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. Author manuscript; available in PMC: 2023 Feb 1.
Published in final edited form as: Biol Psychiatry. 2021 Aug 17;91(3):273–282. doi: 10.1016/j.biopsych.2021.08.003

Developmental timing of trauma in women predicts unique extracellular vesicle proteome signatures

Kathleen E Morrison 1, Anaïs F Stenson 2, Ruth Marx-Rattner 1, Sierra Carter 3, Vasiliki Michopoulos 4, Charles F Gillespie 4, Abigail Powers 4, Weiliang Huang 5, Maureen A Kane 5, Tanja Jovanovic 2, Tracy L Bale 1,*
PMCID: PMC9219961  NIHMSID: NIHMS1751643  PMID: 34715991

Abstract

Background:

Exposure to traumatic events is a risk factor for negative physical and mental health outcomes. However, the underlying biological mechanisms that perpetuate these lasting effects are not known.

Methods:

We investigated the impact and timing of sexual trauma, a specific type of interpersonal violence, experienced during key developmental windows of childhood, adolescence, or adulthood on adult health outcomes and associated biomarkers, including circulating cell-free mitochondrial DNA (ccf-mtDNA) levels and extracellular vesicles (EV), in a predominantly Black cohort of women (n = 101).

Results:

Significant changes in both biomarkers examined, ccf-mtDNA levels and EV proteome, were specific to developmental timing of sexual trauma. Specifically, we identified a large number of keratin-related proteins from EVs unique to the adolescent sexual trauma group. Remarkably, the majority of these keratin proteins belong to a 17q21 gene cluster that suggests a potential local epigenetic regulatory mechanism altered by adolescent trauma to impact keratinocyte EV secretion or its protein cargo. These results, along with changes in fearpotentiated startle and skin conductance detected in these women, suggest that sexual violence experienced during the specific developmental window of adolescence may involve unique programming of the skin, the body’s largest stress organ.

Conclusions:

Together, these descriptive studies provide novel insight into distinct biological processes altered by trauma experienced during specific developmental windows. Future studies will be required to mechanistically link these biological processes to health outcomes.

Keywords: trauma, extracellular vesicle, development, keratin, proteomics, biomarker

Introduction

Trauma exposure early in life is associated with negative health outcomes for the brain and body, including inflammation, hypertension, metabolic disorders, and accelerated aging, in addition to being a potent risk factor for neuropsychiatric disease (1–5). The developmental timing and type of trauma also impact health outcomes, where exposure to trauma prior to age 14 has differential effects on the brain compared to later adolescence (6). Evidence also suggests that adversity experienced around the pubertal transition, a key period of brain maturation, is one of the strongest predictors of lifelong neuropsychiatric disease risk for women (7). Interpersonal trauma leads to higher rates of PTSD, mood disorders, increased suicidal ideation, and poor autonomic nervous system functioning compared to non-interpersonal trauma (8–10). Although differential effects of trauma timing and type are well documented, we know little about the underlying biological changes that lead to a lifetime of risk or resilience to the negative consequences of trauma.

To identify potential biological mechanisms, we compared women with exposure to acute instances of sexual trauma, a particularly harmful form of interpersonal violence, to women who have not experienced this specific trauma type. We investigated the association between the developmental window during which the sexual trauma occurred and the biological signatures. Three developmental windows – childhood, adolescence, and adulthood – were examined based on the well-established differences in central and peripheral nervous system developmental during these windows (11–17). Our main goal in this study was to investigate accessible circulating biological signals that were uniquely impacted by the developmental timing of sexual trauma. We focused on two biological signals: circulating cell-free mitochondrial DNA (ccf-mtDNA) and extracellular vesicles (EVs). Alterations to ccf-mtDNA levels have been associated with major depressive disorder, suicidality, and systemic inflammation (18–23). Similarly, EVs have known significant interactions with the immune system, have been well described in diseases such as cancers, and have recently been implicated in intergenerational transmission of trauma (24–27).

