Progress in Neuropsychopharmacology & Biological Psychiatry xxx (xxxx) xxxx

Contents lists available at ScienceDirect

Progress in Neuropsychopharmacology
& Biological Psychiatry
journal homepage: www.elsevier.com/locate/pnp

Polymorphism of interleukin-1β and interleukin-1 receptor antagonist genes

in children with autism spectrum disorders
⁎
Khaled Saada, , Alam-Eldin M. Abdallaha, Ahmed A. Abdel-Rahmanb, Abdulrahman A. Al-Atramc,
Yasser F. Abdel-Raheema, Eman Fathallah Gada, Mohamed Gamil M. Abo-Elelaa,
Yasser M. Elserogyb, Amira Elhoufeyd,e, Dalia A. Nigmf, Eman M. Nagiub Abdelsalamf,
Thamer A.M. Alruwailig
a
Department of Pediatrics, Faculty of Medicine, Assiut University, Assiut, Egypt
b
Department of Neuropsychiatry, Faculty of Medicine, Assiut University, Assiut, Egypt
c
Department of Psychiatry, College of Medicine, Majmaah University, Majmaah, Saudi Arabia
d
Department of Community Health Nursing, Faculty of Nursing, Assiut University, Egypt
e
Department of Community Health Nursing, Alddrab University College, Jazan University, Saudi Arabia
f
Clinical Pathology Department, Faculty of Medicine, Assiut University, Egypt
g
Department of Pediatrics, Faculty of Medicine, Aljouf University, Saudi Arabia

A R T I C LE I N FO A B S T R A C T

Keywords: In this study, we first investigated interleukin-1 beta (IL-1β) and IL-1 receptor antagonist (IL-1RA) levels in a
Autism cohort of Egyptian children with autism spectrum disorder (ASD) and in healthy controls. Second, we examined
Children the single-nucleotide polymorphisms (SNPs) at positions −31 and − 511 of the IL-1β gene promoter and IL1RA
Interleukin-1 beta and assessed the association between IL1B and IL1RA polymorphisms with ASD. We examined IL1β promoter
Polymorphism
polymorphism at −511 (IL-1β-511) and − 31 (IL-1β-31) and IL1RA gene polymorphism in 80 children with
ASD and 60 healthy children. The children with ASD had significantly higher levels of IL-1β and IL-1RA than the
controls. The children with ASD also had significantly higher frequencies of homozygous (CC) and heterozygous
(TC) genotype variants of IL-1β-511, and IL-1RA than the controls. Moreover, the frequency of the IL-1β-511
allele (C) was higher in the ASD group than in the controls (p = .001). The homozygous and heterozygous
variants of IL-1RA allele II were also significantly higher in the ASD group than in the control group. There was
no significant association between the IL-1β-31 genotype and autism classes. However, there were significant
differences in the distribution of the IL-1RA heterogeneous genotype and allele II among children with severe
autism. The inflammatory role of cytokines has been implicated in a variety of neuropsychiatric pathologies,
including autism. Our data show alterations in the IL-1β system, with abnormally increased serum levels of IL-1β
and IL-1RA in the children with ASD. Further, polymorphisms in the IL-1β-511 and IL-1RA genotype variants
correlated positively with autism severity and behavioral abnormalities. IL-1β-511 and IL-1RA gene poly-
morphisms could impact ASD risk and may be used as potential biomarkers of ASD. Variations in the IL-1β and
IL-1RA systems may have a role in the pathophysiology of ASD.

1. Introduction repetitive and limited behavioral activities. The etiopathogenesis of

Autism spectrum disorder (ASD) is a genetically highly hetero-
geneous developmental disorder that starts before the age of 3 years.
ASD is usually manifested by multiple emotional, social reciprocity, and
verbal and nonverbal communication abnormalities, in addition to
ASD includes many genetic, environmental, and immunological factors
(Bjørklund et al., 2018, 2019, 2020). The interaction between the ge-
netic, inflammatory, and immunological causes of ASD has become the
focus of many investigations in the past 20 years (Bjørklund et al.,
2016, 2019). No established etiologies have explained ASD; however, it

