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Increased inflammasome activity in markedly ill psychiatric patients: An explorative study

  • Author Footnotes
    1 These authors contributed equally.
    Ulrika Hylén
    Correspondence
    Corresponding author at: Universitetssjukvårdens forskningscentrum, S-huset van 2, Universitetssjukhuset, SE-70185 Örebro, Sweden.
    Footnotes
    1 These authors contributed equally.
    Affiliations
    University Health Care Research Center, Faculty of Medicine and Health, Örebro University, Sweden

    School of Medical Sciences, Faculty of Medicine and Health, Örebro University, Örebro, Sweden

    Inflammatory Response and Infection Susceptibility Centre, (iRiSC), Örebro University, Örebro, Sweden
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  • Author Footnotes
    1 These authors contributed equally.
    Daniel Eklund
    Footnotes
    1 These authors contributed equally.
    Affiliations
    School of Medical Sciences, Faculty of Medicine and Health, Örebro University, Örebro, Sweden

    Inflammatory Response and Infection Susceptibility Centre, (iRiSC), Örebro University, Örebro, Sweden
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  • Mats Humble
    Affiliations
    University Health Care Research Center, Faculty of Medicine and Health, Örebro University, Sweden

    School of Medical Sciences, Faculty of Medicine and Health, Örebro University, Örebro, Sweden

    Inflammatory Response and Infection Susceptibility Centre, (iRiSC), Örebro University, Örebro, Sweden
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  • Jakub Bartoszek
    Affiliations
    School of Medical Sciences, Faculty of Medicine and Health, Örebro University, Örebro, Sweden
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  • Author Footnotes
    2 Shared last authors.
    Eva Särndahl
    Footnotes
    2 Shared last authors.
    Affiliations
    School of Medical Sciences, Faculty of Medicine and Health, Örebro University, Örebro, Sweden

    Inflammatory Response and Infection Susceptibility Centre, (iRiSC), Örebro University, Örebro, Sweden
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  • Author Footnotes
    2 Shared last authors.
    Susanne Bejerot
    Footnotes
    2 Shared last authors.
    Affiliations
    University Health Care Research Center, Faculty of Medicine and Health, Örebro University, Sweden

    School of Medical Sciences, Faculty of Medicine and Health, Örebro University, Örebro, Sweden

    Inflammatory Response and Infection Susceptibility Centre, (iRiSC), Örebro University, Örebro, Sweden
    Search for articles by this author
  • Author Footnotes
    1 These authors contributed equally.
    2 Shared last authors.
Open AccessPublished:November 25, 2019DOI:https://doi.org/10.1016/j.jneuroim.2019.577119

      Highlights

      • Patients fulfilled the diagnostic criteria for several psychiatric diagnoses.
      • Patients displayed increased expression of inflammasome genes.
      • Patients had increased plasma levels of cytokines, irrespective of diagnosis.

      Abstract

      The aim of this study was to investigate inflammatory perturbations in 40 patients with severe and complex psychiatric disorders by studying the activity of the NLRP3 inflammasome, with a trans-diagnostic approach. Gene expression of CASP1, NLRP3, PYCARD, IL1B, IL1RN, TNF showed a significant increase in the patient group compared to a matched control group. Plasma levels of IL1Ra, IL-18, TNF, IL-6 and CRP were increased in the patient group. Within the patient group, increased gene expression of inflammatory markers correlated with increased disease severity. The findings support the inflammation hypothesis for markedly ill psychiatric patients across diagnostic groups.

      Graphical abstract

      1. Introduction

      Each psychiatric diagnosis has its own unique characteristics upon which the psychiatric diagnostic system and most research is based. However, psychiatric patients with severe, enduring, and disabling disorders often also meet the diagnostic criteria for multiple unrelated psychiatric and medical disorders (
      • Plana-Ripoll O.
      • Pedersen C.B.
      • Holtz Y.
      • Benros M.E.
      • Dalsgaard S.
      • de Jonge P.
      • et al.
      Exploring comorbidity within mental disorders among a Danish National Population.
      ;
      • De Hert M.
      • Correll C.U.
      • Bobes J.
      • Cetkovich-Bakmas M.
      • Cohen D.
      • Asai I.
      • et al.
      Physical illness in patients with severe mental disorders. I. Prevalence, impact of medications and disparities in health care.
      ). Thus, it is challenging to pinpoint the primary diagnosis and new disorders may emerge over time (
      • Fava G.A.
      • Tossani E.
      • Bech P.
      • Berrocal C.
      • Chouinard G.
      • Csillag C.
      • et al.
      Emerging clinical trends and perspectives on comorbid patterns of mental disorders in research.
      ). In an attempt to describe these complex conditions, new diagnostic categories, such as schizo-obsessive disorder (
      • Scotti-Muzzi E.
      • Saide O.L.
      Schizo-obsessive spectrum disorders: an update.
      ) and an autistic subtype of obsessive-compulsive disorder (
      • Bejerot S.
      An autistic dimension: a proposed subtype of obsessive-compulsive disorder.
      ) have been proposed. Moreover, although non-established diagnoses such as non-suicidal self-injury disorder (
      • Zetterqvist M.
      The DSM-5 diagnosis of nonsuicidal self-injury disorder: a review of the empirical literature.
      ) and Pediatric Acute-Onset Neuropsychiatric Syndrome (PANS) (
      • Swedo S.E.
      • Leonard H.L.
      • Garvey M.
      • Mittleman B.
      • Allen A.J.
      • Perlmutter S.
      • et al.
      Pediatric autoimmune neuropsychiatric disorders associated with streptococcal infections: clinical description of the first 50 cases.
      ) are recognized as entities in their own right, they are primarily characterized as a comorbidity to another primary disorder. Since disorder symptoms, their etiologies, and their biomarkers often overlap between different psychiatric diagnoses, the use of trans-diagnostic approaches is justified. In search of relevant biological markers and common pathophysiological mechanisms, markedly ill patients with complex psychiatric symptomatology should be included in research on these complex conditions.

