Abstract

   The pathophysiology of major depressive disorder (MDD) is highly
   heterogeneous. Previous evidence at the DNA level as well as on the
   serum protein level suggests that the role of inflammation in MDD
   pathology is stronger in patients with hyperphagia during an active
   episode. Which inflammatory pathways differ in MDD patients with
   hyperphagia inflammatory pathways in terms of gene expression is
   unknown. We analyzed whole-blood gene expression profiles of 881
   current MDD cases and 331 controls from the Netherlands Study of
   Depression and Anxiety (NESDA). The MDD patients were stratified
   according to patients with hyperphagia (characterized by increased
   appetite and/or weight, N = 246) or hypophagia (characterized by
   decreased appetite and/or weight, N = 342). Using results of
   differential gene expression analysis between controls and the MDD
   subgroups, enrichment of curated inflammatory pathways was estimated.
   The majority of the pathways were significantly (FDR < 0.1) enriched in
   the expression profiles of MDD cases with hyperphagia, including top
   pathways related to factors responsible for the onset of inflammatory
   response (‘caspase’, ‘GATA3’, ‘NFAT’, and ‘inflammasomes’ pathways).
   Only two pathways (‘adaptive immune system’ and ‘IL-8- and
   CXCR2-mediated signaling’) were enriched in the MDD with hypophagia
   subgroup, these were also enriched in the total current MDD group and
   the group with hyperphagia. This confirms the importance of
   inflammation in MDD pathology of patients with hyperphagia, and
   suggests that distinguishing more uniform MDD phenotypes can help in
   finding their pathophysiological basis.

   Subject terms: Genetics, Genomics

Introduction

   Major depressive disorder (MDD) is one of the leading causes of
   morbidity worldwide with a high socioeconomic burden^[30]1,[31]2. MDD
   is a heterogeneous disorder for which many etiological factors have
   been suggested, for instance low-grade inflammatory
   activation^[32]3,[33]4. Meta-analyses of peripheral inflammatory marker
   studies report elevated levels of C-reactive protein (CRP),
   interleukin(IL)-6, tumor necrosis factor (TNF)-α, the soluble IL-2
   receptor and lowered levels of interferon(IFN)-γ in MDD
   patients^[34]5–[35]8 compared to controls. For some inflammatory
   markers, large between-study heterogeneities and small to moderate
   effect sizes were observed in these meta-analyses, which may partially
   be attributable to the phenotypic heterogeneity of MDD.

   Previous evidence^[36]9–[37]12 suggests that the link with inflammation
   is stronger, or even specific, for MDD patients with some of the
   so-called “reversed neurovegetative symptoms”, such as hyperphagia,
   weight gain, hypersomnia and fatigue. Recent results from the
   Netherlands Study of Depression and Anxiety (NESDA) on ~800 MDD
   patients showed that among reversed neurovegetative symptoms ‘increased
   appetite’ was the most strongly associated with CRP and TNF-α
   levels^[38]13. In line with this are the results of Simmons et
   al.^[39]14, who subgrouped 53 current MDD patients solely on direction
   of appetite change and found elevated levels of IL-1RA, IL-6 and CRP
   levels in patients with increased appetite and no differences between
   patients whose appetite is decreased as compared to controls.

   Results from genetic studies displayed similar patterns. In a recent
   genetic study based on >25,000 samples from the Psychiatric Genomics
   Consortium, MDD cases were stratified according to symptoms of appetite
   and/or weight change during the index episode: increased appetite
   and/or weight (15.8%) vs. decreased appetite and/or weight (45.2%).
   Results showed that the genetic overlap between MDD and CRP levels was
   enhanced in MDD cases with increased appetite and/or weight, who
   carried a significantly higher number of risk variants for CRP levels.
   No associations were found for MDD cases with decreased appetite and/or
   weight^[40]15.

   At the transcriptome level, several case-control studies have been
   carried out. In NESDA, 129 differentially expressed genes in current
   MDD cases were found that were enriched for several pathways among
   which downregulation of the natural killer cell mediated cytotoxicity
   pathway and upregulation of IL-6 mediated signaling pathways^[41]16.
   Another study reported that 90 upregulated genes were significantly
   enriched for pathways related to response to infection and innate
   immune system and 75 downregulated genes were significantly enriched
   for pathways related to T-cell functioning and the adaptive immune
   system^[42]17. Hori et al.^[43]18 detected 317 differentially expressed
   genes in which the synaptic transmission pathway was over-represented.
   Mostafavi et al.^[44]19 analyzed RNA sequence data and did not find
   differentially expressed genes between recurrent MDD patients and
   controls. However, enrichment analyses yielded enrichment of the
   interferon α/β-signaling pathway. The Young Finns Study carried out
   immunological and inflammatory processes gene set enrichment analysis
   in genome-wide expression profiles in participants with a chronic,
   high-severity depression score as compared to other participants from
   this population-based cohort^[45]20. Enriched pathways included IL-1R
   mediated signaling events, Toll-like receptor pathway and Ras signaling
   pathway.

   As opposed to studies examining the DNA and serum protein level, no
   gene expression studies have examined potential differential patterns
   for MDD subtypes so far. The aim of this study was to examine
   enrichment of inflammatory pathways in gene expression profiles of
   subgroups of current MDD cases stratified according to symptoms of
   change in appetite and/or weight. We hypothesize that the more
   prominent role of inflammation in the MDD with hyperphagia subgroup is
   also seen at the level of gene expression, and aim at further
   specifying the inflammatory pathways that could be involved at gene
   expression level.

