Serum Levels of Interleukin-6 and Tumor Necrosis Factor Alpha in Children With Attention-Deficit Hyperactivity Disorder.

BACKGROUND: Attention-deficit hyperactivity disorder (ADHD) is a common disorder in children, but its etiology and pathogenesis are still unclear. AIMS: The aims of this study were to measure the level of serum interleukin-6 (IL-6) and tumor necrosis factor alpha (TNF-α) as markers of immune system involvement in children with ADHD, and to study their correlation with symptoms severity of ADHD.
MATERIALS AND METHODS: The study was conducted on 80 children diagnosed as ADHD based on the criteria adapted from the Diagnostic and Statistical Manual of Mental Disorders, 5th Edition. Eighty healthy children of matched age and sex served as a control group. All children enrolled in the study were subjected to history taking, clinical examination, and psychometric tests. Assay for serum IL-6 and TNF-α for all patients and controls was performed using enzyme-linked immunosorbent assay.
RESULTS: The mean serum level of IL-6 was 26.11 ± 11.14 and 6.23 ± 2.52 in children with ADHD and controls, respectively. Children with ADHD showed significantly higher serum IL-6 levels than the control group (P = 0.001). Serum IL-6 showed no significant correlation with the intelligence quotient (IQ) or the Abbreviated Conners' Rating Scale scores for parents. However, TNF-α showed no significant differences between the two groups and no significant correlation with the IQ or the Abbreviated Conners' Rating Scale scores for parents.
CONCLUSION: Serum IL-6 levels were significantly higher in children with ADHD compared to controls; however, the IL-6 levels did not correlate with ADHD symptoms severity. Increased IL-6 levels may contribute to the etiology of ADHD. Copyright:

INTRODUCTION

Attention-deficit/hyperactivity disorder (ADHD) is a neurodevelopmental disorder that causes hyperactivity, impulsivity, and attention problems.[1] It affects primarily children and teenagers, but it can continue into adulthood. Boys are more likely to develop ADHD than girls.[2] Genetic and environmental factors are involved in the pathogenesis of ADHD, but the exact causes and mechanisms of ADHD are not completely understood to date.[3] The association between ADHD and autoimmune diseases was reported in some studies. This suggests the possible immune mechanism underlying ADHD.[45] Previous studies reported abnormalities in the dopaminergic, noradrenergic, and/or serotonergic systems.[6] These abnormalities might be associated with structural brain abnormalities mainly in the prefrontal cortex, striatum, and cerebellum.[7] It was reported that the immune system may be involved in the pathogenesis of various developmental disorders including ADHD.[8]

Donfrancesco et al.[9] showed the immune involvement in ADHD by detecting neuronal anti-Yo antibody in patients with this disorder.

Other studies found antibasal ganglia antibody (ABGA) and antistreptolysin O antibody (ASO) are more significantly frequent in ADHD.[1011]

It has been proposed that alterations in various inflammatory cytokines have been related to ADHD symptoms severity. Recently, it was reported that serum levels of interleukin-6 (IL-6) and IL-10 are significantly higher in children with ADHD.[9] Also, previous studies reported significant elevation of many cytokines, including IL-2, IL-5, IL-10, and TNF-β, in the cerebrospinal fluid of children with ADHD.[12]

Alterations in the concentration of cytokines might regulate the basal ganglia and play a role in the dopamine synthesis in the brain, which is implicated in ADHD.[13]

We aimed to measure serum levels of IL-6 and tumor necrosis factor alpha (TNF-α) concentration in children with ADHD as markers of immune system activation, and to study the possible correlation with symptoms severity of ADHD.

MATERIALS AND METHODS

This case-control prospective study was conducted in 80 children with ADHD aged 5–14 years, recruited from a pediatric neuropsychiatric outpatient clinic, Al Hada and Taif military hospitals, Saudi Arabia, and Benha University Hospital, Benha, Egypt. The study was conducted during the period from July 2018 to July 2019 after obtaining full informed consent.

All cases of ADHD were diagnosed based on the criteria from the Diagnostic and Statistical Manual of Mental Disorders, 5th Edition.[14]

The exclusion criteria were:

(1) Chronic illness, for example, epilepsy, diabetes mellitus; renal, or hepatic,

(2) History of recent trauma or infection

(3) Vaccination

(4) Autistic spectrum disorder or intellectual disability, and/or

(5) Comorbid disorders, for example, depression.

The study included 80 healthy children matched for age, sex, nutritional status, and body mass index who served as a control group.