Samples for this descriptive study were derived from a biobank generated by the Grady Trauma Project (GTP), which is a large-scale study of the impacts of trauma-related risk factors for PTSD and related behavioral and physical health comorbidities in a high risk, highly trauma-exposed urban population of primarily Black Americans in Atlanta, GA, USA (28). Results from GTP have characterized genetic (29–31), epigenetic (32,33), physiological (31,34,35), and neurobiological (36,37) mechanisms underlying PTSD. Importantly, all women included in this study had generally high levels of trauma exposure. We assessed whether sexual trauma experienced during these specific developmental windows was associated with different circulating biological signatures and differentiated trauma-related health outcomes.

Methods and Materials

Full procedure details are provided in the Supplement.

Subjects

Blood plasma samples from were selected from a biobank generated by a larger study of risk factors for PTSD. The samples were from 101 women ages 18 to 45 (95 African American/Black, 4 Caucasian/White, 1 mixed, 1 other). Detailed demographic data are in the Supplement and Table S1. Participants were approached in the waiting rooms of a large urban tertiary care center in Atlanta, GA, which serves a low-income population with high trauma exposure (34). All participants provided written informed consent. The Institutional Review Boards of Emory University and Grady Memorial Hospital approved all procedures.

Clinical Assessment

Lifetime trauma history and PTSD symptoms were measured via the Traumatic Events Inventory (TEI; (38)) and the Modified PTSD Symptom Scale (PSS; (39)), respectively, as previously (40). Samples were selected from women who reported on the TEI that they had experienced sexual assault or abuse during only one of the following age ranges: before age 14 (<14, N = 25), between the ages of 14–17 (14–17, N = 25), at age 17 or later (>17, N = 24), or no sexual trauma (NST, N = 27) (Figure 1). To control for the impact of PTSD, groups were matched for PSS score at the time of sample collection.

Figure 1. Overview of sample selection and methods.

Figure 1.

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Samples were drawn from a pre-existing biobank at the Grady Trauma Project. Adult women were recruited through the Grady Trauma Project and were assessed for lifetime trauma history and current PTSD symptoms. Samples and data for analysis were selected based on reported sexual trauma before age 14, between the ages of 14–17, later than age 17, or not at all. Importantly, samples from participants in each trauma group were matched for current PTSD symptoms, which permitted the separation of the impact of past trauma and current symptoms on outcomes. Blood plasma circulating cell-free mitochondrial DNA (ccf-mtDNA) and extracellular vesicles (EVs) were analyzed. Created with BioRender.com

Anthropometric Measurements

Height (m), weight (kg), waist circumference (cm), and blood pressure (systolic and diastolic) were measured.

Blood Collection

Fasting whole blood specimens were obtained by venipuncture between 0800–0900 hours by experienced nurses at the Clinical Interactions Network within the Atlanta Clinical and Translational Science Institute.

Fear Conditioning Paradigm

Differential fear conditioning was assessed with a fear-potentiated startle experimental paradigm that has been used successfully in adult trauma populations (41). The protocol included three trial types: 1) startle noise alone (NA); 2) a reinforced conditioned stimulus (CS+) that was paired with the aversive unconditioned stimulus (US); and 3) a second CS that was not paired with the US (CS-). Both CSs were colored shapes presented on a computer monitor using Superlab presentation software (Cedrus, Inc.) for 6 seconds prior to the startle probe. The US was an aversive 250-ms airblast with an intensity of 140 psi directed at the larynx, as has been shown in our previous studies to consistently produce robust fear-potentiated startle (34,41).

Analysis of Fear-Potentiated Startle

The acoustic startle response data were acquired using the electromyography (EMG) module of the Biopac MP150 for Windows (Biopac Systems, Inc., Aero Camino, CA). The eyeblink component of the acoustic startle response was measured by EMG recordings of the right orbicularis oculi muscle with two 5-mm Ag/AgCl electrodes (34). The startle probe was a 108-dB [A] SPL, 40-ms burst of broadband noise, delivered binaurally through headphones. Fear-potentiated startle was calculated as a difference score by subtracting average startle magnitude to the NA trials from average startle magnitude to the CS+ and CS-.

