Microglia-mediated neuroimmune suppression in PTSD is associated with anhedonia

Significance

Using advanced neuroimaging techniques, we show that individuals with posttraumatic stress disorder (PTSD) have a suppressed response to a direct immune insult compared to those without PTSD. Imaging a protein in the brain that is present on glial cells before and after lipopolysaccharide administration, we found that the immune system of individuals with PTSD is compromised relative to controls. This finding suggests that treatments to restore the immune response and regulation of related systems should be considered in future clinical trials.

Abstract

Dynamic brain immune function in individuals with posttraumatic stress disorder is rarely studied, despite evidence of peripheral immune dysfunction. Positron emission tomography brain imaging using the radiotracer [¹¹C]PBR28 was used to measure the 18-kDa translocator protein (TSPO), a microglial marker, at baseline and 3 h after administration of lipopolysaccharide (LPS), a potent immune activator. Data were acquired in 15 individuals with PTSD and 15 age-matched controls. The PTSD group exhibited a significantly lower magnitude LPS-induced increase in TSPO availability in an a priori prefrontal-limbic circuit compared to controls. Greater anhedonic symptoms in the PTSD group were associated with a more suppressed neuroimmune response. In addition, while a reduced granulocyte–macrophage colony-stimulating factor response to LPS was observed in the PTSD group, other measured cytokine responses and self-reported sickness symptoms did not differ between groups; these findings highlight group differences in central–peripheral immune system relationships. The results of this study provide evidence of a suppressed microglia-mediated neuroimmune response to a direct immune system insult in individuals with PTSD that is associated with the severity of symptoms. They also provide further support to an emerging literature challenging traditional concepts of microglial and immune function in psychiatric disease.

Over half of the population will experience a traumatic event during their lifetime, with up to 10% developing symptoms consistent with posttraumatic stress disorder (1–3). The multifaceted phenotype of PTSD contributes to the complicated understanding of this disorder’s pathophysiology (4, 5). Increasing evidence suggests that a dysfunctional immune system may play a critical role in the development and maintenance of PTSD symptoms. Most of this knowledge was gleaned from studies of the peripheral immune system (6), which have generally found relationships between trauma exposure or PTSD symptom severity and elevated peripheral cytokine levels (7, 8). Additionally, PTSD is a known risk factor for immune-related chronic diseases (2, 9). Some of these studies indicate a close relationship between the severity of trauma history and PTSD symptoms, with worse symptoms and test scores from immune-related illnesses (10, 11). These findings motivated investigation of anti-inflammatory treatments (6). However, a more complicated picture has since developed, with evidence emerging of suppressed peripheral and neuroimmune markers in PTSD (12, 13).

Due to the difficulty of probing the central immune system in people, much of our knowledge has come from preclinical studies, which are extraordinarily useful at guiding our understanding of the underlying neuropathology but lack translatability regarding nuances of the illness. Rodent models of PTSD mimic many of the distinct anxiety or depressive phenotypes of PTSD but have limitations in the symptom overlap and complexity of the illness (14). Preclinical findings suggest a role for microglia-mediated cytokine signaling including chemokine receptor 1 (CX3CR1), ionized calcium binding adaptor molecule (IBA1), and other proinflammatory cytokines in the corticolimbic system in PTSD models (15, 16). Within these models, microglia-driven cytokine changes have been found to correlate with depressive symptoms (17–19). Investigations into glia cells in preclinical PTSD models with a lipopolysaccharide (LPS) (20) immune challenge demonstrate significant changes in the microglial cytoarchitecture and chemokine signaling (21–23). Though there is certainty around a role of microglia in fear and memory learning through mediators of synaptic pruning and neuroimmune signaling, there remains a knowledge gap regarding human central immune system function in PTSD (24, 25). Postmortem brain data from individuals with PTSD demonstrate RNA transcriptome changes in central levels of inflammatory cytokines, but these do not always correlate with peripheral biomarker changes (26). These findings highlight the need to directly investigate the central immune system in living people with PTSD. Additionally, though many studies show differences in the immune system between control and PTSD groups at a single timepoint (4), few examine changes in the central immune system response to an acute stressor. This is important considering evidence that environmental stressors can trigger heightened threat perceptions associated with PTSD (27, 28), which potentially have adverse effects on overall immune system function and health.

