Major depressive disorder (MDD) is a heterogeneous condition, hardly captured by single biomarkers. Transcriptomic profiles are important indicators for genes and pathways involved in MDD and its phenotypes. We performed whole-transcriptome and pathway analyses on data from 140 individuals (106 with MDD and 34 controls) from the observational, case-control, BIOmarkers in DEPression (BIODEP) study. We further divided MDD participants based on a) inflammation, measured with serum high-sensitivity C-reactive protein (hsCRP, mg/L) (n=39 CRP<1, n=31 CRP1-3, n=35 CRP>3), and b) exposure/response to antidepressants (n=47 non-responders, n=37 responders, n=22 unmedicated). We performed whole-blood RNA- sequencing using Illumina NextSeq 550 and statistical analyses with Deseq2 (RUV-corrected). We observe several transcripts differentially expressed (p<.05, fold-change>|1.2|, ranging from n=393 to n=1107) in all comparisons, both in the CRP-based and treatment-based groupings. Subsequent analyses with Ingenuity Pathway Analysis (p<.05, z-scores>|2|) reveal various immune-related pathways primarily involved in group differences. This involvement reflects the gradient of serum CRP levels within different MDD groups (progressive activation in CRP<1, 1- 3, >3) and is associated with non-responders and unmedicated MDD (both activated vs. responders). Interestingly, immune-related pathways are differentially activated also in all the three MDD treatment-based subgroups, in the MDD CRP 1-3 and >3, and in the whole-MDD (all cases together) vs. controls; while the main pathways involved in the MDD CRP<1 vs. controls are cell cycle-related. Our findings demonstrate different transcriptomic pathways associatedwith MDD and different MDD phenotypes, which could ultimately inform more tailored approaches to clinical management.

Major depressive disorder (MDD) is associated with inflammation, and 25-30% of patients show low-grade inflammation (C-Reactive Protein (CRP)>3 mg/L). Sex hormones act on inflammation and might contribute to sex-specific differences in MDD. We investigated the interplay between sex hormones and inflammation within-sexes. We measured serum high-sensitivity CRP, testosterone, 17β-estradiol (E2), progesterone, sex-hormone binding globulin (SHBG), gonadotropins levels; we calculated testosterone to E2 ratio (T/E2), free androgen and estradiol indexes (FAI, FEI) in 64 controls and 178 MDD patients. We performed One-way ANOVA with Bonferroni's correction, Pearson's correlations, and linear regressions to investigate sex hormones in controls (25 males, 39 females), patients with CRP<3 mg/L (CRP-) (53 males, 72 females), and patients with CRP>3 mg/L (CRP+) (19 males, 34 females). In males, CRP+ patients showed lower testosterone than controls (p=0.001), lower testosterone (p=0.013), T/E2 (p<0.001) and higher FEI (p=0.015) than CRP- patients. In patients only, CRP negatively correlated with testosterone (r=-. 312, p< 0.005), T/E2 (r=-.274, p< 0.05), and progesterone (r=-223, p<0.05), with testosterone (p=0.020) and T/E2 (p=0.042) predicting CRP (R2 =0.214 p=0.001). In females, CRP+ patients showed lower SHBG than controls (p=0.033) and CRP- patients (p=0.034), which became statistical trends when co-variating for ovulatory phases. In patients only, CRP negatively correlated with testosterone (r=-.285 p=0.003) and SHBG (r=-292 p=0.003), with SHBG (p=0.012) predicting CRP (R2 =.115 p=0.002). MDD patients with inflammation show sex hormone imbalance. Future research is needed to understand this association and hormonal therapies' potential.

