Plasma circulating cell-free mitochondrial DNA in social anxiety disorder

Objective: To investigate plasma levels of circulating cell-free mitochondrial DNA (ccf-mtDNA) in patients with social anxiety disorder (SAD) and healthy controls (HC). Methods: In this study, 88 participants (46 patients with SAD and 42 HCs) were enrolled and both ccf-mtDNA and peripheral blood mononuclear cells (PBMC) mtDNA copy number (mtDNA-cn) were measured at up to three times per individual (9 – 11 weeks apart). SAD patients also received cognitive behavioral therapy (CBT) between the second and third time-point. Results: SAD patients had significantly lower ccf-mtDNA compared to HCs at all time points, but ccf-mtDNA did not change significantly after CBT, and was not associated with severity of anxiety symptoms. Plasma ccf-mtDNA did not significantly correlate with PBMC mtDNA-cn in patients. Conclusion: This is the first report of lower ccf-mtDNA in patients with an anxiety disorder. Our findings could reflect a more chronic illness course in SAD patients with prolonged periods of psychological stress leading to decreased levels of ccf-mtDNA, but future longitudinal studies are needed to confirm or refute this hypothesis.


Introduction
Mitochondrial DNA (mtDNA) is released from the cell into the systemic circulation in response to oxidative stress or apoptotic cell death (McArthur et al., 2018;Nakahira et al., 2011). Such circulating cell-free mtDNA (ccf-mtDNA) may potentiate inflammatory responses (Zhang et al., 2010) but could also have antibacterial effects (Yousefi et al., 2008) and be involved in cell-to-cell communications, as reviewed elsewhere (Trumpff et al., 2021). Ccf-mtDNA is elevated in plasma in a range of somatic conditions such as traumatic injury (Gu et al., 2013), inflammatory disorders (Nakahira et al., 2013), sepsis (Dwivedi et al., 2012), and myocardial infarction (Bliksoen et al., 2012). Recent studies suggest that ccf-mtDNA may be a biomarker of not only physiological stress, but also psychological stress. Plasma ccf-mtDNA is acutely increased after exposure to socio-evaluative challenges (Hummel et al., 2018;Trumpff et al., 2019), and higher ccf-mtDNA levels have also been associated with hypothalamic-pituitary-axis (HPA) hyperactivity (Lindqvist et al., 2016). Despite this, previous studies on psychiatric patients have reported mixed results with high (Lindqvist et al., 2016, low Kageyama et al., 2018), or unchanged (Jeong et al., 2020;Stertz et al., 2015) ccf-mtDNA levels in cases vs controls.
In addition to measuring mtDNA outside the mitochondria (i.e. ccf-mtDNA), intracellular mtDNA-copy number (mtDNA-cn) can also be quantified. We have previously shown that intracellular and extracellular mtDNA do not correlate significantly, suggesting that they reflect different aspects of mitochondrial or cellular physiology . Despite evidence for a link between psychological stress and ccf-mtDNA, no previous studies have measured this biomarker in patients with anxiety disorders, nor has ccf-mtDNA been related to intracellular mtDNA-cn in this psychiatric population. In the current study, including 46 patients with social anxiety disorder (SAD), plasma ccf-mtDNA and peripheral blood mononuclear cells (PBMC) mtDNA-cn were measured twice before, and once after cognitive behavioral therapy (CBT). Further, plasma ccf-mtDNA was collected twice from 42 matched healthy controls. We investigated between-group differences, and treatment related effects in ccf-mtDNA as well as associations between plasma ccf-mtDNA, PBMC mtDNA-cn and symptom severity within the SAD group.

Ethical considerations
The study was pre-registered at clinicaltrials.gov (#NCT01312571) and approved by the regional ethics committee at Umeå University, Sweden (2018-349-32). All subjects provided written informed consent to participate in the study.

