Increased circulating IL-18 levels in severe mental disorders indicate systemic inflammasome activation

TLDR: In this article, the authors found a link between systemic inflammasome activation/dysregulation and cholesterol load in severe mental illnesses (SMI) using a large patient cohort (n=1632; including 737 SCZ and 895 BD), and healthy controls (CTRL).

Abstract: Background Schizophrenia (SCZ) and bipolar disorder (BD) are severe mental illnesses (SMI) that are part of a psychosis continuum, and dysregulated innate immune responses have been suggested to be involved in their pathophysiology. However, disease-specific immune mechanisms in SMI are not known yet. Recently, dyslipidemia has been linked to systemic inflammasome activation, and elevated atherogenic lipid ratios have been shown to correlate with circulating levels of inflammatory biomarkers in SMI. It is, however, not yet known if increased systemic cholesterol load leads to inflammasome activation in these patients. Methods We tested the hypothesis that patients with SCZ and BD display higher circulating levels compared to healthy individuals of key members of the IL-18 system using a large patient cohort (n=1632; including 737 SCZ and 895 BD), and healthy controls (CTRL; n=1070). In addition, we assessed associations with coronary artery disease risk factors in SMI, focusing on relevant inflammasome-related, neuroendocrine, and lipid markers. Results We report higher baseline levels of circulating IL-18 system components (IL-18, IL-18BPA) as well as increased expression of inflammasome-related genes (NLRP3 and NLRC4) in the blood of patients relative to CTRL. We demonstrate a cholesterol dyslipidemia pattern in psychotic disorders, and report correlations between levels of blood cholesterol species and the expression of inflammasome system elements in SMI. Conclusions Based on these results, we suggest a link between systemic inflammasome activation/dysregulation and cholesterol load in SMI. Our findings further the understanding of possible underlying inflammatory and metabolic mechanisms and may expose important therapeutic targets in SMI.

Full text

1
Increased circulating IL-18 levels in severe mental disorders indicate systemic
inflammasome activation
Attila Szabo,
1,2
Kevin S. O`Connell,
1
Thor Ueland,
3,4,5
Mashhood A. Sheikh,
3
Ingrid
Agartz,
6,7,8
Dimitrios Andreou,
6,7
Pål Aukrust,
3,4,9
Birgitte Boye,
10,11
Erlend Bøen,
11
Ole
Kristian Drange,
12,13,14
Torbjørn Elvsåshagen,
1,4,15
John Abel Engh,
1,2
Sigrun Hope,
1,16
Margrethe Collier Høegh,
1
Inge Joa,
17,18
Erik Johnsen,
19,20,21
Rune Andreas Kroken,
19,20,21
Trine Vik Lagerberg,
1
Tove Lekva,
3
Ulrik Fredrik Malt,
4
Ingrid Melle,
1,4
Gunnar Morken,
12,14
Terje Nærland,
4,22,23
Vidar Martin Steen,
20,21
Kjetil Sørensen,
24
Kirsten Wedervang-Resell,
1
Melissa Auten Weibell,
17,18
Lars T. Westlye,
1,22,25
Nils Eiel Steen,
1
Ole Andreassen,
1
Srdjan
Djurovic
2,20
1
Norwegian Centre for Mental Disorders Research, NORMENT, Division of Mental Health
and Addiction, Oslo University Hospital, Oslo, Norway
2
Department of Medical Genetics, Oslo University Hospital, Oslo, Norway
3
Research Institute of Internal Medicine, Oslo University Hospital Rikshospitalet, Oslo,
Norway
4
Institute of Clinical Medicine, University of Oslo, Oslo, Norway
5
K.G. Jebsen Thrombosis Research and Expertise Center, University of Tromsø, Tromsø,
Norway
6
Norwegian Centre for Mental Disorders Research, NORMENT, Institute of Clinical
Medicine, University of Oslo, Oslo, Norway
7
Centre for Psychiatry Research, Department of Clinical Neuroscience, Karolinska Institutet
& Stockholm Health Care Services, Stockholm Region, Stockholm, Sweden
8
Department of Psychiatric Research, Diakonhjemmet Hospital, Oslo, Norway
9
Section of Clinical Immunology and Infectious Diseases, Oslo University Hospital
Rikshospitalet, Oslo, Norway
10
Institute of Basic Medical Sciences, University of Oslo, Oslo, Norway
11
Psychosomatic and Consultation-Liason Psychiatry, Division of Mental Health and
Addiction, Oslo University Hospital, Oslo, Norway
12
Department of Mental Health, Norwegian University of Science and Technology, NTNU,
Trondheim, Norway
13
Department of Østmarka, Division of Mental Health, St. Olavs University Hospital,
Trondheim, Norway
. CC-BY-NC-ND 4.0 International licenseIt is made available under a
is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity. (which was not certified by peer review)
The copyright holder for this preprint this version posted May 31, 2021. ; https://doi.org/10.1101/2021.05.28.21258013doi: medRxiv preprint
NOTE: This preprint reports new research that has not been certified by peer review and should not be used to guide clinical practice.

