Serotonin transporter gene polymorphisms: Relation with platelet serotonin level in patients with primary Sjogren's syndrome

Journal of Neuroimmunology 282 (2015) 104–109

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Serotonin transporter gene polymorphisms: Relation with platelet serotonin level in patients with primary Sjogren's syndrome J. Markeljevic a, H. Sarac b,c,⁎, N. Bozina d, N. Henigsberg c, M. Simic e, L. Cicin Sain f a

Medical School University of Zagreb, Department of Internal Medicine, University Hospital Sestre milosrdnice, HR-Vinogradska cesta 29, 10000 Zagreb, Croatia Department of Neurology, University Clinical Hospital Centre Zagreb, HR-Kispaticeva 12, 10000 Zagreb, Croatia Croatian Institute for Brain Research, Medical School University of Zagreb, HR-Salata 12, 10000 Zagreb, Croatia d Medical School University of Zagreb, University Hospital Zagreb, Department of Laboratory Diagnostic, HR-Kispaticeva 12, 10000 Zagreb, Croatia e Institute of Immunology, HR-Rockefellerova 2, HR-10000 Zagreb, Croatia f Rudjer Boskovic Institute, Laboratory for Neurochemistry and Molecular Neurobiology, Department of Molecular Biology, HR-Bijenicka 54, 10000 Zagreb, Croatia b c

a r t i c l e

i n f o

Article history: Received 7 February 2014 Received in revised form 26 March 2015 Accepted 1 April 2015 Keywords: Serotonin transporter polymorphism 5-HTTLPR/rs25531 5-HTTVNTRin2 Platelet serotonin Sjogren syndrome

a b s t r a c t Significantly lower platelet serotonin level (PSL) in patients with primary Sjogren's syndrome (pSS) than in healthy controls has been reported in our prior studies. In the present report, we demonstrated effect of functional polymorphisms in the serotonin transporter gene (5-HTT) on PSL. We describe a group of 61 pSS patients and 100 healthy individuals subjects, who received PSL measurement in our prior study. All subjects were genotyped for the promoter 5-HTTLPR (L/S), rs25531 (A/G) and intronic 5-HTTVNTRin2 (l/s) polymorphisms. Overall, the presence of 5HTTVNTRin2 ss genotype was associated with significantly lower PSL in pSS patients, not in healthy controls. Reduced PSL in pSS patients is in line with hypothesis of association between chronic immunoinflammation and 5-HT system dysregulation, identifying additional mechanisms such as altered 5-HT transport as potential genetic factor contributing to PSL depletion. © 2015 Elsevier B.V. All rights reserved.

1. Introduction Significantly decreased platelet serotonin (5-HT, 5-hydroxytryptamine) level (PSL) has been reported in rheumatoid arthritis (RA), systemic lupus erythematosus (SLE), systemic sclerosis (SSc), and mixed connective tissue disease (MCTD) (Meyerhoff and Dorsch, 1981; Zeller et al., 1983; Klimiuk et al., 1989). In prior study of 61 patients with primary Sjogren's syndrome (pSS), we reported lower PSL in pSS patient than in healthy individuals (Sarac et al., 2012). Recently, we have reported a significant reduction in pSL in a group of 22 pSS patients with frequent episodic tension-type headache (FETH). These patients have had significantly decreased PSL and more common cerebral white matter signal hyperintensities (SH) than healthy controls, which seem to be associated to increased platelet serotonin release, signifying a more widespread cerebral vasculopathy in pSS patients than in healthy individuals (Šarac et al., 2013). It is our great impression that reduced PSL is general finding in inflammatory rheumatic disorder spectrum, including pSS, signifying the role of the 5-HT in the pathogenesis of pSS. An inverse correlation between PSL and clinical disease activity was found in SLE, the lowest levels occurring in active stage of the disease (Zeller et al., 1983). Absence of ⁎ Corresponding author at: University Hospital Zagreb, HR-Kispaticeva 12, 10000, Croatia. E-mail address: [email protected] (H. Sarac).

