Cytokine Expression Profiles in Children and Adolescents with Tic Disorders.

doi:10.21203/rs.3.rs-3813261/v1

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Cytokine Expression Profiles in Children and Adolescents with Tic Disorders.

https://doi.org/10.21203/rs.3.rs-3813261/v1

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The etiology of tic disorders (TDs) is not precisely known, although several lines of evidence suggest involvement of the immune system in pathogenesis. Here, we aimed to determine the expression levels of pro-inflammatory and anti-inflammatory cytokines in children with TD and compare them with those of healthy controls. Furthermore, we also evaluated their association with clinical variables in the TD group. Within the study period, 88 children with tic disorders and 111 healthy control children were enrolled. Most children with tic disorders were diagnosed with Tourette’s disorder (n = 47, 53.4%) or persistent motor tic disorder (n = 39, 44.3%), while the remainder (n = 2, 2.3%) were diagnosed with persistent vocal tic disorder. We found that children with tic disorders had significantly elevated levels of IL-1β, TNF-α, IL-6 and IL-4 expression, while healthy control cases presented with increased levels of IL-17. Our findings provide a molecular landscape of cytokine expression in children with TD, which may suggest a proinflammatory state not affected by the presence of comorbidity and symptom severity. Delineating the contribution of alterations in the immune system to the pathogenesis of tic disorders will have critical implications for diagnosis and therapeutic interventions.

Health sciences/Diseases

Health sciences/Diseases/Psychiatric disorders

Health sciences/Health care/Quality of life

Health sciences/Risk factors

Tic disorders

Cytokines

Immune system

Inflammation

Tourette syndrome

Tics are defined as “sudden, rapid, recurrent, nonrhythmic motor movements and/or vocalizations, generally preceded by premonitory somatosensory urges.” The DSM-5 defines five tic disorders: provisional tic disorder, persistent motor or vocal tic disorder, Tourette’s disorder (also known as Tourette syndrome), other specified tic disorder, and unspecified tic disorder ¹. Epidemiological studies suggest that most tics among children involve males (male to female ratio is 4:1) and are transient, while persistent/chronic cases involve symptoms lasting more than 12 months. Children may display either simple (affecting one muscle group/body part) or complex (affecting several muscle groups/body parts or purposeful movements) motor/vocal tics. The prevalence of Tic disorders among children may be 3.0–4.0%, while that of Tourette’s Disorder may vary between 0.3-1.0% ^1–3. Tics start mostly in preschool or early school years with motor tics preceding vocal tics, and symptoms wax and wane to peak around puberty and recede in adolescence. Only 5.0–10.0% of cases continue into adulthood. Importantly, isolated cases of tic disorders (TD) among children are rare. Obsessions/compulsions (22.0–66.0%), attention deficit/hyperactivity disorder (ADHD, 55.0–60.0%), autism spectrum disorder (ASD, up to 22.8%), impulse/anger control problems (25.0–75.0%), and academic/neuropsychological problems frequently accompany TD ^1,3. The etiology of tic disorders is complex and multifactorial, probably including multiple genetic and non-genetic factors, such as environmental exposures and immune-mediated mechanisms. Consequently, this complexity was suggested to contribute to the heterogeneous disease phenotypes^1–4.