Abbreviations: Autism Diagnostic Interview-Revised, (ADI-R); Autism spectrum disorder, (ASD); Childhood Autism Rating Scale, (CARS); Diagnostic and Statistical
Manual of Mental Disorders, (DSM-V); Enzyme Amplified Sensitivity Immunoassay, (EASIA); Enzyme-linked immunosorbent assay, (ELISA); Hardy–Weinberg es-
timates, (HWE); IL-1 receptor antagonist, (IL-1RA); Interleukin-1 beta, (IL-1β); Tumor necrosis factor alpha, (TNF-α)
⁎
Corresponding author at: Department of Pediatrics, Faculty of Medicine, Assiut University, Assiut 71516, Egypt.
E-mail address: khaled.ali@med.au.edu.eg (K. Saad).

https://doi.org/10.1016/j.pnpbp.2020.109999
Received 27 April 2020; Received in revised form 28 May 2020; Accepted 1 June 2020
0278-5846/ © 2020 Elsevier Inc. All rights reserved.

Please cite this article as: Khaled Saad, et al., Progress in Neuropsychopharmacology & Biological Psychiatry,
https://doi.org/10.1016/j.pnpbp.2020.109999
K. Saad, et al.
can be attributed to several genetic and immunological factors. More-
over, increasing evidence from animal and human studies on ASD
suggests immune disturbance (Bjørklund et al., 2016).
Inflammatory markers [interleukin-1 beta (IL-1β), IL-6, and tumor
necrosis factor-alpha (TNF-α)] have been studied in autistic children
(Goines and Ashwood, 2013; Bjørklund et al., 2016; Saghazadeh et al.,
2019; Bjørklund et al., 2020). The abnormal expression of many in-
flammatory cytokines in the serum, brain, and gastrointestinal tract has
been reported in ASD. Cytokine imbalances in early life could influence
neural activities and mediate the abnormal behavior in autistic patients
(Goines and Ashwood, 2013). IL-1β is a major proinflammatory cyto-
kine expressed in the immune responses during the early crucial de-
velopmental stages. It is mainly released from the activated microglia
and has receptors throughout the nervous system (Mazahery et al.,
2020). In the peripheral tissues, IL-1β activates the vascular endothelial
and local immune cells with induction of inflammation. Moreover, IL-
1β enhances IL-6 production and finally increases the acute-phase re-
sponse in hepatic cells. In cases with febrile illness, IL-1β crosses the
blood-brain barrier, stimulating the hypothalamus to increase IL-1β
expression, with alterations in neuroendocrine functions (Goines and
Ashwood, 2013; Mazahery et al., 2020). Central and peripheral IL-1β
decreases neurogenesis and increases anxiety, stress, and abnormal
social interaction (Masi et al., 2017). In experimental animals, the ad-
ministration of IL-1 receptor antagonist (IL-1RA) lowered IL-1β–in-
duced impaired social interaction (Bluthé et al., 1991; Tsai et al., 2010;
Masi et al., 2017). The genes for IL-1β and its receptor are linked to
many psychiatric disorders, including ASD and schizophrenia
(Mazahery et al., 2020). Significantly elevated IL-1β levels and skewed
IL-1β responses following stimulation have been reported in children
with ASD. Moreover, polymorphisms in the IL-1β genes and their re-
ceptors have been reported in cognitive disorders (Tsai et al., 2010;
Masi et al., 2017; Mazahery et al., 2020). There is a significant corre-
lation between IL-1β and impaired behavior and regressive outcomes in
autistic children (Goines and Ashwood, 2013; Masi et al., 2017). As the
data on IL-1β, IL-1RA, and their gene mutations in autistic children are
inadequate and entirely absent in studies on Egyptian children, we
aimed first to investigate the IL-1β and IL-1RA levels in a cohort of
Egyptian children with ASD and in healthy controls. Second, we ex-
amined the single-nucleotide polymorphisms (SNPs) at positions −31
and − 511 of the IL-1β gene promoter and IL-1RA and to evaluate any
correlation of these polymorphisms with ASD.
2. Patients and methods
This was a case-controlled study undertaken at Assiut University
Hospital, Assiut, Egypt. The Assiut University Hospital Ethical Scientific
Committee approved our protocol and all procedures in this study. All
procedures were conducted following the Code of Ethics of the World
Medical Association for experiments involving humans of 2000. All
