Accepted Manuscript

Are there differences in lipid peroxidation and immune

biomarkers between major depression and bipolar disorder:
Effects of melancholia, atypical depression, severity of illness,
episode number, suicidal ideation and prior suicide attempts

Magdalena Sowa-Kućma, Krzysztof Styczeń, Marcin Siwek,

Paulina Misztak, Rafał J. Nowak, Dominika Dudek, Janusz K.
Rybakowski, Gabriel Nowak, Michael Maes

PII: S0278-5846(17)30474-8
DOI: doi: 10.1016/j.pnpbp.2017.08.024
Reference: PNP 9213
To appear in: Progress in Neuropsychopharmacology & Biological Psychiatry
Received date: 14 June 2017
Revised date: 9 August 2017
Accepted date: 30 August 2017

Please cite this article as: Magdalena Sowa-Kućma, Krzysztof Styczeń, Marcin Siwek,
Paulina Misztak, Rafał J. Nowak, Dominika Dudek, Janusz K. Rybakowski, Gabriel
Nowak, Michael Maes , Are there differences in lipid peroxidation and immune
biomarkers between major depression and bipolar disorder: Effects of melancholia,
atypical depression, severity of illness, episode number, suicidal ideation and prior suicide
attempts. The address for the corresponding author was captured as affiliation for all
authors. Please check if appropriate. Pnp(2017), doi: 10.1016/j.pnpbp.2017.08.024

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 ACCEPTED MANUSCRIPT

Research Paper

Are there differences in lipid peroxidation and immune biomarkers between major depression

and bipolar disorder: effects of melancholia, atypical depression, severity of illness, episode

number, suicidal ideation and prior suicide attempts.

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Magdalena Sowa-Kućma1,# , Krzysztof Styczeń2 , Marcin Siwek2 , Paulina Misztak1,3 , Rafał J.

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Nowak4 , Dominika Dudek2 , Janusz K. Rybakowski5 , Gabriel Nowak1,3,# , Michael Maes6,7,8,9

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1. Institute of Pharmacology, Polish Academy of Sciences, Laboratory of Trace Elements

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Neurobiology, Department of Neurobiology, Smetna Street 12, 31-343 Krakow, Poland;
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2. Department of Affective Disorders, Chair of Psychiatry, Jagiellonian University Medical

College, Kopernika 21a, 31-501 Krakow, Poland;

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3. Department of Pharmacobiology, Jagiellonian University Medical College, Medyczna 9, 30-

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688 Krakow, Poland;

4. Department of Drug Management, Jagiellonian University Medical College, Grzegórzecka 20,

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31-531 Krakow, Poland;

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5. Department of Adult Psychiatry, Poznań University of Medical Sciences, Szpitalna 27/33, 60-

572 Poznań, Poland;

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6. Department of Psychiatry, Faculty of Medicine, Chulalongkorn University, Bangkok,

Thailand;

7. IMPACT Strategic Research Centre, Deakin University, School of Medicine and Barwon

Health, Geelong, VIC, Australia;

8. Department of Psychiatry, Medical University of Plovdiv, Plovdiv, Bulgaria

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9. Revitalis, Waalre, the Netherlands

#Corresponding authors:

Magdalena Sowa-Kućma, Ph.D.

Institute of Pahrmacology

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Polish Academy of Sciences

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Smetna Street 12, Krakow, Poland

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sowa@if-pan.krakow.pl

https://scholar.google.pl/citations?user=ane1nEcAAAAJ&hl=pl

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Gabriel Nowak, Ph.D.

Institute of Pahrmacology
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Polish Academy of Sciences

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Smetna Street 12, Krakow, Poland

nowak@if-pan.krakow.pl
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https://scholar.google.pl/citations?user=oqCwzOMAAAAJ&hl=pl
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Abstract

Background: There is evidence that major depression (MDD) and bipolar disorder (BD) are

accompanied by activated immune & oxidative (I&O) pathways.

Methods: To compare I&O biomarkers between MDD and BD we assessed serum levels of

thiobarbituric acid reactive substances (TBARS; a lipid peroxidation marker), soluble

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interleukin-2 receptor (sIL-2R), sIL-6R, IL-α, sIL-1R antagonist (sIL-1RA), tumor necrosis

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factor receptor 60 kDa/80 kDa (sTNFR60/R80) in 114 MDD and 133 BD patients, and 50

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healthy controls. We computed z-unit weighted indices reflecting the 5 cytokine receptor levels

(zCytR), cell-mediated immunity (zCMI) and I&O pathways (zCMI+TBARS).

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Results: There are no significant differences in biomarkers between MDD and BD. BD/MDD
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with atypical features is characterized by increased sIL-6R and TBARS, whereas melancholia is

associated with higher TBARS and lower sTNFR60 levels. Severity of illness, as measured with
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the Hamilton Depression Rating Scale, is correlated with increased sIL-6R, sTNFR80, TBARS,
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zCytR and zCMI+TBARS. The number of episodes the year prior to blood sampling is positively

associated with sTNFR80, TBARS, zCMI, zCMI+TBARS, while number of hospitalizations is

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positively associated with sIL-1RA. Prior suicidal attempts are associated with increased sIL-
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1RA, IL-1α, zCMI, TBARS and zCMI+TBARS, while TBARS is associated with current

suicidal ideation.
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Conclusions: There are no I&O biomarker differences between MDD and BD. Atypical

depression is associated with increased IL-6 trans-signaling and lipid peroxidation. Severity of

depression, number of episodes and suicidal attempts are associated with activated I&O

pathways. Increased TBARS is the single best predictor of BD/MDD, atypical depression,

melancholia and current suicidal ideation.

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Key words: major depression, bipolar disorder, melancholia, immune, cytokines, oxidative

stress

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1. Introduction

There is now evidence that major affective disorders (MAFD), namely major depression

(MDD) and bipolar disorder (BD), are characterized by multiple alterations in neuro-immune

and neuro-oxidative pathways (Maes et al., 1995b; 2011; Berk et al., 2011; 2013; Moylan et al.,

2014). Already in the 1990ties it was shown that BD, either the acute phase of depression or

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mania, and MDD are accompanied by signs of a Thelper (Th)-1 shift and inflammation (Maes et

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al., 1990; 1991; 1994b; 1995). Both MDD and BD are accompanied by increased levels of

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soluble interleukin (IL)-2 receptor (sIL-2R), pro-inflammatory cytokines, including IL-6 and its

receptor, the sIL-6R; positive acute phase proteins, including haptoglobin, fibrinogen, alpha 1-

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acid-glycoprotein and hemopexin, and complement factors (Maes et al., 1990; 1995a; 1995b;
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1995; 1997a; 1997b;Wadee et al., 2002; Ortiz-Dominguez et al., 2007; Pandey et al., 2015).

Increased serum IL-6 coupled with increased sIL-6R levels indicate that pro-inflammatory IL-6
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trans-signaling may be activated in MDD and BD (Maes et al., 1995b; 2014). More recent
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findings indicate that other pro-inflammatory cytokines and immune variables are elevated in

both MAFD subtypes, including tumor necrosis factor (TNF)-α (Mikova et al., 2001; O’Brien et
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al., 2006; Kim et al., 2007) and plasma levels of sTNF receptor p55/60 (R1) and p75/80 (R2)
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(Ortiz-Dominguez et al., 2007; Barbosa et al., 2011; Grassi-Oliviera et al., 2009; Moughrabi et

al., 2014; Rizavi et al., 2016; Pandey et al., 2015). The sTNFRs may bind TNF-α, thereby acting
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as decoy receptors attenuating TNFα signaling (Selinsky et al., 1998; Su et al., 1998). There is

also evidence that IL-1β production and serum levels of its soluble receptor antagonist (sIL-

1RA) are elevated in both BD and MDD (Maes et al., 1991; 1995c; 1997a; Levine et al., 1999;

Monfrim et al., 2014; Pandey et al., 2015).

Meta-analyses have consolidated the abovementioned findings in both MAFD subtypes,

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showing that MDD is accompanied by macrophagic (M1) and Th-1 activation as indicated by

increased IL-1β, sIL-1RA, IL-6, TNFα, and sIL-2R levels, while T regulatory (Treg) and Th2

cytokines are not significantly altered (Howren et al., 2009; Dowlati et al., 2009; Liu et al., 2012;

Hiles et al., 2012; Valkanova et al., 2013; Haapakoski et al., 2015; Kohler et al., 2017). In BD,

meta-analyses showed increased sIL-1RA, sIL-6R, TNFα, sTNFR60 and sIL-2R levels,

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indicating monocytic and Th-1 activation, but additionally signs of Treg and Th-2 cell activation

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may be present (Modabbernia et al., 2013; Munkholm et al., 2013).