Analysis of Skin Conductance Response

Electrodermal activity (EDA) was collected using the EDA module for Biopac and was assessed using two Ag/AgCl electrodes. Skin conductance response (SCR) was defined as the average increase from 3 to 6 seconds after each CS onset subtracting skin conductance levels during the 1 second pre-CS onset. SCR data were square-root transformed and averaged for the CS+ and CS-. Low and non-responders were not removed from the dataset, as has been advocated recently in the literature (42).

Extracellular Vesicles

Extracellular Vesicle Isolation

Extracellular vesicles (EVs) were isolated from 250 μl of blood plasma. Plasma was mixed with 500 μl of freshly filtered phosphate buffered saline. Following a series of centrifuge steps, EVs were extracted using the IZON qEVoriginal 70 nm columns in the IZON Automated Fraction Collector. Prior to downstream analyses, the EVs were concentrated using Microcon 30K centrifugal filter devices.

Nanoparticle Tracking Analysis

Fifty microliters of concentrated EVs were sent to the University of North Carolina at Chapel Hill Nanomedicines Characterization Core Facility for nanoparticle characterization as previously (27). All samples were run on a NanoSight NS500 to determine the characteristics of EV particles at the Center for Nanotechnology in Drug Delivery at the University of North Carolina.

Small RNA Sequencing

Small RNA was isolated from concentrated EVs. Using Trizol LS, the upper aqueous phase containing RNA was used in the Qiagen miRNeasy kit. Illumina TruSeq v2.5 chemistry was used for sequencing prepared libraries, with 36 bp single end read length.

Proteomics

A subset of samples was utilized for proteomic analysis (n = 10/group). Individual EV samples were subject to processing, measurement, and analysis as previously described (27,43–48).

Circulating cell-free mitochondrial DNA

DNA was extracted using the QIAmp DNA Blood Mini Kit (Qiagen #51106) with modified use. Blood plasma was thawed to room temperature. Following a brief centrifuge, 150 μl of plasma was removed to a new tube and mixed with 150 μl of 100% ethanol. The mixture was briefly vortexed and centrifuged before being pipetted into the QIAamp Mini spin column. From there, manufacturer instructions were followed. All spins were conducted at 20,000 × g at room temperature. Final DNA sample was eluted in 100 μl of Tris EDTA and stored at −20 °C.

Quantitative real-time PCR was conducted, using a standard curve to determine copy number of genes of interest. The standard curve was generated using IDT gBlocks (sequences in Table S2). Isolated circulating DNA (4 μl) was mixed with 5 μl of PowerUP SYBR Green Master Mix, and 0.5 μl of 10 uM each of forward and reverse primer (Table S3). The qPCR reaction was completed in triplicate on a ThermoFisher QuantStudio5. Copy number was calculated using the standard curve.

Statistics

Two analyses were performed for all data. The sexual trauma group (ST, comprised of all subjects from the <14 years, 14–17 years, and >17 years groups) was compared to the no sexual trauma (NST) group by two-tailed t-test to examine the impact of sexual trauma at any age. Data were also analyzed by 1-way ANOVA with four groups (NST, <14 years, 14–17 years, and >17 years) to examine the impact of the timing of sexual trauma. A Tukey post-hoc or Fisher’s least significant difference post-hoc test was performed when appropriate. The ST group was compared to the NST by two-tailed t-test to examine the impact of sexual trauma at any age on EV-derived small RNA and proteomic outcomes. To identify differentially abundant proteins and small RNA from extracellular vesicles among all four groups, the Benjamini Hochbert FDR correction was applied and an adjusted p-value was used in analysis using the limma package in R (version 3.83.3, (49)). Heatmaps were generated using the ComplexHeatmap package in R (version 4.0, (50)). Correlations between PSS and circulating biomarkers were performed using the corrplot package in R (version 0.84, (51)).