Childhood trauma (CT) is associated with increased inflammation, independently, and comorbidly with, depression. There is also evidence for differential inflammatory gene expression in depression treatment response. Previous analysis identified a trend for the role of CT on NR3C1 expression, however, differences were attributable to depression treatment response. The present work explores the role of CT in inflammation and depression treatment response. The BIOmarkers in DEPression study recruited 170 participants as controls (n=40), responders (n=36), treatment-resistant depressed (TRD) (n=58), or untreated-depressed (UD)(n=36). The Hamilton Depression Rating Scale measured depression. CT was categorised as none-low (n=90), or moderate-severe trauma (n=80) according to the Childhood Trauma Questionnaire. mRNA quantitative polymerase chain reaction (qPCR) measured 7 candidate inflammatory gene expression: A2M, FKBP5, IL6, MIF, SGK1, STAT1, and TNF. Univariate analyses explored inflammatory gene expression differences across CT and treatment response groups. CT did not play a significant role in the differential expression; depression treatment response groups were significantly associated with increased A2M (p<.001), FKBP5 (p<.001), IL6 (p<.001), MIF (p<.001), SGK1 (p<.001) and TNF (p<.001). Responders showed highest mean A2M (.821, SE=.015) and IL-6 expression (.820, SE=.012); TRD showed highest FKBP5 (.819, SE=.010) and TNF (.840, SE=.008); UD participants showed the highest MIF (.832, SE=.013), SGK1 (.801, SE=.008) and STAT1 (.800, SE=.014). Despite a previous trend for CT in NR3C1 expression, increases in further inflammatory gene expression were driven by differences in depression treatment response, with no effect of CT.

Sex hormones have biological effects on inflammation, and these might contribute to the sex -specific features of depression. C -reactive protein (CRP) is the most widely used inflammatory biomarker and consistent evidence shows a significant proportion (20 -30 %) of patients with major depressive disorder (MDD) have CRP levels above 3 mg/L, a threshold indicating at least low-grade inflammation. Here, we investigate the interplay between sex hormones and CRP in the cross-sectional, observational Biomarkers in Depression Study. We measured serum high -sensitivity (hs-)CRP, in 64 healthy controls and 178 MDD patients, subdivided into those with hs-CRP below 3 mg/L (low -CRP; 53 males, 72 females) and with hs-CRP above 3 mg/L (high -CRP; 19 males, 34 females). We also measured interleukin-6, testosterone, 17-8-estradiol (E2), progesterone, sex -hormone binding globulin (SHBG), follicle -stimulating and luteinising hormones, and calculated testosterone -to -E2 ratio (T/E2), free androgen and estradiol indexes (FAI, FEI), and testosterone secretion index. In males, high -CRP patients had lower testosterone than controls ( p = 0.001), and lower testosterone ( p = 0.013), T/E2 ( p < 0.001), and higher FEI ( p = 0.015) than low -CRP patients. In females, high -CRP patients showed lower SHGB levels than controls ( p = 0.033) and low -CRP patients ( p = 0.034). The differences in testosterone, T/E2 ratio, and FEI levels in males survived the Benjamini-Hochberg FDR correction. In linear regression analyses, testosterone (8 = -1.069 p = 0.033) predicted CRP concentrations (R 2 = 0.252 p = 0.002) in male patients, and SHBG predicted CRP levels (8 = -0.628 p = 0.009, R 2 = 0.172 p = 0.003) in female patients. These findings may guide future research investigating interactions between gonadal and immune systems in depression, and the potential of hormonal therapies in MDD with inflammation.