      1.1 Inflammation in psychiatric disorders

      Inflammation is proposed to be a common pathophysiological mechanism for psychiatric disorders (
      • Miller A.H.
      • Raison C.L.
      The role of inflammation in depression: from evolutionary imperative to modern treatment target.
      ;
      • Prata J.
      • Santos S.G.
      • Almeida M.I.
      • Coelho R.
      • Barbosa M.A.
      Bridging autism Spectrum disorders and schizophrenia through inflammation and biomarkers - pre-clinical and clinical investigations.
      ). Depression, bipolar disorder, schizophrenia, and autism spectrum disorder (ASD) are all associated with elevated levels of inflammatory cytokines and chemokines in peripheral blood (
      • Miller A.H.
      • Raison C.L.
      The role of inflammation in depression: from evolutionary imperative to modern treatment target.
      ;
      • Rodrigues-Amorim D.
      • Rivera-Baltanas T.
      • Spuch C.
      • Caruncho H.J.
      • Gonzalez-Fernandez A.
      • Olivares J.M.
      • et al.
      Cytokines dysregulation in schizophrenia: a systematic review of psychoneuroimmune relationship.
      ;
      • Masi A.
      • Quintana D.S.
      • Glozier N.
      • Lloyd A.R.
      • Hickie I.B.
      • Guastella A.J.
      Cytokine aberrations in autism spectrum disorder: a systematic review and meta-analysis.
      ;
      • Marazziti D.
      • Mucci F.
      • Fontenelle L.F.
      Immune system and obsessive-compulsive disorder.
      ). Inflammation can be caused in the brain by activated microglia cells that release cytokines, prostaglandins, and glutamate (
      • Wohleb E.S.
      • Delpech J.C.
      Dynamic cross-talk between microglia and peripheral monocytes underlies stress-induced neuroinflammation and behavioral consequences.
      ;
      • Tay T.L.
      • Bechade C.
      • D'Andrea I.
      • St-Pierre M.K.
      • Henry M.S.
      • Roumier A.
      • et al.
      Microglia gone rogue: impacts on psychiatric disorders across the lifespan.
      ). Indication of such neuroinflammation has been observed in patients with schizophrenia and in patients with ASD by showing microglia activation in imaging and post-mortem studies (
      • Prata J.
      • Santos S.G.
      • Almeida M.I.
      • Coelho R.
      • Barbosa M.A.
      Bridging autism Spectrum disorders and schizophrenia through inflammation and biomarkers - pre-clinical and clinical investigations.
      ). An inflammatory response is initiated upon the recognition of exogenous or endogenous threats through the activation of pattern-recognition receptors. This large class of receptors includes NLRP3, which forms a protein complex known as the inflammasome, responsible for the production of IL-1 family of cytokines (
      • Martinon F.
      • Burns K.
      • Tschopp J.
      The inflammasome: a molecular platform triggering activation of inflammatory caspases and processing of proIL-beta.
      ). In humans, the activity of the NLRP3 inflammasome and subsequent cytokine release is tightly regulated by both transcriptional and post-transcriptional checkpoints. Furthermore, the outcome of inflammasome activation is also affected by a number of common polymorphisms, including polymorphisms in the genes of NLRP3 and IL1B, as well as in the gene encoding regulatory protein CARD8 (
      • Verma D.
      • Särndahl E.
      • Andersson H.
      • Eriksson P.
      • Fredrikson M.
      • Jönsson J.-I.
      • et al.
      The Q705K polymorphism in NLRP3 is a gain-of-function alteration leading to excessive interleukin-1β and IL-18 production.
      ;
      • Sahdo B.
      • Fransén K.
      • Asfaw Idosa B.
      • Eriksson P.
      • Söderquist B.
      • Kelly A.
      • et al.
      Cytokine profile in a cohort of healthy blood donors carrying polymorphisms in genes encoding the NLRP3 inflammasome.
      ;
      • Landvik N.E.
      • Tekpli X.
      • Anmarkrud K.H.
      • Haugen A.
      • Zienolddiny S.
      Molecular characterization of a cancer-related single nucleotide polymorphism in the pro-inflammatory interleukin-1B gene.
      ). Both NLRP3 and inflammasome activation have been suggested as essential components in local responses initiated by microglia and have also been suggested to contribute to neuroinflammation and the subsequent development of psychiatric disorders (
      • Herman F.J.
      • Pasinetti G.M.
      Principles of inflammasome priming and inhibition: implications for psychiatric disorders.
      ). Furthermore, studies have shown an increased expression and activity in the NPLR3 inflammasome system and in circulating immune cells in patients with depression (
      • Alcocer-Gomez E.
      • Casas-Barquero N.
      • Williams M.R.
      • Romero-Guillena S.L.
      • Canadas-Lozano D.
      • Bullon P.
      • et al.
      Antidepressants induce autophagy dependent-NLRP3-inflammasome inhibition in major depressive disorder.
      ), bipolar disorder (
      • Ortiz-Dominguez A.
      • Hernandez M.E.
      • Berlanga C.
      • Gutierrez-Mora D.
      • Moreno J.
      • Heinze G.
      • et al.
      Immune variations in bipolar disorder: phasic differences.
      ), and ASD (
      • Saresella M.
      • Piancone F.
      • Marventano I.
      • Zoppis M.
      • Hernis A.
      • Zanette M.
      • et al.
      Multiple inflammasome complexes are activated in autistic spectrum disorders.
      ), however, studies on other psychiatric disorders are lacking. Based on these findings, an increased peripheral activity in the NLRP3 inflammasome system may serve as a common denominator in psychiatric conditions with a high occurrence of comorbidity.
      The aim of this study, therefore, is to explore the connections between inflammatory perturbations and psychiatric disorders in markedly ill patients with substantial comorbidity, representing psychiatric patients in natural settings.