All patients and controls were subjected to:

(1) Complete history taking through the clinical interviews with stress on obstetric, developmental, psychiatric, and family history, and

(2) Thorough physical examination including developmental, neurologic, and psychometric evaluation.

Conners’ Parent Rating Scale (Fifth Edition)

It is used for the assessment of ADHD and its comorbid disorders in children and adolescents.[15] We scored items related to hyperactivity, inattention, oppositional, and impulsivity. Conners’ Parent Rating Scale consists of 43 items, based on parents’ observations of their child’s behavior. Ratings on the questionnaire ranged from 0 to 3 (0 for never or rarely present, 1 for sometimes present, 2 if frequently present, and 3 for very often present). A total score is derived from the scale and the cutoff score of 15 has been established. On the basis of gender and age, raw scores were converted to T-scores.[16]

Conners’ Abbreviated Parent-Teacher Rating Scale for ADHD

The abbreviated form of the Conners’ Parent-Teacher Rating Scale (CPRS-HI) consists of 10 items.[17] It includes the hyperactivity index (HI) from the long versions of this scale to assess the severity of symptoms of ADHD. An Arabic version of Conners’ Parent Rating Scale was used.[18]

An Arabic version of the Wechsler Intelligence Scale for Children (WISC) is used for assessment of intelligent quotient (IQ).[19] It consists of 11 subtests: six subtests of verbal intelligence and five subtests of nonverbal and performance intelligence. Scores derived from these subtests include the full-scale IQ, verbal IQ, and performance IQ. Children with an IQ < 70 were excluded.[20]

Measurement of cytokines

IL-6 and TNF-α were measured by a multiplex sandwich ELISA (Aushon BiosSystems, Billerica, MA) according to the manufacturer’s instructions. Blood samples were obtained from the antecubital vein in vacutainers as a morning sample at 8 am of the interview day after overnight fasting for 12h.[21] Immediately, serum samples were separated by centrifuge, coded, frozen, and stored at –80°C until time of analysis. The reported limits of detection of these assays were 0.2 pg/mL and 0.8 pg/mL for IL-6 and TNF-α, respectively.[2223]

Statistical analysis

Data and variables were analyzed using SPSS, version 10.0, software (Chicago, IL). Descriptive data were presented as mean ± standard deviation (SD) for continuous normally distributed variables. However, median and IQR for continuous data not following normal distribution and categorical variables were presented as numbers and percentages. Student t test was used for comparison between continuous normally distributed data, whereas the Mann–Whitney U test was used for comparing serum IL-6 and TNF-α levels in children with ADHD and controls. A chi-square test was used for comparison between studied groups qualitative data.

The correlation between cytokines and ADHD symptoms severity was assessed using the Pearson correlation coefficient. The P value of <0.05 is considered significant.

RESULTS

This case-control study was conducted on 80 children with ADHD and 80 healthy controls. There were 55 boys and 25 girls with ages ranging from 5 to 14 years (mean = 8.7 ± 2.3 years) and boy to girl ratio of 2.2. Patients showed no significant difference regard to age, sex, and residence [Table 1].

Table 1

Demographic and clinical data in patients and controls

	Children with ADHD	Controls	P value
Age in years
Range	5–14	5–14	0.68
Mean + SD	8.7 ± 2.3	8.2 ± 2.7
Sex, n (%)
Male	55 (68.75%)	51 (63.75%)	0.35
Female	25 (31.25%)	29 (36.25%)
Residence, n (%)
Urban	50 (62.5%)	46 (57.5%)	0.47
Rural	30 (27.5%)	34 (42.5%)
IQ (WISC)
Range	70–95	85–100	<0.001*
Mean	80.36 ± 7.47	90.11 ± 5.63
Abbreviated Conners’ Rating Scale
scores
Range	19–26	3–11	<0.001*
Mean	18 ± 4.12	5.76 ± 1.7
Sex distribution across ADHD subtypes
ADHD subtypes (total no.)	Male, n (%)	Female, n (%)	P value
Combined (45)	36 (65%)	9 (36%)
Inattentive (22)	13 (23.6%)	10 (40%)	0.65
Hyperactive (13)	6 (10.9%)	6 (24%)

Children with ADHD were mainly of the combined type (65%). However, the inattentive type was reported in 23.6% and the hyperactive type in (10.9%). Regarding sex distribution among the three types of ADHD, males were mainly of the combined type whereas females were mainly of the inattentive type [Table 1].

There was no significant difference between the three types regarding sex. IQ was significantly lower in children with ADHD than controls. The Abbreviated CPRS for ADHD symptoms was 19–26 in children with ADHD and 3–11 in controls.

The HI score was significantly higher in children with ADHD compared to controls.