Methods

Study sample

   Participants were part of the NESDA, an ongoing longitudinal cohort
   study into the long-term course and consequences of depressive and
   anxiety disorders. A description of the study rationale, design, and
   methods is given elsewhere^[46]21. Briefly, from 2004 to 2007, 2981
   participants between the ages of 18 and 65 were recruited from the
   community (19.0%), primary care (54.0%), and specialized mental health
   care settings (27.0%). These participants were healthy controls or had
   a current or prior history of depressive and/or an anxiety disorder.
   Participants were not included when they had a primary diagnosis of
   bipolar, psychotic, obsessive compulsive or severe addictive disorder
   and did not speak fluently Dutch. The Ethical Committee of all
   participating universities approved the NESDA project and all
   participants provided written informed consent.

   Data collection included an extensive interview, blood collection,
   self-reported questionnaires and medical assessments. For the current
   study, we selected an analytical sample of 1847 participants with data
   on MDD diagnostic status and gene expression.

MDD status and stratification

   A Diagnostic and Statistical Manual of Mental Disorders (DSM)-IV MDD
   diagnosis was established with the use of the composite interview
   diagnostic instrument (CIDI)—lifetime version 2.1^[47]22. The
   analytical sample included 881 patients with current MDD (i.e.,
   diagnosed with major depression in the 6 months prior to baseline), 635
   participants with remitted MDD (i.e., lifetime diagnosis of MDD but not
   current) and 331 healthy controls (i.e., participants without a history
   of MDD and/or an anxiety disorder).

   The current MDD group was stratified according to change in appetite
   and/or weight during a major depressive episode consistently with
   previous studies^[48]15,[49]23. MDD symptom-level data ascertained by
   the CIDI interview for the most severe episode in lifetime were
   retrieved. Data on symptoms of change in appetite and were
   disaggregated to code separately for increase, decrease and both
   increase/decrease. Cases were assigned “increased” or “decreased” in
   appetite and/or weight when an increase or decrease in at least one of
   the symptoms was observed. The other symptom was in the same direction
   of change, no change of both increase and decrease. This resulted in
   three subgroups; 246 MDD patients with increased appetite and/or weight
   (27.9%), 341 with decreased appetite and/or weight (38.7%), and a third
   group comprising 294 participants (33.4%) who reported no change, both
   directions or opposite directions in appetite and/or weight change (see
   Supplementary Table [50]1 summarizing the steps in stratification).
   Since previous research indicated that the direction of change in
   appetite is the most discriminating symptom in terms of inflammatory
   alterations^[51]13, and since change in weight commonly parallels those
   in appetite, we labeled the MDD subgroup with increased appetite and/or
   weight “MDD with hyperphagia” (85.8% of the cases experienced increase
   in appetite), and the MDD subgroup with decreased appetite and/or
   weight “MDD with hypophagia” (88.3% of the cases experienced decrease
   in appetite). In summary, the stratification of MDD cases is based on
   appetite change and/or weight change.

   Note that our stratification procedure based on symptoms of appetite
   and/or weight increase and decrease has similarities with the DSM
   specifier of respectively the atypical and melancholic subtypes
   regarding these individual symptoms^[52]24. However, these are not
   interchangeable since we have not taken the core symptoms of mood
   reactivity and anhedonia and other reversed neurovegetative symptoms
   into account. Experiencing appetite and/or weight change are not
   prerequisites for having the atypical or melancholic depression
   diagnosis. Since previous evidence suggests that heightened
   inflammation is not specific for the entire atypical subtype of MDD but
   rather individual atypical-like symptoms^[53]13, we have applied
   stratification only on endorsement of these two symptoms, like has been
   done previously^[54]15,[55]23.

   The present paper focused a priori on the examination of the current
   MDD with hyperphagia and the current MDD with hypophagia subgroups,
   which have been previously shown to have similar higher severity but
   divergent clinical, biological, and genetic
   profiles^[56]13,[57]15,[58]23. Participants reporting no appetite
   and/or weight change have been shown instead to have low symptoms
   severity^[59]15. Participants with remitted MDD were not stratified
   because remitted MDD have been previously shown to have smaller gene
   expression differences from controls, as opposed to the current MDD
   group^[60]16. Also, there was no difference seen in circulating CRP
   concentrations, TNF-α and IL-6 levels between remitted MDD patients and
   controls^[61]25. For these reasons the main results reported concerns
   only the two hyper- and hypophagia subgroups and the overall current
   MDD group considered as benchmark. Nevertheless, the model of the
   differential gene expression analysis appropriately included all four
   MDD (sub)groups ((1) remitted MDD patients, (2) current MDD patients
   with hyperphagia, (3) current MDD patients with hypophagia, and (4)
   current MDD patients without hyperphagia and hypophagia). Results for
   the subgroup of patients without hyperphagia and hypophagia, and
   remitted MDD, are not further discussed.

Other characteristics

   Use of medication was based on drug container inspection of all
   medications used in the past month, classified according to the World
   Health Organization Anatomical Therapeutic Chemical (ATC)
   classification; Use of anti-inflammatory medication (M01A, M01B, A07EB,
   A07EC) and antidepressant medication (N06AA, N06AB, N06AX) were
   assessed. Depression severity scores were assessed with the Inventory
   Depressive Symptomatology (IDS)^[62]26. This 30-item self-rated
   questionnaire assesses the presence of all symptom domains of a major
   depressive episode in the past seven days on a 0–3 scale (not
   severe–severe). Finally, BMI (in kg/m^2) and current smoking status at
   baseline were also measured.

