Transduction of Psychological Stress Into Systematic Inflammation by Mitochondrial DNA Signaling
Phase: NA
Conditions: Acute Inflammatory Response to Psychological Stress
Sponsor: University of Pittsburgh

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Study Protocol  
 
 
 
 
 
 
Official Title: Transduction of Psychological Stress into Systematic 
Inflammation by Mitochondrial DNA Signaling (Brief Title: Biological 
Response to Brief Psychological Challenge)   
 
 
ClinicalTrials.gov ID (NCT number): NCT04078035 
 
 
 
Protocol Date: 10/08/2020 
 
 
 
 

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Scientific Background 
 
Psychosocial stress is pro-inflammatory and confers increased risk for chronic inflammatory and 
mental health disorders. However, the mechanism by which stress is transduced into 
inflammation is not fully understood. Emerging evidence shows that stress can affect the 
function and integrity of mitochondria – unique bacteria-derived organelles with their own 
genome – and that “stressed” mitochondria release immunogenic molecules that trigger 
inflammation systemically. These new results position mitochondria and the release of 
mitochondria-derived signaling molecules (“mitokines”) at the center of the stress-inflammation 
cascade. 
 
Under conditions of psychosocial stress, mitochondria release mitochondrial DNA (mtDNA), 
which is a pro-inflammatory molecule. Bacteria-derived mtDNA contains molecular markers that 
make it particularly immunogenic. In humans, our preliminary work provides initial evidence that 
acute socio-evaluative laboratory stress is sufficient to induce a robust and selective elevation in 
serum ccf-mtDNA after 30 minutes. Moreover, in cultured human cells, we find that the stress 
mediator glucocorticoid promotes mtDNA release from mitochondria. However, the extent to 
which stress-induced increases in ccf-mtDNA contribute to the delayed increase of circulating 
proinflammatory mediators after acute psychological stress in humans remains unknown. 
 
In the current study, we proposed a controlled trial to better characterize stress-induced 
regulation of ccf-mtDNA release and examine its association with the increase in 
proinflammatory mediators that is known to follow acute psychological stress and though to 
contribute to physical health risk. 
 
Preliminary work relevant to the Aims of the proposed study: To test whether mtDNA is released 
in response to psychological stress, we conducted a preliminary uncontrolled laboratory study 
assessing levels of ccf-mtDNA before and after an acute (5-min) socio-evaluative stressor on 
two separate occasions. The stressor was chosen for its ability to evoke glucocorticoid hormone 
release and reliably stimulate increases in circulating markers of inflammation, which typically 
peak 1-2 hours post-stress. We developed a rigorous approach to quantify serum levels of two 
mtDNA genes (CYTB and ND1) in 50 healthy midlife individuals (mean age = 50 years, range: 
41-58). Serum was collected at baseline (pre) and immediately (post) and 30 minutes after 
stress (+30 min). DNA was isolated and analyzed by a blinded operator and normalized to a 
standard curve. 
 
Ccf-mtDNA levels dramatically increased +30 min after the stressor, exhibiting a large effect 
size (η2 = .556, P < 0.0001, Figures in Research Plan), with 93% of participants showing an 
increase in ccf-mtDNA (CYTB). This result was replicated at a second visit, yielding similar 
results and a comparable effect size. Analyses of the second amplicon ND1 confirmed these 
findings at both sessions. Findings provide robust initial evidence for psychological stress-
induced increases in the highly immunogenic molecule, ccf-mtDNA, but did not establish the 
time kinetics, the maximal amplitude of this response, or the association of this response with 
later increases in inflammatory mediators. These are gaps in our knowledge that will be 
examined in the proposed study. 
 
