Schizophrenia risk proteins ZNF804A and NT5C2 interact in cortical neurons.

The zinc finger protein 804A (ZNF804A) and the 5'-nucleotidase cytosolic II (NT5C2) genes are amongst the first schizophrenia susceptibility genes to have been identified in large-scale genome-wide association studies. ZNF804A has been implicated in the regulation of neuronal morphology and is required for activity-dependent changes to dendritic spines. Conversely, NT5C2 has been shown to regulate 5' adenosine monophosphate-activated protein kinase activity and has been implicated in protein synthesis in human neural progenitor cells. Schizophrenia risk genotype is associated with reduced levels of both NT5C2 and ZNF804A in the developing brain, and a yeast two-hybrid screening suggests that their encoded proteins physically interact. However, it remains unknown whether this interaction also occurs in cortical neurons and whether they could jointly regulate neuronal function. Here, we show that ZNF804A and NT5C2 colocalise and interact in HEK293T cells and that their rodent homologues, ZFP804A and NT5C2, colocalise and form a protein complex in cortical neurons. Knockdown of the Zfp804a or Nt5c2 genes resulted in a redistribution of both proteins, suggesting that both proteins influence the subcellular targeting of each other. The identified interaction between ZNF804A/ZFP804A and NT5C2 suggests a shared biological pathway pertinent to schizophrenia susceptibility within a neuronal cell type thought to be central to the neurobiology of the disorder, providing a better understanding of its genetic landscape.

Identifying FDA-approved drugs with multimodal properties against COVID-19 using a data-driven approach and a lung organoid model of SARS-CoV-2 entry.

BACKGROUND: Vaccination programs have been launched worldwide to halt the spread of COVID-19. However, the identification of existing, safe compounds with combined treatment and prophylactic properties would be beneficial to individuals who are waiting to be vaccinated, particularly in less economically developed countries, where vaccine availability may be initially limited. METHODS: We used a data-driven approach, combining results from the screening of a large transcriptomic database (L1000) and molecular docking analyses, with in vitro tests using a lung organoid model of SARS-CoV-2 entry, to identify drugs with putative multimodal properties against COVID-19. RESULTS: Out of thousands of FDA-approved drugs considered, we observed that atorvastatin was the most promising candidate, as its effects negatively correlated with the transcriptional changes associated with infection. Atorvastatin was further predicted to bind to SARS-CoV-2's main protease and RNA-dependent RNA polymerase, and was shown to inhibit viral entry in our lung organoid model. CONCLUSIONS: Small clinical studies reported that general statin use, and specifically, atorvastatin use, are associated with protective effects against COVID-19. Our study corroborrates these findings and supports the investigation of atorvastatin in larger clinical studies. Ultimately, our framework demonstrates one promising way to fast-track the identification of compounds for COVID-19, which could similarly be applied when tackling future pandemics.

Telomere length in patients with obesity submitted to bariatric surgery: A systematic review.

BACKGROUND: Patients with obesity show evidence of increased levels of inflammation, oxidative stress and premature ageing. Telomere length (TL) is a key marker of cellular ageing, and patients with obesity often present shorter TL. Bariatric surgery (BS) is currently the most effective treatment for severe obesity. The aim of this systematic review was to explore whether the beneficial health effects observed after surgery in obese patients correspond to a restoration in TL or slower rates of shortening. As a secondary aim, we evaluated, at baseline and post-surgery, the relationship between TL and different factors that could play a role in TL changes along time. METHODS: Searches for relevant articles were performed in MEDLINE, Web of Knowledge and SCOPUS. Prospective longitudinal studies that evaluated leukocyte TL in adult patients who had undergone BS were included. Data were extracted and evaluated by two independent researchers. The protocol was registered in PROSPERO with the number CRD42020197711. RESULTS: Seven studies based on independent samples that fulfilled our inclusion criteria were included. Obese patients showed shorter telomeres compared to healthy individuals. Long-term studies (>2 years) seem to suggest an improvement in TL after surgery presumably due to the improvement of the inflammatory and oxidative levels of the patients induced by weight loss. CONCLUSION: Studies seem to point towards a beneficial long-term effect of BS on TL recovery. However, the scarce number of studies and the heterogeneity in the variables analysed in the different cohorts make it difficult to draw a firm conclusion. More studies are needed to evaluate long-term changes to TL following BS.