caregivers of all participants provided their informed written consent.
The study was conducted at Assiut University Hospital from June 2018
to May 2019 (Number: 113–4-2018).
2.1. Patients
The study sample size was calculated using G*Power 3.1.9.2 soft-
ware. The size effect of 0.5, significance level of α = 0.05, and statis-
tical power of 1 - β = 0.95 were considered. The power analysis in-
dicated that 100 participants were required, i.e., 50 per group. We
included 80 children with ASD and 60 controls. The inclusion criteria
included a confirmed diagnosis of ASD. All patients were recruited from
Assiut University Hospitals. The 60 controls were age- and sex-matched
healthy children. All controls were also free from any psychiatric dis-
orders and other exclusion criteria.
2
Progress in Neuropsychopharmacology & Biological Psychiatry xxx (xxxx) xxxx
2.2. Exclusion criteria
We excluded any participant with evidence of other genetic and
neurological disorders, e.g., epilepsy, phenylketonuria, neurocutaneous
syndromes, and cerebral palsy. We also excluded any participants with
autoimmune disorders, chronic systemic, or hepatic or renal diseases.
2.3. Clinical and psychiatric assessment
Detailed medical history-taking and physical examination were
performed for all patients and included a family history of similar
conditions and the time of ASD diagnosis. Two senior psychiatrists es-
tablished the diagnosis of ASD before patients were recruited. For di-
agnosing autism, we used the fifth edition of the Diagnostic and
Statistical Manual of Mental Disorders (DSM-V) (American Psychiatric
Association, 2013), and the Autism Diagnostic Interview-Revised (ADI-
R) (Lord et al., 1994). We carried out two-parent interviews: one for
diagnosing autism and the other for evaluating autism severity using
the Childhood Autism Rating Scale (CARS) (Schopler et al., 2010).
2.4. Laboratory investigations
IL-1β was assayed using a solid-phase Enzyme Amplified Sensitivity
Immunoassay (EASIA, Biosource) on a microtiter plate for quantitative
measurement of serum human IL-1β. IL-1RA was measured using a
Human IL-1 RII Quantikine ELISA (enzyme-linked immunosorbent
assay) Kit (R&D Systems, DR1B00). The results are expressed as pico-
gram per milliliter (pg/ml).
2.5. Molecular analysis
We analyzed the IL-1 gene polymorphisms at the transcriptional
start site of the IL-1β genes: IL-1β −511 (C/T), IL-1β −31 (C/T), and
IL-1RA, in all patients and controls.
2.6. DNA extraction
Genomic DNA was extracted from blood samples using a QIAamp
DNA Mini Kit (Qiagen, Hilden, Germany) on a QIAcube extractor and
stored at −70 °C until tested.
2.7. Genotyping
Genomic DNA was extracted from leukocytes using a QIAamp DNA
extraction kit (Qiagen). All patients and controls were genotyped for
analysis of IL1β gene polymorphisms at positions −31 (IL-1β-31)
and − 511 (IL-1β-511) by polymerase chain reaction-restriction frag-
ment length polymorphism (PCR-RFLP). The sense and anti-sense
primer sequences of the IL-1β-31C > T polymorphism (rs1143627)
were 5′-AGAAGCTTCCACCAATACTC-3′ and 5′-AGCACCTAGTTGTAA
GGAAG-3′, respectively, and that for the IL-1β-511 T > C poly-
morphism (rs16944) were: 5′-GCCTGAACCCTGCATACCGT-3′ and
5′-GCCAATAGCCCTCCCTGTCT-3′, respectively (TIB Molbiol, Berlin,
Germany). IL1RA polymorphisms were coded as allele 1 (four repeats),
allele 2 (two repeats), allele 3 (five repeats), allele 4 (3 repeats), and
allele 5 (six repeats). Table 1 lists the PCR primers used in the study.
2.8. Statistical analysis
The IL-1β genotype and allele frequencies in all participants were
assessed using the allele counting method. Hardy–Weinberg estimates
(HWE) for the genotype frequencies were calculated using Haploview
software. The HWE were tested using the χ2 test. Odds ratios (OR) were
calculated, and the 95% confidence interval (95% CI) was estimated
using Fisher's exact test. A p-value of < 0.05 was considered significant.
SPSS version 22 was used for data collection and analysis.
K. Saad, et al. Progress in Neuropsychopharmacology & Biological Psychiatry xxx (xxxx) xxxx