Results

Circulating Biological Signatures

Circulating cell free mitochondrial DNA (ccf-mtDNA) was associated with sexual trauma (p < 0.05) in a timing-specific manner (Figure 2A, p < 0.05). Women who experienced sexual trauma at age 14–17 had significantly more ccf-mtDNA than women who did not experience sexual trauma, women who experienced it at age <14, and age >17 (all p < 0.05). This was specific to this age group, as there was no difference between women who did not experience sexual trauma and those that experienced it at age <14 or age >17. As confirmation of absence of cellular contamination, there was no association with group in the nearly undetectable level of nuclear DNA (Figure 2B). There was no association of age at the time of blood collection with ccf-mtDNA copy number (Figure S1).

Figure 2. Timing-specific sexual trauma was associated with dramatic alterations in blood plasma-derived extracellular vesicles and ccf-mtDNA levels.

Figure 2.

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(A) Circulating cell-free mitochondrial DNA (ccf-mtDNA) levels were significantly higher in women who experienced sexual trauma (t(66) = 2.061, p = 0.0432, η2 = 0.0605) in a timing-specific manner (F(3, 64) = 5.387, p = 0.0023, η2 = 0.2016). Specifically, adult women who experienced sexual trauma between ages 14–17 had significantly more ccf-mtDNA than women who did not experience sexual trauma (p = 0.0007), women who experienced it at age <14 (p = 0.0296), and age >17 (p = 0.0019). There was no difference between women who did not experience sexual trauma and those that experienced it at age <14 (p = 0.2267) or age >17 (p = 0.7150). (B) No circulating nuclear DNA (nDNA) was detected in these samples, supporting that a lack of contamination of cell lysis occurred in sample processing (F(3, 64) = 2.006, p = 0.1219, η2 = 0.0860). (C,D) Extracellular vesicles (EVs) isolated from the blood plasma of adult women were measured by Nanosight for (C) concentration and (D) mean size. There was no impact of sexual trauma on concentration (t(97) = 1.413, p = 0.1609, η2 = 0.0202) or mean size (t(97) = 1.123, p = 0.2641, η2 = 0.0128). (E-H) EVs compared by proteomics analysis showed dramatically altered protein content between groups. (E) All detected proteins are displayed in the heatmap that represents the average abundance of each protein between groups. There were 102 proteins that were significantly different between women who experienced sexual trauma and those who did not. Of those proteins, three different categories were overrepresented – keratin-related proteins, immunoglobulins (Ig), and proteins related to EV function. (F) Proteins critical for EV functions, annexins and galectins, were significantly decreased in EVs collected from women with prior sexual trauma experience. (G) The age at which sexual trauma occurred was associated with a unique profile of protein expression. Proteins that were impacted in abundance based on the developmental timing of sexual trauma are shown. (H) A large group of keratin-related proteins were robustly and significantly elevated in EVs from women who experienced sexual trauma specifically between ages 14–17. The majority of these, which are denoted in red and linked to the chromosome schematic, belong to a previously identified 17q21 gene cluster on chromosome 17. NST – no sexual trauma, ST – sexual trauma, #p < 0.05 for t-test between NST and ST; *p < 0.05 compared to NST on Tukey post-hoc test following a significant one-way ANOVA (NST, <14, 14–17, >17); +Bonferonni-adjusted p < 0.05 between NST and women who experienced sexual trauma at age 14–17

Particle analysis of EVs showed that sexual trauma was not associated with EV concentration (Figure 2C) or mean size (Figure 2D). We found no overall association of sexual trauma exposure on small RNA EV content (Figure S2). Hierarchical clustering showed that the expression of small RNA was highly variable and therefore not distinguishable across groups, and analysis for differentially expressed small RNA yielded no differentially expressed small RNA.

In contrast, the EV proteome was dramatically different when compared by age-specific exposure to sexual trauma. All detected proteins are shown in the heatmap, where it is clear that sexual trauma was associated with altered protein abundances (Figure 2E). Specifically, 102 proteins were significantly different between women who had not experienced sexual trauma and those who did (Supplemental File 1). Of those proteins, three different categories were overrepresented, including keratin proteins, immunoglobulin proteins, and EV-related proteins. Important EV-functioning proteins annexin A1, annexin A2, galectin-3, and galectin-7 were significantly lower in all women who had experienced trauma compared to those who had not (Figure 2F). When protein abundance was examined based on the age at which women experienced sexual trauma, there was a distinct profile of differentially abundant proteins based on the age of trauma exposure (Figure 2G). Keratin-related proteins were uniquely and widely increased in women who had experienced sexual trauma at age 14–17 (Figure 4H). Strikingly, a large group of keratin-associated proteins and cuticular proteins were only present in EVs from these women, the majority of which belong to a previously identified 17q21 gene cluster (52– 55).

