Inflammatory cytokines in vitro production are associated with Ala16Val superoxide dismutase gene polymorphism of peripheral blood mononuclear cells

Cytokine 60 (2012) 30–33

Contents lists available at SciVerse ScienceDirect

Cytokine journal homepage: www.journals.elsevier.com/cytokine

Short Communication

Inflammatory cytokines in vitro production are associated with Ala16Val superoxide dismutase gene polymorphism of peripheral blood mononuclear cells Marco Aurélio Echart Montano a,1, Ivana Beatrice Mânica da Cruz b,c,1, Marta Maria Medeiros Frescura Duarte d, Cristina da Costa Krewer c, Maria Izabel de Ugalde Marques da Rocha b, Maria Fernanda Mânica-Cattani c, Felix Alexandre Antunes Soares c, Guilherme Rosa e, Angélica Francesca Maris f, Francielle Garghetti Battiston a, Alexis Trott a,⇑, Juan Pablo Barrio Lera g a

Laboratory of Molecular Aspects Associated to Genetic Diseases, University of Western Santa Catarina, Unoesc, Brazil Department of Morphology, Federal University of Santa Maria, UFSM, Brazil c Center of Natural and Exact Sciences, Federal University of Santa Maria, UFSM, Brazil d Lutheran University of Brazil, Santa Maria, Brazil e Laboratory of Human Motricity Biosciences, UNIRIO, Brazil f Federal University of Santa Catarina, UFSC, Brazil g Institute of Biomedicine, University of Leon, Spain b

a r t i c l e

i n f o

Article history: Received 31 January 2012 Received in revised form 17 May 2012 Accepted 18 May 2012 Available online 9 June 2012 Keywords: PBMCs Metabolic dysfunctions SOD2 polymorphism Proinflammatory cytokines Obesity

a b s t r a c t Obesity is considered a chronic low-grade inflammatory state associated with a chronic oxidative stress caused by superoxide production (O2 ). The superoxide dismutase manganese dependent (SOD2) catalyzes O2 in H2O2 into mitochondria and is encoded by a single gene that presents a common polymorphism that results in the replacement of alanine (A) with a valine (V) in the 16 codon. This polymorphism has been implicated in a decreased efficiency of SOD2 transport into targeted mitochondria in V allele carriers. Previous studies described an association between VV genotype and metabolic diseases, including obesity and diabetes. However, the causal mechanisms to explain this association need to be more elucidated. We postulated that the polymorphism could influence the inflammatory response. To test our hypothesis, we evaluated the in vitro cytokines production by human peripheral blood mononuclear cells (PBMCs) carrier’s different Ala16Val-SOD2 genotypes (IL-1, IL-6, IL-10, TNF-a, IFN-c). Additionally, we evaluated if the culture medium glucose, enriched insulin, could influence the cytokine production. Higher levels of proinflammatory cytokines were observed in VV-PBMCs when compared to AA-PBMCs. However, the culture medium glucose and enriched insulin did not affect cytokine production. The results suggest that Ala16Val-SOD2 gene polymorphism could trigger the PBMCs proinflammatory cytokines level. However, discerning if a similar mechanism occurs in fat cells is an open question. Ó 2012 Elsevier Ltd. All rights reserved.

1. Introduction Chronic diseases such as obesity, type II diabetes and atherosclerosis are related to a chronic inflammatory state; the imbalance of inflammatory cytokines can affect cellular function [1]. ROS are capable of interfering with various redox-sensitive intracellular signal transduction cascades and regulating the expression of over

⇑ Corresponding author. Address: Laboratory of Molecular Aspects Associated with Genetic Diseases, University of Western Santa Catarina, Av. Oiapoc, 211, São Miguel do Oeste, 89900-000, SC, Brazil. Tel.: +55 49 36311075; fax: +55 49 36311000. E-mail address: [email protected] (A. Trott). 1 These authors contributed equally to this study. 1043-4666/$ - see front matter Ó 2012 Elsevier Ltd. All rights reserved. http://dx.doi.org/10.1016/j.cyto.2012.05.022