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There is also evidence that MDD and BD are accompanied by lipid peroxidation coupled

with lowered levels of antioxidants, which protect against lipid peroxidation, including high-

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density lipoprotein (HDL) cholesterol, vitamin E and paraoxonase 1 activities (Maes et al.,
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1994a; 1997c; 1999; 2000; Peet et al. 1998; Bilici et al., 2001; Sobczak et al., 2004; Tsuboi et al.,

2006; Bortolasci et al., 2014; Nunes et al., 2015; Moreira et al., 2017). Increased levels of lipid
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peroxidation as measured with plasma levels of thiobarbituric acid reactive substances (TBARS)
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or malondialdehyde (MDA) have frequently been described in depression (Bilici et al., 2001;

Khanzode et al., 2003; Ozcan et al., 2004; Sarandol et al., 2007; Maes et al., 2013a; 2013b) and
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bipolar disorder (Ranjekar et al., 2003; Machado-Vieira et al., 2007; Kunz et al., 2008;
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Bengesser et al., 2015; Chowdhury et al., 2017). Different meta-analyses in MAFD have

confirmed increased MDA/TBARS levels in both MAFD subtypes (Andreazza et al., 2008;
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Brown et al., 2014; Liu et al., 2015).

Based on these (and other) results, BD and MDD are now conceptualized as neuro-

immune-oxidative disorders with secondary neurotoxicity and excitotoxicity coupled with

neuronal damage, disorders in synaptic plasticity and neurogenesis, changes in neurotransmitter

signalling and receptor expression, and lowered neuroprotection (Maes et al., 2009; Berk et al.,
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2011; Maes et al., 2011; Moylan et al., 2014).

Previously, we have shown that atypical and melancholic depression and the acute phase

of BD and MDD are associated with different immune biomarkers (Siwek et al., 2016a; Sowa-

Kucma et al., submitted). Nevertheless, there is a paucity of data directly comparing neuro-

immune and neuro-oxidative pathways among MDD and BD. This knowledge is important to

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futher examine whether both phenotypes are biologically continuous categories, lying within a

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spectrum of pathophysiological alterations, or whether BD and MDD are dichotomous categories

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with distint pathophysiologies (Akiskal, 2003; Benazzi, 2005). Previous research showed some

differences between both MAFD subgroups with the number of depressive symptoms being

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higher among patients with bipolar (BP)1, followed by BP2 and MDD (Moreno et al., 2012;
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Perlis et al., 2006), while BD patients have an earlier age at onset than MDD patients (Moreno et

al., 2012; Perlis et al., 2006). BD and especially BP2 patients are overrepresented among
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attempted and committed suicides, with a trend toward higher rates of prior suicide attempts in
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those with BP2 versus BP1 disorder (Rihmer, 2007).

Thus, the aims of this study are to compare immune and oxidative biomarkers, including
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TBARS, sIL-2R, sIL-6R, IL-α, sIL-1RA, sTNF-R1 and sTNF-R2 among MDD and BD patients
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and to examine whether these biomarkers are associated with MAFD characteristics including

subtypes of depression, number of episodes, age at onset, suicidal ideation or previous suicidal
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attempts.

2. Subjects and Methods

2.1. Participants
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This is a case-control study, which included 50 healthy controls and 247 MAFD patients,

namely 114 patients with MDD and 133 with BD, comprising 69 bipolar (BP)1 and 64 BP2

patients. Both males and females were included. All participants were of Caucasian ethnicity and

Polish nationality and aged 21–70 years old. We previously published studies on the

comparisons between BD patients and controls (Siwek et al., 2016a,b) and between MDD

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patients and controls (Sowa-Kucma et al., submitted). Here we combine both data sets in order to

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examine differences in biomarkers between MDD and BD and their relationships with MAFD

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characteristics. Patients were in- or outpatients admitted to the Department of Psychiatry,

University Hospital Krakow, Krakow, Poland, during the period 21 September 2009 to 30 July

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2013. The diagnoses BD or MDD were made by a psychiatrist using the semi-structured clinical
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interview for DSM-IV interview (SCID) axis I Disorders and according to DSM-IV-TR

diagnostic criteria (American Psychiatric Association, 2000). Both BD and MDD participants
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were recruited in the acute phase of depression or in the euthymic phase. Healthy controls were
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recruited by word of mouth from hospital staff and from friends and relatives of hospital staff.

Excluded were patients with axis-1 DSM-IV-TR diagnoses other than BD or MDD, including
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schizophrenia, psychoactive substance dependence (except tobacco use disorder), schizoaffective

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disorder, and psycho-organic disorders. We excluded healthy controls for any current or life-time

axis-I diagnoses, including MDD and BD. Patients and controls were excluded for a) axis-II
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DSM-IV-TR personality disorders; b) obesity, defined as a body mass index (BMI) > 30; c)

acute inflammatory responses due to for example infections, wounds or injuries within one

month prior to inclusion in the study; d) major medical diseases, including primary

adrenocortical insufficiency, (auto)immune diseases, renal failure, chronic pancreatitis, primary

hypoaldosteronism, carcinomas, hypoparathyroidism, hyperthyroidism, megaloblastic anaemia

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due to iron deficiency, thalasemia, hemochromatosis, liver cirrhosis, Wilson’s disease, nephrotic

syndrome and burns; e) use of medications such as hydralazine; nonsteroidal anti-inflammatory

drugs (such as ibuprofen, acetylsalicylic acid and indomethacin); fluorochinolones; tetracyclines;

chelating agents; glucocorticosteroids; calcium and iron; and f) pregnant or breast feeding

women. MDD and BD patients were treated with monotherapy or combinatorial treatments,

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including selective serotonin reuptake inhibitors (SSRIs), selective noradrenaline reuptake

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inhibitors (SNRIs), tricyclic antidepressants (TCAs), mirtazapine, atypical antipsychotics,

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lithium, lamotrigine; quetiapine, olanzapine, lamotrigine, carbamazepine, and depakine. The

study was approved by the bioethical committee of the Jagiellonian University, Krakow, Poland

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(decision number KBET/77/B/2009; 25.06.2009) and all subjects gave written informed consent
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prior to participating in this study.
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2.2. Methods
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We measured severity of depression using the Hamilton Depression Rating Scale

(HDRS) (Hamilton, 1960). A semi-structured questionnaire was used to collect socio-

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demographic data (including age, gender, marital status, employment status) and to screen for
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clinical data, including duration of illness, age at onset, number of lifetime depressive and manic

episodes and total number of episodes. At the same time, current suicidal ideation (last week)
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was screened as active suicidal thoughts with or without intent to act, a specific plan to commit

suicide and preparatory acts. A yes answer was rated as 1 and when no ideation was present a 0

score was assigned. We also assessed the number of prior suicide attempts by any means. The

DSM-IV-TR was used to make the diagnoses of melancholia and atypical depression and

psychotic features. We also assessed use of tobacco as present or not present.