One approach used to examine the central immune system in vivo is positron emission tomography (PET) brain imaging with the radiotracer [¹¹C]PBR28, which selectively binds to the 18-kDa translocator protein (TSPO) (29, 30). TSPO is expressed predominantly on microglia but is also present on astrocytes and endothelial cells. Within neuropsychiatric disorders, it is thought that the main cellular contributions are typically from microglia. These tools have been used to measure TSPO availability in neurologic and psychiatric disorders (31–36), including PTSD (13). Somewhat counterintuitively, we previously found significantly lower levels of TSPO in a group of individuals with PTSD as compared to controls (13). Consistent with the literature (8, 28, 37) we also found elevated peripheral inflammatory levels of c-reactive protein (CRP) in individuals with PTSD as compared to controls. However, elevated CRP was correlated with lower prefrontal-limbic TSPO levels. This pivotal study in human subjects suggested a suppressed neuroimmune system in PTSD; however, the study measured TSPO availability at a single timepoint and thus did not shed light on dynamic neuroimmune function. The present study aimed to capture dynamics of neuroimmune function by measuring TSPO availability both before and after intravenous administration of LPS, a potent immune activator, using our previously published paradigm (38). Our investigation builds upon our previous study (13), focusing on TSPO availability in the prefrontal-limbic circuit composed of the amygdala, hippocampus, insula, and ventromedial prefrontal cortex (vmPFC), which were found to be lower compared to controls and were associated with more severe PTSD symptoms. Additionally, these regions are implicated in neuroimmune (39), neuroendocrine (40), and transcriptomic (41) neural circuits involved in the pathophysiology of PTSD. Based on our previous finding of lower TSPO levels (13), we hypothesized a lower magnitude neuroimmune system response, i.e., a smaller increase in TSPO availability in response to LPS administration, in individuals with PTSD relative to controls. We further hypothesized that individuals with more severe PTSD would have a more suppressed central immune response.

In this study, we present evidence of a suppressed central microglia response, measured as a lower magnitude LPS-induced increase in prefrontal-limbic TSPO availability, in individuals with PTSD compared to controls. Furthermore, individuals with PTSD who reported greater clinical anhedonia exhibited more suppressed neuroimmune responses. Interestingly, many of the peripheral cytokines and subjective sickness symptoms in response to LPS were similar across both groups with one exception. Specifically, the PTSD cohort had a significantly lower peripheral response of the granulocyte–macrophage colony-stimulating factor (GM-CSF) relative to controls. Finally, across all participants, the CRP response was inversely correlated to the TSPO response. We now demonstrate that individuals with PTSD exhibit a suppressed neuroimmune response to a direct immune system insult compared to individuals without PTSD and that greater symptom severity is associated with a more blunted response.

Results

Demographic and Clinical Characteristics.

Fifteen individuals who met DSM-5 criteria for PTSD participated in the study (9M and 6F). Fifteen age-matched individuals also participated in the study (11M and 4F); 10 control participants experienced a criterion A trauma but were without PTSD symptoms. Participant characteristics and injection parameters (Table 1) did not differ between the PTSD vs. control group except for the rs6971 genotype, where the PTSD group had disproportionately more HAB compared to the control group; accordingly, the rs6971 genotype was adjusted for in analyses. Within trauma type, there was a higher prevalence of sudden traumatic loss and military trauma in the PTSD group. There was no difference in injected LPS dose between groups.

Table 1.