2
14
Department of Psychiatry, St Olav University Hospital, Trondheim, Norway
15
Department of Neurology, Oslo University Hospital, Oslo, Norway
16
Department of Neuro Habilitation, Oslo University Hospital Ullevål, Oslo, Norway
17
TIPS, Centre for Clinical Research in Psychosis, Stavanger University Hospital, Stavanger,
Norway
18
Network for Medical Sciences, Faculty of Health, University of Stavanger, Stavanger,
Norway
19
Division of Psychiatry, Haukeland University Hospital, Bergen, Norway
20
NORMENT, Department of Clinical Science, University of Bergen, Bergen, Norway
21
Dr. Einar Martens Research Group for Biological Psychiatry, Department of Medical
Genetics, Haukeland University Hospital, Bergen, Norway
22
K.G. Jebsen Center for Neurodevelopmental Disorders, Oslo, Norway
23
Department of Rare Disorders and Disabilities, Oslo University Hospital, Oslo, Norway
24
Department of Psychiatry, St. Olav’s University Hospital, Trondheim, Norway
25
Department of Psychology, University of Oslo, Oslo, Norway
Author correspondence: Attila Szabo, Ph.D., NORMENT KG Jebsen Centre for Psychosis
Research, and Division of Mental Health and Addiction, Institute of Clinical Medicine, Bygg
49, Ullevål sykehus, P.O. box 4956 Nydalen, 0424 Oslo, Norway. (Email:
attila.szabo@medisin.uio.no); Prof. Srdjan Djurovic, Ph.D., Department of Medical Genetics,
Oslo University Hospital, Bygg 25, Kirkeveien 166, 0450 Oslo, Norway. (Email:
srdjan.djurovic@medisin.uio.no)
Running title: Systemic immune dysregulation in psychotic disorders
Keywords: psychosis, schizophrenia, bipolar disorder, inflammasome, inflammation,
dyslipidemia
. CC-BY-NC-ND 4.0 International licenseIt is made available under a
is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity. (which was not certified by peer review)
The copyright holder for this preprint this version posted May 31, 2021. ; https://doi.org/10.1101/2021.05.28.21258013doi: medRxiv preprint

