Healthy young women with serotonin transporter SS polymorphism show a pro-inflammatory bias under resting and stress conditions

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Abstract

The study of functionally relevant biological effects of serotonin transporter gene promoter region (5-HTTLPR) polymorphisms is especially important given the current controversy about the clinical relevance of these polymorphisms. Here we report an intrinsic immunobiological difference between individuals carrying two short (SS) versus long (LL) 5-HTTLPR alleles, that is observed in healthy subjects reporting low exposure to life stress. Given that 5-HTTLPR polymorphisms are thought to influence susceptibility to depression and are associated with robust neurobiological effects, that depression is associated with higher pro-inflammatory and lower anti-inflammatory cytokines, and that acute stressors increase circulating concentrations of pro-inflammatory cytokines, we hypothesized that compared to LL individuals, SS individuals may show a pro-inflammatory bias under resting conditions and/or during stress. 15 LL and 11 SS individuals participated in the Trier Social Stress Test (TSST). Serum IL-6 and IL-10 were quantified at baseline and 30, 60, 90, and 120 min after beginning the 20-min stress test. Compared to LL individuals, SS individuals showed a higher IL-6/IL-10 ratio at baseline and during stress. Importantly, this pro-inflammatory bias was observed despite both groups being healthy, reporting similar intensities of stress and negative emotionality during the TSST, and reporting similar low exposures to early and recent life stress. To our knowledge, this is the first report of a pro-inflammatory bias/phenotype in individuals carrying the SS genotype of 5-HTTLPR. Thus, healthy SS individuals may be chronically exposed to a pro-inflammatory physiological burden under resting and stress conditions, which could increase their vulnerability to disorders like depression and other diseases that can be facilitated/exacerbated by a chronic pro-inflammatory state.

Introduction

With a lifetime prevalence of over 15% (Schatzberg, 2005), major depressive disorder (MDD) is one of the most common, and most devastating, of psychiatric disorders. Stressful life events, especially those that involve loss or a threat to social standing, are clearly associated with MDD onset (Brown and Harris, 1978, Kendler et al., 2005). However, most individuals faced with such stressors recover without becoming depressed. Therefore, an important question that remains to be answered is: what makes some people more vulnerable than others? Twin studies show that one strong predictor of who will develop MDD after a stressful life event is the presence of a depressed identical twin (Kendler et al., 2005). These studies support the hypothesis that genetic factors modulate our vulnerability to life stressors, and recent work has identified genes that may confer vulnerability to depression after stressor exposure.

One such gene is the serotonin transporter, SLC6A4. A functional polymorphism in the promoter region of this gene (5-HTTLPR) results in a “short” allele variant (SS), with decreased mRNA for the presynaptic serotonin transporter, and a corresponding 50% decrease in serotonin reuptake (Lesch et al., 1996, Richell et al., 2005, Murphy et al., 2008). Studies have shown that although SS individuals may be no more likely than others to be diagnosed with depression overall (Munafo et al., 2009), they may be at increased risk of developing depression after stressful life events (Caspi et al., 2003), or chronic illness (Otte et al., 2007). Attempts to replicate these findings have yielded both positive and negative results, and a recent, highly publicized meta-analysis concluded that the moderation may not exist at all (Risch et al., 2009). However, many attempts at replication of the original findings (Caspi et al., 2003) have been confounded by inconsistent assessment of early and recent life stressors (Monroe and Reid, 2008). Studies relying on structured interviews to assess the level of exposure to life stressors have consistently yielded positive findings, while most negative studies have used self-report questionnaires (Uher and McGuffin, 2008), which have been shown to have substantially less validity and reliability (Dohrenwend, 2006). A large body of research suggests that the 5-HTTLPR SS genotype may be a marker for stress-sensitivity more broadly: for example, SS individuals are more likely to attempt suicide, develop PTSD, or abuse substances after life stress (Roy et al., 2007, Kilpatrick et al., 2007, Brody et al., 2009). The association between serotonin transporter deficiency and stress-sensitivity also holds true in studies of both non-human primates and rodents. Infant macaques who carry the rhesus 5-HTTLPR S-allele show higher emotional distress, more agitation, and an exaggerated HPA response to maternal separation (Champoux et al., 2002, Barr et al., 2004), while serotonin transporter deficient mice consistently show an anxious phenotype, with exaggerated neuroendocrine and behavioral startle responses to stressful stimuli (reviewed in Monroe and Reid, 2008).