Both mitochondrial and nuclear genomic fragments can be found in blood. Prior evidence shows 
that exhaustive physical exercise is followed by increases in both ccf-mtDNA and nuclear-
derived ccf-nDNA, possibly due to tissue damage. To ascertain whether psychological stress-
induced ccf-mtDNA could result from unspecific cellular damage and non-specific bulk cellular 

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material release, we also assessed two nuclear gene markers in our preliminary work. In 
contrast to ccf-mtDNA, ccf-nDNA (GUSB) was unchanged at +30 min on both occasions of 
testing. Accordingly, at +30 min after stress the ratio of circulating mitochondrial to nuclear 
genome mtDNA/nDNA rose by >2-fold.  
 
The proposed study will examine physiologic responses to acute psychological challenge in the 
laboratory among healthy adults. It is widely accepted that there is an increase in circulating 
markers of inflammation following a single bout of laboratory stress. This increase in systemic 
inflammation is believed to contribute to the damaging health effect of psychological stress. 
However, to date the biological mechanisms by which psychological stress is transduced into 
inflammation are unclear. Our preliminary evidence suggests that mitochondrion may play a 
role, with stress-induced increases in circulating levels of mitochondria- derived signaling 
molecules that are known to modulate immune cell function and the production of pro-
inflammatory cytokines. 
 
To test this possibility, we plan to conduct a crossover experimental trial examining 
physiological responses to an evaluative speech task under laboratory conditions. We have 
previously used this task to induce physiological arousal. We plan to recruit 60 non-smoking 
volunteers (50% female, aged 20-50 years) and test them on two occasions separated by at 
least a month. On one occasion they will be exposed to the speech task. On the other occasion, 
they will rest quietly for the same period. Conditions will be counterbalanced. At both visits 
cardiovascular responses (heart rate, blood pressure, and heart rate variability) will be assessed 
as measures of autonomic activation before, during and after the task period. Participants will 
also have an intravenous catheter inserted and blood drawn at ten time points over the two-hour 
testing period on each occasion. Blood samples will be sent to laboratories at the University of 
Pittsburgh and at Columbia University for the assessment of mitochondria-derived signalling 
molecules, inflammatory markers, cortisol and catecholamine levels. 
 
Study Objectives 
Our overarching hypothesis is that psychosocial stress regulates ccf-mtDNA release, which, in 
turn, signals a proinflammatory response. We examine this hypothesis in parallel human and 
cell-based studies. The current Clinical Trial only covers Aim 1 of the parent grant, which is the 
human study. 
 
Specific Aim 1 (human stress study). Determine the temporal relationship between 
psychological stress, ccf-mtDNA, and systemic inflammation. We hypothesize that 
socioevaluative stress will trigger robust and transient ccf-mtDNA release compared to a non-
stressed condition. To test this, 60 volunteers (50% female; aged 20-50) will be tested on two 
occasions separated by at least 1 month. On one occasion, they will be exposed to the a socio-
evaluative speech stressor known to elicit increases in circulating markers of inflammation at 
40-90 minutes. On the other occasion, they will rest quietly for the same period of time. 
Conditions will be counterbalanced across subjects and blood drawn at ten time points over two 
hours. We expect that the stress task will induce a specific increase in ccf-mtDNA, which will 
statistically mediate subsequent peak circulating IL-6 and TNF-α levels. To validate that the 
stress task was associated with physiological arousal, we will examine task-related changes in 
heart rate, blood pressure, heart rate variability, and circulating levels of cortisol, epinephrine, 
and norepinephrine. This study will establish the kinetics and magnitude of psychological stress-
induced ccf-mtDNA release, and whether ccf-mtDNA mediates the inflammatory response to 
acute stress in humans. 