   Differences in baseline characteristics between (1) all current MDD
   cases and controls and (2) the hyper- and hypophagia subgroups and
   controls were tested using χ^2-tests or analyses of (co)variance
   (followed by Tukey’s test for pairwise comparisons) were performed.

Gene expression measurement and analyses

   Serial venous samples were obtained (8–10 am, after overnight fasting)
   in one 7-mL heparin-coated tube (Greiner Bio-One, Monroe, North
   Carolina). Between 10 and 60 min after blood draw, 2.5 mL of blood was
   transferred into a PAXgene tube (Qiagen, Valencia, California). This
   tube was kept at room temperature for a minimum of 2 h and then stored
   at −20 °C. Total RNA was extracted at the VU University Medical Center
   (Amsterdam) according to the manufacturer’s protocol (Qiagen). Gene
   expression in peripheral venous blood was assayed at RUCDR Infinite
   Biologics ([63]http://www.rucdr.org). Samples were randomized to plates
   and gene expression profiles were determined using Affymetrix U219
   arrays (96-well format) and the GeneTitan System as per the
   manufacturer’s protocol (see Jansen et al.^[64]16 for further details).
   Expression values were obtained using RNA normalization implemented in
   Affymetrix Power Tools (APT, v 1.12.0). Gene expression levels were
   measured with 45 574 probe sets and was summarized to gene level by
   calculating the mean expression level of the probe sets targeting the
   same gene. This resulted in 18,364 genes available for analysis. Gene
   expression analyses were corrected for several covariates (age, sex,
   smoking status, RNA measurement plate, RNA quality measure, RNA
   measurement well, blood draw lab, days from blood extraction to RNA
   hybridization, red blood cell count, gene expression-DNA mismatch, hour
   of blood extraction, month of blood extraction; For each of the 18,364
   genes, a linear model was fit with gene expression level as the
   dependent variable and MDD status as the independent variable, while
   including the mentioned covariates as independent variables as well.
   Correction for multiple testing was done according to the
   Benjamini-Hochberg procedure^[65]27. These data were used to generate
   profiles of differentially expressed genes, as compared to healthy
   controls, of MDD (sub)groups to be used as input for pathway enrichment
   analyses. While the statistical power to detect single genes
   differentially expressed at genome-wide significant level may be
   substantially reduced after stratification, the joint effect of genes
   in a pathway could be detected more reliably.

Selection of inflammatory pathways

   We identified inflammatory pathways involving cytokines by a search of
   the curated canonical pathways indexed by the Molecular Signature
   Database (MSigDB;
   [66]http://software.broadinstitute.org/gsea/msigdb/search.jsp), limited
   to Homo sapiens. At the time of search (December 2017), MSigDB
   contained 1329 canonical pathways. First, an initial search was done
   using the MSigDB search engine, with the following key words:
   “inflammation OR inflammatory OR cytokine OR cytokines OR interleukin
   OR interleukins OR IL OR interleukin-* OR chemokine OR chemokines OR
   interferon OR IFN* OR interferons OR TNF OR NLRP*”. This resulted in
   164 pathways. Second, pathways were removed when there were no
   cytokines involved and when the pathway concerned a specific disease. A
   total number of 19 pathways were removed, which are listed in
   Supplementary Table [67]2. Finally, 17 inflammatory pathways involving
   cytokines were not tagged by the key words and were therefore included.
   The final selection includes 162 pathways, containing 3460 unique
   genes, from multiple online pathway databases (63 from BioCarta, 23
   from KEGG, 31 from Reactome, 36 from PID, and 9 from others), which are
   listed in Supplementary Table [68]3. Note that the 162 tested pathways
   are not distinct pathways since they are originating from different
   databases and the genes within these pathways overlap to some extent.
   The final number of significantly enriched pathways is rather a
   reflection of the strength of involvement of inflammation than the
   exact pathways involved.

Pathway enrichment analysis

   The P values generated in the differential gene expression analyses
   were used as input for enrichment analyses, for each group separately
   (all MDD, MDD with hyperphagia and MDD with hypophagia). For each of
   the 162 inflammatory pathways, we performed a one-sided Wilcoxon
   rank-sum test, which does not assume a known distribution and tests for
   a location shift between the P values inside the pathway and the P
   values outside the pathway. We purposely included all genes in the
   pathway enrichment analyses, regardless of the significance level of
   the genes in the differential gene expression analyses. This method is
   particularly useful because it includes subthreshold signal; if
   non-significantly differentially expressed genes belonging to one
   pathway are relatively clustered towards a higher ranking in P values
   compared to remaining genes, enrichment signal could be noticeable.
   Correction for multiple testing was performed using the
   Benjamini-Hochberg procedure^[69]27. Subsequently, we verified whether
   the enrichment results were confirmed after estimation of
   permutation-based FDRs. If correlations between gene expression
   patterns across different pathways have led to false positives, the
   number of pathways with an FDR < 0.1 is lower when the
   permutation-based FDR is applied. This was applied as follows: For each
   of the 1000 permutations, the P values obtained from differential gene
   expression were permuted among gene, and enrichment analysis was
   performed using the permuted P values. For each observed P value from
   the real observed pathway enrichment analyses a corresponding FDR was
   estimated. This was done by dividing the average number of P values
   obtained from the permutation-based enrichment analyses lower than the
   observed P value per permutation by the total number of observed P
   values lower than the observed P value.