The table displays notable participant demographics and imaging parameters

	HC (N = 15)	PTSD (N = 15)	Test of difference	P value
	Mean (SD)	Mean (SD)
	or	or
	N (%)	N (%)
Age	30.1 (8.3)	32.2 (10.4)	0.92	0.18
Sex (N, % male)	11 (73.3%)	9 (60.0%)	0.6	0.44
Race/ethnicity			2.79	0.43
White	7 (46.7%)	7 (46.7%)
Black	3 (20.0%)	5 (33.4%)
Hispanic	5 (33.3%)	2 (13.3%)
Asian	0 (0.0%)	1 (6.7%)
Years of education	14.2 (1.6)	14.0 (2.6)	0.24	0.4
# (%) High-affinity binders(HAB), rs6971 genotype	8 (53.3%)	14 (93.3%)	6.14	0.013
Criterion A trauma	10 (66%)	15 (100%)	14.13	0.17
Sudden traumatic loss	1 (6.7%)	4 (26.7%)
Military combat	0 (0%)	3 (20.0%)
Physical abuse	1 (6.7%)	2 (13.3%)
Assaultive violence	1 (6.7%)	3 (20.0%)
Motor vehicle accident	2 (13.3%)	1 (6.7%)
Sexual abuse	1 (6.7%)	1 (6.7%)
Witnessed violence	2 (13.3%)	1 (6.7%)
Witnessed death	1 (6.7%)	0 (0%)
Natural disaster	1 (6.7%)	0 (0%)
None	5 (33.3%)	0 (0%)
Age of index trauma		20.4 (7.0)	–	–
Years since index trauma	–	10.9 (7.3)	–	–
Substance use			0.32	0.61
Tobacco (number of users)	7 (46.7%)	6 (40.0%)	0.14	0.71
Alcohol (number of users)	6 (40.0%)	7 (46.7%)	0.13	0.72
Cannabis (number of users)	3 (20.0%)	5 (33.3%)	0.68	0.41
Symptom scores
CAPS-5 total * N = 12	0.6 (1.5)	32 (11)	10.95	<0.001
PCL-5 (on scan day)*	0.4 (0.9)	43.7 (13.3)	12.56	<0.001
Injection Parameters Scan 1
Injection dose (MBq)	493.7 (180.9)	554.1 (161.7)	0.96	0.17
Injected mass (μg/Kg)	0.0176 (0.027)	0.0123 (0.008)	0.71	0.24
Injection Parameters Scan 2
Injection dose (MBq)	554.6 (168.6)	538.7 (173.1)	0.47	0.32
Injected mass (μg/Kg)	0.0174 (0.028)	0.0108 (0.092)	0.89	0.19
LPS dose (ng/kg)	1.0 (0.01)	1.0 (0.02)	0.49	0.31

No group differences were observed, except for the percent of high binders, where there are significantly more high binders in the PTSD group.

PTSD Is Associated with Lower TSPO Response to LPS Administration.

A repeated measures ANOVA revealed that the PTSD group exhibited a significantly lower magnitude LPS-induced increase in TSPO availability (29%) relative to the control group (42%) in the a priori prefrontal-limbic circuit (P = 0.021; Cohen d = 0.89) (Fig. 1, Table 2, and SI Appendix, Table S1). This finding remained statistically significant (P = 0.003) after removing from the analysis the individual on selective serotonin reuptake inhibitor treatment, as well as after adjustment for cannabis use (P = 0.025); cannabis use was unrelated to LPS-induced changes in TSPO availability (P = 0.64). Consistent with our prior findings (13), the PTSD group exhibited lower prefrontal-limbic [¹¹C]PBR28 V_T at baseline (P = 0.073; Cohen d = 0.68). In the PTSD group, greater baseline severity of anhedonia symptoms on the clinical administered PTSD scale (CAPS) was associated with a more “blunted” neuroimmune TSPO response to LPS (r = −0.425, P < 0.001) (Fig. 2). No significant correlation was observed between PTSD Checklist for DSM-5 (PCL-5) or PTSD Checklist for DSM-IV (PCL-IV) conversion) and [¹¹C]PBR28 V_T for the PTSD group, or full CAPS scores. No significant correlations were found between [¹¹C]PBR28 V_T and CAPS scores in the control group.

Fig. 1.

[¹¹C]PBR28 V_T percent change values from four individual brain regions and the composite prefrontal-limbic circuit demonstrate a consistently reduced change in the PTSD group as compared to controls. The group average [¹¹C]PBR28 V_T percent change values are shown for the composite circuit (P = 0.021, d = 0.89), amygdala (P = 0.022, d = 0.89), hippocampus (P = 0.008, d = 1.04), insula (P = 0.098, d = 0.63), and vmPFC (P = 0.039, d = 0.79) in controls (blue) and people with PTSD (red). **P < 0.01, *P < 0.05, and ^#P < 0.1.

Table 2.

The table displays the four individual regions, which make up the composite prefrontal limbic circuit, and the group averages for each volume of distribution for the regions both pre- and post-LPS administration

	Control pre- LPS (ML/cm3)	Control post-LPS (ML/cm3)	% Change	PTSD pre-LPS (ML/cm3)	PTSD post-LPS (ML/cm3)	% Change	Significance
Amygdala	5.11 ± 0.85	7.20 ± 1.4	42.0 ± 19 %	4.75 ± 1.4	6.07 ± 2.1	27.9 ± 18 %	P = 0.022
Hippocampus	4.89 ± 0.78	6.82 ± 0.87	42.9 ± 16 %	4.38 ± 1.1	5.66 ± 1.8	29.4 ± 15 %	P = 0.008
lnsula	5.07 ± 0.85	7.14 ± 1.3	40.7 ± 12 %	4.45 ± 1.1	7.14 ± 2.1	31.9 ± 14 %	P = 0.098
vmPFC	5.33 ± 0.91	7.73 ± 1.6	45.1 ± 17 %	5.33 ± 1.2	7.73 ± 1.8	27.2 ± 21 %	P = 0.039

Fig. 2.