There was a significantly higher HI score in patients with ADHD compared with controls [Table 1]. Our results showed significant differences between the two groups in serum IL-6 concentration, with the ADHD group having higher levels than the control group [Table 1]. However, TNF-α showed no significant differences between the two groups. Also, TNF-α showed no significant correlation with the IQ or the Abbreviated Conners’ Rating Scale scores for parents.

The median serum IL-6 levels were 23.7 and 6.4 in children with ADHD and controls, respectively, whereas the interquartile range was 8.47 (17.2–25.49) and 3.8 (5–8.8), respectively. Children with ADHD showed significantly higher serum IL-6 levels than control group [Table 2].

Table 2

Serum IL-6 and TNF-α levels in patients and controls

Variable	Children with ADHD	Controls	P value
IL-6 (pg/mL)
Range	4.2–56.2	1.24–13.34
Mean	26.11 ± 11.14	6.23 ± 2.52	0.001*
Median	23.7	6.4
IQR	8.47 (17.2–25.49)	3.8 (5–8.8)
Normal IL-6 level, n (%)	22 (27.5%)	50 (62.5%)	0.001**
Low IL-6 level, n (%)	3 (3.75%)	30 (62.5%)
Elevated IL-6 level, n (%)	55 (68.75%)	0
TNF-α (pg/mL)
Range	1.14–6.64	1.48–7.65
Mean	1.13 ± 0.65	1.18 ± 0.94	0.34
Median	1.24	1.74
IQR	2.2 (5–8.7)	2.4 (4–7.8)
Normal TNF-α level, n (%)	58 (72.5%)	68 (85%)
Low TNF-α level, n (%)	20 (25%)	12 (15%)	0.16
Elevated TNF-α level, n (%)	2 (2.5%)	0

IQR = the interquartile range

*P value using Mann–Whitney U test is significant when <0.05.

**P value using chi[2] test and is significant when <0.05.

Serum IL-6 showed no significant correlation with the IQ or the Abbreviated Conners’ Rating Scale scores for parents.

DISCUSSION

ADHD is one of the most common neurobehavioral disorders in children. There is a growing body of evidence that abnormality in the immune systems may account for the pathogenesis of ADHD.[4] In our study, we investigated the role of cytokines in immune system activation as a mechanism of ADHD through measurement of serum IL-6 and TNF-α in a cohort of children with ADHD compared to healthy controls.

To date, seven studies (Donfrancesco et al.,[9] Darwish,[24] O’Shea et al.,[25] Hariri et al.,[26] Oades et al.,[27] Oades et al.,[28] and Oades[29]) have assessed the correlation between serum cytokines levels and childhood ADHD. In our study, children with ADHD showed a significantly higher serum level of IL-6 compared to normal healthy children of matched age, sex, and residence. However, TNF-α showed no significant differences between children with ADHD and controls.

In agreement with our findings, there are three previous studies that reported higher serum levels of IL-6 in children with ADHD compared to controls with no significant difference in serum levels of TNF-α between patients and controls.[924,28]

Donfrancesco et al.[9] reported that children with ADHD had significantly higher serum levels of IL-6 and IL-10 than normal children; however, other cytokines (IL-2, IL-4, IL-17, IFN-g, and TNF-α) showed an insignificant difference between patients and controls.[9]

Also, Oades et al. (2010) found a higher serum level of IL-6 in children with ADHD than normal controls although it was statistically insignificant. Also, they found nonsignificantly higher serum levels of other cytokines (IL-2, IFN-g, IL-16, IL-10, and IL-13) in children with ADHD than normal control.[28] Darwish[24] found significantly higher serum level of IL-6 in children with ADHD than normal controls.

Our study did not reveal a significant correlation between IL-6 and symptoms of ADHD although it was reported by other authors.[30] Also, they found a significant correlation between TNF-α and hyperactive-impulsive symptoms.

The reports on peripheral cytokines levels are mixed, but overall they conclude a low-grade inflammatory process in patients with ADHD.[22]

The significantly elevated serum IL-6 concentration suggests the involvement of the immune system in children with ADHD. T-helper lymphocytes release cytokines that can cross the blood-brain barrier. Also, cytokines can be endogenously produced in the brain. Of these cytokines is IL-6 that is produced by leukocytes, astrocytes, and microglia. IL-6 is an inflammatory mediator that can influence synaptic plasticity, neuromodulation, myelination, demyelination, and neurogenesis, which may contribute to ADHD symptoms. Furthermore, it affects neurotransmitter release, reaction time, and working memory, which are deficits in ADHD.[3132]