   Finally, in order to check whether gene expression in inflammatory
   pathways has a major role compared to other pathways, we also performed
   enrichment analyses including all 1329 canonical pathways present in
   the MSigDB. These 1329 canonical pathways concern inflammatory as well
   non-inflammatory pathways.

BMI, inflammation, antidepressant use and MDD with hyperphagia

   Since MDD with hyperphagia patients had higher BMI as compared to all
   other participants, the enrichment of the 162 inflammatory pathways in
   this subgroup was re-estimated after additional BMI adjustment of the
   gene expression analysis. Previous studies consistently identified
   significant genetic correlations between MDD and BMI, indicating
   potential shared etiological mechanisms^[70]15,[71]28. Intriguingly,
   the genetic overlap with BMI has been shown to be especially strong in
   MDD with hyperphagia^[72]15. This strong connection at the genetic
   level is also reflected at the phenotype level^[73]13. Furthermore,
   analyses based on Mendelian Randomization principles suggested that BMI
   is causal for MDD, or BMI is genetically related with a causal factor
   for MDD^[74]28,[75]29. So, inflammation may represent an important
   factor in a causal pathway with obesity as a general underlying route.
   Thereby, adjusting for BMI may result in overadjustment of the
   associations between inflammation and depression. As an alternative,
   examining to what extent BMI plays an explaining role seems more
   appropriate.

   Finally, it is suggested that some antidepressants exert an
   anti-inflammatory effect^[76]30,[77]31. In order to check whether
   antidepressant use is of influence, we have again performed the
   differential gene expression analysis with exclusion of cases using
   antidepressant medication.

   All statistical analyses were performed in R software version
   3.4.1^[78]32.

Results

   The sample about which is reported below consisted of 881 patients with
   current MDD and 331 healthy controls (65.3% females, mean age 41.1).
   Stratification of the current MDD group according to change in appetite
   and/or weight during a major depressive episode resulted in 246
   participants with MDD hyperphagia and 341 participants with MDD with
   hypophagia. As compared to controls, MDD cases were more likely to be
   female, younger, current smokers, and to use antidepressant and
   anti-inflammatory medications, and had a higher IDS score. The subgroup
   MDD with hyperphagia contained significantly more females, less current
   smokers, and had a significantly higher BMI than the subgroup MDD with
   hypophagia. Age and IDS score did not differ between these subgroups
   (Table [79]1).

Table 1.

   Sample characteristics (N = 1212)
   Healthy controls all MDD P value (all MDD vs. controls) MDD with
   hyperphagia MDD with hypophagia P value (overall)
   N 331 881 246 341
   Female, N (%) 199 (60.1) 593 (67.3) <0.05^a 185 (75.2)^d 228 (66.9)^d
   <0.001^a
   Age, mean (sd.) 42.6 (14.6) 40.6 (12.2) <0.05^b 41.1 (11.7) 40.0
   (12.1)^e <0.05^c
   BMI, mean (sd.) 25.5 (4.7) 25.9 (5.4) 0.18^b 28.4 (6.0)^d,e 24.4
   (4.9)^d,e <0.0001^c
   IDS, mean (sd.) 8.2 (7.4) 32.3 (12.3) <0.0001^b 33.8 (11.6)^e 32.4
   (13.3)^e <0.0001^c
   Current anxiety diagnosis, N (%) 0 (0) 582 (66.1) <0.0001^a 170 (69.1)
   221 (64.8) <0.0001^a
   Anti-inflammatory drug use, N (%) 6 (1.8) 42 (4.8) <0.05^a 14 (5.7) 14
   (4.1) <0.05^a
   Current smoking, N (%) 90 (27.2) 406 (46.1) <0.0001^a 84 (34.1)^d 199
   (58.4)^d <0.0001^a
   Antidepressant medication use, N (%) 2 (0.6) 398 (45.2) <0.0001^a 103
   (42.9) 157 (46.0) <0.0001^a
   [80]Open in a new tab

   P value in the fourth column is for testing difference between total
   current MDD group and controls. P value in the seventh column is for
   testing differences between the MDD subgroups with increased and
   decreased appetite and/or weight change and controls and posthoc
   pairwise comparison

   BMI body mass index, IDS inventory of depressive symptomatology, MDD
   major depressive disorder

   ^aχ^2-test

   ^bTwo-sided t-test

   ^cANOVA

   ^dDifferent from the other appetite and/or weight change subgroup

   ^eDifferent from controls

Inflammatory pathway enrichment analyses

   Using genome-wide whole-blood gene expression measurements, we
   performed differential gene expression analysis between all patients
   with current MDD, MDD with hyperphagia, and MDD with hypophagia vs.
   controls. There were 50, 263, and 308 differentially expressed genes
   (FDR < 0.1) in MDD with hyperphagia subgroup, MDD with hypophagia
   subgroup and the total current MDD group, respectively. Supplementary
   Table [81]4 shows the results of the differential gene expression
   analysis. Overlap of differentially expressed genes between the groups
   is shown in the Venn diagram in Supplementary Fig. [82]1. While the
   difference in the number of significantly differentially expressed
   genes may be due to the different sample size of the subgroups, the
   strength and the direction of effects across subgroups were highly
   consistent (r = 0.77; except for three genes, the directions of the
   effects were all the same between the two MDD subgroups).