Figure 4. Sexual trauma experience was associated with increased body weight.

Figure 4.

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(A) Sexual trauma was associated with an increased body weight in adulthood, regardless of the age at which sexual trauma occurred (t(83) = 2.1930, p = 0.0311, η2 = 0.0548; F(3, 81) = 2.5010, p = 0.0652, η2 = 0.0848). (B) There was no significant association of sexual trauma with waist circumference (t(77) = 1.9, p = 0.0612, η2 = 0.0448; F(3, 75) = 1.339, p = 0.2680, η2 = 0.0508). (C) Systolic (t(84) = 0.8107, p = 0.4199, η2 = 0.0078; F(3, 82) = 0.5079, p = 0.6779, η2 = 0.0182) and (D) diastolic (t(84)=1.380, p = 0.1714, η2 = 0.0221; F(3, 82) = 1.003, p = 0.3958, η2 = 0.0354) blood pressure were not associated with prior experience of sexual trauma. NST – no sexual trauma; ST – sexual trauma; #p < 0.05 for t-test between NST and ST

Fear Conditioning Results

Sexual trauma was associated with a lasting impact on FPS to CS+ in a timing-specific manner (Figure 3B, p < 0.05). Women who experienced sexual trauma at age 14–17 showed significantly increased startle compared to all other groups (all p < 0.05). This finding was specific to fear-potentiated startle to the CS+, as there was no association of prior sexual trauma with baseline startle to noise alone trials (t(50) = 0.3009, p = 0.7648, η2 = 0.0018; F(3, 48) = 0.2331, p = 0.8729, η2 = 0.0144) or FPS to CS-, although there was a similar trend towards timing-specificity (Figure 3C, p = 0.0512). Skin conductance response (SCR) during fear conditioning was also greater for the CS+ compared to the CS- (F(1, 40) = 4.571, p = 0.039). There was no overall association of sexual trauma with SCR either during the CS+ (Figure 3D) or the CS- (Figure 3E).

Figure 3. Age of sexual trauma exposure was associated with increased fear responding.

Figure 3.

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Physiological measurements associated with autonomic nervous system functioning were undertaken during a fear conditioning task. (A) Schematic of fear conditioning task. The fear conditioning paradigm resulted in successful startle potentiation, i.e. increased startle magnitude during CS+ relative to noise alone (F(1,51) = 36.829, p < 0.0001). Fear-potentiated startle (FPS, difference score between CS and NA) was greater for the CS+ compared to the CS- (F(1, 51) = 4.603, p = 0.037). (B) Prior experience with sexual trauma was associated with an increase in fear-potentiated startle to a noise probe in the presence of an airblast reinforced conditioned stimulus (CS+) in a timing-specific manner (F(3, 48) = 4.4150, p = 0.0056, η2 = 0.2289). Adult women who experienced sexual trauma at age 14–17 had a heightened fear-potentiated startle response relative to all other groups (NST: p = 0.0137; <14: p = 0.0005; >17: p = 0.0390). This finding was specific to fear-potentiated startle to the CS+, as there was no association of prior sexual trauma with baseline startle to noise alone trials (t(50) = 0.3009, p = 0.7648, η2 = 0.0018; F(3, 48) = 0.2331, p = 0.8729, η2 = 0.0144) or (C) FPS to CS-, although there was a similar trend towards timing-specificity (t(50) = 0.6606, p = 0.5119, η2 = 0.0086; F(3, 48) = 2.778, p = 0.0512, η2 = 0.1479). (D,E) Skin conductance response (SCR) during the fear conditioning paradigm was measured. (D) SCR in the presence of the CS+ did not differ between women with and without prior sexual trauma (t(39) = 0.8032, p = 0.4267, η2 = 0.0163; F(3, 37) = 0.4265, p = 0.7352, η2 = 0.0334). (E) Similarly, there was no main effect of sexual trauma on SCR in the presence of the CS- (t(39) = 0.8521, p = 0.3993, η2 = 0.0183; F(3, 37) = 2.1660, p = 0.1084, η2 = 0.1494). NST – no sexual trauma, ST – sexual trauma, SCR – skin conductance response, CS – conditioned stimulus. *p < 0.05 compared to NST on LSD post-hoc test following a significant one-way ANOVA (NST, <14, 14–17, >17)

