Novel DNA Methylation Sites Influence GPR15 Expression In

biomolecules

Article Novel DNA Methylation Sites Inﬂuence GPR15 Expression in Relation to Smoking

Tina Haase 1,2, Christian Müller 1,2, Julia Krause 1,2, Caroline Röthemeier 1, Justus Stenzig 2,3, Sonja Kunze 4,5, Melanie Waldenberger 2,4,5, Thomas Münzel 2,6,7,8, Norbert Pfeiffer 9, Philipp S. Wild 2,8,10,11, Matthias Michal 12, Federico Marini 13 ID , Mahir Karakas 1,2, Karl J. Lackner 2,14, Stefan Blankenberg 1,2 and Tanja Zeller 1,2,* 1 Clinic for General and Interventional Cardiology, University Heart Center Hamburg, 20246 Hamburg, Germany; [email protected] (T.H.); [email protected] (C.M.); [email protected] (J.K.); [email protected] (C.R.); [email protected] (M.K.); [email protected] (S.B.) 2 German Centre for Cardiovascular Research (DZHK), 13316 Berlin, Germany; [email protected] (J.S.); [email protected] (M.W.); [email protected] (T.M.); [email protected] (P.S.W.); [email protected] (K.J.L.) 3 Institute of Experimental Pharmacology and Toxicology, University Medical Center Hamburg-Eppendorf (UKE), 20246 Hamburg, Germany 4 Research Unit of Molecular Epidemiology, Helmholtz Zentrum München, German Research Center for Environmental Health, 85764 Neuherberg, Germany; [email protected] 5 Institute of Epidemiology II, Helmholtz Zentrum München, German Research Center for Environmental Health, 85764 Neuherberg, Germany 6 Center for Cardiology, Cardiology I, University Medical Center Mainz, Johannes Gutenberg University-Mainz, 55131 Mainz, Germany 7 Center for Thrombosis and Hemostasis, University Medical Center Mainz, Johannes Gutenberg-University Mainz, 55131 Mainz, Germany 8 Center for Translational Vascular Biology (CTVB), University Medical Center Mainz, Johannes Gutenberg-University Mainz, 55131 Mainz, Germany 9 Department of Ophthalmology, University Medical Center of the Johannes Gutenberg-University Mainz, 55131 Mainz, Germany; [email protected] 10 Preventive Cardiology and Preventive Medicine, Center for Cardiology, University Medical Center of the Johannes Gutenberg-University Mainz, 55131 Mainz, Germany 11 Center for Thrombosis and Hemostasis, University Medical Center of the Johannes Gutenberg-University Mainz, 55131 Mainz, Germany 12 Department of Psychosomatic Medicine and Psychotherapy, University Medical Center of the Johannes Gutenberg-University Mainz, 55131 Mainz, Germany; [email protected] 13 University Medical Center, Institute of Medical Biostatistics, Epidemiology and Informatics (IMBEI), 55131 Mainz, Germany; [email protected] 14 Institute of Clinical Chemistry and Laboratory Medicine, University Medical Center, Johannes Gutenberg University Mainz, 55131 Mainz, Germany * Correspondence: [email protected]; Tel.: +49-(0)-40-7410-56575

Received: 16 July 2018; Accepted: 9 August 2018; Published: 20 August 2018

Abstract: Smoking is a major risk factor for cardiovascular diseases and has been implicated in the regulation of the G protein-coupled receptor 15 (GPR15) by affecting CpG methylation. The G protein-coupled receptor 15 is involved in angiogenesis and inflammation. An effect on GPR15 gene regulation has been shown for the CpG site CpG3.98251294. We aimed to analyze the effect of smoking on GPR15 expression and methylation sites spanning the GPR15 locus. DNA methylation of nine GPR15 CpG sites was measured in leukocytes from 1291 population-based individuals using the EpiTYPER. Monocytic GPR15 expression was measured by qPCR at baseline and five-years follow up. GPR15 gene expression was upregulated in smokers (beta (ß) = −2.699, p-value (p) = 1.02 × 10−77) and strongly correlated with smoking exposure (ß = −0.063, p = 2.95 × 10−34). Smoking cessation within five years reduced GPR15 expression about 19% (p = 9.65 × 10−5) with decreasing GPR15 expression

Biomolecules 2018, 8, 74; doi:10.3390/biom8030074 www.mdpi.com/journal/biomolecules Biomolecules 2018, 8, 74 2 of 10

over time (ß = 0.031, p = 3.81 × 10−6). Additionally, three novel CpG sites within GPR15 affected by smoking were identified. For CpG3.98251047, DNA methylation increased steadily after smoking cessation (ß = 0.123, p = 1.67 × 10−3) and strongly correlated with changes in GPR15 expression (ß = 0.036, p = 4.86 × 10−5). Three novel GPR15 CpG sites were identified in relation to smoking and GPR15 expression. Our results provide novel insights in the regulation of GPR15, which possibly linked smoking to inflammation and disease progression.