3
ABSTRACT
Background: Schizophrenia (SCZ) and bipolar disorder (BD) are severe mental illnesses
(SMI) that are part of a psychosis continuum, and dysregulated innate immune responses have
been suggested to be involved in their pathophysiology. However, disease-specific immune
mechanisms in SMI are not known yet. Recently, dyslipidemia has been linked to systemic
inflammasome activation, and elevated atherogenic lipid ratios have been shown to correlate
with circulating levels of inflammatory biomarkers in SMI. It is, however, not yet known if
increased systemic cholesterol load leads to inflammasome activation in these patients.
Methods: We tested the hypothesis that patients with SCZ and BD display higher circulating
levels compared to healthy individuals of key members of the IL-18 system using a large
patient cohort (n=1632; including 737 SCZ and 895 BD), and healthy controls (CTRL;
n=1070). In addition, we assessed associations with coronary artery disease risk factors in
SMI, focusing on relevant inflammasome-related, neuroendocrine, and lipid markers.
Results: We report higher baseline levels of circulating IL-18 system components (IL-18, IL-
18BPA) as well as increased expression of inflammasome-related genes (NLRP3 and NLRC4)
in the blood of patients relative to CTRL. We demonstrate a cholesterol dyslipidemia pattern
in psychotic disorders, and report correlations between levels of blood cholesterol species and
the expression of inflammasome system elements in SMI.
Conclusions: Based on these results, we suggest a link between systemic inflammasome
activation/dysregulation and cholesterol load in SMI. Our findings further the understanding
of possible underlying inflammatory and metabolic mechanisms and may expose important
therapeutic targets in SMI.
. CC-BY-NC-ND 4.0 International licenseIt is made available under a
is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity. (which was not certified by peer review)
The copyright holder for this preprint this version posted May 31, 2021. ; https://doi.org/10.1101/2021.05.28.21258013doi: medRxiv preprint

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1. INTRODUCTION
Schizophrenia (SCZ) and bipolar disorder (BD) are severe mental disorders with high
heritability that adversely impact the individual with large costs to society.
1,2
These psychotic
disorders are suggested to be part of a psychosis continuum,
3-5
and dysregulated immune
responses, inflammation and autoimmunity have been implicated in their pathophysiology.
6,7
Recent genome-wide association studies (GWAS) of both SCZ and BD have reported genetic
loci in immune function-related regions.
8-10
Still, the involved specific immune-related
mechanisms are not yet clarified. Recent evidence links psychosis to sterile inflammation of
the brain or to systemic inflammatory processes that affect the central nervous system.
11-13
This is supported by dysregulated systemic markers of inflammation and immune activation,
with correlations to clinical indices of disease severity.
14-16
Host innate immune responses to microorganisms are predominantly based on
germline-encoded pattern recognition receptors (PRRs) that recognize pathogen-associated
molecular patterns (PAMPs), which are ancient, evolutionally conserved microbial motifs
shared by many phylogenetic microbial taxa.
17
PRRs can also be activated by endogenous
non-microbial signals, such as damage-associated molecular patterns (DAMPs). The
consequent sterile inflammation can either resolve the initial insult or lead to disease.
11
A
subfamily of PRRs, the nucleotide-binding leucine-rich repeat (LRR)-containing proteins
(NLRs; also known as NOD-like receptors) have emerged as a key family of sensors and
regulators responding to microbial PAMPs, as well as to endogenous DAMPs produced under
nonmicrobial/noninfectious inflammatory conditions by host cells.
18
Upon ligand
(PAMP/DAMP) binding, NLR proteins assemble with the adaptor protein ASC which then
mediates the activation of caspase-1 and the subsequent production of the pro-inflammatory
. CC-BY-NC-ND 4.0 International licenseIt is made available under a
is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity. (which was not certified by peer review)
The copyright holder for this preprint this version posted May 31, 2021. ; https://doi.org/10.1101/2021.05.28.21258013doi: medRxiv preprint

5
cytokines IL-1
β
and IL-18.
19,20
This process is referred to as inflammasome activation, in
which other factors, such as the macrophage migration inhibitory factor (MIF) are also
critically involved.
21
Two of the NLRs, NLRP3 and NLRC4, have been showed to mediate
sterile inflammation in the brain (targeting astrocytes and microglia) and have been suggested
to be involved in psychotic disorders, but also linked to autoimmunity and cardiovascular
diseases.
22-24
However, systemic inflammasome status and potential activation has not yet
been explored in SMI.
NLRP3 and NLRC4, as most of the NLR family members, can be triggered by various
endogenous signals, such as uric acid or cholesterol crystals.
18
Dyslipidemia and high blood
cholesterol have been linked to systemic inflammasome activation in circulating immune cells
and in the vascular endothelium implicating inflammasome dysregulation in atherosclerosis
and increased coronary artery disease (CAD) risk.
25-28
Dyslipidemia and elevated atherogenic
lipid ratios correlate with circulating levels of inflammatory biomarkers in psychotic
disorders, but it is unknown if enhanced systemic cholesterol load leads to inflammasome
activation in these patients.
29,30
In the present study, we tested the hypothesis that patients with SCZ and BD have
higher circulating levels of key members of the IL-18 system using a large SMI cohort
(n=1632) including SCZ (n=737) and BD (n=895), relative to healthy controls (CTRL)
(n=1070). Furthermore, we assessed associations with CAD risk factors in SCZ and BD,
focusing on relevant inflammasome-related, neuroendocrine, and lipid markers.
2. METHODS AND MATERIALS
2.1 Sample characteristics
The study sample (n

