Early-life adversity is associated with differential gene expression in response to acute psychological stress: preliminary findings

Objective Exposure to early-life adversity (ELA) can result in long-term changes to physiological systems, which predispose individuals to negative health outcomes. This biological embedding of stress-responsive systems may operate via dysregulation of physiological resources in response to common stressors. The present study used a novel experimental design to test how young adults’ exposure to ELA influence neuroendocrine and inflammatory responses to acute stress. Materials and methods Participants were 12 males (mean age= 21.25), half of whom endorsed at least three significant adverse events up to age 18 years (‘ELA group’), and half who confirmed zero (‘controls’). Using a randomized within-subjects, between-groups experimental design, we induced acute psychosocial stress (Trier Social Stress Test, TSST), and included a no-stress control condition one week apart. During these sessions, we obtained repeated measurements of physiological reactivity, gene expression of NR3C1, FKBP5 and NFKB1, and plasma levels of pro-inflammatory cytokines (IL-1β, IL-6, IL-8 and TNFα) over a 4-hour window post-test. Results The ELA group evinced significantly higher cortisol response and lower NR3C1 gene expression in response to the TSST compared with controls, while no differences were observed in the no-stress condition. Cortisol and group status interacted such that increase in cortisol predicted increase in both NR3C1 and NFKB1 expression among controls, but decrease in the ELA group. For pro-inflammatory cytokines, only IL-6 increased significantly in response to the TSST, with no differences between the two groups. Conclusion Overall, we provide preliminary findings for the biological embedding of stress via a dynamic and dysregulated pattern evidenced in response to acute psychosocial stress. ELA may program physiological systems in a maladaptive manner more likely to manifest during times of duress, predisposing individuals to the negative health consequences of everyday stressors. Future studies with larger sample size including both males and females are needed to replicate these findings.

endocrine illness (for example, asthma, diabetes, thyroid disease or pituitary gland disorders 153 confirmed by self-report and physical examination), were currently non-smokers and were not 154 using medication on a regular basis, including psychiatric medication. The final sample included 155 12 men, 6 of whom experienced early adversity (i.e., 'ELA group') and 6 who did not (i.e.,

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The study was approved by the Ethics Committee at the Pennsylvania State University and all

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Participants made two visits to the CRC during weekdays, one week apart, on the same day.

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Testing was scheduled to begin at 11:00am and end by 4:15pm. We used a randomized 165 counter-balanced order for the two sessions (i.e., TSST and no-stress control conditions) blind 166 to participants and lab personnel. Lab personnel were also blind to group status. Participants 167 were given specific instructions to refrain from excessive physical activity on the day of the 168 testing, consuming alcohol for 12 hours before their arrival, and eating and drinking (besides 169 water) for 2 hours prior to the testing session. After arrival and consent, trained nurses 170 completed a physical examination and inserted an IV catheter into the antecubital vein 30 171 minutes after arrival (30 minutes prior to testing). The TSST session was scheduled to begin at 172 12:00pm to minimize the effects of circadian changes in cortisol, and was carried out as 173 described previously [41]. Briefly, the TSST consists of a free speech and a mental arithmetic 174 task of 10 minutes duration performed in front of a panel of two committee members (mixed 175 gender) with a camera and microphone situated between the interviewers. Participants were 176 told that they would play the role of an interviewee for a job and have 5 minutes to make an 8 177 argument for their candidacy. After 5 minutes, the second task emphasizing cognitive load 178 commenced. In this task, participants were asked to count backwards from 1,687 in multiples of 179 13. If a mistake was made, they were instructed to start again from the beginning. In the no-180 stress control condition, participants were instructed to sit in a room, read magazines, and to 181 refrain from any stressful activities (e.g., cell-phone use was restricted). After the second blood 182 draw, approximately 60 minutes after the TSST session and 90 minutes after the first baseline 183 measure in the no-stress control condition, participants were administered a set of 184 questionnaires. These questionnaires were administered in both sessions and the average 185 score was calculated before analyses (see below for details). Considering the long time-frame 186 of the study and the repeated collection of multiple blood samples, a standardized low-calorie 187 meal was provided after the third blood draw (approximately at 1:45pm  (Fig 1). Given known changes in immune cell redistribution and composition in response to 212 acute stress [20], complete blood count with differential was measured within 24 hours by Quest

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Diagnostics using additional 4 ml EDTA collection tubes.

