http://informahealthcare.com/iht ISSN: 0895-8378 (print), 1091-7691 (electronic) Inhal Toxicol, Early Online: 1–6 ! 2015 Informa Healthcare USA, Inc. DOI: 10.3109/08958378.2015.1040139
RESEARCH ARTICLE
Treg responses are associated with PM2.5-induced exacerbation of viral myocarditis Yuquan Xie1, Changyi Gong2, Liang Bo2, Shuo Jiang2, Haidong Kan2, Weimin Song2, Jinzhuo Zhao2, and Yigang Li1 Department of Cardiology, Xinhua Hospital, Shanghai Jiao Tong University, School of Medicine, Shanghai, China and 2Department of Environmental Health, School of Public Health and Key Laboratory of Public Health Safety, Fudan University, Shanghai, China
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Abstract
Keywords
The adverse cardiovascular events induced by ambient fine particles (PM2.5) are paid more attention in the world. The current study was conducted to explore the mechanisms of T regulatory cells (Treg) responses in PM2.5-induced exacerbation of viral myocarditis. The male BALB/c mice were administered an intratracheal (i.t.) instillation of 10 mg/kg b.w. PM2.5 suspension. Twenty-four hours later, the mice were injected intraperitoneally (i.p.) with 100 ml of coxsackievirus B3 (CVB3) diluted in Eagle’s minimal essential medium (EMEM). Seven days after the treatment, serum, splenetic, and cardiac tissues were examined. The results showed that pre-exposure to PM2.5 aggravated the cardiac inflammation in the CVB3-infected mice along with an increase of Treg cells in the spleen. The mRNA expressions of interleukin-6 (IL-6), TNF-a, transforming growth factor-b (TGF-b), and Foxp3 were up-regulated in the PM2.5-pretreated mice than that in the CVB3-treated mice. Similar results were found in the sera. In addition, compared with the CVB3-treated mice, the cardiac protein expression of TGF-b increased in the PM2.5-pretreated mice. These results demonstrated that preexposure to PM2.5 exacerbated virus-induced myocarditis possibly through the depression of the immune response and increase of inflammation in myocardium through the Treg responses.
Inflammation, PM2.5, treg cell, viral myocarditis
Introduction The association between ambient fine particulate matter (PM2.5) and cardiovascular diseases is well known in the world (Hoek et al., 2002; Miller et al., 2007; Pope et al., 2004, 2006). Epidemiological studies have reported that ambient PM2.5 is a risk factor in the development of cardiovascular diseases through the mechanisms that may include pulmonary and systemic inflammation, accelerated atherosclerosis, and altered cardiac autonomic functions (Pope et al., 2004). However, to date, the underlying pathophysiological mechanisms connecting PM2.5 and cardiovascular diseases remain unclear. Previous studies have shown that inflammatory response (Sun et al., 2005), oxidative DNA damage (Lee et al., 2014), and the recruitment (Becker & Soukup, 2003) and differentiation (Zhao et al., 2012) of immune cells are the main mechanisms linking PM2.5 and cardiovascular diseases. Immune system plays a vital role during the progress of inflammation and oxidative stress. T helper 17 cell (Th17)
Address for correspondence: Jinzhuo Zhao, PhD, Department of Environmental Health, School of Public Health, Fudan University, 331 Building 8, 130 Dong’an Road, Shanghai 200032, China. Tel: +86 21 54237085. Fax: +86 21 54237085. E-mail: [email protected] Yigang Li, Ph.D., Department of Cardiology, Xinhua Hospital, Shanghai Jiao Tong University, School of Medicine, Shanghai 200092, China. Tel: +86 21 25077308. Fax: +86 21 25077308. E-mail: [email protected]
History Received 13 January 2015 Revised 24 March 2015 Accepted 8 April 2015 Published online 7 May 2015
and regulatory T (Treg) cells are subsets of the CD4+ T cell compartment which are vital modulators of the innate and adaptive immune response in many immune diseases (Chauhan et al., 2009; Ratajczak et al., 2010). The imbalance of Th17 and Treg cells plays prominent roles in immune functions: Th17 cells are key players in the pathogenesis of autoimmune diseases, which could exert pro-inflammatory function and protect cells against bacterial infections. On the contrary, Treg cells could restrain excessive effector of T-cell responses. These two subsets have been extensively analyzed in the development of inflammatory and autoimmune diseases (Homey, 2006; Sakaguchi, 2004). It has known that IL-6 and Treg-produced cytokine transforming growth factorb (TGF-b) were necessary for murine Th17 cell differentiation (Bettelli et al., 2006). Myocarditis, usually caused by viral infection in the heart, is a common cardiac disease that is responsible for millions of cases annually worldwide. Coxsackievirus B3 (CVB3) infection has been shown to cause acute or chronic myocarditis (Tam, 2006). The direct cytopathic effect and immune or autoimmunal response triggered by CVB3 infection has been a dominant cause of myocyte injury (Huber, 2006; Liu & Mason, 2001). Our previous study has reported that the increase of Th17 cells and Treg cells is associated with the cardiac injury in CVB3-induced viral myocarditis (Xie et al., 2011). Recently, the environmental pollution has also been considered a risk factor for myocarditis. The development of
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myocarditis is associated not only with virus infection but also with the environmental pollutant exposure (Bae et al., 2010). Our previous study indicated that pre-exposure to PM2.5 exacerbated virus-induced myocarditis possibly through the increase in Th17-mediated inflammation and viral replication (Xie et al., 2013). Therefore, in the current study, we attempt to further explore the effects of ambient PM2.5 on viral myocarditis. Treg cells belong to a unique lineage of T cells that play an important role in the modulation of immune responses and the reduction of deleterious immune activation owing to their immunoregulatory and immunosuppressive functions (Sakaguchi et al., 2006). So far, few studies were conducted to explore the relationship between air pollution and Treg cells (Dutta et al., 2012; Nadeau et al., 2010). Meanwhile, the precise molecular and cellular pathways leading to the immunological impairment caused by air pollution remain largely unknown. Especially, the role of Tregs responses in PM2.5-induced cardiac injury has not yet been elucidated. It has been reported that higher average polycyclic aromatic hydrocarbons (PAH) exposure was significantly associated with the impaired Treg function (Hew et al., 2014). The current study aims to explore the mechanism of Treg responses in PM2.5-exacerbated inflammatory responses in acute viral myocarditis model.