Recruitment procedures
Subject recruitment procedures have been described in detail elsewhere (Mansson et al., 2019(Mansson et al., , 2022. This is a secondary analysis of a study originally designed to test blood and brain imaging biomarkers of response to CBT. Blood was sampled at three separate time points with a 9-11 week interval. We used repeated baseline assessments (separated by 11 weeks; pre 1 and pre 2 ) to account for confounds related to time and measurements including regression to the mean and repeated testing. All SAD patients underwent 9 weeks of CBT delivered via the Internet, whereafter blood sampling was repeated at post-treatment (Post-tx).
All SAD patients were over 18 years of age and had no neurological disorders, no concurrent psychological treatment and if treated with a psychotropic medication, they agreed to maintain a stable dose at least 3 months before enrollment. A diagnosis of SAD was determined using both the Mini-International Neuropsychiatric Interview (M.I.N.I.) and the Structured Clinical Interview for DSM-IV-Axis I disorders. Exclusion criteria were severe depression (Montgomery Åsberg Depression Rating Scale score > 34), current bipolar disorder, psychotic disorder, alcohol or substance use disorder or antisocial personality disorder. Four (8.7%) of the SAD patients had a concurrent but stable dose of an SSRI during the study period. In addition to patients, we also recruited a matched (i. e., on age, sex, education and handedness) group of healthy controls without any previous or current psychiatric disorders (as determined by a modified M.I.N.I. interview). There were no significant patient-control differences with regards to sex, age, BMI, or smoking (all p's > 0.20).

Blood sampling
Blood samples were collected in the morning after a night of fasting. Subjects received a mobile phone text message the day before sampling reminding about fasting instructions. Subjects rested 15 min before blood draws. Whole blood was drawn in a 8 ml BD Vacutainer® CPT™ Mononuclear Cell Preparation Tube-Sodium Citrate (Becton Dickinson). Plasma and mononuclear cells (PBMCs) were separated according to manufacturer's protocol and was stored at − 80 • C. Briefly, 15 min after venipuncture, the CPT tube was centrifuged at 1500xg for 20 min at 20 • C. After immediate separation of plasma and PBMCs, both tubes (the PBMC tube with 14 ml PBS) were centrifuged for 15 min at 300xg and 20 • C. The plasma was separated from the pellet, aliquoted and immediately frozen at − 80 • C. PBMCs were again, in two equal-sized aliquots, washed in PBS (5 min 300xg), and after removal of PBS immediately frozen at − 80 • C.

Plasma circulating cell-free mitochondria DNA assay
In order to analyze the concentration of ccf-mtDNA in plasma samples from patients and controls, quantitative PCR was used, as previously described (Lindqvist et al., 2016. Briefly, DNA was isolated using the QIAmp 96 DNA Blood Kit (Qiagen, Valencia, CA, USA) according to the manufacturer's instruction for Blood and body-fluid protocol after an initial centrifugation at 10 000 x g for 10 min. 200 µl of plasma was used for each isolation and the samples were eluted in the same volume, 200 µl. The amount of ccf-mtDNA was analyzed using SYBR Green technology and primers specific for NADH dehydrogenase 2 (ND2, accession number KJ676545) on a LC480 LightCycler (Roche, Mannheim, Germany). Primer sequences were as follows: CACACT-CATCACAGCGCTAA (fwd) and GGATTATGGATGCGGTTGCT (rev). 5 µl of template, 1 µl of each primer (10 μM), 10 µl SYBR MIX (2 ×, Sensi-FAST, Bioline, London, UK) and 3 µl of nuclease-free water. Each reaction was run in triplicate using the following program: Initial denaturation at 95 • C for 10 min, followed by 45 cycles consisting of 95 • C in 10 s for melting, 65 • C for 10 s annealing and 72 • C for 11 s extension. The program ended with a melting curve analysis measuring fluorescence continuously from 60 • to 97 • C.
Finally, the concentration was calculated and results given as number of mitochondrial units per µl plasma (Lindqvist et al., 2016). Plasma ccf-mtDNA samples were available from all 46 SAD patients at all time-points. Forty-two samples were available from HCs at the first time-point, and 41 samples at second time-point 11 weeks later.