Table 1
PCR primers and restriction enzyme sizes of IL-1B and IL-1RN genes.
Primer Primer sequence Genotype Restriction enzyme Cutting size

IL-1β-511 5′-GCCTGAACCCTGCATACCGT-3′ T/T Ava I 156

5′-GCCAATAGCCCTCCCTGTCT-3′ C/T 156, 88, 68
C/C 88, 68
IL-1β-31 5′-AGA AGCTTCCACCAATACTC-3′ T/T Alu I 137, 97, 5
5′-AGCACCTAGTTGTAAGGAAG-3′ C/T 234, 137, 97, 5
C/C 234, 5
IL-1RN 5′-CTCAGCAACACTCCTAT-3′ Five repeats – 500
5′-TCCTGGTCTGCAGGTAA-3′ Four repeats 411
Three repeats 325
Two repeats 240

Table 2 Table 3
Demographic data ADI-R scores, classification of autism of the studied group. Distribution of IL-1β and IL-1RA genotypes, alleles in patients with ASD and
controls.
ASD children Control p-value
(N = 80) (N = 60) Genotype Patient group (80) (N, Control group (60) (N, P-value
%) %)
Age (years)
Range 3.5–10.5 3–9.5 0.57 IL-1β (−511) T > C genotype
Mean ± SD 4.8 ± 2.4 4.2 ± 1.9
• TT (Wild type) 4 (5%) 11 (18.3%) –
• CC (Mutant) 31 (38.75%) 18 (30%) 0.032*
•
Gender [Number (%)]
TC (Hetero) 45 (56.25%) 31 (51.7%) 0.039*
Males 65 (81.25) 50 (83.3) 0.81
Females 15 (18.75) 10 (16.7) Allele frequency
BMI (kg/m2) 27.6 ± 4.8 27.5 ± 4.2 0.28
• TC 56 (35%) 88 (73.3%) –
Serum IL-1β (pg/ml)
Serum IL-1RA (pg/ml)
29.4 ± 1.3
119.2 ± 28.6
3.4 ± 1.3
79.4 ± 34.3
< 0.0001*
0.001*
• 104 (65%) 32 (26.7%) 0.001*

IL-1β (−31) C > T genotype

Age at diagnosis [Number (%)]
• CC (Wild type) 20 (25%) 14 (23.3%) –
Before three years 54 (67.5) – –
• TT (Mutant) 25 (31.2%) 19 (31.7%) 0.63
After three years 26 (32.5) – –
• CT (Hetero) 35 (43.8%) 27 (45%) 0.54
Childhood Autism Rating Scale (CARS) Allele frequency
Severe (≥37) 32 (40) – –
• CT 84 (52.5%) 66 (55%) –
Mild/moderate (≤36.5)
Range
48 (60)
30 to 55.5
–
–
–
–
• 76 (47.5%) 54 (45%) 0.63

IL-1RA (VNTR) genotype

• I/III/II(Wild
Mean ± SD 37.2 ± 5.3 – –
type) 30 (37.5%) 30 (50%) –
Autism Diagnostic Interview-Revised” (ADI-R)
• I/II (Hetero)
(mutant) 10 (12.5%) 5 (8.3%) 0.04*
ADI-R Communication
ADI-R Stereotyped behavior
18.2 ± 6.2
3.5 ± 1.7
–
–
–
–
• 40 (50%) 25 (41.7%) 0.008*

Allele frequency
• III
ADI-R Social interaction 12.4 ± 5.3 – –
107 (66.9%) 91 (75.8%) –
•
ADI-R Play 5.8 ± 2.1 – –
53 (33.1%) 29 (24.2%) 0.01*
*Significant.
*Significant.