Rather than focusing solely on specific negative health outcomes (such as depression) that might result from a long-term pattern of stress-sensitivity, recent research has sought to understand the physiologic mechanisms by which 5-HTTLPR associated health-vulnerability might be conferred. Studies have confirmed that the S-allele is associated with robust increases in amygdala reactivity (Hariri et al., 2005, Munafo et al., 2008), as well as with decreased cingulate–amygdala functional coupling (Pezawas et al., 2005). This evidence suggests that downstream effects of the S-allele, such as increased risk of depression after life stressors, might be preceded by stereotypic alterations in brain physiology. Here we explore psychological stress-related inflammation as another avenue by which the short allele might exert physiological effects that might, downstream, increase vulnerability to post-stressor depression.

Enhanced inflammation during acute or short-term stress responses is likely to have adaptive effects by increasing immuno-protection to defend the organism from the actions of the stressor (e.g., wound inflicted by a predator) (Dhabhar, 2009). Healthy individuals show elevated inflammatory cytokines during and following exposure to stress (Altemus et al., 2001, Steptoe et al., 2007). However, vulnerable individuals, such as those suffering from depression, may exhibit exaggerated inflammatory responses during stress (Pace et al., 2006). This is thought to induce excessive, and potentially detrimental pro-inflammatory physiological changes that contribute to the development of pathology. Several converging lines of evidence suggest that depression and systemic inflammation are linked (Pollak and Yirmiya, 2002, Dantzer et al., 2008, Miller et al., 2009, Raison et al., 2009). First, individuals with major depression have higher levels of circulating inflammatory markers, including pro-inflammatory cytokines, than non-depressed individuals (Tsao et al., 2006, Dantzer et al., 2008, Capuron et al., 2008, Cizza et al., 2008). These elevations are found in both serum and cerebrospinal fluid (CSF), have been associated with a history of treatment non-responsiveness (Maes et al., 1997), and are known to decrease following successful treatment with SSRI (Kenis and Maes, 2002, Dantzer et al., 2008). Second, non-depressed individuals given pro-inflammatory cytokines, both in experimental and therapeutic contexts, develop depressive symptoms (Reichenberg et al., 2001, Capuron et al., 2000, Miller, 2009, Miller et al., 2009, Raison et al., 2009). 20–50% of individuals who receive long-term interferon therapy develop clinical depression, and such depressive episodes are responsive to SSRI treatment (Capuron et al., 2002a). Third, new lines of evidence suggest that depression may be associated not only with increases in inflammatory cytokines such as IL-6, but also with impaired production of anti-inflammatory cytokines such as IL-10. In healthy individuals, there is a regulated balance between pro- and anti-inflammatory cytokines. For example, IL-6 mediates the early phase of the inflammatory process, and then induces the release of IL-10 that exerts immuno-regulatory effects and resolves inflammation (Daftarian et al., 1996, Ogawa et al., 2008). It has been hypothesized that this regulatory loop is disrupted in individuals with depression, with one recent study showing both decreased IL-10 and an increased ratio of IL-6 to IL-10 in depressed individuals (Dhabhar et al., 2009), resulting in an overall bias towards inflammation. Despite these promising lines of evidence, no investigation has examined whether currently healthy individuals who may have a genetic predisposition towards depression after life stressors (such as SS homozygotes) show an abnormal inflammatory response to stress, with systemic inflammatory dysfunction preceding, or contributing to, overt psychiatric symptoms.

