Blunted response of tinnitus patients to experimental stress. Reprinted with permission from (Hebert and Lupien 2007)
In tinnitus patients, the post-TSST cortisol peak is reduced, and it occurs later than in the control subjects.
Interestingly, after a challenge with 0.5 mg of dexamethasone, tinnitus patients experienced deeper and longer-lasting suppression of cortisol production than the control subjects, suggesting increased glucocorticoid sensitivity and augmented HPA axis feedback (Simoens and Hebert 2012). Measurement of cortisol responses seems to be a promising step toward development of reliable set of tinnitus biomarkers.
6.2.2 Catecholamines: Dopamine, Epinephrine (Adrenaline), and Norepinephrine
Catecholamines are monoamines derived from tyrosine with a catechol side chain. Catecholamines comprise epinephrine (adrenaline), norepinephrine (noradrenaline), and dopamine. Adrenaline and noradrenaline are synthesized in response to sympathoadrenal medullary axis activation.
Two studies investigated the systemic concentrations and various correlations between blood/urine catecholamines and tinnitus-related distress; however, no conclusive correlations have been shown between the catecholamine concentrations and psychometric scores (Kim et al. 2014; Savastano et al. 2007). It is possible that the future classification of tinnitus subtypes, which will take under account all the comorbid conditions, may facilitate the use of catecholamines as tinnitus biomarkers.
6.3 Immune Biomarkers of Stress
To understand the biological basis of the immune biomarkers of stress, we have to turn back into the past. Until evolutionarily recent times (about 200 years ago), people’s life expectancy was rather short and was on average 30 years (Finch 2012). The developments in medicine and technology occurring in the past two centuries, contributed to the enormous extension of human life expectancy by roughly two-and-a half-fold. While today, the life expectancy is restricted by neurodegenerative, cancer, and heart diseases, in the past, the main life expectancy-restricting factors were infectious diseases.
The evolutionary process positively selected individuals that upon being wounded (which was connected with emotional stress, imminent infection, and a consequent danger of serious illness and death), quickly responded with an inflammatory reaction. Also the sickness behavior was evolutionarily driven, ensuring that the infected individual kept away from the group to not to spread the infection further, and was vigilant regarding possible sources of infection (Miller and Raison 2016). The sickness behavior is also a feature of depression whereas vigilant behavior characterizes anxiety disorders.
Also until recent times, commensal microorganisms and parasites, such as helminths, kept the “background” systemic inflammatory reaction under control, by activating regulatory T cells, inducing production of anti-inflammatory cytokines, such as interleukin-10 (IL-10) or transforming growth factor beta (TGF-beta) (Finlay et al. 2014). What it meant for people was that they would have low “spontaneous” or “background” inflammation.
In the past, being wounded belonged to the major and most common emotional stressors. Stress not only affects the neuroendocrine system but also immediately activates the white blood cells to produce proinflammatory interleukin-6 (IL-6) and tumor necrosis factor alpha (TNF alpha). In addition, stress activates special proteases (caspase-1) to cleave the inactive form of already produced proinflammatory interleukin-1 beta (IL-1 beta) into its active form. As a result, when a person is wounded, strong inflammatory reaction supported by emotional stress is mounted in an attempt to eliminate the dangerous pathogen.
Our contemporary, highly hygienic living condition, prevent the modern people from the contact with commensal organisms. As a result, the inflammatory background, for which our ancestors were positively selected, can no longer be mitigated. This is believed to be the cause for ever increasing incidence of allergies, autoimmune reactions, and other proinflammatory conditions.
The pathogen-host defense hypothesis of depression is a novel notion in the field that explains the origin of depressive symptoms as well as the even increasing incidence of depression in the industrialized world. According to this hypothesis, individuals with the increased inflammatory background (i.e., producing proinflammatory cytokines or C-reactive protein) will react to it with the sickness behavior that includes social withdrawal (as in depression) and increased vigilance (as in anxiety) (Fig. 6.2). Of note—depressive and anxiety symptoms are very often diagnosed in patients with tinnitus—see Chap. 3 (Zirke et al. 2013a, b).