http://dx.doi.org/10.1016/j.jneuroim.2015.04.002 0165-5728/© 2015 Elsevier B.V. All rights reserved.

correlation between decreased PSL and increased release of other platelet granule constituents in RA and SLE was found (Meyerhoff and Dorsch, 1981). In a prior study, we have documented no correlation between magnitude of PSL reduction and clinical disease activity (Sarac et al., 2012), indicating different biochemical (Sellam et al., 2009) and/or genetic (Emamian et al., 2006) features of pSS, as pSS patients demonstrate a systemic inflammatory reaction of low-grade intensity as previously suggested by Valtýsdótir et al. (2001). It is well known that the actions of 5HT are primarily terminated by the membrane 5-HT transporter (5-HTT) (Lesch et al., 1993; Rammamoorthy et al., 1993; Brenner et al., 2007), and that 5-HTT gene (SLC6A4) could be a common genetic modulator of 5-HT transport. Functional polymorphism of the serotonin transporter-linked promoter region (5-HTTLPR) consists of the presence or absence of 44 base-pair (bp) segment producing either a long (L) variant which enhances basal activity of the 5-HTT gene, or the short (S) variant (Heils et al., 1996). In the proximity of this locus, additional polymorphism rs25531 is located the nearest of the 5-HTTLPR segment, and they can be considered as two independent polymorphisms (Bonvicini et al., 2010). This single nucleotide polymorphism (SNP), according to the repeat architecture (Nakamura et al., 2000), results in an A to G substitutes (alleles) that has been shown to modulate transcriptional efficacy. The G allele of rs25531 is in phase with the 5-HTTLPR long (L) allele and mitigates transcriptional efficacy more than does the 5-HTTLPR short (S) allele. Therefore, the modulation of the 5-HTTLPR by rs25531 results in haplotypes

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with high (LA) or low (LG, SA or SG) transcriptional efficacy (Hu et al., 2006; Martin et al., 2007). Another functional polymorphism in the 5-HTT gene is the intron 2 variable-number-of-tandem-repeats region (5-HTTVNTRin2) with two common repeats; the short (s), with 9 or 10 repeats, and long (l) with 12 repeats, alleles are purported to modulate transcription, with 12 repeat allele having stronger properties (MacKenzie and Quinn, 1999). Studies investigated relation between the plasma 5-HT level and the uptake ability of 5-HTT (Heils et al., 1996; Lesch et al., 1996), demonstrated that the 5-HT uptake depends on the plasma 5-HT level (Ramamoorthy and Blakely, 1999). Overall, significant heterogeneity was observed among studies investigating the effects of the 5-HTT polymorphisms on PSL in healthy subjects and thus did not support the hypothesis that the 5-HTT variants have an effect on PSL (Pivac et al., 2009; Greenberg et al., 1999; Sookoian et al., 2007; Stoltenberg et al., 2002). This is inconsistent with data obtained in vitro showing that the 5-HTT number and 5-HT uptake in cells carrying the SS variant are lower that those carrying LL variant (Heils et al., 1996; Lesch et al., 1996) and in migraine patients carriers of 5-HTTLPR SS genotype (Juhasz et al., 2003). In the present study we searched for potential functional consequences of both promoter and intronic 2 VNTR polymorphisms of the 5-HTT gene on PSL in pSS patients and healthy individuals. We documented significant association between PSL and 5-HTT gene variants, indicating that 5-HT system may play an important role in the pathogenesis of pSS. We analyzed the study sample consisting of pSS patients and healthy subjects who had already received PSL measurement in our prior studies (Sarac et al., 2012; Šarac et al., 2013). In reviewing the literature, no data was found on the influence of the 5-HTTLPR, rs 25531 and 5-HTTVNTRin2 polymorphisms of 5-HTT gene on PSL in patients with pSS. In addition, this study also produced results which corroborate the findings of a great deal of the previous work, which did not demonstrate association between 5-HTTLPR variants and platelet 5-HT concentration in the group of healthy individuals (Pivac et al., 2009; Greenberg et al., 1999; Sookoian et al., 2007).