3. Results
significantly higher frequencies of the IL-1β-511 (C) allele than the
controls (OR: 1.8, 95% CI: 1.13–2.45; p = .001). The autistic children
Here, we included 80 autistic children and 60 healthy age- and sex-
also had significantly elevated heterozygous and homozygous genotype
matched controls. Table 2 shows the patient demographics, including
variants of IL-1RA as compared to the control group (OR: 3.19, 95% CI:
age at ASD diagnosis, CARS scores, autism classification, body mass
1.16–16.32; p = .04; OR: 1.89, 95% CI: 1.13–3.77; p = .008, respec-
index (BMI), and serum IL-1β and IL-1RA results. The patients were
tively). The ASD group also had significantly higher frequencies of IL-
3.5–10 years old (mean ± SD, 4.8 ± 2.4 years), and there were 65
1RA allele (II) compared to the control group (OR: 1.33, 95% CI:
boys (81.25%); the male-to-female ratio was 4.3:1. More than two-
1.12–2.85; p = .01). However, neither the alleles nor the genotypes of
thirds of the autistic children (67.5%) were diagnosed earlier than the
IL-1β-31 showed any significant difference between the ASD patients
age of 3 years. The CARS scores were 30–55.5 points (mean ± SD,
and controls (p > .05; Table 3).
37.2 ± 5.3 points). Sixty percent of the patients had mild/moderate
To identify the relationship between IL1β polymorphisms and
autism, while 40% had severe autism (Table 2). Table 2 shows the mean
autism class, we compared the results of the severe autism, mild/
ADI-R scores of our patients. The autistic children had significantly
moderate autism, and control groups. The homozygous variant geno-
higher levels of IL-1β (p < .0001) and IL-1RA (p = .001) than the
type of IL-1β-511 was significantly higher in both autism classes as
healthy controls (Table 2). Compared mild/moderate cases, patients
compared to the controls (severe autism, p = .038; mild/moderate
with severe ASD had significantly elevated serum levels of IL-1β and IL-
autism, p = .042; Table 4). Furthermore, the variant allele (C) was
1RA. However, the values reached significant levels for serum IL-1RA
significantly higher in both autism groups than in the controls
only (Table 3). Table 3 shows the data of the IL-1β and IL-1RA (variable
(p < .0001). Our results showed no significant correlations between
number of tandem repeats, VNTR) genotypes and alleles in all partici-
the IL-1β-31 genotypes and autism classes. However, the IL-1RA het-
pants. Here, we report for the first time that the frequencies of the
erozygous genotype and allele II distributions in the patients with se-
homozygous (CC) and heterozygous (TC) genotype variants of IL-1β-
vere autism were significantly different when compared with the con-
511 were significantly higher in the ASD children than in the controls
trols (OR: 3.11, 95% CI: 2.63–24.23; p = .045; OR: 1.86, 95% CI:
(OR: 2.36, 95% CI: 1.06–5.18; p = .032; OR: 2.04, 95% CI: 1.06–38;
1.17–4.22; p = .03, respectively; Table 4).
p = .039, respectively). Moreover, the autistic children had

3
K. Saad, et al. Progress in Neuropsychopharmacology & Biological Psychiatry xxx (xxxx) xxxx

Table 4
Association of IL-1B-31, IL-1B-511, and IL-1RA polymorphisms and classification of autism.
Genotype Severe autism (32) Mild/moderate autism (48) Control group (60) P1-value P2-value

IL-1β (−511) T > C genotype

• TT (Wild type) 2 (6.3%) 2 (4.2%) 11 (18.3%) – –
• CC (Mutant) 13 (40.6%) 18 (37.5%) 18 (30%) 0.038* 0.042*
• TC (Hetero) 17 (52.1%) 28 (58.3%) 31 (51.7%) 0.39 0.31

Allele frequency
• TC 25 (39%) 31 (32.3%) 88 (73.3%) – –
• 39 (61%) 65 (67.7%) 32 (26.7%) < 0.0001* < 0.0001*

IL-1β (−31) C > T genotype

• CC (Wild type) 7 (21.9%) 13 (27%) 14 (23.3%) – –
• TT (Mutant) 10 (31.2%) 15 (31.3%) 19 (31.7%) 0.89 0.36
• CT (Hetero) 15 (46.9%) 20 (41.7%) 27 (45%) 0.59 0.66

Allele frequency
• CT 34 (53.1%) 50 (52.1%) 66 (55%) – –
• 30 (46.9%) 46 (47.9%) 54 (45%) 0.63 0.85

IL-1RA (VNTR) genotype

• I/III/II(Wild type) 11 (34.4%) 19 (39.6%) 30 (50%) – –
• I/II (Hetero)
(mutant) 3 (9.3%) 5 (10.4%) 5 (8.3%) 0.08 0.11
• 18 (56.3%) 24 (50%) 25 (41.7%) 0.045* 0.07

Allele frequency
• III 38 (59.4%) 69 (71.9%) 91 (75.8%) – –
•
Serum IL-1β (pg/ml)
26 (40.6%)
30.2 ± 3.1
27 (28.1%)
28.2 ± 3.1
29 (24.2%)
3.4 ± 1.3
0.03*
< 0.0001*
0.87
< 0.0001*
Serum IL-1RA (pg/ml) 131.5 ± 28.6 108.8 ± 30.9 79.4 ± 34.3 0.042* 0.001*

P1 = severe autism versus controls, P2 = mild/ moderate autism versus controls, *Significant.