The evolutionary theory of inflammation and depression. Early evolutionary pressures derived from human interactions with pathogens, predators and human conspecifics (such as rivals) resulted in an inflammatory bias that included an integrated suite of immunological and behavioral responses that conserved energy for fighting infection and healing wounds, while maintaining vigilance against attack. This inflammatory bias is believed to have been held in check during much of human evolution by exposure to minimally pathogenic, tolerogenic organisms in traditional (that is, rural) environments that engendered immunological responses characterized by the production of the anti-inflammatory cytokines. In modern times, sanitized urban environments of more developed societies are rife with psychological challenges but generally lacking in the types of infectious challenges that were primary sources of morbidity and mortality across most of human evolution. In the absence of traditional immunological checks and balances, the psychological challenges of the modern world instigate ancestral immunological and behavioral repertoires that represent a decided liability, such as high rates of various inflammation-related disorders including depression. Reprinted with permission from (Miller and Raison 2016)
Pathogen-host Defense Hypothesis of Depression
Our ancestors were evolutionarily selected by exposure to pathogens because of proinflammatory immune response and specific behavior induced by this response (social isolation and vigilance).
Interleukin-1 beta (IL-1 beta), Interleukin-6 (IL-6), and tumor necrosis factor alpha (TNF alpha) belong to a group of cytokines commonly associated with the process of inflammation (so-called proinflammatory cytokines).
Cytokines are peptides used for communication between the cells and comprise interferons, lymphokines (e.g., interleukins), chemokines, and tumor necrosis factors. Cytokines are released from the producing cells and act on cells with an appropriate receptor by inducing signal transduction, which is cytokine-, receptor- and cell type-specific. First reports about cytokines date form the late fifties in the last century and regard interferons produced in response to viral infection. In the eighties, the cytokine discovery picked up enormously whereas presently, the field comprises several hundreds of molecules (for more detail, see Horst Ibelgaufts’ cytokine encyclopedia “COPE” under www.cells-talk.com).
For decades, cytokines were considered to be strictly connected with the immune system; however, presently we know that also the nonimmune cells are also capable to synthesize, release, and react to cytokines. A very important issue regarding cytokines in the field of biomarkers is to realize that the local production, and release of cytokines by specific tissues, e.g., the brain or the lymph node (local changes), must not necessarily change the levels of cytokines in blood (systemic changes). Short-term and long-term stresses act differently on the immune system and consequently on the release of cytokines.
Earlier, cytokines were considered to be strictly connected with the immune system; however, by now we know that nonimmune cells are also capable to synthesize, release and react to cytokines.
18.104.22.168 Interleukin-1 Beta
Interleukin-1 beta (IL-1 beta) is a small peptide (17.5 kDa) produced in an inactive form and activated by cleavage with caspase 1. IL-1 beta was cloned and characterized in 1984 by the group of Charles A. Dinarello (Dinarello 1994). Dinarello was scientifically captivated by the pyrogenic (inducing fever) properties of interleukin-1 beta—a clear link between the immune and neuronal systems (Dinarello 1999). IL-1 beta has another important property—it is a major mediator of fatigue in various unrelated diseases (Roerink et al. 2017). In addition, increased concentrations of IL-1 beta in blood in individuals who were exposed to acute experimental stress were observed. The increases could be seen already 30 min after the stressor application (Brydon et al. 2005). The source of stress-induced blood IL-1 beta was identified as peripheral blood mononuclear cells.
Interestingly, in the subjects with post-traumatic stress disorder (PTSD), spontaneous production of IL-1 beta by peripheral blood mononuclear cells was greater than in control subjects (Gola et al. 2013). Moreover, patients with major depression have lower concentrations of IL-1 beta in blood than the non-depressed subjects (Hernandez et al. 2013). The influence of IL-1 beta on the anxiety behavior was evidenced by using animal knock-out or knock-down models, which demonstrated that reducing the ability of cells to react to IL-1 beta, correlates with abolishment of anxiety behavior (Murray et al. 2013; Wohleb et al. 2014).
In 2014, our group used several psychometric instruments to assess the tinnitus-related distress in 30 patients with chronic tinnitus and correlated the scores with the concentrations of selected cytokines in serum (Szczepek et al. 2014). We found significant positive correlation between the scores of visual analogue scale testing the awareness of tinnitus and IL-1 beta concentration (see Fig. 6.3). Unfortunately, we did not measure anxiety symptoms in the patients studied. This was so far the only attempt to use this cytokine as a possible biomarker. The time and more experiments will demonstrate the usefulness of determining the blood concentration of IL-1 beta for tinnitus diagnostics and monitoring.