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3′, reverse 5′-GGA CCG CAA GGT GGG CGG GA-3′. Amplification was performed in a 7500 Real Time PCR System in 96-well plates under the following PCR conditions: 50 °C for 2 min and 95 °C for 15 s and 62.50 °C for 1 min 30 s. In order to acquire controls, genotype sequencing (3130xl Genetic Analyzer, Applied Biosystems) was performed. Analysis of the 5-HTTVNTRin2 was performed according to a method described previously (Ito et al., 2002). For the PCT reaction, the following primer pair was used: forward 5′-GTCAGTATCACAGGCTGCGAG-3′, reverse 5′-TGTTCCTAGTCTTACGCCAGTG-3′. The l (12 repeats) and s (10 or 9 repeats) allele are represented by 300 bp and 267 or 250 bp PCR products, respectively. Data on PSL measurement for study sample were obtained in our prior study (Sarac et al., 2012), where the measurement of PSL has been conducted by the ortho-phthaldialdehyde-enhanced fluorimetry, as described previously (Jernej et al., 2000). 2.3. Statistical analysis Results are expressed as means ± standard deviation (SD) or medians with range of values. The normality assumption was examined by Shapiro–Wilk test. The differences in the genotype and allele frequencies were evaluated using a X2 test. Results were evaluated using one-way or two-way analysis of variance (ANOVA), followed by the Pearson's coefficient of correlation. Statistical analyses were performed using Statistica Version 8.0 for Windows (StatSoft Inc. Tulsa, Oklahoma, USA). The level of significance was set to P b 0.05. 3. Results We have examined the association of PSL with two 5-HTT gene polymorphisms, 5-HTT gene-linked polymorphic region (5-HTTLPR) in the promoter, rs25531 and intron 2 variable number of tandem repeats (5-HTTVNTRin2), in a sample of 61 pSS patients and 100 healthy medication-free Caucasian healthy controls of Croatian origin. The descriptive characteristics for the entire sample are shown in Table 1.

2. Materials and methods 3.1. The 5-HTTLPR/rs25531 triallelic structure 2.1. Population sample This study combines our previous results on PSL in pSS patients (Sarac et al., 2012) with the results from a new investigation on 5-HTT gene polymorphisms in the same cohort of patients and in healthy controls. The original sample included 61 individuals (55 females, 6 males) who met the criteria for pSS (Vitali et al., 2002). Patients had been recruited over the three years period (from 2009 to 2012) at three clinical sites: the University of Zagreb School of Medicine, Department of Internal Medicine, University Hospital Zagreb and University Hospital Sisters of Mercy, Zagreb, and the Croatian Institute for Brain Research, Zagreb. The community control group consisted of 100 healthy Caucasian subjects of Croatian origin (74 females, 26 males), recruited from the blood donors at the Croatian Institute for Transfusion Medicine, Zagreb, whose healthy status was checked by written questionnaire. The study protocol was approved by the multi-centre research ethics committee, and the informed consent of all participating subjects was obtained. 2.2. Experimental procedures Genomic DNA was extracted from leukocytes (EDTA-anticoagulated blood) by salting out method (Miller et al., 1988). PCR amplification of the 5-HTTLPR was performed as previously described (Rauch et al., 2002) with some modifications (Bozina et al., 2007). First the S and L alleles were discriminated. Recently, 5-HTTLPR is considered to be triallelic, the L allele is subdivided as LG and LA by a common SNP, rs25531, whereas the LG is functionally behaving like an S allele (S′) and LA is behaving like an L allele (L′) (Hu et al., 2006). The following primers were used: forward 5′-ATG CCA GCA CCT AAC CCC TAA TGT-

