Correction: Do multiple experimenters improve the reproducibility of animal studies?

[This corrects the article DOI: 10.1371/journal.pbio.3001564.].

Do multiple experimenters improve the reproducibility of animal studies?

The credibility of scientific research has been seriously questioned by the widely claimed "reproducibility crisis". In light of this crisis, there is a growing awareness that the rigorous standardisation of experimental conditions may contribute to poor reproducibility of animal studies. Instead, systematic heterogenisation has been proposed as a tool to enhance reproducibility, but a real-life test across multiple independent laboratories is still pending. The aim of this study was therefore to test whether heterogenisation of experimental conditions by using multiple experimenters improves the reproducibility of research findings compared to standardised conditions with only one experimenter. To this end, we replicated the same animal experiment in 3 independent laboratories, each employing both a heterogenised and a standardised design. Whereas in the standardised design, all animals were tested by a single experimenter; in the heterogenised design, 3 different experimenters were involved in testing the animals. In contrast to our expectation, the inclusion of multiple experimenters in the heterogenised design did not improve the reproducibility of the results across the 3 laboratories. Interestingly, however, a variance component analysis indicated that the variation introduced by the different experimenters was not as high as the variation introduced by the laboratories, probably explaining why this heterogenisation strategy did not bring the anticipated success. Even more interestingly, for the majority of outcome measures, the remaining residual variation was identified as an important source of variance accounting for 41% (CI95 [34%, 49%]) to 72% (CI95 [58%, 88%]) of the observed total variance. Despite some uncertainty surrounding the estimated numbers, these findings argue for systematically including biological variation rather than eliminating it in animal studies and call for future research on effective improvement strategies.

Social Defeat Modulates T Helper Cell Percentages in Stress Susceptible and Resilient Mice.

Altered adaptive immunity involving T lymphocytes has been found in depressed patients and in stress-induced depression-like behavior in animal models. Peripheral T cells play important roles in homeostasis and function of the central nervous system and thus modulate behavior. However, the T cell phenotype and function associated with susceptibility and resilience to depression remain largely unknown. Here, we characterized splenic T cells in susceptible and resilient mice after 10 days of social defeat stress (SDS). We found equally decreased T cell frequencies and comparably altered expression levels of genes associated with T helper (Th) cell function in resilient and susceptible mice. Interleukin (IL)-17 producing CD4+ and CD8+ T cell numbers in the spleen were significantly increased in susceptible mice. These animals further exhibited significantly reduced numbers of regulatory T cells (Treg) and decreased gene expression levels of TGF-ß. Mice with enhanced Th17 differentiation induced by conditional deletion of PPARγ in CD4+ cells (CD4-PPARγKO), an inhibitor of Th17 development, were equally susceptible to SDS when compared to CD4-PPARγWT controls. These data indicate that enhanced Th17 differentiation alone does not alter stress vulnerability. Thus, SDS promotes Th17 cell and suppresses Treg cell differentiation predominantly in susceptible mice with yet unknown effects in immune responses after stress exposure.

Environmental enrichment and physical exercise revert behavioral and electrophysiological impairments caused by reduced adult neurogenesis.

It is well known that adult neurogenesis occurs in two distinct regions, the subgranular zone of the dentate gyrus and the subventricular zone along the walls of the lateral ventricles. Until now, the contribution of these newly born neurons to behavior and cognition is still uncertain. The current study tested the functional impacts of diminished hippocampal neurogenesis on emotional and cognitive functions in transgenic Gfap-tk mice. Our results showed that anxiety-related behavior evaluated both in the elevated plus maze as well as in the open field, social interaction in the sociability test, and spatial working memory in the spontaneous alternation test were not affected. On the other hand, recognition and emotional memory in the object recognition test and contextual fear conditioning, and hippocampal long-term potentiation were impaired in transgenic mice. Furthermore, we evaluated whether environmental enrichment together with physical exercise could improve or even restore the level of adult neurogenesis, as well as the behavioral functions. Our results clearly demonstrated that environmental enrichment together with physical exercise successfully elevated the overall number of progenitor cells and young neurons in the dentate gyrus of transgenic mice. Furthermore, it led to a significant improvement in object recognition memory and contextual fear conditioning, and reverted impairments in hippocampal long-term potentiation. Thus, our results confirm the importance of adult neurogenesis for learning and memory processes and for hippocampal circuitry in general. Environmental enrichment and physical exercise beneficially influenced adult neurogenesis after it had been disrupted and most importantly recovered cognitive functions and long-term potentiation. © 2016 Wiley Periodicals, Inc.

