Marie-Laure Ancelin
email: marie-laure.ancelin@inserm.fr

Jacqueline Scali
email: jacqueline.scali@inserm.fr

Joanna Norton
email: joanna.norton@inserm.fr

Karen Ritchie
email: karen.ritchie@inserm.fr

Anne- Marie Dupuy
email: am-dupuy@chu-montpellier.fr

Isabelle Chaudieu
email: isabelle.chaudieu@inserm.fr

Joanne Ryan
email: joanne.ryan@inserm.fr

The effect of an adverse psychological environment on salivary cortisol levels in the elderly differs by 5-HTTLPR genotype

Keywords: Stress response, adverse events, trauma, depression, hypothalamic-pituitary-adrenal axis, serotonin transporter-linked promoter region

Background: An adverse psychological environment (e.g. stressful events or depression) has been shown to influence basal cortisol levels and cortisol response to stress. This differs depending on the adverse stimuli, but also varies across individuals and may be influenced by genetic predisposition. An insertion/deletion polymorphism in the serotonin transporter gene (5-HTTLPR) is a strong candidate in this regard.

Objective: To investigate how stressful life events and depression are associated with diurnal cortisol levels in community-dwelling elderly and determine whether this varies according to genetic variability in the 5-HTTLPR.

Methods: This population-based study included 334 subjects aged 65 and older (mean (SD) = 76.5 (6.3)). Diurnal cortisol was measured on two separate days, under quiet (basal) and stressful conditions.

The number of recent major stressful events experienced during the past year was assessed from a 12item validated questionnaire as an index of cumulative recent stressful events. Lifetime trauma was evaluated using the validated Watson's PTSD inventory, which evaluates the most severe traumatic or frightening experience according to DSM criteria. Depression was defined as having a Mini-International Neuropsychiatric Interview (MINI) diagnosis of current major depressive disorder or high levels of depressive symptoms (Center for Epidemiologic Studies-Depression Scale ≥16). 5-HTTLPR genotyping was performed on blood samples.

Results: Exposure to stressful life events was associated with lower basal evening cortisol levels overall, and in the participants with the 5-HTTLPR L allele but not the SS genotype. The greatest effects (over 50% decrease, p<0.001) were observed for the LL participants having experienced multiple recent stressful events or severe lifetime traumas. Participants with the L allele also had higher evening cortisol stress response. Conversely, depression tended to be associated with a 42% higher basal morning cortisol in the SS participants specifically, but did not modify the association between stressful events and cortisol levels.

Conclusion:

An adverse psychological environment is associated with basal cortisol levels and cortisol stress response, but this differs according to 5-HTTLPR genotype.

Introduction

One of the most consistent findings in the biology of psychiatric disorders is altered activity of the corticotropic hypothalamic-pituitary-adrenal (HPA) axis, but this varies according to the disorder and across individuals [START_REF] Lupien | Effects of stress throughout the lifespan on the brain, behaviour and cognition[END_REF]. Major depressive disorder has often been associated with enhanced cortisol release in concert with a reduced feedback sensitivity of the HPA axis [START_REF] Belvederi Murri | HPA axis and aging in depression: systematic review and meta-analysis[END_REF][START_REF] Stetler | Depression and hypothalamic-pituitary-adrenal activation: a quantitative summary of four decades of research[END_REF], while an opposing picture has been described in posttraumatic stress disorder (PTSD) or trauma-exposed individuals [START_REF] Morris | Relations among posttraumatic stress disorder, comorbid major depression, and HPA function: a systematic review and meta-analysis[END_REF]. Cortisol secretion can be influenced by a number of factors, e.g. sampling conditions (time of sampling, basal vs. stress condition, experimental stressor vs. naturalistic protocol), as well as other moderators such as age, sex, comorbidity, and genetic sensitivity to environmental stress [START_REF] Belvederi Murri | HPA axis and aging in depression: systematic review and meta-analysis[END_REF][START_REF] Franz | Genetic and environmental influences on cortisol regulation across days and contexts in middle-aged men[END_REF][START_REF] Kudielka | Salivary cortisol in ambulatory assessment--some dos, some don'ts, and some open questions[END_REF][START_REF] Miller | The serotonin transporter gene-linked polymorphic region (5-HTTLPR) and cortisol stress reactivity: a meta-analysis[END_REF].

A genetic variant within the promoter region of the serotonin transporter (5-HTTLPR) has been associated with differential psychological sensitivity to stressful experiences. The short (S) allele of the 5-HTTLPR is associated with lower expression and decreased activity of the serotonin transporter and could modulate both risk for depression and psychopathological HPA axis signaling. Two recent metaanalyses reported a robust link between the short (S) form, reporting lifetime stress experience, and resulting depression [START_REF] Sharpley | An update on the interaction between the serotonin transporter promoter variant (5-HTTLPR), stress and depression, plus an exploration of non-confirming findings[END_REF], but no significant association between 5-HTTLPR and PTSD when compared to trauma-exposed controls [START_REF] Navarro-Mateu | Meta-analyses of the 5-HTTLPR polymorphisms and post-traumatic stress disorder[END_REF]. The influences of serotonergic neurotransmission on HPA-axis regulation and the link between serotonergic signaling and 5-HTTLPR genotype and HPA axis functioning have been largely documented from both human and animal studies [START_REF] Andrews | Programming of the hypothalamo-pituitary-adrenal axis: serotonergic involvement[END_REF][START_REF] Fuller | Serotonin receptors and neuroendocrine responses[END_REF][START_REF] Li | Reduction of 5hydroxytryptamine (5-HT)(1A)-mediated temperature and neuroendocrine responses and 5-HT(1A) binding sites in 5-HT transporter knockout mice[END_REF][START_REF] Porter | Corticosteroid-serotonin interactions in depression: a review of the human evidence[END_REF][START_REF] Vazquez | Regulation of corticoid and serotonin receptor brain system following early life exposure of glucocorticoids: long term implications for the neurobiology of mood[END_REF]. Several experimental studies have investigated the association between 5-HTTLPR genotype and HPA-axis reactivity to standardized laboratory stress tasks in healthy individuals with mixed results. A recent meta-analysis of 11 studies reported a weak but significant association between the homozygous SS genotype and enhanced cortisol reactivity to laboratory-imposed stressor [START_REF] Miller | The serotonin transporter gene-linked polymorphic region (5-HTTLPR) and cortisol stress reactivity: a meta-analysis[END_REF], although the unique study in older adults included in this metaanalysis reported an inverse association [START_REF] Mueller | Interaction of serotonin transporter gene-linked polymorphic region and stressful life events predicts cortisol stress response[END_REF]. Studies outside the laboratory are more limited and inconsistent and may depend on population age and moderation by psychopathological environment, e.g. life experiences and depressive symptomatology. They have been performed with populations at elevated risk for depression and adversity (self-rated). Two studies did not observe a moderating effect of 5-HTTLPR genotype on the association between psychosocial adversity and morning cortisol levels in adolescents [START_REF] Goodyer | Serotonin transporter genotype, morning cortisol and subsequent depression in adolescents[END_REF] or between recent stressful life events and awakening or evening cortisol levels in middle-aged adults [START_REF] Vinberg | Variations in 5-HTTLPR: relation to familiar risk of affective disorder, life events, neuroticism and cortisol[END_REF]. However, when considering cumulative risk exposure, a moderate association was found with cortisol area under the curve (AUC), moderated by 5-HTTLPR genotype in youth at elevated family risk for depression [START_REF] Willner | Diurnal cortisol rhythms in youth from risky families: effects of cumulative risk exposure and variation in the serotonin transporter gene[END_REF]. Among LL youth, greater cumulative risk was associated with a lower AUC, whereas among those carrying the short allele, there was a trend for an inverse association.

So far, no study has examined whether cortisol levels in response to various adverse psychological environment (real-life stress, trauma or depression), could differ according to 5-HTTLPR vulnerability. This could be particularly critical in older people, given that they have an accumulation of lifetime stressful events and show specific depression symptoms and etiologic factors as well as ageassociated neurobiological changes [START_REF] Fiske | Depression in older adults[END_REF]. With increasing age, the HPA axis might also become more vulnerable to dysregulation [START_REF] Belvederi Murri | HPA axis and aging in depression: systematic review and meta-analysis[END_REF][START_REF] Otte | A meta-analysis of cortisol response to challenge in human aging: importance of gender[END_REF]. Age has been frequently evoked as a potential source of inconsistent findings for the associations between 5-HTTLPR and depression [START_REF] Uher | The moderation by the serotonin transporter gene of environmental adversity in the aetiology of mental illness: review and methodological analysis[END_REF] and PTSD [START_REF] Navarro-Mateu | Meta-analyses of the 5-HTTLPR polymorphisms and post-traumatic stress disorder[END_REF]. Further, unlike in younger populations where the S allele is a risk factor, the LL genotype appears a risk factor for mental and physical distress in elderly people highly exposed to chronic disorders and severe stressors [START_REF] Grabe | Update on the 2005 paper: moderation of mental and physical distress by polymorphisms in the 5-HT transporter gene by interacting with social stressors and chronic disease burden[END_REF] as well as increased cortisol reactivity to laboratory stress tasks [START_REF] Mueller | Interaction of serotonin transporter gene-linked polymorphic region and stressful life events predicts cortisol stress response[END_REF].