Peripheral blood mononuclear cells mitochondria DNA copy number assay
Genomic DNA was extracted from one of the aliquots of PBMCs using DNeasy® Blood & Tissue Kit (Qiagen), with a modified protocol to reduce DNA shearing. Briefly, cell lysis was done at 37 • C for 3 h, vortex was avoided, and centrifugations were performed at 6000xg. Copy number of mitochondrial DNA per nuclear genome (i.e. per cell) was determined using real-time quantitative PCR (qPCR) according to published protocols (Kumar et al., 2018) where the relative amount of mitochondrial gene tRNA-Leu(UUR), to the nuclear single-copy gene β2-microglobulin (B2M) (M/S) was determined using a standard curve. In brief, each DNA sample (4.0 ng) was assessed for tRNA-Leu(UUR) and B2M in triplicate within the same 384-well plate, amplified by using Power SYBR Green. The reaction was performed on QuantStudio 7 Flex (Applied Biosystems; Life Technologies, Carlsbad, CA, USA) with the following conditions: 95 • C for 10 min, followed by 39 repeats of 95 • C for 15 s and 60 • C for 1 min, followed by a dissociation stage to monitor amplification specificity. A standard curve of pooled DNA from these patients (10 ng/µl, 2.0 ng/ µl, 0.40 ng/µl, 0.080 ng/µl, 0.016 ng/µl) was run on each of the three 384-well plates to determine the M/S ratio for each sample. The 0.016 ng/µl standard samples had Ct~22 for tRNA-Leu and Ct~31 for B2M. The correlation coefficients of the standard curves were above 0.99 for each primer set and 384-plate. The inter-plate coefficient of variation (CV) of M/S ratio was 8% calculated from four patient samples run in all plates. The success rate was 100% (i.e. Ct standard deviation < 0.3 between triplicates and a Ct value within the standard curve). PBMC mtDNA-cn samples were available for SAD patients only (i.e., 45 samples at the first time point, 46 samples at the second and third time point). All three time points per individual were assayed in the same 384-well plate.

Statistics
All analyses were performed using the STATA statistical software v15.1 (STATA Corporation, College Station, TX, USA). Natural logarithm transformed values of ccf-mtDNA and mtDNA-cn were entered in separate linear regression models to investigate differences between SAD patients and healthy controls, changes across time, and test associations between biomarkers and clinical outcomes. Generalized estimating equations (GEE) with exchangeable correlation structure were used to evaluate panel data on treatment effects across time. Delta scores from pre-to posttreatment (i.e., the difference between post-treatment score, and the average of pre 1 and pre 2 ) were created for both mtDNA and clinical anxiety scores and used to estimate associations between mtDNA and anxiety symptoms. Nonparametric bootstrapping with replacement (×1000) was used to estimate confidence intervals, and permutations (×1000) were used to test the significance of the effects. Regression models including estimates of PBMC mtDNA-cn included cell type (proportion of lymphocytes to monocytes) as a covariate. Detailed outputs from all statistical analyses are available at our GitHub repository (https://github.com/neuronssonlab/ Lindqvist_etal_2023_Psychoneuroendocrinology).
No significant correlations between ccf-mtDNA and severity of social anxiety or depressive symptoms were noted (data not shown). Further, ccf-mtDNA did not significantly change during 9 weeks of CBT (Z = 1.21, permuted p = 0.311), and SAD patients still had significantly lower post treatment ccf-mtDNA compared to controls at the second baseline ( pre 2 ) (β = 0.46, 95% CI 0.29, 0.64; Adj-R 2 = 21%, permuted p < 0.001). While social anxiety scores were markedly reduced from pre-to posttreatment (see Table 1), symptom improvement was not associated with changes in ccf-mtDNA (permuted p = 0.948). PBMC mtDNA-cn did not change significantly during 9 weeks of CBT (Z = 1.38, permuted p = 0.295).

