Association between resting-state functional connectivity of amygdala subregions and peripheral pro-inammation cytokines levels in bipolar disorder

Background: The pathophysiological mechanisms of mood disorders including bipolar disorder (BD) are not completely known, and systemic inammation and immune dysregulation are considered as risk factors of it. Previous neuroimaging studies have proved metabolic, structural and functional abnormalities of the amygdala in BD patients, suggesting the vital role of amygdala in BD patients. This study aimed to test the underlying neural mechanism of inammation-induced functional connectivity (FC) in the amygdala subregions of BD patients. Methods: Resting-state functional MRI (rs-fMRI) was used to delineate the amygdala FC from two pairs of amygdala seed regions (the bilateral lateral and medial amygdala) in 51 unmedicated BD patients and 69 healthy controls (HCs). The levels of pro-inammatory cytokines including interleukin (IL)-1β, IL-6 and tumor necrosis factor (TNF)-α were measured in the serum. The correlation between abnormal levels of pro-inammatory cytokines and FC values were calculated in BD patients. Results: The BD group exhibited decreased FC between the right medial amygdala and bilateral medial frontal cortex (MFC), and decreased FC between the left medial amygdala and the left temporal pole (TP), right orbital inferior frontal gyrus compared with HCs. The BD patients had higher levels of TNF-α than HCs. Correlation analysis showed negative correlation between the TNF-α level and abnormal FC of the right medial amygdala-bilateral MFC; and negative correlation between TNF-α levels and abnormal FC of the left medial amygdala-left TP in BD group. Conclusions: These ndings suggest that dysfunctional and immune dysregulation between the amygdala and the frontotemporal circuitry might play a critical role in the pathogenesis of BD.