      2. Method

      2.1 Patients and controls

      In this case-control study, 40 psychiatric patients (ages 11–47 years) and 40 controls were included. The psychiatric patients were primarily diagnosed with schizophrenia spectrum disorders (SSD) (including psychotic disorder, schizophrenia, and schizoaffective disorder), ASD, obsessive-compulsive disorder (OCD), or non-suicidal self-injury disorder (NSSID). Psychiatric patients were recruited from psychiatric clinics in Örebro County and the surrounding areas. The inclusion criteria for psychiatric patients was being diagnosed with a psychiatric disorder (SSD, ASD, OCD, or NSSID), while having neurological autoimmune disorders and/or an ongoing infection at the time of blood sampling were the only exclusion criteria.
      Controls were recruited through flyers or word of mouth and must have lived in a demographically comparable region. Inclusion criteria for the control was based on age, sex, and ethnicity matched to the psychiatric patients. Exclusion criteria included being diagnosed with any psychiatric or medical disorders, however, one of the controls had a diagnosis of psoriasis, one had a diagnosis of asthma and one had a diagnosis allergy. Diagnostic interviews were performed with the controls to exclude individuals with current or previous psychiatric disorder. None of the participants were related to one another. All psychiatric patients were recruited between November 2016 and June 2018 and controls were recruited between January 2017 and June 2018. All controls were reimbursed (58€) for their participation. (Table 1).
      Table 1Demographics of participants.
      ControlsPsychiatric patients
      n = 40n = 40
      General parameters:
      Sex (m/f)15/2515/25
      Age, median (range)27 (11–45)27 (11–47)
      BMI, mean (SD)23.0 (2.6)24.4 (5.8)
      Educational level ≤ 9 years013
      Educational level > 9 years3819
      Age at onset13 (4–33)
      Medications (n):
      SRI15
      MAO inhibitors2
      Central stimulants2
      Antipsychotics (other than clozapine)18
      Clozapine4
      Lithium4
      Anticonvulsants9
      GABA-ergic sedatives10
      Histamine blockers12
      Anticholinergics4
      Antibiotics1
      Medical diagnoses:
      Asthma12
      Allergy11
      Diabetes type II-1
      Coeliac1
      Hypothyroidism2
      Gastric bypass surgery2
      Epilepsy1
      Psoriasis1
      Brain malformation1
      SRI: serotonin reuptake inhibitors (includes SSRI and SNRI).

      2.2 Diagnostic assessment prior to inclusion

      All patients had been diagnosed according to the DSM-IV by a clinical psychiatrist prior to being enrolled in the study. In addition, all psychiatric and medical records were available to the researchers throughout the study, including blood pressure, heart rate, and laboratory testing. The diagnostic criteria for ASD include an interview on childhood behavior with a parent of the subject in addition to an extensive assessment by a psychiatrist and a psychologist.