In the genotyping analyses, we did detect rs25531 G allele in 8 (6.56%) pSS patients and in 19 (10.11%) healthy subjects, and the short 5-HTTLPR allele, S allele was found in 44 (36.00%) pSS patients and in 74 (39.36%) healthy subjects. 5-HTTLPR S allele and rs25531 G minor allele frequency (MAF) in the European origin population is 40% and 6%, respectively. Distribution of clustered haplotype frequencies of pSS patients and healthy controls are shown in Fig. 1. The genotype LGLG has been detected only in one healthy subject, not in Sjogren's patients, which is consistent with previous studies (Bonvicini et al., 2010; Mrazek et al., 2009) showing that rs25531 G allele occurs very rarely on haplotypes with the S version of the promoter variant. The results revealed no differences for 5-HTTLPR L allele (X2 = 1.56; p = 0.21)

Table 1 Characteristic of subjects. Characteristic Number of subjects Sex, female/male Age, years, median (range) Disease duration, years, median (range) Duration of sicca symptoms, median (range) ESSDAI score, median (range) Positive anti-Ro/SSA antibodies, N (%) Positive anti-La/SSB antibodies, N (%) Antinuclear antibodies (ANA), N (%) PSL, mean (SD) μg 5HT/109 platelets

pSS patients 61 55/6 59.4 (34–80) 5.54 (1–19) 8.21 (1–33) 6 (1–16) 23 (37.7) 16 (26.5) 36 (59.0) 384 (209)

Controls 100 74/26 38.0 (19–66) na na na na na na 464 (157)

pSS = primary Sjogren's syndrome; ESSDAI = European League Against Rheumatism (EULAR) Sjogren's syndrome Disease Activity Index; PSL = platelet 5-HT level; N = number of subjects; na = not applicable.

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found in genotypes ll, ls or ss frequencies in 30 (49.18%), 20 (36.06%) and 11 (14.75%) Sjogren's patients, and in 32 (33.33%), 51 (53.13%) and 13 (13.54%) healthy subjects, respectively. 3.3. Association analysis of the platelet 5-HT level and 5-HTTLPR/rs25531 variants

Fig. 1. Distribution of haplotypes of 5-HTTLPR, rs25531 polymorphisms in pSS patients and healthy individuals. Each column represents number of subjects per group in respect to clustered phased haplotypes L′L′, L′S′ and S′S′.

and for S allele (X2 = 1.62; p = 0.44) frequencies, between two study groups. No evidence of association was observed for the rs25531 alleles and genotype frequencies. In addition, we analyzed, estimated and phased haplotype distribution transforming the classification into a triallelic/biallelic model according to allele expression. The triallelic LA–LG–S system of the 5HTTLPR polymorphism provided six phased haplotypes: SASA, LGSA, LGLG as S′S′; LASA, as L′S′; and LALA as (L′L′) (Hu et al., 2006). As regards the estimated phased haplotypes, no significant (X2 = 1,73; p = 0.42) differences were found in LALA, LASA, LALG, SALG or SASA frequency in 20 (32.79%), 24 (39.34%), 6 (9.84%), 0 (0%), 2 (3.28%) and 9 (14.75%) Sjogren's subject, and in 26 (27.66%), 31 (32.98%), 12 (12.77%), 1 (1.06%) 5 (5.32%) and 19 (20.21%) healthy subjects, respectively. The frequencies of long (L′) (57.38%) and short (S′) (42.62%) haplotypes in pSS patients, and L′ (50.53%) and S′ (49.47%) haplotypes in healthy subjects were not significantly different (X2 = 1.56; p = 0.212). Concerning the estimated haplotype analyses, pSS patients were more frequently the carriers of clustered phased SS′ haplotype (26.6% vs. 18.03% in healthy subject), although difference has not yielded statistical significance (X2 = 1.97; p = 0.37). No significant differences have been found in clustered phased haplotype frequencies L′L′, L′S′ or S′S′. Additional analysis of subjects according to the presence of homozygous L′L′ genotype vs. the combined L′S′ and S′S′ (X2 = 0.46; p = 0.5), or the homozygous S′S genotype vs. L′L′ and L′S′ genotype, revealed no significant differences in the frequency of these genotypes. 3.2. The 5-HTTVNTRin2 variants The results of the genotype distribution for the 5-HTTVNTRin2 polymorphism (Fig. 2) showed borderline differences between two study populations in allele s frequencies (X2 = 3.76; p = 0.052). No significant (X2 = 4.77; p = 0.09) differences between two study groups were