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The assays of the immune biomarkers and TBARS have been described in detail

previously (Siwek et al., 2016b). In brief, fasting blood was sampled around 8.00 a.m. and

centrifuged at 1800 x g for 30 minutes. We stored serum aliquots at -80 o C until thawed for

biomarker assay. Immune biomarkers were assayed using an enzyme-linked immunoassay

(ELISA) employing commercially available high sensitive kits for IL-1α (RayBiotech, Norcross,

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GA, USA); sIL-1RA, sIL-2R and sIL-6R80 (R&D Systems, Minneapolis, MN, USA) and sTNF-

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R1 and sTNF-R2 (eBioscience, Hatfield, UK). Appropriate volumes of standards and samples

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were dispensed into 96-well plates coated with human anti-sIL-1RA, sIL-2R, sIL-6R, sTNF-R1

or sTNF-R2 antibodies and incubated. After extensive washing, an enzyme-linked polyclonal

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antibody specific for examined proteins was added and incubated. Following a wash to remove
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any unbound antibody-enzyme reagent, a substrate solution was pipetted to the wells and color

develops in proportion to the amount of specific protein bound in the initial step. The reaction
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was stopped by adding a stop solution, and the absorbance was determined at a wavelength of
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450 nm. Lipid peroxidation levels were measured using TBARS Assay kit (Cayman Chemical,

Ann Arbor, MI, USA) according to the manufacturer’s recommendations. In this method,
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malondialdehyde (MDA) reacts with thiobarbituric acid (TBA) under high temperature (90-
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100o C) and acidic conditions, generating the MDA-TBA adduct. The MDA-TBA adduct was

determined fluorometrically at an excitation wavelenght of 530 nm and an emission wavelenght

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of 550 nm. All the measurements were performed using the Synergy 2 multi-mode microplate

reader and Gen5 Software (BioTek, Winooski, VT, USA). All assays were performed in

duplicate. The detection limits were IL-1α: 0.412 pg/ml, sIL-1RA: 18.3 pg/ml, sIL-2R: 10 pg/ml,

sIL-6R: 15.1 pg/ml, sTNF-R60: 0.05 ng/ml, sTNF-R60: 0.10 ng/ml and TBARS: 0.0625

nmol/ml. The intra- and inter-assay coefficients of variation were <7.5% for all analytes.
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2.3. Statistics

Analyses of contingency tables (Χ2 -tests) were employed to check associations between

two sets of nominal variables. Analyses of variance (ANOVAs) were used to assess intergroup

differences in scale variables, including age, HDRS and biomarker data. Multivariate general

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linear model (GLM) analyses were employed to examine the effects of explanatory variables

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(including diagnosis) on dependent variables (including immune biomarkers and TBARS) while

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adjusting for confounding variables (including age, sex and tobacco use). If there were

significant effects of explanatory variables, we performed tests for between-subjects effects to

check which dependent variables were

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significantly associated with the predictors.
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Consequently, we used parameter estimates to evaluate the impact of significant continuous

explanatory variables, while estimated marginal means obtained by GLM analyses were
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employed to evaluate differences among nominal variables using protected least significant
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differences (LSD). Automatic binary logistic regression analyses were carried out with different

classifications (namely MAFD versus controls, melancholia versus no melancholia, atypical

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versus no atypical depression, suicidal ideation versus no suicidal ideation) as dependent

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variables and the biomarker and staging data (including number of episodes and

hospitalizations), age, sex and tobacco use as explanatory variables. Ln transformations of the
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biomarker data were employed to normalize data distributions, which were assessed with the

Kolmogorov-Smirnov test. We transformed the immune and TBARS data into z scores and

consequently computed z-unit weighted composite scores (Kanchanatawan et al., 2017) to make

indices of the 5 cytokine receptors (zCytR = zLnsIL-6R + zLnsIL-1RA + zLnsIL-2R +

zLnsTNFR60 + zLnsTNFR80), cell-mediated immunity (zCMI = zCytR + zLnIL-1α) and

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immune-oxidative stress (zCMI+TBARS = zCMI + zLnTBARS). All regression analyses were

checked for collinearity using tolerance and variance inflation factor (VIF) values. We used the

IBM-SPSS Statistics (Windows ver 22) to analyze all data. All tests were 2-tailed and a p-value

of 0.05 was used for statistical significance.

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3. Results

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3.1. Descriptive statistics

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Table 1 shows the descriptive statistics in normal controls and MDD and BD patients.

Patients with MDD were somewhat older than BD patients, while there were no significant

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differences in sex ratio between the three study groups. There was a minor difference in marital
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status between controls and MDD patients, whereas significantly more BD patients are

unemployed as compared to controls. There were significantly more MDD and BD patients who
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smoked as compared to normal controls. There were no significant differences in the incidence
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of melancholic, atypical and psychotic features between MDD and BD, whilst also the HDRS

score was not significantly different between the groups. Suicidal ideation was significantly
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more present in MDD than BD, whereas the number of prior suicide attempts was significantly
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greater in BD patients. Using ANOVAs without adjustment for confounding variables, we found

that IL-1RA and TBARS levels were significantly higher in MAFD patients than in controls,
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while there were no significant differences in other biomarkers.

In the same table we also show the results of BP1 and BP2 patients analyzed by means of

univariate ANOVAs or analyses of contigency tables without correction for false discovery rates

and without controlling for confounding variables. BP1 patients were younger than BP2 and

MDD patients (F=8.24, df=2/244, p<0.001) and showed a lower HDRS score that MDD and
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BD2 patients (F=4.82, df=2/239, p=0.009). The prevalence of melancholia was somewhat lower

in BP1 patients than in MDD and BP2 patients (Χ2 =10.73, df=2, p=0.005), while suicidal

ideation was higher in BP1 than in the other 2 groups (Χ2 =12.15, df=2, p=0.002). The other

variables, including the immune markers, did not significantly differ between BP1 and BP2.

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3.2. Effects of diagnoses on immune-oxidative biomarkers

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Table 2 shows the impact of the clinical diagnoses on the biomarkers. Multivariate GLM

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analysis #1 shows a significant effect of MAFD on the biomarkers, whilst tests for between-

subject analyses followed by post-hoc analyses performed on the estimated marginal means (see

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Table 3) shows higher sIL-1RA, TBARS, zCytR, zCMI and zCMI+TBARS in MAFD patients
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compared to controls (after controlling for confounding variables). Consequently, we have

examined the effects of diagnosis, categorized as MDD, BD and HC (see multivariate regression
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#2). This analysis showed that diagnosis had a significant effect on the immune-oxidative
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measurements after controlling for possible effects of age, sex and tobacco use. Tests for

between-subjects effects and protected post-hoc tests performed on the estimated marginal
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means (see Table 3) shows that sIL-1RA, TBARS, zCytR, zCMI and zCMI+TBARS are
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significantly higher in MDD and BD as compared with controls. There were no significant

differences between MDD and BD in sIL-1RA (p=0.785), TBARS (p=0.851), zCytR (p=0.436),
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zCMI (p=0.735) and zCMI+TBARS (p=0.797). In order to examine possible differences in the

biomarkers between MDD, BP1, BP2 and controls we have examined the effects of this

categorization on the biomarkers in another multivariate GLM analysis. Post-hoc analyses

performed on the estimated marginal means obtained by this analysis did not show significant

differences in any of the biomarkers between MDD, BP1 and BP2.

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Consequently, we have analyzed the effects of melancholia on the biomarkers in the

patient group and toward this end we have entered melancholia, MAFD and the interaction

MAFD X melancholia in a GLM analysis (see multivariate regression #3) showing that the

interaction term was significant. Tests for between-subject effects showed that the latter was

significant for IL-1α, sTNFR80, TBARS and zCMI+TBARS. Table 3 shows that these 4

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biomarkers were relatively lowered in MDD+melancholia versus MDD, while they were

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increased in BD+melancholia versus BD. For example, the zCMI+TBARS index increases from

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healthy controls (mean ±SE=-1.64 ±0.49) to MDD+melancholia (-0.04 ±0.56) and BD (0.02

±0.34), to MDD (0.43 ±0.36) and to BD+melancholia (1.43 ±0.54), although none of the

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pairwise comparisons was significant even at the p=0.05 level.
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In multivariate regression #4, we have assessed the effects of atypical depression on the

biomarkers in the patient group and toward this end we have entered atypical depression, MAFD
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and their interaction in the analysis. There was no significant difference between BD and MDD
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and also the interaction term was not significant, whilst there was a significant effect of atypical

depression on the biomarkers. Tests for between-subject effects and estimated marginal means
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(See Table 3) show higher sIL-6R and TBARS levels in MAFD with atypical features than in
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MAFD without these features.

We have also examined the effects of psychotic symptoms using the latter as an
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explanatory variable in multivariate GLM analysis. We were, however, unable to find a

significant association between psychotic symptoms and biomarkers (F=1.51, df=7/180,

p=0.166).