Studies conducted in the last three decades suggest that immune mechanisms may play a role in the development of TD. For instance, various studies suggest that children with TD may have elevated levels of proinflammatory cytokines such as TNF-α, IL-1β, IL-2, IL-6, IL-8, IL-12, IL-17, and IFN-γ ⁵. TD among children was found to be associated with autoimmune disorders such as asthma, allergic rhinitis, allergic conjunctivitis and possibly eczema and food allergies ⁶. Furthermore, TD and related syndromes have been shown to display abnormalities in microglial functioning ⁷. Consistent with a role of immune signaling in TD-related disorders, injection of soluble IL-2 and IL-6 receptors was found to induce motor stereotypies in rats, which correlated with their deposition in the striatum, thalamus, and various cortical regions ^8,9. Exposure to maternal acute/chronic inflammation in utero was found to be associated with various neurodevelopmental disorders, including TD, in the offspring ¹⁰. Cytokine levels among children with TD may be related to both comorbidities and symptom severity ^11,12. Moreover, dopamine itself was shown to mediate the pathogenesis of TD and exert immunomodulatory effects ¹³. In agreement with this observation, atypical antipsychotics, which are used to manage tics, were found to display peripheral effects on the immune system ¹⁴. Despite the accumulating evidence of immune mechanisms in TD among children and adolescents, to the best of our knowledge, no study has evaluated cytokine expression levels among Turkish children with TD. Previously, one study from Turkey reported changes in cytokine levels among children with OCD ¹⁵.

Herein, we examined the expression levels of IL-1β, IL-1α, IL-4, IL-6, IL-17, TNF-α, and TGF-β in peripheral blood mononuclear cells (PBMCs) in children with tics and healthy controls and determine the correlations between cytokine levels and clinical features to determine their contributions in children and adolescents with tic disorders.

Study centers, sampling, and ethics

This study was conducted at the Child and Adolescent Psychiatry Departments from seven centers and involved patients followed up with TD diagnosis in these centers (persistent (chronic) motor or vocal tic disorder [307.22, ICD-10 F95.1] and Tourette’s disorder [307.23, ICD-10 F95.2] as per DSM-5) ¹. The diagnoses of potentially eligible patients were corroborated with clinical interviews. The inclusion criteria for the TD group were a diagnosis of persistent motor/vocal tic disorder or Tourette’s disorder without known genetic syndromes and/or chronic neurological/medical conditions. Patients with intellectual disabilities (according to developmental history, mental status examination and academic achievement), autism spectrum disorders, PANDAS/PANSS (Pediatric Autoimmune Neuropsychiatric Disorders Associated with Streptococci/Pediatric Acute Onset Neuropsychiatric Syndrome) and psychosis were excluded. Informed consent was obtained from the parents of the children included in the study, and the children provided verbal/written assent. The study was carried out in accordance with the Declaration of Helsinki and local laws and regulations. The study protocol was approved by the Institutional Review Board (IRB) of Inonu University (No:2014/ 237). The control participants were recruited from children coming for routine control visits in the departments of pediatrics in the study centers. They were matched in terms of gender and age to children with TD. Inclusion criteria for control children were lack of lifetime psychopathology in psychiatric interviews. The exclusion criteria for both children with TD and controls were acute respiratory or infectious disease, history of cardiovascular, endocrine, neurological, or other disorders/factors known to affect inflammatory indices, and history of receiving steroids and intravenous gamma globulin within the last 6 months prior to the study visit.

Psychometric evaluations

Children’s Depression Inventory (CDI) is a 27-item self-report scale was developed to evaluate subjective symptoms of depression among 6- to 17-year-old children. The Turkish version was previously found to be valid and reliable. CDI was completed by children ≥ 8 years old in this study. Children scoring above 19 were accepted to display clinically significant depressive symptoms as per the reliability and validity study ^16,17. State-Trait Anxiety Inventory for Children (STAI-C) is a 40-item self-report scale was developed to evaluate symptoms of state and trait anxiety ^18,19. The STAI-C was completed by children ≥ 8 years old in this study. Children scoring above the median on the STAI-C-Trait were considered to have significant trait anxiety. Maudsley Obsessive Compulsive Symptom Checklist (MOCSL) is a 30-item self-report scale evaluates subjectively reported obsessive symptoms ²⁰. MOCSL was completed by children ≥ 8 years old in this study. Yale Global Tic Severity Scale (YGTSS) is a semi-structured, clinician-applied interview assesses the severity of tics for the past week. Motor and vocal tics are scored separately between 0 and 5 according to frequency, number, severity, complexity, and disability. Higher scores indicate greater severity, and finally, a clinical impairment score is added to form a total score ²¹.