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Study Design & Methods 
We propose a crossover experimental trial examining physiological responses to a socio-
evaluative speech task under laboratory conditions. Participants will be tested on two occasions 
separated by at least 1 month. On one occasion, they will be exposed to the speech task. On 
the other occasion, they will rest quietly for the same period of time with identical assessment in 
the absence of the stressor. This control session will allow us to assess and control for any 
possible effects of the passage of time (e.g., circadian variation in cortisol levels), simply being 
assessed (e.g., discomfort), and the intravenous catheter. Conditions will be counterbalanced in 
randomized starting order across subjects and blood drawn at ten time points over two hours to 
assess ccf-mtDNA, markers of inflammation (IL-6 and TNF-α), and levels of cortisol, 
epinephrine and norepinephrine. Heart rate (HR), blood pressure (BP), and heart rate variability 
(HRV) will be assessed as indices of autonomic activation before, during and after the stressor. 
The speech task is a widely used, highly effective way to investigate stress responses in a 
laboratory setting. It induces significant increases in cortisol, indices of autonomic activation, 
and circulating IL-6 and TNFα. Our preliminary data provides novel evidence that this stressor 
also results in increased cc-mtDNA, which may mediate the increases in circulating levels of IL-
6 and TNF-α that peak 40-90 minutes post-stress. 
 
Recruitment: Participants who respond to study advertisements or who are identified by 
Pitt+Me ( a research registry) will be scheduled for a screening telephone call to determine 
eligibility. After the initial screener by Pitt+Me, eligible volunteers from these sources will be 
contacted by study staff by text message, telephone, or email. A screening call will be 
scheduled at which time a member of the research staff will explain the study and administer a 
telephone screening to assess inclusion/exclusion criteria. If eligible, the staff member will solicit 
the person’s interest in participating. If eligible and interested, male participants will be 
scheduled for the first laboratory visit and female participants will be asked the predicted date of 
their next menses so that we may contact them and their laboratory visit can be scheduled 
during the luteal phase (21-28 days post-menstruation) of their menstrual cycle. Prior to each 
visit, participants will be asked to abstain from alcohol and vigorous exercise for 24hrs, from 
non-prescription drugs for two days before testing and from food and caffeine for 3 hrs. 
 
Once the first visit has been scheduled, participants will be mailed/emailed confirmation of the 
visit, including instructions regarding where to park, public transportation and where to come. 
This mailing will also include a reminder of the visit preparation instructions (e.g., refrain from 
alcohol). 
 
Methods: Once the first visit has been scheduled, participants will be mailed/emailed 
confirmation of the visit, including instructions regarding where to park, public transportation and 
where to come. This mailing will also include a reminder of the visit preparation instructions 
(e.g., refrain from alcohol). 
 
Two days before the first laboratory visit, participants will receive a reminder telephone call. On 
this call, subjects will be screened to determine if they are eligible for the blood draw the 
following day. Participants who endorse in the past two weeks (1) having an infection, cold or 
flu, (2) taking antibiotics or glucocorticoids, or (3) having received a vaccination or tattoo will be 
rescheduled. Participants will also be asked about presence and severity of current symptoms 
of upper respiratory illness. If they score in a range that is consistent with possible infection, the 

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visit will be rescheduled. Participants who have taken non-steroidal anti-inflammatories in the 
past 24 hours will also be rescheduled. If they pass these screenings, standby participants will 
be confirmed t come in if the primary participant has to be rescheduled. 
 
Participants will attend two laboratory visits at the Behavioral Physiology Laboratory, University 
of Pittsburgh, scheduled at least one month apart. The first visit will last approximately 4 hours 
and the second visit approximately 3.5 hours. Both visits will be scheduled in the afternoon 
starting between 1 and 3 pm to control for diurnal variation in the biological measures of 
interest. Participants will be asked to avoid eating or drinking anything except water from 12pm 
on both days of testing. At the first visit, the project coordinator will meet with the volunteer to 
explain the study and obtain written informed consent according to the University of Pittsburgh 
Institutional Review Board guidelines.  
 
Following consent, the research nurse will conduct a medical history and medication use 
screen, and record the participants resting heart rate, blood pressure, height, weight, and body 
fat. In the event that participants are identified who are ineligible for the study (e.g., due to 
exclusionary medical or psychiatric conditions or taking excluded medications, with a resting 
blood pressure >140/90, weight < 110 lbs or BMI equal to or greater than 30), they will be 
excused from the study at this point. 
 