Telomere length as a predictor of emotional processing in the brain.

Shorter telomere length (TL) has been associated with the development of mood disorders as well as abnormalities in brain morphology. However, so far, no studies have considered the role TL may have on brain function during tasks relevant to mood disorders. In this study, we examine the relationship between TL and functional brain activation and connectivity, while participants (n = 112) perform a functional magnetic resonance imaging (fMRI) facial affect recognition task. Additionally, because variation in TL has a substantial genetic component we calculated polygenic risk scores for TL to test if they predict face-related functional brain activation. First, our results showed that TL was positively associated with increased activation in the amygdala and cuneus, as well as increased connectivity from posterior regions of the face network to the ventral prefrontal cortex. Second, polygenic risk scores for TL show a positive association with medial prefrontal cortex activation. The data support the view that TL and genetic loading for shorter telomeres, influence the function of brain regions known to be involved in emotional processing.

The genome-wide expression effects of escitalopram and its relationship to neurogenesis, hippocampal volume, and antidepressant response.

Antidepressant-induced hippocampal neurogenesis (AHN) is hypothesized to contribute to increases in hippocampal volume among major depressive disorder patients after long-term treatment. Furthermore, rodent studies suggest AHN may be the cellular mechanism mediating the therapeutic benefits of antidepressants. Here, we perform the first investigation of genome-wide expression changes associated with AHN in human cells. We identify gene expression networks significantly activated during AHN, and we perform gene set analyses to probe the molecular relationship between AHN, hippocampal volume, and antidepressant response. The latter were achieved using genome-wide association summary data collected from 30,717 individuals as part of the ENIGMA Consortium (genetic predictors of hippocampal volume dataset), and data collected from 1,222 major depressed patients as part of the NEWMEDS Project (genetic predictors of response to antidepressants dataset). Our results showed that the selective serotonin reuptake inhibitor, escitalopram evoked AHN in human cells; dose-dependently increasing the differentiation of cells into neuroblasts, as well as increasing gliogenesis. Activated genome-wide expression networks relate to axon and microtubule formation, and ribosomal biogenesis. Gene set analysis revealed that gene expression changes associated with AHN were nominally enriched for genes predictive of hippocampal volume, but not for genes predictive of therapeutic response.

The duration of lithium use and biological ageing: telomere length, frailty, metabolomic age and all-cause mortality.

Lithium is an established first-line treatment for bipolar disorder. Beyond its therapeutic effect as a mood stabiliser, lithium exhibits potential anti-ageing effects. This study aimed to examine the relationship between the duration of lithium use, biological ageing and mortality. The UK Biobank is an observational study of middle-aged and older adults. We tested associations between the duration of lithium use (number of prescriptions, total duration of use and duration of the first prescription period) and telomere length, frailty, metabolomic age (MileAge) delta, pulse rate and all-cause mortality. Five hundred ninety-one individuals (mean age = 57.49 years; 55% females) had been prescribed lithium. There was no evidence that the number of prescriptions (ß = - 0.022, 95% CI - 0.081 to 0.037, p = 0.47), the total duration of use (ß = - 0.005, 95% CI - 0.023 to 0.013, p = 0.57) or the duration of the first prescription period (ß = - 0.018, 95% CI - 0.051 to 0.015, p = 0.29) correlated with telomere length. There was also no evidence that the duration of lithium use correlated with frailty or MileAge delta. However, a higher prescription count and a longer duration of use was associated with a lower pulse rate. The duration of lithium use did not predict all-cause mortality. We observed no evidence of associations between the duration of lithium use and biological ageing markers, including telomere length. Our findings suggest that the potential anti-ageing effects of lithium do not differ by the duration of use.