Transcriptomic profiles are important indicators for molecular mechanisms and pathways involved in major depressive disorder (MDD) and its different phenotypes, such as immunometabolic depression. We performed whole-transcriptome and pathway analyses on 139 individuals from the observational, case-control, BIOmarkers in DEPression (BIODEP) study, 105 with MDD and 34 controls. We divided MDD participants based on levels of inflammation, as measured by serum high-sensitivity C-reactive protein (CRP), in n = 39 'not inflamed' (CRP < 1 mg/L), n = 31 with 'elevated CRP' (1-3 mg/L), and n = 35 with 'low-grade inflammation' (>3 mg/L). We performed whole-blood RNA sequencing using Illumina NextSeq 550 and statistical analyses with the Deseq2 package for R statistics (RUV-corrected) and subsequent pathway analyses with Ingenuity Pathway Analysis. Immunometabolic pathways were activated in individuals with CRP > 1 mg/L, although surprisingly the CRP 1-3 group showed stronger immune activation than the CRP > 3 group. The main pathways identified in the comparison between CRP < 1 group and controls were cell-cycle-related, which may be protective against immunometabolic abnormalities in this 'non-inflamed' depressed group. We further divided MDD participants based on exposure and response to antidepressants (n = 47 non-responders, n = 37 responders, and n = 22 unmedicated), and identified specific immunomodulatory and neuroprotective pathways in responders (especially vs. non-responders), which could be relevant to treatment response. In further subgroup analyses, we found that the specific transcriptional profile of responders is independent of CRP levels, and that the inhibition of cell-cycle-related pathways in MDD with CRP < 1 mg/L is present only in those who are currently depressed, and not in the responders. The present study demonstrates immunometabolic and cell-cycle-related transcriptomic pathways associated with MDD and different (CRP-based and treatment-based) MDD phenotypes, while shedding light on potential molecular mechanisms that could prevent or facilitate an individual's trajectory toward immunometabolic depression and/or treatment-non-responsive depression. The recognition and integration of these mechanisms will facilitate a precision-medicine approach in MDD.

Obesity and lack of physical activity (PA) are suggested to contribute to development of depression, seemingly ameliorated by integration of regular PA in post-bariatric surgery care. Effects of pre-surgery PA on depressive symptoms and weight loss remain unclear in long-term follow-up post-surgery. We examined associations between PA and depressive symptoms and weight loss over 3-years in obese bariatric surgery patients with and without major depressive disorder (MDD). Data obtained from a sub-group of participants from the bariatric surgery and depression (BARIDEP) study reported PA at baseline, 6-months, and 3-years post-surgery follow-up; 23 MDD (age=45.82±2.61; BMI=48.01±1.49 kg/m2) and 25 controls (age(±se)=44.28±2.08; BMI=49.75±2.33 kg/m2). PA measured using International Physical Activity Questionnaire (IPAQ) and depressive symptoms with Hamilton Depression Scale (HAM- D-17). Metabolic equivalent (MET) scores (weighted sum of walking, moderate, and vigorous PA) were lower in MDD than controls, before (W=354.5, p<0.01), 6-months (W=405.5, p<0.05), but not 3-years post-surgery. The total sample, baseline HAM-D-17 was correlated with baseline MET score (r=-0.361, p=0.016), PA days (r=-0.310, p=0.032), and time walking (r=- 0.422, p=0.004). 3-year HAM-D-17 was not correlated with any 3-year IPAQ domain. No baseline IPAQ domains predicted 3-year HAM-D-17 score. Weight loss post-surgery was not correlated with baseline or 3-year followup IPAQ scores in (MDD, controls, total) groups. We found gradual improvement in PA levels in patients with MDD 3-years following bariatric surgery, but no association with depressive symptom improvement nor weight loss

OBJECTIVE:Electronic Health Record (EHR) systems are digital platforms in clinical practice used to collect patients' clinical information related to their health status and represents a useful storage of real-world data. EHRs have a potential role in research studies, in particular, in platform trials. Platform trials are innovative trial designs including multiple trial arms (conducted simultaneously and/or sequentially) on different treatments under a single master protocol. However, the use of EHRs in research comes with important challenges such as incompleteness of records and the need to translate trial eligibility criteria into interoperable queries. In this paper, we aim to review and to describe our proposed innovative methods to tackle some of the most important challenges identified. This work is part of the Innovative Medicines Initiative (IMI) EU Patient-cEntric clinicAl tRial pLatforms (EU-PEARL) project's work package 3 (WP3), whose objective is to deliver tools and guidance for EHR-based protocol feasibility assessment, clinical site selection, and patient pre-screening in platform trials, investing in the building of a data-driven clinical network framework that can execute these complex innovative designs for which feasibility assessments are critically important.METHODS:ISO standards and relevant references informed a readiness survey, producing 354 criteria with corresponding questions selected and harmonised through a 7-round scoring process (0-1) in stakeholder meetings, with 85% of consensus being the threshold of acceptance for a criterium/question. ATLAS cohort definition and Cohort Diagnostics were mainly used to create the trial feasibility eligibility (I/E) criteria as executable interoperable queries.RESULTS:The WP3/EU-PEARL group developed a readiness survey (eSurvey) for an efficient selection of clinical sites with suitable EHRs, consisting of yes-or-no questions, and a set-up of interoperable proxy queries using physicians' defined trial criteria. Both actions facilitate recruiting trial participants and alignment between study costs/timelines and data-driven recruitment potential.CONCLUSION:The eSurvey will help create an archive of clinical sites with mature EHR systems suitable to participate in clinical trials/platform trials, and the interoperable proxy queries of trial eligibility criteria will help identify the number of potential participants. Ultimately, these tools will contribute to the production of EHR-based protocol design.