Next, the blood eligibility assessment will be repeated to confirm that the participant adhered to 
pre-visit instructions (e.g., no non-prescription medications in the prior 2 days) and did not 
present with new symptoms of upper respiratory infection. In the event that they are no longer 
eligible for the blood draw that day, the visit will be rescheduled. Participants who are eligible for 
the blood draw will next complete demographic questionnaires on the computer. It is estimated 
that these questionnaires will take 30 minutes to complete. 
 
At both visits, participants will be situated upright in a comfortable chair, where an intravenous 
catheter will be inserted into the antecubital fossa of one arm for collection of blood samples. 
Next, a blood pressure cuff (Dinamap) will be placed on the opposite arm from the catheter. 
Research staff will then apply 11 skin surface electrodes to permit the assessment of heart rate 
variability [HRV]. Electrodes will be applied to the skin (on the back of the neck, upper chest, 
shoulders, ribs, and calf area below the knee). The electrodes are the same as those used to 
record a person's EKG. HRV will be derived from the EKG signal. Following instrumentation, 
participants will sit quietly for a 30-minute baseline (habituation) period before completing either 
the speech task or a rest period. Following this, participants will watch a wildlife documentary 
and complete questionnaires for a 120-min recovery period. Blood samples will be collected at 
mins. -5 (end of baseline/habituation), and +5, 10, 20, 30, 45, 60, 75, 90, and 120 post the task 
period. Blood pressure will be recorded twice prior to each of the 10 blood draws. Participants 
will also complete the Brief Profile of mood State questionnaire on 4 occasions, immediately 
pre- and post-task, and at 60- and 120-minutes post task. This 39-item adjective checklist 
provides a measure of emotional state. These time points were chosen to capture both acute 
and delayed neuroendocrine and inflammatory changes, as well as recovery patterns following 
standard schedules in the literature. 
 
At one visit, participants will be exposed to the speech task. This is a widely used, highly 
effective way to investigate stress responses in a laboratory setting. It induces significant 
increases in cortisol, indices of autonomic activation, and circulating levels of inflammatory 
mediators. Our preliminary data provides novel evidence that this stressor also results in 
increased cc-mtDNA. Participants will be told that they will give a speech, recorded by a video 
camera and in front of an audience (two research staff) trained to assess non-verbal behaviors. 

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Participants will be given 2 minutes to prepare for the speech defending themselves against an 
alleged transgression and 3 minutes to deliver it. At the end of the recovery period following the 
task, all participants will be thoroughly debriefed. 
 
At the other visit, participants will rest quietly for the same period of time with identical 
assessment in the absence of the stressor. This control session will allow us to assess and 
control for any possible effects of the passage of time (e.g., circadian variation in cortisol levels), 
simply being assessed (e.g., discomfort), and the intravenous catheter. Conditions will be 
counterbalanced in randomized starting order across subjects. 
 
Following completion of the second study visit, participants will be thanked for taking part and 
receive their payment. 
 
 
Eligibility Criteria  
 
Inclusion Criteria: Men and premenopausal women between 20 and 50 years of age (equally 
distributed across decades of age - e.g., 10 men and 10 women aged 20-30) who meet the 
following criteria: 
 
1. Generally healthy 
2. Non-smokers/illicit drug users 
3. Blood pressure below 140/90 
4. Weight > 110 lbs 
5. BMI < 30 
6. Fluent in English 
7. Women -- regular menstrual cycles over the past 12 months (defined as 21-35 days in length) 
8. Able and willing to give informed consent 
9. Willing to abstain from alcohol and vigorous exercise for 24 hours, from food and drinks 
(other than water) for 3 hours and from non-prescription medications (other than oral 
contraception) for 2 days before testing. 
10. Willing to attend two laboratory stress testing sessions, give blood though an intravenous 
catheter, undergo medical evaluation and complete psychosocial questionnaires. 
 