SMAD dependent signaling plays a detrimental role in a fly model of SMARCB1-deficiency and the biology of atypical teratoid/rhabdoid tumors.

Atypical teratoid/rhabdoid tumors (ATRT) are highly malignant brain tumors arising in young children. The majority of ATRT is characterized by inactivation of the chromatin remodeling complex member SMARCB1 (INI1/hSNF5). Little is known, however, on downstream pathways involved in the detrimental effects of SMARCB1 deficiency which might also represent targets for treatment. Using Drosophila melanogaster and the Gal4-UAS system, modifier screens were performed in order to identify the role of SMAD dependent signaling in the lethal phenotype associated with knockdown of snr1, the fly homolog of SMARCB1. Expression and functional role of human homologs was next investigated in ATRT tumor samples and SMARCB1-deficient rhabdoid tumor cells. The lethal phenotype associated with snr1 knockdown in Drosophila melanogaster could be shifted to later stages of development upon additional knockdown of several decapentaplegic pathway members including Smox, and Med. Similarly, the transforming growth factor beta (TGFbeta) receptor type I kinase inhibitor SB431542 ameliorated the detrimental effect of snr1 knockdown in the fruit fly. Examination of homologs of candidate decapentaplegic pathway members in human SMARCB1-deficent ATRT samples revealed SMAD3 and SMAD6 to be over-expressed. In SMARCB1-deficent rhabdoid tumor cells, siRNA-mediated silencing of SMAD3 or SMAD6 expression reduced TGFbeta signaling activity and resulted in decreased proliferation. Similar results were obtained upon pharmacological inhibition of TGFbeta signaling using SB431542. Our data suggest that SMAD dependent signaling is involved in the detrimental effects of SMARCB1-deficiency and provide a rationale for the investigation of TGFbeta targeted treatments in ATRT.

Deficiencies of the T and natural killer cell system in major depressive disorder: T regulatory cell defects are associated with inflammatory monocyte activation.

BACKGROUND: In a previous study, we found an up-regulated inflammatory monocyte gene expression profile in major depressive disorder (MDD) patients aged ⩾ 28 years and a down-regulated inflammatory gene expression profile in MDD patients aged<28 years. In the same sample of patients, we aimed to investigate immune dysregulation in the lymphocyte arm of the immune system, particularly in the context of the described monocyte (de-)activation states. METHODS: From deep frozen leukocytes, circulating percentages of monocytes, lymphocytes, B, T, and natural killer (NK) cells, and various functional subsets of T and T helper (Th) cells (Th1, Th2, Th17, and natural T regulatory cells) were measured in N=50 MDD patients and N=58 age- and gender-matched healthy controls (HC). In addition, serum levels of interleukin (IL)-6, sCD25, IL-7, IL-3, SCF, IGF-BP2, and EGF were evaluated. RESULTS: MDD patients were in general characterized by an impaired maturation of Th2 cells, Th17 cells, and NK cells and by decreased serum levels of IL-7 and sCD25. MDD patients aged ⩾ 28 years additionally exhibited decreased percentages of CD4(+)CD25(high)FoxP3(+) T regulatory cells, next to signs of the above described partial T cell defects. Natural T regulatory cells were inversely associated with the pro-inflammatory state of the monocytes (r=-.311; p=.034) that characterized this patient subgroup. CONCLUSIONS: Deficiencies of the NK and T (regulatory) cell system and inflammatory monocyte immune activation co-occur as partly interrelated phenomena within the same MDD patients.

S100B Serum Levels Predict Treatment Response in Patients with Melancholic Depression.