Individual data for N = 15 participants in the PTSD group demonstrating that a smaller percent change in [¹¹C]PBR28 V_T values following LPS administration was significantly associated with worse anhedonia scores from the CAPS (r = −0.45; P < 0.001) using linear regression analysis. Gray shading indicates the 95% CI for the linear regression.

C-reactive Protein Levels.

CRP data were available for a subset (n = 10 PTSD and n = 4 controls) of the full sample. Greater LPS-induced increase in CRP was associated with a less robust increase of TSPO availability in the subset of both the PTSD and control groups for which CRP data were available (Spearman rank correlation, r = −0.58, P = 0.029; Fig. 3). This association was especially strong in the PTSD group (Spearman rank correlation r = −0.72, P = 0.019). Consistent with our previous study, higher baseline CRP levels were associated with greater severity of negative affect symptoms (worsening negative emotional state including fear, horror, guilt, and shame) (Spearman rank correlation r = −0.61, P = 0.015).

Fig. 3.

Percent change in peripheral CRP values for the PTSD group, N = 10 (red), and controls (blue), N = 4, as compared to change in [¹¹C]PBR28 V_T values. Greater CRP change was significantly associated with a smaller [¹¹C]PBR28 V_T change across all data in both groups (r = −0.58, P = 0.02) using linear regression analysis with Spearman correlation. Within just the PTSD cohort, the results remain significant with an inverse relationship between CRP change and [¹¹C]PBR28 V_T change, r = −0.72, P = 0.19.

Scan-Day Cytokine Levels.

Relative to controls, individuals with PTSD had a significantly reduced GM-CSF cytokine response after LPS (P = 0.007) (Fig. 4). Other cytokines including tumor necrosis factor-α (TNF-α), interleukin-1β (IL-1β), interferon-γ (IFN-γ), interleukin-4 (IL-4), interleukin-6 (IL-6), interleukin-10 (IL-10), interleukin-12p70 (IL-12p70), interleukin-17 (IL-17), interleukin-8 (IL-8), and interleukin (IL-1A) and monocyte chemoattractant protein-1 (MCP-1) were explored but there were no significant differences in the LPS-induced change in cytokine levels between groups (SI Appendix, Fig. S1 and Table S2), though most cytokines demonstrated consistent trends of showing lower magnitude responses in the PTSD relative to control group. There were no significant relationships between the TSPO response and the change in other cytokines.

Fig. 4.

Peripherally drawn and analyzed values for GM-CSF concentration in pg/nl plotted over the time relative to LPS administration, where LPS was administered at time = 0 min. The baseline was drawn 10 min prior to LPS administration, which was after the first [¹¹C]PBR28 scan. The control group (blue, N = 15) has a significantly higher change and peak than the PTSD group (red, N = 15, P = 0.007). Error bands represent SE of mean values for each group average at a given time point.

Sickness Symptoms.

LPS is known for its ability to produce sickness behaviors in preclinical (23) and clinical (38) studies, and it is thought that these symptoms result from the effect on the brain of the increases in blood serum levels of cytokines. Participants were asked to rate sickness symptoms (fatigue, headache, muscle pain, and shivering) on a scale from 0 (least) to 10 (most) before and at various time points after LPS administration. Self-reported sickness symptoms significantly increased from baseline, consistent with previous studies (38), but there was no difference between groups in the degree of change (SI Appendix, Table S3). Other symptoms known to be associated with LPS administration were measured on a visual analog scale from 0% (not at all) to 100% (the most) (SI Appendix, Table S3). Most individuals reported wanting to remain alone and felt more fatigued following LPS administration but again there was no significant difference in the change or maximum symptoms reported between groups, indicating that the PTSD and control groups experienced similar levels of sickness symptoms.

Materials and Methods

Participant Recruitment.