=

2702) consisted of 1070 healthy controls (CTRL), 737 SCZ
spectrum disorder patients (544 schizophrenia, 153 schizoaffective, 40 schizophreniform), or
. CC-BY-NC-ND 4.0 International licenseIt is made available under a
is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity. (which was not certified by peer review)
The copyright holder for this preprint this version posted May 31, 2021. ; https://doi.org/10.1101/2021.05.28.21258013doi: medRxiv preprint

Frequently asked questions

Q1. What contributions have the authors mentioned in the paper "Increased circulating il-18 levels in severe mental disorders indicate systemic inflammasome activation" ?

In this paper, Szabo et al. found that schizophrenia and bipolar disorder can be linked to systemic inflammatory processes that affect the central nervous system. 

Q2. What have the authors stated for future works in "Increased circulating il-18 levels in severe mental disorders indicate systemic inflammasome activation" ?

However, evaluation of triggers for inflammasome activation in SMI will have to be further evaluated in forthcoming studies. Based on these results, the authors suggest a 14 link between systemic inflammasome activation/dysregulation and cholesterol load in psychotic disorder patients. Their findings further the understanding of possible underlying inflammatory and metabolic mechanisms in SMI, and may expose important therapeutic targets in severe mental disorders. 

Q3. What is the role of IL-18 in the inflammatory process?

Since circulating total cholesterol can form crystals that can cause systemic inflammasome activation (by serving as DAMPs) and thereby the systemic release of IL-18,2528 while HDL-cholesterol has been shown to inhibit inflammasome activation and to lower total cholesterol,43,44 their results raise the possibility that chronically increased blood cholesterol may contribute to a lipid-driven sterile inflammation in SCZ. 

Q4. How many probes were used in the analysis?

Global gene expression analyses were performed with Illumina HumanHT-12 v4 Expression BeadChip (Illumina, Inc.) consisting of ~47,000 probes. 

Q5. What is the role of the adaptor protein in the activation of caspase-1?

18 Upon ligand (PAMP/DAMP) binding, NLR proteins assemble with the adaptor protein ASC which then mediates the activation of caspase-1 and the subsequent production of the pro-inflammatory5cytokines IL-1β and IL-18.19,20 

Q6. What is the source of IL-18 and IL-1 in BD?

Besides circulating immune cells, systemic source of IL-18 and IL-1β can also be the liver38 or the vascular endothelium39,40 following tissue-specific inflammasome activation. 

Q7. How many tests were applied to the IL-18 system?

To correct for multiple testing the authors applied8a Bonferroni-corrected significance level of p<9.259 x 10-4 (Four IL-18 system components, three cholesterol levels and 11 probes across three diagnostic groupings = 54 tests; 0.05/54). 

Q8. What is the main source of circulating MIF in SMI?

the observed very small effect sizes suggest that the major source of circulating MIF is probably not immune cells in SMI. 

Q9. What are the main findings of this study?

Their findings further the understanding of possible underlying inflammatory and metabolic mechanisms in SMI, and may expose important therapeutic targets in severe mental disorders. 

Q10. What is the main source of IL-18 in BD?

Although this may suggest that circulating immune cells are not the major source of the enhanced plasma levels of IL-18 and IL-18BPA in their study, inflammasome activation only induces the cleavage and release of the mature IL-18 protein, but not IL-18 mRNA synthesis.