BACKGROUND: There is an ongoing search for biomarkers in psychiatry, for example, as diagnostic tools or predictors of treatment response. The neurotrophic factor S100 calcium binding protein B (S100B) has been discussed as a possible predictor of antidepressant response in patients with major depression, but also as a possible biomarker of an acute depressive state. The aim of the present study was to study the association of serum S100B levels with antidepressant treatment response and depression severity in melancholically depressed inpatients. METHODS: After a wash-out period of 1 week, 40 inpatients with melancholic depression were treated with either venlafaxine or imipramine. S100B levels and Hamilton Depression Rating Scale (HAM-D) scores were assessed at baseline, after 7 weeks of treatment, and after 6 months. RESULTS: Patients with high S100B levels at baseline showed a markedly better treatment response defined as relative reduction in HAM-D scores than those with low baseline S100B levels after 7 weeks (P=.002) and 6 months (P=.003). In linear regression models, S100B was a significant predictor for treatment response at both time points. It is of interest to note that nonresponders were detected with a predictive value of 85% and a false negative rate of 7.5%. S100B levels were not associated with depression severity and did not change with clinical improvement. CONCLUSIONS: Low S100B levels predict nonresponse to venlafaxine and imipramine with high precision. Future studies have to show which treatments are effective in patients with low levels of S100B so that this biomarker will help to reduce patients' burden of nonresponding to frequently used antidepressants.

Normal cerebellar development in S100B-deficient mice.

The calcium-binding protein S100B has been shown to support neuron proliferation, migration and neurite growth in vitro, while the significance of S100B for neuronal development in vivo is controversial. We have investigated the effect of S100B deficiency on cerebellar development in S100B knockout mice at an age of 5 and 10 days after birth (P5 and P10). This time range covers important developmental steps in the cerebellum such as granule cell proliferation and migration, as well as dendritic growth of Purkinje cells. Bergmann glial cells contain a particularly high concentration of S100B and serve as scaffold for both migrating granule cells and growing Purkinje cell dendrites. This renders the postnatal cerebellum ideal as a model system to study the importance of S100B for glial and neuronal development. We measured the length of Bergmann glial processes, the width of the external granule cell layer as a measure of granule cell proliferation, the decrease in width of the external granule cell layer between P5 and P10 as a measure of granule cell migration, and the length of Purkinje cell dendrites in wild-type and S100B knockout mice. None of these parameters showed significant differences between wild-type and knockout mice. In addition, wild-type and knockout mice performed equally in locomotor behaviour tests. The results indicate that S100B-deficient mice have normal development of the cerebellum and no severe impairment of motor function.

Clinical characteristics of inflammation-associated depression: Monocyte gene expression is age-related in major depressive disorder.

Increased inflammatory activation might only be present in a subgroup of depressed individuals in which immune processes are especially relevant to disease development. We aimed to analyze demographic, depression, and trauma characteristics of major depressive disorder (MDD) patients with regard to inflammatory monocyte gene expression. Fifty-six naturalistically treated MDD patients (32 ± 12 years) and 57 healthy controls (HC; 31 ± 11 years) were analyzed by the Inventory of Depressive Symptomatology (IDS) and by the Childhood Trauma Questionnaire (CTQ). We determined the expression of 38 inflammatory and immune activation genes including the glucocorticoid receptor (GR)α and GRß genes in purified CD14(+) monocytes using quantitative-polymerase chain reaction (RT-qPCR). Monocyte gene expression was age-dependent, particularly in MDD patients. Increased monocyte gene expression and decreased GRα/ß ratio were only present in MDD patients aged ⩾ 28 years. Post hoc analyses of monocyte immune activation in patients <28 years showed two subgroups: a subgroup with a severe course of depression (recurrent type, onset <15 years) - additionally characterized by panic/arousal symptoms and childhood trauma - that had a monocyte gene expression similar to HC, and a second subgroup with a milder course of the disorder (73% first episode depression, onset ⩾15 years) - additionally characterized by the absence of panic symptoms - that exhibited a strongly reduced inflammatory monocyte activation compared to HC. In conclusion, monocyte immune activation was not uniformly raised in MDD patients but was increased only in patients of 28 years and older.

Monocyte activation, brain-derived neurotrophic factor (BDNF), and S100B in bipolar offspring: a follow-up study from adolescence into adulthood.