The aim of this study was to examine diurnal cortisol levels in community-dwelling elderly under basal and stressful naturalistic conditions, in response to an adverse psychological environment (stressful life events including recent and major life traumas, and late-life depression), and to determine whether this varies according to 5-HTTLPR genotype.

Subjects and Methods

Participants

The data were derived from a longitudinal study of neuropsychiatric disorders in communitydwelling French elderly, the Esprit study [START_REF] Ritchie | Prevalence of DSM-IV psychiatric disorder in the French elderly population[END_REF]. Eligible participants, who were at least 65 years of age and non-institutionalized, were recruited by random selection from the electoral rolls between 1999 and 2001. Ethics approval for the study was given by the national ethics committee and written informed consent was obtained from all participants. The study was based on a random sample of 360 non-demented participants who had a complete set of salivary cortisol samples, collected at 4 time points, under both quiet and stressful conditions (see below), with a typical eucortisolemic pattern (including decline throughout the day), and were not being treated with medications likely to modify cortisol levels. Of these, 19 participants were missing 5-HTTLPR genotyping data, 1 did not have a depression assessment, and 6 failed to provide information on recent stressful events (RSE). This left 334 participants in the analyses (mean (SD) age = 76.5 (6.3) years). Compared to the overall Esprit sample (n=1855), the participants included in the present analysis were younger, less frequently women and less likely to have depression, cognitive impairment, and cardiovascular ischemic pathologies (p<0.003). They did not differ however regarding other characteristics, particularly recent stressful events (RSE, p=0.14) and lifetime traumas (p=0.27).

Cortisol measurement

Salivary cortisol was measured over the course of the day in participants who were not being treated with medication likely to modify cortisol levels (e.g. glucocorticoids, benzodiazepines, and hormonal treatment for women), as described previously [START_REF] Beluche | Persistence of abnormal cortisol levels in elderly persons after recovery from major depression[END_REF]. Saliva samples were not collected from participants with dementia. Participants were instructed not to drink, eat or smoke for at least 30 min before saliva collection. As cortisol levels increase shortly after awakening and to avoid potential interference with sleep-wake transition [START_REF] Kudielka | Salivary cortisol in ambulatory assessment--some dos, some don'ts, and some open questions[END_REF], participants were asked to collect the first sample in the morning, but that it needed to be 1 hour or more after awakening (morning cortisol). Subsequent samples were then collected 3 (midday cortisol), 7 (afternoon cortisol), and 14 hours (evening cortisol) after the first morning sample (the last sample being collected before midnight to eliminate early cortisol increase occurring during the nocturnal phase). To maximize compliance, the participants were first shown by the lay interviewer at the examination center how to perform salivary collection and were instructed to carefully write down the exact times when collections were made in a diary to be returned with the samples. Furthermore, as in other naturalistic studies, participants were allowed to decide their own wake and sleep times. Participants were encouraged to carry on their normal daily activities with limited physical exertion in order to maximize ecological validity. Samples were taken under two contrasting conditions; at the hospital ("stressful situation"), with the first sampling at their arrival at the examination center, just prior to a lengthy clinical examination which involved various recognized psychosocial stressors (e.g. psychiatric examination, cognitive testing, clinical evaluation, and blood collection) and a subsequent quiet day at home with normal daily activities (baseline condition) [START_REF] Belvederi Murri | HPA axis and aging in depression: systematic review and meta-analysis[END_REF][START_REF] Franz | Genetic and environmental influences on cortisol regulation across days and contexts in middle-aged men[END_REF].

The participants were aware of the duration and global content of the examination. Maximal contrasting conditions were thus obtained, while avoiding novelty or anticipatory effects on the baseline measures.

None of the participants reported any additional stressors on the days they performed sampling. The stress versus basal conditions were associated with a highly significant increase in cortisol levels (p<0.0001), as published previously (Ancelin et al., 2013a;[START_REF] Beluche | Persistence of abnormal cortisol levels in elderly persons after recovery from major depression[END_REF][START_REF] Chaudieu | Abnormal reactions to environmental stress in elderly persons with anxiety disorders: evidence from a population study of diurnal cortisol changes[END_REF].

Cortisol levels were determined from saliva collection [START_REF] Hellhammer | Measurement of salivary cortisol under psychological stimulation[END_REF] by direct radioimmunoassay (Diagnostic Systems Laboratories-Webster, Texas). Intra-assay as well as interassay coefficients of variation averaged 5%. Individuals with a typical eucortisolemic pattern are those displaying cortisol decline throughout the day, thus with a negative slope of the regression of the four log-transformed cortisol values on the sampling time. The individuals with missing data and those who did not show a negative slope were excluded as previously reported [START_REF] Lupien | How to measure stress in humans? in: Centre for Studies on Human Stress©[END_REF]. They did not differ significantly regarding main characteristics from those retained in the study.

Adverse psychological environment

Exposure to recent stressful events (RSE) during the past year was assessed using the Gospel Oak questionnaire [START_REF] Harwood | The prevalence of diagnoses, impairments, disabilities and handicaps in a population of elderly people living in a defined geographical area: the Gospel Oak project[END_REF]. This 12-item list of major life events included bereavement, rupture of close relationships, severe illness and serious financial or judicial problems. The number of stressful events experienced during the past year was used as an index of cumulative recent stressful events. Severe lifetime traumatic events and symptom severity were assessed using the validated Watson's PTSD Inventory which evaluates the most severe lifetime traumatic event or frightening experience according to DSM criteria [START_REF] Watson | The PTSD interview: rationale, description, reliability, and concurrent validity of a DSM-III-based technique[END_REF]. This questionnaire can provide measures of the severity of the disorder for every symptom and allow the measurement of subsyndromic PTSD as described previously [START_REF] Chaudieu | Late-life health consequences of exposure to trauma in a general elderly population: the mediating role of reexperiencing posttraumatic symptoms[END_REF].

The diagnosis of lifetime major depression and anxiety disorder was made by psychologists and psychiatric nurses according to DSM-IV criteria and using the Mini-International Neuropsychiatric Interview (MINI, French version 5.00), a standardized psychiatric examination validated in the general population [START_REF] Sheehan | The Mini-International Neuropsychiatric Interview (M.I.N.I.): the development and validation of a structured diagnostic psychiatric interview for DSM-IV and ICD-10[END_REF] and with high inter-rater reliability [START_REF] Lecrubier | The Mini International Neuropsychiatric Interview (MINI). A short diagnostic structured interview: reliability and validity according to the CIDI[END_REF]. The interviewers were initially trained for a 3 month period under the supervision of psychiatrists from the Department of Adult Psychiatry at Montpellier University Hospital and interviewer drift was minimized. The positive cases were reviewed by a panel of independent psychiatrists as described previously [START_REF] Ancelin | Gender and genotype modulation of the association between lipid levels and depressive symptomatology in community-dwelling elderly (the ESPRIT study)[END_REF][START_REF] Ritchie | Late-onset agoraphobia: general population incidence and evidence for a clinical sub-type[END_REF]. The Center for Epidemiologic Studies-Depression Scale (CES-D), validated in the elderly, was used to evaluate current depressive symptomatology [START_REF] Radloff | The CES-D scale: a self-report depression scale for research in the general population[END_REF].

In older adults, late-life depression covers a range of mild to severe depressive symptoms which does not always correspond to the DSM criteria for major depression, although it has devastating consequences on physical and social functioning, disability, and mortality [START_REF] Fiske | Depression in older adults[END_REF].To adequately capture this construct, we based our late-life depression assessment on two criteria.

Participants with either a MINI diagnosis of current major depression or high levels of depressive symptomatology (CES-D>16) were defined as having a clinical level of late-life depression (Dep), i.e. levels of psychopathology which would warrant clinical intervention [START_REF] Ancelin | Gender and genotype modulation of the association between lipid levels and depressive symptomatology in community-dwelling elderly (the ESPRIT study)[END_REF].

5-HTTLPR genotyping

Blood samples were collected after the baseline clinical interview, enabling DNA extraction and 5-HTTLPR genotyping as described previously [START_REF] Ritchie | Association of adverse childhood environment and 5-HTTLPR genotype with late-life depression[END_REF]. To verify the accuracy of the data, replicate independent genotyping was also performed using DNA extracted from buccal samples as described previously (Ancelin et al., 2013a).

Socio-demographic and clinical variables

The standardized interview included information on socio-demographic characteristics, physical health, and medical history of the participants. Weight and height were measured during clinical examination and body mass index (BMI) was calculated and expressed as kg/m 2 . Detailed medical questionnaires (with additional information from general practitioners) were used to obtain information on history of cardiovascular ischemic pathologies (angina pectoris, myocardial infarction, stroke, cardiovascular surgery, and arteritis). Global cognitive function was evaluated using the Mini-Mental State Examination (MMSE), a score <26 indicating cognitive impairment [START_REF] Folstein | Mini-mental state". A practical method for grading the cognitive state of patients for the clinician[END_REF].

Dementia was diagnosed by a neurologist as part of a standardized examination and validated by a panel of independent neurologists, as described previously (Ancelin et al., 2013b).

Statistical Analysis

All data were cleaned following standard procedures. Due to the exponential distribution of raw diurnal cortisol data, cortisol values were log-transformed. Given the non-fixed time sampling protocol, cortisol levels were calculated at fixed times from the regression of the four-cortisol values on the sampling times, for each participant and on two different days (basal and stressful situation).