4. Discussion in autistic children than in healthy groups. Furthermore, experimental

We investigated IL-1β and IL-1RA levels in a cohort of Egyptian
children with ASD and in healthy controls. IL-1β has structural simi-
larity to fibroblast growth factors, which are essential for the early
stages of embryonic neural development (Barksby et al., 2007; Stevens
et al., 2010). IL-1β stimulates the inflammatory response by activating
both lymphocyte and macrophage functions. Moreover, it increases the
expression of adhesion molecules assisting tissue infiltration by in-
flammatory cells from the circulation, leading to a chronic in-
flammatory state (Deckmann et al., 2018). IL-1β also plays a key role in
the expression of inflammatory mediators and in the induction of T-
helper 17 cells (Th17) response (Manzardo et al., 2012). IL-1RA an-
tagonizes the effects of IL-1β and other cytokines, e.g., IL-1α, therefore
the estimation of IL-1RA levels may play a role in the negative feedback
regulation in the case of IL-1β increase (Suzuki et al., 2011). The human
brain recognizes the effect of inflammatory cytokines such as IL-1β, IL-
1α, and TNF-α as molecular signals of illness. Moreover, IL-1β and
other proinflammatory cytokines cross the blood-brain barrier, acting
on the hypothalamus and promoting illness and febrile behavior
(Dantzer, 2009). The mechanism of action of illness and febrile beha-
viors and its similarities to the expression of some psychiatric behaviors
have led to the theory that proinflammatory cytokines can participate
in the pathophysiology of many psychiatric diseases, such as ASD
(Dantzer, 2009). In the last 20 years, extensive research has explored
how the inflammatory pathways affect the brain and behavior through
the immune system (Capuron and Miller, 2011; Goines and Ashwood,
2013). IL-1β and its genes and the associated receptors and multiple
psychiatric diseases such as ASD and schizophrenia have been linked, as
inflammation is a crucial factor in these psychopathological disorders
(Capuron and Miller, 2011; Goines and Ashwood, 2013; Saghazadeh
et al., 2019). Moreover, the effects of cytokines on neurogenesis, neu-
rotransmitter roles, endocrine activity, and modification of brain cir-
cuitry can influence behavior (Capuron and Miller, 2011). Here, we
show that ASD patients had significantly increased serum levels of IL-1β
(p < .0001) and IL-1RA (p = .001) than the controls. Our findings
agree with previous studies (Ashwood et al., 2011; Suzuki et al., 2011;
Manzardo et al., 2012), which reported significantly higher IL-1β levels
studies on animal models of ASD have reported increased IL-1β levels in
the hippocampus and whole-brain homogenate (Theije et al., 2014;
Hegazy et al., 2015).
A previous meta-analysis investigated serum IL-1RA levels in nine
studies involving 519 patients with ASD (Saghazadeh et al., 2019). The
authors found that blood IL-1RA levels were comparable between ASD
patients and the controls (p = .62) (Saghazadeh et al., 2019). Our re-
sults show that children with severe autism have significantly higher
serum IL-1RA levels compared to children with mild and moderate
autism. This result has been reported only in adults with ASD
(Emanuele et al., 2010). We found that IL-1RA levels had a significant
positive correlation with IL-1β (p < .05). We also examined the as-
sociations between IL-1β levels and the ADI-R and CARS behavioral
outcomes, and found that increased IL-1β levels had significant positive
associations with ADI-R parameters [communication (p < .05) and
stereotyped behavior (p < .05)] and CARS parameters [social inter-
action (p < .05), non-verbal communication (p < .01), total CARS
scores (p < .01), and autism severity (p < .01)]. Suzuki et al., 2011,
reported a significant positive correlation between IL-1β and IL-1RA.
They hypothesized that IL-1RA might increase in ASD as a negative
feedback regulation due to the increased levels of IL-1β (Suzuki et al.,
2011). Increased IL-1RA in ASD may be an attempt to compensate for
the higher levels of IL-1β and may not be beneficial. The administration
of IL-1RA during the critical neurodevelopmental windows can affect
the neurogenesis, memory, behavior, and brain morphology processes
negatively (Goines and Ashwood, 2013; Spulber et al., 2011). In line
with our results, previous studies have reported significant associations
between IL-1β levels and abnormal autistic behavior (Ashwood et al.,
2011; Emanuele et al., 2010; Goines and Ashwood, 2013). Increased IL-
1β levels have been positively associated with impaired stereotyped
behavior (Ashwood et al., 2011), impaired social interactions (Ashwood
et al., 2011), autism severity (Emanuele et al., 2010), and diminishing
memory and learning (Goines and Ashwood, 2013).
In the present study, our novel results show that IL-1β-511 hetero-
zygous (OR 2.04) and homozygous (OR: 2.36) genetic variants are
significantly associated with increased autism susceptibility. Moreover,
the variant (C) allele has 1.7-fold increased susceptibility to autism (OR:

4
K. Saad, et al.
1.7, 95% CI: 1.13–2.45; p = .001). Further, the IL-1RA heterozygous
and homozygous variants were associated more with autism risk than in
the controls (OR: 1.89 and 3.19, respectively). Moreover, the IL-1RA
mutant allele (II) has 1.33-fold increased susceptibility to severe autism
(OR: 1.33, 95% CI: 1.12–2.85; p = .01). However, the IL-1 β-31 gen-
otypes and alleles did not show any significant autism risk.
To our knowledge, IL-1β-511, IL-1β-31, and IL-1RA gene poly-
morphisms in children with ASD have not been investigated. A very
recent study by Hylén et al., 2020, involved a group of 40 adult patients
with different psychiatric disorders; eight of the patients were autistic.
In that study, the ASD group had significantly higher levels of IL-1RA
than the healthy control adults (p = .001). However, IL-1β serum levels
in the two groups were comparable. In line with our findings, the
homozygous and heterozygous IL-1β-31 variants in that study were not
significantly different between the adults with ASD and other psy-
chiatric disorders and the controls (Hylén et al., 2020). Previous studies
(Estes and McAllister, 2016; Krakowiak et al., 2017) have suggested
that cytokine profiles at birth, including elevated IL-1β, are associated
with autism diagnosis at the age of 2–5 years and correlate with ASD
symptom severity. Increased expression of IL-1β and other proin-
flammatory cytokines might indicate prenatal immune abnormalities,
and their connection with autism and the risk of cognitive-develop-
mental abnormalities suggests the likelihood of a global impact of early
cytokine dysregulation in children with ASD (Estes and McAllister,
2016; Krakowiak et al., 2017).
5. Conclusion
The inflammatory role of cytokines is implicated in a variety of
neuropsychiatric pathologies, including ASD. Our data show that au-
tistic children have alterations in the IL-1β system, with abnormally
increased levels of IL-1β and IL-1RA. In addition, polymorphisms in the
IL-1β-511 and IL-1RA genotype variants have positive correlations with
autism severity and behavioral abnormalities. IL-1β-511 and IL-1RA
polymorphisms may significantly impact ASD risk and could be used as
potential biomarkers for ASD. Variations in the IL-1β and IL-1RA sys-
tems may contribute to the pathophysiology of ASD.
Funding
This research received funding from Majmaah University, Kingdom
of Saudi Arabia (219–2018).
Authors' statement
They state that the article is original, has not been submitted for
publication in other journals and has not yet been published either
wholly or in part. They state that they are responsible for the research
that they have designed and carried out; that they have participated in
drafting and revising the manuscript submitted, whose contents they
approve.
Declaration of Competing Interest
The authors declare that they have no competing interests.
All authors read and agreed on the final version of the manuscript.
References
American Psychiatric Association, 2013. Diagnostic and Statistical Manual of Mental
Disorders 5. American Psychiatric Association, Washington, DC.
Progress in Neuropsychopharmacology & Biological Psychiatry xxx (xxxx) xxxx
Ashwood, P., Krakowiak, P., Hertz-Picciotto, I., Hansen, R., Pessah, I., Van de Water, J.,
2011. Elevated plasma cytokines in autism spectrum disorders provide evidence of