200 genes that lead, among other things, to the production of proinflammatory cytokines [2–4]. The superoxide radical is an important ROS constantly produced by the human metabolism; the level of this molecule is controlled by superoxide dismutase enzymes. The superoxide dismutase manganese dependent (SOD2 or MnSOD) activity occurs just into mitochondria whereas the other SODs (Cu/Zn dependent) present activity in cytosol and extracellular matrix. MnSOD dismutate the O2 to H2O2, which is then converted to non-toxic H2O and O2 by glutathione peroxidase (GPX1) and catalase enzymes [5]. The human MnSOD gene presents a polymorphism localized in an exon 2 Val16Ala variation (rs 4880) that results in a valine to alanine substitution, causing a conformational change from a b-sheet to an a-helix, inducing a 30–40% increase in

M.A.E. Montano et al. / Cytokine 60 (2012) 30–33

mitochondrial MnSOD activity [6]. The MnSOD Val16Ala variation has been reported to increase the risk of oxidative stress-related pathological conditions related to both homozygous genotypes (AA and VV) due a MnSOD imbalance. The V allele presents higher superoxide radical levels than the A allele due its lower efficiency to dismutate this molecule into H2O2 [6]. The VV genotype has been related to metabolic disorders and their complications such as diabetes or diabetic cardiovascular diseases [7,8], and diabetic nephropathy [9], retinopathy [10]an increase in plasmatic oxided-LDL levels [11] and in oxidative stress biomarkers, mainly in hypercholesterolemic patients [12]. An association between the VV genotype and obesity [13] was also observed in a previous study. A complementary in vitro study showed that when activated by phytohaemagglutinin (PHA), the T-cell carrier VV genotype presented lower viability and higher lipid peroxidation levels when compared to AA T-cells [14]. These results showed a potential influence of Ala16Val–MnSOD polymorphism on inflammatory immune response corroborating previous evidences that suggested SOD2 role on inflammation response [15]. Therefore, we hypothesized that a MnSOD imbalance could affect the in vitro lymphocyte proinflammatory cytokines production by PBMCs carriers, different Ala16Val–MnSOD genotypes in a regular culture medium, and a glucose and insulin-enriched culture medium. These treatments were performed because the PBMCs of metabolic disorders, such as those found in obese and diabetic patients, are more exposed to hyperglycemia and hyperinsulinemia states that can interfere with immune cell functions including cytokines productions [16]. 2. Materials and methods 2.1. Subjects and Ala16Val–MnSOD polymorphism analysis The research study is part of a project approved by the Ethics Committee of the Universidade Federal de Santa Maria. All blood cell donors signed a consent form. To perform this study we used a similar methodology described in Montagner et al. [14] and Costa et al. [17]. Briefly, we selected 200 healthy adults without cardiovascular medical history, hypertension, obesity or other metabolic diseases or morbidity that could affect the results. A DNA extraction was performed from the peripheral blood collection to perform the Ala16Val–MnSOD polymorphism genotyping using the Polymerase Chain Reaction followed by the Restriction Fragment Length Polymorphism (PCR-RFLP) techniques. From these volunteers, we also excluded those who were smokers, were using chronic medication, vitamin supplements or taking hormonal contraceptives. Because Montano et al. [13] showed an association between the Ala16Val polymorphism and dietary behaviour, we selected volunteers with similar lifestyle behaviours regarding diet and physical activity to avoid possible environmental interferences. Additionally, we asked volunteers to avoid consuming antioxidant-containing foods for 24 h before blood collection. The foods that were not consumed by volunteers included salads, fruits and juices. Three subjects of each genotype, who were considered to have the ideal profile, were selected as blood donors for the in vitro assays performed in this study. 2.2. PBMCs culture and treatments PBMCs were obtained from the blood of the volunteers by centrifugation with Ficoll–Histopaque to isolate the mononuclear cells. The PBMCs were immediately transferred to culture tubes at 5.0  104 cells concentration. RPMI 1640 supplemented with 10% fetal calf serum (FCS), PHA and 1% penicillin/streptomycin was used added to the PBMCs culture medium. The PBMCs were