   All the P values generated in the differential gene expression analysis
   were used as input for ‘genome-wide’ enrichment analyses. For 162
   inflammatory pathways, we tested whether the median P value was lower
   than the median P value of the genes outside the pathway (“Methods”).
   Most pathways (n = 32, at FDR < 0.1) were enriched in the MDD with
   hyperphagia subgroup, including pathways related to inflammasomes,
   apoptosis and cytokine signaling, namely IL-2, IL-3, IL-4, IL-5, IL-6,
   IL-8, IL-12, and IL-22. The most strongly enriched pathways are the
   ‘PID caspase pathway, ‘BioCarta GATA3 pathway’, and ‘IL-3, IL-5 and
   GM-CSF signaling pathway’. Only two pathways were enriched for the MDD
   with hypophagia subgroup (i.e., ‘PID IL-8 CXCR2 pathway’ and ‘Reactome
   adaptive immune system’), and fourteen pathways for the overall current
   MDD group. The most strongly enriched pathways for the overall current
   MDD group are ‘KEGG natural killer cell mediated cytotoxicity’, ‘KEGG T
   cell receptor signaling pathway’ and ‘PID IL-8 CXCR2 pathway’. At a
   stricter FDR of <0.05, 21 pathways were enriched in the MDD with
   hyperphagia subgroup, none in the MDD with hypophagia subgroup and ten
   in the overall current MDD group.

   Table [83]2 presents 35 out of the 162 pathways that are significantly
   enriched in at least one MDD (sub)group (FDR < 0.1) and P values
   obtained from these analyses. Enrichment analyses were repeated after
   an additional permutation-based correction, taking into account
   correlations between gene expression profiles of pathways, providing
   substantially overlapping results. Only the results of the enrichment
   analysis of all MDD cases vs. controls differed. The ‘Reactome NLRP3
   inflammasome pathway’ reached the significance threshold in in the
   permutation-based analyses.

Table 2.

   35 enriched pathways at FDR < 0.1 in at least one group of MDD patients
   as compared to controls in enrichment analyses of inflammatory pathways
   P value
   Pathway All MDD MDD with hyperphagia MDD with hypophagia
   KEGG natural killer cell mediated cytotoxicity 7.94E-6 5.20E-4 0.0138
   KEGG T cell receptor signaling 1.39E-4 2.09E-3 0.0411
   PID IL-8 CXCR2 1.39E-4 2.94E-3 5.79E-4
   Reactome adaptive immune system 3.70E-4 4.25E-3 4.98E-4
   Reactome immune system 5.60E-4 2.07E-3 3.98E-3
   PID IL-8 CXCR1 5.71E-4 6.19E-3 2.32E-3
   Reactome signaling by interleukins 6.93E-4 1.22E-3 0.0344
   PID CXCR4 pathway 1.41E-3 4.80E-3 7.66E-3
   Reactome IL-3, IL-5 and GM-CSF signaling 2.80E-3 4.70E-4 8.10E-3
   Reactome apoptosis 4.85E-3 0.0147 0.0101
   Reactome inflammasomes 6.08E-3 1.71E-3 0.149
   BioCarta natural killer cells pathway 2.62E-3 0.0637 4.17E-3
   PID aMb2 neutrophils pathway 7.69E-3 0.0230 9.63E-3
   PID CD8 TCR downstream pathway 8.42E-3 0.0283 0.0380
   PID caspase pathway 0.0188 3.76E-4 0.0558
   BioCarta GATA3 pathway 0.0280 4.10E-4 0.0359
   PID NFAT transcription factor pathway 0.0297 5.10E-4 0.154
   BioCarta PAR1 pathway 0.203 1.73E-3 0.127
   Reactome the NLRP3 inflammasome 0.0101 3.06E-3 0.0240
   BioCarta IL22BP pathway 0.0749 4.11E-3 7.54E-3
   KEGG apoptosis 0.0452 5.10E-3 0.169
   PID IL-4 2pathway 0.474 5.48E-3 0.170
   ST interleukin 4 pathway 0.110 5.67E-3 0.0101
   Reactome IL-6 signaling 0.143 6.13E-3 0.107
   PID angiopoietin receptor pathway 0.0136 6.29E-3 0.0284
   Reactome nucleotide-binding domain leucine-rich repeat containing
   receptor NLR signaling pathways 0.0520 7.73E-3 0.161
   Reactome cytokine signaling in immune system 0.0129 8.36E-3 0.128
   BioCarta IL2RB pathway 0.123 0.0125 0.208
   BioCarta mitochondria pathway 0.303 0.0155 0.224
   BioCarta IL-6 pathway 0.497 0.0162 0.182
   BioCarta TNFR1 pathway 0.389 0.0167 0.403
   KEGG JAK-STAT signaling pathway 0.173 0.0173 0.322
   Reactome extrinsic pathway for apoptosis 0.0147 0.0174 0.308
   PID IL-2 STAT5 pathway 0.137 0.0181 0.180
   PID IL-12 2pathway 0.0206 0.0190 0.0372
   [84]Open in a new tab

   The values represent the raw P values obtained from the enrichment
   analyses. Bold values correspond to an FDR < 0.1

   The raw P values and corresponding FDRs of the enrichment analyses of
   all 162 inflammatory pathways are listed in Supplementary Table [85]5.
   Supplementary Table [86]6 presents, of the 25% percentile genes with
   the lowest P value within each pathway, the percentage of genes with a
   positive effect size in the differential gene expression analysis