Integrating human endogenous retroviruses into transcriptome-wide association studies highlights novel risk factors for major psychiatric conditions.

Human endogenous retroviruses (HERVs) are repetitive elements previously implicated in major psychiatric conditions, but their role in aetiology remains unclear. Here, we perform specialised transcriptome-wide association studies that consider HERV expression quantified to precise genomic locations, using RNA sequencing and genetic data from 792 post-mortem brain samples. In Europeans, we identify 1238 HERVs with expression regulated in cis, of which 26 represent expression signals associated with psychiatric disorders, with ten being conditionally independent from neighbouring expression signals. Of these, five are additionally significant in fine-mapping analyses and thus are considered high confidence risk HERVs. These include two HERV expression signatures specific to schizophrenia risk, one shared between schizophrenia and bipolar disorder, and one specific to major depressive disorder. No robust signatures are identified for autism spectrum conditions or attention deficit hyperactivity disorder in Europeans, or for any psychiatric trait in other ancestries, although this is likely a result of relatively limited statistical power. Ultimately, our study highlights extensive HERV expression and regulation in the adult cortex, including in association with psychiatric disorder risk, therefore providing a rationale for exploring neurological HERV expression in complex neuropsychiatric traits.

Allele-specific expression of the serotonin transporter and its transcription factors following lamotrigine treatment in vitro.

Lamotrigine, a mood stabilizer used clinically in the treatment of bipolar disorder, is thought to exert actions on the serotonin system. However lamotrigine's exact mechanism of action remains unclear. The current study investigated whether lamotrigine might exert its effects through altering the expression of the serotonin transporter (5-HTT) gene and its regulatory transcription factors Y box binding protein 1 (YB-1) and CCCTC-binding factor (CTCF). We further considered whether functional variable number tandem repeat (VNTR) polymorphisms in the promoter region of 5-HTT, (5-HTTLPR) and within intron 2 (Stin2) of the gene, moderated any putative gene expression changes. The study employed an in vitro design carried out in human lymphoblastoid cell lines (LCLs) to investigate the effects of lamotrigine treatment at 0.04, 0.2, and 0.4 mM doses for 24 hr on the mRNA expression of 5-HTT, YB-1, and CTCF. LCLs were selected based on combinations of haplotypes of the two VNTRs in the serotonin transporter gene; creating low-expressing and high-expressing LCL groups. Ubiquitin C (UBC) and topoisomerase I (TOP1) genes were found to be the most stably expressed housekeeping genes in drug-treated LCLs. Subsequently, quantitative PCR revealed that higher doses of lamotrigine significantly lowered 5-HTT expression and increased CTCF expression. Haplotype-specific differences in CTCF expression were found in response to lamotrigine, with strongest expression changes observed in the high-expressing LCLs. These data provide an allele-specific in vitro model for examining the molecular targets of lamotrigine, and support the important role of the serotonin transporter gene in its clinical mechanism of action.

A single-cell transposable element atlas of human cell identity.

Single cell RNA sequencing (scRNA-seq) is revolutionizing the study of complex biological systems. However, most sequencing studies overlook the contribution of transposable element (TE) expression to the transcriptome. In both scRNA-seq and bulk tissue RNA sequencing (RNA-seq), quantification of TE expression is challenging due to repetitive sequence content and poorly characterized TE gene models. Here, we developed a tool and analysis pipeline for Single cell Transposable Element Locus Level Analysis of scRNA Sequencing (Stellarscope) that reassigns multi-mapped reads to specific genomic loci using an expectation-maximization algorithm. Using Stellarscope, we built an atlas of TE expression in human PBMCs. We found that locus-specific TEs delineate cell types and define new cell subsets not identified by standard mRNA expression profiles. Altogether, this study provides comprehensive insights into the influence of transposable elements in human biology.