Sample collection, qRT‒PCR and ELISA assays

10 mL of blood were collected into PAXGene Vacutainer tubes (Qiagen, Hilden, Germany), and RNA was isolated using the PAXGene Blood RNA Kit as described by the manufacturer. RNA integrity and quality were confirmed using an Agilent 2100 Bioanalyzer (Agilent Inc., Santa Clara, CA, USA). mRNA expression profile of cytokines was determined by using using QuantiTect Primer assays as described before ²². To evaluate the concordance between serum cytokine levels and mRNA expression data, serum samples from children with TD and healthy control children were isolated and stored at − 80°C. Serum concentrations of IL-4 (Catalog #: D4050), IL-1α (Catalog #: DLA50), IL-1β (Catalog #: DLB50), TGF-β (Catalog #: DB100B), IL-17 (Catalog #: D1700), IL-6 (Catalog #: D6050) and TNF-α (Catalog #: DTA00D) were measured by using Quantikine enzyme-linked immunosorbent assays according to the protocol of the manufacturer (R&D Systems, Minneapolis, MN, USA). Statistical analysis to evaluate the significance of serum cytokine levels in different groups was conducted by means of Student’s t-test module on GraphPad Prism 10.0.2 software (San Diego, CA, USA). In all statistical analyses, p < 0.05 was considered significant.

Statistical analyses

Data were entered into a database prepared with Statistical Program for Social Sciences (SPSS™, IBM Inc., Armonk, NY) Version 22.0. Analyses were conducted with SPSS and with Jamovi (The Jamovi Project, https://www.jamovi.org) Version 2.3.21. Nominal data were summarized as counts and frequencies, while quantitative data were summarized as the means and standard deviations or medians and interquartile ranges (IQRs) depending on normality and outliers. Comparisons between groups were conducted with chi-square tests for nominal variables. Yates’, Fisher’s, and likelihood ratio corrections were used as needed. Bivariate correlations between symptom severities and cytokine expression levels were evaluated using Spearman correlation analyses. Multivariate analysis of variance (MANOVA) with follow-up ANOVA was used to evaluate the effects of diagnosis on cytokine expression levels. P was set at 0.05 (two-tailed). Bonferroni corrections were used to adjust for multiple comparisons (Gaetano J. Holm‒Bonferroni sequential correction: An Excel ^TM calculator. 2018. https://www.researchgate.net./profile/Justin_Gaetano2). Effect sizes for significant findings were also reported.

Within the study period, 88 children with tic disorders (n = 64, 72.7% male) and 111 control children (n = 74, 66.7% male) were enrolled. The mean ages of children with tic disorders and control children were 11.2 (SD = 3.1) and 12.0 (SD = 3.2) years, respectively. The groups did not differ significantly in terms of gender (χ2 = 0.6, p = 0.444, Yates’ correction) or mean age (t [197] =-1.9, p = 0.063, Student’s t test for independent groups). Other sociodemographic and familial variables of children with tic disorders and control children are illustrated in Table 1.

