This study demonstrated that there were no differences between BD- and BD+ in terms of sTNF-R1 and sIL-6R concentrations. However, sTNF-R1 and sIL-6R concentrations were higher in both BD- and BD+ in comparison to WC.

Our findings are in line with a previous study reporting higher concentrations of sTNF-R1 in euthymic BD patients compared to controls [18, 19]. It has been suggested that soluble cytokine receptors may better reflect the activity of the cytokines in question, given that they are detectable in plasma even when cytokine concentrations are undetectable [18]. Thus, it is plausible to think of soluble cytokine receptors as more reliable markers of activity. In the light of this evidence, both sTNF-R1 and sIL-6R can be considered as markers reflecting pro-inflammatory activity. Both TNF-α and IL-6 are primary regulators of pro-inflammatory process due to their initiating roles in the inflammatory cascade of the acute phase response [7]. They have the ability of affecting various cells and therefore are regarded as possessing pleiotropic properties. It has been demonstrated that the degradation of tryptophan (TRP) by the indoleamine 2,3-dioxygenase (IDO) enzyme is mainly induced by interferon gamma (IFN-γ), but also, to a degree, by TNF-α and IL-6 [20]. Induced IDO activity causes degradation of TRP, which serves as a precursor for serotonin. Moreover, induced IDO activity leads to the formation of neurotoxic metabolites, 3-hydroxykynurenine and quinolinic acid [21]. Several studies have shown an association between IDO activation and mood disorder, particularly unipolar depression [20]. Recently, the TNF-α system has been postulated as a target for understanding the pathophysiology of BD [6, 22]. TNF-α exerts its effects via the cell surface receptors called TNFR1 and TNFR2. The effect of TNFR2 is negligible to TNFR1, since TNFR1 is expressed in most cells, whereas TNFR2 is expressed only in hematopoietic cells. TNFR1 mediates apoptosis, cytokine production and activation of nuclear factor kappa B (NF-κB), which induces transcription of cell survival genes (i.e. cellular inhibitors of apoptosis (cIAP), B-cell lymphoma 2 (Bcl-2) family) [6]. It has been postulated that there is a shift towards apoptosis during mood episodes and that this shift arises from increased TNF-α concentration and cessation of NF-κB neuronal translocation due to decreased brain-derived neurotrophic factor (BDNF) and nerve growth factor (NGF) [22]. On balance, the TNF-α system may be considered an important factor playing a role in the regulation of neuroplasticity. Moreover, the notion of TNF-α as a trait marker of BD has some validity given evidence of higher TNF-α concentration in patients with BD than in healthy controls, evidence of effects of TNF-α antagonists on mood symptoms and effects of psychotropics on TNF-α [5, 6]. However, it is still premature to count TNF-α as a trait marker of BD due to the influence of several confounding factors, particularly the use of medication, on the immune system. Likewise, the results of our study should be interpreted carefully considering medication use as a potential confounder [23]. Controlling for this was not possible, as only patients used medication, resulting in collinearity. Recent studies showing no difference between medicated euthymic BD patients and well controls in terms of TNF-α and IL-6 concentrations contradict the current findings [8, 10, 11, 24]. The conclusion is that additional studies evaluating inflammatory markers in the euthymic state, ideally comparing medication-free BD patients and matched controls in large samples, are still needed in order to clarify if ongoing inflammation is a trait in BD.