Stress response ( was calculated for each participant and for all the four samples across the day as the ratio of (stress cortisol levelbasal cortisol level)/ basal cortisol level; and expressed as %, as published previously [START_REF] Beluche | Persistence of abnormal cortisol levels in elderly persons after recovery from major depression[END_REF][START_REF] Chaudieu | Abnormal reactions to environmental stress in elderly persons with anxiety disorders: evidence from a population study of diurnal cortisol changes[END_REF]. Chi-squared tests were used to compare the distribution of 5-HTTLPR genotypes with those predicted under the Hardy-Weinberg equilibrium.

The primary analyses concerned the average cortisol concentrations at the four times of the day (morning, midday, afternoon and evening) on basal conditions and then under stress condition (expressed as stress response). Subsequent multivariate analyses focused on morning and evening times which constitute very common HPA axis indicators and the most contrasting conditions of the diurnal cycle (at which cortisol levels reached the daily zenith and nadir, respectively), and displaying distinct characteristics. Morning cortisol levels show a much higher heritability whereas evening cortisol has a greater environmental influence and could also be differently influenced by depression and stressful life events [START_REF] Belvederi Murri | HPA axis and aging in depression: systematic review and meta-analysis[END_REF][START_REF] Franz | Genetic and environmental influences on cortisol regulation across days and contexts in middle-aged men[END_REF][START_REF] Miller | If it goes up, must it come down? Chronic stress and the hypothalamic-pituitary-adrenocortical axis in humans[END_REF][START_REF] Morris | Relations among posttraumatic stress disorder, comorbid major depression, and HPA function: a systematic review and meta-analysis[END_REF]. Bivariate associations between cortisol levels and exposure to stressful events (RSE or severe trauma) or Dep were evaluated using ANCOVA adjusted for age and sex. Multivariate models were then generated combining the different adverse environmental factors (stressful events and Dep), to determine the adjusted associations. Further adjustment was made for other covariates known to influence cortisol, based on prior studies, notably BMI (Incollingo [START_REF] Rodriguez | Hypothalamic-pituitary-adrenal axis dysregulation and cortisol activity in obesity: A systematic review[END_REF], a history of major depression [START_REF] Beluche | Persistence of abnormal cortisol levels in elderly persons after recovery from major depression[END_REF], current phobia and generalized anxiety disorder [START_REF] Chaudieu | Abnormal reactions to environmental stress in elderly persons with anxiety disorders: evidence from a population study of diurnal cortisol changes[END_REF]. SAS (v9.4, SAS Institute, Inc., North Carolina) was used for the statistical analyses with a significance level of p<0.05.

Results

Participant characteristics

Baseline characteristics of the 334 participants are summarized in Table 1. Forty percent reported at least one RSE in the last year, 57% experienced severe traumatic events during their life, and 14.4% currently had Dep of whom 4.2% had major depression. One quarter of the participants were homozygous carriers of the S allele and 26.3% were LL. The 5-HTTLPR genotype frequency did not significantly deviate from Hardy-Weinberg equilibrium (p=0.75). The socio-demographic and clinical characteristics of SS, SL, and LL participants did not differ significantly (p≥0.25).

Cortisol levels according to recent stressful events

Fig. 1 shows the diurnal patterns of basal cortisol levels (adjusted for age and sex) as a function of RSE among all participants and according to the 5-HTTLPR genotype. Overall and for the participants with the SL and LL genotype, a difference in cortisol levels was observed in the afternoon and evening between the participants reporting RSE and those who did not (see Supplementary Table S1 for full details). Lower cortisol levels were seen with a higher number of RSE. Conversely, for the SS participants, no significant differences were found at any time.

In a similar manner to the basal cortisol levels, RSE were not associated with morning stress response, irrespective of the 5-HTTLPR genotype whereas the evening stress response (evening) appeared to significantly increase with RSE in LL (p=0.005) and SL (p=0.007) participants, but not for those with the SS (p=0.43) genotype (Supplementary Fig. S1). The highest evening (37.8%) was observed for the LL participants having reported at least two RSE, compared with 3.3% in the LL participants having reported no RSE in the last year.

Cortisol levels according to lifetime trauma and re-experiencing symptoms

Data on lifetime traumatic events were also available for 288 participants of whom 57% reported a trauma according to DSM IV criteria, at a median age of 24 years (ranging from 5-82 years). Of these traumatized participants, 30.5% had reported re-experiencing symptoms, the most common and clinically relevant symptom associated with trauma in PTSD (subclinical PTSD). In a model adjusted for age and sex, significant associations were only found for evening time; cortisol levels were 45.1% lower in the LL participants with re-experiencing symptoms compared to non-traumatized LL participants (p=0.015) whereas no significant differences were observed for the SL and SS participants (see Supplementary Table S1). When further adjusting for RSE, the same pattern was observed, evening cortisol levels being 47.8% lower specifically in the LL participants with re-experiencing symptoms compared to non-traumatized LL participants (p=0.006) (Supplementary Figure S2). The evening stress response was also significantly higher for the LL participants having reported reexperiencing symptoms (evening=36.9%) compared to 14.6% in non-traumatized participants (p=0.019) whereas no significant differences were found for morning stress response (Supplementary Fig. S3).

Cortisol levels according to Dep

Fig. 2 shows the diurnal pattern of basal cortisol levels (adjusted for age and sex) as a function of Dep in the overall sample and according to 5-HTTLPR genotype. Cortisol levels across the day were not significantly different between the depressed and non-depressed participants in the overall sample (p>0.15) and in the SL group (p>0.72). For the SS participants, morning cortisol levels were 40.6% higher (non-log transformed scale) in the depressed compared with the non-depressed participants (p=0.059) (see Supplementary Table S1). For the LL participants, evening cortisol levels specifically were 46.4% lower (p=0.011) in depressed compared to non-depressed participants. Further adjustment for RSE (Fig. 3) had very little effect on the association between Dep and morning cortisol levels in SS participants (42.3% higher, p=0.05) as well as further adjusting for past major depression (p=0.047).

However, among LL participants, there was no longer an association between Dep and evening cortisol levels after inclusion of RSE (p=0.13).

In this model, Dep was not associated with cortisol stress response irrespective of the 5-HTTLPR genotype (Supplementary Table S1 and Fig. S4). Hence, despite higher basal morning cortisol levels, the depressed SS participants largely maintained their capacity to further increase cortisol levels under stress conditions, in a similar manner to the non-depressed SS participants.

The influence of RSE and lifetime trauma on cortisol levels, accounting for Dep

We next sought to determine whether accounting for Dep, modified the associations between RSE or lifetime trauma and cortisol. Further adjusting for Dep in the analyses described in § 3.2, did not modify the pattern of associations for cortisol basal levels and stress response regardless of the time (see Supplementary Table S1). More particularly, in the LL participants, evening basal cortisol levels were 28.1% (non-log transformed scale) and 54.2%, lower for those reporting one and at least two RSE respectively, compared to those who did not (p=0.0097) (Fig. 4B). For participants with the SL genotype evening cortisol levels were also significantly lower (p=0.0005), by 35.2% and 53.3% respectively, whereas no significant difference was observed for the SS participants (p=0.76). The same data were also found in the most complete multivariable model adjusted for age, sex, Dep and lifetime trauma (see Supplementary Table S1).

The results with lifetime trauma (cf. § 3.3) were also unchanged after further adjusting for Dep; LL participants with re-experiencing compared to those without had 51% lower evening cortisol levels specifically (p≤0.006), as well as higher evening stress response (p=0.02), irrespective of whether RSE was in the model (see Supplementary Table S1). Further adjusting for BMI, a history of major depression, or anxiety disorder did not change these patterns (data not shown).

Discussion

To our knowledge, this is the first study to investigate the impact of different adverse psychological environments (recent stressful events, major life trauma, and Dep) on diurnal cortisol levels as a function of 5-HTTLPR genotype. These results suggest that stressful events and Dep can independently influence cortisol levels and the cortisol stress response, even when accounting for other factors such as comorbidity, however the associations vary according to genotype. While the SS genotype tended to be associated with higher morning cortisol levels in depressed participants, a lower evening cortisol was observed in the participants with LL and SL genotypes having being exposed to stressful environment, independently of Dep. The strongest associations were found for the homozygous LL participants having reported multiple RSE or who experienced severe lifetime trauma.