Background
Bipolar disorder (BD) is a chronic, recurrent and disabling psychiatric illness that causes cognitive impairment and dysfunction and reduces quality of life, and is associated with high morbidity and mortality [1,2]. Early and accurate diagnosis of BD is di cult in clinical practice, and it is often misdiagnosed as unipolar depression as the onset of BD is commonly characterized by nonspeci c symptoms, mood lability or a depressive episode. Although it has been studied for over decades, the pathophysiology of BD remained unclear. Dysfunction of innate immune system that leads to neuroin ammation has been considered as risk factor in the pathophysiology of numerous psychiatric illnesses including BD [3][4][5][6].
Resting-state functional magnetic resonance imaging (rs-fMRI) is a primary noninvasive neuroimaging technique that can re ect the changes of brain function status and resting state functional connectivity (rs-FC), and is a fundamental tool in re ecting the correlations of activities between different brain regions, helping to understand brain regions with altered connectivity and function in mental disorders [7,8]. Previous neuroimaging studies have reported that the amygdala abnormity, including reduced gray matter volume [9], broken white matter integrity, decreased FC [10] and lower activation [11], ais Page 4/23 associated with the pathophysiology of BD. The amygdala is a complex consisting of structural and functional heterogeneous nuclei, tucked in the medial temporal lobe and comparatively small in size. It plays a central role in regulating emotions and mediating fear responses [12,13]. The amygdala complex is grouped into three distinct subregions cytoarchitectonically: centromedial, basolateral, and super cial [12,14]. As there several studies that discussed different functions of centromedial (including the central and medial nuclei) and basolateral (including the lateral, basolateral, basomedial, and basoventral nuclei) amygdala, but not the super cial region [14][15][16], this study focused on the centromedial and basolateral subregions that are similar to the medial and lateral masks according to the Brainnetome atlas [14]. The lateral amygdala receives sensory information and involves assessment of emotional content of sensory information, while the medial amygdala is regarded as the major output station of the entire amygdala and involves enhancement of attention allocation and determine the salience of input [13,17,18].
Existing studies have put forwarded the changes in amygdala FC that are related to BD accompanied by the frontal lobe (including the dorsolateral prefrontal cortex [19], prefrontal cortex [20], and inferior frontal gyrus [IFG] [21]), supplementary motor area [22] and anterior hippocampus [23]. Among these studies, only one study used the amygdala subregions FC, but through region of interest (ROI)-to-ROI analyses instead of whole-brain approach based on subregions seed masks of each subregion [23]. However, the functional connectivity between the amygdala subregions and the brain areas has not been examined in detailed manner in BD.
Cytokines act as regulators of host responses to infection, in ammation, trauma, and immune responses [24]. During the process of in ammation, proin ammatory cytokines access the central nervous system and interact with cytokine network in the brain to virtually in uence each and every aspect of brain function that is relevant to the behavior including neurotransmitter metabolism, neuroendocrine function, synaptic plasticity, mood regulation, motor activity, motivation, anxiety and panic [25]. Immune system dysfunction leads to the occurrence of neuroin ammation in BD and increasingly implicated in the pathophysiology of this mental disorder [26,27]. Several studies have shown elevated proin ammatory cytokines including but not limited to IL-6, IL-1β, and TNF-α in patients with BD [4,28], particular in acute mania [29][30][31][32] and depressive episodes [29][30][31][32]. Previous meta-analyses have reported that IL-1β, IL-6, and TNF-α are the most common abnormal cytokines in BD patients [3,29,33]. Some MRI studies have revealed abnormal structure and dysfunction in speci c brain regions (such as amygdala, IFG, medial frontal cortex [MFC], and temporal lobe [TP]) and its correlation with IL-1β, IL-6, and TNF-α in the peripheral area, and might contribute to the symptoms of fear, anxiety and emotional disturbances. This might help us to better understand the role of IL-1β, IL-6, and TNF-α, and its relation to the pathophysiology of mood disorder [32,[34][35][36]. A rs-fMRI study revealed that higher IL-6 receptor levels are associated with connectivity abnormalities between the medial prefrontal cortex and amygdala involved in BD [37].
In our study, the whole-brain FC of each amygdala subdivision in BD patients BD and HCs was calculated, and the levels of IL-1β, IL-6, and TNF-α were obtained in the peripheral blood of these participants. This study aimed to explore the neural mechanism of in ammation induced in the amygdala subdivisions FC Page 5/23 in patients with BD. We hypothesized that both the amygdala subdivisions FC and pro-in ammatory cytokines levels are abnormal, and the amygdala dysfunction is linked to the changes in cytokines. The exclusion criteria included patients with other Axis-I psychiatric disorders as assessed by two experienced psychiatrists (Y.J. and S.Z., with 21 and 6 years of experienced clinical psychiatry, respectively) through SCID-P, a history of electroconvulsive therapy, other neurological disorders, any history of organic brain disorder, autoimmune/immune diseases, mental retardation, pregnancy, alcohol/substance abuse, cardiovascular diseases or presence of any concurrent and major physical illnesses as reported by laboratory examination or self-reported by patients in the experienced psychiatrists' interview and when conversing with patients. Those patients who currently took antiin ammatory medications or immunomodulators (i.e., statins or metformin) were excluded from the study. At the time of testing, all patients were either medication-naive, or were not medicated for at least six months. In addition, 70 right-handed healthy controls (HCs) were recruited via local advertisements. They were carefully screened through a diagnostic interview, the Structured Clinical Interview for DSM-V Nonpatient Edition (SCID-NP), to rule out the presence of current or past history of psychiatric illnesses. Further exclusion criteria for HCs include history of psychiatric illnesses in rst-degree relatives, current or past signi cant medical or neurological illnesses. This study was approved by the Ethics Committee of First A liated Hospital of Jinan University, Guangzhou, China. All subjects were right-handed and signed a written informed consent form after full writing and verbal explanation of the study. Two senior clinical psychiatrists have con rmed that all subjects had the ability to participate in the examination.

MR imaging data acquisition and preprocessing
All MRI data were obtained by using a GE Discovery MR 750 3.0T System with an 8-channel phased array head coil. Subjects were scanned in a supine, head-rst position with symmetrically placed cushions on both sides of the head to decrease motion. During the scanning, participants were instructed to relax with their eyes closed without falling asleep; and after the experiment, each participant con rmed not having fallen asleep.