Thirty individuals participated in this study and were recruited from 2015 to 2023 via advertisements in public forums or through clinician referral. Written informed consent was obtained from all participants after a complete explanation of study procedures. A structured clinical interview for the Diagnostic and Statistical Manual of Mental Disorders-5 (42) was administered to all participants to screen for history of trauma, PTSD among trauma exposed individuals, presence of comorbid major depressive disorder in the PTSD group and exclusion of all other psychiatric diagnoses, including alcohol use disorder in both PTSD and control groups. Clinical characterization of PTSD (n = 15) was performed using the CAPS (DSM-IV n = 4, DSM-5, n = 11), which was administered by trained clinical research assistants under the supervision of a licensed clinical psychologist. Individuals in the control group with trauma exposure were also administered the CAPS, to confirm they did not meet diagnostic criteria for PTSD. Social drinking, and tobacco and cannabis use were permitted and quantified using the past month Timeline Followback (43). Current use of psychotropic medications other than antipsychotics and anticonvulsant mood stabilizers was permitted in the PTSD group and was determined at screening and confirmed on scan day (total n = 1, selective serotonin reuptake inhibitor; n = 1, norepinephrine-dopamine reuptake inhibitor). Medical examination was performed, and clinical laboratory tests were reviewed by the study physicians to confirm the absence of significant medical illness. All participants were screened for the TSPO rs6971 genotype (NeoGenomics Laboratories, Houston, TX), and individuals with homozygous T/T at the rs6971 locus (also called low-affinity binders) were excluded, based on previous work establishing the effect of TSPO rs6971 genotype on affinity of [¹¹C]PBR28 for TSPO (44). All screening and investigation procedures were in accordance with Code of Federal Regulations Title 45, Part 46 policy on protection of human subjects in research and were approved and overseen by the Yale University Institutional Human Investigation Committee and the Yale New Haven Hospital Radiation Safety Committee.

Trauma History Assessment.

Trauma history was assessed in all participants using the structured clinical interview for posttraumatic stress disorder (DSM-IV) or structured clinical interview for Trauma and Stress Related Disorders Module (DSM-5). The history of particular types of trauma as listed in Table 1 was documented via self-report using the Life Events Checklist (45), a validated tool to assess trauma history, and the childhood trauma questionnaire short form (CTQ) (46) a validated assessment of history of traumatic events prior to 18 y of age. To examine baseline PTSD symptom clusters associated with LPS-induced changes in TSPO availability, we computed sum scores of CAPS measures that assessed the seven symptom dimensions comprising the hybrid model of PTSD symptom structure (47)—intrusions, avoidance, negative affect, anhedonia, externalizing behaviors, anxious arousal, and dysphoric arousal.

Imaging Acquisition.

Synthesis of [¹¹C]PBR28 was achieved at a high molar activity of 557.9 ± 337.3 MBq/nmol using previously published methods (48). Participants received a 1-min intravenous bolus injection of 523.7 ± 190.9 MBq (first scan) and 554.1 ± 161.7 MBq (second scan) [¹¹C]PBR28 and were scanned for 120 min following injection on the high-resolution research tomograph (HRRT; Siemens, Medical Solutions, Knoxville, TN). A T1-weighted structural magnetic structural image (49) was acquired with a sagittal gradient-echo (MPRAGE) sequence (Siemens 3.0 T Prisma Fit; 176 sagittal slices, thickness = 1 mm, TR = 2,530 ms, TE = 2.26 ms, flip angle = 7°, FoV = 256 mm, and matrix size = 256 × 256, 1 × 1 × 1 mm³ voxels) was also obtained in all individuals for coregistration of PET images and for definition of anatomical regions of interest. All participants were scanned with [¹¹C]PBR28 twice on the same day, before and 3 h after intravenous injection of LPS, as previously validated (38).

Cytokine Analysis.

Plasma samples to measure levels of cytokines including TNF-α, IL-1β, IFN-γ, IL-4, IL-6, IL-10, IL-12p70, IL-17, IL-8, and IL-1A and monocyte chemoattractant protein-1 (MCP-1) were taken 10 min prior to LPS administration and then at 30- to 60-min intervals following LPS injection for the next 240 min (4 h). The cytokines were measured using the MILLIPLEX panel assay (MillporeSigma, Burlington, MA). Measurements below the assay-specific detection limit were imputed using chained equations, as the assay-specific detection limit normalized by a factor of 2 or √2, which has previously shown to be appropriate for normal and log-normal distributions, respectively, of data with values below the detection limit (50).

Lipopolysaccharide Administration.

LPS (NIH Clinical Center Reference Endotoxin Escherichia coli serotype O:113) was administered at a dose of 1.0 ng/kg, intravenous, approximately 3 h prior to the second radiotracer injection and scan. Vital signs (temperature, blood pressure, and heart rate) were recorded before and 60, 90, 120, 180, 240, and 300 min (incomplete subject data) after LPS administration. Sickness symptoms (fatigue, headache, muscle pain, and shivering) were assessed on a scale from 0 (least) to 4 (most) relative to LPS administration (t = 0 min) at −10, 60, 90, 120, 180, and 240 min. A visual analog scale of 0 (least) −100 (most) was used to evaluate symptoms such as alertness, energy, focus, pep, and social interest relative to LPS administration at −10, 60, 90, and 180 min.