   CD cluster of differentiation, CXCR chemokine receptor, GM-CSF
   granulocyte-macrophage colony-stimulating factor, IL interleukin,
   IL22BP interleukin-22 binding protein, IL2RB interleukin-2 receptor
   beta subunit, JAK Janus Kinase, KEGG Kyoto Encyclopedia of Genes and
   Genomes, NFAT nuclear factor of activated T cells, NLR
   nucleotide-binding domain leucine-rich repeat, NLRP3 nucleotide-binding
   domain leucine-rich repeat containing family pyrin domain-containing-3,
   PAR1 protease-activated receptors, ST signaling transduction KE, STAT
   signal transducer and activator of transcription, TCR T cell receptor,
   TNFR1 tumor necrosis factor receptor 1

   The Venn diagram in Fig. [87]1 shows the overlap between the enriched
   pathways at an FDR < 0.1 in the three groups of MDD patients. In
   general, it could be said that inflammatory pathways were more strongly
   involved in the MDD subtype endorsing hyperphagia. There are 21
   inflammatory pathways uniquely enriched at FDR < 0.1 in the MDD with
   hyperphagia subgroup, which are listed in the lower 21 rows of Table
   [88]2. Noteworthy, among these pathways are two pathways on IL-6
   mediated signaling, two on IL-4 mediated signaling and four
   apoptosis-related pathways (i.e., ‘PID caspase pathway’, ‘KEGG
   apoptosis’, ‘BioCarta mitochondria pathway’, ‘Reactome extrinsic
   pathway for apoptosis’). The majority of pathways enriched in the total
   MDD group were also enriched in the MDD with hyperphagia subgroup. Only
   three pathways (i.e., downstream signaling of naïve CD8+ T cells, aMb2
   integrin, and natural killer cells) were uniquely enriched when
   considering together all current MDD cases. The only two enriched
   pathways in MDD cases with hypophagia (i.e., adaptive immune system and
   IL-8 CXCR2 signaling pathway) were also enriched in both analyses of
   the subgroup of MDD with hyperphagia and the total current MDD group.

Fig. 1.

   [89]Fig. 1
   [90]Open in a new tab

   Number of enriched inflammatory gene expression pathways per group at
   FDR < 0.1

Enrichment analyses using all canonical pathways

   Enrichment analyses using all 1329 canonical pathways were performed to
   test whether inflammation pathways indeed show up importantly as
   compared to the other canonical pathways. Supplementary Table [91]7
   shows the 32 out of the 1329 pathways that are significantly enriched
   in at least one MDD (sub)group as compared to controls (FDR < 0.1). For
   the total MDD group, 27 pathways were enriched, of which 9 were part of
   the 162 inflammatory pathways we initially selected. Similar to the
   main enrichment analysis, the most strongly associated pathway was the
   ‘KEGG natural killer cell mediated cytotoxicity’. Note that the
   expression profiles of the total group of current MDD cases are
   enriched for many pathways regarding T cell signaling. The majority of
   canonical pathways enriched for the MDD with hyperphagia subgroup (five
   out of nine) were inflammatory pathways. The most strongly enriched
   pathway for this subgroup is ‘BioCarta caspase pathway’, which was not
   selected as an inflammatory pathway in the main analysis. The top
   pathways in the main enrichment analysis did survive the multiple
   testing penalty in this analysis including all canonical pathways. No
   enriched pathway was seen in MDD with hypophagia. These results confirm
   that even when tested together with all kinds of different biological
   pathways, inflammation still plays a prominent role in the gene
   expression profiles of the MDD with hyperphagia subgroup.

BMI, inflammation, antidepressant use, and MDD with hyperphagia

   In order to examine the impact of BMI on the association between
   inflammatory gene expression and MDD with hyperphagia, we repeated for
   this subgroup the pathway enrichment analysis after correcting the gene
   expression analysis for BMI.

   A relative attenuation of the signals was shown at the gene level as
   well as at the pathway level. The mean effect sizes of genes that were
   significantly differently expressed in MDD with hyperphagia as compared
   to controls were reduced by 13.1% after BMI adjustment. Of the 32
   initially enriched pathways in MDD with hyperphagia, 22 were still
   nominally significant after BMI adjustment. Five pathways were enriched
   at an FDR < 0.1, namely ‘PID NFAT transcription factor pathway’, ‘PID
   IL-8 CXCR2 pathway’, ‘PID caspase pathway’, ‘PID CXCR4 pathway’ and
   ‘BioCarta GATA3 pathway’. Except for ‘Reactome IL-3, IL-5 and GM-CSF
   signaling’ and ‘KEGG natural killer cell mediated cytotoxicity’, the
   most strongly associated pathways in the inflammatory pathways
   enrichment analysis were also enriched after BMI adjustment. The
   results of the inflammatory pathway enrichment analysis after BMI
   adjustment are listed in Supplementary Table [92]8.

   Additional differential gene expression analysis after exclusion of
   antidepressant users suggests no major influence of its use within the
   subgroup of MDD with hyperphagia. The beta-beta plot in Supplementary
   Figs. [93]2 and [94]3 show the consistencies between the two
   differential gene expression analyses. Spearman ρ between the effect
   sizes of all genes with and without these antidepressant using cases
   was 0.75 and 1 of the significantly differentially expressed genes
   (n = 6 at FDR < 0.1). This is in line with NESDA’s previous
   transcriptomic study that found no effect of antidepressant use in
   expression profiles of currently depressed cases^[95]16.