Finally, we have also examined the effects of the acute phase of depression by entering

the HDRS score or categories split according to a HDRS threshold of 16. Table 2 presents data
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on the HDRS continuous score showing that in MAFD patients there was a significant impact of

HDRS on the biomarkers. Tests for between-subject effects showed that HDRS was significantly

and positively associated with sIL-6R, sTNFR80, zCytR, TBARS, and zCMI+TBARS. Also

diagnostic categories (healthy controls versus MAFD with HDRS<16 and MAFD with

HDRS≥16) had a significant effect on the biomarkers (F=5.03, df=14/444, p<0.001). Tests for

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between-subject effects and post-hoc analyses showed significantly higher sIL-6R (p=0.040),

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sTNFR60 (p=0.017), TBARS (p=0.002) and zCMI+TBARS (p=0.046) in MAFD patients with

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MAFD≥16, versus those with HDRS<16. We also used an univariate automatic stepwise

regression with the HDRS score as dependent variable and all biomarkers, number of depressive

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and total number of episodes and hopitalizations including those of last year, age, sex and
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tobacco use as explanatory variables. We found that 16% of the variance in the HDRS score

(F=9.64, df=3/152, p<0.001) was predicted by TBARS (t=+4.36, p<0.001), sTNFR80 (t=+2.95,
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p=0.004) and sTNFR60 (t=+2.02, p=0.045).

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3.3. Effects of confounding variables

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Table 2 regression #1 shows that tobacco use had a significant effect on the biomarkers,
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whereas age and sex were not significant. Tests for between-subject effects shows that tobacco

use has a significant enhancing effect on sTFR60, namely no tobacco use -0.05 (±0.11) and
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tobacco use 0.20 (±0.11), and sIL-2R, namely no tobacco use -0.21 (±0.10) and tobacco use

+0.23 (±0.10) (expressed as z-scores). Although the effects are minimal we have controlled all

our results for possible effects of tobacco use (and age and sex). We also examined whether there

are any effects of the psychotropic drugs used by the patients. Thus, we could not find any

significant effects of the drug state of the patients on the biomarkers when entered in regression
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#1 Table 2, including SSRIs (n=74; F=1.05, df=7/221, p=0.398); SNRIs (n=51; F=1.46,

df=7/221, p=0.184); lithium (n=17; F=0.53, df=7/221, p=0.814); lamotrigine (n=18; F=1.12,

df=7/221, p=0.351); TCAs (n=13; F=0.75, df=7/220, p=0.629); and atypical antipsychotics

(n=11; F=1.39, df=7/214, p=0.211).

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3.4. Staging characteristics

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Table 4 shows the results of multivariate general linear model (GLM) analysis with

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staging characteristics as dependent variables and diagnosis as explanatory variable, namely

MDD versus BP1 versus BP2, while controlling for sex and tobacco use. There was a significant

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effect of diagnosis on the staging characteristics, whereas sex was not significant (p=0.09). Tests
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for between-subject effects show significant effects of diagnosis on the 6 staging variables.

Estimated marginal means and protected post-hoc tests show a higher number of episodes the
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year prior to the study and number of lifetime episodes in BP1 and BP2 patients as compared to
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MDD patients. The number of hospitalizations the year prior to the study and number of lifetime

hospitalizations was significantly greater in BP1 than in MDD and BP2. The number of
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depressive episodes was significantly higher in BP2 than MDD patients, while age at onset was
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significantly lower in BP1 than MDD patients.

Table 2, regressions #5 and #6 show that the numbers of hospitalizations and episodes the
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year prior to the study were significantly associated with the biomarkers. Tests for between-

subject effects showed that the number of episodes the year prior to the study was significantly

associated with increased levels of sTNFR80, zCMI, TBARS, zCMI+TBARS, while the number

of hospitalization was associated with increased sIL-1RA levels. When entered alone, the

number of lifetime depressive episodes showed a significant effect (F=2.09, df=7/198, p=0.046),
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while tests of between-subject effects showed significant positive associations with sTNFR80

(F=7.71, df=1/204, p=0.006), TBARS (F=4.55, df=1/204, p=0.034) and zCMI+TBARS (F=6.26,

df=1/204, p=0.013).

3.5. Best predictors of diagnostic groups

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Table 5 shows the results of binary automatic logistic regression analyses with diagnoses

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as dependent variable and biomarkers combined with staging variables as explanatory variables,

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while controlling for age, sex and tobacco use. We found that MAFD (versus controls, as

reference group) was significantly predicted by increased levels of TBARS, zCytR and tobacco

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use (regression #1). Regression #2 shows that three explanatory variables increased risk towards
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melancholia (versus non melancholia patients), namely sTNFR60, TBARS and number of

hospitalizations the year prior to the study. Regression #3 shows that atypical depression (versus
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patients without atypical depression) was positively associated with TBARS and number of
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lifetime depressive episodes, and negatively with age.

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3.6. Prior suicide attempts and suicidal ideation

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Table 3, regression #6 shows that there was a significant effect of prior suicide attempts

on the biomarkers (after considering the effects of age, sex and tobacco use). Tests for between-
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subject effects showed positive associations between prior suicide attempts and sIL-1RA, IL-1α,

TBARS, zCMI and zCMI+TBARS. Table 5 shows the results of two different stepwise logistic

regression analyses with suicidal ideation (versus no suicidal ideation as reference group) and

diagnoses together with staging characteristics and/or biomarkers as explanatory variables.

Regression #4 shows that current suicidal ideation is predicted by psychotic, melancholic and
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atypical features, MDD and number of hospitalizations the year prior to the study. Regression #5

shows that suicidal ideation is predicted by TBARS, number of hospitalization the year prior to

the study and MAFD.

3.7. Intertwined associations between TBARS and immune variables

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We have also examined the associations between TBARS and the immune variables.

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Table 6, regressions #1 and #2 show different models predicting TBARS. Regression #1

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considers the immune biomarkers, staging characteristics, age, sex and tobacco use as

explanatory variables. We found that 18.2% of the variance in TBARS is predicted by number of

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episodes the year prior to blood sampling, tobacco use, age, sIL-1RA and sIL-6R (all positively)
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and sIL-2R and sTNFR60 (both inversely). Regression #2 also included MAFD and depression

subtypes as predictor variables and shows that 18.0% of the variance in TBARS is explained by
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MAFD, number of episodes the year prior to blood sampling, sIL-6R (positively) and sTNFR60
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(inversely).

Table 6, regression #3 shows that 8.5% of the variance in sIL-1RA levels is explained by
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TBARS and number of hospitalizations the year prior to the study. Regression #4 shows that sIL-
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6R is associated with TBARS only, while regression #5 shows that sIL-2R levels are predicted

by TBARS, tobacco use, number of episodes last year (all positively) and number lifetime manic
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episodes (inversely). Regression #6 shows that sTNFR60 levels are predicted by melancholia,

tobacco use and TBARS. Regression #7 shows that 9.7% of the variance in sTNFR80 was

explained by age at onset and tobacco use (both inversely) and number of episodes last year

(positively).
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4. Discussion

The first major finding of this study is that MAFD is accompanied by increased sIL-1RA

and TBARS levels, and composite indices of circulating cytokine receptor levels coupled with

IL-1α and/or TBARS. These results corroborate previous research that MDD and BD are

characterized by increased IL-1β production and serum sIL-1RA levels (Maes et al., 1991;

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1995c; Levine et al., 1999; Monfrim et al., 2014; Tsai et al., 2014; Pandey et al., 2015). IL-1

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expressing cells (including monocytes, hepatocytes, epithelial cells, keratinocytes and

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adipocytes) produce sIL-1RA, which may be released following triggers, including IL-1β, viral

products, IL-6 and IL-4-related mechanisms and some acute phase proteins (Sone et al., 1994;

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Kay and Calabrese, 2004; Perrier et al., 2006). As reviewed previously, increased IL-1β
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signaling may explain the onset of depressive behaviors, neuroinflammation and

neuroprogression (Maes et al., 2012c). Interestingly, we found that MAFD is not accompanied
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by changes in serum levels of IL-1α, which usually acts as an autocrine growth factor reaching
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the serum following cellular necrosis (Maes et al., 2012c; Dinarello, 2011). Shelton et al. (2011)

reported increased IL-1α gene expression in postmortem brains of MDD patients. The results
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also corroborate previous research showing increased levels of TBARS, MDA or IgM responses
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directed to MDA in MDD and BD (Bilici et al., 2001; Khanzode et al., 2003; Ozcan et al., 2004;

Sarandol et al., 2007; Maes et al., 2013a; 2013b; Ranjekar et al., 2003; Manchado-Vieira et al.,
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2007; Kunz et al., 2008; Bengesser et al., 2015; Chowdhury et al., 2017). Our findings also

consolidate the results of various meta-analyses showing increased TBARS or MDA levels in

MAFD (Andreazza et al., 2008; Brown et al., 2014; Liu et al., 2015). Increased levels of

TBARS, MDA or IgM responses to MDA indicate increased lipid peroxidation and as such the

results are in agreement with findings that MAFD is accompanied by many other signs of lipid
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peroxidation, including increased levels of oxidized LDL and increased 4-hydroxynenonal, and

lowered levels of antioxidants or antioxidant enzymes, which protect against lipid peroxidation,

including HDL-cholesterol, vitamin E and PON1 (Maes et al., 1997c; Sobczak et al., 2004;

Bortolasci et al., 2014; Nunes et al., 2015; Moreira et al., 2017).