Transcriptome-wide association study of HIV-1 acquisition identifies HERC1 as a susceptibility gene.

The host genetic factors conferring protection against HIV type 1 (HIV-1) acquisition remain elusive, and in particular the contributions of common genetic variants. Here, we performed the largest genome-wide association meta-analysis of HIV-1 acquisition, which included 7,303 HIV-1-positive individuals and 587,343 population controls. We identified 25 independent genetic loci with suggestive association, of which one was genome-wide significant within the major histocompatibility complex (MHC) locus. After exclusion of the MHC signal, linkage disequilibrium score regression analyses revealed a SNP heritability of 21% and genetic correlations with behavioral factors. A transcriptome-wide association study identified 15 susceptibility genes, including HERC1, UEVLD, and HIST1H4K. Convergent evidence from conditional analyses and fine-mapping identified HERC1 downregulation in immune cells as a robust mechanism associated with HIV-1 acquisition. Functional studies on HERC1 and other identified candidates, as well as larger genetic studies, have the potential to further our understanding of the host mechanisms associated with protection against HIV-1.

Antiretroviral drug activity and potential for pre-exposure prophylaxis against COVID-19 and HIV infection.

COVID-19 is the disease caused by SARS-CoV-2 which has led to 2,643,000 deaths worldwide, a number which is rapidly increasing. Urgent studies to identify new antiviral drugs, repurpose existing drugs, or identify drugs that can target the overactive immune response are ongoing. Antiretroviral drugs (ARVs) have been tested in past human coronavirus infections, and also against SARS-CoV-2, but a trial of lopinavir and ritonavir failed to show any clinical benefit in COVID-19. However, there is limited data as to the course of COVID-19 in people living with HIV, with some studies showing a decreased mortality for those taking certain ARV regimens. We hypothesized that ARVs other than lopinavir and ritonavir might be responsible for some protection against the progression of COVID-19. Here, we used chemoinformatic analyses to predict which ARVs would bind and potentially inhibit the SARS-CoV-2 main protease (Mpro) or RNA-dependent-RNA-polymerase (RdRp) enzymes in silico. The drugs predicted to bind the SARS-CoV-2 Mpro included the protease inhibitors atazanavir and indinavir. The ARVs predicted to bind the catalytic site of the RdRp included Nucleoside Reverse Transcriptase Inhibitors, abacavir, emtricitabine, zidovudine, and tenofovir. Existing or new combinations of antiretroviral drugs could potentially prevent or ameliorate the course of COVID-19 if shown to inhibit SARS-CoV-2 in vitro and in clinical trials. Further studies are needed to establish the activity of ARVs for treatment or prevention of SARS-CoV-2 infection .Communicated by Ramaswamy H. Sarma.

Relationship between CRP and depression: A genetically sensitive study in Sri Lanka.

BACKGROUND: Previous studies have shown associations between major depression and C-reactive protein (CRP) levels. Few studies have considered the extent to which shared genetic and environmental factors contribute to this association, nor have they considered the relationship outside of European populations. We examined the association between CRP levels and depression and their aetiology in a Sri Lankan population. METHODS: Data were collected from 2577 twins and 899 singletons in Colombo, Sri Lanka. Depression symptoms were assessed using the revised Beck Depression Inventory (BDI-II). High-sensitive CRP blood levels were assessed using immunoturbidimetry. Linear regressions were performed to test the association between CRP and depression. The heritability of CRP levels was estimated using Structural Equation Modelling. RESULTS: CRP was significantly associated with BMI (p < 0.01) but not depression (p > 0.05). In males, variance in CRP levels was explained by shared environment (51% 95%CIs: 13-62) and non-shared environment (45% 95%CIs: 36-54). In contrast, in females, CRP variance was explained by genetic (41% 95%CIs: 10-52) and non-shared environment (56% 95%CIs: 47-67). A genetic correlation between CRP and BMI was observed in females only. LIMITATIONS: CRP level was based on a single data collection point, longer term data collection would give a more accurate picture of an individual's state of inflammation. CONCLUSIONS: The lack of association between depression and CRP strengthens the hypothesis that inflammation might contribute to the development of some, but not all types of depression. CRP levels were moderated by the environment, suggesting interventions aimed at reducing CRP levels and risk for inflammatory conditions, particularly in males.

