differentiation to either Th1 or Th2 cells is significantly influenced by the relative expression of T-bet and GATA-3, respectively (Zhu et al., 2006; Jenner et al., 2009). T-bet acts as a key regulator of Th1 cell fate determination and directly activates IFN-␥ and suppresses IL-4 (Szabo et al., 2002; Djuretic et al., 2007), whereas GATA3 acts in the opposite manner to activate IL-4 and suppress IFN-␥ (Zheng and Flavell, 1997; Chang and Aune, 2007).
L. Xiang et al. / Molecular Immunology 60 (2014) 129–134
Both factors exert their roles at gene loci that encode Th1 and Th2 cytokines, respectively. There was a trend toward downregulation on T-bet mRNA expression one week post-marathon compared to pre-marathon. The small sample size of this subset may have contributed to failure to detect a statistically significant decrease or it may reflect less effect of the stressor on T-bet expression. However, a significant increase of GATA3 expression was detected after the event. Thus, the T-bet/GATA3 mRNA ratio was significantly decreased post-marathon compared to pre-marathon. These results further contribute to understanding the molecular mechanisms associated with the reported stress-induced Th1/Th2 imbalance (Salicru et al., 2007; Xiang and Marshall, 2011; Xiang et al., 2012). Sixteen participants had been recruited in this study including 5 female and 11 male. It will be important to do future studies to search for effects of gender, race and age. This small sample size (n = 16) was predominantly male (69%), all Caucasians and a relatively young age. A larger sample is needed for a meaningful analysis in future investigation. We are only proposing that Th1/Th2 reduction lingers one week post-marathon and a potential mechanism to this is reduced mRNA expression as opposed to post transcriptional modifications. The current study provides intriguing data to suggest that strenuous exercise can cause a prolonged Th1/Th2 imbalance and that transcription factor gene expression associated with Th1 and Th2 cytokine production is affected. This may contribute to an increased susceptibility to post-race infections. Our study suggests that a mechanism for Th1/Th2 imbalance in this physical stress model (Rehm et al., 2013) is an altered cascade including IFN-␥/IL-4 mRNA ratio, T-bet/GATA3 mRNA ratio and the other Th2 related genes which were detected by PCR Array. Psychological stress can increase the production of stress hormones which are potential regulators in immune balance (Salicru et al., 2007). Previous studies by our group have shown the effects of stress level doses of dexamethasone (cortisol analog) on in vitro cultures of PBMC from normal individuals and demonstrated a shift in the secreted supernatant cytokine balance toward a Th2 predominant profile (Agarwal and Marshall, 1998, 2001). Using a focused PCR Array that allows screening of relevant genes, we have added additional data to further characterize the physiological basis of this physical stress-induced immune imbalance. Given that the ultimate clinical risks likely involve more pathways than just Th1/Th2 imbalances (such as altered regulatory T cell and other T cell subpopulations) in different individuals, the PCR Array technology may become a useful screening tool for categorizing and characterizing differing immune dysfunction profiles in specific patient populations as well as providing possible monitoring of effects that could help design and predict efficacy of therapeutic interventions. This would allow tailoring of these interventions for prophylaxis and/or early therapeutic interventions. Further definition of these mechanisms will require additional investigations in larger, more diverse populations. References Agarwal, S.K., Marshall, G.D., 1998. Glucocorticoid-induced type 1/type 2 cytokine alterations in humans: a model for stress-related immune dysfunction. J. Interferon Cytokine Res. 18, 1059–1068. Agarwal, S.K., Marshall, G.D., 2001. Dexamethasone promotes type 2 cytokine production primarily through inhibition of type 1 cytokines. J. Interferon Cytokine Res. 21, 147–155. Agnello, D., Lankford, C.S., Bream, J., Morinobu, A., Gadina, M., O’Shea, J.J., Frucht, D.M., 2003. Cytokines and transcription factors that regulate T helper cell differentiation: new players and new insights. J. Clin. Immunol. 23, 147–161. Akira, S., Isshiki, H., Sugita, H., Tanabe, O., Kinoshita, S., Nishio, Y., Nakajima, T., Hirano, T., Kishimoto, T., 1990. A nuclear factor for IL-6 expression (NF-IL6) is a member of a C/EBP family. EMBO J. 9, 1897–1906. Alegre, M.L., Frauwirth, K.A., 2001. Thompson CB: T-cell regulation by CD28 and CTLA-4. Nat. Rev. Immunol. 1, 220–228.