Values of PSL measured in patients with pSS and healthy controls in relation to the genotypes and alleles of 5-HTTLPR and rs25531 are shown in Table 2. The association analysis of the PSL, 5-HTTLPR, and rs 25531 variants was carried out using one-way ANOVA to evaluate the potential relationship between the clustered phased haplotypes L'L', L′S′ and S′S′ genotypes on PSL in each study group. No significant association was observed between clustered phased haplotypes and PSL in Sjogren's group (p = 0.49), neither in healthy population (p = 0.89). There were also no significant differences in PSL between two study groups in relation to clustered phased haplotypes L′/L′, L′/S′ and S′/S′ genotypes (p = 0.41, p = 0.19, p = 0.12). 3.4. Association analysis of the platelet 5-HT level and 5-HTTVNTRin2 variants Values of PSL measured in patients with pSS and healthy controls in relation to the genotype of 5-HTTVNTRin2 polymorphism are shown in Table 3. The association analysis of the PSL and 5-HTTVNTRin2 variants was carried out using one-way ANOVA to evaluate the potential relationship between PSL and the genotypes ll, ls and ss within each study group. Significant differences (p = 0.0056) in PSL between pSS patients and healthy subjects have been observed in relation to 5-HTTVNTRin2 ss genotype. There were no significant associations between PSL and 5HTTVNTRin2 variants within each study group. 4. Discussion The present study represents the first report of the association of PSL with 5-HTT gene polymorphisms in pSS. Although decreased PSL has long been recognized in all examined inflammatory rheumatic disorders, including RA, SLE, SSc and MCTD, only recently we have recognized significantly lower PSL in pSS patients. In reviewing the literature, no data was found on the effects of 5-HTT gene variants on PSL in inflammatory rheumatic disorders, including pSS. On this basis, we used association approach to test the effect of 5HTTLPR, rs25531 and 5-HTTVNTRin2 variants of the 5-HTT gene for phenotypic variance in PSL, in a sample of patients with pSS and healthy subjects. The major finding of this study was the detection of ss variant of the 5-HTTVNTRin2 which seems to exercise the significant influence on PSL regulation in pSS patients, not in healthy individuals. To date, only one study has investigated the effects of the 5-HTTLPR and 5-HTTVNTRin2 on PSL (Coutinho et al., 2004), and the results of that study were in line with a data on a significant effect on PSL of each 5-HTT gene marker, 5-HTTLPR and 5-HTTVNTRin2, as well as of haplotypes of both markers, with a major contribution of the L.Stin2.10 haplotype. Two studies have shown that the S allele at 5-HTTLPR Table 2 Platelet serotonin level expressed as ng 5HT/109 platelets in patients with primary Sjogren's syndrome and healthy controls subdivided according to 5-HTTLPR and rs25531 polymorphisms.

Fig. 2. Distribution of genotypes of 5-HTTVNTRin2 polymorphisms in pSS patients and healthy individuals. Each column represents number of subjects per group in respect to genotypes ll, ls, and ss.

Haplotypes (5-HTTLPR)

Patients platelet 5-HT level

N

Controls platelet 5-HT level

N

p-Value

L′L′ L′S′ S′S′

438 ± 204 344 ± 204 398 ± 176

(20) (30) (11)

455 ± 151 483 ± 161 483 ± 146

(26) (43) (25)

0.4097 0.1971 0.1235

5-HTTLPR = serotonin transporter gene-linked polymorphic region; mean ± SD; N = number of subjects, 5-HT = serotonin (5-hydroxytryptamine); p-value — patients vs respective control.