Materials and methods Particle collection and process The ambient PM2.5 was collected on glass fiber filters using a Thermo Anderson G-2.5 air sampler (Model GV 2630 Series, Thermo Fisher Scientific, Inc, Waltham, MA) in a nonindustrial district in Shanghai, China. The filters were heated for 24 h at 200 C before sampling. After sampling, the collection of PM2.5 was conducted as previous study (Xie et al., 2013). The filters were cut into small pieces and immerged in 0.9% saline followed by sonicating 3 10 min with a sonicator (Model JL-120DT, Fu Rui, Beijing, China). Then, the PM2.5 suspension was treated by vacuum-freeze dry to get the concentrated components. Before instillation, the PM2.5 was diluted with sterilized 0.9% saline according to the experimental concentration and stored at 4 C. Virus The CVB3 (Nancy strain) was prepared by passage through HeLa cell cultures. Viruses were harvested by freeze-thawing and were stored at 80 C. The virus titer was determined by median tissue culture infective dose (TCID50 ¼ 10, Nancy strain) in 100 ml of Eagle’s minimal essential medium (EMEM, GIBCO, Waltham, MA) (Chen et al., 2011). Mice Forty male Balb/c mice (4–6 weeks old) were purchased from the Joint Ventures Sipper BK Experimental Animal Company (Shanghai, China). Immediately after the arrival, the mice were randomly divided into four groups. The mice, which were specific pathogen free (SPF), were housed in macrolon cages in an animal room at constant temperature (21 ± 1 C) and relative humidity (60%) under a regular light/dark
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(12:12) cycle. Food and water were freely available. The protocols and the use of animals were approved by and in accordance with the Fudan University Animal Care and Use Committee. All animal experiments were performed in accordance with the National Institute of Health (NIH) Guide for the Care and Use of Laboratory Animals. PM2.5 and virus treatments The treatment for four groups of mice was as follows: (1) control: the mice were instilled with 0.9% saline alone. Twenty-four hours later, the mice were then injected i.p. with 100 ml of EMEM. (2) PM2.5: the mice were intratracheally instilled with PM2.5 particles (10.0 mg/kg b.w.) of 0.15 ml/ 100 g b.w. Twenty-four hours later, the mice were injected i.p. with 100 ml of EMEM. (3) Virus: the mice were instilled with 0.9% saline. Twenty-four hours later, the mice were then injected i.p. with 100 ml of CVB3 diluted in EMEM. (4) PM2.5 and virus: the mice were instilled with PM2.5 particles (10.0 mg/kg b.w.). Twenty-four hours later, the mice were then treated with 100 ml of CVB3. After 7 d of virus injection, all the mice were euthanized by overdose isoflurane inhalation. Then, the heart and spleen tissues were isolated from the mice for further analyses. Flow cytometric analysis After 7 d of virus injection, there were one mouse dead in the CVB3 group, two in the CVB3 + PM2.5 group, and zero in the control and the PM2.5 group. Eight mice for each group were used for final analysis. The spleen was taken for Treg cells determination. The splenocytes were isolated and suspended in RPMI 1640 containing 10% fetal bovine sera. Then, red blood cells were lysed by 3 min incubation in ACK lysis buffer (TIANGEN, Shanghai, China). The cells were collected and resuspended at a density of 1.0 106/ml. The stain and the detection were conducted as previous study (Xie et al., 2013). The cells were stained with FITC-labeled antimouse CD4 (eBiosciences, San Diego, CA). After washing, fixing, and permeabilizing according to the instructions of the manufacturer, the cells were stained intracellularly with APCconjugated anti-mouse Foxp3 (eBiosciences, San Diego, CA). After incubation at 4 C for 30 min, the samples were finally washed in staining buffer and measured by flow cytometer with a FACSCalibur machine. The data were analyzed with Cell Quest software (BD Biosciences, San Jose, CA). Quantification by real time-PCR The frozen cardiac tissue samples were homogenized using 1 ml of Trizol reagent (Invitrogen, Waltham, MA) per 100 mg samples. Total RNA was extracted from the tissue according to the protocol of the manufacturer. RNA pellets were dissolved in 100 ml diethylpyrocarbonate (DEPC)-treated water and the total RNA concentration was determined by spectrophotometric analysis at 260 nm. Total RNA was converted into cDNA using M-MuLV reverse transcriptase (Fermentas, Hanover, MD), cDNA was amplified by SYBR green master mix (TaKaRa, Liaoning, China) using an ABI7500 thermocycler (Applied Biosystems, Foster City, CA). The 2DCT method (Livak & Schmittgen, 2001) was
The association between Treg response and PM2.5
DOI: 10.3109/08958378.2015.1040139
Table 1. Primers sequences used for real time-PCR reactions. Genes
Primer pairs
Upper: 5 -ATCCAGTTGCCTTCTTGGGACTGA-30 Lower: 50 -TAAGCCTCCGACTTGTGAAGTGGT -30 TNF-a Upper: 50 -CAACGGCATGGATCTCAAAGAC-30 Lower: 50 -AGATAGCAAATCGGCTGACGGT-30 TGF-b Upper: 50 -GCAACAACGCCATCTATG-30 Lower: 50 -CAAGGTAACGCCAGGAAT-30 Foxp3 Upper: 50 -ACACCCAGGAAAGACAGCAACC-30 Lower: 50 -CCTCGAAGACCTTCTCACAACC-30 GAPDH Upper: 50 -AGTATGATGACATCAAGAAGG-30 Lower: 50 -ATGGTATTCAAGAGAGTAGGG-30 IL-6
0
3
and CVB exposure induced the decrease of CD4+ T cells. When compared with the treatment of CVB3 alone, preexposure to PM2.5 caused the significant decrease of CD4+ T cells (p50.05). Compared with those in the control group, the splenic percentages of CD4+Foxp3+ cells in the PM2.5-treated group (p50.05), CVB3-treated group (p50.01), and PM2.5 + CVB3 group (p50.01) were markedly higher, suggesting that PM2.5 induced the increase of CD4+Foxp3+ cells in spleen. Additionally, the percentages of CD4+Foxp3+ cells were higher in the PM2.5-pretreated group than that in the CVB3 group.