OBJECTIVES: There is increasing evidence that both immune and neurochemical alterations are involved in the pathogenesis of bipolar disorder; however, their precise role remains unclear. In this study, we aimed to evaluate neuro-immune changes in a prospective study on children of patients with bipolar disorder. METHODS: Bipolar offspring, from the prospective Dutch bipolar offspring study (n = 140), were evaluated cross-sectionally within a longitudinal context at adolescence, young adulthood, and adulthood. We examined the expression of 44 inflammation-related genes in monocytes, the cytokines pentraxin 3 (PTX3), chemokine ligand 2 (CCL2), and interleukin-1ß (IL-1ß), and brain-derived neurotrophic factor (BDNF) and S100 calcium binding protein B (S100B) in the serum of bipolar offspring and healthy controls. RESULTS: During adolescence, bipolar offspring showed increased inflammatory gene expression in monocytes, high serum PTX3 levels, but normal CCL2 levels. BDNF levels were decreased, while S100B levels were normal. During young adulthood, monocyte activation remained, although to a lesser degree. Serum PTX3 levels remained high, and signs of monocyte migration became apparent through increased CCL2 levels. BDNF and S100B levels were not measured. At adulthood, circulating monocytes had lost their activation state, but CCL2 levels remained increased. Both BDNF and S100B were now increased. Abnormalities were independent of psychopathology state at all stages. CONCLUSIONS: This study suggests an aberrant neuro-immune state in bipolar offspring, which followed a dynamic course from adolescence into adulthood and was present irrespective of lifetime or future mood disorders. We therefore assumed that the aberrant neuro-immune state reflects a general state of vulnerability for mood disorders rather than being of direct predictive value.

Serotonin transporter gene methylation is associated with hippocampal gray matter volume.

BACKGROUND: The serotonin transporter (5-HTT) and the 5-HTTLPR/rs25531 polymorphisms in its gene (SLC6A4) have been associated with depression, increased stress-response, and brain structural alterations such as reduced hippocampal volumes. Recently, epigenetic processes including SLC6A4 promoter methylation were shown to be affected by stress, trauma, or maltreatment and are regarded to be involved in the etiology of affective disorders. However, neurobiological correlates of SLC6A4 promoter methylation have never been studied or compared to genotype effects by means of human neuroimaging hitherto METHODS: Healthy subjects were recruited in two independent samples (N = 94, N = 95) to obtain structural gray matter images processed by voxel-based morphometry (VBM8), focusing on hippocampal, amygdala, and anterior cingulate gyrus gray matter structure. SLC6A4 promoter methylation within an AluJb element and 5-HTTLPR/rs25531 genotypes were analyzed in view of a possible impact on local gray matter volume RESULTS: Strong associations of AluJb methylation and hippocampal gray matter volumes emerged within each sample separately, which in the combined sample withstood most conservative alpha-corrections for the entire brain. The amygdala, insula, and caudate nucleus showed similar associations. The 5-HTTLPR/rs25531 showed no main effect on gray matter, and the effect of methylation rates on hippocampal structure was comparable among the genotype groups CONCLUSIONS: Methylation within the AluJb appears to have strong effects on hippocampal gray matter volumes, indicating that epigenetic processes can alter brain structures crucially involved in stress-related disorders. Different ways of regulating SLC6A4 expression might involve exonization or transcription factor binding as potentially underlying mechanisms, which, however, is speculative and warrants further investigation.

Monoamine oxidase A gene DNA hypomethylation - a risk factor for panic disorder?

The monoamine oxidase A (MAOA) gene has been suggested as a prime candidate in the pathogenesis of panic disorder. In the present study, DNA methylation patterns in the MAOA regulatory and exon 1/intron 1 region were investigated for association with panic disorder with particular attention to possible effects of gender and environmental factors. Sixty-five patients with panic disorder (44 females, 21 males) and 65 healthy controls were analysed for DNA methylation status at 42 MAOA CpG sites via direct sequencing of sodium bisulfate treated DNA extracted from blood cells. The occurrence of recent positive and negative life events was ascertained. Male subjects showed no or only very minor methylation with some evidence for relative hypomethylation at one CpG site in intron 1 in patients compared to controls. Female patients exhibited significantly lower methylation than healthy controls at 10 MAOA CpG sites in the promoter as well as in exon/intron 1, with significance surviving correction for multiple testing at four CpG sites (p≤0.001). Furthermore, in female subjects the occurrence of negative life events was associated with relatively decreased methylation, while positive life events were associated with increased methylation. The present pilot data suggest a potential role of MAOA gene hypomethylation in the pathogenesis of panic disorder particularly in female patients, possibly mediating a detrimental influence of negative life events. Future studies are warranted to replicate the present finding in independent samples, preferably in a longitudinal design.