Keywords: GPR15; smoking; biomarker; DNA methylation

1. Introduction Cigarette smoking severely increases the risk for life-threatening diseases such as cardiovascular diseases, cancer, and chronic respiratory diseases with over 7 million deaths attributed to tobacco worldwide [1]. Cigarette smoke contains more than 5000 compounds with many of them, upon entering the blood stream, negatively affecting organs and cells [2]. However, the exact molecular mechanisms showing how tobacco substances influence disease onset and progression remain elusive. Within the last several years, the field of epigenetics has emerged and has provided new approaches and insights into the regulation of gene expression. It was shown that the modification of a cytosine followed by a guanine nucleotide (CpG) to 5-methylcytosine in the gene promoter region may result in a decrease of transcriptional activity of the corresponding gene [3]. Several studies have been published by focusing on the effect of cigarette smoke on methylation and changes in methylation status in response to smoking have been shown for several genes such as AHRR and F2RL3 [4,5]. These genes have been implicated in cell proliferation, immune response, and detoxification, which can contribute to the pathogenesis of smoking-associated diseases [6]. The CpG site CpG3.98251294 was identified in relation to smoking within the GPR15 gene, which encodes the G protein-coupled receptor 15 [7–9]. Smoking was associated with decreased CpG3.98251294 methylation and increased G protein-coupled receptor 15 gene (GPR15) expression [8–11]. This effect was slowly reversible after smoking cessation [7,11,12]. Additionally, CpG3.98251294 methylation correlated with increased cumulative smoking exposure [13]. The transmembrane receptor GPR15 is mainly expressed on T cells and acts as a coreceptor for human immunodeficiency virus and simian immunodeficiency virus [14–16]. The G protein-coupled receptor 15 has also been shown to be involved in angiogenesis and inflammation [17–22], which indicated a role of GPR15 in smoking-associated diseases such as cardiovascular diseases and cancer. Hence, GPR15 poses an interesting new candidate gene of smoking-related diseases and smoking-induced molecular pathways. However, to date, the influence of smoking on only one CpG site (CpG3.98251294) within the GPR15 gene region has been described [7–11,13]. The aim of the present study was to analyze the association between smoking, GPR15 methylation and expression and to gain further knowledge of additional DNA methylation sites spanning the GPR15 locus. This study identified three novel DNA methylation sites within the GPR15 locus related to smoking as well as GPR15 expression.

2. Materials and Methods

2.1. Study Participants In this study, 1291 individuals in the population-based Gutenberg Health Study were analyzed [23]. RNA and DNA were collected, as described previously [24]. Self-reported smoking statuses were classiﬁed as follows: current smokers (including occasional smokers), ex smokers (smoking cessation at least six weeks before study participation), and never smokers. Cumulative smoking exposure was evaluated by pack years (one pack year = smoking of 20 cigarettes per day Biomolecules 2018, 8, 74 3 of 10 for one year) for current smokers and ex smokers. For longitudinal gene expression (follow up), participants visited the same study center ﬁve years after baseline recruitment. Written informed consent was obtained from all study participants. The study protocols and sampling design were approved by the local ethics committee of the Medical Chamber of Rhineland-Palatinate, Germany (ethical approval code 837.020.07 (5555)).

2.2. RNA and DNA Isolation Monocytic RNA was isolated as described previously [24]. Blood was collected using the Vacutainer CPT Cell Preparation Tube System (BD Biosciences, San Jose, CA, USA) and monocytes were enriched by negative selection with the Rosette Sep Monocyte Enrichment Cocktail (StemCell Technologies, Vancouver, BC, Canada), which leads to 72% to 85% of enriched monocytes. Total RNA was isolated using Trizol extraction and puriﬁcation by silica-based columns. Genomic DNA from leukocytes was extracted as described by Miller et al. from buffy-coated ethylenediamine tetraacetic acid blood samples [24,25].

2.3. Analysis of GPR15 Gene Expression Monocytic GPR15 mRNA expression was measured by real-time quantitative polymerase chain reaction (qPCR) using the 7900 TaqMan system (Applied Biosystems, Vancouver, BC, Canada). RNA was reverse transcribed using High-Capacity cDNA Reverse Transcription Kit (Applied Biosystems), according to manufacturers’ protocols. For GPR15 gene expression analyses, the GPR15 Hs00922903_s1 gene expression assay was used (Applied Biosystems). Quantiﬁcation of the housekeeping gene GAPDH (Hs99999905_m1, Applied Biosystems) as an internal control was performed for each sample. Expression of GPR15 mRNA was normalized to GAPDH mRNA expression by calculating ∆Ct values.