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Detection of cardiac IL-6, TNF-a, TGF-b, and Foxp3 mRNA used to normalize transcription to GAPDH mRNA and the fold induction was calculated relative to control. The PCR primers for IL-6, TNF-a, TGF-b, and Foxp3 were designed using the primer design software. The primer pairs are shown in Table 1. Western blot The total proteins of cardiac tissue were lysed in radioimmunoprecipitation assay lysis buffer, which contained a protease and phosphatase inhibitor cocktail (AG Scientific, San Diego, CA). Protein concentration was quantified using a bicinchoninic (BCA) protein assay kit (Pierce, Rockford, IL). Samples containing 50 mg of proteins were separated on an 8% to 12% SDS-PAGE gel and transferred to polyvinylidene difluoride membranes. Themembranes were blocked for 2 h at room temperature with 5% BSA, reacted with anti-TGF-b antibody (1:500, Santa Cruz Biotechnology, Santa Cruz, CA) and anti-GAPDH antibody (1:1000, Kangchen Biotech, Shanghai, China) at 4 C overnight, and then incubated with a secondary HRP-conjugated antibody (1:2000, Kangchen Biotech, Shanghai, China) for 1 h at room temperature. Then, the blots were detected with ECL (Thermo Fisher Scientific, Inc, Waltham, MA). The protein bands on the X-ray film were scanned, and the band density was calculated by Quantity One software (Bio-Rad, Berkeley, CA). The GAPDH was used as control to calculate the expression of the proteins. Cytokine detection in serum The serum was collected after centrifugation and stored at 80 C. The levels of IL-6, TNF-a, and TGF-b in serum were measured by ELISA kits according to the instructions of the manufacturer (Dakewe Biotech Co. Ltd., Beijing, China). Statistical analysis The differences among the control, PM2.5, CVB3 and PM2.5 + CVB3 groups were analyzed by one-way analysis of variance (ANOVA) with Dunnett’s or Tukey post-test. The statistical software was SPSS16.0 (SPSS Inc., Chicago, IL). Probability value p50.05 was considered significant.
Results Proportional changes of CD4+Foxp3+ cells The percentages of Treg cells in the spleen were determined by flow cytometric analysis (Figure 1A and B). The PM2.5
The mRNA expressions of cardiac IL-6, TNF-a, TGF-b, and Foxp3 in the mice were determined by RT-PCR. The inflammation-related cytokines IL-6 (Figure 2A) and TNF-a (Figure 2B) significantly increased in the exposure group than those in control. The mRNA expression of Treg-related cytokine TGF-b (Figure 2C) and relative transcriptional factor Foxp3 (Figure 2D) were up-regulated in the CVB3 group and the PM2.5-pretreated group. However, the PM2.5 exposure alone just induced the increase of TGF-b (p50.05) but not Foxp3. Compared with the CVB3 treatment alone, the PM2.5 + CVB3 treatment induced greater increase in the expressions of IL-6, TNF-a, TGF-b, and Foxp3 (p50.05). Cardiac protein expression of TGF- To further explore whether myocardial injury was exacerbated by PM2.5 exposure, the protein expressions of TGF-b were determined by Western blot (Figure 3A and B). The protein levels of cardiac TGF-b were elevated in the mice after exposure to PM2.5 (p50.05) or CVB3 (p50.01) when compared with the control. In addition, the expression of TGF-b protein was markedly higher in the PM2.5-pretreated group than that in the CVB3-treated group (p50.05). The levels of cytokines in sera The serum levels of inflammation-related cytokines IL-6, TNF-a, and TGF-b were detected (Figure 4). The PM2.5 treatment alone just induced the increase in IL-6 (p50.05). However, no similar result was observed in TNF-a and TGFb. When compared with the control, the CVB3 or PM2.5 + CVB3 exposure induced the increase of IL-6 and TGF-b. To TNF-a, there was a significantly increase in the PM2.5 + CVB3-treated mice (p50.05) but not in the CVB3and PM2.5-treated mice. In comparison, the levels of IL-6, TNF-a, and TGF-b in PM2.5-pretreated mice were significantly higher than those in the CVB3-treated mice.
Discussion Recently, the severe PM2.5 air pollution is paid more attention in China. In 2012, the Chinese government has established the air quality standard for PM2.5 (the 24-hour average standard is 75 mg/m3), which is around two times higher than the standard of American Environmental Protection Agency (EPA). Because of the limitation of technique and the huge difficult to control the PM2.5 pollution, the standard will be
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Figure 1. The percentages of CD4+ T cells and CD4+Foxp3+ cells (Treg) were investigated by flow cytometry in mice. Representative pictures for CD4+Foxp3+ cells gated on CD4+ T cells in the groups of spleen (n ¼ 8 for every group). Numbers in upper right quadrants indicate the percentages of CD4+Foxp3+ in the spleen (A). Statistical analysis showed the variation of CD4+ T cell and CD4+Foxp3+ cells (B). The differences among control, PM2.5 group, CVB3 group, and PM2.5 + CVB3 group were made by one-way analysis of variance (ANOVA). **p50.01, *p50.05 versus the control group. Significant difference (#p50.05, ##p50.01) between CVB3 and PM2.5+ CVB3 groups. Figure 2. The mRNA expression of IL-6 (A), TNF-a (B), TGF-b (C), and Foxp3 (D) in cardiac tissues was determined by real-time PCR (n ¼ 8). Quantification of these cytokines mRNA level normalized to GAPDH. Data are expressed as mean ± SD. **p50.01, *p50.05 versus the control group. Significant difference (#p50.05) between CVB3 and PM2.5 + CVB3 groups.