Anthropometric Measurements

Exposure to sexual trauma in the context of a broadly traumatic environment was associated with minimal impacts on anthropometric measurements (Figure 4). Adult women exposed to sexual trauma had greater body weight (Figure 4A, p < 0.05), although this was not specific to when in development the sexual trauma occurred. There was no significant association of sexual trauma with waist circumference (Figure 4B). Exposure to sexual trauma was not associated with changes in blood pressure measurements. Individually, systolic (Figure 4C) and diastolic (Figure 4D) blood pressure were not associated with sexual trauma experience.

PTSD Symptoms

To control for the impact of PTSD, groups were matched for PSS score (Figure S3). Therefore, the study was planned so that each group was balanced in terms of the score on the PSS at the time the sample was collected. As such, there were predictably no associations between PSS severity and sexual trauma experience (t(98) = 0.3686, p = 0.7132, η2 = 0.0014; F(3, 96) = 0.1824, p = 0.9081, η2 = 0.0057). Furthermore, there were no correlations between PSS and any of the circulating biological signals (ccf-mtDNA and any protein detected in proteomics, p > 0.05).

Discussion

Interpersonal trauma experience is associated with negative health outcomes across the lifespan, including an increased risk for neuropsychiatric disease. We investigated the developmental timing-specificity of sexual trauma, an extremely salient and discrete interpersonal trauma, in producing unique physiological and biological outcomes in adulthood in a predominantly Black cohort of adult women. We assessed whether sexual trauma experienced during specific periods of brain maturation and development – childhood, adolescence, or adulthood – differentiated or uniquely informed these biological signatures. Altogether, our findings point to unique biological consequences that depended on the developmental timing of an acutely traumatic event. Most notable was that sexual trauma during the adolescent window produced the most distinct signal.

Our main goal was to investigate easily accessible circulating biological signals within blood plasma that were uniquely impacted by the developmental timing of sexual trauma. One relatively recent biological signal associated with neuropsychiatric diseases and intersecting with the immune system is circulating cell-free mitochondrial DNA (ccf-mtDNA), which are fragments of mitochondrial DNA found unassociated with any cell in the blood stream. The exact role of circulating ccf-mtDNA is not well understood, however, studies have shown that it is an indicator of cell damage and appears to reflect levels of systemic inflammation (18,19). Recent association studies found that ccf-mtDNA is increased in the plasma of patients with major depressive disorder, those exhibiting suicidality, and in subjects exposed to acute stressors (20–22). Further, patients with major depression who responded positively to antidepressant treatment had a concomitant drop in ccf-mtDNA (20). In our current study, we found that ccf-mtDNA was significantly increased in the plasma of adult women who had previously experienced sexual trauma. Interestingly, this association of sexual trauma with ccf-mtDNA levels was timing-specific, where women who had experienced sexual trauma between the ages 14–17 had significantly higher ccf-mtDNA levels in adulthood than all other women. While this single timepoint provides only a snapshot, and there may be other factors involved such as physical activity and somatic disorders, these data are striking in their group specificity. As the investigation of ccf-mtDNA as a biomarker is relatively new within the field of neuropsychiatry, there remain many outstanding questions concerning the biological mechanisms that may explain these effects (23).