The greatest difference in cortisol levels was seen for elderly individuals reporting both multiple RSE and Dep, compared with non-depressed individuals with no RSE (LL had 80% lower evening cortisol (non-log transformed scale), p<0.0001; SS had 126% higher morning cortisol, p=0.003). However the low number of participants in this subgroup precluded drawing definite conclusions. These data provide further support for the complex interplay between HPA axis functioning and serotonergic signaling with possible modulation according to adverse psychological environment, e.g. depression or trauma. In line with this, a large meta-analysis of case-control studies have reported heightened basal cortisol levels throughout the diurnal cycle in depressed patients compared to healthy controls [START_REF] Belvederi Murri | HPA axis and aging in depression: systematic review and meta-analysis[END_REF]. Conversely, lowered evening cortisol levels, but not morning levels, have consistently been reported in trauma-exposed individuals (see for meta-analysis [START_REF] Morris | Relations among posttraumatic stress disorder, comorbid major depression, and HPA function: a systematic review and meta-analysis[END_REF]). Hence, in depressed people, morning cortisol levels specifically appeared to be increased, and independently of trauma, whereas for evening cortisol this may vary according to lifetime traumatic experience [START_REF] Morris | Relations among posttraumatic stress disorder, comorbid major depression, and HPA function: a systematic review and meta-analysis[END_REF]. Our findings thus concur with these meta-analyses and add novel information regarding moderation by 5-HTTLPR vulnerability. Heightened morning basal cortisol levels being found only in depressed SS participants, while evening cortisol levels were specifically lowered, and in a dose (RSE number)-dependent manner, in trauma-exposed participants with the L allele. For the SL participants the effects were only significant with recent stressful events, whereas for the LL homozygous both recent and distant past traumatic events (subclinical PTSD) were associated with lower evening cortisol levels and higher stress response. Our data suggests that the LL elderly participants could be vulnerable not only to the short-term but also lasting consequences of stressful events on neuroendocrine function, and possibly to the subsequent development of chronic PTSD symptoms. A recent meta-analysis did not support a direct effect of 5-HTTLPR polymorphisms on PTSD, but the authors reported that several characteristics, e.g. stressor, comorbidity, and population characteristics, especially age and ethnicity could moderate the associations [START_REF] Navarro-Mateu | Meta-analyses of the 5-HTTLPR polymorphisms and post-traumatic stress disorder[END_REF]. Consistently, Grabe et al. reported that in community-dwelling older European/ Caucasian people, the risk of lifetime PTSD increased with the number of L alleles as well as the number of traumatic events [START_REF] Grabe | Serotonin transporter gene (SLC6A4) promoter polymorphisms and the susceptibility to posttraumatic stress disorder in the general population[END_REF]. The same group also reported a switch from the S allele to the LL genotype for the risk of mental and physical distress in older adults according to the number of chronic diseases [START_REF] Grabe | Update on the 2005 paper: moderation of mental and physical distress by polymorphisms in the 5-HT transporter gene by interacting with social stressors and chronic disease burden[END_REF].

The regulation of the HPA axis is complex and influenced by multiple factors, e.g. exposure to different stressors, person-dependent factors, and heritable factors. Twin studies show a much higher heritability of morning cortisol than evening cortisol levels (60% vs. 8%), with the latter having a greater environmental influence [START_REF] Franz | Genetic and environmental influences on cortisol regulation across days and contexts in middle-aged men[END_REF]. Our data also support a greater effect of stressful environment on evening cortisol levels as well as stress response. Further, they suggest that increased morning cortisol in the SS depressed participants may be under genetic control. HPA axis functioning and cortisol levels could be influenced by various genes involved in corticosteroid signaling. Specifically, the corticosteroid receptors have been involved in a large range of neurobiological correlates that underlie depression, e.g. HPA axis hyperactivity, glucocorticoid resistance, and changes in neural plasticity and neurogenesis [START_REF] Anacker | The glucocorticoid receptor: pivot of depression and of antidepressant treatment?[END_REF]. Antidepressants have been shown to impact all of these mechanisms and to modulate receptor function, providing further support that these receptors may play a pivotal role in the neurobiological disturbances that contribute to depression [START_REF] Anacker | The glucocorticoid receptor: pivot of depression and of antidepressant treatment?[END_REF].

Cortisol hypersecretion is thought to characterize the short-term effect of stress with initial activation of the HPA axis, whereas hypo-secretion may develop in the long-term [START_REF] Miller | If it goes up, must it come down? Chronic stress and the hypothalamic-pituitary-adrenocortical axis in humans[END_REF][START_REF] Morris | Relations among posttraumatic stress disorder, comorbid major depression, and HPA function: a systematic review and meta-analysis[END_REF] and has been associated with experiencing a high degree of chronic stress [START_REF] Bremmer | Major depression in late life is associated with both hypo-and hypercortisolemia[END_REF]. Our findings suggest that in the general elderly population, cortisol hypersecretion may be a reflection of current psychological load (depression), whereas hyposecretion could reflect early as well as recent exposure to stressful events. The exact time when RSE occurred during the past year was not known and we cannot ascertain whether this could reflect short-lasting attenuated HPA axis activity in the context of high cumulative risk or leading to a more persistent stress effect. Furthermore, we demonstrate that this is dependent on 5-HTTLPR genotype. A moderate association between cumulative risk exposure and cortisol AUC, moderated by 5-HTTLPR genotype was previously reported in 138 multiethnic youth at elevated family risk for depression [START_REF] Willner | Diurnal cortisol rhythms in youth from risky families: effects of cumulative risk exposure and variation in the serotonin transporter gene[END_REF]. Among LL youth, greater cumulative risk was associated with a lower AUC, whereas among those carrying the short allele, there was a trend for an inverse association. Our study extends this finding to elderly general population and further suggests that early trauma could have a long lasting effect on evening cortisol in LL individuals, specifically and independently of Dep.

Limitations to our study include the bias introduced from selecting community-dwelling participants who were in relatively good physical and mental health which may have decreased the overall power of the study, possibly underestimating the associations found. Bias from population stratification needs to be considered. However, prior genotyping data of these participants indicated that less than 1% were non-Caucasian (Ancelin et al., 2013a) and genotype frequency was similar with that already published in white Europeans [START_REF] Miller | The serotonin transporter gene-linked polymorphic region (5-HTTLPR) and cortisol stress reactivity: a meta-analysis[END_REF]. Although this study in the elderly population was limited to only one day on two conditions but with four salivary cortisol measures, and despite exclusion of the participants with missing data and an atypical pattern, the basal characteristics of cortisol are similar to previous studies in older adults with more frequent sampling [START_REF] Ice | Diurnal cycles of salivary cortisol in older adults[END_REF].

Data related to life events were retrospective, which may introduce recall bias, but participants diagnosed with probable/possible dementia were excluded. Finally, since multiple analyses have been performed we cannot exclude that some associations were due to chance. However, many of the associations between stressful life events and participants with the L allele remained significant even after applying overly conservative multiple testing correction. On the other hand, further studies are needed to replicate our findings with depressive symptomatology which may be underpowered due to the small number of Dep cases.

Strengths are that it was the first population-based study involving 334 elderly people having complete data on 5-HTTLPR and diurnal cortisol levels under basal and stressful conditions. Cortisol levels were measured under naturalistic conditions and using a non-fixed time-sampling protocol [START_REF] Belvederi Murri | HPA axis and aging in depression: systematic review and meta-analysis[END_REF][START_REF] Franz | Genetic and environmental influences on cortisol regulation across days and contexts in middle-aged men[END_REF], known to improve compliance in the elderly [START_REF] Jacobs | Electronic monitoring of salivary cortisol sampling compliance in daily life[END_REF][START_REF] Kraemer | Design decisions to optimize reliability of daytime cortisol slopes in an older population[END_REF], and compliance rates were excellent, with the systematic return of saliva samples by all the participants. Thus cortisol measures most likely reflect their "normal/everyday" levels previously reported in the elderly [START_REF] Ice | Diurnal cycles of salivary cortisol in older adults[END_REF]. Dep was assessed by trained staff using two distinct measures validated in the general population, including a structured diagnostic interview [START_REF] Radloff | The CES-D scale: a self-report depression scale for research in the general population[END_REF][START_REF] Sheehan | The Mini-International Neuropsychiatric Interview (M.I.N.I.): the development and validation of a structured diagnostic psychiatric interview for DSM-IV and ICD-10[END_REF] according to DSM-IV criteria. RSE and lifetime traumatisms (DSM criteria) were evaluated using validated questionnaires. We controlled for important stress-related covariates thus minimizing any confounding or moderating effect, which contrasts with previous studies which have predominantly presented minimally adjusted analyses.

Finally, we were able to control for accuracy through duplicate samples collected at different time and with independent genotyping.

In conclusion, this study is the first to provide a thorough investigation of the extent to which different adverse psychological environments can influence diurnal cortisol levels and cortisol stress response, and how this varies according to the environmental sensitivity of 5-HTTLPR polymorphism.

We have shown that older adults can display a differential pattern of HPA dysregulation according to their psychopathological characteristics and 5-HTTLPR genetic vulnerability. Stressful environment was associated with significantly lower basal evening cortisol levels and heightened stress response in L allele carriers and Dep tended to be associated with a higher basal morning cortisol in the SS individuals. Furthermore, this suggests that there is a difference in the biological effects of acute stress versus chronic stress or trauma and could also complement the current debate on potential mechanisms mediating vulnerability for the development of psychiatric disorders as a function of 5-HTTLPR genotype.

linked polymorphic region (5-HTTLPR) [corrected]. Dev Psychopathol 26, 999-1019. a Except for BMI and education (n=332), MMSE (n=331), current anxiety disorder (n=320), past major depression (n=307), and lifetime traumatic events (n=288). b Number of RSE during the past year assessed using the Gospel Oak questionnaire [START_REF] Harwood | The prevalence of diagnoses, impairments, disabilities and handicaps in a population of elderly people living in a defined geographical area: the Gospel Oak project[END_REF]. c Severe lifetime traumatic events were assessed with Watson's PTSD Inventory according to DSM criteria [START_REF] Watson | The PTSD interview: rationale, description, reliability, and concurrent validity of a DSM-III-based technique[END_REF]. d Having a MINI diagnosis of current major depression or high levels of depressive symptomatology (CES-D>16). e Diagnosis of major depression and anxiety disorders (generalized anxiety disorder, phobias) according to DSM-IV criteria and using the MINI [START_REF] Sheehan | The Mini-International Neuropsychiatric Interview (M.I.N.I.): the development and validation of a structured diagnostic psychiatric interview for DSM-IV and ICD-10[END_REF]. f History of cardiovascular ischemic pathologies (angina pectoris, myocardial infarction, stroke, cardiovascular surgery, arteritis).