31

maintained in a suspended culture medium at 37 oC in a 5% humidified CO2 atmosphere for 72 h. PBMCs carriers with different Ala16Val–MnSOD obtained from these cultures were centrifuged for 10 min (3 rpm) and transferred to tubes with an HBSS buffer containing 118 mM NaCl, 4.6 mM KCl, 10 mM D-glucose, 20 mM HEPES, 0.4 mM CaCl2, and pH 7.4. The cells were washed twofold with an HBSS buffer and transferred to the same buffer with and without glucose and insulin supplementation. The PBMCs with different genotypes were grouped in the follow treatments: HBSS buffer only, containing 10 mM D-glucose (H); HSBB enriched with an additional 5 mM glucose (G); HSBB enriched with 5 mU insulin (I) and finally, HSBB enriched with both glucose (5 mM) and insulin (5 mM) (GI). The PBMCs were maintained in these treatments for six hours. Since the same number of cells was placed in each treatment and we used a basic HSBB buffer as a medium to guarantee the survival of cells, we assume that in the period of treatment the cellular proliferation was low and cell viability was similar with minimal interference on the results. We tested this assumption from the lactate dehydrogenase enzyme evaluation measured by spectrophotometry. Additionally, we measured to discover if the glucose present in the HSBB buffer was differentially used by PBMC Ala16Val–MnSOD carriers. The glucose concentrations in the HSBB buffer medium and in the PBMCs were evaluated by spectrophotometry assay using a commercial kit (Glucose PAP Labtest, Lagoa Santa, Brazil). The final glucose level that indirectly indicates glucose utilization from the medium buffer culture was calculated by subtracting the glucose value obtained after six hours by the glucose measure before the treatments exposition. The results were transformed by percentage considering as 100% the glucose level found in basal moment (0 h). During a six hour period of treatments, we analysed the levels of the proinflammatory cytokines interleukin 1 (IL-1), interleukin 6 (IL-6), tumor necrosis factor alpha (TNF-a), gamma interferon (IFN-c) using the ELISA capture, according to the manufacturer’s instructions (Biomyx Technology, San Diego, CA). 2.3. Statistics analysis All analyses were carried out using the SPSS statistical software, version 18.0 (SPSS Inc., Chicago, IL). The results were presented in mean ± standard deviation. The LDH, cytokine, as well as glucose levels were compared among different PBMC Ala16Val–MnSOD carriers with or without HSBB glucose and insulin, were compared among genotypes using an analysis of two-way variance followed by Tukey post hoc test. All p values were two-tailed. The alpha value considered to be statistically significant was p = 0.05. 3. Results and discussion Initially, we sought to analyze LDH enzyme activity, to evaluate the treatment’s effect on PBMCs viability. We found no difference in the LDH levels among treatments before or after six hours (LDL Mean in 0 h = 7.3 ± 1.0 U/l; LDL Mean in 6 h = 7.8 ± 1.7U/l). Therefore, we assumed that the allotted time for treatment, as well as the glucose and insulin supplementation added in HSBB medium, did not induce a toxicity state. The glucose level in the PBMCs carrier is different Ala16Val– MnSOD genotypes with and without medium glucose and enriched insulin were also measured (Fig. 1). The glucose levels were similar among PBMCs carriers different from Ala16Val–MnSOD genotypes in a medium without glucose and insulin supplementation. However in the presence of these molecules the percentage of glucose

32

M.A.E. Montano et al. / Cytokine 60 (2012) 30–33 Table 1 Comparison of cytokines levels among peripheral blood mononuclear cells with different Ala16Val-SOD2 genotypes.

Fig. 1. The percentage of glucose after a six-hour cell culture with and without glucose and insulin supplementation of PBMCs carriers is different from the Ala16Val SOD2 genotypes (AA, AV, VV). H = culture without glucose/insulin supplementation; G = glucose supplementation (5 mM); I = insulin supplementation (5 mM), GI = glucose plus insulin supplementation. For each PBMCs genotype, different letters (a b c) indicate significant differences at p > 0.001 from the analysis of the variance followed by the Tukey post hoc test.

after 6 h compared to basal glucose (0 h) was differentially modified in the same ways as Ala16Val genotypes (p < 0.0001). AA-PBMCs and VV-PBMCs presented higher glucose levels when enriched with glucose than cells with and without insulin and glucose plus insulin. Nevertheless, in the heterozygous PBMCs, the glucose supplementation (with and without insulin) decreased the percentage of glucose after 6 h. Despite the methodological limitation in the glucose measurement performed here by a spectrophotometry assay, our data suggest that the PBMCs mitochondrial oxidative metabolism present some regulation effect on energetic metabolism when more glucose occurs and with more insulin availability to the cell. There is a close relation between energetic metabolism that produces ATP from glucose and oxygen and the production of ROS (superoxide ion) that is metabolized by the SOD2 enzyme. As the PBMCs did not need to present GLUT receptors to obtain glucose of medium we can postulated that the impairment in the SOD2 efficiency observed in the V allele could to affect the glucose use for the cells. However, the mechanism that this phenomenon occurs is an open question. The SOD2 is a key enzyme that protects the energy-generating mitochondria from oxidative damage. Therefore, the potential involvement of oxidative metabolism from differential SOD2 efficiency genetically determined on cell energetic metabolism corroborate previous investigations that described effects of nutritional status on mitochondrial physiology of PBMCs especially lymphocytes [18,19]. These studies also consider that nutritional diseases such as obesity, hyperglycaemia, and insulin resistance affect directly the lymphocyte mitochondrial oxidative phosphorylation and other mitochondrial metabolic responses. Our results also corroborate the potential involvement of Ala16Val SOD2 polymorphism on susceptibility risk to developed obesity, diabetes and their metabolic alterations, like LDL oxidized and triglycerides [11,13,20] and clinical complications as retinopathy [10] previously described in the literature. We also analyzed the potential effect of Ala16Val SOD2 polymorphism on the production of PBMCs cytokines involving