Another recently identified and highly informative biological signal found in circulation is extracellular vesicles (EVs). EVs are cell-derived membranous particles that carry a variety of cargo, including membrane-bound and cytosolic proteins and nucleic acids. EVs have been recognized as an important mode of systemic cellular communication, as every tissue in the body secretes EVs, EVs travel in circulation to specific tissues, and EVs and their content are taken up by cells through unique protein signals to impact local cellular functions. EVs come in a variety of types and sizes, including microvesicles and exosomes. The type of EV can signify what intracellular compartment it was derived from, its content, and the mode of its cellular secretion (56,57). Similar to ccf-mtDNA, EVs have significant interaction with the immune system and have been well described in diseases such as cancers (24–26). We isolated EVs from blood plasma and examined the potential lasting effects of prior sexual trauma on EV-associated proteome and miRNA content.

In the EV proteomic analysis, we found robust and significant associations of EV proteins with sexual trauma exposure. Overall, EVs from women who had previously experienced sexual trauma had profound reductions in 4 key proteins known to broadly regulate EV function and secretion (58,59). Specifically, EV protein levels of annexin-A1 and -A2 and galectin-3 and −7 were significantly reduced, to nearly undetectable levels in some cases. Combined with potential EV size differences we noted, this finding supports a potential dramatic change in EV type or EV secretion from specific tissue(s).

We further compared our EV proteomics data with available published EV proteomics data and found a surprising shared overlap with secretion associated with EVs previously isolated from keratinocytes (60). Relatedly, we detected many keratin-related proteins in EV samples from women with prior sexual trauma history. Until recently, keratin proteins were often discarded from proteomics analysis as potential contaminants. However, examination of cell-type specific EV release has reproducibly demonstrated high levels of keratin and keratin-associated proteins isolated with EVs from gut, hair, and skin sources (60–62). Further, blood samples from all subjects in this cohort were collected in the same manner and in the same time period, and all samples were subsequently processed in the lab in an identical manner by an investigator blinded to sample group. We also noted that a large number of the keratins, specifically that of cuticular and keratin-associated protein types, were dramatically increased specifically in, and in many cases were only detectable in, EVs from women who had experienced sexual trauma between 14–17. Keratin-associated proteins (KAPs) are cysteine-rich proteins produced by the hair, forming the hair fiber. Cuticular keratins are cytoskeletal proteins found in the hair and skin and are involved in keratinocyte differentiation (63). We detected more than 12 KAPs and 4 cuticular keratin proteins robustly increased uniquely in the 14–17 group. Keratin type II cytoskeletal-7 was also highly expressed and only in EVs from this group. This keratin is associated with cellular injury, wound healing in the skin, and epithelial expression in other tissues (64). Overall, the top EV protein candidates, particularly in the women who experienced sexual trauma from 14–17, are affiliated with keratinocytes and aging keratinocytes (e.g., galectin-7, annexinA1, keratins, serpins, HSPB1, prelamin, H2A).

Skin is the largest organ, a protective barrier with important wound healing and immune functions, is highly innervated, and is a major component of the physiological stress response (65,66). Skin has long been ignored as a stress tissue, but recent studies have begun to address the unique connection between the brain and skin, as well as the critical function the skin serves as an autonomic endpoint (66). In addition, skin immune cells and keratinocytes secrete cytokines in response to stress, providing another interactive measure for EV release and signaling to other tissues as to organismal homeostasis (67). A number of hypothalamic-derived stress hormones interact with keratinocytes to activate the local skin stress response (65,68). Thus, these EVs, acting as biomarkers, may support a ‘premature’ aging process at the level of the skin in women who experienced sexual trauma, specifically around puberty when the skin is highly plastic (69,70). Importantly, the majority of the keratin proteins uniquely found in EVs from women who had experienced sexual trauma between the ages of 14–17, both keratin-associated and keratin cuticular type, are part of a 17q21 gene cluster (52–55). This suggests a potential local epigenetic regulatory mechanism altered by adolescent trauma to impact keratinocyte EV secretion or its protein cargo. This gene cluster is also proximal to another 17q21 gene cluster that includes stress-regulatory genes such as CRHR1 and MAPT, and additional genes that have been strongly associated with PTSD in large GWAS studies (71,72). These significant changes detected in EV proteins specific to age of sexual trauma experience (14–17 yrs) point to a novel programming of keratinocytes that occurs during unique windows of development when these cells may be more vulnerable to epigenetic reprogramming.