2.4. Analysis of DNA Methylation Analysis of GPR15 DNA methylation was carried out with the EpiTYPER MassARRAY technology (Agena Bioscience, San Diego, CA, USA), according to the manufacturer’s protocols, which allowed region-specific quantification of CpG sites [26]. Genomic DNA was bisulfite modified to convert un-methylated cytosines into uracils. One µg of DNA was treated with sodium bisulfite using the EZ methylation kit (Zymo Research, Irvine, CA, USA), according to the manufacturers’ protocols. Ten ng of bisulfite DNA was amplified by polymerase chain reaction (PCR) using the following primers: 50-aggaagagagGTTTTTTGGTGATGGATTTAGAAGA-30 and 50-cagtaatacgactcactatagggag aaggctTAAACAAAAAAATAAACAACCCCAA-30, which results in a 523 bp-fragment. Subsequently, DNA was reverse transcribed, fragmented, and analyzed by mass spectrometry. The GPR15 gene includes 15 CpG sites. Nine of these CpG sites (CpG3.98250924 to CpG3.98251294) were located within the 523 bp-fragment and were labeled, according to Saffery et al. [27]. Six of these CpG sites could be analyzed. Due to the low mass of the cleavage product, which leads to unreliable methylation values, CpG3.98250924 and CpG3.98251081 could not be covered by the assay. CpG3.98251268 was not included in the analyses since it was fully methylated in 90% of the samples. CpG3.98251294 corresponds to the previously described cg19859270 site. The GPR15 locus including the CpG sites CpG3.98250924 to CpG3.98251294 is depicted in Figure S1. More detailed information on the mass fragments detected by the EpiTYPER are given in Table S1.

2.5. Statistical Analysis Associations between cigarette smoking status, GPR15 mRNA expression, and GPR15 DNA methylation were calculated by linear mixed regression models and adjusted for age and sex. Changes in GPR15 gene expression between the baseline and a five-years follow up visit were analyzed by a paired t-test. The threshold for statistical significance was set at p-value (p ≤ 0.05). Bonferroni correction was performed for linear mixed regression models to adjust for multiple testing. Statistical Biomolecules 2018, 8, 74 4 of 10 analyses were performed and figures were prepared using the R version 3.4.3 [28] and GraphPad prism version 6.05 for Windows (GraphPad Software, La Jolla, CA, USA). Biomolecules 2018, 8, x FOR PEER REVIEW 4 of 10 3. Results 3. Results 3.1. Characteristics of the Study Population 3.1. Characteristics of the Study Population Baseline characteristics of the study population are provided in Table1. Out of the 1291 individuals, Baseline characteristics of the study population are provided in Table 1. Out of the 1291 46% were never smokers, 37% were ex smokers, and 17% were current smokers. Smoking cessation was individuals, 46% were never smokers, 37% were ex smokers, and 17% were current smokers. 18.6 years for ex smokers when calculating the mean value. Current smokers differed from never smokers Smoking cessation was 18.6 years for ex smokers when calculating the mean value. Current smokers in termsdiffered of sex from (8% never women smokers compared in terms to 27%)of sex and (8% hypertension women compared (6% compared to 27%) and to hypertension 23%). (6% compared to 23%). Table 1. Characteristics of study individuals. Table 1. Characteristics of study individuals. Characteristic All Never Smokers Ex Smokers Current Smokers Never Ex Current Characteristic All n (%) 1291 593 (46)Smokers 477Smokers (37) 221Smokers (17) Females, n (%) 643 (50) 354 (27) 187 (15) 102 (8) n (%) 1291 593 (46) 477 (37) 221 (17) Age, years 55 (46–64) 57 (45–66) 57 (48–65) 50 (45–57) Females, n (%) 643 (50) 354 (27) 187 (15) 102 (8) Time since quitting, 18 (7–28) NA 18 (7–28) NA yearsAge, years 55 (46–64) 57 (45–66) 57 (48–65) 50 (45–57) TimePack since years quitting, years 0.1 (0.0–3.6) 18 (7–28) NA NA 1.6 (0.6–3.7)18 (7–28) 23.1 (11.7–36.0) NA ContinuousPack variables years are described by 0.1 median (0.0–3.6) values (25th percentile NA to 75th1.6 percentile). (0.6–3.7) Dichotomous 23.1 (11.7–36.0) variables are presentedContinuous as total variables numbers are (%). described NA = data by not median available. values (25th percentile to 75th percentile). Dichotomous variables are presented as total numbers (%). NA = data not available 3.2. Smoking Increases GPR15 mRNA Expression 3.2. Smoking Increases GPR15 mRNA Expression Gene expression was measured to determine the effect of smoking status on GPR15 expression. Gene expression was measured to determine the effect of smoking status on GPR15 expression. − × −77 GPR15GPR15mRNA mRNA expression expression strongly strongly correlated correlated with with current current smoking smoking (beta(beta (ß) = −2.699,2.699, p =p 1.02= 1.02 × 10−7710) ) and wasand 7.6-foldwas 7.6-fold higher higher in current in current smokers smokers when comparedwhen compared to never to smokers never smokers (Figure1 a).(Figure Furthermore, 1a). the effectFurthermore, of cumulative the effect smoking of cumula exposuretive smoking as indicated exposure as by indicated the number by the of number pack years of pack significantly years correlatedsignificantly with GPR15 correlatedmRNA with expressionGPR15 mRNA levels expression (ß = − levels0.0631, (ß p= =−0.0631, 5.95 × p 10= 5.95−34 )× (Figure10−34) (Figure1b). 1b).