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Whole blood samples were collected in 10 mL EDTA blood tubes via an IV catheter into the 215 antecubital vein, and immediately centrifuged for 10 minutes at 1500g prior to collection of 216 plasma. PBMCs were immediately isolated through density-gradient centrifugation using Ficoll. findings confirm previous studies indicating increased stress and anxiety levels in individuals 343 exposed to ELA, compared with non-exposed individuals.  p<0.001, η 2 = 0.46), and a trend towards a higher cortisol response in the TSST relative to no-stress (ΔAUCi: F=3.58, p=0.088) (Fig 2). Notably, no significant differences were observed 364 between the ELA and control groups in the no-stress condition (Time , indicating increased physiological reactivity to acute stress in young adults 367 exposed to early adversity, compared with non-exposed individuals. to the TSST relative to the no-stress condition (F=5.85, p=0.026) (Fig 2). No differences were 400 observed between the ELA and control groups in response to the TSST relative to no-stress 401 using either AUCi PCA components.

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Exploratory analyses of each pro-inflammatory cytokine revealed a significant within-  whether the responses differ between the ELA and control groups. Specifically, we used cortisol 414 slope increase from baseline to peak levels (from 30 minutes prior to testing to 15 minutes after 415 stress onset) to predict summary changes in gene expression and cytokines in response to the 416 TSST (Fig 4). AUCi summary measure, which was then standardized for ease of comparison across genes.

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R 2 shown are from models with cortisol slope as the only predictor.

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In the whole sample, cortisol increase did not predict significant changes in gene expression

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To our knowledge, this is the first investigation of stress-induced gene expression and pro-465 inflammatory cytokines changes within-individuals, comparing stratified groups of ELA-exposed 466 and control individuals. By comparing a validated laboratory-based stressor to a no-stress 467 condition within the same individuals, we were able to disentangle the effects of acute stress 468 from noisy measurements in the same individuals. Further, this design allowed us to distinctly 469 identify if/when differences between ELA-exposed and control individuals were context 470 dependent (i.e. manifesting only during stress). Results provide preliminary evidence in humans 471 of a dysregulated pattern of NR3C1, FKBP5 and NFKB1 gene expression activation as a 472 consequence of ELA. Importantly, these changes manifest more acutely in the presence of 473 stress-induced cortisol release as compared to a no-stress resting condition.

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As predicted by previous research, the ELA group evince higher cortisol response and lower 475 NR3C1 gene expression in response to the TSST compared with controls, with no difference 476 between groups in the no-stress condition. Moreover, cortisol-induced changes in gene 477 expression revealed a decoupling between the stress-induced cortisol release and nuclear 478 signaling in the ELA group. Cortisol reactivity was associated with increased NR3C1 and 479 NFKB1 expression in the control group, but in the ELA group these associations were blunted.

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However, contrary to expectation, stress-induced cortisol release did not predict increased pro-514 inflammatory profile among individuals exposed to ELA.

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The methodological strengths of this study include a laboratory-based within-subjects 516 experimental design, which allows stronger causal inferences. We collected repeated 517 measurements over a relatively long time scale to document changes in gene expression and 518 pro-inflammatory cytokines. Our within-subjects, between-groups design, combined with four 519 repeated measurements in each session, reduced biological variability and increased power to 520 detect true associations. Finally, we tested the moderating effects of ELA, which enables tests 521 of potential programming of biological systems.

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In conclusion, ELA may program physiological systems in a maladaptive manner more likely