Table 1

Sociodemographic and familial features of children with tic disorders and control children
N (%)		Tic Disorder (n = 88)	Control (n = 111)	χ 2/dF or t	P*	E.S.**
Educational status	Not attending school	1 (1.1)	3 (2.7)	12.7/4	0.013	0.25
	Kindergarten/preschool	6 (6.8)	1 (0.9)
	Primary school	34 (38.6)	26 (23.4)
	Secondary school	28 (31.8)	51 (45.9)
	High school	19 (21.6)	30 (27.0)
Maternal age (years)		37.9 (6.7)	38.7 (7.1)	-0.8/197	0.419	-
Maternal education	Primary school or lower	47 (53.4)	47 (42.7)	4.0/3	0.266	-
	Secondary school	10 (11.4)	9 (8.2)
	High school	14 (15.9)	23 (20.9)
	University or higher	17 (19.3)	31 (28.2)
Maternal vocation	Housewife	70 (79.5)	75 (67.6)	7.8/4	0.167	-
	Worker	2 (2.3)	5 (4.5)
	Civil servant	12 (13.6)	25 (22.5)
	Artisan	3 (3.4)	4 (3.6)
	Retired	1 (1.1)	0 (0.0)
Paternal age (years)		42.0 (6.5)	41.3 (10.2)	0.6/197	0.581	-
Paternal education	Primary school or lower	29 (33.0)	32 (28.8)	0.8/3	0.841	-
	Secondary school	9 (10.2)	9 (8.1)
	High school	23 (26.1)	32 (28.8)
	University or higher	27 (30.7)	38 (34.2)
Paternal vocation	Jobless	3 (3.4)	1 (0.9)	7.0/4	0.222	-
	Worker	23 (26.1)	23 (20.7)
	Civil servant	27 (30.7)	36 (32.4)
	Artisan	30 (34.1)	40 (36.0)
	Retired	5 (5.7)	7 (6.3)
Family status	Intact/Nuclear	85 (96.6)	97 (87.4)	5.3/1	0.023	0.16
Family status	Separated/widowed/divorced	3 (3.4)	14 (12.6)	5.3/1	0.023	0.16
Maternal psychopathology present		14 (15.9)	11 (9.9)	1.1/1	0.292	-
Paternal psychopathology present		7 (8.0)	6 (5.4)	0.2/1	0.664	-

*Chi square test with likelihood ratio, Yates’ corrections, Fisher’s exact test and t test for independent groups, **Phi and Cramer’s V.

Control healthy children were significantly more likely to attend secondary and high schools than children with tic disorders, while they were less likely to live in intact, nuclear families. Apart from those, no significant differences emerged between groups. Most children with tic disorders were diagnosed with Tourette’s disorder (n = 47, 53.4%) or persistent motor tic disorder (n = 39, 44.3%), while the remainder (n = 2, 2.3%) were diagnosed with persistent vocal tic disorder. According to evaluations with the Yale Global Tic Disorder Severity Scale, mean scores for motor and vocal tics were 13.7 (SD = 4.9) and 5.2 (SD = 6.2), respectively. The mean global impairment and total scores were 29.2 (SD = 11.4) and 48.1 (SD = 18.6), respectively. Most children were classified as having either moderate (n = 40, 45.5%) or significant (n = 32, 36.4%) tics, while children with mild (n = 9, 10.2%) and severe (n = 7, 8.0%) tics were rarer.

Additionally, most of the children had either one (n = 47, 53.4%) or two (n = 9, 10.2%) comorbid diagnoses (Median = 1.0, IQR = 1.0). The most common comorbid diagnoses were attention deficit/hyperactivity disorder (ADHD, n = 38, 43.2%), anxiety disorder (n = 13, 14.8%) and obsessive-compulsive disorder (OCD, n = 8, 9.1%). Of note, one child each (1.1%) was diagnosed with trichotillomania, childhood onset speech fluency disorder, enuresis, and oppositional defiant disorder. Most children were receiving pharmacological treatment (n = 72, 81.8%) with either one (n = 41, 46.6%) or two (n = 31, 35.2%) agents. The most common psychopharmacological treatments were aripiprazole (n = 42, 48.9%), methylphenidate (n = 15, 17.0%) and atomoxetine/risperidone/haloperidol (n = 12, 13.6%, each). Eight of the children (9.1%) were treated with SSRIs, while only one (1.1%) received olanzapine treatment. Self-reported symptoms of anxiety, depression, and obsessive-compulsive disorder among children with tic disorders and control children are listed in Table 2.