Functional Image Preprocessing
The preprocessing was carried out using the Data Processing Assistant for Resting-State fMRI (DPABI_V3.0, http://restfmri.net/forum/DPABI) [38], and this is based on Statistical Parametric Mapping (SPM12, http://www. l.ion.ucl.ac.uk/spm/). For each subject, the rst 10 images of the rs-fMRI dataset were discarded to ensure steady-state longitudinal magnetization. The remaining 200 images were slicetime corrected initially and then were realigned to the rst image for correcting the inter-TR head motion.
This realignment correction provides a record of the head motion within the rs-fMRI scan. All subjects should not have more than 2 mm maximum displacement in any plane, 2° of angular motion as well as 0.2 mm in the mean frame-wise displacement (FD) [39]. The individual T1 structural images were segmented (white matter, gray matter, and cerebrospinal uid) using a segmentation toolbox. The DARTEL toolbox was then used to create a study speci c template for accurate normalization. The resting-state functional images were co-registered with the structural images and transformed into the standard Montreal Neurological Institute (MNI) space, resliced to a voxel size of 3 × 3 × 3 mm³ resolution and smoothened using a 6 mm full width at half maximum (FWHM) Gaussian kernel. The data were removed the linear trend and passed through the band-pass lter of 0.01-0.1 Hz. The global mean signal regression is commonly used in the eld of rs-fMRI, and it could afford decreased dependencies on head motion [40] and increased tissue sensitivity [41]. The signals from white matter and cerebrospinal uid were regressed out to reduce both respiratory and cardiac effects [42]. The whole brain functional connectivity estimates were in uenced by head motion and the head motion could be misinterpreted as neuronal effects easily [43]. Several spurious covariates and their temporal derivatives were then regressed out from the time course of each voxel, including the signals of the brain global mean, white matter, and cerebrospinal uid as well as the Friston-24 parameters of head motion (including the 6 head motion parameters, 6 head motion parameters at one time point before, and the 12 corresponding squared items) [44].

Resting-state Functional Connectivity
Consistent with the previous studies of amygdala FC [14], four seed masks (the bilateral lateral amygdala and medial amygdala) were selected using Brainnetome atlas (http://atlas.brainnetome.org/bnatlas.php) [45]. The time course of the masks was correlated against all other voxels within the whole brain.
Individual rs-FC maps of the lateral amygdala, and medial amygdala were generated by calculating the Pearson's correlation coe cients between the mean time series of the masks and the time series of each voxel in the whole brain. The subject-level correlation maps were then converted to the z-value maps using Fisher's transformation to improve the normality. For all the subjects, four z-score maps that represent the intrinsic FC of the four amygdala masks were nally obtained.

Pro-in ammatory Cytokines Measures
Blood samples from BD patients and HCs were obtained in the morning under fasting condition, followed by abstaining by alcoholic beverages for at least one day prior to testing and then were processed (then frozen) by the technicians. Through venipuncture, four milliliters of fasting blood were drawn from each subject into a sterile vacuum tube (Becton & Dickinson 367812; Becton Dickinson, Franklin Lakes, NJ, USA), clotted for 30 min, and then centrifuged at 1,000 g for 15 min at 4 ℃. The serum was centrifuged again (3,000 rpm for 10 min at 4 ℃), and stored at -80 ℃ until use. Before testing, the serum should be melted on ice at low temperature, and the testing process was carried out at room temperature in accordance with the requirements of the kit to ensure effective combining of antigen and antibody. The levels of pro-in ammatory cytokines, including IL-6, IL-1β, and TNF-α were determined from the serum by the Bio-Plex Pro Human Cytokine Assay kit (Bio-Rad) according to the manufacturer's directions using a Bio-Plex 200 array reader (Bio-Rad). Bio-Plex Manager Software, version 6.1 was used for data acquisition (Bio-Rad). The intra-assay coe cients of variation (CVs) were 3.58% for IL-1β, 2.97% for IL-6, and 3.66% for TNF-α. A reliable detection range was provided with the lower limit of quantitation (LLOQ) and the upper limit of quantitation (ULOQ). The detection ranges (LLOQ-ULOQ) were 0.057-1027.514 pg/ml for IL-1β, 0.11-1997.01 pg/ml for IL-6, and 0.69-13336.72 pg/ml for TNF-α. No immune variables were below the limits of assay detection.