(a) (b)

FigureFigure 1. Smoking 1. Smoking increases increases G G protein-coupled protein-coupled receptorreceptor 15 15 (GPR15 (GPR15) mRNA) mRNA expression. expression. (a) GPR15 (a) GPR15 mRNA expression is depicted for never smokers, ex smokers, and current smokers. It was mRNA expression is depicted for never smokers, ex smokers, and current smokers. It was signiﬁcantly significantly associated with current smoking (beta (ß) = −2.699, p-value (p) = 1.02 × 10−77). n = 191–542. associated with current smoking (beta (ß) = −2.699, p-value (p) = 1.02 × 10−77). n = 191–542; (b) GPR15 (b) GPR15 mRNA expression levels depending on cumulative smoking exposure are plotted for mRNAcurrent expression smokers levels and ex depending smokers combined. on cumulative They smokingcorrelated exposuresignificantly are with plotted the number for current of pack smokers and exyears smokers smoked combined. (ß = –0.0631, They p = 5.95 correlated × 10−34). One signiﬁcantly pack equals with smoking the number20 cigarettes ofpack per day years for one smoked −34 (ß = –0.0631,year. n =p 6-509.= 5.95 The× 10linear ).mixed One regression pack equals model smoking was adjusted 20 cigarettes for age per and day sex. for Box one plot year. whiskersn = 6-509. The linearrepresent mixed 10–90th regression percentile. model GPR15 was mRNA adjusted expression for age is depicted and sex. as BoxΔCt values plot whiskers normalized represent to 10–90thGAPDH percentile. mRNAGPR15 expression.mRNA Lower expression ΔCt values is depictedindicate higher as ∆Ct GPR15 values mRNA normalized expression. to GAPDH mRNA expression. Lower ∆Ct values indicate higher GPR15 mRNA expression. 3.3. Smoking Cessation Alters GPR15 Expression To determine the longitudinal effect of smoking behavior on GPR15 expression, gene expression levels at baseline and after a follow up time of five years were compared. Smokers who quit smoking Biomolecules 2018, 8, 74 5 of 10

3.3. Smoking Cessation Alters GPR15 Expression To determine the longitudinal effect of smoking behavior on GPR15 expression, gene expression levels atBiomolecules baseline 2018 and, 8, x after FOR PEER a follow REVIEW up time of five years were compared. Smokers who quit5 of 10 smoking within five years (n = 39) showed a significant decrease in GPR15 expression (ß = 1.182, p = 9.65 × 10−5) in Figurewithin2a, which five years indicated (n = 39) showed that the a changesignificant in decrease smoking in GPR15 behavior expression affected (ß =GPR15 1.182, p mRNA= 9.65 × 10 expression.−5) Furthermore,in Figure the 2a, time which since indicated smoking that the cessation change in significantly smoking behavior correlated affectedwith GPR15GPR15 mRNAmRNA expression. expression Furthermore, the time since smoking cessation significantly correlated with GPR15 mRNA (ß = 0.031, p = 3.81 × 10−6) (Figure2b), which shows that, within the first years after smoking cessation, expression (ß = 0.031, p = 3.81 × 10−6) (Figure 2b), which shows that, within the first years after smoking GPR15 mRNAcessation, expression GPR15 mRNA decreased expression more decreased rapidly more (Figure rapidly S2). (Figure S2).

(a)

(b)

