Serum level of soluble interleukin 6 receptor is a useful biomarker for identification of treatment‐resistant major depressive disorder

Abstract Aim A substantial proportion of major depressive disorder patients are treatment‐resistant to antidepressant therapy, who require augmentation drugs, or other treatments including electroconvulsive therapy or transcranial magnetic stimulation. Identifying treatment‐resistant major depressive disorder patients before the actual administration of antidepressant is, however, often difficult. Accordingly, the serum biomarker to identify treatment‐resistant patients will be helpful in clinical settings. This study aims to clarify the appropriate biomarkers for identification of treatment‐resistant major depressive disorder. Method Given that immune‐inflammatory processes are involved in the pathogenesis of major depressive disorder, it is possible that certain cytokine‐related molecules could serve as clinically useful biomarkers of treatment‐resistant major depressive disorder patients. In this study, we measured serum levels of tumor necrosis factor‐α, interleukin 6, and soluble interleukin 6 receptor after major depressive disorder patients underwent antidepressant therapy. Results The serum level of soluble interleukin 6 receptor, but not interleukin 6 or tumor necrosis factor‐α, was significantly higher in treatment‐resistant major depressive disorder patients than in remitted patients, suggesting that serum soluble interleukin 6 receptor could be a good biomarker of treatment‐resistant major depressive disorder. Receiver operating characteristic analysis confirmed that serum soluble interleukin‐6 receptor level measurement was useful for identification of treatment‐resistant major depressive disorder patients. Multiple regression analysis using the serum levels of the aforementioned cytokines as explanatory variables and the Quick Inventory of Depressive Symptomatology‐Self Report score (QIDS‐SR16) as a target variable showed that only serum soluble interleukin‐6 receptor level could explain the severity of major depressive disorder. Conclusion Based on these results, we recommend measurement of serum soluble interleukin‐6 receptor level to discriminate treatment‐resistant major depressive disorder patients. High serum soluble interleukin‐6 receptor level is associated with the pathogenesis of treatment‐resistant major depressive disorder, suggesting the involvement of the interleukin 6 trans‐signaling system in onset of treatment‐resistant major depressive disorder.


| INTRODUC TI ON
Major depressive disorder (MDD) is a leading cause of disability with the highest disability-adjusted life year (DALY) value among all diseases and disorders, and thus places a significant burden on society. 1,2 A substantial proportion of MDD patients are treatment-resistant, that is, they do not enjoy satisfactory therapeutic outcomes in response to first-line or subsequent attempts at pharmacological therapies. 3,4 Hence, more efficacious treatment for such patients is urgently required.
The definition of treatment-resistant depression is controversial. In actual clinical settings, patients who receive antidepressants are evaluated for therapeutic outcome using rating scales based on subjective indexes; the relationships between the answers to each question are relative, and some degree of arbitrariness cannot be entirely eliminated. Given the importance of highly reproducible means for evaluating the effect of longitudinal treatment, more objective measures must be developed to evaluate the outcome of pharmacotherapy in MDD patients.
Recently, it has become clear that the pathogenesis of MDD involves immune-inflammatory processes, and the cytokines interleukin-6 (IL-6) and tumor necrosis factor-α (TNF-α) have been implicated in induction of depressive symptoms. [5][6][7][8][9] In addition, soluble IL-6 receptor (sIL-6R), which is produced by cleavage before the membrane-spanning region of IL-6 receptor, 10,11 may also play a role in the onset of depressive symptoms. 12,13 We investigated the usefulness of the serum levels of IL-6, TNF-α, and sIL-6R as biomarkers for discriminating treatment-resistant MDD patients from remitted patients.
The results revealed that serum sIL-6R level is a biomarker of treatment-resistant MDD. Because sIL-6R can stimulate trans-signaling by forming a ligand-receptor complex with IL-6 after binding to gp130, our findings suggest a possible role for the IL-6 trans-signaling system in the onset of treatment-resistant MDD.

| Definition of treatment-resistant and remitted MDD patients
A definition of treatment-resistant MDD patient is crucial for selection of the study population. Treatment resistance can be partial or full, and the definition should rely on an operational algorithm. It is not currently known how many adequately delivered antidepressant trials are necessary in order to declare a patient treatment-resistant. Modest resistance involves inadequate response to a single antidepressant trial, whereas greater resistance involves failures of two monotherapy trials or one or more augmentation trials. [14][15][16] However, in usual clinical settings, if an episode of depression is not adequately treated by at least two adequate trials of medications from different classes of antidepressants, treatment resistance can be clinically declared. In this study, patients who had not achieved remission after receiving two or more medications from different classes of antidepressants for at least 12 weeks were defined as treatment-resistant. On the other hand, remitted patients were defined as patients who had attained remission after receiving one or two antidepressant medication. A score of ≤5 on the Quick Inventory of Depressive Symptomatology-Self Report (QIDS-SR 16 ) equivalent to ≤7 on the 17-item Hamilton Rating Scale for Depression (HRSD 17 ) was defined as remission. disorder patients. High serum soluble interleukin-6 receptor level is associated with the pathogenesis of treatment-resistant major depressive disorder, suggesting the involvement of the interleukin 6 trans-signaling system in onset of treatment-resistant major depressive disorder.