      2.3 Diagnostic assessments within this study

      For diagnostic validation, each participant was interviewed by a senior psychiatrist for approximately three to four hours, during which a brief motor skills test and a cognitive test were performed. Validated rating scales were used. For general diagnosis, the Mini International Neuropsychiatric Interview (M.I.N.I. version 7 and MINI-KID for children under 15 years) was used (
      • Sheehan D.V.
      • Lecrubier Y.
      • Sheehan K.H.
      • Amorim P.
      • Janavs J.
      • Weiller E.
      • et al.
      The Mini-international neuropsychiatric interview (M.I.N.I.): the development and validation of a structured diagnostic psychiatric interview for DSM-IV and ICD-10.
      ;
      • Sheehan D.V.
      • Sheehan K.H.
      • Shytle R.D.
      • Janavs J.
      • Bannon Y.
      • Rogers J.E.
      • et al.
      Reliability and validity of the Mini international neuropsychiatric interview for children and adolescents (MINI-KID).
      ). In addition, level of depression was assessed using the Patient Health Questionnaire (PHQ-9) (
      • Kroenke K.
      • Spitzer R.L.
      • Williams J.B.
      The PHQ-9: validity of a brief depression severity measure.
      ) and number of personality disorder traits were evaluated with The Personality Inventory for DSM-5, brief form (PID-5-BF) (
      • Quilty L.C.
      • Ayearst L.
      • Chmielewski M.
      • Pollock B.G.
      • Bagby R.M.
      The psychometric properties of the personality inventory for DSM-5 in an APA DSM-5 field trial sample.
      ). To confirm the severity of an ASD diagnosis, we used the Clinician-Rated Severity of Autism Spectrum and Social Communication Disorders, a DSM-5 derived measure (
      • American Psychiatric Association, APA
      Diagnostic and Statistical Manual of Mental Disorders.
      ). In order to confirm an OCD diagnosis, the Global Obsessive-Compulsive Scale (NIMH-GOCS) was used (
      • Tek C.
      • Uluğ B.
      • Rezaki B.G.
      • Tanriverdi N.
      • Mercan S.
      • Demir B.
      • et al.
      Yale-Brown obsessive compulsive scale and US National Institute of Mental Health global obsessive compulsive scale in Turkish: reliability and validity.
      ). For confirmation of SSD, the Clinician-Rated Dimensions of Psychosis Symptom Severity was given (
      • American Psychiatric Association, APA
      Diagnostic and Statistical Manual of Mental Disorders.
      ) and NSSID was established with the Clinician Administered Non Suicidal Self-Injury Disorder Index (CANDI) (
      • Gratz K.L.
      • Dixon-Gordon K.L.
      • Chapman A.L.
      • Tull M.T.
      Diagnosis and characterization of DSM-5 nonsuicidal self-injury disorder using the clinician-administered nonsuicidal self-injury disorder index.
      ) and the Deliberate Self-Harm Inventory (DSHI-9) (
      • Latimer S.
      • Meade T.
      • Tennant A.
      Measuring engagement in deliberate self-harm behaviours: psychometric evaluation of six scales.
      ). An assessment of health and disability was made using the self-administered, 12-item version of the WHO Disability Assessment Schedule (WHODAS 2.0). The complex method of scoring was used, for which an algorithm is utilized to calculate a total score (0 = no disability and 100 = full disability), with the resulting score indicating the percent of disability (
      • Gold L.H.
      DSM-5 and the assessment of functioning: the World Health Organization disability assessment schedule 2.0 (WHODAS 2.0).
      ). Global severity was determined with the Clinical Global Impression Severity (CGI-S) and the Global Assessment of Functioning (GAF) (
      • Jones S.H.
      • Thornicroft G.
      • Coffey M.
      • Dunn G.
      A brief mental health outcome scale-reliability and validity of the global assessment of functioning (GAF).
      ). CGI-S is a single question measurement of severity, (1 = not at all ill and 7 = extremely ill). The GAF measures overall psychological disturbance with separate ratings for symptoms and disability (1 = severely impaired and 100 = extremely high functioning). We assessed signs of neuroinflammation with the PsychoNeuroInflammatory related Signs and Symptoms Inventory (PNISSI), which is an unpublished questionnaire developed for this study. Each patient was then designated to a primary diagnostic group: SSD, ASD, OCD, or NSSID. (Table 2).
      Table 2Diagnostic assessments.
      n = 14n = 8n = 10n = 8
      Number of psychiatric diagnoses (median, range)6 (2−10)3 (1−10)5 (1−11)6 (3−13)
      Current depression, n (%)7 (50)4 (50)6 (60)8 (100)
      PHQ-9 (median, range)21 (5–26)16 (4–23)14 (4–20)21 (0–25)
      PID-5-BF (median, range)32 (16–49)26 (7–30)23 (9–51)32 (16–43)
      DSHI-9 (median, range)1 (0–27)0 (0−13)1 (0−12)30 (22–48)
      WHODAS 2.0 (median, range)65 (41–100)83 (27–100)73 (58–85)64 (33–81)
      CGI-S (median, range)5 (3–7)5.5 (3–7)5 (1–6)5 (2–6)
      GAF (median, range)40 (11–61)46 (22–60)45 (35–85)42 (25–51)
      M.I.N.I.: Mini-International Neuropsychiatric Interview.
      WHODAS 2.0: WHO Disability Assessment Schedule; Summary score (0 indicates no disability and 100 means maximal disability).
      PHQ-9: Patient Health Questionnaire (PHQ) (range 0–27), Scores of 5, 10, 15, and 20 represent cut-points for mild, moderate, moderately severe, and severe depression).
      PID-5-BF: The Personality Inventory for DSM-5 Short Form.
      DSHI-9: Deliberate Self-Harm Inventory – 9 item version; maximum score 54.
      CGI-S: Clinical Global Impression Scale - Severity.
      GAF: Global Assessment of Functioning.

      2.4 Psychiatric subject psychiatric and medical information

      The included psychiatric patients were all currently (or had previously been) markedly ill (GAF mean 43, CGI-S mean 4.7), with a median age at onset of illness of 13 years. For all future calculations, age of onset is defined as; early onset <13 y (n = 21), late onset ≥13 y (n = 16), and missing data (n = 3). Eighteen of the 40 psychiatric patients were being treated in psychiatric inpatient care because their illness was recently exacerbated. Another nine psychiatric patients were assessed as having a recent exacerbation, but were not in need of inpatient care, eight had a chronic course, and another two were newly diagnosed with a disorder. Exacerbations in illness, treated at either an inpatient ward or an outpatient clinic, were defined as an acute course, while symptom stability was categorized as chronic course. The psychiatric comorbidity of the patients was substantial and 25 of the patients fulfilled the criteria for major depression, even though this was not a target diagnosis. Furthermore, the majority of the patients met the diagnostic criteria for a least five different psychiatric diagnoses, eight patients fulfilled six or more diagnoses, and one patient met the criteria for 11 diagnoses. Four patients had an intellectually disability and another seven of the patients had medical diagnoses (celiac disease, type II diabetes, asthma, hypothyroidism, or elevated blood pressure (160/95, 160/100, 190/110)). The history of medical illnesses in psychiatric patients and controls are shown in Supplementary Table S1. Laboratory testing, including blood status, electrolytes, lever function (alanine transaminase, ALT; aspartate transaminase, AST), and thyroid function, were available for the psychiatric patients (nine were missing). One psychiatric subject had a low thyroid test, one had low hemoglobin levels, three psychiatric patients had high leukocyte counts (range 11.4–12.3), one had high ALT and AST, another two had only high ALT, and one patient had a high creatinine concentration. Two psychiatric patients had elevated blood glucose levels, one of whom had a diagnosis of diabetes. All other laboratory tests were normal (information available on request). The median overall dysfunction and disability as measured by the WHODAS was 69%. Thirty of the psychiatric patients were on permanent sick leave, seven patients were students, and three had a job on the open market.

      2.5 Ethics and consent

      All participants received verbal and written information about the study and signed an informed consent prior to being enrolled in the study. The study was approved by the Regional Ethical Review Board in Uppsala, Sweden (2016/091).

      2.6 Sample collection and preparation

      Peripheral whole blood was collected from all participants between 8 and 10 a.m., following an overnight fast. A total of 86 mL was drawn in eight 10 mL ethylenediaminetetraacetic acid (EDTA) tubes, one four mL EDTA tube, and one PAXgene Blood RNA tube. The four mL EDTA tubes were placed on ice directly after the sampling.