implemented in 2016. So the PM2.5 pollution is still a risk factor threatening people’s health. Because of its small size, ambient PM2.5 is possibly inhaled deeply into the lungs, with a portion depositing in the alveoli and potentially entering pulmonary circulation and presumably systemic circulation. However, the biological mechanisms linking PM2.5 and cardiovascular diseases are still unclear. In the past decades, researches focused on the mechanisms about the changes in heart rate (Pope et al., 1999), the changes of
cardiac function (Pekkanen et al., 2002), plasma viscosity (Peters et al., 1997), vasomotor dysfunction (Mills et al., 2007), and inflammatory response (Peters et al., 2001). Nevertheless, the immune mechanisms between ambient particulate matter and cardiovascular disease are paid little attention. The occurrence of viral myocarditis exhibits an increase in recent years in the world. Our previous study indicated that ambient PM2.5 could be a risk factor in exacerbating the
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DOI: 10.3109/08958378.2015.1040139
Figure 3. The protein expression of TGF-b in cardiac tissue. (A) Representative bands for protein expression of cardiac TGF-b in different groups (n ¼ 8). (B) Statistical analysis for the levels of TGF-b protein. **p50.01, *p50.05 versus the control group. Significant difference (#p50.05) between CVB3 and PM2.5 + CVB3 groups.
Figure 4. The levels of inflammation-related cytokines IL-6, TNF-a, and TGF-b in the sera of mice (n ¼ 8). One-way ANOVA was used to analyze the data. **p50.01, *p50.05 versus the control group. Significant difference (#p50.05) between CVB3 and PM2.5 + CVB3 groups.
viral myocarditis. The immune impairment involving the Th17 was associated with the development of myocarditis (Xie et al., 2013). Considering the relationship between Th17 and Treg cells, the current study further explored the role of Treg responses in viral myocarditis. The results found that pre-exposure to PM2.5 might induce the differentiation of Treg cells and in turn increase the expression of Treg-related cytokines, transcriptional factor, and inflammation-related cytokines in the spleen, serum, and cardiac tissue of mice, suggesting that ambient PM2.5 might impair the immune function and exacerbate the cardiac inflammatory response via mediate the Treg responses. Treg cells belong to a T cell subset that can inhibit effector T cell responses. Foxp3 is recognized as the exclusive marker of Treg cells. Treg cells are important for maintaining immune tolerance and protection from autoimmune diseases.
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The Treg cells exert their regulatory activity either by cellto-cell contact or by the release of suppressive cytokines such as TGF-b and interleukin-10 (IL-10). Previous study provided the evidence that the rise of Treg cells may indicate a defense against inflammatory manifestations and chronic infection in biomass users (Dutta et al., 2012). Therefore, the upregulation of Treg cell is not only a protective mechanism but also an external chemistry-induced immune impairment. In the current study, the increase of Treg cells suggested that ambient PM2.5 activated the differentiation of Treg cell in order to protect heart against intensive inflammatory response. A prior consistent finding showed that biomass users display a rise in Treg cells because they usually were exposed to biomass smoke during daily household cooking (Dutta et al., 2012). Moreover, a significant association between PAH exposure and impairment of immune function in both asthmatic and non-asthmatic was reported (Hew et al., 2014). However, this study showed a decrease of Treg cells, which might be explained by the different constituents between PAH and ambient PM2.5. Moreover, other study found that maternal tobacco smoke exposure during pregnancy correlates with a decrease of children’s cord blood Treg cell numbers and subsequent allergy risk (Herberth et al., 2014). The possible reason for the different results between our study and previous studies are as follows: first, the different components between ambient PM2.5 and tobacco smoke and PAH. The tobacco smoke is a much more complex mixture than PM2.5 and it mainly tends to induce allergic diseases (Callesen et al., 2014), whereas high concentration of PM2.5 susceptibly promotes the Treg cell differentiation and depresses the immune response. Meanwhile, the dose of PM2.5 and the subjects were obviously different. Second, the increase of the Treg cells and Treg-related cytokine TGF-b was associated with the immunosuppressive response, hereby our study showed an immunosuppressive response. Certainly, TGF-b was a regulatory cytokine with a pleiotropic function in T-cell development, homeostasis, and tolerance (Mesa et al., 2010). Treg cells played an important role in the regulation of T-cell-mediated immune responses through the suppression of T-cell proliferation and secretion of inhibitory cytokines, such as IL-10 and TGF-b (Foks et al., 2014). The maintenance of long-term Treg cell activation could contribute to the progressive loss of CD4+ T cell immune function and promote the development of myocarditis. Supporting our study, prior study reported that simian immunodeficiency virus (SIV) infection of Macaques induced an early immunosuppressive response that correlated with a marked increase in frequency of CD4+Foxp3+ (Treg) cells and TGF-b, indicating that Treg cells might possibly contribute to viral persistence by prematurely limiting the antiviral (Estes et al., 2006). Our results indicated that PM2.5 induced the increase of Treg cells may contribute to viral persistence by prematurely limiting the antiviral immune response.
Conclusion In summary, these finding suggested that PM2.5 may exacerbate the CVB3-induced increase of Treg responses, and the later may have a primary role in the failure of the host immune system to eradicate viral infection. The study
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indicated that PM2.5-induced increase of Treg cells could suppress the immune responses in turn cause the development of cardiac injury.