Table 2

Self-reported anxiety, depression, and obsessive-compulsive disorder symptoms among children with tic disorder and control children
Median (IQR)	Tic Disorder (n = 88)	Control (n = 111)	Z	P*	E.S.
State-Trait Anxiety Inventory for Children- State	33.0 (10.3)	31.0 (10.0)	-1.8	0.072	0.13
State-Trait Anxiety Inventory for Children- Trait	36.0 (12.0)	35.0 (10.0)	-1.3	0.189	-
Children’s Depression Inventory	10.0 (10.0)	8.0 (8.0)	-1.6	0.108	-
Maudsley Obsessive Compulsive Symptom Checklist	16.0 (9.0)	14.5 (10.0)	-2.1	0.036	14.9
*Mann‒Whitney U test, E.S.: Effect Size

In bivariate comparisons, children with tic disorder tended to have elevated levels of state anxiety and obsessive-compulsive symptom scores. Children with Tourette Disorder, Persistent Motor and Vocal tic disorders did not differ significantly in terms of self-reported symptoms (Mann‒Whitney U test, p > 0.05). Bivariate correlations of psychometric measures between the YGTSS- Total Tic Score and Global Impairment score revealed that only global impairment correlated significantly with the MoCA score (Spearman’s rank order correlation rho = 0.003, rho’= 0.021, Holm‒Bonferroni corrected). Less than half of the TD children had trait (n = 36, 40.9%) or state (n = 35, 39.8%) anxiety above the median, while only ten (11.4%) reported clinically significant depressive symptoms.

Finally, we compared children with tic disorders and control children in terms of the expression of cytokines (i.e., IL-1α, IL-1β, TNF-α, IL-6, TGF-β, IL-17, and IL-4) by means of qRT‒PCR. Apart from TNF-α (p = 0.159) and IL-6 (p = 0.198) among children with tics and IL-17 among controls (p = 0.129, Kolmogorov‒Smirnov test with Lilliefors correction), none of the cytokines conformed to assumptions of normality. Covariance matrices of dependent variables were not equal across groups (Box’s M = 218.0, F = 7.5, p = 0.000), and only the error variances for IL-17 were equal (p = 0.393, Levene’s test). Therefore, interleukin levels across groups were compared with multivariate analysis of variance (MANOVA) with Pillai’s trace method. According to MANOVA, the groups differed significantly in terms of interleukin levels (F [7.0, 191.0] = 13.2, p = 0.000, partial η2 = 0.33). The results of univariate ANOVAs and interleukin levels across groups are illustrated in Table 3. In addition, we evaluated the serum levels of cytokines in children with tic disorders and healthy control children by using ELISA to evaluate the concordance of qPCR results with serum protein levels. As shown in Fig. S1, serum IL-1β, TNF-α, IL-6, and IL-4 levels were higher in children with tic disorders than in healthy controls, in line with the qRT‒PCR data. Furthermore, we could not detect any significant change in IL-1α and TGF-β levels in concordance with the qRT‒PCR results. These results may indicate that the increased expression of cytokines in children with tic disorders is a consequence of reprogrammed gene expression in PBMCs. Although we could not detect a significant alteration in IL-17 serum levels, we observed a trend for lower IL-17 serum levels in children with tic disorders (Fig. S1). Hence, this finding may suggest that cellular mechanisms other than transcriptional regulation could mediate the secretion and stability of IL-17.

Table 3

Interleukin levels in children with tic disorders and control children
Mean (SD)	Tic Disorder (n = 88)	Control (n = 111)	F	P*	Partial η2
IL-1α	1.7 (0.8)	1.8 (0.7)	0.3	0.562	0.00
IL-1β	2.1 (1.2)	1.9 (0.8)	4.2	0.042	0.02
TNF-α	2.3 (1.1)	1.7 (0.7)	20.0	0.000	0.09
IL-6	3.1 (1.9)	1.7 (0.6)	55.2	0.000	0.22
TGF-β	1.7 (1.0)	1.7 (0.6)	0.00	0.961	0.00
IL-17	1.8 (0.9)	2.2 (0.8)	10.4	0.001	0.05
IL-4	2.0 (1.1)	1.6 (0.7)	10.1	0.002	0.05

*ANOVA with Bonferroni correction, IL: interleukin, TNF: tumor necrosis factor, TGF: tumor growth factor.