K E Y W O R D S
cytokines, IL-6 trans-signaling system, major depressive disorder, soluble IL-6 receptor, treatment-resistant depression

| Statistical analysis
Serum levels of TNF-α, IL-6, and sIL6-R were compared between treatment-resistant and remitted patients. Differences among groups were analyzed by IBM SPSS. Data are presented as mean ± SEM P < .05 was considered to represent a statistically significant difference.
Receiver operating characteristic (ROC) analysis was performed; a criterion of area under the curve (AUC) > .7 was used to determine treatment efficiency.
Multiple regression analysis was performed in SPSS by a stepwise selection method in which QIDS-SR 16 score was used as a dependent variable, and individual cytokine levels were used as independent variables. Correlation analysis was also performed between serum sIL-6R level and each category of QIDS-SR 16 score, and factor analysis was performed based on the correlation-coefficient matrix among categories of QIDS-SR 16 .  Figure 1A).
In ROC analysis, serum sIL-6R level yielded the only significant AUC (=0.789), which was the highest among the cytokines measured ( Figure 2). Based on this analysis, the cutoff serum sIL-6R level for discriminating treatment-resistant from remitted patients was 35.80 ng/mL, with sensitivity and specificity of 0.68 and 0.71, respectively.
In multiple regression analysis, only sIL-6R was extracted as an explanatory variable for QIDS-SR 16 score (Figure 3). The coefficient of determination was 0.278, which was not high, so we performed a correlation analysis between the score in each QIDS-SR 16 category and serum sIL-6R level. We detected a significant correlation between serum sIL-6R level and scores related to abnormality of appetite and loss of interest (Table 2). Sorrowful feelings and suicidal ideation had low correlation coefficients with serum sIL-6R level,

| DISCUSS IONS
The number of MDD patients has been increasing around the world, causing substantial impairment of individual patients' functions, as well as a significant economic burden to society. 18  Here, we demonstrated that serum sIL-6R concentration is significantly higher in treatment-resistant than remitted MDD patients.
Our results show that serum sIL-6R level is a potentially useful biomarker for identifying treatment-resistant patients. In the future, physicians could develop effective treatments through experiments targeting patients with higher serum concentrations of sIL-6R.
In regard to the underlying mechanisms of MDD, depressive symptoms are associated with sustained activation of innate immune system, leading to increased production of proinflammatory cytokines. [19][20][21] Proinflammatory cytokines such as IL-6 and TNF-α pathological synaptic pruning, which may induce structural and functional brain changes, 27 ultimately leading to depressive symptoms and maladaptive behaviors. 28,29 The IL-6 trans-signaling system is involved in IL-6 production in microglia and neurons; consistent with this, lipopolysaccharide injection can induce maladaptive behaviors. 12 Inflammatory cytokines including IL-6 are known to contribute to the pathogenesis of rheumatoid arthritis, and monoclonal antibody such as tocilizumab is effective to some patients but not all rheumatoid arthritis with high remission rate. It is essential to identify the rheumatoid arthritis patients who will respond to anti-IL-6 therapy. There are papers reporting biomarkers to identify those rheumatoid arthritis patients with good response using sIL-6R levels, in which serum sIL-6R level, but not IL-6 level, reportedly predicts clinical remission in patients with rheumatoid arthritis treated with IL-6 receptor antibody (tocilizumab). 30,31 Our finding of the higher serum sIL-6R level of treatment-resistant MDD patients may share the similar inflammatory process caused by cytokines including IL-6 in rheumatoid arthritis and MDD.
Because IL-6 trans-signaling causes microglial activation in the brain, 32 it is possible that a high serum sIL-6R level leads to the onset of treatment-resistant MDD.

Khandaker et al reported the association between a functional
IL-6R genetic variant and risk of depression, suggesting IL-6R Asp358 Ala variant is associated with decreased risk of severe depression causing decreased serum CRP level in spite of the increased serum IL-6 level, consistent with an anti-inflammatory effect downstream of IL-6. They concluded that the IL-6/ IL-6R pathways are involved with pathogenesis of severe depression. 33 Further study is required to determine the threshold serum sIL-6R level necessary for treatment resistance. Since inflammatory process is regarded as the common process between MDD and somatic diseases including rheumatoid arthritis and cardiovascular diseases, 34  trans-signaling, which might affect the brain function without peripheral proinflammatory process oriented with higher serum IL-6 and sIL-6R together with higher CRP. We cannot conclude whether the treatment-resistant MDD patients classified in our study are matching with the low-grade systemic inflammation as reflected by elevated CRP. 35 We do not know how much serum sIL-6R penetrates the blood-brain barrier, whether sIL-6R concentrations in the brain and periphery are correlated, or whether they are regulated through the same or similar mechanisms.

ACK N OWLED G M ENTS
The authors wish to thank Prof. Tadamitsu Kishimoto, the discoverer of IL-6 and professor of Immunology Frontier Research Center (IFReC), Osaka University, for his timely support and helpful advice, and N. Ohgoshi for her valuable contributions to sample preparation and coordination among the researchers involved in this study.

CO N FLI C T O F I NTE R E S T
The authors declare no conflict of interest.

AUTH O R CO NTR I B UTI O N S
KY involved in conception and design of the study, acquisition and analysis of data, drafting the manuscript and figures, and project administration. TH involved in conception and design of the study, analysis of data, and drafting the figures. MT: involved in supervision and validation.

DATA R E P O S I TO RY
All relevant data are included in Supporting Information.

A PPROVA L O F TH E R E S E A RCH PROTO CO L BY A N I N S TITUTI O N A L R E V I E WER B OA R D
The study was reviewed and approved by Clinical Research Ethical Committee of Kensho-kai Medical Corporation (No. 1201-05-005708).

I N FO R M E D CO N CE NT
The patients/participants provided their written informed consent to participate in this study.

R EG I S TRY A N D TH E R EG I S TR ATI O N N O. O F TH E S TU DY/ TR I A L
N/A.