Dendritic Cells: Neglected Modulators of Peripheral Immune Responses and Neuroinflammation in Mood Disorders?

Affective disorders (AD) including major depressive disorder (MDD) and bipolar disorder (BD) are common mood disorders associated with increased disability and poor health outcomes. Altered immune responses characterized by increased serum levels of pro-inflammatory cytokines and neuroinflammation are common findings in patients with AD and in corresponding animal models. Dendritic cells (DCs) represent a heterogeneous population of myeloid cells that orchestrate innate and adaptive immune responses and self-tolerance. Upon sensing exogenous and endogenous danger signals, mature DCs secrete proinflammatory factors, acquire migratory and antigen presenting capacities and thus contribute to neuroinflammation in trauma, autoimmunity, and neurodegenerative diseases. However, little is known about the involvement of DCs in the pathogenesis of AD. In this review, we summarize the current knowledge on DCs in peripheral immune responses and neuroinflammation in MDD and BD. In addition, we consider the impact of DCs on neuroinflammation and behavior in animal models of AD. Finally, we will discuss therapeutic perspectives targeting DCs and their effector molecules in mood disorders.

Deficiency of the palmitoyl acyltransferase ZDHHC7 impacts brain and behavior of mice in a sex-specific manner.

The palmitoyl acyltransferase ZDHHC7 belongs to the DHHC family responsible for the covalent attachment of palmitic acid (palmitoylation) to target proteins. Among synaptic proteins, its main targets are sex steroid receptors such as the estrogen receptors. When palmitoylated, these couple to membrane microdomains and elicit non-genomic rapid responses. Such coupling is found particularly in cortico-limbic brain areas which impact structure, function, and behavioral outcomes. Thus far, the functional role of ZDHHC7 has not been investigated in this context. To directly analyze an impact of ZDHHC7 on brain anatomy, microstructure, connectivity, function, and behavior, we generated a mutant mouse in which the Zdhhc7 gene is constitutively inactivated. Male and female Zdhhc7-/- mice were phenotypically compared with wild-type mice using behavioral tests, electrophysiology, protein analyses, and neuroimaging with diffusion tensor-based fiber tractography. Zdhhc7-deficiency impaired excitatory transmission, synaptic plasticity at hippocampal Schaffer collateral CA1 synapses, and hippocampal structural connectivity in both sexes in similar manners. Effects on both sexes but in different manners appeared in medial prefrontal cortical synaptic transmission and in hippocampal microstructures. Finally, Zdhhc7-deficiency affected anxiety-related behaviors exclusively in females. Our data demonstrated the importance of Zdhhc7 for assembling proper brain structure, function, and behavior on a system level in mice in a sex-related manner. Given the prominent role of sex-specificity also in humans and associated mental disorders, Zdhhc7-/- mice might provide a promising model for in-depth investigation of potentially underlying sex-specifically altered mechanisms.

Environmental enrichment counteracts Alzheimer's neurovascular dysfunction in TgCRND8 mice.

We and others have recently demonstrated that cognitive and physical stimulation in form of environmental enrichment reduces cerebral beta-amyloid (Abeta) deposition in transgenic mouse models of Alzheimer's disease. This effect was independent from amyloid precursor protein (APP) expression or processing and rather a consequence of enhanced clearance of Abeta. However, the detailed mechanisms remain unclear. In the present study, we show that environmental enrichment in TgCRND8 mice (carrying human APP(Swedish+Indiana)) affect the neurovascular unit by increased angiogenesis and differential regulation of Abeta receptor/transporter molecules, namely up-regulation of LRP1, ApoE and A2M as well as down-regulation of RAGE so that brain to blood Abeta clearance is facilitated. These results suggest a hitherto unknown effect of environmental enrichment counteracting the vascular dysfunction in Alzheimer diseased brain.

Effects of environmental enrichment on exploration, anxiety, and memory in female TgCRND8 Alzheimer mice.