According to univariate ANOVAs, children with tic disorders had significantly elevated levels of IL-1β, TNF-α, IL-6 and IL-4 expression, while controls had significantly elevated levels of IL-17 expression. The type of TD did not affect cytokine expression levels (Mann‒Whitney U test, p > 0.05). Among children with tic disorders, state anxiety scores correlated significantly and positively with IL-17 levels (rho = 0.32, p = 0.006), while among control children, state anxiety correlated significantly and negatively with IL-6 levels (rho=- 0.25, p = 0.026). Moreover, self-reported obsessive compulsive symptom scores correlated significantly and negatively with IL-4 levels (rho= -0.22, p = 0.049) among controls. Apart from those, no significant correlations emerged between interleukin levels and self-reported anxiety, depression, and obsessive-compulsive disorder symptoms. YGTSS-total tic and impairment scores did not correlate significantly with cytokine levels.

This multicenter, cross-sectional, case‒control study evaluated the expression levels of cytokines in peripheral blood mononuclear cells of children diagnosed with Tic disorders and compared them with controls. Complex neurobiological and genetic mechanisms, alteration of iron metabolism, and environmental factors are thought to interact with each other in the etiology of TD, and immune dysfunction may be a promising avenue of research in the onset and development of tic disorders ^4,23–27. In our study, children with tic disorders had significantly elevated levels of IL-1β, TNF-α, IL-6 and IL-4 expression, while controls had significantly elevated levels of IL-17 expression. There were no significant differences between the TD groups in terms of cytokine levels. Among children with tic disorders, state anxiety scores correlated significantly and positively with IL-17 expression levels. The gender ratios of children with TD in our sample, their mean age and features of comorbid diagnoses conform to those reported in previous studies ^2,3. Henceforth, children with TD in our study reported significantly elevated levels of obsessive-compulsive symptoms compared to controls. Reflecting the effectiveness of atypical antipsychotics in the management of TD, almost half of our sample group was prescribed aripiprazole while more than four-fifths were receiving psychopharmacological treatment either for TD or for comorbid conditions ²⁸. Cytokines are multifunctional pleiotropic proteins that play crucial roles in cell-to-cell communication and cellular activation. Besides, many cytokines can act as neuromodulators and their expression is strictly regulated at peripheral and central nervous systems by means of neurotransmitters ²⁹. A recent meta-analysis found elevated levels of increased levels of proinflammatory cytokines in pediatric patients with Tourette syndrome, including TNF-α and IL-6 ⁵. Consistent with this analysis, we found elevated levels of IL-1β, TNF-α, IL-6 and IL-4 expression among children with TD, while state anxiety among those children correlated positively with IL-17 levels. Among these cytokines, IL-1β, TNF-α, IL-6 and IL-17 are pro-inflammatory, while IL-4 is mainly anti-inflammatory. Underscoring this observation, IL-6 is produced by most cell lines in the CNS, is neurotrophic for midbrain dopaminergic neurons as well as cholinergic neurons in the basal forebrain and septum, increases dopaminergic and serotonergic activity in the hippocampus and prefrontal cortex, regulates neuronal excitability and sleep, and elevated levels were reported in various psychopathologies and neurodevelopmental disorders, including TD ³⁰. IL-6 levels are also known to be reduced after treatment with antipsychotics ³¹. In fact, Tao et al. reported increased IL-6 serum concentration in children with TD compared with control group, which includes 1724 patients and 550 healthy control subjects ³². In contrast to our results and previous reports, they found lower IL-4, IL-10 and TNF-α levels in TD patients ^33,34. Similarly, Matz et al. also reported decreased TNF-α levels in children with Tourette’s syndrome with no alteration of IL-6 ³⁵. However, Bos-Veneman et al. did not detect any significant difference between IL-4, IL-10 and TNF-α levels between patients and healthy controls ¹². The inconsistency in these studies may be due to different methodologies to determine serum cytokine levels, medications, presence of comorbidities, maternal autoimmunity, or age of patients ^32,36. TNF-α regulates the activation of microglia and astrocytes, regulation of blood‒brain barrier permeability, glutamatergic neurotransmission, and synaptic plasticity ³⁷. In fact, preclinical studies suggest that it may affect serotonergic and dopaminergic neurotransmission ³⁸. An increase in TNF-α levels would increase the permeability of the blood‒brain barrier and lead to an enhanced autoimmune response, along with greater dopamine release in the basal ganglia, which in turn contribute to the clinical symptoms of TS and related disorders ^39–41. It is worth noting that TNF-α and IL-6 were shown to be significantly increased in the peripheral blood of patients with Tourette’s syndrome in a recent meta-analysis ⁵. IL-17 is a pro-inflammatory cytokine that acts synergistically with other pro-inflammatory cytokines and plays a role in neurotoxicity and autoimmunity ⁴². Increased IL-17 levels have been previously reported in neurodegenerative, neurodevelopmental, and psychiatric disorders ^43,44. Therefore, higher IL-17 levels were reported to cause cell loss among dopaminergic neurons, while dopaminergic neurotransmission was found to modulate IL-17 secretion ^45,46. Surprisingly, we found reduced IL-17 expression in children with tic disorders compared with healthy control subjects. IL-4 is an anti-inflammatory cytokine that has paradoxical effects in the central nervous system. It may protect neurons against sepsis, ischemia and multiple sclerosis while contributing to the degeneration of dopaminergic neurons in the substantia nigra in the presence of inflammation ⁴⁷. Moreover, IL-4 levels may play a role in synaptic homeostasis, and exposure to elevated levels in utero may increase the risk of neurodevelopmental disorders in the fetus ⁴⁸. TGF-β is an immunosuppressive cytokine that also plays a role in neurodevelopment and synapse formation ⁴⁹. It was reported that lack of TGF-β led to disorganization in the extracellular matrix, neurodegeneration, microgliosis, reductions in synaptophysin, and defects in glutamatergic and GABAergic synapses ⁵⁰. On the other hand, overexpression of TGF-β was also found to disrupt the extracellular matrix and lead to seizures/motor incoordination and behavioral abnormalities ⁵¹. Although TGF-β overexpression has been demonstrated to induce different behavioral consequences depending on the developmental stage, children with tic disorders in our study did not display changes in TGF-β expression levels compared to healthy controls.