      2.7 Extraction of mRNA, conversion to cDNA and qPCR

      RNA was extracted from whole blood using the PAXgene system and qPCR was performed using the TaqMan Fast Universal PCR Master Mix (Applied Biosystems) and the TaqMan Gene Expression Assays (Applied Biosystems) on 15 genes (Table S2). The geometric mean for HPRT1 and TBP was used to normalize the expression levels of the genes of interest and the relative quantity was determined using a serially four-fold dilution standard curve from stimulated peripheral blood mononuclear cells.

      2.8 Protein measures

      Levels of cytokines and chemokines were measured in plasma using reagents and instrumentation for electrochemiluminescence (ECL) multiplex assay (U-Plex Biomarker 1 - Human) from Meso Scale Diagnostics (MSD), Rockville, MD.

      2.9 Digital immunoassay for quantification of IL-1β

      To enable quantification of low levels of circulating IL-1β in plasma, the SIMOA (Single Molecule Array) digital immunoassay platform was used (Quanterix, Lexington, MA, USA).

      2.10 Analysis of functional single-nucleotide polymorphisms (SNPs)

      Genotyping was performed for functional polymorphisms in NLRP3 (Q705K, rs35829419), CARD8 (C10X, rs2043211), and the promotor region of IL1B (T-31C, rs1143627) using TaqMan® SNP genotyping assays.
      For details for all laboratory analyses used, see supplementary file 1.

      2.11 Statistical analysis

      The quantitative data were not normally distributed, therefore a Mann Whitney U test was used to test the significance between psychiatric and control patients. Allele and genotype frequency for the analyzed SNPs were conducted using Fisher's exact test. A Spearman's rank-order correlation test was used to assess the relationship between the immunological markers and the CGI-S, GAF, and WHODAS. Conditional logistic regression was used to control for BMI as a confounding variable. A probability level of p < .05 set as the threshold for statistical significance. To account for multiple comparisons, the Benjamini-Hochberg procedure with a false discovery rate of 0.05 was used (
      • Benjamini Y.
      • Hochberg Y.
      Controlling the false discovery rate: a practical and powerful approach to multiple testing.
      ) for the cytokine analyses. Statistics were conducted using SPSSs version 22 and Graph Pad Prism 7.

      3. Results

      Psychiatric patients reported a more frequent history of inflammatory related physical illnesses than did the controls (41 vs 23). Similarly, the first-degree family members of the psychiatric patients reported more inflammatory related physical illnesses than the first-degree family members of the controls (55 vs 37) (See Table S1).
      Number of gene expression, cytokine levels, and functional polymorphisms associated with the NLRP3 inflammasome were analyzed along with general markers of inflammation to examine the connections between inflammatory perturbations and psychiatric disorders.

      3.1 Inflammasome related gene expression in patients with psychiatric disorders

      Gene expression of inflammasome components NLRP3, CASP1, and PYCARD were analyzed to investigate the priming state of circulating immune cells related to the NLRP3 inflammasome. Significant increases were found in psychiatric patients compared to controls for all three genes, CASP1 (p =.001), NLRP3 (p =.043), and PYCARD (p =.048) (Fig. 1A ). Similarly, IL1B also showed a significant increase in expression in the psychiatric patients (p =.001). However, IL18 levels did not differ between groups and neither IL33 nor IL37 could be quantified due to low expression levels. The IL-1 receptor antagonist gene (IL1RN) was, however, significantly increased among psychiatric patients (p =.001) (Fig. 1B). To examine more general markers of inflammation, the gene expression of TNF, IL6, and IL10 were analyzed, of which TNF was significantly increased compared to controls (p =.031) (Fig. 1C). When correcting for multiple comparisons, the differences between the psychiatric subject group and the control group remained significant for the genes CASP1, IL1B, and IL1RN. There was also a trend for increased levels for the genes NLRP3 (p =.077) and PYCARD (p =.073) among psychiatric patients.
      Fig. 1
      Fig. 1The priming state of circulating immune cells with respect to the NLRP3 inflammasome. Normalized gene expression of the inflammasome components NLRP3, CASP1, and PYCARD (A), members of the IL-1 family IL1B, IL18, and IL1RN (B), and general markers of inflammation TNF, IL6, and IL10 (C) were analyzed in peripheral whole blood sampled by PAXgene tubes. The blood was drawn in the morning after an overnight fast from 40 markedly ill psychiatric patients (red) with a mix of psychiatric disorders and 40 healthy controls (blue) matched for sex, age, and ethnicity. Following RNA isolation and reverse transcription, the gene expression was performed by qPCR. The figure shows the relative expression levels, quantified using the relative standard curve method and normalized by the selected reference genes (geometric mean of HPRTI and TBP). The bars show the mean expression (+/− SD). *p < .05, **p < .01, *** p < .001.

      3.2 Cytokine levels in patients with psychiatric disorders

      To examine whether the increased levels of gene expression translated into higher levels of circulating inflammasome-mediated cytokines, IL-1β, IL-18, and IL-1Ra were analyzed. For the IL-1 family cytokines, the plasma levels were significant higher in psychiatric patients compared to controls with respect to IL-1Ra (p <.001) and IL-18 (p =.001) (Fig. 2A ). However, there were no significant differences found in IL-1β levels. Psychiatric patients also had higher levels of the general inflammatory cytokines TNF (p <.001) and IL-6 (p <.001), but not IL-8. Significant differences were also seen between psychiatric patients and controls in hs-CRP (p =.009) (Fig. 2B), however, the levels of IL-10 were too low to be detected. When correcting for multiple comparisons, the differences between the groups remained significant for the cytokines presented above.
      Fig. 2
      Fig. 2Circulating plasma levels for the IL-1 family cytokines and general inflammatory cytokines. Cytokines IL-1β, IL-18, and IL-1Ra of the IL-1 family (A) and general inflammatory cytokines TNF and IL-6 (B), were analyzed in plasma from 40 markedly ill psychiatric patients (red) with a mix of psychiatric disorders and 40 healthy controls (blue) matched for sex, age, and ethnicity. Plasma was obtained from blood drawn into EDTA tubes in the morning after an overnight fast. Samples were run in duplicates in a multiplex ECL assay and the signals obtained were correlated to a standard curve of known concentrations for all analytes except IL-1β. IL-1β was instead quantified in duplicates using digital immunoassay, also using a standard curve of known concentrations for quantification. The bars show the concentration as pg/ml (+/− SD). * p < .05, **p < .01, *** p < .001.