133
Barrett, B., Hayney, M.S., Muller, D., Rakel, D., Ward, A., Obasi, C.N., Brown, R., Zhang, Z., Zgierska, A., Gern, J., West, R., Ewers, T., Barlow, S., Gassman, M., Coe, C.L., 2012. Meditation or exercise for preventing acute respiratory infection: a randomized controlled trial. Ann. Fam. Med. 10, 337–346. Bashian, G.G., Braun, C.M., Huang, S.K., Kagey-Sobotka, A., Lichtenstein, L.M., Essayan, D.M., 1997. Differential regulation of human, antigen-specific Th1 and Th2 responses by the B-7 homologues, CD80 and CD86. Am. J. Respir. Cell Mol. Biol. 17, 235–242. Chang, S., Aune, T.M., 2007. Dynamic changes in histone-methylation ‘marks’ across the locus encoding interferon-gamma during the differentiation of T helper type 2 cells. Nat. Immunol. 8, 723–731. Djuretic, I.M., Levanon, D., Negreanu, V., Groner, Y., Rao, A., Ansel, K.M., 2007. Transcription factors T-bet and Runx3 cooperate to activate Ifng and silence II4 in T helper type 1 cells. Nat. Immunol. 8, 145–153. Fahlman, M.M., Engels, H.J., 2005. Mucosal IgA and URTI in American college football players: a year longitudinal study. Med. Sci. Sports Exerc. 37, 374–380. Glaser, R., Kiecolt-Glaser, J.K., 2005. Stress-induced immune dysfunction: implications for health. Nat. Rev. Immunol. 5, 243–251. Gleeson, M., Bishop, N., Oliveira, M., Tauler, P., 2013. Influence of training load on upper respiratory tract infection incidence and antigen-stimulated cytokine production.Scand. J. Med. Sci. Sports 23, 451–457. Hansel, N.N., Cheadle, C., Diette, G.B., Wright, J., Thompson, K.M., Barnes, K.C., Georas, S.N., 2008. Analysis of CD4+ T-cell gene expression in allergic subjects using two different microarray platforms. Allergy 63, 366–369. Huber, R., Pietsch, D., Panterodt, T., Brand, K., 2012. Regulation of C/EBP and resulting functions in cells of the monocytic lineage. Cell Signal. 24, 1287–1296. Jenner, R.G., Townsend, M.J., Jackson, I., Sun, K., Bouwman, R.D., Young, R.A., Glimcher, L.H., Lord, G.M., 2009. The transcription factors T-bet and GATA-3 control alternative pathways of T-cell differentiation through a shared set of target genes. Proc. Natl. Acad. Sci. U. S. A. 106, 17876–17881. Kapasi, Z.F., McRae, M.L., Ahmed, R., 2005. Suppression of viral specific primary Tcell response following intense physical exercise in young but not old mice. J. Appl. Physiol. 98, 663–671. Kidd, P., 2003. Th1/Th2 balance: the hypothesis, its limitations, and implications for health and disease. Altern. Med. Rev. 8, 223–246. Kohut, M.L., Boehm, G.W., Moynihan, J.A., 2001. Prolonged exercise suppresses antigen-specific cytokine response to upper respiratory infection. J. Appl. Physiol. 90, 678–684. Marshall, G.D., 2011. The adverse effects of psychological stress on immunoregulatory balance: applications to human inflammatory diseases. Immunol. Allergy Clin. North Am. 31, 133–140. Matsuzaki, J., Tsuji, T., Imazeki, I., Ikeda, H., Nishimura, T., 2005. Immunosteroid as a regulator for Th1/Th2 balance: its possible role in autoimmune diseases. Autoimmunity 38, 369–375. Nieman, D.C., Johanssen, L.M., Lee, J.W., 1989. Infectious episodes in runners before and after a roadrace. J. Sports Med. Phys. Fit. 29, 289–296. Nieman, D.C., Johanssen, L.M., Lee, J.W., Arabatzis, K., 1990. Infectious episodes in runners before and after the Los Angeles Marathon. J. Sports Med. Phys. Fit. 30, 316–328. Nieman, D.C., Pedersen, B.K., 1999. Exercise and immune function. Recent developments. Sports Med. 27, 73–80. Peters, E.M., Bateman, E.D., 1983. Ultramarathon running and upper respiratory tract infections. An epidemiological survey. S. Afr. Med. J. 64, 582–584. Poli, V., Mancini, F.P., Cortese, R., 1990. IL-6DBP, a nuclear protein involved in interleukin-6 signal