We found that MAFD is strongly predicted by increased TBARS levels coupled with the

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composite index of the 5 cytokine receptors, although TBARS was a stronger predictor of

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MAFD versus the cytokine receptors. Previously, many studies and meta-analyses have reported

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that MDD and BD are accompanied by increased levels of sIL-2R, sIL-6R, sTNFR60 and

sTNFR80 (Maes, 2011; Modabbernia et al., 2013; Munkholm et al., 2013; Liu et al., 2012;

Goldsmith et al., 2016).

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Interestingly, while the current results indicate activation of different immune pathways

in MAFD, our findings also indicate increased negative immunoregulatory effects in MAFD.
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Indeed, increased levels of sIL-1RA, sIL-2R, sTNFR60 and sTNFR80 have immunosuppressive
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effects. sIL-1RA functions as an endogenous inhibitor of IL-1β and IL-1α signaling and as such

is a key molecule in the resolution of inflammation and promotion of tissue repair in disorders
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accompanied by increased IL-1β (Arend and Guthridge, 2000). Both sTNFRs function as decoy
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receptors thereby increasing negative immunoregulatory effects on TNFα signaling (Selinsky et

al., 1998; Su et al., 1998). T cell activation is accompanied by an increased release of the sIL-2R,
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which consequently may act as a negative immunoregulator by binding IL-2 and thus lowering

IL-2 signaling (Caruso et al., 1993; Witkowska, 2005). Thus, elevated sIL-1RA, sIL-2R,

sTNFR60 and sTNFR80 levels indicate cell-mediated immune activation as well as increased

negative immunoregulation and anti-inflammatory activity. This response in soluble receptor

levels is most likely part of the compensatory (anti)inflammatory reflex system (CIRS), which is
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a counter reflex response attenuating an overzealous immune system thereby lowering IL-1, IL-2

and TNFα signaling (Maes et al., 2012a).

The second major finding of this study is that there were no significant differences in any

of the immune variables, composite indices of circulating cytokine receptor levels and cell-

mediated immunity and TBARS between BD and MDD and between BP1 and BP2. These

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findings extend those of Maes et al. (1995b) showing no significant differences in sIL-6R, sIL-

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2R, IL-6 and soluble transferrin receptor between major depressed and bipolar patients. O’Brien

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et al. (2006) found increased TNF-α in BD and MDD patients versus controls, but no differences

between both MAFD subtypes. Another study found that both MDD and the manic phase of BD

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are accompanied by increased levels of acute phase proteins, including haptoglobin, fibrinogen
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and α1-acid glycoprotein and hemopexin, indicating comparable levels of inflammation between

the two MAFD subtypes (Maes et al., 1997b). Both MDD and the manic phases of BD are
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accompanied by elevated levels of complement factors, including complement C3 (Maes et al.,

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1997b; Wadee et al., 2002). Also, different studies and meta-analyses show that both MDD and

BD are associated with increased plasma levels of sTNFR60 and sTNFR80 (Ortiz-Dominguez et
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al., 2007; Barbosa et al., 2011; Grassi-Oliveira et al., 2009; Moughrabi et al., 2014; Rizavi et al.,
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2016; Pandey et al., 2015), sIL-2R, sIL-6R, sIL-1RA (Modabbernia et al., 2013; Munkholm et

al., 2013; Maes et al., 1995; Mikova et al., 2001; Haapakoski et al., 2015; Kohler et al., 2017)
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and TBARS/MDA (Andreazza et al., 2008; Liu et al., 2012). A recent meta-analysis showed that

there are no major differences cytokine signatures among BD and MDD (Goldsmith et al., 2016).

In the current study, atypical features in MAFD were accompanied by increased sIL-6R

and TBARS levels. Rudolf et al. (2014) reported increased IL-6 levels in atypical depression, but

not in typical depression. Since both MDD and BD are accompanied by increased IL-6 (Maes et
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al., 1995a; 1995b; Pandey et al., 2015), the presence of increased sIL-6R levels may indicate that

IL-6 (trans-) signaling is enhanced especially in MAFD with atypical features. The sIL-6R

comprises the extracellular part of the IL-6R and can bind IL-6 forming a IL-6 + sIL-6R complex

in the serum, which can bind to gp130 on cells not expressing the IL-6R thereby inducing IL-6

trans-signaling in cells which are normally non-responsive to IL-6 (Rose-John, 2012; Maes et al.,

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2015b). IL-6 trans-signaling is pro-inflammatory, contrasting classical IL-6 signaling, which is

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more protective (Maes et al., 2015). Furthermore, increased lipid peroxidation in MAFD with

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atypical features may further shape this phenotype. We found that MAFD with melancholic

features is characterized by increased TBARS and sTNFR60 levels indicating that this phenotype

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is accompanied by increased lipid peroxidation and changes in TNF-α signaling.
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Interestingly, we found that TBARS was significantly associated with immune variables

indicating that signs of immune activation (namely increased sIL-1RA and sIL-6R levels) and
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lowered immunosuppression (as indicated by sIL-2R and sTNFR60) are associated with TBARS.
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Phrased differently, increased TBARS is associated with increased monocytic (and maybe Th-1)

activity. Previously, it was reviewed that there are, in mood disorders, intertwined associations
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between oxidative pathways and cell-mediated immunity (Moylan et al., 2014).

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The third major finding of this study is that there were significant positive associations

between severity of illness and increasing levels of sIL-1RA, sTNFR80, TBARS, and the
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composite indices of the 5 cytokine receptor levels and immune-oxidative pathways and that sIL-

6R, sTNFR60, TBARS and zCMI+TBARS levels were higher in acute depressed MAFD

patients (HDRS≥16) than in the partial remission phase. These findings suggest that the acute

phases of MAFD may present with increased immune-oxidative alterations, versus euthymic

phases. In a meta-analysis, Goldsmith et al. (2016) found that IL-6 levels may be increased in
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euthymic BD, but not MDD, while IL-1β and sIL-2R may be higher in euthymic BD patients.

Nevertheless, based on our findings we conclude that euthymic and acute phases of MAFD are

accompanied by activated immune-oxidative pathways and that further increases in these

pathways are associated with increased illness severity.

A fourth major finding of this study is that the number of episodes (depressive and

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manic) the year prior to the study was significantly associated with increased sTNFR80 and

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TBARS levels and higher composite indices of cell-mediated immunity and immune-oxidative

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pathways, while the number of hospitalizations the year prior to the study was associated with

increased sIL-1RA levels. This indicates that recurrent episodes (or its consequences, such as

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hospitalizations) are associated with more severe immune alterations. Previous research in MDD
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found positive correlations between number of lifetime depressive episodes and pro-

inflammatory cytokines and neopterin, an index of cell-mediated immunity (Maes et al., 2012b).
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Celik et al. (2010) reported that in major depression, serum neopterin concentrations were higher
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in patients who had experienced two or more episodes versus first-episode patients and healthy

controls. Parturients who had experienced a lifetime history of major depression had exaggerated
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increases in serum sIL-1RA and IL-6 the first few days after delivery as compared to parturients
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without a lifetime history of major depression (Maes et al., 2001). Overall, the results of these

studies suggest that a lifetime history of major depression and the number of depressive episodes
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is associated with a sensitization of the immune system (Maes et al., 2001; 2012b). These

findings are in agreement with preclinical research showing that induced responses in pro-

inflammatory cytokines are greater in interferon-γ or carrageenan-sensitized rodents (Blanque et

al., 1998). Nevertheless, the results of the current study show that the combined effects of
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number of depressive and manic episodes the year prior to blood sampling is more closely

associated with the immune status than a life- long history of depressive episodes.