Figure captions

Fig. 1 .

 1 Fig. 1. Diurnal basal cortisol secretion as a function of recent traumatic events (RSE) in the whole sample (n=334) and according to the 5-HTTLPR genotype a,b .

Fig. 2 .

 2 Fig. 2. Diurnal basal cortisol secretion as a function of current depression (Dep) in the overall sample (n=334) and according to the 5-HTTLPR genotype a,b .

Fig. 3 .

 3 Fig. 3. Morning (A) and evening (B) basal cortisol as a function of current depression (Dep) and according to 5-HTTLPR genotype (n=334) a,b .

Fig. 4 .

 4 Fig. 4. Morning (A) and evening (B) basal cortisol as a function of recent stressful events (RSE) according to 5-HTTLPR genotype and accounting for depression (n=334) a,b .

Fig. 1 *

 1 Fig.1

Table 1

 1 Characteristics of the 334 a participants

	Characteristic
		Mean (SD)
	Age (years)	76.5 (6.3)
	Body Mass Index (kg/m 2 )	25.3 (3.5)
		% (n)
	Sex (female)	49.4% (165)
	< 12 years of education (%)	51.5% (332)
	Recent stressful events (RSE) b
	0	60.5% (202)
	1	22.7% (76)
	≥ 2	16.8% (56)
	Lifetime traumatic events c
	No Trauma	43.0% (124)
	Trauma without re-experiencing	39.6% (114)
	Trauma with reexperiencing	17.4% (50)
	Depression d	14.4% (48)
	Past major depression e	26.4% (81)
	Current antidepressant use	4.5% (15)
	Current anxiety disorders e	7.8% (25)
	Cardiovascular ischemic pathologies f	8.4% (28)
	Cognitive impairment	8.5% (28)
	(MMSE score <26)
	5-HTTLPR genotype
	SS	24.6% (82)
	SL	49.1% (164)
	LL	26.3% (88)

Funding

The ESPRIT project is financed by the Agence Nationale de la Recherche (ANR) (grant 07 LVIE 004), and an unconditional grant from Novartis. The funders had no role in the design and conduct of the study; in data collection, management, analysis, interpretation of the data; or writing the report preparation, review, or approval of the manuscript.

Disclosure statement

The authors declare no conflict of interest to disclose. No Trauma

ANNEX: SUPPLEMENTARY INFORMATION

Trauma no re-exp.

Trauma + re-exp p=0.20 p=0.57 p=0.17


Marie-Laure Ancelin
email: marie-laure.ancelin@inserm.fr

Jacqueline Scali

Joanna L Norton

Karen A Ritchie

Anne-Marie Dupuy

Isabelle Chaudieu

Joanne Ryan

S1 Ancelin

Heterogeneity in HPA axis dysregulation and serotonergic vulnerability to depression

Keywords: Cortisol, depression, elderly, hypothalamic-pituitary-adrenal axis, serotonin transporterlinked promoter region, stress

à la diffusion de documents scientifiques de niveau recherche, publiés ou non, émanant des établissements d'enseignement et de recherche français ou étrangers, des laboratoires publics ou privés.

Introduction

In the past, clinicians have made the distinction between 'endogenous' and 'reactive' depression in accordance with the empirical observation that depressive episodes may be triggered in some patients by environmental stressors, but not in others, where it was attributable to genetic vulnerability. The most recent meta-analysis confirmed a robust link between the short (S) form of the serotonin transporter gene

(5-HTTLPR), experiencing stress, and resulting depression [START_REF] Sharpley | An update on the interaction between the serotonin transporter promoter variant (5-HTTLPR), stress and depression, plus an exploration of non-confirming findings[END_REF]. Around 35% of these studies however, failed to show any significant association or found opposite results, with carriers of the long (L) form being at a higher risk of depression following life stress. The studies reporting opposing findings did not appear to be flawed by reduced power or methodological weaknesses, and the results were independent of study design, depression measure and type of stressful event [START_REF] Sharpley | An update on the interaction between the serotonin transporter promoter variant (5-HTTLPR), stress and depression, plus an exploration of non-confirming findings[END_REF].

Age has been frequently evoked as a potential source of inconsistent findings for depression (Uher and McGuffin, 2008) and post-traumatic stress disorder [START_REF] Navarro-Mateu | Meta-analyses of the 5-HTTLPR polymorphisms and post-traumatic stress disorder[END_REF]. Further, unlike in younger populations where the S allele is a risk factor, the LL genotype appears a risk factor for mental and physical distress in elderly people highly exposed to chronic disorders and severe stressors [START_REF] Grabe | Update on the 2005 paper: moderation of mental and physical distress by polymorphisms in the 5-HT transporter gene by interacting with social stressors and chronic disease burden[END_REF]. This may explain why there is no generally accepted gene-dose model. Another important source of heterogeneity pointed by Sharpley et al. and potential limitation of previous studies is the method used to assess stress, i.e. whether extrinsic stress was self-reported or objectively recognized [START_REF] Sharpley | An update on the interaction between the serotonin transporter promoter variant (5-HTTLPR), stress and depression, plus an exploration of non-confirming findings[END_REF]. They also raised the possibility of different neurobiological underpinnings and pathways for depression but, so far, intrinsic biological stress measures have not been examined.

The hypothalamic-pituitary-adrenal (HPA) axis is one of the principal stress signaling pathways, and results in releases of cortisol [START_REF] Kudielka | Salivary cortisol in ambulatory assessment--some dos, some don'ts, and some open questions[END_REF]. Cortisol secretion is not only a good indicator of HPA responsivity and differential biological response to short-term versus long-term effect of stress (Miller et al., 2007;Morris et al., 2012) but it also constitutes one important neurobiological characteristic of depression [START_REF] Stetler | Depression and hypothalamic-pituitary-adrenal activation: a quantitative summary of four decades of research[END_REF]. Heightened diurnal cortisol levels have been frequently reported in depressed individuals (Belvederi Murri et al., 2014), and remain high after recovery from major depression (Beluche et al., 2009), suggesting it may constitute a trait marker for vulnerability to depression. Conversely, exposure to traumatic events has been associated with lower evening cortisol (Chaudieu et al., 2008;Morris et al., 2012). There is also a link between serotonergic signaling and HPA axis functioning (Vazquez et al., 2012), and the 5-HTTLPR gene has been shown to influence cortisol reactivity in young adults exposed to acute psychosocial stress, whereas the unique study in elderly reported an inverse association [START_REF] Miller | The serotonin transporter genelinked polymorphic region (5-HTTLPR) and cortisol stress reactivity: a meta-analysis[END_REF]. So far, no study has examined the impact of both an adverse environment and corticotropic axis dysfunctioning, which could represent different aspects of stress with distinct biological effects [START_REF] Grabe | Update on the 2005 paper: moderation of mental and physical distress by polymorphisms in the 5-HT transporter gene by interacting with social stressors and chronic disease burden[END_REF] and consequences for depression risk. Whether this could differ as a function of 5-HTTLPR genotype, yielding contradictory findings has also not been investigated.

This study evaluated whether both recent stressful events and cortisol levels are associated with late-life depression and if this differs according to 5-HTTLPR genotype, while taking into account sociodemographic and stress-related factors, as well as past major depression.

Subjects and Methods

Eligible participants, who were at least 65 years of age and non-institutionalized, were recruited by random selection from the electoral rolls between 1999 and 2001 [START_REF] Ritchie | Association of adverse childhood environment and 5-HTTLPR genotype with late-life depression[END_REF]. The standardized interview in the Esprit study included information on socio-demographic characteristics, physical health, and medical history of the participants. This study was based on a random sample of 344 non-demented participants who underwent depression assessment, responded to all questions concerning their experience of recent stressful events, had complete diurnal cortisol samples, and agreed to provide blood samples for 5-HTTLPR genotyping. Repeated diurnal salivary cortisol samples and measures were taken, as published previously (see Supplemental Methods for details). Genotyping of 5-HTTLPR and the A/G polymorphism (rs25531) within the promoter region were performed as described previously [START_REF] Ritchie | Association of adverse childhood environment and 5-HTTLPR genotype with late-life depression[END_REF], which was validated with independent genotyping of matched buccal DNA samples. Major depression and anxiety disorder were diagnosed according to DSM-IV criteria using the Mini International Neuropsychiatric Interview (MINI, French version 5.00), a standardized psychiatric examination validated in the general population [START_REF] Sheehan | The Mini-International Neuropsychiatric Interview (M.I.N.I.): the development and validation of a structured diagnostic psychiatric interview for DSM-IV and ICD-10[END_REF]. Caselevel late-life depression was defined as a MINI diagnosis of current major depression or high levels of depressive symptomatology (score ≥16) on the Center for Epidemiologic Studies-Depression Scale [START_REF] Radloff | The CES-D scale: a self-report depression scale for research in the general population[END_REF]. Exposure to stressful events during the past year was assessed using the validated Gospel Oak questionnaire (Harwood et al., 1998). The national ethics committee approved the study and all participants provided written informed consent.