      3.3 Functional polymorphisms in inflammasome related genes

      The observed increased gene expression of inflammasome components coupled with the increased circulating levels of the IL-1 family cytokines motivated the analysis of the presence of functional SNPs (specifically IL1B, NLRP3, and CARD8), previously associated with increased inflammasome activity and cytokine release (
      • Verma D.
      • Särndahl E.
      • Andersson H.
      • Eriksson P.
      • Fredrikson M.
      • Jönsson J.-I.
      • et al.
      The Q705K polymorphism in NLRP3 is a gain-of-function alteration leading to excessive interleukin-1β and IL-18 production.
      ;
      • Sahdo B.
      • Fransén K.
      • Asfaw Idosa B.
      • Eriksson P.
      • Söderquist B.
      • Kelly A.
      • et al.
      Cytokine profile in a cohort of healthy blood donors carrying polymorphisms in genes encoding the NLRP3 inflammasome.
      ). However, there were no significant differences in allele frequency between healthy controls and patients with severe psychiatric illness (Table S3).

      3.4 Correlations between trans-diagnostic measures of severity and inflammatory markers

      In the current study, general measures of severity in the patient groups included GAF (separately for symptoms and disability), CGI-S, and WHODAS, irrespective of diagnosis. Within the group of 40 psychiatric patients, these measures correlated with several of the gene expression markers. GAF symptoms were negatively correlated with IL18 (−0.39; p =.012), IL10 (−0.33; p =.043), and CASP1 (−0.32; p =.047) and GAF disability was negatively correlated with IL18 (−0.35; p =.025). CGI-S was positively correlated with IL18 (0.44; p =.004), PYCARD (0.34; p =.035), IL1RN (0.33; p =.037), and IL1B (0.32; p =.046) and the WHODAS was positively correlated with CASP1 (0.36; p =.037). The only correlation with circulating cytokines that emerged was between the WHODAS and IL-1Ra (0.35; p =.045). Contrarily, none of the inflammatory markers were correlated with general measures of function in the controls.

      3.5 Inflammatory markers among the different psychiatric diagnostic groups

      Post-hoc analyses were performed to examine possible differences between the four diagnostic groups. Differential findings emerged in subgroup analyses of gene expression levels, comparing the psychiatric patients in each primary diagnostic group with the controls. Patients with ASD had significantly elevated levels of three inflammation related genes (CASP1, IL1RN, and TNF), while CASP1 was higher in patients with SSD and TNF was higher in patients with OCD, when compared to controls (Table S4).
      Similarly, when comparing plasma cytokine levels between the diagnostic groups and controls, patients with SSD had higher levels in a majority of the investigated cytokines (i.e. IL-18, IL-1Ra, TNF, IL-6, and hs-CRP; Table S2). Moreover, the plasma levels of IL-18, IL-1Ra, and TNF were higher in patients with OCD and hs-CRP was increased in patients with ASD when compared to the control group (Table S5).

      3.6 Confounders

      After adjusting for BMI, the gene expression difference between patients and controls remained significant for CASP1 and IL1RN, but not for the other genes. For the circulating cytokines, the group difference remained significant for IL-18, TNF, and IL-6 after adjustment for BMI.
      In post-hoc analyses, neither comorbid depression nor use of psychotropic medication (yes/no), disease course (acute vs. chronic), or age at onset (early vs. late) had an effect on the obtained results.