Prolactin, Estradiol and Testosterone Differentially Impact Human Hippocampal Neurogenesis in an In Vitro Model.

Previous studies have indicated that sex hormones such as prolactin, estradiol and testosterone may play a role in the modulation of adult hippocampal neurogenesis (AHN) in rodents and non-human primates, but so far there has been no investigation of their impact on human hippocampal neurogenesis. Here, we quantify the expression levels of the relevant receptors in human post-mortem hippocampal tissue and a human hippocampal progenitor cell (HPC) line. Secondly, we investigate how these hormones modulate hippocampal neurogenesis using a human in vitro cellular model. Human female HPCs were cultured with biologically relevant concentrations of either prolactin, estradiol or testosterone. Bromodeoxyuridine (BrdU) incorporation, immunocytochemistry (ICC) and high-throughput analyses were used to quantify markers determining cell fate after HPCs were either maintained in a proliferative state or allowed to differentiate in the presence of these hormones. In proliferating cells, estrogen and testosterone increased cell density but had no clear effect on markers of proliferation or cell death to account for this. In differentiating cells, a 3-day treatment of prolactin elicited a transient effect, whereby it increased the proportion of microtubule-associated protein 2 (MAP2)-positive and Doublecortin (DCX)-positive cells, but this effect was not apparent after 7-days. At this timepoint we instead observe a decrease in proliferation. Overall, our study demonstrates relatively minor, and possibly short-term effects of sex hormones on hippocampal neurogenesis in human cells. Further work will be needed to understand if our results differ to previous animal research due to species-specific differences, or whether it relates to limitations of our in vitro model.

Reconsidering the reasons for heightened inflammation in major depressive disorder.

BACKGROUND: Increased circulating pro-inflammatory markers have repeatedly been associated with major depressive disorder (MDD). However, it remains unclear whether inflammation represents a causal mechanism for MDD, or whether the association is influenced by confounding factors such as body mass index (BMI). METHODS: To better understand this complex relationship, we generated polygenic risk scores (PRS) for MDD and BMI in a population cohort and attempted to isolate the impact these potential risk factors have on adulthood inflammation. Peripheral blood samples were collected as part of the South East London Community Health study, where we generated individualized PRS for MDD and BMI and quantified inflammatory markers using multiplex ELISA-based technology. We performed linear regressions to investigate the effects of PRS for MDD and BMI on inflammatory marker levels. RESULTS: Out of 35 inflammatory markers, we found a nominal effect of PRS for MDD on interleukin-10. We also found a significant positive effect of BMI on nine inflammatory markers, of which the two most strongly affected markers, interleukin-6 (IL-6) and C-reactive protein (CRP), were also nominally predicted by BMI PRS. LIMITATIONS: The study utilized a cross-sectional design with a moderately sized sample. CONCLUSIONS: Our findings suggest there may not be a shared genetic mechanism contributing to MDD and higher inflammatory marker levels. However, there may be shared genetic etiology between BMI and adulthood levels of CRP and IL-6. Therefore, polygenic risk scores for BMI may represent a useful indicator for heightened levels of inflammation in adulthood.

Genetic risk for severe COVID-19 correlates with lower inflammatory marker levels in a SARS-CoV-2-negative cohort.