The association between the 5-HTTLPR allelic frequency and depression was examined by logistic regression analysis, using the conventional biallelic and the triallelic model further considering the rs25531 polymorphism within the promoter. Previous association studies have suggested dominant, codominant, and recessive models of the 5-HTTLPR S (or S') allele without a clear consensus (Uher and McGuffin, 2008). To overcome this problem, we compared both possible allele groups separately by aggregating samples according to an S (or S') recessive model (having two alleles), and an S (or S')

dominant model (having at least one allele). We also examined whether there was a statistical significant interaction between genotype (5-HTTLPR) and stress-related variables on the risk of depression. In keeping with the original aim of our study, we then stratified analysis by 5-HTTLPR, and generated multivariable models to determine the association between stress-related variables (morning and evening cortisol levels, recent stressful events), and depression. SAS (v9.4, SAS Institute, NC, USA) was used for the statistical analyses with a significance level of p <0.05.

Results

Participant characteristics are summarized in Table 1 and4.5% currently used antidepressants. The mean (SD) age of first onset depression was 48.3 (16.6) years and 43% of the participants with past depression had recurrent episodes. There were no significant differences in socio-demographic or clinical characteristics across genotype groups. In logistic regression models adjusted for age and sex, experiencing a recent stressful event and a history of major depression were both associated with a >2fold increased risk of clinical depression in the whole sample, but cortisol levels were not associated with depression risk (Table 2A). There was some evidence that 5-HTTLPR (+/-rs25531) genotype modified the association between stress-related factors and depression risk, in particular for recent stressful events, although the multiplicative interaction term failed to reach statistical significance (p<0.10 in both the dominant and recessive models) (Tables S1 andS2).

After stratification by genotype, the associations of recent stressful events and depression with depression more than doubled in strength for L homozygous individuals, but were clearly not significant for individuals homozygous for the S allele (Table 2B). A distinct pattern was found when examining cortisol levels. Higher morning cortisol levels were associated with a 4-fold increased risk of depression in the SS individuals only. Conversely, a negative association between evening cortisol and depression risk was found specifically for LL participants. In multivariable models combining the significant stressrelated risk factors, evening cortisol levels were no longer significantly associated with depression in LL participants, after accounting for recent stress (evening cortisol: OR=0.57, 95% CI=0.29-1.11, p=0.10; recent stressful events: OR=5.16, 95% CI=1. 35-19.78, p=0.017). These findings remained similar after inclusion of past major depression (Table S3A) and were not modified when taking into account recurrent episodes or current antidepressant use. Additional variables shown in Table 1 were not significantly associated with depression (all p ≥0.15). The associations remained consistent when examining the triallelic model, and in addition recent stressful events were associated with depression among S'L' heterozygotes (Table S3B).

Discussion

To our knowledge, this is the first study to investigate the impact of stress on depression, using both extrinsic (self-report of stressful events) and intrinsic (cortisol secretion) stress indicators. Our findings show differential stress-related susceptibility to late-life depression, with some indication that this might also vary depending on 5-HTTLPR genotype, although the GxE interaction term failed to reach the 5% significance level. In stratified analysis, the risk of depression was significantly associated with higher morning cortisol levels specifically in the SS participants and this was independent of stressful events. In the LL participants however, stressful events and past major depression were significantly associated with increased depression risk. These significant associations were independent of sociodemographic characteristics and comorbidity. No stress-related factors were significantly associated with depression in the SL individuals despite double the number of participants in this group compared with the SS or LL participants. This may explain why in this study and others, there is no clear conclusion regarding the inheritance model of the 5-HTTLPR polymorphism (morning cortisol suggests a recessive model but past major depression a dominant one).

These data provide further support for the complex interplay between HPA axis functioning and serotonergic signaling with possible modulation according to adverse psychological environment, e.g. depression or trauma. In line with this, a large meta-analysis of case-control studies have reported heightened basal cortisol levels throughout the diurnal cycle in depressed patients compared to healthy controls (Belvederi Murri et al., 2014). Conversely, lowered evening cortisol levels, but not morning levels, have consistently been reported in trauma-exposed individuals (see for meta-analysis (Morris et al., 2012)). Hence, in depressed people, morning cortisol levels specifically appeared to be increased, and independently of trauma, whereas for evening cortisol this may vary according to lifetime traumatic experience (Morris et al., 2012).

Cortisol hypersecretion is thought to characterize the short-term effect of stress with initial activation of the HPA axis, whereas hypo-secretion may develop in the long-term (Miller et al., 2007;Morris et al., 2012) and has been associated with experiencing a high degree of chronic stress or recurrent depressive episodes [START_REF] Bremmer | Major depression in late life is associated with both hypo-and hypercortisolemia[END_REF]. In the elderly, late-life depressive symptoms have been found

to be associated with both hyperactivity and hypoactivity of the HPA axis, with distinct characteristics, e.g. low cortisol levels being specifically associated with recurrent depression [START_REF] Bremmer | Major depression in late life is associated with both hypo-and hypercortisolemia[END_REF].

However these studies were limited by only one cortisol sample and they did not take into account stressful life events. This could be particularly critical considering that elderly people are more likely to accumulate stressful experiences with potential influences on cortisol secretion, and that with increasing age the HPA axis could be more vulnerable to dysregulation (Belvederi Murri et al., 2014).

Our findings in the general elderly population concur with these previous reports; cortisol hypersecretion reflecting current psychological load, whereas hyposecretion possibly reflecting exposure history and chronic stress. Furthermore, we present some preliminary evidence that this might depend on 5-HTTLPR genotype, which would also be supported by some prior research findings. Lowered evening cortisol in LL depressed participants could actually reflect or be a consequence of adversity, as the former was not significant after multivariable adjustment. This is consistent with our previous findings in this Esprit population of a significant association between exposure to traumas (Chaudieu et al., 2008) and lower evening cortisol levels. It may be that LL participants are more vulnerable to intrinsically stress related environmental factors which could reflect 'reactive' depression, however this hypothesis could not be directly tested in our study. The specific association of past depression, as well as early life events [START_REF] Ritchie | Association of adverse childhood environment and 5-HTTLPR genotype with late-life depression[END_REF] with current depression in the LL participants, also supports this hypothesis.

Conversely, heightened morning cortisol could represent a biological correlate or even a causal factor for late-life depression in SS individuals and might be a marker of 'endogenous' depression. In contrast, SL individuals appeared resilient to both intrinsically and extrinsically stress-related factors in terms of depression risk. A schematic model was drawn to illustrate these findings (Figure S1). Equivalent results

were found for both S'S' and L'L' homozygous. For the S'L' heterozygotes, recent stressful events were associated with depression, but to a much weaker extent than for L'L' participants. A number of allelic variants have been described in the 5-HTTLPR polymorphisms in humans and it is currently not clear whether the magnitude of any association may be affected by this variability and if rs25531 has any impact on the functionality of the short 5-HTTLPR allele (Uher and McGuffin, 2008). Caution however must also be taken in the interpretation of these findings, given that the interaction terms did not reach statistical significance and the effect sizes were modest. It would be interesting to now try and replicate these findings in a very large independent cohort.

The regulation of the HPA axis is complex and influenced by multiple factors, e.g. exposure to different stressors, person-dependent factors, and heritable factors. Twin studies show a much higher heritability of morning cortisol than evening cortisol levels (60% vs. 8%), with the latter having a greater environmental influence (Franz et al., 2010). Our data would also support this and further suggests that increased morning cortisol in the SS depressed participants may be under genetic control. The set-point of the HPA axis could be influenced by genes involved in corticosteroid signaling. The corticosteroid receptors have been involved in a large range of neurobiological correlates that underlie depression, e.g.

HPA axis hyperactivity, glucocorticoid resistance, and changes in neural plasticity and neurogenesis [START_REF] Anacker | The glucocorticoid receptor: pivot of depression and of antidepressant treatment?[END_REF]. Antidepressants have been shown to impact all of these mechanisms and to modulate receptor function, providing further support that these receptors may play a pivotal role in the neurobiological disturbances that contribute to depression [START_REF] Anacker | The glucocorticoid receptor: pivot of depression and of antidepressant treatment?[END_REF].

Although the size of our study was relatively large for a study of this kind (with data both on diurnal cortisol secretion and 5-HTTLPR genotyping), the stratified analysis included groups of less than 100 individuals, thus potentially limiting the overall power of the study. Data related to recent stressful events were retrospective, but participants diagnosed with possible dementia were excluded to minimize recall bias. Strengths of our study are that it was population-based and involved 344 elderly people with 5-HTTLPR genotyping data (including information on the rs25531 variant) as well as complete diurnal cortisol secretion and validated measures of recent stressful events and depression. We were not able to consider atypical depression subtypes but we controlled for key stress-related covariates thus minimizing any confounding as well as for genotyping accuracy through duplicate samples collected at different times and with independent genotyping, to ensure the integrity of this data. Finally, since multiple analyses have been performed and we stratified analysis by 5-HTTLPR genotype, we cannot exclude that some associations were due to chance. Further studies are needed to replicate our findings.