Discussion
We show that ccf-mtDNA is significantly lower in patients with SAD compared to healthy controls. This difference was statistically significant across three separate time points and was not significantly affected by CBT treatment.
Although there are previous reports of ccf-mtDNA in mood disorders Jeong et al., 2020;Kageyama et al., 2018;Lindqvist et al., 2018;Stertz et al., 2015), this is the first study to measure this biomarker in patients with anxiety disorders. Previous studies of mood disorders have produced variable results of high (Lindqvist et al., 2016, low Kageyama et al., 2018), or unchanged (Jeong et al., 2020;Stertz et al., 2015) plasma ccf-mtDNA in patients compared to healthy controls. Low ccf-mtDNA in SAD patients could reflect a trait rather than a state because this biomarker showed temporal stability and was not related to psychological treatment or symptom severity. Acute and chronic stress may have differential, and perhaps even opposite, effects on mitochondrial biology (Picard and McEwen, 2018). Acute stress leads to a robust increase in plasma ccf-mtDNA (Hummel et al., 2018;Trumpff et al., 2019), but less is known about ccf-mtDNA dynamics after longer periods of mental illness and associated stress. Our findings of lower ccf-mtDNA in SAD may reflect a more chronic disease course with prolonged periods of psychological stress leading to adaptive biological changes and a subsequent decrease in ccf-mtDNA, but future studies should investigate this hypothesis.
We did not detect any association between ccf-mtDNA and SAD symptomatology, or symptom changes after CBT. Saliva mtDNA-cn has previously been related to severity of anxiety symptoms in a sample of adolescents (Tymofiyeva et al., 2018). In another study, higher levels of blood leukocyte mtDNA-cn was associated with a lifetime history of an anxiety disorder (Tyrka et al., 2016). Also, we detected no correlation between ccf-mtDNA and PBMC mtDNA-cn, which is consistent with a previous study from our group on patients with depression , and further supports the notion that these two biomarkers represent different aspects of mitochondrial biology and cellular function. While PBMC mtDNA-cn reflects intracellular mtDNA from white blood cells, ccf-mtDNA could be derived from almost any cell tissue, hence one would expect that these two markers would not significantly intercorrelate.
A major strength of the present study is that we used repeated sampling with two baseline assessments separated by 11 weeks. There are, however, several potentially confounding factors that could have influenced our results. For instance some (Hummel et al., 2018;Stawski et al., 2017), but not all (Beiter et al., 2011), studies have reported that physical activity acutely increases ccf-mtDNA. SAD patients may have a more sedentary lifestyle compared to healthy volunteers potentially leading to lower levels of ccf-mtDNA. On the other hand, blood sampling was standardized for all subjects with a night of fasting and 15 min rest before the blood draw. Moreover, there is no evidence that regular exercise training would be associated with higher baseline ccf-mtDNA, in fact there is some preliminary results suggesting the opposite (Nasi et al., 2016). There are also some indications that ccf-mtDNA levels may be dependent on various factors related to blood sampling, processing and mtDNA extraction techniques (Randeu et al., 2022;Wong et al., 2016), although this was not likely a confounder in the present study since all samples were handled identically. We found an association between ccf-mtDNA across different time points, but other studies have reported high intra-individual variation in ccf-mtDNA over time (Trumpff et al., 2021). Within-person stability over time is indeed a crucial issue if this biomarker was ever to be introduced in the psychiatric clinic, and more studies are needed to examine this issue.
In conclusion, we found evidence for lower ccf-mtDNA in SAD patients compared to matched healthy controls. These findings are novel but need to be replicated in future large-scale longitudinal studies.

Funding sources
Dr. Lindqvist is funded by the Swedish Research Council (grant number #2020-01428) and Swedish governmental funding of clinical research (ALF). Dr. Furmark receives funding from the Swedish Research Council (#2020-02426), The Swedish Brain Foundation (#FO2016-0106) and Riksbankens Jubileumsfond (#P17-0639:1). Dr. Lavebratt is funded by the Swedish Research Council (#2014-10171), Table 1 Demographic characteristics, symptom severity and ccf-mtDNA in social anxiety disordered patients and matched healthy controls. Abbreviations: ccf-mtDNA, circulating cell-free mitochondrial DNA; LSAS-SR, Liebowitz Social Anxiety Scale, Self-reports; MADRS-S, Montgomery-Åsberg Depression Rating Scale, Self-reports; SAD, social anxiety disorder; Post-tx, Posttreatment; Note: Units for ccf-mtDNA is copies/microliter plasma. Time-points Pre 1 and Pre 2 were baseline assessments separated by 11 weeks for all participants. Timepoints Pre 2 and Post-tx were separated by 9 weeks of treatment for all SAD patients.
the Swedish Brain Foundation (#FO2020-0305) and the regional agreement on clinical research between Region Stockholm and Karolinska Institutet [SLL20190589]. Dr. Månsson receives funding from the Swedish Research Council (#2018-06729), and StratNeuro at Karolinska Institutet.

Declarations of interest
All authors declare no conflict of interest. Fig. 1. Patient-control differences across three time-points. Mean and SD values of log-transformed ccf-mtDNA in patients with social anxiety (SAD) and healthy controls (HC) at the first baseline (Pre 1 ), second baseline (Pre 2 ), and at post-treatment. * p < 0.001 Pre 1 in HC vs pre 1 in SAD patients. ** p < 0.001 Pre 2 HC vs Pre 2 in SAD patients and Pre 2 in HC vs post-treatment in SAD patients.