Compelling evidence demonstrates that some individuals suffering from major depressive disorder (MDD) exhibit increased levels of inflammation. Most studies focus on inflammation-related proteins, such as serum or plasma C-reactive protein (CRP). However, the immune-related modifications associated with MDD may be not entirely captured by CRP alone. Analysing mRNA gene expression levels, we aimed to identify broader molecular immune-related phenotypes of MDD. We examined 168 individuals from the non-interventional, case–control, BIODEP study, 128 with a diagnosis of MDD and 40 healthy controls. Individuals with MDD were further divided according to serum high-sensitivity (hs)CRP levels (n = 59 with CRP <1, n = 33 with CRP 1–3 and n = 36 with CRP >3 mg/L). We isolated RNA from whole blood and performed gene expression analyses using RT-qPCR. We measured the expression of 16 immune-related candidate genes: A2M, AQP4, CCL2, CXCL12, CRP, FKBP5, IL-1-beta, IL-6, ISG15, MIF, GR, P2RX7, SGK1, STAT1, TNF-alpha and USP18. Nine of the 16 candidate genes were differentially expressed in MDD cases vs. controls, with no differences between CRP-based groups. Only CRP mRNA was clearly associated with serum CRP. In contrast, plasma (proteins) IL-6, IL-7, IL-8, IL-10, IL-12/IL-23p40, IL-16, IL-17A, IFN-gamma and TNF-alpha, and neutrophils counts, were all differentially regulated between CRP-based groups (higher in CRP >3 vs. CRP <1 and/or controls), reflecting the gradient of CRP values. Secondary analyses on MDD individuals and controls with CRP values <1 mg/L (usually interpreted as 'no inflammation') confirmed MDD cases still had significantly different mRNA expression of immune-related genes compared with controls. These findings corroborate an immune-related molecular activation in MDD, which appears to be independent of serum CRP levels. Additional biological mechanisms may then be required to translate this mRNA signature into inflammation at protein and cellular levels. Understanding these mechanisms will help to uncover the true immune abnormalities in depression, opening new paths for diagnosis and treatment.

We recruited obese individuals with or without comorbid depression before undergoing bariatric surgery for the longitudinal bariatric surgery and depression (BARIDEP) study. Participants were followed-up 3-years post-surgery with blood samples and depressive symptom assessment. Based on Hamilton Depression (HAM-D) scale scores participants were classed as controls (HAM-D<8), persistent depression (HAM-D≥8), remission (HAM-D≥8 at baseline and <8 at follow-up), or new depression (HAM-D<8 at baseline and ≥8 at follow-up). Inflammatory cytokines were measured in serum using Meso Scale Discovery immunoassay. Participants who had depression at baseline had significantly higher serum tumour necrosis factor (TNF) and interleukin 6 (IL-6) at 3-year follow-up compared with controls. Those with persistent depression had significantly higher TNF compared with participants with newly diagnosed depression. IL-6 levels were correlated with body mass index (BMI) only in the control group. Our data show persistent higher levels of inflammation in individuals with persistent depression which is not present in individuals who developed depression post-surgery, suggesting a difference in the mechanisms leading to depression between these two groups. Furthermore, pro-inflammatory cytokine levels are not correlated with BMI in individuals with depression indicating the inflammation may be independent of weight.