Correlation between the concentration of circulating IL-1 beta and psychometric scores. Shown are the regression lines indicating significant correlations between the concentration of circulating IL-1 beta and the scores representing three VAS scale “awareness” of the patients with chronic tinnitus. Dotted lines represent 95% confidence interval (CI). Shown are the regression lines indicating significant correlations between the concentration of circulating tumor necrosis factor α and the scores of VAS “loudness,” total PSQ, and PSQ subscales “tension” and “joy.” Dotted lines represent 95% confidence interval (CI). Reprinted with permission from (Szczepek et al. 2014)
We found significant positive correlation between the scores of visual analogue scale testing the awareness of tinnitus and IL-1 beta concentration (r = 0.466, p < 0.05).
Interleukin-6 (IL-6) is a 24 kDa peptide cloned in 1986 (Hirano 2014) that can be produced by ample types of cells and that induces various responses in numerous cells and tissues. Acting via IL-6 receptor complex and JAK-STAT signal transduction pathway as well as other routes (Hirano 1998) IL-6 mediates several effects such as antibody production by B cells, T- and B-cell activation, growth and differentiation of diverse cells, growth of some tumors and also fever by acting on specific neurons in the brain. In the recent years, the key role of IL-6 in many noninfectious but inflammatory diseases as well as in cancer started to be recognized (Ho et al. 2015). In addition, the anti-IL-6 or anti-IL-6 receptor strategies have been developed. At the same time, presence and the concentration of IL-6 in blood began to be used as a biomarker for many illnesses such as multiple myeloma (Kyle 1995), colorectal carcinoma (Xu et al. 2016), rheumatoid arthritis (da Mota et al. 2009), and many other.
Important for the tinnitus research was the discovery of positive correlation between the presence of IL-6 in blood and the psychosocial stress. For instance, psychologically traumatized women were found to have an increased blood concentration of IL-6. The concentration went down after successful stress reduction therapy (Gallegos et al. 2015). Moreover, both types of stress, acute and chronic, were associated with overproduction of IL-6 in blood.
The normal baseline production of IL-6 undergoes circadian regulation (see Chap. 4 for circadian regulation) (Vgontzas et al. 2005). Physiologically increased levels of IL-6 at the end of the day mediate sleepiness, whereas the modulation of the systemic IL-6 concentration induces disturbances in sleep pattern. Reversely, disturbances in sleep pattern are accompanied by abnormal diurnal secretion of IL-6. Interestingly, the IL-6-dependent sleepiness can be modulated by cortisol, producing deep sleep when cortisol level is low (healthy pattern) or shallow sleep and the feeling of tiredness (sickness behavior) when cortisol is elevated (Vgontzas et al. 2005).
The fact, that the elevated concentrations of circulating IL-6 were found in persons with depression, made the IL-6 the recent target molecule in major depression clinical research (Hodes et al. 2016). The already existing biologics developed against IL-6 (e.g., siltuximab) or against IL-6 receptor (e.g., tocilizumab) are being presently used in the clinical trials with patients suffering with major depression (Hodes et al. 2016). These biologics have to our best knowledge not yet been tried in patients with tinnitus.
What do we know about IL-6 in patients with tinnitus? In an early prospective study with a “before-after” design, we determined the concentration of IL-6 at three time points in serum of tinnitus patients who decided to do relaxation training, another group that was not doing the relaxation and in subjects without tinnitus (Weber et al. 2002). We found that the relaxation exercises corresponded with an improvement of stress management and with lowering the concentrations of circulating IL-6. Our next attempt to use the IL-6 as a biomarker was a recent pilot study, where we used validated psychometric instruments to assess tinnitus-related distress (Szczepek et al. 2014). Unfortunately, the detected levels of IL-6 were below the detectability thresholds of ELISA and, therefore, could not be used for further analyses. More studies with larger sample and more sensitive detection methods are necessary to confirm or reject the usefulness of IL-6 as a biomarker in tinnitus.
Performing relaxation exercises corresponded with an improvement of stress management and with lowering the concentrations of circulating IL-6 in the blood of tinnitus patients.
22.214.171.124 Tumor Necrosis Factor Alpha
Tumor necrosis factor alpha (TNF alpha) is another small, 17 kDa cytokine that can be produced and secreted by many cell types. It was cloned and described by a group of Bharat B. Aggarwal in 1984 (Aggarwal et al. 1984). Similarly to IL-1 beta and IL-6, TNF alpha is a pyrogenic cytokine, and it also induces the sickness behavior. Three types of TNF alpha receptor have been characterized to date—they all bind TNF alpha but mediate various functions (see Fig. 6.4), from the cell survival and tissue regeneration to the cell death (Kalliolias and Ivashkiv 2016). Experimental animal models enabled detailed understanding of molecular mechanisms linking the presence of TNF alpha with depressive symptoms (Fig. 6.5) with a key finding being that chronic mild stress induces production of proinflammatory cytokines such as TNF alpha, and this in turn contributes to the development of depression (Kubera et al. 2011).