Declaration of interest The authors report that they have no conflicts of interests. This work was supported by grants from the National Natural Science Foundation of China (Nos. 81001229 and 81172617).
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RESEARCH ARTICLE
Treg responses are associated with PM2.5-induced exacerbation of viral myocarditis Yuquan Xie1, Changyi Gong2, Liang Bo2, Shuo Jiang2, Haidong Kan2, Weimin Song2, Jinzhuo Zhao2, and Yigang Li1 Department of Cardiology, Xinhua Hospital, Shanghai Jiao Tong University, School of Medicine, Shanghai, China and 2Department of Environmental Health, School of Public Health and Key Laboratory of Public Health Safety, Fudan University, Shanghai, China
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Abstract
Keywords
The adverse cardiovascular events induced by ambient fine particles (PM2.5) are paid more attention in the world. The current study was conducted to explore the mechanisms of T regulatory cells (Treg) responses in PM2.5-induced exacerbation of viral myocarditis. The male BALB/c mice were administered an intratracheal (i.t.) instillation of 10 mg/kg b.w. PM2.5 suspension. Twenty-four hours later, the mice were injected intraperitoneally (i.p.) with 100 ml of coxsackievirus B3 (CVB3) diluted in Eagle’s minimal essential medium (EMEM). Seven days after the treatment, serum, splenetic, and cardiac tissues were examined. The results showed that pre-exposure to PM2.5 aggravated the cardiac inflammation in the CVB3-infected mice along with an increase of Treg cells in the spleen. The mRNA expressions of interleukin-6 (IL-6), TNF-a, transforming growth factor-b (TGF-b), and Foxp3 were up-regulated in the PM2.5-pretreated mice than that in the CVB3-treated mice. Similar results were found in the sera. In addition, compared with the CVB3-treated mice, the cardiac protein expression of TGF-b increased in the PM2.5-pretreated mice. These results demonstrated that preexposure to PM2.5 exacerbated virus-induced myocarditis possibly through the depression of the immune response and increase of inflammation in myocardium through the Treg responses.
Inflammation, PM2.5, treg cell, viral myocarditis
Introduction The association between ambient fine particulate matter (PM2.5) and cardiovascular diseases is well known in the world (Hoek et al., 2002; Miller et al., 2007; Pope et al., 2004, 2006). Epidemiological studies have reported that ambient PM2.5 is a risk factor in the development of cardiovascular diseases through the mechanisms that may include pulmonary and systemic inflammation, accelerated atherosclerosis, and altered cardiac autonomic functions (Pope et al., 2004). However, to date, the underlying pathophysiological mechanisms connecting PM2.5 and cardiovascular diseases remain unclear. Previous studies have shown that inflammatory response (Sun et al., 2005), oxidative DNA damage (Lee et al., 2014), and the recruitment (Becker & Soukup, 2003) and differentiation (Zhao et al., 2012) of immune cells are the main mechanisms linking PM2.5 and cardiovascular diseases. Immune system plays a vital role during the progress of inflammation and oxidative stress. T helper 17 cell (Th17)
Address for correspondence: Jinzhuo Zhao, PhD, Department of Environmental Health, School of Public Health, Fudan University, 331 Building 8, 130 Dong’an Road, Shanghai 200032, China. Tel: +86 21 54237085. Fax: +86 21 54237085. E-mail: [email protected] Yigang Li, Ph.D., Department of Cardiology, Xinhua Hospital, Shanghai Jiao Tong University, School of Medicine, Shanghai 200092, China. Tel: +86 21 25077308. Fax: +86 21 25077308. E-mail: [email protected]
History Received 13 January 2015 Revised 24 March 2015 Accepted 8 April 2015 Published online 7 May 2015
and regulatory T (Treg) cells are subsets of the CD4+ T cell compartment which are vital modulators of the innate and adaptive immune response in many immune diseases (Chauhan et al., 2009; Ratajczak et al., 2010). The imbalance of Th17 and Treg cells plays prominent roles in immune functions: Th17 cells are key players in the pathogenesis of autoimmune diseases, which could exert pro-inflammatory function and protect cells against bacterial infections. On the contrary, Treg cells could restrain excessive effector of T-cell responses. These two subsets have been extensively analyzed in the development of inflammatory and autoimmune diseases (Homey, 2006; Sakaguchi, 2004). It has known that IL-6 and Treg-produced cytokine transforming growth factorb (TGF-b) were necessary for murine Th17 cell differentiation (Bettelli et al., 2006). Myocarditis, usually caused by viral infection in the heart, is a common cardiac disease that is responsible for millions of cases annually worldwide. Coxsackievirus B3 (CVB3) infection has been shown to cause acute or chronic myocarditis (Tam, 2006). The direct cytopathic effect and immune or autoimmunal response triggered by CVB3 infection has been a dominant cause of myocyte injury (Huber, 2006; Liu & Mason, 2001). Our previous study has reported that the increase of Th17 cells and Treg cells is associated with the cardiac injury in CVB3-induced viral myocarditis (Xie et al., 2011). Recently, the environmental pollution has also been considered a risk factor for myocarditis. The development of
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myocarditis is associated not only with virus infection but also with the environmental pollutant exposure (Bae et al., 2010). Our previous study indicated that pre-exposure to PM2.5 exacerbated virus-induced myocarditis possibly through the increase in Th17-mediated inflammation and viral replication (Xie et al., 2013). Therefore, in the current study, we attempt to further explore the effects of ambient PM2.5 on viral myocarditis. Treg cells belong to a unique lineage of T cells that play an important role in the modulation of immune responses and the reduction of deleterious immune activation owing to their immunoregulatory and immunosuppressive functions (Sakaguchi et al., 2006). So far, few studies were conducted to explore the relationship between air pollution and Treg cells (Dutta et al., 2012; Nadeau et al., 2010). Meanwhile, the precise molecular and cellular pathways leading to the immunological impairment caused by air pollution remain largely unknown. Especially, the role of Tregs responses in PM2.5-induced cardiac injury has not yet been elucidated. It has been reported that higher average polycyclic aromatic hydrocarbons (PAH) exposure was significantly associated with the impaired Treg function (Hew et al., 2014). The current study aims to explore the mechanism of Treg responses in PM2.5-exacerbated inflammatory responses in acute viral myocarditis model.