Statistical analysis
Independent-sample t-test (normal variable) and Mann-Whitney U test (non-normal variable) were used to compare the demographic data (except gender) and levels of serum pro-in ammatory cytokines between the two groups with SPSS 19.0 software (SPSS, Chicago, IL, USA). A chi-squared test was performed to compare gender distribution. All tests were two-tailed, and p < 0.05 was considered to be statistically signi cant. One-sample t-test was performed on z-score maps for each mask to demonstrate withingroup FC spatial distribution of each seed for BD patients and HCs, and the signi cant level was set at p < 0.05 (uncorrected). Then the two-sample t-test was performed to assess signi cant differences of the whole brain FC in each region between BD patients and HCs within the union mask of one-sample t-test results of both groups. Age, gender, years of education and the mean FD were included as nuisance covariates in the whole brain FC and cytokine group comparisons. Gaussian random eld (GRF) theory was used for cluster-level multiple comparison correction (voxel p value < 0.001; cluster p value < 0.05, GRF corrected). When statistically signi cant group differences were observed in the brain regions and pro-in ammatory cytokines levels, Spearman correlation analysis was performed to compute the correlation between FC values and pro-in ammatory cytokine levels both in BD and HC groups. Also, Spearman correlation coe cients were calculated between the clinical variables and abnormal FC values, and abnormal pro-in ammatory cytokines levels in the BD group. These clinical variables included onset age of illness, number of episodes, duration of illness, 24-item HDRS scores and YMRS scores. Further, multiple linear regression was adopted to model the relationship between FC values and proin ammatory cytokines levels after adjusting for potential confounders including demographic variables, number of cigarettes, body mass index (BMI), and BD subgroup variable ("1" for BD I and "2" for BD II). The signi cant level was set as p < 0.05.

Demographic and clinical characteristics
The demographic and clinical data of all study participants were presented in Table 1. Four patients in BD (three BD I and one BD II) group and one HC were excluded from further analyses because of excessive head motion. Finally, 51 patients with BD (12 BD and 39 BD ) and 69 HCs were included in the analysis.
The 24-item HDRS score was 23.14 (9.94), and the YMRS score was 8.57 (9.13) for the BD group. There were no signi cant differences between the BD group and the HCs group in gender, age, BMI, or FD parameters (p > 0.05).  (Fig. 1).

FC changes between-group
Decreased FC between the right medial amygdala and the bilateral MFC and decreased FC between the left medial amygdala and the left TP, the right orbital IFG were observed in the BD group when compared to the HC group. There was no aberrant FC between the right/left lateral amygdala and the cerebral regions in the BD group was observed when compared to the HC group (Table 2 and Fig. 2).

Group Differences Of Pro-in ammatory Cytokines Levels
The levels of IL-6, IL-1β, and TNF-α of the two groups were shown in Table 1. The measurements of serum pro-in ammatory cytokines levels were completed in 51 BD patients and 69 HCs. No signi cant differences were observed in sex, age, and FD parameter between the two groups. Compared with HCs, BD patients showed increased TNF-α levels (p < 0.05). No statistical differences of IL-6 and IL-1β were observed between the two groups (p > 0.05) ( Table 1).

Correlation Analyses
For BD patients, the FC values in the right medial amygdala-bilateral MFC showed a negative correlation with TNF-α (r = -0.314, p = 0.025) level; and the FC values in the left medial amygdala-left TP showed a negative correlation with TNF-α (r = -0.330, p = 0.018) level (Fig. 3). For HC group, no signi cant correlations between the FC values in the right medial amygdala-bilateral MFC and TNF-α (r = 0.048, p = 0.696), and between the FC values in the left medial amygdala-left TP and TNF-α (r < 0.001, p = 0.997) were observed.
For the correlations between FC values and clinical variables, including onset age of illness, number of episodes, duration of illness, 24-item HDRS score, and YMRS score, and between levels of TNF-α and clinical variables in BD patients, no signi cant correlation of survival was observed when Spearman correlation analysis was performed (p > 0.05). After