      4. Discussion

      In the current study of markedly ill psychiatric patients diagnosed with either SSD, ASD, OCD, or NSSID with extensive comorbidity compared to matched controls, we measured gene expression, cytokine levels, and functional polymorphisms associated with the NLRP3 inflammasome. In addition, we measured more general cytokines associated with inflammatory responses. In line with previous research, our results showed group differences in most of the biomarkers, suggesting an ongoing immune dysregulation in psychiatric patients (
      • Marazziti D.
      • Mucci F.
      • Fontenelle L.F.
      Immune system and obsessive-compulsive disorder.
      ;
      • Masi A.
      • Glozier N.
      • Dale R.
      • Guastella A.J.
      The immune system, cytokines, and biomarkers in autism Spectrum disorder.
      ;
      • Khandaker G.M.
      • Cousins L.
      • Deakin J.
      • Lennox B.R.
      • Yolken R.
      • Jones P.B.
      Inflammation and immunity in schizophrenia: implications for pathophysiology and treatment.
      ). To our knowledge, this is the first study examining the priming state of the NLRP3 inflammasome in psychiatric patients with extensive comorbidity. This is also the first study that, to our knowledge, has investigated signs of inflammation in patients that fulfil the diagnostic criteria for NSSID. As opposed to previous research, we have included a wide range of psychiatric diagnoses and age groups, but the patients are largely homogenous regarding symptom severity and ability to function in the daily life, representing a typical patient in psychiatric care.
      The patients had longstanding illnesses, but unexpectedly the most common diagnosis was depression, which was neither an exclusion nor an inclusion criterion. Interestingly, our analysis did not support depression as a contributor to the elevation of the cytokines. Furthermore, most of the psychiatric patients were treated with psychotropic medication. Although we cannot exclude a drug effect on the measured biomarkers, the post-hoc analysis did not support that effect.
      Increased expression and activity of the inflammasome has been shown to promote inflammation in a large number of diseases in humans, including multiple sclerosis, Alzheimer's disease, and Parkinson's disease (
      • Guo H.
      • Callaway J.B.
      • Ting J.P.Y.
      Inflammasomes: mechanism of action, role in disease, and therapeutics.
      ). Previous studies measuring inflammasome-related gene expression have mainly focused on depression and stress-related mental disorders. Increased mRNA levels of CASP1 and NLRP3 were found in depressed patients compared to controls (
      • Alcocer-Gomez E.
      • de Miguel M.
      • Casas-Barquero N.
      • Nunez-Vasco J.
      • Sanchez-Alcazar J.A.
      • Fernandez-Rodriguez A.
      • et al.
      NLRP3 inflammasome is activated in mononuclear blood cells from patients with major depressive disorder.
      ). Furthermore, a study of mitochondrial fractions of post-mortem brain tissue from patients with either bipolar disorder or schizophrenia showed increased levels of CASP1, NLRP3, and PYCARD in those with bipolar disorder, while only CASP1 was elevated in those with schizophrenia (
      • Kim H.K.
      • Andreazza A.C.
      • Elmi N.
      • Chen W.
      • Young L.T.
      Nod-like receptor pyrin containing 3 (NLRP3) in the post-mortem frontal cortex from patients with bipolar disorder: a potential mediator between mitochondria and immune-activation.
      ). The only study published including patients with ASD also showed increased levels of CASP1 (
      • Saresella M.
      • Piancone F.
      • Marventano I.
      • Zoppis M.
      • Hernis A.
      • Zanette M.
      • et al.
      Multiple inflammasome complexes are activated in autistic spectrum disorders.
      ). These earlier studies on patients with individual diagnoses support the findings in the current study, which demonstrated increased mRNA levels of the three major NLRP3 inflammasome-related genes (NLRP3, CASP1, PYCARD), suggesting that immune cells of markedly ill psychiatric patients are primed with respect to the NLRP3 inflammasome. Furthermore, increased expression of PYCARD was significantly correlated with increased disease severity (
      • Dunlop B.W.
      • Gray J.
      • Rapaport M.H.
      Transdiagnostic clinical global impression scoring for routine clinical settings.
      ). Induced expression of these inflammasome components is believed to be a crucial regulatory step required for subsequent inflammasome activation, in which translated proteins (NRLP3, ASC, and caspase-1) assemble into a complete inflammasome (
      • Herman F.J.
      • Pasinetti G.M.
      Principles of inflammasome priming and inhibition: implications for psychiatric disorders.
      ).
      The primary function of the assembled NLRP3 inflammasome is to generate active forms of the cytokines IL-1β and IL-18 through the proteolytic activity of caspase-1 ((
      • Herman F.J.
      • Pasinetti G.M.
      Principles of inflammasome priming and inhibition: implications for psychiatric disorders.
      ;
      • Coll R.C.
      • O'Neill L.
      • Schroder K.
      Questions and controversies in innate immune research: what is the physiological role of NLRP3?.
      ). Both expression of and protein levels of these IL-1 family cytokines have previously been shown to be elevated in patients with particular psychiatric disorders, such as SSD (
      • Rodrigues-Amorim D.
      • Rivera-Baltanas T.
      • Spuch C.
      • Caruncho H.J.
      • Gonzalez-Fernandez A.
      • Olivares J.M.
      • et al.
      Cytokines dysregulation in schizophrenia: a systematic review of psychoneuroimmune relationship.
      ;
      • Al-Hakeim H.K.
      • Al-Rammahi D.A.
      • Al-Dujaili A.H.
      IL-6, IL-18, sIL-2R, and TNFalpha proinflammatory markers in depression and schizophrenia patients who are free of overt inflammation.
      ) and ASD (
      • Masi A.
      • Quintana D.S.
      • Glozier N.
      • Lloyd A.R.
      • Hickie I.B.
      • Guastella A.J.
      Cytokine aberrations in autism spectrum disorder: a systematic review and meta-analysis.
      ). In the current study, the mRNA levels of IL1B, but not IL18, were increased in patients compared to controls. However, this did not directly translate into different protein levels, where instead, IL-18 plasma levels showed a significant difference but IL-1β did not differ between patients and controls. This could be partially explained by the fact that gene expression of IL1B is readily induced upon activation of pattern-recognition receptors, while gene expression of IL18 is constitutively expressed in circulating immune cells and requires type I interferon to signal for further induction of gene transcription (
      • Zhu Q.
      • Kanneganti T.D.
      Cutting edge: distinct regulatory mechanisms control Proinflammatory cytokines IL-18 and IL-1beta.
      ). Furthermore, IL-18 shows a protein expression pattern that is more widely distributed among tissues compared to IL-1β (www.proteinatlas.org), suggesting that increased levels of circulating IL-18 in patients might have non-immune cell origin. Support for the increased activity in the IL-1 family pathway is further strengthened by the findings that IL-1Ra, the IL-1 receptor antagonist, showed increased levels of both gene expression and circulating protein in psychiatric patients. This is similar to previous studies in SSD patients, in which increased levels of IL-1Ra were shown (
      • Rodrigues-Amorim D.
      • Rivera-Baltanas T.
      • Spuch C.
      • Caruncho H.J.
      • Gonzalez-Fernandez A.
      • Olivares J.M.
      • et al.
      Cytokines dysregulation in schizophrenia: a systematic review of psychoneuroimmune relationship.
      ;
      • Morch R.H.
      • Dieset I.
      • Faerden A.
      • Hope S.
      • Aas M.
      • Nerhus M.
      • et al.
      Persistent increase in TNF and IL-1 markers in severe mental disorders suggests trait-related inflammation: a one year follow-up study.
      ), although previous studies of ASD patients have not shown any such differences (
      • Prata J.
      • Santos S.G.
      • Almeida M.I.
      • Coelho R.
      • Barbosa M.A.
      Bridging autism Spectrum disorders and schizophrenia through inflammation and biomarkers - pre-clinical and clinical investigations.
      ;
      • Masi A.
      • Quintana D.S.
      • Glozier N.
      • Lloyd A.R.
      • Hickie I.B.
      • Guastella A.J.
      Cytokine aberrations in autism spectrum disorder: a systematic review and meta-analysis.
      ). Within the psychiatric subject group, increased expression of all three IL-1 cytokines was correlated with increased disease severity. Taken together, the results from circulating cytokines suggest that increased activity in the IL-1 family pathway in markedly ill psychiatric patients.
      Elevated levels of the IL-1 family cytokines are a hallmark of certain auto-inflammatory disorders and are strongly associated with mutations in NLRP3 and CARD8, which are related to increased inflammasome activity (28-30). Therefore, it is plausible that the increased levels of mRNA and circulating proteins in the IL-1 family pathway in our study may be due to the presence of such common SNPs. However, we were unable to find any differences in the distribution of the SNPs between psychiatric and controls.
      Another novel finding in the current study is that of increased levels of one of the more general markers of inflammation when gene expression of TNF, IL6, and IL10 were analyzed, namely that TNF was significantly increased in psychiatric compared to controls. Similar to previous research on schizophrenia (
      • Rodrigues-Amorim D.
      • Rivera-Baltanas T.
      • Spuch C.
      • Caruncho H.J.
      • Gonzalez-Fernandez A.
      • Olivares J.M.
      • et al.
      Cytokines dysregulation in schizophrenia: a systematic review of psychoneuroimmune relationship.
      ) and autism (
      • Masi A.
      • Glozier N.
      • Dale R.
      • Guastella A.J.
      The immune system, cytokines, and biomarkers in autism Spectrum disorder.
      ), the general protein markers of inflammation, TNF, IL-6, and CRP were increased in the psychiatric group compared to the control group. Data from patients with OCD, however, are scarce and show inconsistent results (
      • Marazziti D.
      • Mucci F.
      • Fontenelle L.F.
      Immune system and obsessive-compulsive disorder.
      ). Of note, there was no correlation between disease severity and increased plasma concentrations nor gene expression levels of these general proinflammatory markers.
      Finally, in our limited sub-group analysis, differences in mRNA levels regarding CASP1, IL1RN, and TNF, were found in patients with SSD, ASD, and OCD when compared to controls. Increased levels of circulating cytokines were most pronounced in patients with SSD and OCD, where IL-1Ra, IL-18, and TNF, were elevated. Even though these diagnoses differ to some extent in their inflammatory pattern, the current sub-group analyses strengthen the idea that severely ill psychiatric patients display perturbations in the inflammasome and IL-1 family pathway. Further studies are needed to determine if these perturbations are directly involved in the pathophysiology of psychiatric disorder in general or if inflammasome priming and subsequent production of IL-1 family cytokines is a secondary effect of other, primary mechanisms.
      In conclusion, the data shows that circulating immune cells from individuals with psychiatric disorders with comorbidities, irrespective of the specific diagnosis, are primed with respect to the NLRP3 inflammasome. Additionally, psychiatric patients also showed increased cytokine levels when compared to matched controls. Together, this supports the inflammation hypothesis for markedly ill psychiatric patients across diagnostic groups.