Figure 2. Effect of smoking cessation on G protein-coupled receptor 15 (GPR15) mRNA expression. Figure 2. Effect of smoking cessation on G protein-coupled receptor 15 (GPR15) mRNA expression. (a) GPR15 mRNA expression was compared between baseline (BL) and the five-years follow up (FU) GPR15 (a) visit.mRNA Quitting expression smoking significantly was compared decreased between GPR15 mRNA baseline expression (BL) and about the 19% five-years (p) = 9.65 follow × 10−5). up (FU) −5 visit. Quittingn = 39, paired smoking t-test. significantly (b) Time course decreased of GPR15 GPR15mRNA expressionmRNA expression after smoking about cessation. 19%( Timep) = 9.65since× 10 ). n = 39, pairedsmokingt -test.cessation (b) significantly Time course correlated of GPR15 withmRNA GPR15 mRNA expression expression after (ß smoking = 0.031, p cessation. = 3.81 × 10−6 Time). n since smoking= 20–186, cessation linear significantly mixed regression correlated model withadjustedGPR15 for agemRNA and sex. expression Box plot whiskers (ß = 0.031, representp = 3.81 10–× 10−6). n = 20–186,90th percentile. linear mixed GPR15 regression mRNA expression model is adjusted depicted as for Δ ageCt values and normalized sex. Box plotto GAPDH whiskers mRNA represent expression. Lower ΔCt values indicate higher GPR15 mRNA expression. 10–90th percentile. GPR15 mRNA expression is depicted as ∆Ct values normalized to GAPDH mRNA expression.3.4. Novel LowerGPR15 ∆DNACt values Methylation indicate Sites higher AssociatedGPR15 withmRNA Smoking expression. In previous studies, only the GPR15 CpG3.98251294 site had been described in relation to 3.4. Novel GPR15 DNA Methylation Sites Associated with Smoking smoking and GPR15 expression [7–11,13]. To enhance the understanding of the molecular Inmechanisms previous studies,regulating only GPR15 the expressionGPR15 CpG3.98251294 and to identify new site GPR15 hadbeen CpG describedsites in relation in relation to to smoking, DNA methylation of nine additional CpG sites within the GPR15 gene was measured. smoking and GPR15 expression [7–11,13]. To enhance the understanding of the molecular mechanisms Out of nine sites, DNA methylation of CpG3.98251047, CpG3.98251179, and CpG3.98251219 regulatingshowedGPR15 strongexpression associations andwith tosmoking identify (ß = new−3.376,GPR15 p = 1.37CpG × 10−6; sites ß = −4.655, in relation p = 1.78 to× 10 smoking,−4; and ß DNA methylation= −3.609, of ninep = 2.24 additional × 10−3, respectively) CpG sites within(Figure the3). ForGPR15 sitesgene CpG3.98251063, was measured. CpG3.98251070, and OutCpG3.98251294, of nine sites, no DNAassociation methylation with the smoking of CpG3.98251047, status was identified CpG3.98251179, in our analyses. and CpG3.98251219 showed strong associations with smoking (ß = −3.376, p = 1.37 × 10−6; ß = −4.655, p = 1.78 × 10−4; and ß = −3.609, p = 2.24 × 10−3, respectively) (Figure3). For sites CpG3.98251063, CpG3.98251070, and CpG3.98251294, no association with the smoking status was identified in our analyses. Biomolecules 2018, 8, 74 6 of 10 BiomoleculesBiomolecules 2018 2018, 8, ,8 x, x FOR FOR PEER PEER REVIEW REVIEW 66 of of 10 10

Figure 3. Decreased G protein-coupled receptor 15 (GPR15) DNA methylation in smokers. Methylation FigureFigure 3.3. DecreasedDecreased GG protein-coupledprotein-coupled receptorreceptor 15 (GPR15) DNA methylationmethylation inin smokers.smokers. GPR15 of MethylationMethylationDNA negatively of of GPR15 GPR15 DNA correlatedDNA negativelynegatively with correlated smokingcorrelated inwithwith the smokin threeg newly in the identiﬁed threethree newlynewly sites identifiedidentified CpG3.98251047, sites sites CpG3.98251179,CpG3.98251047,CpG3.98251047, and CpG3.98251219CpG3.98251179,CpG3.98251179, and (andn = CpG3.98251219CpG3.98251219 114–582). Linear (n = mixed 114–582). regression LinearLinear mixedmixed model regressionregression adjusted model formodel age and sex withadjustedadjusted a signiﬁcance for for age age and and threshold sex sex with with ofaa significancesignificance 0.0083 after threthre Bonferronisholdshold of 0.0083 correction, after BonferroniBonferroni the box plot correction,correction, whiskers the the representbox box 10–90thplotplot percentile. whiskers whiskers represent represent 10–90th 10–90th percentile. percentile.

AsAs expected, expected, followingfollowing thethe decreasedecrease inin GPR15, mRNA expressionexpression afterafter smokingsmoking cessationcessation As(Figure expected, 2) and followingGPR15 DNA the methylation decrease at insiteGPR15 CpG3.98251047, mRNA co expressionrrelated with aftertime since smoking quitting cessation in (Figure 2) and GPR15 DNA methylation at site CpG3.98251047 correlated with time since quitting in (Figureex2) smokers and GPR15 (ß = 0.123,DNA p = methylation 1.67 × 10−3) (Figure at site 4), CpG3.98251047 which showed increasing correlated levels with of timeGPR15 since methylation. quitting in ex ex smokers (ß = 0.123, p = 1.67 × 10−3) (Figure 4), which showed increasing levels of GPR15 methylation. For CpG sites CpG3.98251179−3 and CpG3.98251219, no significant change over time could be smokersFor (ßCpG = 0.123, sites pCpG3.98251179= 1.67 × 10 )and (Figure CpG3.98251219,4), which showed no significant increasing change levels over of GPR15time couldmethylation. be observed. For CpGobserved. sites CpG3.98251179 and CpG3.98251219, no signiﬁcant change over time could be observed.