J. Markeljevic et al. / Journal of Neuroimmunology 282 (2015) 104–109 Table 3 Platelet serotonin level expressed as ng 5HT/109 platelets in patients with primary Sjogren's syndrome and healthy controls subdivided according to 5-HTTVNTRin2 polymorphism. Genotypes 5-HTTNTRin2

Patients platelet 5-HT level

N

Controls platelet 5-HT level

N

p-Value

ll ls ss

390 ± 196 399 ± 250 331 ± 140

30 22 9

456 ± 151 462 ± 167 507 ± 139

32 52 12

0.4361 0.0589 0.0056

5-HTTVNTRin2 = serotonin transporter gene variable number of tandem repeats intron 2 region; mean ± SD; N = number of subjects; 5-HT = serotonin (5-hydroxytryptamine); p-value — patients vs respective control.

influences responses to mild stress and/or to acute tryptophan depletion (Firk and Markus, 2009; Roiser et al., 2006). This study demonstrates that 5-HTTVNTRin2 ss genotype can be associated with significantly decreased PSL in a large group of clinically well-characterized pSS patients in comparisons to healthy controls. Likewise, borderline significance was apparent for 5-HTTVNTRin2 ls genotype, and no other associations, including ll genotype and allele-based comparisons did not reach the level of statistical significance. Other studies have reported findings contradictory to those of the present study regarding the allele and genotype frequencies of 5-HTTLPR, rs 25531 and 5-HTTVNTRin2 variants in healthy subjects. Several studies failed to demonstrate a functional relationship between 5HTTLPR variants and PSL in healthy individuals and thus did not support the hypothesis that the 5-HTT variants have a direct and sole effect on PSL (Pivac et al., 2009; Stoltenberg et al., 2002; Greenberg et al., 1999; Sookoian et al., 2007). Significantly higher proportion of 5-HTTVNTRin2 short (s) allele among patients with major depressive disorder (MDD) in comparison to healthy controls has been reported by Bozina et al. (2006). Less frequent than expected, 5-HTTLPR SS genotype was found in patients with treatment-resistant depression (Kishida et al., 2007) in a European origin population. Conversely, Bonvicini et al. (2010) found more frequent 5HTTLPR SS genotype (19%) in depressed patients, closely resembling the results of an excess of the S allele in prior studies (Smits et al., 2004; Holsboer, 2008; Kiyohara and Yoshimasu, 2009) as well as the data obtained in research conducted by Smits et al. (2004), in European descendent depressive patients (21%–28%). In the present study, we observed that two groups differ with the borderline significance in a frequency of the 5-HTTVNTRin2 lower expression s allele, whereas the healthy individuals have been carriers of s allele more frequently than pSS patients. There have been no significant differences in the allele or genotype frequencies of the 5-HTTLPR between two study groups. Our data support the hypothesis that the 5-HTTVNTRin2 genotype may represent a marker susceptibility factor for decreased PSL in pSS patients. Sjogren's patients could be distinguished from the healthy individuals on a biological level, including PSL and 5-HTTVNTRin2 ss polymorphism. However, the role of the serotonin system in the etiology and pathogenesis of pSS is not clearly defined. Considering both, our results and available literature reports, it seems that decreased PSL is a general finding in inflammatory rheumatic disorders. This could reflect increased 5-HT secretion during platelet activation in the acute stage of the disease (Gresele, 1991). Direct measuring of platelet release reaction in acute stage of SLE showed no relationship with magnitude of PSL depletion (Meyerhoff and Dorsch, 1981), and mean PSL of those with inactive disease still remained lower than that of the healthy controls, suggesting that increased secretion of platelet granule constituents cannot provide the sole explanation for reduced PSL. The absence of correlation between PSL values and clinical disease activity in pSS patients (Sarac et al., 2012; Šarac et al., 2013), as contrasted to RA, SLE and SSc, may indicate distinct features of pSS, as patients with pSS demonstrate a systemic inflammatory reaction of low-grade intensity as previously suggested (Valtýsdótir et al., 2001). Indirect lines of evidence suggest that the association of reduced PSL with 5-HT system alteration may represent a causal relationship. We demonstrated here that additional mechanisms, such as impaired 5-HT transport can contribute to PSL reduction.