The fifth major finding of this study is that, in MAFD, the number of previous suicide

attempts is significantly associated with IL-1α, sIL-1RA, TBARS, and the composite indices of

cell-mediated immunity and immune-oxidative pathways. In addition, current suicidal ideation

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was associated with increased TBARS levels. There are now three meta-analyses reporting

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relationships between suicidal behaviors and cytokines. The first (Chang et al., 2016) showed

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that lowered levels of cytokines, including IL-8 (Isung et al., 2012), and lowered levels of fish oil

nutrients are weak predictors of suicide attempts. Another meta-analysis reported that suicidal

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patients showed lowered plasma IL-2 but no differences in IL-6, TNF-α, and IFN-γ among
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suicidal patients, non-suicidal patients and controls (Ducasse et al., 2015). Black and Miller

(2015) reported that serum and brain levels of IL-1β and IL-6 levels were most robustly
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associated with suicidal behaviors. To the best of our knowledge, our paper is the first to report
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increased TBARS/MDA levels in association with suicidal ideation and prior suicidal attempts.

Another study showed that patients with prior suicidal attempts had significantly higher levels of
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lipid hydroperoxides, but not MDA, and a lowered total radical trapping antioxidant parameter as
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compared to patients without suicide attempts (Vargas et al., 2013), indicating that increased

lipid peroxides and lowered antioxidant defenses are associated with suicidal behaviors. In
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addition, no significant associations were found between a history of suicide attempts and

inflammatory biomarkers, including IL-6, TNF-α, C-reactive protein (CRP), fibrinogen, and

erythrocyte sedimentation rate (Vargas et al., 2013). All in all, these findings show that suicidal

behaviors in MAFD are more related to lipid peroxidation than to immune-inflammatory

mechanisms.
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Another question is to what extent cytokines or their receptors can pass the blood-brain

barrier (BBB) thereby affecting brain functions. For example, IL-1 and sIL-1RA (and IL-1)

use a saturable transport system from the blood compartment to the central nervous system

(Banks et al., 1995a). Immune cells including activated T cells (producing IL-2 and IL-2R) may

especially under pathological conditions cross the BBB (Prinz et al., 2008; Banks, 2009).

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Interestingly, the sTNFR2 may block the entry of TNF into the brain (Banks et al., 1995b).

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More importantly, as discussed above elevated levels of these receptors indicate activation of

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cell-mediated immunity and thus increased levels of their pro-inflammatory cytokine

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counterparts. Increased levels of these cytokines may disrupt the integrity of the BBB thereby

promoting cytokine and immune cell trafficking through the BBB and thus neuroinflammatory
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disorders (Banks, 2009; Fiorentino et al., 2016). Furthermore, there are many more paths

whereby peripheral cytokines can modulate brain functions (Goehler et al., 2000). In addition, a
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peripheral increase in reactive oxygen species and oxidative stress (as indicated by increased
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MDA levels) may activate apoptotic pathways and disrupt the BBB (Qu et al., 2016).
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The results of the current study should be interpreted with regards to its strengths and

limitations. First, this is a cross-sectional study and therefore no causal modeling is possible.
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Second, it would have been more complete if we had assayed other cytokines, acute phase
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proteins and complement factors. Third, the current findings may not be generalized to

adolescents or an elderly population (more than 70 years old). Fourth, we only included patients

with a BMI < 30 kg/m2 in order to minimize effects of BMI on our immune measurements and

therefore the results cannot be generalized to obese patients. Indeed, we have previously

determined that there are no significant effects of BMI (range < 30 kg/m2 ) on the cytokine

receptors measured here in normal controls and pathological samples as well (including
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depression, bipolar disorder, rheumatoid arthritis, lupus erythematosus, schizophrenia). The

major strengths are that the patients are well-phenotyped and that results are controlled for

relevant confounding variables.

In conclusion: while MAFD is characterized by intertwined increments in immune and

oxidative pathways, there are no immune-oxidative biomarker differences between MDD and

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BD. Atypical features are associated with increased IL-6 trans-signaling and lipid peroxidation,

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whereas melancholic features are predicted by increased lipid peroxidation and attenuated

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immunosuppressive effects through lowered sTNFR60 levels. Severity of depression and number

of depressive and manic episodes the year prior to the study were associated with increased IL-6

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trans-signaling, cell-mediated immune activation and lipid peroxidation. Prior suicidal attempts
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are associated with increased IL-1 signaling, cell-mediated immune activation and lipid

peroxidation, whilst the latter increases risk towards suicidal ideation. Increased TBARS is the
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single best predictor of MAFD, atypical depression, melancholia and current suicidal ideation.
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5. Conclusions
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There are no I&O biomarker differences between MDD and BD. Atypical depression is
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associated with increased IL-6 trans-signaling and lipid peroxidation. Severity of depression,

number of episodes and suicidal attempts are associated with activated I&O pathways. Increased
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TBARS is the single best predictor of BD/MDD, atypical depression, melancholia and current

suicidal ideation.

Contributors
M.S.-K., M.S., D.D. and G.N. designed the study; K.S., M.S. and D.D. recruited study
participants and made diagnosis; K. S. collected blood samples for biochemical analysis; M.S.-
K. and P.M. performed all biochemical analysis; M.S.-K., K.S., M.S. and R.J.N. prepared
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database; M.M. with input from M.S. and M.S.-K. performed statistical analysis; M.M., M.S.-
K., M.S., D.D., J.K.R. and G.N. created the manuscript; G.N. supervised the project. All authors
contributed to and have approved the final version of the manuscript.

Conflict of interest

The authors do not report any conflict of interest.

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Acknowledgements

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This study was partially supported by a grant no. POIG 01.01.02-12-004/09 (Task 3.2) financed

by the European Regional Development Fund and Funds for the Statutory Activity of the

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Institute of Pharmacology, Polish Academy of Sciences and Jagiellonian University Medical
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College, Kraków, Poland.
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Table 1. Socio-demographic, clinical and biomarker data in patients with major depression
(MDD) and bipolar disorder (BD), also subdivided in to those with bipolar (BP)1 and BD2, and
healthy controls (HC).

Variables HC MDD BD BP2 BP1 F/Χ2 df p

(n=50) (n=114) (n=133) (n=64) (n=69)
A B C

Age (years) 45.8 49.4 44.3 46.8 42.0 5.48 2/294 0.005

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(12.4) (10.7) C (12.9) B (10.2) (14.6)

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Sex (F/M) 36/14 73/41 87/46 47/17 40/29 1.02 2 0.601
Employed 9/41 B,C 67/47 A 70/63 A 33/31 37/32 24.28 2 <0.001
(N/Y)

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Tobacco use 45/5 B,C 59/54 A 68/65 A 38/26 30/39 25.17 2 <0.001
(N/Y)
Marital status 21/29 B 24/88 A 43/90 18/46 25/44 7.71 2 0.021

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Atypical - 91/21 110/23 48/16 62/7 0.09 1 0.767

depression
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(N/Y)
Melancholic - 74/37 97/35 39/25 58/10 1.34 1 0.246
features (N/Y)
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Psychotic - 108/4 119/13 57/5 62/6 3.68 1 0.055

features (N/Y)
Suicidal - 56/55 88/44 36/28 52/16 6.57 1 0.010
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ideation (N/Y)
# suicidal - 0.57 0.91 0.81 1.02 6.40 1/221 0.041
attempts (1.10) (1.35) (1.31) (1.38)
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HDRS score - 13.0 11.3 13.7 9.2 1.86 1/240 0.173

(9.0) (9.7) (9.5) (9.4)
sIL-1RA 3.30 5.27 5.39 5.7 (6.0) 5.1 4.10 2/282 0.018
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(ng/mL)* (2.45) (3.66) (5.71) (3.1)

IL-1α (pg/mL) 2.72 3.24 2.62 3.1 (5.2) 2.1 0.48 2/288- 0.619
* (2.42) (6.86) (3.88) (1.8)
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sIL-2R 783.7 884.5 914.7 916 913 1.65 2/290 0.195