BACKGROUND Research implicates inflammation in the vicious cycle between depression and obesity, yet few longitudinal studies exist. The rapid weight loss induced by bariatric surgery is known to improve depressive symptoms dramatically, but preoperative depression diagnosis may also increase the risk for poor weight loss. Therefore, we investigated longitudinal associations between depression and inflammatory markers and their effect on weight loss and clinical outcomes in bariatric patients. METHODS This longitudinal observational study of 85 patients with obesity undergoing bariatric surgery included 41 cases with depression and 44 controls. Before and 6 months after surgery, we assessed depression by clinical interview and measured serum high-sensitivity C-reactive protein (hsCRP) and inflammatory cytokines, including interleukin (IL)-6 and IL-10. RESULTS Before surgery, depression diagnosis was associated with significantly higher serum hsCRP, IL-6, and IL-6/10 ratio levels after controlling for confounders. Six months after surgery, patients with pre-existing depression still had significantly higher inflammation despite demonstrating similar weight loss to controls. Hierarchical regression showed higher baseline hsCRP levels predicted poorer weight loss (β = -0.28, p = 0.01) but had no effect on depression severity at follow-up (β = -0.02, p = 0.9). Instead, more severe baseline depressive symptoms and childhood emotional abuse predicted greater depression severity after surgery (β = 0.81, p < 0.001; and β = 0.31, p = 0.001, respectively). CONCLUSIONS Depression was significantly associated with higher inflammation beyond the effect of obesity and other confounders. Higher inflammation at baseline predicted poorer weight loss 6 months after surgery, regardless of depression diagnosis. Increased inflammation, rather than depression, may drive poor weight loss outcomes among bariatric patients.

AbstractBackgroundDepression and overweight are each associated with abnormal immune system activation. We sought to disentangle the extent to which depressive symptoms and overweight status contributed to increased inflammation and abnormal cortisol levels.MethodsParticipants were recruited through the Wellcome Trust NIMA Consortium. The sample of 216 participants consisted of 69 overweight patients with depression; 35 overweight controls; 55 normal-weight patients with depression and 57 normal-weight controls. Peripheral inflammation was measured as high-sensitivity C-Reactive Protein (hsCRP) in serum. Salivary cortisol was collected at multiple points throughout the day to measure cortisol awakening response and diurnal cortisol levels.ResultsOverweight patients with depression had significantly higher hsCRP compared with overweight controls (p = 0.042), normal-weight depressed patients (p < 0.001) and normal-weight controls (p < 0.001), after controlling for age and gender. Multivariable logistic regression showed that comorbid depression and overweight significantly increased the risk of clinically elevated hsCRP levels ⩾3 mg/L (OR 2.44, 1.28–3.94). In a separate multivariable logistic regression model, overweight status contributed most to the risk of having hsCRP levels ⩾3 mg/L (OR 1.52, 0.7–2.41), while depression also contributed a significant risk (OR 1.09, 0.27–2). There were no significant differences between groups in cortisol awakening response and diurnal cortisol levels.ConclusionComorbid depression and overweight status are associated with increased hsCRP, and the coexistence of these conditions amplified the risk of clinically elevated hsCRP levels. Overweight status contributed most to the risk of clinically elevated hsCRP levels, but depression also contributed to a significant risk. We observed no differences in cortisol levels between groups.

Background: Hypothalamic-pituitary-adrenal (HPA) axis dysregulation has been suggested to play a role in the association between depression and obesity. The study aimed to investigate differences in cortisol levels in in-dividuals with obesity with and without depression and the role of perceived stress on these differences.Methods: Saliva samples were collected at awakening, 15-, 30-and 60-minutes post-awakening from 66 in-dividuals with obesity (30 with major depressive disorder and 36 without major depressive disorder). Salivary cortisol was analysed using ELISA technique. Linear Mixed Models were used for group differences in cortisol awakening response (CAR) with adjustment for socio-demographic confounders and binge eating.Results: Individuals with obesity and depression had lower CAR compared with individuals with obesity without depression (8 =-0.44; p = 0.036). When controlling for perceived stress, CAR was no longer influenced by depression (8 =-0.09; p = 0.75), but individuals with moderate/high stress had lower CAR compared with those with low stress (8 =-0.63; p = 0.036).Conclusions: Our results suggest that differences in CAR between individuals with obesity with and without depression could be due to higher levels of perceived stress in the depressed subjects.