Discussion

   This study evaluated enrichment of inflammatory pathways among mean
   gene expression profiles of two MDD subgroups that can be distinguished
   based on the direction of appetite and/or weight change (hyperphagia
   and hypophagia) during an episode, compared to controls. The large
   majority of enriched inflammatory pathways were found only when
   focusing on the MDD with hyperphagia subgroup. Almost all the
   inflammatory pathways that were found to be enriched in the pooled
   group of patients with MDD were also detected in the MDD with
   hyperphagia subgroup, despite the substantial reduction in sample size.
   This suggests that the inflammatory signal in the pooled group of
   patients with MDD may be driven by this subgroup. These findings
   complement previous serum proteomic and genetic studies showing,
   respectively, higher circulating levels of inflammatory markers and
   increased genetic risk for high CRP levels in MDD patients with
   hyperphagia^[96]9–[97]12,[98]14,[99]15. The current study indicates
   that, also at the transcriptome level, inflammation is especially
   pronounced in individuals who experience hyperphagia during their
   current depressive episode. Moreover, we show which inflammatory
   pathways are most prominent. Conclusions on the directions of this
   involvement, however, cannot be drawn.

   In the MDD with hyperphagia subgroup, there were three pathways
   associated in the main inflammatory pathway analysis, the inflammatory
   pathway enrichment analysis after BMI adjustment as well as in the
   canonical pathway enrichment analysis. These robustly enriched pathways
   are the ‘BioCarta GATA3 pathway’, ‘PID NFAT transcription factor
   pathway’, and the ‘PID caspase pathway’. These pathways include several
   transcription factors (GATA3, NFAT) and enzymes (caspases) that have a
   major role in the onset of the inflammatory response. For instance,
   caspase-1 activates the pro-inflammatory cytokines IL-1β and IL-18, by
   cleaving their precursors. Caspase-1 have been shown to be associated
   with depression-like behavior in mice before^[100]33,[101]34 and
   increased gene expression of its coding gene were observed in blood
   cells of untreated depressed patients^[102]35. Caspase-1 is also a
   downstream target of the inflammasome complex^[103]36, groups of
   protein complexes that are triggered by molecular patterns induced by
   physiological and psychosocial stress. According to the inflammasome
   hypothesis^[104]36, psychological stress-induced activation of the
   NLRP3 inflammasome regulates caspase-1 activation and pro-inflammatory
   cytokine secretion, which causes subsequent development of
   depression^[105]36,[106]37. Interestingly, in our study two pathways
   related to inflammasomes (‘Reactome inflammasomes’ and ‘Reactome NLRP3
   inflammasome’) were strongly enriched in the MDD with hyperphagia
   subgroup. It is suggested that the NLRP3 inflammasome is involved in
   comorbid diseases such as obesity^[107]36,[108]38,[109]39. In mice,
   targeting the NLRP3 inflammasome has already been successful in
   reducing depressive-like behavior^[110]40. To our knowledge, there are
   no studies suggesting the NLRP3 inflammasome is specifically involved
   in depression with hyperphagia. Furthermore, caspase-1 is suggested to
   contribute to glucocorticoid resistance and, subsequently, increases
   levels of glucocorticoids (e.g., cortisol). These are reflective signs
   of HPA-axis hyperactivation, one of the most studied biological risk
   factors associated with MDD^[111]4. If HPA-axis activation associated
   with MDD involves caspase-1 in the molecular mechanisms, enrichment of
   these pathways might relate to that.

   Among the pathways uniquely enriched in the MDD with hyperphagia were
   several linked to signaling of IL-2 or its receptor, IL-4, TNF and
   IL-6, which is in line with earlier findings showing that IL-6 blood
   concentrations were especially higher in MDD patients with
   hyperphagia^[112]13,[113]14. Other enriched pathways in the MDD with
   hyperphagia subgroup were related to apoptosis. Increased apoptosis and
   disrupted proliferation of peripheral mononuclear blood cells in
   depressed patients compared to controls is previously
   suggested^[114]41–[115]45.

   There were two enriched pathways in expression profiles of MDD with
   hypophagia, which were also enriched in the two other groups (i.e.,
   ‘Reactome adaptive immune system’ and ‘PID IL-8 CXCR2 pathway’). Three
   pathways were uniquely enriched in overall current MDD group (i.e.,
   ‘BioCarta natural killer cells pathway’, ‘PID a[M]b[2] neutrophils
   pathway’, and ‘PID downstream signaling of naïve CD8 +T cells’). The
   latter three may be enriched because of greater power due to the
   increase in sample size in the total MDD group compared to the MDD
   subgroups. These five pathways are likely to be generally involved in
   MDD and are related to the innate immune system as well as the adaptive
   immune system. The involvement of activation of the innate immune
   system in MDDs pathogenesis (e.g., pro-inflammatory cytokine secretion)
   has been widely reported^[116]46. Innate immune suppression, also in
   the form of decreased natural killer cell mediated cytotoxicity, has
   been associated with MDD before^[117]16,[118]45,[119]47. Impairments in
   the adaptive immune system in relation to MDD have also been
   reported^[120]17,[121]44.

   The present results confirm also that BMI may play a role in the
   relationship between inflammation and MDD with hyperphagia. After
   adjustment for BMI, the pathway signals were relatively attenuated,
   although the top pathways (‘PID NFAT transcription factor pathway’,
   ‘PID IL-8 CXCR2 pathway’, ‘PID caspase pathway’, ‘PID CXCR4 pathway’,
   and ‘BioCarta GATA3 pathway’) remained significantly associated. The
   pathways ‘Reactome immune system’, ‘KEGG JAK-STAT signaling pathway’,
   and ‘KEGG apoptosis’ are found to be significantly enriched in obesity
   gene expression analyses^[122]48–[123]50 and were strongly attenuated
   after BMI correction (FDR: 0.034 to 0.453; 0.094 to 0.426; and 0.049 to
   0.203, respectively), indicating that these pathways especially show
   overlap between obesity and depression.