Arterial Input Function Measurement.

An arterial catheter was placed in the radial artery contralateral to [¹¹C]PBR28 injection site for arterial sampling throughout the duration of the scan. Blood samples were acquired to measure plasma radioactivity concentration either manually every 10 s (n = 8) or continuously for the first 3 min using an automated blood sampler (PBS-101, Veenstra Instruments, Joure, Netherlands) and then at 3, 5, 8, 12, 15, 20, 25, 30, 40, 50, 60, 75, 90, 105, and 120 min in manual samples analyzed with a cross-calibrated well counter (1480 Wizard, Perkin-Elmer, Waltham, MA), with merging of plasma radioactivity concentrations measured by automated and manual sampling. Fraction of unmetabolized [¹¹C]PBR28 was measured using high-performance liquid chromatography as previously described in plasma samples taken at 3, 15, 30, 60, and 90 min. The metabolite-corrected arterial input function was calculated as the product of the plasma radioactivity and unmetabolized [¹¹C]PBR28 fraction in plasma.

Imaging Processing and Analysis.

PET data were acquired in list-mode and time-binned as follows: 6 × 0.5-min, 3 × 1-min, 2 × 2-min, and 22 × 5-min. Data were then reconstructed using the MOLAR algorithm (51), with correction for participant head motion (Polaris Vicra Optical Tracking System, NDI Systems, Waterloo, ON, Canada), attenuation, scatter, randoms, and dead time.

The following automatic anatomical labeling (52)-defined regions of interest (53) were defined in template Montreal Neurological Institute space and transformed first onto each individual participant’s MRI and then onto a 0 to 10 min summed PET image: amygdala, anterior cingulate cortex (ACC), caudate, cerebellum, hippocampus, insula, occipital cortex, orbitofrontal cortex, parietal cortex, putamen, temporal cortex, thalamus, and ventromedial prefrontal cortex (vmPFC).

The Multilinear Analysis method (MA1 with t* = 30 min) with metabolite-corrected arterial input function was used to estimate regional [¹¹C]PBR28 volume of distribution or V_T (54), defined as the equilibrium ratio of tissue to plasma radioactivity (55).

Statistical Analysis.

Data analyses proceeded in six steps. First, demographic, clinical, rs6971, and imaging parameters were compared between the PTSD and control groups using independent-samples t tests and χ² tests. Second, a repeated-measures ANOVA was conducted to examine the association between group (PTSD vs. control) and pre- to post-LPS-induced changes in TSPO availability in an a priori-defined corticolimbic circuit, which was calculated based on the weighted gray matter volume average of TSPO availability in the amygdala, hippocampus, insula, and vmPFC. Group and rs6971 genotype were entered as between-subject factors and TSPO availability pre- and post-LPS administration as within-subject variables. Third, planned secondary repeated-measures ANOVAs were then conducted to examine pre- to post-LPS changes in the four brain regions that comprise this circuit. Fourth, to determine whether certain PTSD symptom clusters were associated with changes in TSPO availability pre- and post-LPS administration in the PTSD group, we conducted a multivariable linear regression that included intrusion, avoidance, negative alterations in cognitions and mood, and alterations in arousal and reactivity entered as independent variables and LPS-induced changes in TSPO availability as the dependent variable. Fifth, a series of repeated-measures ANOVAs were conducted to explore changes in cytokine levels and sickness symptoms over time, as described above. Sixth, exploratory Spearman rank correlations were computed for the available dataset (a subset of both group samples) and stratified by group (PTSD and control) to examine associations between pre- to post-LPS changes in CRP levels and TSPO availability.

Discussion

This study provides direct evidence of suppressed central immune response to an acute stressor in living individuals with PTSD. Direct measurement of the central response is important given previous evidence demonstrating PTSD biology and pathology often deviate from expectations (56–59), particularly when examining the links between cortisol and inflammation (59, 60). Here, we found a lower magnitude LPS-induced increase in TSPO availability in individuals with PTSD relative to controls, indicating a suppressed neuroimmune response. Using a cross-sectional study design, we previously found evidence of lower microglial levels, indicated by lower TSPO availability in PTSD individuals vs. controls, which was associated with greater severity of PTSD symptoms (13). We now extend these findings to suggest suppressed microglial function in people with PTSD following LPS administration. In addition, while there was a smaller magnitude LPS-induced change in GM-CSF in the PTSD group, the changes in other cytokines examined and severity of sickness symptoms did not differ between groups. These findings indicate a crucial role for neuroimmune dysfunction in the pathophysiology of PTSD.