Materials and methods Particle collection and process The ambient PM2.5 was collected on glass fiber filters using a Thermo Anderson G-2.5 air sampler (Model GV 2630 Series, Thermo Fisher Scientific, Inc, Waltham, MA) in a nonindustrial district in Shanghai, China. The filters were heated for 24 h at 200 C before sampling. After sampling, the collection of PM2.5 was conducted as previous study (Xie et al., 2013). The filters were cut into small pieces and immerged in 0.9% saline followed by sonicating 3 10 min with a sonicator (Model JL-120DT, Fu Rui, Beijing, China). Then, the PM2.5 suspension was treated by vacuum-freeze dry to get the concentrated components. Before instillation, the PM2.5 was diluted with sterilized 0.9% saline according to the experimental concentration and stored at 4 C. Virus The CVB3 (Nancy strain) was prepared by passage through HeLa cell cultures. Viruses were harvested by freeze-thawing and were stored at 80 C. The virus titer was determined by median tissue culture infective dose (TCID50 ¼ 10, Nancy strain) in 100 ml of Eagle’s minimal essential medium (EMEM, GIBCO, Waltham, MA) (Chen et al., 2011). Mice Forty male Balb/c mice (4–6 weeks old) were purchased from the Joint Ventures Sipper BK Experimental Animal Company (Shanghai, China). Immediately after the arrival, the mice were randomly divided into four groups. The mice, which were specific pathogen free (SPF), were housed in macrolon cages in an animal room at constant temperature (21 ± 1 C) and relative humidity (60%) under a regular light/dark
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(12:12) cycle. Food and water were freely available. The protocols and the use of animals were approved by and in accordance with the Fudan University Animal Care and Use Committee. All animal experiments were performed in accordance with the National Institute of Health (NIH) Guide for the Care and Use of Laboratory Animals. PM2.5 and virus treatments The treatment for four groups of mice was as follows: (1) control: the mice were instilled with 0.9% saline alone. Twenty-four hours later, the mice were then injected i.p. with 100 ml of EMEM. (2) PM2.5: the mice were intratracheally instilled with PM2.5 particles (10.0 mg/kg b.w.) of 0.15 ml/ 100 g b.w. Twenty-four hours later, the mice were injected i.p. with 100 ml of EMEM. (3) Virus: the mice were instilled with 0.9% saline. Twenty-four hours later, the mice were then injected i.p. with 100 ml of CVB3 diluted in EMEM. (4) PM2.5 and virus: the mice were instilled with PM2.5 particles (10.0 mg/kg b.w.). Twenty-four hours later, the mice were then treated with 100 ml of CVB3. After 7 d of virus injection, all the mice were euthanized by overdose isoflurane inhalation. Then, the heart and spleen tissues were isolated from the mice for further analyses. Flow cytometric analysis After 7 d of virus injection, there were one mouse dead in the CVB3 group, two in the CVB3 + PM2.5 group, and zero in the control and the PM2.5 group. Eight mice for each group were used for final analysis. The spleen was taken for Treg cells determination. The splenocytes were isolated and suspended in RPMI 1640 containing 10% fetal bovine sera. Then, red blood cells were lysed by 3 min incubation in ACK lysis buffer (TIANGEN, Shanghai, China). The cells were collected and resuspended at a density of 1.0 106/ml. The stain and the detection were conducted as previous study (Xie et al., 2013). The cells were stained with FITC-labeled antimouse CD4 (eBiosciences, San Diego, CA). After washing, fixing, and permeabilizing according to the instructions of the manufacturer, the cells were stained intracellularly with APCconjugated anti-mouse Foxp3 (eBiosciences, San Diego, CA). After incubation at 4 C for 30 min, the samples were finally washed in staining buffer and measured by flow cytometer with a FACSCalibur machine. The data were analyzed with Cell Quest software (BD Biosciences, San Jose, CA). Quantification by real time-PCR The frozen cardiac tissue samples were homogenized using 1 ml of Trizol reagent (Invitrogen, Waltham, MA) per 100 mg samples. Total RNA was extracted from the tissue according to the protocol of the manufacturer. RNA pellets were dissolved in 100 ml diethylpyrocarbonate (DEPC)-treated water and the total RNA concentration was determined by spectrophotometric analysis at 260 nm. Total RNA was converted into cDNA using M-MuLV reverse transcriptase (Fermentas, Hanover, MD), cDNA was amplified by SYBR green master mix (TaKaRa, Liaoning, China) using an ABI7500 thermocycler (Applied Biosystems, Foster City, CA). The 2DCT method (Livak & Schmittgen, 2001) was
The association between Treg response and PM2.5
DOI: 10.3109/08958378.2015.1040139
Table 1. Primers sequences used for real time-PCR reactions. Genes
Primer pairs
Upper: 5 -ATCCAGTTGCCTTCTTGGGACTGA-30 Lower: 50 -TAAGCCTCCGACTTGTGAAGTGGT -30 TNF-a Upper: 50 -CAACGGCATGGATCTCAAAGAC-30 Lower: 50 -AGATAGCAAATCGGCTGACGGT-30 TGF-b Upper: 50 -GCAACAACGCCATCTATG-30 Lower: 50 -CAAGGTAACGCCAGGAAT-30 Foxp3 Upper: 50 -ACACCCAGGAAAGACAGCAACC-30 Lower: 50 -CCTCGAAGACCTTCTCACAACC-30 GAPDH Upper: 50 -AGTATGATGACATCAAGAAGG-30 Lower: 50 -ATGGTATTCAAGAGAGTAGGG-30 IL-6
0
3
and CVB exposure induced the decrease of CD4+ T cells. When compared with the treatment of CVB3 alone, preexposure to PM2.5 caused the significant decrease of CD4+ T cells (p50.05). Compared with those in the control group, the splenic percentages of CD4+Foxp3+ cells in the PM2.5-treated group (p50.05), CVB3-treated group (p50.01), and PM2.5 + CVB3 group (p50.01) were markedly higher, suggesting that PM2.5 induced the increase of CD4+Foxp3+ cells in spleen. Additionally, the percentages of CD4+Foxp3+ cells were higher in the PM2.5-pretreated group than that in the CVB3 group.