IL-6 and other cytokines are normally present in the brain at low levels. Increased production of IL-6 can cause activation of microglial cells and glutamate-induced toxicity in the brain. The activated microglial cells release IL-6 as well as other pro-inflammatory cytokines.[3132] In addition, IL-6 plays a role in brain development and function via its effect on neurogenesis, synapse formation, and myelination, and this, in turn, could be one of the possible mechanisms for the pathogenesis of ADHD.[3132] IL-6 causes a decrease in cell survival and neuronal differentiation with consequent inhibition of neurogenesis.[33] Previous studies showed structural brain changes in children with ADHD in the form of smaller volume of the whole brain and gray matter, and cortical thinning, in the prefrontal cortex, and the basal ganglia.[3435] These changes could be related to the inhibitory effect of IL-6 on neurogenesis.

Altered maturation of these specific brain areas may result in persistent neural changes, thereby increasing the risk of ADHD in children. There is compelling evidence that inflammatory cytokines activate neuroimmune mechanisms that involve behaviorally and emotionally relevant brain circuits.[936]

In animals studies, allergen exposure has been found to stimulate limbic brain regions as well as avoidance behavior, increased anxiety, and reduced social behavior.[37] In humans, altered neuronal activity of the anterior cingulate cortex and the prefrontal cortex during a chronic (allergic) episode has been shown by means of functional magnetic resonance imaging.[38] Increased IL-6 can cause an imbalance between norepinephrine and dopamine in the brain through its effect on the metabolism of neurotransmitters.

Animal studies showed an increase in norepinephrine and a decline in dopamine levels on the administration of IL-6.[39] Similar changes in norepinephrine and dopamine were found in the prefrontal cortex in patients with ADHD.[40] Previous studies reported that IL-6 is increased in different neurologic and psychiatric disorders such as depression, multiple sclerosis, and autism. This suggests the role of inflammation in some neuropsychiatric disorders.[2241] Interestingly, children with ADHD on stimulant medications showed normalization of the elevated cytokines levels.[27]

In our study, we did not find any correlation between IL-6 levels and Conners’ scale scores. Although increased IL-6 could be a possible mechanism for ADHD, other factors are involved in the pathogenesis of ADHD.

Darwish[24] reported similar findings and also Vogel et al.[42] found no association between IL-6 and ADHD symptoms in adults with affective disorders. However, ADHD symptoms were positively correlated with increased IL-13 and IL-16 levels as reported by other authors. Symptoms of inattention were correlated with IL-13 levels, whereas symptoms of hyperactivity were correlated with IL-16.[42]

Although we reported no significant changes in TNF-α between children with ADHD and controls, Cortesea et al.[30] found a significant correlation between TNF-α and hyperactive-impulsive symptoms. Also, O’Shea et al.[25] found significantly higher levels of TNFR1 (the receptor for TNF-α) in children with ADHD compared to healthy controls.

Although TNF-alpha is not involved in ADHD-affected adults. (Corominas-Roso et al.[43]), elevated levels of TNF-α were noted in animal studies. An important role of TNF-α in the etiopathogenesis of ADHD may explain significant connections between TNF-α gene polymorphism and ADHD.[4243]

In our study, the increased IL-6 in children with ADHD could be related to either genetic or environmental factors. The most important gene involved in the development of AHDH is DOCK2 that is also involved in cytokine regulation. So, increased IL-6 could be related to genetic factors.[43]

A crucial question in our finding is “Why IL-6 blood levels high, whereas those of TNF-alpha is not?” This could be explained by the fact that not every tissue expresses every cytokine. IL-6 is produced by several types of brain cells and is known to be very important for the central nervous system.[44] Moreover, animal models strongly suggest that IL-6 may play a role in the neuropathology and that it is a clear target of strategic therapies.[3245] Our study is a cross-sectional one that does not allow us to infer any causal relationship between the increase in cytokines levels and ADHD.

This study provides a rationale for a prospective, larger sample, longitudinal studies to gain insight into inflammatory processes underpinning the link between ADHD and inflammatory cytokines. This line of research has the potential to lead to novel, pathophysiologically based management strategies for children with ADHD.

CONCLUSIONS

We can conclude that serum IL-6 levels were significantly higher in children with ADHD compared to controls; however, the IL-6 levels did not correlate with ADHD symptoms severity. Elevated levels of IL-6 in children with ADHD point to the immune pathogenesis of ADHD. Increased IL-6 levels may contribute to the etiology of ADHD. However, TNF-α did not show any significant changes or correlations in children with ADHD.

Financial support and sponsorship

Nil.

Conflicts of interest

There are no conflicts of interest.