Our previous finding of lower baseline TSPO availability in individuals with PTSD (13) challenged the idea of a heightened neuroimmune system as central to stress-related pathophysiology; however, our finding was cross-sectional in nature and thus did not provide direct insight into the dynamics of this system. Examining the within-subject response to an acute stressor, such as LPS, provides direct evidence of a neuroimmune function deficiency. This dysfunction may contribute to the pathophysiology of PTSD, including the high comorbidity between PTSD and immune-related illnesses such as myocardial infarction, stroke, and vascular disease (8, 37, 61, 62). Lower TSPO at baseline and a lower magnitude TSPO response to a stressor may also indicate a problem in TSPO function itself. Importantly, the magnitude of TSPO reduction following LPS administration was significantly larger than the baseline between-group differences. Furthermore, no correlation between the baseline and change following LPS was observed, indicating these two are not directly related in either the control or PTSD group. TSPO are located on the outer mitochondrial membrane and are thought to play a fundamental role in cellular energy metabolism, mitochondria function, and innate immune microglia processes (63). Importantly, TSPO deficiency was shown to inhibit microglial activation induced by LPS and suppress mitochondrial function resulting in overall microglial metabolic deficits (64). Though the current finding was noted in the entire prefrontal-limbic circuit, it is important to note that three out of four individual regions were statistically significant, with the largest LPS-induced TSPO change observed in the hippocampus. This finding may be related in part to the greater relative vulnerability of the hippocampus in the brain to injury (65), but is also of particular interest given its role in PTSD (66), and memory formation and reconsolidation (67).

Another notable finding in this study is that greater severity of anhedonic symptoms in the PTSD group was associated with a more suppressed neuroimmune response. This finding resonates with our previous finding that lower levels of microglia were associated with greater severity of PTSD symptoms. Reductions in microglia levels at baseline and in response to stress likely inhibit neuronal resilience and even response to antidepressant treatment, especially as many rely on microglial release of brain-derived neurotrophic factor (BDNF) and cytokines. For example, in rodents, inhibition of microglia blocks the antidepressant effects of ketamine (68, 69). Microglial function and promotion of neurotrophic factors are therefore both essential for a positive response to either an insult or to treatment. Overall, our findings suggest that greater severity of PTSD may be associated with both suppressed microglia levels and function and again highlight that therapeutic interventions targeted at restoring neuroimmune function may be beneficial.

Though we observed these differences centrally, we did not see significant group differences in most peripheral biomarkers studied or in self-reported sickness symptoms in response to the LPS challenge. The notable exception was in the LPS-induced response of GM-CSF, which was significantly lower in people with PTSD vs. controls. This is intriguing, as GM-CSF is the primary cytokine to increase peripheral macrophage proliferation at the initiation of an immune response and is the primary driver of monocytes across the blood–brain barrier. Furthermore, this cytokine is highly expressed in microglia and macrophages (70). Interestingly, suppressed monocyte invasion has been correlated with reductions in microglial activity (71). GM-CSF is a profound activator of the peripheral and central immune response and necessary for adequate immune function but has a significantly suppressed response in individuals with PTSD.

There were no group differences observed in peripheral levels of proinflammatory cytokine IL-1β, which is a primary product of many peripheral inflammatory signaling pathways. Though differing findings have been reported in the literature, meta-analyses have found that greater severity of PTSD symptoms is associated with higher levels of IL-6, (72) (a precursor to CRP, discussed below) and IL-1β relative to trauma-exposed individuals without PTSD and healthy controls (57, 73, 74). Furthermore, individuals with PTSD routinely have lower cortisol levels than those without PTSD symptoms, resulting in a hyperinflammatory baseline state due to the reduction in cortisol-suppressed inflammation (75). However, on an individual cytokine level, findings among individuals with PTSD have been mixed. An inverse association between trauma exposure and TNF-α has been reported (76) but not with PTSD (57). In addition, we found that individuals with PTSD did not have significantly different sickness symptoms in response to LPS as compared to what was experienced by control individuals (SI Appendix, Table S3). No group difference in sickness symptoms is supported by the lack of group differences in the proinflammatory cytokines.