Discussion
We performed whole brain amygdala subregions rs-FC and measured the levels of pro-in ammatory cytokines including IL-6, IL-1β, and TNF-α in the serum of BD patients and HCs. The primary ndings of this study were as follows: (i). decreased FC was observed between the right medial amygdala and the bilateral MFC, and decreased FC between the left medial amygdala and the left TP, the right orbital IFG in BD group; (ii). increased TNF-α levels were observed in BD patients than that in HCs; (iii). negative correlation was observed between the TNF-α levels and abnormal FC in the right medial amygdalabilateral MFC; and negative correlation between the TNF-α levels and abnormal FC in the left medial amygdala-left TP was observed in BD patients. To the best of our knowledge, this is the rst study to assess the association between the amygdala subregion rs-FC and in ammatory cytokines levels in BD patients, and this assists us to raise awareness with regard to BD from the perspective of intrinsic brain activity and immune system.
In the present study, patients with BD showed decreased FC between the right medial amygdala and the bilateral MFC, suggesting a disturbed connectivity between the amygdala and MFC in BD patients. The MFC has been assumed to play a role in emotional regulation by generating preventive responses preceding the emotional events [46]. Previous rs-fMRI studies have shown decreased FC of the MFC and IFG in patients with BD, and this has shed light on the underlying pathophysiology and presentation of impaired emotion regulation and cognitive control [47][48][49][50]. Furthermore, another rs-fMRI study using voxel-mirrored homotopic connectivity (VMHC) has investigated that lower VMHC in the MFC in BD patients. VMHC, is a common rs-fMRI method, that measures the functional connection between each voxel in one hemisphere and the mirror voxel in the other hemisphere, suggesting substantial impairment of interhemispheric coordination in BD patients [51]. Study of regional function in BD patients showed decreased activity in the MFC [52] and IFG regions [53]. Recent task-based fMRI investigation found that patients with BD had neural dysfunction in the MFC, implicating appraisal and expression of emotions [54]. Vai et al have reported that successful antidepressant chronotherapeutics can enhance fronto-limbic connectivity (MFC-amygdala) in emotional processing neural responses in BD patients [55]. Postmortem studies have revealed dendritic atrophy, oligodendrocyte loss, and gray matter loss in the MFC in BD patients, raising the possibility of abnormal synaptic interactions between the mPFC, amygdala, and OFC among other regions. This may contribute to the disturbances in emotional regulation characteristics of BD [56,57]. Taken together, our ndings of decreased FC between the right medial amygdala and the bilateral MFC might show an abnormal brain integration in default processing of emotional information, displaying the clinical sign of emotional dysregulation in patients with BD.
The TP covering the anterior-most part of the temporal lobe, like a cap lies between the orbital frontal cortex and the amygdala, receives and sends the connections to both regions, playing an important role in socioemotional processing [58]. In our study, decreased FC was found between the left medial amygdala and the left TP, suggesting disrupted connectivity between the amygdala and the TP. Another fMRI study of BD also exhibited decreased FC from the amygdala to the TP, and this was consistent with our results [59]. Other several rs-fMRI studies have shown decreased FC of the TP in participants with psychiatric symptoms [60][61][62]. Other studies have shown TP abnormalities, including decreased gray matter density [63], reduced gray matter volume [64,65], lower mean diffusivity (re ects the microstructural integrity of white matter) [66], and decreased degree centrality (re ects the functional connections) [67] in BD patients. This suggested that the abnormal structure and connectivity alterations might underlie the mood uctuations and cognitive and emotional dysregulation that characterize BD. One task-based fMRI research on viewing affective or neutral faces tasks showed that both euthymic and manic BD patients have displayed reduced activation than HCs in the TP [68]. A magnetoencephalography study investigated implicit emotional tasks that showed abnormal activation in the temporal cortices in BD [69]. Thus, our ndings of de cient FC between the amygdala and the TP might be related to emotional dysregulation of BD, explaining the neurobiological processes that underlie impaired emotion processing in BD.
Cytokines play a signi cant role in stimulating and maintaining the immune processes, and strong evidence has indicated that TNF-α could mediate the clinical presentation of BD and its neurobiological manifestations [70]. TNF-α has been described as a major in ammatory mediator, that promote the production of other cytokines such as IL-6 and C-reactive protein [71]. In this study, increased TNF-α levels were found in BD patients when compared to those in HCs, and this was consistent with most of the previous studies [3,72]. Moreover, a negative correlation was found between TNF-α levels and abnormal FC of the right medial amygdala-right MFC, and abnormal FC of the left medial amygdala-left TP in BD patients. Chronic in ammatory responses in patients with mood disorders can lead to downstream biological damage, causing damage to BD patients through upcoming actions and effects the neurotransmitter and neuropeptide systems [73]. Karabulut et al have found that TNF-α levels were correlated with Montgomery-Asberg Depression Rating Scale scores and Clinical Global Impression Scale scores in BD patients, suggesting the progression at later stages of BD and possible association with gliosis and neuronal loss [74]. According to an animal study, chronic unpredictable mild stress exposure induced microglia activation and overexpression of cytokine TNF-α within the MFC, and the effects were paralleled with neuronal structural changes. In contrast, chronic administration of antagonists has signi cantly ameliorated the dysregulation of neuronal structure and biochemical parameters within the MFC, and displayed depression-like behaviors [75]. The cytokine (TNF-α) has been shown to cross the rodent blood-brain barrier, rapidly inducing the correlated-sensitive genes within the brain, and altering the neuronal ring rates within the amygdala and other brain regions [76]. Recent literatures have found that in ammatory stimulation leads to changes in the amygdala activity and connectivity, and that the production of stress-induced in ammatory cytokines is related to the enhancement of amygdala reactivity [77,78]. A rs-fMRI study has found that the FC between striatum and prefrontal cortices showed negative association with serum TNF-α levels in major depressive disorder patients [79]. Another fMRI study investigated that elevated levels of depression and serum cytokine levels were associated with increased regional activity in the medial frontal lobe [80]. Evidence was obtained for distinct mechanisms by which the MFC function interacts with the in ammatory cytokine TNF-α, and this can partly explain the chronic in ammatory of impaired mood regulations in BD. This might in turn serve as a target for antiin ammatory treatment strategies to improve emotion de cits in patients with increased in ammation, including BD [75,80]. However, transient elevation of in ammation might be a part of adaptation to the disease. For patients with BD, more research is warranted to determine if the higher TNF-α level and decreased FC of the amygdala-MFC, and -TP were associated with illness or pathogenesis to BD.