   Rather than a simple confounder, the role of BMI may be described in a
   more complex interrelation. It has already been reported that
   especially MDD with increased appetite and/or weight during a
   depressive episode share genetic risk with BMI as well as inflammatory
   marker levels^[124]15,[125]28. Bidirectional Mendelian Randomization
   analyses between BMI and MDD^[126]28 and BMI and depressive
   symptoms^[127]29 provided evidence for a causality of BMI for MDD and
   depressive symptoms but not for a reverse causality. In other words,
   these analyses suggest that BMI is a causal risk factor or associated
   with causal risk factors for MDD and depressive symptoms. In short,
   inflammation is a characteristic of both obesity and a subgroup of MDD
   patients with a higher BMI. It could be hypothesized that a higher BMI
   may induce a chronic inflammatory state, which, in turn contributes to
   development of depression symptomatology. The link with this specific
   symptom may be explained by the negative effect of inflammation on
   important neuroendocrine regulators of energy homeostasis, such as on
   the leptin–melanocortin signaling, which regulates food intake and
   promotes energy expenditure. It is suggested that central inflammation
   may impair the functioning of leptin receptors expressed in the brain,
   resulting in leptin resistance that blunts its anorexigenic effect and
   consequently disinhibits feeding despite increasing circulating leptin
   (a process comparable with insulin resistance in type 2
   diabetes)^[128]38,[129]51. In line with this hypothesis, high leptin
   levels have been found specifically in MDD patients with hyperphagia,
   independently from BMI^[130]52. Furthermore, MDD patients with
   hyperphagia have been shown to carry a higher number of genetic risk
   variants for increased BMI, CRP and leptin levels compared to
   controls^[131]15.

   In addition to BMI, it could be that MDD with hyperphagia share
   underlying mechanisms with bipolar disorder. Serum levels of
   pro-inflammatory cytokines previously associated with MDD were also
   associated with bipolar disorder^[132]53. A probabilistic
   approach^[133]54 suggested that, among the individuals experiencing a
   depressive episode without a prior history of manic episodes,
   individuals with hyperphagia were more probable to have a later
   diagnosis of bipolar disorder.

   The potential causal role inflammation in MDD pathogenesis has
   stimulated the examination of antidepressant effect of
   cytokine-inhibiting medication^[134]55,[135]56. Clinical trials carried
   out so far reported promising results; overall reductions in depressive
   symptoms severity after anti-inflammatory treatment were seen in a
   large meta-analysis^[136]57, although effects were highly heterogeneous
   suggesting the presence of subgroups with differential effect size.
   Also, anti-inflammatory treatment was suggested to be especially
   beneficial for subgroups of patients marked by elevated
   inflammation^[137]55–[138]58. These data underline the need to identify
   subgroups of patients who may benefit most from an anti-inflammatory
   treatment according to a personalized medicine approach. The current
   findings suggest that the subgroup may be marked by symptoms of
   hyperphagia.

   Several limitations of our study should be noted. Analyzing subgroups
   of current MDD patients have come on cost of power. This may have led
   to false positives or truly associated pathways that were not detected.
   However, we have applied permutation-based correction for multiple
   testing additionally, in order to check for false positives, which
   yielded similar results. False positives due to correlations between
   gene expression patterns across different pathways are therefore less
   likely. Also, whole-blood gene expression was measured. Inflammatory
   gene expression profiles in other tissues (e.g., relevant brain areas)
   that are informative about the pathophysiology of MDD might have been
   different. Finally, we did not correct for cortisol levels or HPA-axis
   activity. In NESDA, cases marked by hyperphagia had different cortisol
   levels compared to cases marked by hypophagia. We did not control for
   cortisol levels since we were mainly interested in the MDD with
   hyperphagia subgroup, which was found to have comparable cortisol
   levels to controls^[139]11,[140]59.

   To conclude, inflammatory pathways are enriched in expression profiles
   of MDD patients with hyperphagia. This study further supports the idea
   that there is a distinct depressive subtype, with partly differential
   pathophysiology regarding immuno-metabolic processes. Future research
   will have to show whether this subgroup benefits most from
   anti-inflammatory treatment strategies, according to a personalized
   medicine approach, and system biology approaches will have to shed
   light on the specific transcriptomic pathways involved and identify
   genes driving the association.

Supplementary information

   [141]Supplementary Tables 1-3, 5-8^ (116.9KB, docx)
   [142]Supplementary Table 4^ (5.7MB, xls)
   [143]41398_2019_528_MOESM3_ESM.tif^ (64.4KB, tif)

   Supplementary Figure 1: Number of differentially expressed genes per
   (sub)group of MDD cases at FDR<0.1
   [144]41398_2019_528_MOESM4_ESM.tif^ (47.5KB, tif)

   Supplementary Figure 2: Beta-beta plot of results of the differential
   gene expression analyses including and excluding cases using
   antidepressant medication
   [145]41398_2019_528_MOESM5_ESM.tif^ (26.9KB, tif)

   Supplementary Figure 3: Beta-beta plot of the 6 differentially
   expressed genes including and excluding cases using antidepressant
   medication

Acknowledgements