After we could recently demonstrate a beneficial effect of environmental enrichment on AD-like brain pathology in female TgCRND8 mice [Ambrée O, Leimer U, Herring A, Görtz N, Sachser N, Heneka MT, et al. Reduction of amyloid angiopathy and Abeta plaque burden after enriched housing in TgCRND8 mice: involvement of multiple pathways. Am J Pathol 2006;169:544-52] the present study focuses on the behavioural effects of environmental enrichment with special emphasis on learning and memory performance in this AD model. In the first experiment spontaneous exploration, locomotor activity and anxiety-related behaviour were assessed as the performance in learning tasks can be biased substantially by exploratory behavioural traits. In the second experiment spatial memory in the Barnes maze test and object recognition memory were examined. Regarding exploratory behaviour transgenic mice from standard housing condition were statistically indistinguishable from wild-type controls. Enrichment had comparable effects in both genotypes indicated by higher levels of exploration and locomotor activity. In transgenic mice the elevated plus-maze revealed less anxiety-related behaviour due to enrichment in contrast to wild-type mice that statistically did not differ in anxiety-related behaviour. Concerning learning and memory performance, cognitive deficits of standard housed transgenic mice could be demonstrated in both learning tasks. Surprisingly, in both housing conditions a significantly higher number of transgenic mice refused to explore any objects compared to wild-type mice. Furthermore, the Barnes maze test revealed deficits of the transgenic mice in spatial memory compared to wild-type mice whereas no effect of environmental enrichment was detectable. Thus environmental enrichment increased exploratory behaviour and decreased anxiety-related behaviour but could not clearly ameliorate deficits in learning and memory performance of TgCRND8 mice.

Wheel-running in a transgenic mouse model of Alzheimer's disease: protection or symptom?

Several studies on both humans and animals reveal benefits of physical exercise on brain function and health. A previous study on TgCRND8 mice, a transgenic model of Alzheimer's disease, reported beneficial effects of premorbid onset of long-term access to a running wheel on spatial learning and plaque deposition. Our study investigated the effects of access to a running wheel after the onset of Abeta pathology on behavioural, endocrinological, and neuropathological parameters. From day 80 of age, the time when Abeta deposition becomes apparent, TgCRND8 and wildtype mice were kept with or without running wheel. Home cage behaviour was analysed and cognitive abilities regarding object recognition memory and spatial learning in the Barnes maze were assessed. Our results show that, in comparison to Wt mice, Tg mice were characterised by impaired object recognition memory and spatial learning, increased glucocorticoid levels, hyperactivity in the home cage and high levels of stereotypic behaviour. Access to a running wheel had no effects on cognitive or neuropathological parameters, but reduced the amount of stereotypic behaviour in transgenics significantly. Furthermore, wheel-running was inversely correlated with stereotypic behaviour, suggesting that wheel-running may have stereotypic qualities. In addition, wheel-running positively correlated with plaque burden. Thus, in a phase when plaques are already present in the brain, it may be symptomatic of brain pathology, rather than protective. Whether or not access to a running wheel has beneficial effects on Alzheimer-like pathology and symptoms may therefore strongly depend on the exact time when the wheel is provided during development of the disease.

Alterations of the Innate Immune System in Susceptibility and Resilience After Social Defeat Stress.

Dysregulation of innate immune responses has frequently been reported in stress-associated psychiatric disorders such as major depression. In mice, enhanced circulating cytokine levels as well as altered innate immune cell numbers have been found after stress exposure. In addition, stress-induced recruitment of peripheral monocytes to the brain has been shown to promote anxiety-like behavior. However, it is yet unclear whether specific differences in the innate immune system are associated with stress susceptibility or resilience in mice. Utilizing chronic social defeat, a model of depression and stress vulnerability, we characterized peripheral and brain-invading myeloid cells in stress-susceptible and resilient animals. In all defeated animals, we found reduced percentages of CD11c+ dendritic cells (DCs) by flow cytometry in the spleen when compared to non-defeated controls. Exclusively in susceptible mice conventional DCs of the spleen showed up-regulated expression of MHC class II and co-stimulatory CD80 molecules pointing toward an enhanced maturation phenotype of these cells. Susceptible, but not resilient animals further exhibited an increase in inflammatory Ly6Chi monocytes and higher numbers of spleen-derived CD11b+ cells that produced the proinflammatory cytokine tumor necrosis factor (TNF) upon lipopolysaccharide (LPS) stimulation. Increased percentages of peripheral CD45hi CD11b+ cells immigrated into the brain of defeated mice, regardless of resilience or susceptibility. However, cellular infiltrates in the brain of susceptible mice contained higher percentages of CC chemokine receptor 2 (CCR2+) Ly6Chi monocytes representing an inflammatory phenotype. Thus, we defined specific stress-related immune signatures involving conventional DCs and inflammatory Ly6Chi monocytes in susceptible and resilient mice. Together, our findings suggest an impact of the innate immune system in vulnerability to stress-related disorders such as major depression.