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Detection of cardiac IL-6, TNF-a, TGF-b, and Foxp3 mRNA used to normalize transcription to GAPDH mRNA and the fold induction was calculated relative to control. The PCR primers for IL-6, TNF-a, TGF-b, and Foxp3 were designed using the primer design software. The primer pairs are shown in Table 1. Western blot The total proteins of cardiac tissue were lysed in radioimmunoprecipitation assay lysis buffer, which contained a protease and phosphatase inhibitor cocktail (AG Scientific, San Diego, CA). Protein concentration was quantified using a bicinchoninic (BCA) protein assay kit (Pierce, Rockford, IL). Samples containing 50 mg of proteins were separated on an 8% to 12% SDS-PAGE gel and transferred to polyvinylidene difluoride membranes. Themembranes were blocked for 2 h at room temperature with 5% BSA, reacted with anti-TGF-b antibody (1:500, Santa Cruz Biotechnology, Santa Cruz, CA) and anti-GAPDH antibody (1:1000, Kangchen Biotech, Shanghai, China) at 4 C overnight, and then incubated with a secondary HRP-conjugated antibody (1:2000, Kangchen Biotech, Shanghai, China) for 1 h at room temperature. Then, the blots were detected with ECL (Thermo Fisher Scientific, Inc, Waltham, MA). The protein bands on the X-ray film were scanned, and the band density was calculated by Quantity One software (Bio-Rad, Berkeley, CA). The GAPDH was used as control to calculate the expression of the proteins. Cytokine detection in serum The serum was collected after centrifugation and stored at 80 C. The levels of IL-6, TNF-a, and TGF-b in serum were measured by ELISA kits according to the instructions of the manufacturer (Dakewe Biotech Co. Ltd., Beijing, China). Statistical analysis The differences among the control, PM2.5, CVB3 and PM2.5 + CVB3 groups were analyzed by one-way analysis of variance (ANOVA) with Dunnett’s or Tukey post-test. The statistical software was SPSS16.0 (SPSS Inc., Chicago, IL). Probability value p50.05 was considered significant.
Results Proportional changes of CD4+Foxp3+ cells The percentages of Treg cells in the spleen were determined by flow cytometric analysis (Figure 1A and B). The PM2.5
The mRNA expressions of cardiac IL-6, TNF-a, TGF-b, and Foxp3 in the mice were determined by RT-PCR. The inflammation-related cytokines IL-6 (Figure 2A) and TNF-a (Figure 2B) significantly increased in the exposure group than those in control. The mRNA expression of Treg-related cytokine TGF-b (Figure 2C) and relative transcriptional factor Foxp3 (Figure 2D) were up-regulated in the CVB3 group and the PM2.5-pretreated group. However, the PM2.5 exposure alone just induced the increase of TGF-b (p50.05) but not Foxp3. Compared with the CVB3 treatment alone, the PM2.5 + CVB3 treatment induced greater increase in the expressions of IL-6, TNF-a, TGF-b, and Foxp3 (p50.05). Cardiac protein expression of TGF- To further explore whether myocardial injury was exacerbated by PM2.5 exposure, the protein expressions of TGF-b were determined by Western blot (Figure 3A and B). The protein levels of cardiac TGF-b were elevated in the mice after exposure to PM2.5 (p50.05) or CVB3 (p50.01) when compared with the control. In addition, the expression of TGF-b protein was markedly higher in the PM2.5-pretreated group than that in the CVB3-treated group (p50.05). The levels of cytokines in sera The serum levels of inflammation-related cytokines IL-6, TNF-a, and TGF-b were detected (Figure 4). The PM2.5 treatment alone just induced the increase in IL-6 (p50.05). However, no similar result was observed in TNF-a and TGFb. When compared with the control, the CVB3 or PM2.5 + CVB3 exposure induced the increase of IL-6 and TGF-b. To TNF-a, there was a significantly increase in the PM2.5 + CVB3-treated mice (p50.05) but not in the CVB3and PM2.5-treated mice. In comparison, the levels of IL-6, TNF-a, and TGF-b in PM2.5-pretreated mice were significantly higher than those in the CVB3-treated mice.
Discussion Recently, the severe PM2.5 air pollution is paid more attention in China. In 2012, the Chinese government has established the air quality standard for PM2.5 (the 24-hour average standard is 75 mg/m3), which is around two times higher than the standard of American Environmental Protection Agency (EPA). Because of the limitation of technique and the huge difficult to control the PM2.5 pollution, the standard will be
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Figure 1. The percentages of CD4+ T cells and CD4+Foxp3+ cells (Treg) were investigated by flow cytometry in mice. Representative pictures for CD4+Foxp3+ cells gated on CD4+ T cells in the groups of spleen (n ¼ 8 for every group). Numbers in upper right quadrants indicate the percentages of CD4+Foxp3+ in the spleen (A). Statistical analysis showed the variation of CD4+ T cell and CD4+Foxp3+ cells (B). The differences among control, PM2.5 group, CVB3 group, and PM2.5 + CVB3 group were made by one-way analysis of variance (ANOVA). **p50.01, *p50.05 versus the control group. Significant difference (#p50.05, ##p50.01) between CVB3 and PM2.5+ CVB3 groups. Figure 2. The mRNA expression of IL-6 (A), TNF-a (B), TGF-b (C), and Foxp3 (D) in cardiac tissues was determined by real-time PCR (n ¼ 8). Quantification of these cytokines mRNA level normalized to GAPDH. Data are expressed as mean ± SD. **p50.01, *p50.05 versus the control group. Significant difference (#p50.05) between CVB3 and PM2.5 + CVB3 groups.