Criteria for treatment-resistant depression (TRD) and partially responsive depression (PRD) as subtypes of major depressive disorder (MDD) are not unequivocally defined. In the present document we used a Delphi-method-based consensus approach to define TRD and PRD and to serve as operational criteria for future clinical studies, especially if conducted for regulatory purposes. We reviewed the literature and brought together a group of international experts (including clinicians, academics, researchers, employees of pharmaceutical companies, regulatory bodies representatives, and one person with lived experience) to evaluate the state-of-the-art and main controversies regarding the current classification. We then provided recommendations on how to design clinical trials, and on how to guide research in unmet needs and knowledge gaps. This report will feed into one of the main objectives of the EUropean Patient-cEntric clinicAl tRial pLatforms, Innovative Medicines Initiative (EU-PEARL, IMI) MDD project, to design a protocol for platform trials of new medications for TRD/PRD.

Background: Inflammation is a well-known risk factor for depression. Specifically, patients who do not respond to antidepressant treatment show higher levels of inflammatory biomarkers compared with responders. Thus, several studies have investigated the efficacy of anti-inflammatory add-on treatment in this population. However, major depressive disorder is more prevalent in females than in males, with sex differences present in antidepressant treatment response and in immune system regulation. To explore sex differences in inflammatory profiles and treatment responses, we investigated a cohort of patients with treatment resistant depression (TRD), for which they received an adjunctive, anti-inflammatory treatment with minocycline - the Minocycline in Depression (MINDEP) study. Methods: The MINDEP study is a 4-week double-blind, randomised, placebo-controlled clinical trial (stratified by sex) with 39 TRD participants, which demonstrated the efficacy of minocycline, an antibiotic with antiinflammatory properties, in TRD patients with major depressive disorder (MDD) and evidence of low-grade inflammation measured with C-reactive protein (CRP) >= 3 mg/L. In these secondary analyses, we investigated the differential effects of minocycline in females (N = 22, 10 randomised to minocycline and 12 randomised to placebo) and in males (N = 17, 8 randomised to minocycline and 9 randomised to placebo) on changes in depressive symptoms (Delta-Hamilton Rating Scale for Depression (HAMD)-17), taking also into consideration CRP levels (CRP >= 3 mg/L vs. CRP <3 mg/L). Additionally, we investigated the role of serum IL-6 in predicting treatment response to minocycline, using sex-specific medians of IL-6, in novel exploratory analyses. Results: Sex differences in Delta-HAMD-17 indicate that only females (F =10.49, p = 0.005), but not males (F = 1.64, p = 0.22), presented an effect of CRP levels on the response to minocycline. Also, we detected sex differences in the relationship between serum CRP and IL-6 levels: CRP was strongly correlated with IL-6 in females (Spearman's rho = 0.658, P < 0.001) but not in males (rho = 0.007, p = 0.979). Exploratory analyses found that IL-6 was indeed a better predictor of response than minocycline than CRP, as we found an interaction between study arms and IL-6 groups (above and below the IL-6 sex-specific median) in females (F = 4.435 p = 0.050) and, at trend statistical level, in males (F = 4.258 p = 0.060). Moreover, Delta-HAMD-17 was numerically comparable in the two high-IL-6 group taking minocycline (females, mean 9.20 +/- SD 7.80; males, mean 8.80 +/- SD 5.97), confirming that high IL-6, differently from high CRP, identified responders to minocycline both in males and females. Conclusion: Our findings highlight the need of sex-specific inflammatory biomarkers in predicting antidepressant response to anti-inflammatories in TRD patients, with the possibility of CRP being a relevant predictor of treatment response only for females, and IL-6 being relevant for both sexes.