Figure 4. Methylation of G protein-coupled receptor 15 (GPR15) DNA slowly increased after smoking FigureFigurecessation. 4. Methylation 4. Methylation In ex smokers, of Gof protein-coupledG protein-coupledGPR15 DNA methylation receptor receptor 15 of ( (GPR15CpG3.98251047GPR15) )DNA DNA slowly slowlygradually increased increased rose afterwithin after smoking years smoking cessation.cessation.after Inquitting ex In smokers,ex whereas smokers, CpG3.98251179GPR15 GPR15DNA DNA methylationand methylation CpG3.98251219 of CpG3.98251047 methylation wasgradually gradually not associated rose rose within with within timeyears years after quittingaftersince quitting quitting whereas whereas (n = CpG3.982511799–153). CpG3.98251179 Linear mixed and and regression CpG3.98251219 CpG3.98251219 model adjust methylation methylationed for age was and was not sex not associated with associated a significance with with time time since quitting (n = 9–153). Linear mixed regression model adjusted for age and sex with a significance since quittingthreshold (n of= 0.0083 9–153). after Linear Bonferroni mixed correction, regression Box model plot whiskers adjusted represent for age 10–90th and sex percentile. with a significance threshold of 0.0083 after Bonferroni correction, Box plot whiskers represent 10–90th percentile. threshold of 0.0083 after Bonferroni correction, Box plot whiskers represent 10–90th percentile. 2.5. Decreased Levels of GPR15 DNA Methylation Correlate with Increased GPR15 mRNA Expression 2.5. Decreased Levels of GPR15 DNA Methylation Correlate with Increased GPR15 mRNA Expression 3.5. DecreasedOur Levels data showed of GPR15 decreased DNA Methylationlevels of novel Correlate GPR15 DNA with methylation Increased GPR15 as well mRNA as increased Expression levels of GPR15Our data mRNA showed expression decreased in levels smokers. of novel Consequently, GPR15 DNA GPR15 methylation mRNA as expression well as increased significantly levels Ourofcorrelated GPR15 datashowed mRNA with GPR15 decreasedexpression DNA levelsinmethylation smokers. of novel atConsequently, GPR15sites CpG3.98251047DNA GPR15 methylation mRNA(ß = 0.036,as expression well p = as4.86 increased significantly× 10−5) and levels of GPR15correlatedCpG3.98251179mRNA expressionwith GPR15(ß = 0.024, in DNA smokers. p = methylation 4.72 Consequently,× 10−4) (Figureat sites 5). GPR15CpG3.98251047 Site CpG3.98251219mRNA expression(ß = 0.036,showed p significantly =a 4.86similar × 10pattern.−5 correlated) and with GPR15CpG3.98251179However,DNA it methylationdid (ß not= 0.024, reach pat =statistical sites4.72 × CpG3.98251047 10 −significan4) (Figurece. 5). In Site (ß contrast = CpG3.98251219 0.036, top =never 4.86 showedsmokers,× 10−5 )a and CpG3.98251179similar CpG3.98251179 pattern. (ß = 0.024,However,methylationp = it 4.72 didwas × notsignificantly10 reach−4) (Figure statistical correlated5). significan Site to GPR15 CpG3.98251219ce. mRNA In contrast expression showed to never in acurrent similarsmokers, smokers pattern. CpG3.98251179 (ß = 0.032, However, methylationp = 6.30 × 10 was−3). significantly correlated to GPR15 mRNA expression in current smokers (ß = 0.032, it did not reach statistical significance. In contrast to never smokers, CpG3.98251179 methylation was p = 6.30 × 10−3). significantly correlated to GPR15 mRNA expression in current smokers (ß = 0.032, p = 6.30 × 10−3).