According to the model for staging BD with the use of biomarkers, progression through later stages with social and cognitive dysfunction and persistent subsyndromal symptoms in the inter-episode period is associated with increased oxidative stress and inflammation, which can be demonstrated by high 3-nitrotyrosine and TNF-α concentrations [13, 14]. This model stages BD in five categories according to clinical features: Latent Stage is defined by positive family history of mood disorders, subthreshold mood or anxiety symptoms; Stage 1 is defined by euthymic periods without overt psychiatric symptoms; Stage 2 is defined by symptoms during inter-episodic periods and comorbid psychiatric disorders; Stage 3 is defined by marked impairment in cognition and functioning; Stage 4 is defined by need of a caregiver in daily life due to severe impairment in cognition and functioning [13]. The patients with subsyndromal symptoms in our study had persistent symptoms during euthymia, and experienced a greater number of episodes. Thus, they can be considered as patients presenting at a later stage of illness than the group of patients without subsyndromal symptoms. However, we did not find any differences between these two groups in terms of sTNF-R1 and sIL-6R concentrations. In their evaluation of brain-derived neurotrophic factor and cytokines in early and late stage BD patients, Kauer-Sant’Anna and colleagues demonstrated that TNF-α concentration remained higher in late stage patients even after controlling for subsyndromal symptoms [25]. Consequently, they propose that higher TNF-α concentration in the later stages of illness may be due to the cumulative effect of past mood episodes or the cumulative effect of pro-inflammatory shifts throughout the course of illness, rather than the effect of subsyndromal symptoms. In accordance with this finding, the current analysis confirms that subsyndromal symptoms have minimal effect on cytokine concentrations. Nevertheless, we do not underestimate the cumulative effect of ongoing subsyndromal symptoms in patients with advanced duration of illness. BD+ patients had a greater number of episodes per year, which can be counted as an identifier for poor prognosis in the future. Thus, it is also plausible to think that pro-inflammatory shifts may become more evident later in the course of illness in BD+ compared to BD-. Alternatively, BD- and BD+ could belong to same stage, in contrast to what was hypothesized. An in-depth evaluation of social and cognitive functioning could have provided more variables for empirical staging.

To the best of our knowledge, this is the first study investigating immune changes in subsyndromal BD patients. Although our analyses focused on potential inflammatory markers of subsyndromal symptoms in BD-I, no actual associations were presented between inflammatory markers and specific symptoms. Instead, we relied on group-level comparisons of patients with and without subsyndromal symptoms. To the degree that these groups differ in other aspects other than symptomatology, results could have been confounded. Therefore, follow-up research focusing on comparisons at the symptom level in larger samples is required. Although the study had the benefit of recruiting patients with subsyndromal symptoms with the help of standardized assessments in a specialized mood disorders outpatient unit, the definition for subsyndromal state recommended by the International Society of Bipolar Disorder (ISBD) could have been measured more precisely [26]. However, there was no consensus on the definition of subsyndromal states when the study recruitment was initiated. Moreover, the cross-sectional design of the study limits our further prediction about the effect of subsyndromal symptoms on cytokines in the long term. It is also important to emphasize that we were unable to assess body mass index as a potential confounder. Only soluble receptors of TNF-α and IL-6 were assessed, which are the most studied cytokines in BD and considered as molecular targets [6, 15, 22]. Nevertheless, evaluation of additional inflammatory markers could provide the opportunity of interpreting the findings in more detail. All patients were on medication. As psychotropics (i.e. lithium) may impact on cytokine concentrations [9, 12, 23], this could have confounded the results. Given the fact that almost all patients in this small study had unique, non-overlapping, treatment modalities (i.e. different combinations of medications and/or different doses), it was impossible to group according to treatment modality in detail and describe medication-related effects in the patient sample. Moreover, previous work suggests that medications have different, sometimes interacting effects on immune mediators [23]. Although it could be argued that medication equivalents might have been used in analyses within the patient group, this method can only be applied to antipsychotics. Furthermore, caution must be taken when using this methodology, since medication equivalents reflect efficacy rather than biological mechanisms. Thus, it was decided not to analyze the effect of each separate medication on immune mediators in this relatively small sample. This is a limitation. On the other hand, eliminating this possibly confounding effect is nearly impossible given the fact that augmentation strategies, resulting in patient-unique medication mixes, are routine practice in the treatment of bipolar disorder [15]. The cross-sectional design of the study also prevented us to evaluate the effects of earlier, currently discontinued treatment. Given the possibility of residual confounding by medication, and the difficulties in disentangling confounding by medication within patient-specific polypharmacy approaches, follow-up semi-experimental studies are required to settle this issue. Finally, the sample size of the study was relatively small, which had the inherent risk of type-II error.