implemented in 2016. So the PM2.5 pollution is still a risk factor threatening people’s health. Because of its small size, ambient PM2.5 is possibly inhaled deeply into the lungs, with a portion depositing in the alveoli and potentially entering pulmonary circulation and presumably systemic circulation. However, the biological mechanisms linking PM2.5 and cardiovascular diseases are still unclear. In the past decades, researches focused on the mechanisms about the changes in heart rate (Pope et al., 1999), the changes of
cardiac function (Pekkanen et al., 2002), plasma viscosity (Peters et al., 1997), vasomotor dysfunction (Mills et al., 2007), and inflammatory response (Peters et al., 2001). Nevertheless, the immune mechanisms between ambient particulate matter and cardiovascular disease are paid little attention. The occurrence of viral myocarditis exhibits an increase in recent years in the world. Our previous study indicated that ambient PM2.5 could be a risk factor in exacerbating the
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DOI: 10.3109/08958378.2015.1040139
Figure 3. The protein expression of TGF-b in cardiac tissue. (A) Representative bands for protein expression of cardiac TGF-b in different groups (n ¼ 8). (B) Statistical analysis for the levels of TGF-b protein. **p50.01, *p50.05 versus the control group. Significant difference (#p50.05) between CVB3 and PM2.5 + CVB3 groups.
Figure 4. The levels of inflammation-related cytokines IL-6, TNF-a, and TGF-b in the sera of mice (n ¼ 8). One-way ANOVA was used to analyze the data. **p50.01, *p50.05 versus the control group. Significant difference (#p50.05) between CVB3 and PM2.5 + CVB3 groups.
viral myocarditis. The immune impairment involving the Th17 was associated with the development of myocarditis (Xie et al., 2013). Considering the relationship between Th17 and Treg cells, the current study further explored the role of Treg responses in viral myocarditis. The results found that pre-exposure to PM2.5 might induce the differentiation of Treg cells and in turn increase the expression of Treg-related cytokines, transcriptional factor, and inflammation-related cytokines in the spleen, serum, and cardiac tissue of mice, suggesting that ambient PM2.5 might impair the immune function and exacerbate the cardiac inflammatory response via mediate the Treg responses. Treg cells belong to a T cell subset that can inhibit effector T cell responses. Foxp3 is recognized as the exclusive marker of Treg cells. Treg cells are important for maintaining immune tolerance and protection from autoimmune diseases.
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The Treg cells exert their regulatory activity either by cellto-cell contact or by the release of suppressive cytokines such as TGF-b and interleukin-10 (IL-10). Previous study provided the evidence that the rise of Treg cells may indicate a defense against inflammatory manifestations and chronic infection in biomass users (Dutta et al., 2012). Therefore, the upregulation of Treg cell is not only a protective mechanism but also an external chemistry-induced immune impairment. In the current study, the increase of Treg cells suggested that ambient PM2.5 activated the differentiation of Treg cell in order to protect heart against intensive inflammatory response. A prior consistent finding showed that biomass users display a rise in Treg cells because they usually were exposed to biomass smoke during daily household cooking (Dutta et al., 2012). Moreover, a significant association between PAH exposure and impairment of immune function in both asthmatic and non-asthmatic was reported (Hew et al., 2014). However, this study showed a decrease of Treg cells, which might be explained by the different constituents between PAH and ambient PM2.5. Moreover, other study found that maternal tobacco smoke exposure during pregnancy correlates with a decrease of children’s cord blood Treg cell numbers and subsequent allergy risk (Herberth et al., 2014). The possible reason for the different results between our study and previous studies are as follows: first, the different components between ambient PM2.5 and tobacco smoke and PAH. The tobacco smoke is a much more complex mixture than PM2.5 and it mainly tends to induce allergic diseases (Callesen et al., 2014), whereas high concentration of PM2.5 susceptibly promotes the Treg cell differentiation and depresses the immune response. Meanwhile, the dose of PM2.5 and the subjects were obviously different. Second, the increase of the Treg cells and Treg-related cytokine TGF-b was associated with the immunosuppressive response, hereby our study showed an immunosuppressive response. Certainly, TGF-b was a regulatory cytokine with a pleiotropic function in T-cell development, homeostasis, and tolerance (Mesa et al., 2010). Treg cells played an important role in the regulation of T-cell-mediated immune responses through the suppression of T-cell proliferation and secretion of inhibitory cytokines, such as IL-10 and TGF-b (Foks et al., 2014). The maintenance of long-term Treg cell activation could contribute to the progressive loss of CD4+ T cell immune function and promote the development of myocarditis. Supporting our study, prior study reported that simian immunodeficiency virus (SIV) infection of Macaques induced an early immunosuppressive response that correlated with a marked increase in frequency of CD4+Foxp3+ (Treg) cells and TGF-b, indicating that Treg cells might possibly contribute to viral persistence by prematurely limiting the antiviral (Estes et al., 2006). Our results indicated that PM2.5 induced the increase of Treg cells may contribute to viral persistence by prematurely limiting the antiviral immune response.
Conclusion In summary, these finding suggested that PM2.5 may exacerbate the CVB3-induced increase of Treg responses, and the later may have a primary role in the failure of the host immune system to eradicate viral infection. The study
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indicated that PM2.5-induced increase of Treg cells could suppress the immune responses in turn cause the development of cardiac injury.
Declaration of interest The authors report that they have no conflicts of interests. This work was supported by grants from the National Natural Science Foundation of China (Nos. 81001229 and 81172617).
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