      4.1 Strengths and limitations

      There are some important limitations to this study. First, the sample size is small and therefore comparisons between the different diagnostic groups must be regarded as preliminary findings. Second, the cytokines were only sampled and measured at one-time point, which does not allow any conclusions on deviations over time to be drawn. Some of our cytokine samples were below the limit of detection and, consequently, this loss of data decreased the power of our study. Third, although our ambition was to match the controls closely to the psychiatric patients, there was a difference in BMI between the groups. When adjusting for BMI, the differences between the groups became smaller. However, psychiatric patients are frequently overweight and it is a challenge to match the groups for BMI when also matching to sex, age, and ethnicity. Furthermore, psychotropic medication such as antidepressants and antipsychotics can have an immunomodulatory effect and may cause changes in cytokine levels by normalize or decreasing them (
      • Baumeister D.
      • Ciufolini S.
      • Mondelli V.
      Effects of psychotropic drugs on inflammation: consequence or mediator of therapeutic effects in psychiatric treatment?.
      ). The results in our study showed elevated levels of cytokines despite the fact that the majority of the patients received psychotropic drugs. An additional strength is that our clinical sample was assessed by a clinical psychiatrist and included patients with severe psychiatric symptoms. However, another limitation is that the controls were not examined by a senior psychiatrist and not controlled for medical work-up. However, the controls were recruited simultaneously to the psychiatric patients and were age, gender, and ethnicity matched, and carefully examined for psychiatric disorders, allergies, and other inflammatory disorders, similarly to the psychiatric patients. Finally, as we included patients in markedly symptomatic phases, our results may pertain to the patients' present state rather than disorder related traits. Accordingly, trait-related inflammasome measures remain to be studied.

      Acknowledgements

      The authors also would like to thank Kaya Tursun for technical assistance in analyzing the SNPs.

      Fundings

      The work was supported by research grants awarded from Region Örebro County (ALF-funding and from the Research Committee) and strategical funding from the Faculty of Medicine and Health , Örebro University . The funding sources had no role in study design, data collection, analysis or interpretation of the data.

      Appendix A. Supplementary data

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