In the present study, we examined differences between healthy SS and LL individuals in the balance between pro- versus anti-inflammation under resting state and acute stress conditions. We chose the Trier Social Stress Test (TSST) (Kirschbaum et al., 1993) to induce acute stress because this test creates a controlled “real world” situation that is psychosocially threatening, inducing the sort of social threat noted by Kendler et al. (2005) to be particularly relevant to depression risk in genetically vulnerable subjects. We chose the ratio of circulating IL-6/IL-10 as an index of the balance between pro- and anti-inflammatory factors because it has recently been suggested that an important and relatively under-appreciated mechanism for increased susceptibility to depression and other diseases related to chronic inflammation may be a disruption of the immuno-regulatory balance between pro- and anti-inflammatory cytokines (Dhabhar et al., 2009). Such an immune balance can be shifted towards an inflammatory physiological milieu due to increased concentrations of pro-inflammatory cytokines, such as IL-6, decreased concentrations of anti-inflammatory/immunomodulatory cytokines, such as IL-10, or a combination of the two. Importantly, there may be significant inter-individual differences in the absolute concentrations of pro- or anti-inflammatory factors, many of which exert their opposing effects on the same target cells and tissues. Therefore, examining the ratio of pro- to anti-inflammatory factors provides a useful measure of the net immunological effect that the circulating cytokines and/or other factors are likely to have on their targets, of the overall immune bias of an individual’s physiological milieu, and of immune dysregulation (Dhabhar et al., 2009). Moreover, the balance of pro- and anti-inflammatory cytokines at baseline and immediately after acute stress may be indicative of the body’s ability to return to homeostasis after inflammation. Therefore, we measured circulating concentrations of IL-6, IL-10, and the IL-6/IL-10 ratio under relaxed resting state conditions and over time after the TSST, expecting that SS homozygotes would demonstrate an exaggerated inflammatory milieu under resting conditions, increased inflammatory reactivity during acute stress, or both.

Section snippets

Participants

Participants were 30 healthy, Caucasian women aged 18–25 (mean age: 21.7, SD 2.9) recruited via online advertisements in the San Francisco Bay Area. All potential participants were screened using a phone interview based on the Structured Clinical Interview for DSM-IV (SCID; First et al., 1996). Eligible participants had no indication of any psychiatric disorder within the past year, no substance abuse within the past six months, no more than minimal depressive symptoms (Beck Depression

Participant characteristics

Genotype groups did not differ significantly in terms of age, years of education, Beck Depression Inventory score, or scores on measures of early (CTQ) or recent (LESS) life stress. Additionally, the groups did not differ significantly in baseline heart rate or systolic or diastolic blood pressure (Table 1). Although obese subjects (BMI > 30) were not eligible for the study, LL individuals had significantly lower BMI than SS individuals, t(22) = 2.22, p = 0.037, d = 0.95, an effect which has previously

Discussion

5-HTTLPR genotype significantly affected the ratio of IL-6 to IL-10 under resting conditions and after acute psychosocial stress, with SS individuals exhibiting a significantly higher ratio across timepoints. This inflammatory bias was present despite both groups being medically healthy and reporting similarly low exposure to early and recent life stress, which may be independently associated with inflammation (Cohen et al., 1999, Danese et al., 2007, Danese et al., 2008). All participants

Acknowledgments

This work was supported by National Institute of Mental HealthR01 # MH58147 (J.J.G.), the Carl & Elizabeth Naumann Fund Startup Grant (F.S.D.), NARSAD Young Investigator Award34676 (W.R.), and Grant M01 RR-00070 from the National Center for Research Resources, National Institutes of Health. The authors thank Lin Xiaoyan for performing genotype analyses, Katie Denny for her help in developing the TSST protocol, Cole Shiflett and Michael Mehler for their assistance as Trier panelists, and the

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