Exclusion Criteria: 
1. Reported history of chronic systemic immune, metabolic or mitochondrial diseases, or chronic 
diseases that influence the central nervous, autonomic nervous or neuroendocrine systems, 
e.g., autoimmune disease, chronic infections, cardiovascular disease, diabetes, chronic kidney 
or liver disease, cancer treatment. 
2. Reported psychiatric history of schizophrenia or other psychotic illness, or mood disorder. 
3. Resting blood pressure > 140/90 mmHg at baseline testing. 
4. Weight < 110 lbs 
5. BMI equal to or greater than 30 
6. Report currently taking glucocorticoid, anti-inflammatory, anti-retroviral, immunosuppressant, 
insulin, antiarrhythmic, antihypertensive, oral hypoglycemic, antidepressant, benzodiazepine or 
prescription weight loss medications or other medications known to influence the immune, 
autonomic or neuroendocrine systems. 
7. For women - Post-menopausal or irregular menstrual cycles over the past 12 months. Report 
current pregnancy or lactation. 

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8. Current smokers (defined as having smoked a cigarette in the previous 3 months). 
9. Current illicit drug use (defined as reported use of illicit drugs such as marijuana, cocaine or 
heroin in the previous 3 months). 
10. Not fluent in English (have used English in everyday speaking and reading for at least 10 
years) 
11. Unable or unwilling to give informed consent 
12. Unwilling to abstain from alcohol and vigorous exercise for 24 hours, from food and drinks 
(other than water) for 3 hours and from non-prescription medications (other than oral 
contraception) for 2 days prior to testing. 
 
 
 
 
 
 
Statistical Considerations 
 
For the primary analysis in Aim 1, because we are utilizing a “matched” design (in which each 
subject serves as his/her own control), we base our power calculations on a simple paired t-test 
since power formulas are not available for complex models such as the ones we propose to 
apply to our data. We find that with the proposed sample size n = 60, we have 80% power to 
detect a normalized difference (in terms of Cohen’s d) as small as 0.38. Combined effects sizes 
from a meta-analysis of the literature [1] yield normalized effect sizes for IL-6 and TNF-α at 0.35 
and 0.28, respectively. For ccf-mtDNA, our pilot data give an estimated effect size of 1.13 (see 
Fig. 4), such that we will be sufficiently powered to detect stress-induced changes, and to detect 
statistically significant differences in ccf-mtDNA separately in women and in men. In fact, 
however, we will not have to rely on such a simple analysis, since we are collecting several 
measurements over time and will be modeling the entire trajectory. With multiple observations, 
by taking into account necessary covariates and accounting for the correlation structure among 
repeated observations, we will increase the power of the analysis to detect a change, potentially 
dramatically, and so we expect to be amply powered. For the secondary analyses, again we will 
be applying more sophisticated and powerful analyses, but for reference we base our power 
calculations on Pearson’s correlation analysis and find that with the proposed sample size we 
are powered to detect a “true” correlation coefficient as small as 0.35 (in absolute value), 
typically considered somewhere between a “small” and “medium” effect size. 
 
Univariate and descriptive analysis will be performed on all dependent variables and, if 
necessary, normalizing and/or variance stabilizing transformations will be applied to the data 
before inferential analyses are undertaken. All hypothesis testing will involve two-sided 
alternative hypotheses with the α level set to 0.05. The analytic strategy for addressing Aim 1 
will involve fitting linear mixed models, which allow flexible covariance structures to account for 
correlation of repeated measures data. Specifically, subject and day (nested within subject) will 
be the random effects, and condition (stress/control) along with other covariates will be the fixed 
effects. We will also include time and time^2 (squared) as fixed effects to allow for a nonlinear 
response. The outcome variables will be the various physiological and psychological responses: 

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IL-6, TNF-α, ccf-mtDNA, cortisol, epinephrine, norepinephrine HR, BP, momentary negative 
affect, and HRV.