(a) TNF receptor 1 (TNFR1) signalling is activated by both soluble and transmembrane TNF. TNFR1 bears a death domain that recruits the adaptor protein TNFR1-associated death domain protein (TRADD). Ligation of TNFR1 by soluble TNF or transmembrane TNF leads initially to the assembly of complex I, which activates nuclear factor κB (NFκB) and mitogen-activated protein kinases (MAPKs). TNFR1–complex I signalling induces inflammation, tissue degeneration, cell survival and proliferation, and orchestrates the immune defense against pathogens. Alternative signalling modalities, associated with programmed cell death, can also be activated downstream of TNFR1. The formation of the complexes IIa and IIb (also known as ripoptosome) results in apoptosis, whereas complex IIc (necrosome) induces necroptosis and inflammation. (b) TNFR2 is proposed to be fully activated primarily by transmembrane TNF, in the context of cell-to-cell interactions. TNFR2 recruits TNFR-associated factor 2 (TRAF2) via its TRAF domain, triggering the formation of complex I and the downstream activation of NFκB, MAPKs, and AKT. TNFR2 mediates primarily homeostatic bioactivities including tissue regeneration, cell proliferation and cell survival. This pathway can also initiate inflammatory effects and host defense against pathogens. MLKL, mixed lineage kinase domain-like protein. Reprinted with permission from (Kalliolias and Ivashkiv 2016)
External stress-induced depression-like behavior. External stressors in animals, such as chronic mild stress and learned helplessness, are accompanied by depressive-like behaviors. External stress-induced depression-like behavior is accompanied by peripheral and central inflammation, with increased levels of proinflammatory cytokines, such as interleukin-1β (IL-1β), tumor necrosis factor-α (TNFa) and IL-6. The external triggers and cytokines may induce nuclear factor κB (NFκB), which in turn induces the expression of proinflammatory cytokines, oxidative and nitrosative stress (O&NS) pathways, and cyclooxygenase (COX)-2. Through these pathways, external stress may cause increased amounts of reactive oxygen (ROS) and nitrogen (RNS) species, including O2− and NO that results in the generation of peroxynitrite. COX-2 may generate prostaglandins (PG), such as PGE2 and PGJ2. External stressors also increase the expression of Toll-like receptors (TLR4), which increase the sensitivity to internal stressors, including pathogen associated molecular patterns (PAMPs), i.e., conserved microbial structural motifs, such as lipopolysaccharide (LPS), and damage-associated molecular patterns (DAMPs). External stressors increase glucocorticoids and glutamate release and consequently provoke activation of neuronal N-methyl-d-aspartate (NMDA) receptors. External stressors cause antineurogenic effects by downregulating neurotrophic factors, such as brain-derived neurotrophic factor (BDNF) and VGF. Finally, external stressors cause apoptosis with lowered levels of Bcl-2 and BAG1 (Bcl-2 associated athanogene 1) and increased levels of caspase-3. Cytokines, O&NS, NFκB, COX-2, prostaglandins, e.g., PGE2, excitotoxic glutaminergic effects, apoptotic pathways, and reduced neurotrophic substances contribute to the neurodegenerative processes and reduced neurogenesis which are observed in depression-like behaviors. Reprinted with permission from (Kubera et al. 2011)
Intriguing case report about possible causative contribution of TNF alpha to tinnitus was published recently (Stein et al. 2014). In this report, a patient was diagnosed with Melkersson-Rosenthal syndrome (MRS), accompanying hearing loss and intermittent tinnitus. Upon off-label treatment with TNF alpha blocker (adalimumab), not only the remission of the main MRS symptoms was observed (orofacial edema, facial nerve palsy and furrowed tongue) but also total remission of hearing loss and tinnitus occurred. This case report provides evidence for the following subjects:
Diversity of diseases that have tinnitus as a symptom
Intermittent tinnitus as an underestimated form of tinnitus, during which the precept could be successfully treated
Inflammatory character of diseases associated with tinnitus
Two of our earlier mentioned studies have scrutinized the usefulness of TNF alpha as a biomarker for tinnitus. In the first study, we demonstrated that not only tinnitus-related distress but also TNF alpha decreases in a group of tinnitus patients who performed relaxation exercises but not in those, who did not exercise (Weber et al. 2002). In the second study, using psychometric instruments, we measured the perceived stress and tinnitus-related distress. We found that the concentration of TNF alpha in serum obtained from tinnitus patients significantly correlates with the perceived loudness of tinnitus (as per analogue visual scale) and with the total score of perceived stress questionnaire PSQ (for more on the PSQ questionnaire see Chaps. 7 and 9) (Fig. 6.6).