Biomolecules 2018, 8, 74 7 of 10 Biomolecules 2018, 8, x FOR PEER REVIEW 7 of 10

Figure 5.FigureMethylation 5. Methylation of G protein-coupledof G protein-coupled receptor receptor 15 15 (GPR15 (GPR15)) DNADNA was was associated associated with with GPR15GPR15 mRNA expression.mRNA expression. DNA DNA methylation methylation of CpG3.98251047 of CpG3.98251047 and and CpG3.98251179 CpG3.98251179 significantly significantly correlated correlated with GPR15with mRNAGPR15 mRNA expression. expression. DNA DNA methylation methylation were weredivided divided in tertiles tertiles (T) (T) to todistinguish distinguish low,low, medium,medium, and high and DNAhigh DNA methylation methylation levels. levels. Subj Subjectsects with with lower lower DNA DNAmethylation methylation had higher had GPR15 higher GPR15 mRNAmRNA expression expression levels. levels. n = 32–164,n = 32–164, linear mixed linear regression mixed regression model adjusted model for adjusted age and sex for with age aand significance threshold of 0.0083 after Bonferroni correction, Box plot whiskers represent 10–90th sex with a significance threshold of 0.0083 after Bonferroni correction, Box plot whiskers represent percentile. GPR15 mRNA expression is depicted as ΔCt values normalized to GAPDH mRNA 10–90th percentile. GPR15 mRNA expression is depicted as ∆Ct values normalized to GAPDH mRNA expression. Lower ΔCt values indicate higher GPR15 mRNA expression. expression. Lower ∆Ct values indicate higher GPR15 mRNA expression. 4. Discussion 4. DiscussionThe association between smoking and multiple life-threatening diseases such as cardiovascular Thediseases association has been between well-known smoking [1]. This and is multiple partly caused life-threatening by the promotion diseases of inflammatory such as cardiovascular processes diseases[29]. has In beenthe present well-known study, we [ 1investigated]. This is the partly effects caused of smoking by on the DNA promotion methylation of and inflammatory mRNA expression of the GPR15 locus in the population-based Gutenberg Health Study. Our results are processes [29]. In the present study, we investigated the effects of smoking on DNA methylation and manifold: (i) we identified associations between DNA methylation and smoking status in three mRNA expressionpreviously unstudied of the GPR15 CpGlocus sites inand the (ii) population-based showed that smokers Gutenberg had increased Health levels Study. of Our GPR15 results are manifold:expression, (i) we which identified slowly decreased associations afterbetween smoking ce DNAssation. methylation In addition, and(iii) smokers smoking had status decreased in three previouslymethylation unstudied levels CpG of sitesthe three and novel (ii) showed CpG sites that within smokers the GPR15 had increased gene andlevels (iv) DNA of GPR15 methylationexpression, of which slowlythe novel decreased methylation after sites smoking negatively cessation. correlated In addition,with GPR15 (iii) expression. smokers had decreased methylation levels of theThe three GPR15 novel gene CpG comprises sites within 15 CpG the sitesGPR15 in total.gene However, and (iv) up DNA to now, methylation only CpG3.98251294 of the novel methylation(cg19859270) sites negatively has been correlatedinvestigated with [7–9,13].GPR15 Previousexpression. studies showed decreased CpG3.98251294 methylation in smokers compared to never smokers, which was associated with cumulative smoking The GPR15 gene comprises 15 CpG sites in total. However, up to now, only CpG3.98251294 exposure [7–9,30,31]. In this study, we discovered three novel methylation sites in relation to (cg19859270)smoking: has CpG3.98251047, been investigated CpG3.98251179, [7–9,13]. and Previous CpG3.98251219, studies with showed CpG3.98251047 decreased being CpG3.98251294 the most methylationstrongly in smokers influenced compared by smoking. to neverThe three smokers, novel Cp whichG sites was presented associated an even with stronger cumulative difference smoking in exposureDNA [7– 9methylation,30,31]. In of this 4% study,to 6% between we discovered smokers and three never novel smokers. methylation Contrary to sites CpG3.98251294, in relation to smoking:methylation CpG3.98251047, of none of CpG3.98251179, the three new CpG and sites CpG3.98251219, was significantly withassociated CpG3.98251047 with cumulative being smoking the most stronglyexposure influenced (Table by S2). smoking. The three novel CpG sites presented an even stronger difference in DNA methylationBeside DNA of 4% methylation, to 6% between mRNA smokers expression and of GPR15 never had smokers. been described Contrary to be to influenced CpG3.98251294, by smoking status [10,11,32,33]. Consistently, we showed that current smokers had 7.6-fold higher methylation of none of the three new CpG sites was significantly associated with cumulative smoking GPR15 mRNA expression levels compared to never smokers. GPR15 mRNA levels slowly decreased exposureafter (Table smoking S2). cessation. Additionally, our data showed that GPR15 mRNA expression correlated Besidewith DNAthe number methylation, of pack years. mRNA Our expressionresults implicate of GPR15 that GPR15had beenexpression described depends to on be the influenced amount by smokingof status cigarettes [10, 11smoked,32,33 per]. Consistently, day and the duration we showed of smoking. that current smokers had 7.6-fold higher GPR15 mRNA expressionAnalyzing levels five years compared longitudinal to never gene smokers. expressionGPR15 data, wemRNA examined levels whether slowly smoking decreased changes after smokingGPR15 cessation. mRNA Additionally, expression. ourGPR15 data mRNA showed expression that GPR15 decreasedmRNA after expression smoking correlated cessation. with the numberConsistently, of pack GPR15 years. mRNA Our results expression implicate decreased that withGPR15 increasingexpression time since depends quitting,on which the is amount in line of cigaretteswith smoked current per knowledge day and [7]. the Furthermore, duration of smoking.our data showed that decreasing methylation levels of CpG3.98251047 and CpG3.98251179 were linked to increasing levels of GPR15 mRNA, which Analyzing five years longitudinal gene expression data, we examined whether smoking changes indicated a direct influence of methylation status on GPR15 gene expression. Even though the GPR15 mRNAmethylation expression. sites analyzedGPR15 inmRNA our study expression are not directly decreased located after in the smoking GPR15 cessation.promoter region Consistently, but GPR15 mRNAare within expression the single decreasedexon of GPR15 with and increasing since GPR15 time consists since quitting,of only 1252 which bp, DNA is in methylation line with current in knowledgeclose [ 7proximity]. Furthermore, to the promoter our data can showed influence that transc decreasingription by inhibiting methylation transcription levels of factor CpG3.98251047 binding and CpG3.98251179 were linked to increasing levels of GPR15 mRNA, which indicated a direct influence of methylation status on GPR15 gene expression. Even though the methylation sites analyzed in our study are not directly located in the GPR15 promoter region but are within the single exon of GPR15 and since GPR15 consists of only 1252 bp, DNA methylation in close proximity to the promoter can influence transcription by inhibiting transcription factor binding [34]. Hence, as a Biomolecules 2018, 8, 74 8 of 10 potential mechanism, smoking might decrease GPR15 DNA methylation in the progenitor cells, which could lead to an increase in GPR15 positive cells in the blood, which results in increased GPR15 mRNA expression. Taken together, our data indicate that smoking decreases GPR15 DNA methylation, which, in turn, leads to increased GPR15 mRNA expression. Thereby, GPR15 DNA methylation or GPR15 mRNA expression might have a potential to act as new biomarkers for smoking behavior since factors such as second-hand smoke exposure, irregular smoking behavior, electrical cigarettes, and smokeless tobacco are almost impossible to estimate accurately by questionnaire. The strengths of the presented work are the measurement of previously undescribed CpG methylation sites within the GPR15 gene and its integration with mRNA expression. Our results originate from a large sample size with individuals from a population-based cohort and include longitudinal data. In this study, RNA was only available from enriched monocytes and DNA was available from leukocytes. GPR15 RNA expression levels were low and the detected increase in GPR15 expression in smokers could result from an increase in the number of GPR15 positive cells in the negatively selected monocyte fraction, as suggested by Bauer et al., rather than an upregulation of the gene expression [33]. The measurement of limited numbers of CpG sites, however, is a limitation of this study. Out of 15 CpG sites within GPR15, six sites could be analyzed due to technical limitations in the EpiTYPER amplicon design. Three sites were associated with smoking. Therefore, we cannot exclude the possibility that more methylation sites within the GPR15 gene are present that are also associated with smoking. Contrary to previous results from microarray data [7–9,30,31], the CpG3.98251294 DNA methylation site was neither significantly associated with smoking nor GPR15 expression in our analyses possibly because the EpiTYPER assay is less sensitive [26]. However, microarrays only include a limited selection of CpG sites. Using the EpiTYPER assay, we not only identified three novel CpG sites in relation to smoking, but the alterations in DNA methylation for these sites were even higher compared to Cpg3.98251294 [7–9,30,31]. Kõks et al. also determined DNA methylation of GPR15 with the EpiTYPER assay. Comparably to our results, a different methylation between smokers and non-smokers was shown for CpG3.98251179 but not for CpG3.98251294 [10]. In summary, we identified three novel methylation sites within the GPR15 gene whose methylation was affected by smoking, which led to an altered GPR15 gene expression. These smoking-related changes in GPR15 methylation and expression could perturb immune function and increase the risk for complex diseases with inflammatory pathogenesis, which could contribute to cardiovascular disease.