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However, the absence of association between 5-HTT gene polymorphisms and PSL in healthy controls and the presence of the specific genotype 5HTTVNTRin2 ss cannot provide the sole explanation for lower PSL. The observed PSL depletion may reflect the overall alteration of 5-HT system in pSS. These findings demonstrate genetic heterogeneity in pSS, signifying the 5-HTT gene as a susceptibility factor for reduced PSL in pSS. Reduced PSL may also be a consequence of activation of kynurenine-pathwayenzymes by proinflammatory cytokines, which then redirect tryptophan metabolism and affect 5-HT synthesis (Davies et al., 2010), or other factors liberated from leukocytes (Totani and Evangelista, 2010). Compatible with prior literature on PSL (Mercado and Killic, 2010), our results showed that platelets require the expression of 5-HTT. Our prior study demonstrated that white matter signal hyperintensities (SH) are more common in pSS patients than in healthy individuals, suggesting more widespread cerebral vasculopathy in the pSS patients (Šarac et al., 2013). These findings are in line with the hypothesis of interrelation between increased platelet 5-HT release and pathophysiology of brain tissue damage in pSS patients (Šarac et al., 2013). Cloutier et al. (2012) reported that 5-HT released by platelets helps drive the persistent vascular permeability via 5-HT elsewhere. This effect was mediated by platelet 5-HT accumulated via the 5-HTT and could be inhibited by serotonin-selective reuptake inhibitor antidepressants (SSRI). Authors demonstrated a significant decrease in vascular permeability, compatible with the decreased PSL after treatment with SSRI. The authors demonstrated improvement of vascular permeability in the absence of platelets. In opposition to this, the disease severity in the absence of platelets was only partially reduced, pointing to the uncoupling of permeability and inflammation (Cloutier et al., 2012). Thus, in line with the existing literature reports on platelet 5-HT (Mercado and Killic, 2010) our findings support the hypothesis that platelets require the expression of 5-HTT gene for 5-HT transport. Recently, Patrick and Ames (2014) have provided an evidence that vitamin D upregulates the central 5-HT gene and downregulates the peripheral 5-HT gene. This suggests that adequate level of vitamin D may be required to activate the transcription of the 5-HT-synthesizing gene tryptophan hydroxylase 2 (TPH2) in the brain and suppresses the transcription of TPH1 in peripheral tissues, where when found in excess, promotes inflammation. Previous studies reported that the increased type of I IFN activity observed in pSS patients is a systemic phenomenon and pointed to plasmacytoid dendritic cells (pDCs) as a possible source of this activity (Wildenberg et al., 2008). Dendritic cells, as an important innate immune cells express the presence of different 5-HT receptors including 5-HT4 and 5-HT7, which indicates that the activation of these receptors enhanced the release of the cytokines interleukin-1β (IL-1β) and IL-8 (Idzko et al., 2004). Although DCs expressing 5-HT receptors are considered to play an important role in immune activation, the role of 5-HT system in modulating pDC function in the context of pSS inflammation remains to be determined. The important role of 5-HT in mediating neuroimmunoendocrine regulation in susceptibility/resistance to autoimmune-inflammatory disorders is emerging (Eskandari et al., 2003; Ahern, 2011). Thus, the regulation of PSL in pSS patients might be a complex process, and that the presence of 5-HT, other neurotransmitters, the hormonal status, the immune system and different gene polymorphisms, all might alter PSL in pSS. Thereby, our demonstration that altered 5-HT uptake by platelets is a prerequisite for the decreased PSL implies that antidepressants used to treat depression by targeting 5HTT may also target PSL in pSS. 5. Conclusion We revealed an unexpected role for 5-HT in pSS. Our results show that variants of the 5-HTT gene significantly contribute to decreased PSL in a subset of patients with pSS. The pathogenetic mechanism, however, is unlikely to involve the 5-HTT gene solely. The associated 5-HTT gene probably interacts with other alleles, neurohumoral, immunological and/or