(pg/mL)* (385.4) (405.6) (468.4) (499) (442)
sIL-6R 26619 29734 26098 26056 26136 1.70 2/271 0.185
(pg/mL) * (9427) (22376) (7553) (8078) (7117)
sTNFR60 0.184 0.211 0.236 0.276 0.200 1.67 2/264 0.190
(ng/mL) * (0.067) (0.140) (0.216) (0.305) (0.055)
sTNFR80 1.006 0.929 1.159 1.058 1.249 0.94 2/260 0.391
(ng/mL) * (1.320) (1.215) (1.267) (1.054) (1.432)
TBARS 2.365 3.427 3.363 3.539 3.207 13.23 2/284 <0.001
(nmol/mL) * (1.099) (1.326) (1.293) (1.378) (1.202)
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All results are shown as mean (±SD, standard deviation). Results of analyses of variance (F) and
analyses of contingency tables (Χ2 ) are obtained for analyses performed on HC, MDD and BD.
Possible differences between BP1, BP2 and MDD are described in the text.
$
Marital status: stable relationship + married / single + divorced
HDRS: Hamilton Depression Rating Scale
sIL-1RA: soluble interleukin-1 receptor antagonist (sIL-1RA)
IL-1α: interleukin-1α
sIL-2R: soluble interleukin-2 receptor
sIL-6R: soluble interleukin-6 receptor

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sTNFR60/80: soluble tumor necrosis factor receptor 1 (p60) and 2 (p80)

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TBARS: thiobarbituric reactive substances
* These data are processed in Ln transformation

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Table 2. Results of multivariate general linear model (GLM) analyses with the immune-
oxidative biomarkers as dependent variables and diagnostic classifications into major depression
(MDD), bipolar disorder (BD), major affective disorder (MAFD), and MAFD with melancholic,
atypical and psychotic features, staging characteristics, Hamilton Depression Rating Scale
(HDRS) or number of prior suicidal attempts as explanatory variables, while controlling for sex,
age and tobacco use

Type Test Dependent variables Explanatory F df p

variables
Multivariate #1 sIL-1RA; sTNFR60; HC vs MAFD 4.90 7/222 <0.001

T
sTNFR80; sIL-2R; Sex 0.80 7/222 0.592

IP
IL-1α; zCytR; zCMI; Tobacco use 3.27 7/222 0.003
TBARS; Age 2.01 7/222 0.055

CR
zCMI+TBARS
Between-subject sIL-1RA HC vs MAFD 11.39 1/228 0.001
effects zCytR 7.45 1/228 0.007

US
zCMI 6.51 1/228 0.011
TBARS 19.75 1/228 <0.001
zCMI+TBARS 15.03 1/228 <0.001
Between-subject sTNFR60 Tobacco use 5.52 1/228 0.020
AN
effects sIL-2R 9.03 1/228 0.003

Multivariate #2 sIL-1RA; sIL-6R; HC vs MDD vs BD 2.62 14/444 0.001

M

sTNFR60; sTNFR80; Sex 0.72 7/221 0.654

sIL-2R; IL-1α; Tobacco use 3.24 7/221 0.003
ED

zCytR; zCMI; Age 2.04 7/221 0.052

TBARS;
zCMI+TBARS
PT

Between-subject sIL-1RA HC vs MDD vs BD 5.71 2/227 0.004

effects zCytR HC vs MDD vs BD 4.03 2/227 0.019
zCMI HC vs MDD vs BD 9.85 2/227 <0.001
CE

TBARS HC vs MDD vs BD 7.52 2/227 0.001

zCMI+TBARS HC vs MDD vs BD 3.30 2/227 0.039
AC

Multivariate #3 sIL-1RA; sIL-6R; MDD vs BD 1.28 7/178 0.265

sTNFR60; sTNFR80; Melancholia 1.30 7/178 0.255
sIL-2R; IL-1α; MDD vs BD x 3.10 7/178 0.004
zCytR; zCMI; Melancholia
TBARS;
zCMI+TBARS
Between-subject sTNFR80 MDD vs BD x 5.83 1/184 0.025
effects IL-1α Melancholia 5.13 1/184 0.017
TBARS MDD vs BD x 4.71 1/184 0.031
zCMI+TBARS Melancholia 4.81 1/184 0.030
MDD vs BD x
Melancholia
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MDD vs BD x
Melancholia

Multivariate #4 sIL-1RA; sIL-6R; MDD vs BD 0.788 7/179 0.598

sTNFR60; sTNFR80; Atypical 2.23 7/179 0.034
sIL-2R; IL-1α; MDD vs BD x 0.72 7/179 0.652
zCytR; zCMI; Atypical
TBARS;
zCMI+TBARS
Between-subject sIL-6R Atypical 7.08 1/187 0.008

T
effects TBARS Atypical 9.48 1/187 0.002

IP
Multivariate #5 sIL-1RA; sIL-6R; HDRS 3.51 7/179 0.001

CR
sTNFR60; sTNFR80;
sIL-2R; IL-1α;
zCytR; zCMI;

US
TBARS;
zCMI+TBARS
Between-subject sIL-6R HDRS 5.15 1/185 0.024
AN
effects sTNFR80 HDRS 6.23 1/185 0.013
zCytR HDRS 4.48 1/185 0.036
TBARS HDRS 9.93 1/185 0.002
zCMI+TBARS HDRS 8.01 1/185 0.005
M

Multivariate #5 sIL-1RA; sIL-6R; Episodes last year 3.82 7/214 0.001

ED

sTNFR60; sTNFR80; Hospitalizations last 2.48 7/214 0.018

sIL-2R; IL-1α; year
zCytR; zCMI;
PT

TBARS;
zCMI+TBARS
Between-subject sTNFR80 Episodes last year 10.17 1/220 0.002
CE

effects zCMI Episodes last year 3.98 1/220 0.047

TBARS Episodes last year 13.65 1/220 <0.001
zCMI+TBARS Episodes last year 8.92 1/220 0.003
AC

Between-subject zsIL-1RA Hospitalizations last 11.18 1/220 0.001

effects year

Multivariate #6 sIL-1RA; sIL-6R; Prior suicidal 2.45 7/203 0.020

sTNFR60; sTNFR80; attempts
sIL-2R; IL-1α;
zCytR; zCMI;
TBARS;
zCMI+TBARS
Between-subject sIL-1RA Prior suicidal 6.42 1/209 0.012
effects IL-1α attempts 4.26 1/209 0.040
zCMI Prior suicidal 5.54 1/209 0.020
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TBARS attempts 5.88 1/209 0.016

zCMI+TBARS Prior suicidal 8.98 1/209 0.003
attempts
Prior suicidal
attempts
Prior suicidal
attempts

sIL-1RA: soluble interleukin-1 receptor antagonist (sIL-1RA)

T
IL-1α: interleukin-1α

IP
sIL-2R: soluble interleukin-2 receptor
sIL-6R: soluble interleukin-6 receptor

CR
sTNFR60/80: soluble tumor necrosis factor receptor 1 (p60) and 2 (p80)
TBARS: thiobarbituric reactive substances
zCytR: computed as zLnIL-1RA + zLnsIL-6R + zLnsIL-2R + zLnsTNFR60 + zLnTNFR80

US
zCMI: zCytR + zLnIL-1α
zCMI+TBARS: zCMI + zLnTBARS
HC: healthy controls
HDRS: Hamilton Depression Rating Scale
AN
M
ED
PT
CE
AC
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Table 3. Estimated marginal mean (SE) values (in z scores) obtained after GLM analyses shown
in Table 2
A
Variables Healthy Controls Major depression B Bipolar disorder C
zsIL-1RA -0.522 (0.168) B,C +0.062 (0.104) A +0.101 (0.101) A
zCytR -1.008 (0.411) B,C +0.044 (0.256) A +0.320 (0.0.247) A
zCMI -1.061 (0.461) B,C +0.124 (0.287) A +0.258 (0.277) A
zTBARS -0.605 (0.161) B,C +0.175 (0.100) A +0.149 (0.097) A
zCMI+TBARS -1.666 (0.488) B,C +0.299 (0.303) A +0.406 (0.293) A

T
Variables Healthy controls Major affective

IP
disorders
zsIL-1RA -0.523 (0.082) +0.082 0.073

CR
zCytR -1.014 (0.410) +0.187 (0.179)
zCMI -1.064 0.460 +0.139 0.200
zTBARS -0.605 0.161 +0.161 0.070

US
zCMI+TBARS -1.669 0.487 +0.354 0.212

Variables No Atypical Atypical depression

AN
depression
zsIL-6R -0.066 (0.071) +0.368 (0.149)
zTBARS +0.062 (0.072) +0.572 (0.151)
M