Variables (pg/ml)

Genotypes

Interleukin 1

AA VV AV Total

Mean ± SD 23.9 ± 1.8a 99.0 ± 2.0b 55.9 ± 2.5c 59.6 ± 32.7

Interleukin 6

AA VV AV Total

76.0 ± 2.1a 122.7 ± 2.6b 87.3 ± 2.5c 95.3 ± 2.2

TNF-a

AA VV AV Total

81.1 ± 3.0a 148.3 ± 2.6b 100.0 ± 2.0c 99.9 ± 13.4

IFN-c

AA VV AV Total

80.0 ± 2.0a 199.9 ± 1.1b 158.3±,2.5c 146.0 ± 52.1

Interleukin 10

AA VV AV Total

55.6 ± 2.5a 34.6 ± 2.5b 46.7 ± 2.6c 48.3 ± 10.6

AA, AV and AV = genotypes from Ala16Val SOD2 polymorphism; SD = standard deviation; different letters (a, b, c) indicated significant differences evaluated by Anova One-way followed by Tukey post hoc test. P < 0.0001. Number of subjects analyzed of each genotype: 03.

an inflammatory response. As seen in Table 1, the PBMCs carrier VV genotype presented higher levels of proinflammatory cytokines IL-1, IL-6, TNF-a, IFN-c the level of IL-10 antiinflammatory cytokine was lower in these cells when compared to PBMCs carrier AA genotype. The PBMCs carrier’s heterozygous genotypes presented intermediary cytokine values when compared to AA and VV genotypes indicating the co-dominance nature of this genetic trait on the cytokine levels measure here. Superoxide anions have a proinflammatory role in many diseases and past studies have reported that SOD2 has the potential to be used as an antiinflammatory agent because of its superoxide anion scavenging ability [21]. Our data corroborate the evidences that the suppressive effects of SOD2 are related to inflammatory diseases [15]. Therefore, subjects with less SOD2 efficiency might present more susceptibility to establish the inflammatory process related to the development and maintenance of diseases such as obesity and diabetes. However, if the less efficiency of SOD2, genetically determined by Ala16Val polymorphism presents potential pharmacogenetic effects on the treatment response of inflammatory diseases like obesity, it is an open question that needs to be explored in future studies. Acknowledgments This work was supported by grants and fellowships from CNPq (Nos. 471233/2007-2, 311231/2006-3), CAPES and FAPERGS. References [1] Fitzgerald KA. NLR-containing inflammasomes: central mediators of host defense and inflammation. Eur J Immunol 2010;40:595–653. [2] Min KJ, Jou I, Joe E. Plasminogen-induced IL-1beta and TNF alpha production in microglia is regulated by reactive oxygen species. Biochem Biophys Res Commun 2003;312:969–74. [3] Chen XH, Zhao YP, Xue M, Ji CB, Gao CL, Zhu JG. TNF-alpha induces mitochondrial dysfunction in 3T3-L1 adipocytes. Mol Cell Endocrinol 2010;328:63–9. [4] Kaminski KA, Bonda TA, Korecki J, Musial WJ. Oxidative stress and neutrophil activation—the two keystones of ischemia/reperfusion injury. Int J Cardiol 2002;86:41–59.