Accordingly, our results may support the presence of a pro-inflammatory status among Turkish children with TD, which was not affected by the presence of comorbidity and symptom severity. The lack of effects of comorbidity and symptom severity may be due to the limited sample size. Additionally, the limited number of children with mild or severe symptoms may have affected our power to evaluate the relationships of symptom severity with cytokine levels.

Our results should be evaluated within their limitations. First, our results may be valid only for patients with Tourette’s Disorder and Persistent Motor Tic Disorder receiving treatment at the study centers and those without comorbid chronic neurological/medical conditions. Children with persistent vocal tic disorder were also limited in our sample, and future studies should enroll more patients with this diagnosis. Second, we excluded patients with comorbid intellectual disability/autism spectrum disorder, which may affect our results. Third, we evaluated intellectual disability in our sample according to psychiatric interviews, developmental history, academic achievement and mental status examination, and evaluation of intellectual functioning with valid psychometric measures (e.g., Wechsler Intelligence Scale for Children-Fourth Edition, WISC-IV) may have enriched our results. Fourth, most of our sample was receiving treatment, especially with antipsychotics, which may have affected cytokine levels. Fifth, evaluation of depression, anxiety, and obsessive-compulsive symptoms with clinician-rated measures in addition to personal reports may prove more valuable. Sixth, self-reports of depression, anxiety and obsessive-compulsive symptoms may be subject to reporting and recall bias in addition to shared method variance. Seventh, rather than unstructured clinical interviews, the use of semi-structured interviews (e.g., K-SADS-PL Turkish version) may be preferable in clinical evaluations. Last, in addition to cytokine levels, other measures of inflammation may be added to provide a more comprehensive battery.