Our findings demonstrate differential stress-related susceptibility to late-life depression and the potential that these effects might vary depending on 5-HTTLPR genotype. a The 5-HTTLPR genotype frequency did not significantly deviate from Hardy-Weinberg equilibrium ( 2 =0.10, df=1, p=0.75) and reflected frequencies seen in white Europeans [START_REF] Miller | The serotonin transporter genelinked polymorphic region (5-HTTLPR) and cortisol stress reactivity: a meta-analysis[END_REF].

b Except for body mass index and education (n=332), MMSE (n=331), current anxiety disorder (n=320), and past major depression (n=307).

c Number of recent stressful events during the past year assessed using the validated Gospel Oak questionnaire (Harwood et al., 1998).

d Having a MINI diagnosis of current major depression or high levels of depressive symptomatology (CES-D score ≥ 16).

e Diagnosis of major depression and current anxiety disorders (generalized anxiety disorder and phobia) according to DSM-IV criteria and using the MINI [START_REF] Sheehan | The Mini-International Neuropsychiatric Interview (M.I.N.I.): the development and validation of a structured diagnostic psychiatric interview for DSM-IV and ICD-10[END_REF]).

e History of cardiovascular ischemic pathologies (angina pectoris, myocardial infarction, stroke, cardiovascular surgery, arteritis).

5-HTTLPR p g SS (N=82) SL (N=164) LL (N=88) p g S allele (N=246) p g L allele (N=252)

Recent stressful event d 0.13 0. d At least one recent stressful event during the past year assessed using the validated Gospel Oak questionnaire (Harwood et al., 1998).

e Diagnosis of past major depression according to DSM-IV criteria and using the MINI [START_REF] Sheehan | The Mini-International Neuropsychiatric Interview (M.I.N.I.): the development and validation of a structured diagnostic psychiatric interview for DSM-IV and ICD-10[END_REF].

f Morning and evening basal salivary cortisol were calculated at fixed times (8.00am for morning and 10.00pm for evening cortisol) from the regression of the fourcortisol values on the sampling times, for each participant (see Supplemental Methods).

g p-value for the interaction term between stress-related variable and genotype in the multivariable regression model.

Laboratories-Webster, Texas) [START_REF] Hellhammer | Measurement of salivary cortisol under psychological stimulation[END_REF]. Intra-assay and inter-assay coefficients of variation averaged 5%.

Since the distribution of raw cortisol is typically skewed, and the normal diurnal profile can be approximated by an exponential curve, cortisol values were log-transformed. Cortisol levels were calculated at fixed times (8.00am for morning and 10.00pm for evening cortisol) from the regression of the four-cortisol values on the sampling times, for each participant as published previously (Beluche et al., 2009;Chaudieu et al., 2008). These times constitute very common HPA axis indicators and were chosen as the most contrasting conditions of the diurnal cycle (at which cortisol levels reached their daily zenith and nadir, respectively), and displaying distinct characteristics. Morning cortisol levels show a much higher heritability whereas evening cortisol has a greater environmental influence and could also be differently influenced by depression and stressful life events (Belvederi Murri et al., 2014;Franz et al., 2010;Miller et al., 2007;Morris et al., 2012).

Sheehan, D.V., Lecrubier, Y., Sheehan, K.H., Amorim, P., Janavs, J., Weiller, E., Hergueta, T., Baker, R., Dunbar, G.C., 1998. The Mini-International Neuropsychiatric Interview (M.I.N.I.): the development and validation of a structured diagnostic psychiatric interview for DSM-IV and ICD-10. J Clin Psychiatry 59, 22-33. b At least one recent stressful event during the past year assessed using the validated Gospel Oak questionnaire (Harwood et al., 1998).

c Diagnosis of past major depression according to DSM-IV criteria and using the MINI [START_REF] Sheehan | The Mini-International Neuropsychiatric Interview (M.I.N.I.): the development and validation of a structured diagnostic psychiatric interview for DSM-IV and ICD-10[END_REF].

d Morning and evening basal salivary cortisol were calculated at fixed times (8.00am for morning and 10.00pm for evening cortisol) from the regression of the fourcortisol values on the sampling times, for each participant (see Supplemental Methods).

B. Triallelic model

All S'S' (N=99) S'L' (N=158) L'L' (N=65) L' allele

Recent stressful event b 2. 64 [1.34-5.20 Green arrows indicate associations found in this study (light green is not significant after adjustment) and the purple arrow indicates potential association inferred here. MDD: major depressive disorder; RSE: recent stressful event.

LL participants appeared more vulnerable to stress-related environmental factors (reactive depression), morning cortisol could represent a biological correlate or a causal factor for depression in SS individuals (endogenous depression), whereas SL individuals appeared resilient to both intrinsically and extrinsically stress-related factors.

Figure S1 .

 S1 Figure S1. Hypothetical model for the differential stress-related susceptibility to late-life depression according to 5-HTTLPR genotype.

  This would suggest individual neuroendocrine heterogeneity of depression which may require different clinical management. Uher, R., McGuffin, P., 2008. The moderation by the serotonin transporter gene of environmental adversity in the aetiology of mental illness: review and methodological analysis. Mol Psychiatry 13, 131-46.Vazquez, D.M., Neal, C.R., Jr., Patel, P.D., Kaciroti, N., Lopez, J.F., 2012. Regulation of corticoid and serotonin receptor brain system following early life exposure of glucocorticoids: long term implications for the neurobiology of mood. Psychoneuroendocrinology 37, 421-37. Characteristics of the participants according to their 5-HTTLPR genotype a

	Table 1
	5-HTTLPR	Whole sample	SS	SL	LL	Comparison SS/SL/LL
		(N=334 b )	(N=82)	(N=164)	(N=88)	(global test p-value)
		Mean (SD)				t-test
	Age (years)	76.5 (6.3)	76.3 (6.3)	76.2 (6.3)	77.2 (6.3)	0.53
	Body mass index (kg/m 2 )	25.3 (3.5)	25.5 (3.7)	25.4 (3.5)	24.8 (3.5)	0.42
		% (n)				Chi 2 -test
	Sex (female)	49.4% (165)	48.8% (40)	47.6% (78)	53.4% (47)	0.67
	< 12 years of education	51.5% (171)	54.3% (44)	51.2% (84)	49.4% (43)	0.81
	At least one recent stressful event c	39.5% (132)	35.4% (29)	43.2% (71)	36.4% (32)	0.38
	Clinical level of depression d	14.4% (48)	14.6% (12)	11.6% (19)	19.3% (17)	0.25
	Past major depression d	26.4% (81)	28.4% (21)	22.7% (34)	31.3% (26)	0.32
	Current anxiety disorders e	7.8% (25)	4.9% (4)	9.0% (14)	8.4% (7)	0.53
	Cardiovascular ischemic pathologies f	8.4% (28)	6.1% (5)	7.9% (13)	11.4% (7)	0.44
	Cognitive impairment (MMSE <26)	8.5% (28)	9.9% (8)	8.5% (14)	7.0% (6)	0.80

Abbreviations: CES-D, Center for Epidemiologic Studies-Depression Scale; MINI, Mini International Neuropsychiatric Interview; MMSE, Mini-Mental State Examination; 5-HTTLPR, serotonin transporter gene-linked polymorphic region.

Table 2

 2 Risk of depression a associated with stress-related factors b

	A. In the whole sample (N=334 c )
		Odds Ratio [95% Confidence Interval] (p)
	Recent stressful event d	2.28 [1.18-4.40] (0.014)
	Past major depression e	2.03 [1.01-4.09] (0.047)
	Morning cortisol f	1.45 [0.84-2.50] (0.18)
	Evening cortisol f	0.80 [0.56-1.14] (0.21)
	5-HTTLPR:
	SS vs. SL	1.25 [0.56-2.81] (0.58)
	LL vs. SL	1.87 [0.90-3.88] (0.09)
	SS vs. LL	0.65 [0.27-1.55] (0.33)
	L allele vs. SS	1.03 [0.49-2.14] (0.95)
	S allele vs LL	0.57 [0.29-1.13] (0.11)
	B. According to 5-HTTLPR genotype (biallelic model)
		Odds Ratio [95% Confidence Interval] (p)

  Center for Epidemiologic Studies-Depression Scale; CI, confidence interval; MINI, Mini International Neuropsychiatric Interview; OR, odds ratio; 5-HTTLPR, serotonin transporter gene-linked polymorphic region. Having a MINI diagnosis of current major depression or high levels of depressive symptomatology (CES-D score ≥ 16).

	Abbreviations: CES-D,
		80 [0.19-3.41]	2.26 [0.80-6.34]	6.45 [1.82-22.9] 0.09	1.48 [0.67-3.28]	0.09	3.39 [1.54-7.46]
	Past major depression e 0.19 1.01 [0.43-4.54]	1.41 [0.43-4.62]	4.47 [1.34-14.9] 0.07	1.27 [0.51-3.17]	0.35	2.51 [1.12-5.60]
	Morning cortisol f	0.20 4.09 [1.03-16.2]	0.89 [0.41-1.92]	1.72 [0.57-5.21] 0.76	1.44 [0.76-2.72]	0.12	1.44 [0.76-2.72]
	Evening cortisol f	0.23 1.00 [0.45-2.22]	1.01 [0.58-1.73]	0.46 [0.24-0.87] 0.08	1.05 [0.68-1.62]	0.62	0.74 [0.50-1.11]

a b Logistic regression model adjusted for age and sex. c Except for past major depression; n=307 (74 SS, 150 SL, 83 LL).

Table S1 .