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environmental factors to produce lower PSL in pSS patients, which possibly represent a separate etiopathogenetic subgroup. Our data demonstrate an altered capacity for 5-HT transport in platelets in pSS patients. New data demonstrate an important role of 5-HT outside of the central nervous system in immune signaling. Further large prospective studies are needed to elucidate pathological role of 5-HT alteration in pSS multisystem dysfunction. Funding statement Research funding was provided by the Croatian Ministry of Science, Education and Sport, project Nos. 108-1081874-2416 and 098-1081870-2397. Authors' contributions Study conception and design: Sarac. Acquisition of data: Markeljevic, Sarac, Bozina, Cicin-Sain, Henigsberg, Simic. Analysis and interpretation of data: Bozina, Sarac, Cicin-Sain, Markeljevic, Simic. Drafting, revising and approval of final version: All authors were involved in drafting the article or revising it critically for intellectual content, and all authors approved the final version to be published. Study approval Multi-centre research ethics committee approvals were obtained prior to starting the study, including the Ethics Committee of the Medical School University of Zagreb and study was carried out in accordance with Declaration of Helsinki. Patient consent Written informed consent was obtained from all participants. Competing interests None. Acknowledgments We thank prof. Jadranka Sertic for scientific contribution and laboratory facilities, Viktorija Erdeljic for statistical analysis Mladen Vucic for expert technical assistance in preparing the pictures. We also thank the Croatian Institute for Brain Research, Zagreb for providing facilities for the neurological evaluations of patients. References Ahern, G.P., 2011. 5-HT and the immune system. Curr. Opin. Pharmacol. 11 (1), 29–33 (Feb). Bonvicini, C., Minelli, A., Scassellati, C., Bortolomasi, M., Segala, M., Sartori, R., Giacopuzzi, M., Gennarelli, M., 2010. Serotonin transporter gene polymorphisms and treatmentresistant depression. Prog. Neuropsychopharmacol. Biol. Psychiatry 16 (34(6)), 934–939. Bozina, N., Mihaljević-Peles, A., Sagud, M., Jakovljević, M., Sertic, J., 2006. Serotonin transporter polymorphism in Croatian patients with major depressive disorder. Psychiatr. Danub. 18 (1–2), 83–89. Bozina, N., Medved, V., Kuzman, M.R., Sain, I., Sertic, J., 2007. Association study of olanzapine-induced weight gain and therapeutic response with SERT gene polymorphisms in female schizophrenic patients. J. Psychopharmacol. 21 (7), 728–734. Brenner, B., Harney, J.T., Ahmed, B.A., Jeffus, B.C., Unal, R., Mehta, J.L., Kilic, F., 2007. Plasma serotonin levels and the platelet serotonin transporter. J. Neurochem. 102, 206–215. Cloutier, N., Paré, A., Farndale, R.W., Schumacher, H.R., Nigrovic, P.A., Lacroix, S., Boilard, E., 2012. Platelets can enhance vascular permeability. Blood 9 (120(6)), 1334–1343. Coutinho, A.M., Oliveira, G., Morgadinho, T., Fesel, C., Macedo, T.R., Bento, C., Marques, C., Ataíde, A., Miguel, T., Borges, L., Vicent, A.M., 2004. Variants of the serotonin transporter gene (SLC6A4) significantly contribute to hyperserotonemia in autism. Mol. Psychiatry 9 (3), 264–271.

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