M.A.E. Montano et al. / Cytokine 60 (2012) 30–33 [5] Newsholme P, Haber EP, Hirabara SM, Rebelato EL, Procopio J, Morgam D, et al. Diabetes associated cell stress and dysfunction: role of mitochondrial and nonmitochondrial ROS production and activity. J Phys 2007;583:9–24. [6] Sutton A, Khoury H, Prip-Buus C, Cepanec C, Pessayre D, Degoul F. The Ala16Val genetic dimorphism modulates the import of human manganese superoxide dismutase into rat liver mitochondria. Pharmacogenetics 2003;13:145–57. [7] Chen H, Yu M, Li M, Zhao R, Zhu Q, Zhou W, et al. Polymorphic variations in manganese superoxide dismutase (MnSOD), glutathione peroxidase-1 (GPX1), and catalase (CAT) contribute to elevated plasma triglyceride levels in Chinese patients with type 2 diabetes or diabetic cardiovascular disease. Mol Cell Biochem 2011. http://dx.doi.org/10.1007/s11010-011-1160-3. [8] Jones DA, Prior SL, Tang TS, Bain SC, Hurel SJ, Humphries SE, et al. Association between the rs4880 superoxide dismutase 2 (C > T) gene variant and coronary heart disease in diabetes mellitus. Diabetes Res Clin Pract 2010;90:196–201. [9] Mollsten A, Marklund SL, Wessman M, Svensson M, Forsblom C, Parkkonen M, et al. A functional polymorphism in the manganese superoxide dismutase gene and diabetic nephropathy. Diabetes 2007;56:265–9. [10] Kangas-Kontio T, Vavuli S, Kakko SJ, Penna J, Savolainen ER, Savolainen MJ, et al. Polymorphism of the manganese superoxide dismutase gene but not of vascular endothelial growth factor gene is a risk factor for diabetic retinopathy. Br J Ophthalmol 2009;93:1401–6. [11] Gottlieb MG, Schwanke CH, Santos AF, Jobim PF, Müssel DP, Cruz IBM. Association among oxidized LDL levels, SOD2, apolipoprotein E polymorphisms, and cardiovascular risk factors in a south Brazilian region population. Genet Mol Res 2005;4:691–703. [12] Duarte MM, Moresco RN, Duarte T, Santi A, Bagatini MD, Da Cruz IB, et al. Oxidative stress in hypercholesterolemia and its association with Ala16Val superoxide dismutase gene polymorphism. Clin Biochem 2010;43:1118–23. [13] Montano MA, Barrio Lera JP, Gottlieb MG, Schwanke CH, da Rocha MI, ManicaCattani MF, et al. Association between manganese superoxide dismutase

[14]

[15] [16] [17]

[18]

[19]

[20]

[21]

33

(SOD2) gene polymorphism and elderly obesity. Mol Cell Biochem 2009;328:33–40. Montagner GFF, Sagrillo M, Machado MM, Almeida RC, Mostardeiro CP, Duarte MFF, et al. Toxicological effects of ultraviolet radiation on lymphocyte cells with different manganese superoxide dismutase Ala16Val polymorphism genotypes. Toxicol In Vitro 2010;24:1410–6. Li C, Zhou HM. The role of manganese superoxide dismutase in inflammation defense. Enzyme Res 2011;2011:387176. Fernández-Real JM, Pickup JC. Innate immunity, insulin resistance and type 2 diabetes. Diabetologia 2012;55:273–8. Costa F, Dornelles E, Mânica-Cattani MF, De Souza Filho OC, Sagrillo MR, Garcia LF, da Cruz IB. Influence of Val16Ala SOD2 polymorphism on the in vitro effect of clomiphene citrate in oxidative metabolism. Reprod Biomed 2012;24:474–81. Cortez E, Neves FA, Bernardo AF, Stumbo AC, Carvalho L, Garcia-Souza E, et al. Lymphocytes mitochondrial physiology as biomarker of energy metabolism during fasted and fed conditions. Scient World J 2012;2012:629326. Briet F, Twomey C, Jeejeebhoy KN. Relationship between metabolism and peripheral blood mononuclear cell mitochondrial complex I activity before and after a short-term refeeding in weight-losing cancer patients. Clin Nutr 2003;22:247–53. Nakanishi S, Yamane K, Ohishi W, Nakashima R, Yoneda M, Nojima H, et al. Manganese superoxide dismutase Ala16Val polymorphism is associated with the development of type 2 diabetes in Japanese–Americans. Diabetes Res Clin Pract 2008;81:381–5. Pani G, Colavitti R, Bedogni B, Fusco S, Ferraro D, Borrello S, et al. Mitochondrial superoxide dismutase: a promising target for new anticancer therapies. Curr Med Chem 2004;11:1299–308.