Supplementary Materials: The following are available online at http://www.mdpi.com/2218-273X/8/3/74/s1. Table S1: Mass fragments detected by the EpiTYPER assay, Table S2: Results from linear mixed regression analyses, Figure S1: The GPR15 locus in the UCSC Genome Browser human GRCh37/hg19 assembly, Figure S2: GPR15 mRNA expression after smoking cessation. Availability of primary data is restricted due to limitations in ethical approvals. Author Contributions: Conceptualization, T.H. and T.Z. Methodology, T.H., C.M., J.K., C.R., J.S., M.W., and S.K. Formal Analysis, T.H. and C.M. Resources, P.S.W., M.K., and K.J.L. Data Curation, P.S.W., KJ.L., C.M., and T.H. Writing-Original Draft Preparation, T.H. and T.Z. Writing-Review & Editing, T.H., C.M., J.K., C.R., J.S., S.K., M.W., T.M., N.P., P.S.W., M.M., F.M., M.K., K.J.L., S.B., and T.Z. Visualization, T.H. and C.M. Supervision, T.Z. Project Administration, T.H. and T.Z. Funding Acquisition, T.H. and T.Z. Funding: This research was funded by the German Centre for Cardiovascular Research (DZHK e.V.) grant number [81X2710163_81604/151 (TH), B17-036 SE (MW) and 81Z1710101 (TZ)] and the Research Promotion Fund of the Faculty of Medicine (FFM) of the University Medical Center Hamburg Eppendorf (UKE) to TH. The Gutenberg Health Study is funded through the government of Rhineland-Palatinate (“Stiftung Rheinland-Pfalz für Innovation,” contract AZ 961-386261/733), the research programs “Wissen schafft Zukunft” and “Center for Translational Vascular Biology (CTVB)” of the Johannes Gutenberg-University of Mainz and its contract with Boehringer Ingelheim and PHILIPS Medical Systems including an unrestricted grant for the Gutenberg Health Study. Acknowledgments: The authors thank Simone Schnella for editorial assistance. Conﬂicts of Interest: The authors declare no conﬂict of interest. The funders had no role in the design of the study, in the collection, analyses, or interpretation of data, in the writing of the manuscript, or in the decision to publish the results. Biomolecules 2018, 8, 74 9 of 10

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