Core symptoms of PTSD are related to dysregulated fear processing, including exaggerations in well-validated and reliable physiological measures of fear processing, fear-potentiated startle (FPS) and skin conductance response (SCR) to threat and safety cues (73,74). Interestingly, we found that FPS in women with prior sexual trauma appeared much more varied compared to women who had no sexual trauma exposure, which may suggest phenotypic subgroups in this measure. To this point, women who experienced sexual trauma specifically between the ages 14–17 showed significantly higher FPS responses to threat when compared with all other groups, supportive of potential elevated autonomic tone (75,76). It is possible that threat neurocircuitry development during late adolescence, along with pubertal increases in fear learning, is associated with a increased vulnerability to the impact of sexual trauma (77,78).

We also examined broader health outcomes using physiological measures both indicative of stress and metabolic state and previously linked to lasting consequences of trauma (79,80). Women who had experienced sexual trauma were more likely to have significantly increased body weight compared to women who had not experienced sexual trauma, despite all women in this study having an overall high level of other types of trauma. While previous studies have linked genetic risk for PTSD to body shape in women, which may be relevant to our EV (keratin) results, we found no significant effect of sexual trauma exposure on waist circumference (81). Other key indicators of health, including blood pressure, were broadly unaffected by sexual trauma.

To further understand the relationship between prior sexual trauma and current symptomology, we examined associations between current PTSD symptoms and circulating biological signatures. There was no significant correlation between current PTSD-related symptoms and ccf-mtDNA or EV proteins. The lack of an association supports a conclusion that these biomarkers may be indicative of past trauma and biological programming, and not responsive to current presentation of PTSD symptoms.

There are several limitations of these studies. First, we present here only descriptive associations of biomarker data with health outcomes. Second, these exploratory findings should be replicated in an independent cohort. We are unable at this time to comment on direct or causal relationships between the developmental timing of trauma experience and the adult outcomes measured. Additionally, while the starting sample size was 24–25 per group, we did have smaller sample sizes for some of the physiological measures based on available data, including for fear potentiated startle. Therefore, increased sample sizes would also be beneficial. Future studies will need to examine mechanistic questions using in vitro studies or animal models to more clearly determine the relationship between trauma programming and health risks.

Overall, these studies illuminate the critical importance for understanding the developmental timing of when trauma occurred as a predictor for the lasting influence on unique health and disease outcomes. Importantly, our cohort was comprised primarily of Black women. Rates of sexual trauma are especially high, and result in worse health outcomes, in Black women (82,83). However, we currently lack knowledge as to the biological underpinnings of how such trauma affects women’s health, or biomarkers that might identify individuals at greatest disease risk (28). Identification of unique biomarkers in this population may help predict those at greatest risk and provide novel insight into interventions.

Supplementary Material

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Acknowledgments

This work was supported by National Institutes of Health funding to KEM (HD091376), TJ (MH111682, MH110364, MH098212), and TLB (ES028202, HD097093, MH108286, MH104184). This work was supported, in part, by the University of Maryland School of Pharmacy Mass Spectrometry Center (SOP1841-IQB2014). KEM, AFS, TJ, and TLB conceptualized the study. AFS, SC, VM, and TJ provided plasma samples, as well as analyses related to demographics, trauma exposure, and health outcomes. SC and AP consulted on analyses and interpreted clinical data. CFG served as study physician and examined and recorded health outcomes. AFS and TJ analyzed the fear potentiated startle and skin conductance data. KEM and RMR performed all analysis of blood plasma, except for proteomics, which was performed by WH and MAK. KEM performed final statistical analyses and generated visualizations. KEM and TLB wrote the original draft of the paper. AFS, SC, VM, WH, and TJ provided text, and reviewed and edited the paper.

Footnotes

Disclosures

The authors (KEM, AFS, RMR, SC, VM, CFG, AP, WH, MAK, TJ, TLB) report no biomedical financial interests or potential conflicts of interest.

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NIHMS1751643-supplement-1.pdf (10.2MB, pdf)
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NIHMS1751643-supplement-2.xlsx (38.5KB, xlsx)

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