Limitation
There are several limitations in this study that should be acknowledged. Firstly, patients with BD in mixed subtypes (including BD and BD ) were examined, and it is still unclear as to whether the altered FC and TNF-α is shared by all episodes of the disease or speci c to some subtypes of BD. Secondly, this study lacked longitudinal data and our understanding with regard to the functional role of individual cytokines is also limited. Thirdly, the ndings of TNF showed no survival after applying Bonferonni correction.
Fourthly, in order to avoid excessive measurement costs, all cytokines samples were measured in one experiment, but this may not achieve high reliability and sensitivity of the retest. Fifthly, all smoking information of HCs, that affects the immune system was missed in this study. Finally, a larger sample size was needed to conclude the alterations of TNF-α levels in patients with BD. Also, a structural study is also warranted to con rm the results, of determining whether the changes of FC and TNF-α levels in BD patients has structural basis.

Conclusions
Our ndings of decreased FC between the right medial amygdala-right MFC, and between the left medial amygdala-left TP might re ect the disrupted cortico-amygdala connectivity in patients with BD, which might show correlation with immune dysregulation in BD patients to some extent. These ndings suggest the importance of dysfunctional and immune-dysregulated cortico-amygdala circuitry in the pathogenesis of BD. The FC patterns of the lateral amygdala, medial amygdala in the BD patients and HCs (p < 0.05, uncorrected). The color bar represents functional connection. The T value indicated the intensity of the signi cant difference which was obtained by one-sample t-test. FC, functional connectivity; BD, bipolar disorder; HCs, healthy controls.

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