Altered B Cell Homeostasis in Patients with Major Depressive Disorder and Normalization of CD5 Surface Expression on Regulatory B Cells in Treatment Responders.

Pro-inflammatory activity and cell-mediated immune responses have been widely observed in patients with major depressive disorder (MDD). Besides their well-known function as antibody-producers, B cells play a key role in inflammatory responses by secreting pro- and anti-inflammatory factors. However, homeostasis of specific B cell subsets has not been comprehensively investigated in MDD. In this study, we characterized circulating B cells of distinct developmental steps including transitional, naïve-mature, antigen-experienced switched, and non-switched memory cells, plasmablasts and regulatory B cells by multi-parameter flow cytometry. In a 6-weeks follow-up, circulating B cells were monitored in a small group of therapy responders and non-responders. Frequencies of naïve lgD+CD27- B cells, but not lgD+CD27+ memory B cells, were reduced in severely depressed patients as compared to healthy donors (HD) or mildly to moderately depressed patients. Specifically, B cells with immune-regulatory capacities such as CD1d+CD5+ B cells and CD24+CD38hi transitional B cells were reduced in MDD. Also Bm1-Bm5 classification in MDD revealed reduced Bm2' cells comprising germinal center founder cells as well as transitional B cells. We further found that reduced CD5 surface expression on transitional B cells was associated with severe depression and normalized exclusively in clinical responders. This study demonstrates a compromised peripheral B cell compartment in MDD with a reduction in B cells exhibiting a regulatory phenotype. Recovery of CD5 surface expression on transitional B cells in clinical response, a molecule involved in activation and down-regulation of B cell responses, further points towards a B cell-dependent process in the pathogenesis of MDD.

Association of Serotonin Transporter Gene AluJb Methylation with Major Depression, Amygdala Responsiveness, 5-HTTLPR/rs25531 Polymorphism, and Stress.

DNA methylation profiles of the serotonin transporter gene (SLC6A4) have been shown to alter SLC6A4 expression, drive antidepressant treatment response and modify brain functions. This study investigated whether methylation of an AluJb element in the SLC6A4 promotor was associated with major depressive disorder (MDD), amygdala reactivity to emotional faces, 5-HTTLPR/rs25531 polymorphism, and recent stress. MDD patients (n=122) and healthy controls (HC, n=176) underwent fMRI during an emotional face-matching task. Individual SLC6A4 AluJb methylation profiles were ascertained and associated with MDD, amygdala reactivity, 5-HTTLPR/rs25531, and stress. SLC6A4 AluJb methylation was significantly lower in MDD compared to HC and in stressed compared to less stressed participants. Lower AluJb methylation was particularly found in 5-HTTLPR/rs25531 risk allele carriers under stress and correlated with less depressive episodes. fMRI analysis revealed a significant interaction of AluJb methylation and diagnosis in the amygdala, with MDD patients showing lower AluJb methylation associated with decreased amygdala reactivity. While no joint effect of AluJb methylation and 5-HTTLPR/rs25531 existed, risk allele carriers showed significantly increased bilateral amygdala activation. These findings suggest a role of SLC6A4 AluJb methylation in MDD, amygdala reactivity, and stress reaction, partly interwoven with 5-HTTLPR/rs25531 effects. Patients with low methylation in conjunction with a shorter MDD history and decreased amygdala reactivity might feature a more stress-adaptive epigenetic process, maybe via theoretically possible endogenous antidepressant-like effects. In contrast, patients with higher methylation might possibly suffer from impaired epigenetic adaption to chronic stress. Further, the 5-HTTLPR/rs25531 association with amygdala activation was confirmed in our large sample.