 S1 Logistic regression models a for the association between stress-related variables b and depression c , considering effect modification by 5-

	HTTLPR (biallelic model)
		S recessive (L allele vs. SS)	S dominant (S allele vs. LL)	3 genotype group (LL, SL, SS)
		 coefficient (SE)	p	 coefficient (SE)	p	 coefficient (SE) d Global p-value
	≥ 1 stressful event (E)	-0.16 (0.68)	0.81	1.67 (0.62)	0.007	0.86 (0.52)	0.10
	Genotype (G)	-0.65 (0.50)	0.19	0.09 (0.55)	0.87	1: 0.23 (0.62) 2: 0.74 (0.56)	0.41
	Interaction: G x E	1.33 (0.79)	0.09	-1.22 (0.73)	0.09	1: 0.82 (0.80) 2: -1.03 (0.85)	0.13
	Past major depression (E)	0.14 (0.71)	0.84	1.55 (0.60)	0.01	0.22 (0.59)	0.71
	Genotype (G)	-0.18 (0.46)	0.70	0.007 (0.48)	0.99	1: 0.06 (0.51) 2: 0.20 (0.49)	0.92
	Interaction: G x E	0.76 (0.80)	0.35	-1.35 (0.74)	0.07	1: 1.33 (0.83) 2: -0.08 (0.91)	0.19
	Morning cortisol (E)	1.29 (0.66)	0.05	0.55 (0.57)	0.34	-0.05 (0.39)	0.90
	Genotype (G)	6.60 (4.22)	0.12	0.51 (3.67)	0.89	1: -2.65 (3.83) 2: -7.47 (4.41)	0.23
	Interaction: G x E	-1.14 (0.73)	0.12	-1.96 (0.65)	0.76	1: 0.60 (0.69) 2: 1.35 (0.77)	0.20
	Evening cortisol (E)	-0.07 (0.36)	0.85	-0.63 (0.30)	0.03	0.01 (0.27)	0.97
	Genotype (G)	0.67 (1.44)	0.64	-2.61 (1.23)	0.03	1: 2.73 (1.34) 2: 0.47 (1.58)	0.11
	Interaction: G x E	-0.20 (0.41)	0.62	0.63 (0.37)	0.08	1: -0.65 (0.40) 2: -0.07 (0.45)	0.23

a Adjusted for age and sex. b Full definitions of stress-related variables are provided in Table

1

.

c

Having a MINI diagnosis of current major depression or high levels of depressive symptomatology (CES-D score ≥ 16). d 1: LL vs. SL; 2: SS vs. SL.

Table S2 .

 S2 Logistic regression models a for the association between stress-related variables b and depression c , considering effect modification by

	5-HTTLPR (triallelic model)
		S recessive (L' allele vs. S'S')	S dominant (S' allele vs. L'L')	3 genotype group (L'L', S'L', S'S')
		 coefficient (SE)	p	 coefficient (SE)	p	 coefficient (SE) d global p-value
	≥ 1 stressful event (E)	-0.23 (0.67)	0.73	1.74 (0.69)	0.01	1.75 (0.69)	0.01
	Genotype (G)	-0.29 (0.50)	0.56	0.17 (0.60)	0.78	1: 0.05 (0.65) 2: 0.33 (0.66)	0.84
	Interaction : G x E	1.48 (0.78)	0.058	-1.18 (0.78)	0.13	1: -0.76 (0.84) 2: -1.99 (0.96)	0.11
	Past major depression (E)	0.21 (0.69)	0.77	1.53 (0.66)	0.02	1.53 (0.66)	0.02
	Genotype (G)	0.26 (0.46)	0.57	0.01 (0.51)	0.98	1: 0.13 (0.54) 2: -0.17 (0.60)	0.82
	Interaction : G x E	0.70 (0.79)	0.38	-1.18 (0.78)	0.13	1: -1.06 (0.85) 2: -1.32 (0.95)	0.32
	Morning cortisol (E)	1.28 (0.62)	0.04	0.93 (0.66)	0.16	0.93 (0.66)	0.16
	Genotype (G)	6.85 (4.02)	0.09	3.17 (4.11)	0.44	1: 5.43 (4.25) 2: -2.85 (5.18)	0.10
	Interaction : G x E	-1.11 (0.69)	0.11	-0.66 (0.73)	0.37	1: -1.04 (0.76) 2: 0.35 (0.90)	0.10
	Evening cortisol (E)	-0.09 (0.35)	0.79	-0.45 (0.32)	0.16	-0.45 (0.32)	0.16
	Genotype (G)	0.86 (1.40)	0.54	-1.58 (1.26)	0.21	1: -1.53 (1.36) 2: -1.84 (1.63)	0.43
	Interaction : G x E	-0.13 (0.40)	0.73	0.34 (0.37)	0.36	1: 0.36 (0.41) 2: 0.36 (0.47)	0.63
	a Adjusted for age and sex
	b Full definitions of stress-related variables are provided in Table 1.

c

Having a MINI diagnosis of current major depression or high levels of depressive symptomatology (CES-D score ≥ 16). d 1: L'L' vs. S'L'; 2: S'S' vs. S'L'.

Table S3 .

 S3 Multivariable logistic regression models for the risk of depression a adjusted for age, sex, and relevant stress-related factors, according to 5-

	HTTLPR genotype

a Having a MINI diagnosis of current major depression or high levels of depressive symptomatology (CES-D score ≥ 16).

  n.a. = not applicable; not included in the final multivariable model if p >0.10 in model adjusted for age and sex.

			Odds Ratio [95% Confidence Interval] (p)
	A. Biallelic model	All	SS (N=82)	SL (N=164)	LL (N=83)	L allele
	Recent stressful event b	2.58 [1.30-5.10] (0.007)	n.a. e	n.a.	4.96 [1.16-21.26] (0.031)	3.15 [1.37-7.23] (0.007)
	Past major depression c	2.11 [1.03-4.32] (0.041)	n.a.	n.a.	5.91 [1.46-23.92] (0.013)	2.64 [1.15-6.08] (0.022)
	Morning cortisol (continuous) d	n.a.	4.09 [1.03-16.2] (0.045) n.a.	n.a.	n.a.
	Evening cortisol (continuous) d	n.a.	n.a.	n.a.	0.57 [0.29-1.11] (0.10)	0.83 [0.54-1.29] (0.41)

e

  ] (0.005) n.a. 2.79 [1.05-7.44] (0.040) 7.02 [1.30-37.85] (0.024) 4.10 [1.78-9.42] (0.0009)

	Past major depression c	2.20 [1.08-4.51] (0.030) n.a.	n.a.	5.73 [1.23-26.77] (0.027)	2.69 [1.16-6.27] (0.022)
	Morning cortisol (continuous) d	n.a.	4.09 [1.14-14.7] (0.031)	n.a.	n.a.	n.a.
	Evening cortisol (continuous) d	n.a.	n.a.	n.a.	0.68 [0.31-1.48] (0.33)	n.a.

Funding

The ESPRIT project is financed by the Agence Nationale de la Recherche (ANR) (grant 07 LVIE 004), and an unconditional grant from Novartis. The funders had no role in the design and conduct of the study; in data collection, management, analysis, interpretation of the data; or writing the report preparation, review, or approval of the manuscript.

Disclosure statement

The authors declare no conflict of interest to disclose.

SUPPLEMENTARY MATERIALS

Supplemental Methods

Cortisol measurement

Cortisol measures were taken under naturalistic conditions with participants having the choice of a non-fixed time-sampling protocol (Belvederi Murri et al., 2014;Franz et al., 2010) known to improve compliance in the elderly [START_REF] Jacobs | Electronic monitoring of salivary cortisol sampling compliance in daily life[END_REF][START_REF] Kraemer | Design decisions to optimize reliability of daytime cortisol slopes in an older population[END_REF]. Saliva samples were not collected from participants with dementia. Participants with missing time points, inadequate saliva volume or atypical cortisol baseline profiles (flat pattern or abnormal time peak) were excluded from the study sample. The compliance rates were excellent with the systematic return of saliva samples by all the subjects. Hence, the study was initially based on a random sample of 360 non-demented participants who had complete salivary cortisol samples, with a typical eucortisolemic pattern, and were not being treated with medications likely to modify cortisol levels (e.g. glucocorticoids, benzodiazepines, and hormonal treatment for women), as described previously [START_REF] Ancelin | Angiotensin-converting enzyme gene variants are associated with both cortisol secretion and late-life depression[END_REF]Beluche et al., 2009;Chaudieu et al., 2008). Of these, 19 participants were missing 5-HTTLPR genotype, 1 did not have a depression assessment, and 6 failed to provide information on recent stressful events. This left 334 participants in the analyses. Compared to the overall Esprit sample (n=1855), the participants included in the present analysis were younger, less frequently women and less likely to have depression, cognitive impairment, and cardiovascular ischemic pathologies (p<0.003), but did not differ regarding other characteristics, including recent stressful events (p=0.14).

Participants were instructed not to drink, eat or smoke for at least 30 mins before saliva collection and to start the protocol at least 1h after awakening. Subsequent samples were collected at 3, 7, and 14 h after the first morning sampling and exact times were recorded. Participants carried on their normal daily activities with limited physical exertion in order to maximize ecological validity. They did not report any additional stressors on the day of sampling. and the basal levels were similar to previous studies in the elderly [START_REF] Ice | Diurnal cycles of salivary cortisol in older adults[END_REF]. Salivary cortisol levels were determined by direct radioimmunoassay (Diagnostic Systems