OBJECTIVES: It remains unknown how inflammatory marker levels differ amongst individuals susceptible to coronavirus disease 2019 (COVID-19), prior to severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infection and the onset of the cytokine storm. We used genetic risk scores to model how susceptibility to severe COVID-19 correlates with baseline levels of 35 inflammatory markers, by testing their impact in a SARS-CoV-2-negative population cohort. Because of the established effects of age and body mass index on severe COVID-19 risk, we further considered how these variables interacted with genetic risk to affect inflammatory marker levels. METHODS: We accessed data on 406 SARS-CoV-2-negative individuals as part of a UK population study. Multiplex electrochemiluminescence methods were applied to blood serum, and 35 inflammatory markers were assayed. Corresponding genotype data, alongside results from a large genome-wide association study of severe COVID-19, allowed us to construct genetic risk scores and to test their impact on inflammatory protein levels. RESULTS: Our results revealed that a higher genetic risk for severe COVID-19 was associated with lower blood levels of interferon gamma (IFN-γ), vascular endothelial growth factor D (VEGF-D) and tumor necrosis factor alpha (TNF-α). Inflammatory profiles of those with high genetic risk increasingly diverge from the norm in association with age and obesity. CONCLUSION: Our results support the theory that individuals at risk of severe COVID-19 have a deficient innate immunity marked by reduced levels of inflammatory markers at baseline, including IFN-γ, VEGF-D and TNF-α. We hypothesise that a secondary overactive adaptive immune response may subsequently explain the high levels of cytokines observed in SARS-CoV-2-positive COVID-19 patients.

Lithium treatment and human hippocampal neurogenesis.

Lithium is a first-line treatment for bipolar disorder, where it acts as a mood-stabilizing agent. Although its precise mechanism remains unclear, neuroimaging studies have shown that lithium accumulates in the hippocampus and that chronic use amongst bipolar disorder patients is associated with larger hippocampal volumes. Here, we tested the chronic effects of low (0.75 mM) and high (2.25 mM) doses of lithium on human hippocampal progenitor cells and used immunocytochemistry to investigate the effects of lithium on cell parameters implicated in neurogenesis. Corresponding RNA-sequencing and gene-set enrichment analyses were used to evaluate whether genes affected by lithium in our model overlap with those regulating the volume of specific layers of the dentate gyrus. We observed that high-dose lithium treatment in human hippocampal progenitors increased the generation of neuroblasts (P ≤ 0.01), neurons (P ≤ 0.01), and glia (P ≤ 0.001), alongside the expression of genes, which regulate the volume of the molecular layer of the dentate gyrus. This study provides empirical support that adult hippocampal neurogenesis and gliogenesis are mechanisms that could contribute to the effects of lithium on human hippocampal volume.

Influence of the BDNF Val66Met polymorphism on weight loss after bariatric surgery: a 24-month follow-up.

BACKGROUND: Bariatric surgery is currently the most effective long-term treatment for severe obesity. However, interindividual variation in surgery outcome has been observed, and research suggests a moderating effect of several factors including baseline co-morbidities (e.g., type 2 diabetes [T2D] and genetic factors). No data are currently available on the interaction between T2D and variants in brain derived neurotrophic factor (BDNF) and its effect on weight loss after surgery. OBJECTIVES: To examine the role of the BDNF Val66Met polymorphism (rs6265) and the influence of T2D and their interaction on weight loss after bariatric surgery in a cohort of patients with severe obesity. SETTING: University hospital in Spain. METHODS: The present study evaluated a cohort of 158 patients with obesity submitted to bariatric surgery (Roux-en-Y gastric bypass or sleeve gastrectomy) followed up for 24 months (loss to follow-up: 0%). During the postoperative period, percentage of excess body mass index loss (%EBMIL), percentage of excess weight loss (%EWL), and total weight loss (%TWL) were evaluated. RESULTS: Longitudinal analyses showed a suggestive effect of BDNF genotype on the %EWL (P = .056) and indicated that individuals carrying the methionine (Met) allele may experience a better outcome after bariatric surgery than those with the valine/valine (Val/Val) genotype. We found a negative effect of a T2D diagnosis at baseline on %EBMIL (P = .004). Additionally, we found an interaction between BDNF genotype and T2D on %EWL and %EBMIL (P = .027 and P = .0004, respectively), whereby individuals with the Met allele without T2D displayed a greater %EWL and greater %EBMIL at 12 months and 24 months than their counterparts with T2D or patients with the Val/Val genotype with or without T2D. CONCLUSION: Our data showed an association between the Met variant and greater weight loss after bariatric surgery in patients without T2D. The presence of T2D seems to counteract this positive effect.