Higher blood levels of CRP, an acute phase reactant protein in the inflammatory pathway, are linked with exposure to trauma (77) and an increased risk of developing PTSD (78) or worsening PTSD symptoms (79, 80). Previously, we found higher levels of CRP in people with PTSD relative to controls, which were associated with greater severity of PTSD symptoms, and lower TSPO availability (13). In this study, we add that there is an inverse relationship between the change in CRP and the change in TSPO availability, both following LPS administration, in a subset of both groups. A bigger LPS-induced increase in CRP was associated with a smaller LPS-induced increase in TSPO. Though this correlation is in a subset of the total sample due to limitations with CRP sample collection, the findings were consistent in both groups and stronger in the PTSD group. The inverse relationship between CRP and TSPO availability suggests that the central and peripheral immune systems respond differently to an immune stimulus and may indicate a negative feedback loop between the peripheral and central immune systems. It is intriguing that this association appears to hold in both PTSD and control groups, indicating that this inverse relationship between central and peripheral systems is not PTSD specific. These findings indicate a need to further probe the relationships between central and peripheral immune responses.

It is possible that in people with long-standing PTSD, the central immune response becomes more blunted over time. Indeed, in the current sample, the average length of time since exposure to index traumas was 10 y, and in most (13/15) individuals, the event occurred more than 5 y ago. Interestingly, preclinical studies demonstrate that a history of stress is associated with suppressed LPS-induced microglial activation (81). One potential mechanism is through repeated exposure to endogenous LPS, which is released by the body in times of stress to trigger an inflammatory response that includes upregulation of peripheral and central proinflammatory cytokines. This response starts in the gut but triggers subsequent effects in the central nervous system through microglia-dependent mechanisms. However, preclinical studies demonstrate that microglia with chronic exposure to LPS become less responsive to its inflammatory effects (82). Individuals with PTSD have been shown to have an altered gut microbiota including elevated endogenous LPS (83), which over time may contribute to a blunted immune response. This is supported by preclinical (84) and clinical studies (85, 86) demonstrating that stress can lead to a “leaky gut” allowing LPS to enter the circulatory system (87). The gut–brain axis modulates aspects of the macrophage/microglial response to immune stressors as well as triggering these systemic immune responses. As noted in prior studies, repairing the gut (83) and subsequent gut–brain disequilibrium may aid in reparation of the immune system in PTSD (88).

There are several strengths and limitations of this study that can inform future research. A notable strength is that this study is a clinical application of our previously developed PET paradigm (38) to examine dynamic changes in neuroimmune function. Our current study highlights that we can probe the neuroimmune system in people with PTSD to inform our understanding of central-peripheral relationships and it lays the foundation for longitudinal studies that may track recovery of microglial function. Limitations include the use of a single dose of LPS (1 ng/kg). It is possible that a lower dose may elicit more antidepressant effects (23, 82) whereas a higher dose may allow for greater between-group differentiation in peripheral sickness symptoms. An additional limitation is our lack of systematic measurement of cortisol levels, which may be altered by LPS and are known to be differentially regulated between groups (56). For example, we have previously demonstrated higher brain levels of a cortisol marker, 11β-HSD1, in people with PTSD vs. controls (89), which may indicate higher brain cortisol levels. We previously concluded this may be a compensatory upregulation in response to lower peripheral cortisol levels in those with PTSD (57, 62). However, further studies are needed to examine within-subject associations between peripheral and central cortisol and immune systems in PTSD.

In conclusion, the results of the present study provide evidence demonstrating that PTSD is associated with dynamic suppression of neuroimmune function. They further demonstrate a relationship between anhedonic symptoms of PTSD and neuroimmune suppression, which suggests that restoring neuroimmune function may potentially help mitigate these symptoms and serve as a treatment for PTSD. Recent studies have focused on neuronal disconnection, synaptic pruning, and plasticity as targets for novel therapeutics such as ketamine (90, 91) and most recently MDMA (92) for synaptic reorganization following fear memory processing. Though these are promising treatments, results of the present study suggest that pharmacotherapies targeting TSPO and microglial and neuroimmune system function may be instrumental in treating PTSD, especially since microglia are responsible for many of the neural adaptations noted in prior studies (93), such as synaptic pruning.

Supplementary Material

Appendix 01 (PDF)

Data, Materials, and Software Availability

Anonymized (human image files) data have been deposited in NIMH NDA (Imaging Microglial Activation in PTSD with PET #2713) (94). All other data are included in the manuscript and/or SI Appendix.