Correlations between the concentration of circulating TNF α and psychometric scores. Shown are the regression lines indicating significant correlations between the concentration of circulating TNF alpha and the scores of VAS “loudness,” total PSQ, and PSQ subscales “tension” and “joy.” Dotted lines represent 95% confidence interval (CI). Reprinted with permission from (Szczepek et al. 2014)
One needs caution when designing and interpreting the results from these types of studies—the measured concentration of serum or plasma cytokines is below of what we consider “normal” inflammatory levels, consistent with lack of fever, which is only present then when there is a lot of circulating pyrogenic cytokines. The second problem is the choice of tools available for determination of cytokine concentration. There are several products on the market which can be purchased from various companies and the sensitivity and reproducibility of the tests differ between the products. We recommend using products labeled with “IVD” which stands for “in vitro diagnostic medical device.” These products are rigorously controlled for their quality and licensed for human diagnostics.
6.3.2 Thrombocytes (Platelets)
Thrombocytes (or platelets) are anuclear components of blood and derivative of megakaryocytes residing in the bone marrow. Thrombocytes are indispensable for the repair of damaged blood vessels. Through the process of adhesion, activation, and plug formation, they also initiate the process of blood coagulation and activate circulating white blood cells. Mean platelet volume (MPV) is used to determine the size of thrombocytes whereas platelet count is used to estimate their concentration. Both values are routinely included in the complete blood count (CBC).
Psychosocial or emotional stress was shown to affect the blood homeostasis and even lead to the coronary heart disease by expressing adhesion molecules (e.g., P-selectin) on the thrombocytes, which in turn activate the white blood cells. Bulk of evidence has accumulated in the last two decades to support the notion about acute and chronic stress contributing to platelet activation via HPA axis and elevated concentrations of glucocorticoids (see Chap. 2) or via impaired serotonergic function (Brydon et al. 2006).
Stressful situations contribute to the increase in numbers of thrombocytes (Baltrusch et al. 1990; Jern et al. 1989) and to the decrease of mean thrombocyte volume (Baltrusch et al. 1990; Gogcegoz Gul et al. 2014). Interestingly, few recent clinical studies determined increased numbers of thrombocytes and decreased mean platelet volume (MPV) in the blood of tinnitus patients [Kemal et al. 2016; Sarikaya et al. 2016a, b; Yuksel and Karatas 2016]. However, the studies assessed neither the coronary disease nor the level of stress in the subjects tested. Still, methodical study of the thrombocyte/platelet count and MPV in patients with tinnitus may prove in the near future to be cost-effective and a meaningful biomarker in the diagnosis and monitoring of tinnitus-related distress.
6.4 Neurotransmitters and Neurotrophins as Biomarkers of Stress
6.4.1 Serotonin (5-Hydroxytryptamine (5-HT))
Serotonin is a monoamine that serves as neurotransmitter. Serotonin is produced from a precursor, L-tryptophan and metabolized into 5-hydroxyindoleacetic acid (5-HIAA). Elevated plasma serotonin levels were found to correlate with stress and depressive symptoms (Tyano et al. 2006). Interestingly, in a cohort of 344 tinnitus patients, there were statistically significant more individuals who had elevated blood 5-HIAA serotonin metabolite than the control subjects; however, the only correlation found was between 5-HIAA and tinnitus duration (Kim et al. 2014). Another study connecting tinnitus with serotonin was a blood-based genetic analysis of the serotonin transporter gene polymorphism (promoter region) and various characteristics of tinnitus percept and tinnitus-related distress (Deniz et al. 2010). The authors found that those of tinnitus patients who have ll–type of allele encoding serotonin transporter gene promoter region (5-HTTLPR) are more sensitive to tinnitus-related distress (severity, frequency and duration, discomfort level, attention deficit, and sleep disorder) than those patients who had sl or ss allele. The ll allele correlates functionally with a quicker serotonin reuptake (Greenberg et al. 1999). Larger studies should address feasibility of adding 5-HTTLPR polymorphism into the tinnitus-related distress biomarkers.