Regardless of those limitations, our results support the presence of a pro-inflammatory status among Turkish children with TD, which was not affected by the presence of comorbidity and symptom severity. Future studies may enroll children with diverse age ranges and symptom severities to evaluate inflammatory status among Turkish children with TD. In conclusion, our study adds to the ever-growing evidence base supporting the importance of the immune system in the pathophysiology of TS and related disorders. Recent research is progressively unveiling a complex interplay between neural transmission, immune response regulation and endocrine systems, which might provide further insight into the natural history of TS and its relationships with environmental stressors, especially infections ⁵².

The cross-sectional and correlational nature of our study design precludes hypotheses on causality. However, considering the role of dopamine in the modulation of immune cells via autocrine/paracrine routes as well as the expression of dopamine receptors on lymphocytes, prospective longitudinal studies may evaluate the role of the hypothesized hyperdopaminergic state of children with TD on their immune responses. The role of aberrant immunity in the brain development of children with neurodevelopmental disorders, including those with TD, also awaits further study. This avenue of research requires the development of more valid animal models as well as a larger availability of brain specimens. Once the complex interplay of neurodevelopment, immunity and environmental stressors is elucidated in more detail, the effects of immunotherapy with antibodies or antibody components targeting specific cytokines (e.g., IL-6) may be evaluated in preclinical models. These potential therapies may prevent peripheral cytokine receptors from crossing the blood‒brain barrier and prevent the induction of inflammatory cascades in the periphery with downstream effects. Additionally, the effects of anti-inflammatory agents such as cyclooxygenase inhibitors or n-acetylcysteine may be evaluated as potential treatments for TD.

Understanding the role of immune abnormalities in the pathogenesis of tic disorders may not only aid in our understanding of the mechanism driving this disorder but may also provide biological markers that can be utilized clinically in diagnosis, the determination of symptom severity, and therapeutic interventions. We look forward to future progress in this field, which has the potential to elucidate new pathological mechanisms and therapeutic targets in TS based on neural-immune interactions.

Acknowledgments

None declared.

Compliance with ethics guidelines

Meryem Ozlem Kutuk, Ali Evren Tufan, Fethiye Kilicaslan, Cem Gokcen, Gulen Guler Aksu, Cigdem Yektas, Hasan Kandemir, Fatma Celik, Tuba Mutluer, Ahmet Buber, Mehmet Karadag, Nurdan Coban, Seyma Coskun, Zehra Hangul, Ebru Altintas, Ufuk Acikbas, Aslı Giray, Yeliz Aka, Bilkay Basturkand Ozgur Kutuk declare that they have no conflict of interest. All authors have given approval for this version to be published. The study was approved by the appropriate institutional and/or national research ethics committee and the study was performed in accordance with the ethical standards as laid down in the 1964 Declaration of Helsinki and its later amendments or comparable ethical standards. Informed consent was obtained from all patients for being included in the study.

Data availability

The datasets used and/or analysed during the current study available from the corresponding author on reasonable request.

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No competing interests reported.

SupplementaryFigure1.pptx
Supplementary Figure S1. Altered serum levels of cytokines in Turkish children with tic disorders (TD) and healthy controls. Serum samples from children with TD and healthy control children were evaluated by ELISA. The results are representative of 3 independent measurements and expressed as pg/mL or ng/mL, accordingly. ***, p<0.001, unpaired, two-tailed Student’s t test.

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Cytokine Expression Profiles in Children and Adolescents with Tic Disorders.

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Abstract

Introduction

Materials and methods

Study centers, sampling, and ethics

Psychometric evaluations

Sample collection, qRT‒PCR and ELISA assays

Statistical analyses

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