transduction, defines a new family of leucine zipper proteins related to C/EBP. Cell 63, 643–653. Ramji, D.P., Foka, P., 2002. CCAAT/enhancer-binding proteins: structure, function and regulation. Biochem. J. 365, 561–575. Rehm, K.E., Elci, O.U., Hahn, K., Marshall, G.D., 2013. The impact of self-reported psychological stress levels on changes to peripheral blood immune biomarkers in recreational marathon runners during training and recovery. Neuroimmunomodulation 20, 164–176. Robson-Ansley, P., Howatson, G., Tallent, J., Mitcheson, K., Walshe, I., Toms, C., Du Toit, G., Smith, M., Ansley, L., 2012. Prevalence of allergy and upper respiratory tract symptoms in runners of the London marathon. Med. Sci. Sports Exerc. 44, 999–1004. Romagnani, S., 2006. Regulation of the T cell response. Clin. Exp. Allergy 36, 1357–1366. Salicru, A.N., Sams, C.F., Marshall, G.D., 2007. Cooperative effects of corticosteroids and catecholamines upon immune deviation of the type-1/type-2 cytokine balance in favor of type-2 expression in human peripheral blood mononuclear cells. Brain Behav. Immun. 21, 913–920. Salomon, B., Bluestone, J.A., 2001. Complexities of CD28/B7: CTLA-4 costimulatory pathways in autoimmunity and transplantation. Annu. Rev. Immunol. 19, 225–252. Sharpe, A., Freeman, G.J., 2002. The B7-CD28 superfamily. Nat. Rev. Immunol. 2, 116–126. Shepard, R.J., Shek, P.N., 1996. Impact of physical activity and sport on the immune system. Rev. Environ. Health 11, 133–147. Smith, L.L., 2003. Overtraining, excessive exercise, and altered immunity: is this a T helper-1 versus T helper-2 lymphocyte response? Sports Med. 33, 347–364. Suzuki, K., Nakaji, S., Kurakake, S., Totsuka, M., Sato, K., Kuriyama, T., Fujimoto, H., Shibusawa, K., Machida, K., Sugawara, K., 2003. Exhaustive exercise and type1/type-2 cytokine balance with special focus on interleukin-12 p40/p70. Exerc. Immunol. Rev. 9, 48–57.
134
L. Xiang et al. / Molecular Immunology 60 (2014) 129–134
Suzuki, K., Nakaji, S., Yamada, M., Totsuka, M., Sato, K., Sugawara, K., 2002. Systemic inflammatory response to exhaustive exercise. Cytokine kinetics. Exerc. Immunol. Rev. 8, 6–48. Szabo, S.J., Sullivan, B.M., Stemmann, C., Satoskar, A.R., Sleckman, B.P., Glimcher, L.H., 2002. Distinct effects of T-bet in TH1 lineage commitment and IFN-gamma production in CD4 and CD8 T cells. Science 295, 338–342. Vandesompele, J., De Preter, K., Pattyn, F., Poppe, B., Van Roy, N., De Paepe, A., Speleman, F., 2002. Accurate normalization of real-time quantitative RT-PCR data by geometric averaging of multiple internal control genes. Genome Biol. 3, 1–12. Walsh, N.P., Gleeson, M., Shephard, R.J., Gleeson, M., Woods, J.A., Bishop, N.C., Fleshner, M., Green, C., Pedersen, B.K., Hoffman-Goetz, L., Rogers, C.J., Northoff, H., Abbasi, A., Simon, P., 2011. Position statement. Part one: immune function and exercise. Exerc. Immunol. Rev. 17, 6–63.
Xiang, L., Marshall, G.D., 2011. Immunomodulatory effects of in vitro stress hormones on FoxP3, Th1/Th2 cytokine and costimulatory molecule mRNA expression in human PBMC. Neuroimmunomodulation 18, 1–10. Xiang, L., Del Ben, K., Rehm, K.E., Marshall, G.D., 2012. Effects of acute stress-induced immunomodulation on Th1/Th2 cytokine and catecholamine receptors expression in human peripheral blood cells. Psychoneurobiology 65, 12–19. Zheng, W., Flavell, R.A., 1997. The transcription factor GATA-3 is necessary and sufficient for Th2 cytokine gene expression in CD4 T cells. Cell 89, 587–596. Zhu, J., Yamane, H., Cote-Sierra, J., Guo, L., Paul, W.E., 2006. GATA-3 promotes Th2 responses through three different mechanisms: induction of Th2 cytokine production, selective growth of Th2 cells and inhibition of Th1 cell-specific factors. Cell Res. 16, 3–10.