Serum from Older Adults Increases Apoptosis and Molecular Aging Markers in Human Hippocampal Progenitor Cells.

Age-related alteration in neural stem cell function is linked to neurodegenerative conditions and cognitive decline. In rodents, this can be reversed by exposure to a young systemic milieu and conversely, the old milieu can inhibit stem cell function in young rodents. In this study, we investigated the in vitro effect of the human systemic milieu on human hippocampal progenitor cells (HPCs) using human serum from early adulthood, mid-life and older age. We showed that neuroblast number following serum treatment is predictive of larger dentate gyrus, CA3, CA4 and whole hippocampus volumes and that allogeneic human serum from asymptomatic older individuals induced a two-fold increase in apoptotic cell death of HPCs compared with serum from young adults. General linear models revealed that variability in markers of proliferation and differentiation was partly attributable to use of antihypertensive medication and very mild cognitive decline among older subjects. Finally, using an endophenotype approach and whole-genome expression arrays, we showed upregulation of established and novel ageing molecular hallmarks in response to old serum. Serum from older subjects induced a wide range of cellular and molecular phenotypes, likely reflecting a lifetime of environmental exposures. Our findings support a role for the systemic enviroment in neural stem cell maintenance and are in line with others highlighting a distinction between neurobiological and chronological ageing. Finally, the herein described serum assay can be used by future studies to further analyse the effect of environmental exposures as well as to determine the role of the systemic environment in health and disease.

The Role of Inflammatory Proteins in Anti-Glucocorticoid Therapy for Treatment-Resistant Depression.

BACKGROUND: Optimising treatments for patients with treatment-resistant depression (TRD) is key to reducing the burden of this severe illness. The anti-glucocorticoid medication metyrapone has mixed evidence supporting a role as a possible augmentation treatment in TRD. The degree of treatment resistance in depression has been associated prospectively and retrospectively with elevated inflammation, and inflammatory activity may influence responses to antidepressant treatments. AIMS: To investigate whether levels of pro-inflammatory cytokines are associated with clinical outcomes to metyrapone or placebo. METHODS: A double-blind RCT randomised patients with TRD to 3 weeks of placebo or metyrapone augmentation to ongoing serotonergic antidepressants. No benefit of metyrapone was reported in the primary analysis. The current study assessed levels of pro-inflammatory proteins interleukin-6 (IL-6), tumour necrosis factor (TNFα), c-reactive protein (CRP) and interleukin-10 (IL-10) before randomisation and after treatment as potential moderators and/or mediators of clinical outcomes. RESULTS: The three pro-inflammatory proteins (but not IL-10) were elevated in this sample of patients with TRD compared to a non-affected control group. High pre-treatment IL-6 levels predicted a poorer response in the trial overall but did not moderate response to metyrapone versus placebo. Changes in IL-6 indirectly mediated depression outcome, with metyrapone increasing IL-6 levels and IL-6 increase associated with a poorer outcome on depression. Other inflammatory proteins did not mediate or moderate treatment outcomes. INTERPRETATION: Metyrapone is hypothesised to have a therapeutic effect in depression on the basis of inhibiting the synthesis of cortisol. In this study, metyrapone did not reduce cortisol, possibly due to glucocorticoid system overcompensation). The mediation effect of IL-6 may support this and perhaps help to indicate why the treatment was not effective.