Variables Major Bipolar

depression disorder
ED

Without With No Melancholia

Melancholia Melancholia Melancholia
zIL-1α +0.254 (0.131) -0.282 (0.202) -0.132 0.124 +0.134 0.198
PT

zsTNFR80 +0.007 (0.124) -0.113 (0.192) -0.101 0.118 +0.492 0.188

zTBARS +0.235 (0.114) +0.004 (0.176) +0.041 0.108 +0.439 0.172
zCMI+TBARS +0.466 (0.348) -0.019 (0.537) +0.035 0.330 +1.488 0.526
CE

All data are shown as z scores

sIL-1RA: soluble interleukin-1 receptor antagonist (sIL-1RA)
AC

IL-1α: interleukin-1α
sIL-6R: soluble interleukin-6 receptor
sTNFR80: soluble tumor necrosis factor receptor 2 (p80)
TBARS: thiobarbituric reactive substances
zCytR: computed as zLnIL-1RA + zLnsIL-6R + zLnsIL-2R + zLnsTNFR60 + zLnTNFR80
zCMI: zCytR + zLnIL-1α
zCMI+TBARS: zCMI + zLnTBARS
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Table 4. Results of multivariate general linear model (GLM) analyses with staging
characteristics as dependent variables and diagnosis into major depression (MDD), bipolar 1 or
bipolar 2 (BP1/BP2) as explanatory variables, while controlling for sex and tobacco use

Type Test Dependent variables Explanatory F df p

variables
Multivariate #1 Mood episodes last Diagnosis 11.60 12/410 <0.001
year, Hospitalizations Sex 1.85 6/205 0.090
last year, Depressive Tobacco Use 1.42 6/205 0.208
episodes, All episodes,

T
All hopitalizations,

IP
Age at onset
Between-subject Mood episodes last Diagnosis 17.82 2/210 <0.001

CR
effects year Diagnosis 5.51 2/210 0.005
Hospitalizations last Diagnosis 4.25 2/210 0.015
year Diagnosis 14.96 2/210 <0.001

US
Depressive episodes Diagnosis 10.19 2/210 <0.001
All episodes Diagnosis 4.52 2/210 0.012
All hopitalizations
Age at onset
AN
Multivariate #2 Mood episodes last Diagnosis 11.94 12/408 <0.001
year, Hospitalizations Sex 1.83 6/203 0.094
M

last year, Depressive Tobacco use 1.41 6/203 0.241

episodes, All episodes, Atypical 1.92 6/203 0.079
ED

All hopitalizations, depression 1.27 6/203 0.274

Age at onset Melancholia
PT

Estimated marginal mean (SE) values

Variables MDD A BP2 B BP1 C
CE

Episodes last year 1.31 (0.16) B,C 2.57 (0.20) A 2.21 (0.21) A
Hospitalizations last year 0.63 (0.11) C 0.76 (0.14) C 1.18 (0.15) A,B
Depressive episodes 6.05 (0.61) B 8.52 (0.77) A 7.13 (0.80)
6.34 (0.89) B,C 11.94 (1.13) A 12.29 (1.17) A
AC

All episodes
All hopitalizations 2.07 (0.47) C 3.27 (0.59) C 5.14 (0.61) A,B
Age at onset (years) 34.8 (1.4) C 31.7 (1.8) 28.1 (1.8) A
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Table 5. Results of automatic logistic regression analyses with different classifications, namely
major affective disorder, melancholia and atypical depression and current suicidal ideation as
dependent variables and biomarker and staging data, age, sex and tobacco use as explanatory
variables.

Dependent Model Χ2 Explanatory Wald df p Odds Lower

variables Model df, variables ratio and
p upper
Nagelkerke 95% CI

T
#1 Major 53.16 TBARS 17.80 1 <0.001 2.38 1.59 –

IP
Affective df=3, zCytR 9.75 1 0.002 1.35 3.55
Disorder p<0.001 Tobacco use 10.70 1 0.002 6.02 1.12 –

CR
versus controls 0.342 1.64
1.99 –
18.24

US
#2 15.86 sTNFR60 5.76 1 0.016 1.47 1.07 –
Melancholia df=3, TBARS 5.90 1 0.015 1.56 2.00
versus no p<0.001 Hospitalizations 3.90 1 0.048 1.39 1.09 –
AN
melancholia 0.111 last year 2.23
patients 1.00 –
1.93
M

#3 Atypical 23.84 TBARS 10.78 1 0.001 2.34 1.41 –

depression df=3, Depressive 7.59 1 0.006 1.75 3.90
ED

versus all p<0.001 episodes 4.21 1 0.040 0.96 1.18 –

other patients 0.193 Age 2.60
0.92 –
PT

0.998
#4. Suicidal 70.23 Psychotic 7.60 1 0.006 7.29 1.77 –
CE

ideation df=5, symptoms 9.18 1 0.002 2.77 29.97

p<0.001 Melancholia 18.44 1 <0.001 5.89 1.43 –
0.348 Atypical 12.26 1 <0.001 3.15 5.36
2.62 –
AC

depression 17.49 1 <0.001 2.13

Major Depression 13.24
Hospitalizations 1.66 –
last year 5.99
1.50 –
3.04
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#5. Suicidal 29.29 TBARS 4.78 1 0.029 1.42 1.04 –

ideation df=3, Hospitalizations 15.67 1 <0.001 1.89 1.95
p<0001 last year 10.80 1 0.001 2.71 1.38 –
0.165 Major affective 2.60
Disorder 1.49 –
4.90

95% CI: 95% confidence intervals

TBARS: thiobarbituric reactive substances

T
zCytR: computed as zLnIL-1RA + zLnsIL-6R + zLnsIL-2R + zLnsTNFR60 + zLnsTNFR80

IP
sTNFR60: soluble tumor necrosis factor receptor 1 (p60)

CR
US
AN
M
ED
PT
CE
AC
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Table 6. Results of stepwise, automatic linear regression analyses with thiobarbituric reactive
substances (TBARS) or immune biomarkers as dependent variables and diagnoses and staging
characteristics as explanatory variables, while controlling for tobacco use, age and sex

Dependent Significant t p R 2 (%) F df p

variables explanatory
variables
Episodes last year +9.98 <0.001 18.2% 7.04 7/221 <0.001
Tobacco use +2.88 0.004
sIL-2R -2.12 0.035

T
#1. TBARS sIL-1RA +2.35 0.020

IP
sIL-6R +2.28 0.023
sTNFR60 -2.50 0.013

CR
Age +2.19 0.029
MAFD +3.65 <0.001 18.0% 11.23 4/204 <0.001
Episodes last year +2.60 0.010
#2. TBARS

US
zsTNFR60 -2.44 0.016
zsIL-6R +2.13 0.034
Hospitalizations +3.72 <0.001 8.5% 11.52 2/248 <0.001
#3 sIL-1RA past +2.71 0.007
AN
TBARS
#4. sIL-6R TBARS +3.65 <0.001 5.3% 12.31 1/239 <0.001
M

Tobacco use +3.86 <0.001 11.4% 8.15 4/253 <0.001

TBARS -3.05 0.003
ED

#5. sIL-2R Episodes last year +3.25 0.001

Manic episodes -2.62 0.009
past
PT

Tobacco use +2.99 0.003 7.9% 6.67 3/233 <0.001

#6. sTNF60 Melancholia +2.79 0.006
TBARS -2.47 0.014
CE

Age at onset -3.23 0.001 9.7% 6.79 3/190 0.001

#7. sTNFR80 Tobacco use -2.51 0.013
Episodes last year +2.33 0.021
AC

sIL-2R: soluble interleukin-2 receptor

sIL-1RA: soluble interleukin-1 receptor antagonist (sIL-1RA)
sIL-6R: soluble interleukin-6 receptor
sTNFR60: soluble tumor necrosis factor receptor 1 (p60)
MAFD: major affective disorders
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Highlights
 There are no immune & oxidative (I&O) biomarker differences between major
depression (MDD) and bipolar disorder (BD)
 Atypical depression is associated with increased IL-6 trans-signaling and lipid
peroxidation
 Severity of depression, number of episodes and suicidal attempts are associated with
activated I&O pathways
 Increased TBARS is the single best predictor of BD/MDD, atypical depression,
melancholia and current suicidal ideation

T
IP
CR
US
AN
M
ED
PT
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AC