immune dysfunction and are associated with impaired behavioral outcome. Brain
Behav. Immun. 25 (1), 40–45.
Barksby, H.E., Lea, S.R., Preshaw, P.M., Taylor, J.J., 2007. The expanding family of in-
terleukin-1 cytokines and their role in destructive inflammatory disorders. Clin. Exp.
Immunol. 149 (2), 217–225.
Bjørklund, G., Saad, K., Chirumbolo, S., Kern, J.K., Geier, D.A., Geier, M.R., et al., 2016.
Immune dysfunction and neuroinflammation in autism spectrum disorder. Acta
Neurobiol. Exp. (Wars) 76 (4), 257–268.
Bjørklund, G., Kern, J.K., Urbina, M.A., Saad, K., El-Houfey, A.A., Geier, D.A., et al., 2018.
Cerebral hypoperfusion in autism spectrum disorder. Acta Neurobiol. Exp. (Wars) 78
(1), 21–29.
Bjørklund, G., Waly, M.I., Al-Farsi, Y., Saad, K., Dadar, M., Rahman, M.M., et al., 2019.
The role of vitamins in autism spectrum disorder: what do we know? J. Mol.
Neurosci. 67 (3), 373–387.
Bjørklund, G., Meguid, N.A., El-Bana, M.A., Tinkov, A.A., Saad, K., Dadar, M., et al., 2020.
Oxidative stress in autism spectrum disorder. Mol. Neurobiol. 57 (5), 2314–2332.
Bluthé, R., Parnet, P., Dantzer, R., Kelley, K., 1991. Interleukin-1 receptor antagonist
blocks effects of IL-1α and IL-1β on social behaviour and body weight in mice.
Symptom Res. CAO 9, 151–158.
Capuron, L., Miller, A.H., 2011. Immune system to brain signaling: neuropsycho-
pharmacological implications. Pharmacol. Ther. 130 (2), 226–238.
Dantzer, R., 2009. Cytokine, sickness behavior, and depression. Immunol. Allergy Clin. N.
Am. 29 (2), 247–264.
Deckmann, I., Schwingel, G.B., Fontes-Dutra, M., Bambini-Junior, V., Gottfried, C., 2018.
Neuroimmune alterations in autism: a translational analysis focusing on the animal
model of autism induced by prenatal exposure to valproic acid.
Neuroimmunomodulation. 25 (5–6), 285–299.
Emanuele, E., Orsi, P., Boso, M., Broglia, D., Brondino, N., Barale, F., et al., 2010. Low-
grade endotoxemia in patients with severe autism. Neurosci. Lett. 471 (3), 162–165.
Estes, M.L., McAllister, A.K., 2016. Maternal immune activation: implications for neu-
ropsychiatric disorders. Science 353, 772–777.
Goines, P.E., Ashwood, P., 2013. Cytokine dysregulation in autism spectrum disorders
(ASD): possible role of the environment. Neurotoxicol. Teratol. 36, 67–81.
Hegazy, H.G., Ali, E.H., Elgoly, A.H., 2015. Interplay between pro-inflammatory cyto-
kines and brain oxidative stress biomarkers: evidence of parallels between butyl
paraben intoxication and the valproic acid brain physiopathology in autism rat
model. Cytokine 71, 173–180.
Hylén, U., Eklund, D., Humble, M., Bartoszek, J., Särndahl, E., Bejerot, S., 2020. Increased
inflammasome activity in markedly ill psychiatric patients: an explorative study. J.
Neuroimmunol. 339, 577119.
Krakowiak, P., Goines, P.E., Tancredi, D.J., Ashwood, P., Hansen, R.L., Hertz-Picciotto, I.,
et al., 2017. Neonatal cytokine profiles associated with autism spectrum disorder.
Biol. Psychiatry 81 (5), 442–451.
Lord, C., Rutter, M., Le Couteur, A., 1994. Autism diagnostic interview-revised: a revised
version of a diagnostic interview for caregivers of individuals with possible pervasive
developmental disorders. J. Autism Dev. Disord. 24 (5), 659–685.
Manzardo, A.M., Henkhaus, R., Dhillon, S., Butler, M.G., 2012. Plasma cytokine levels in
children with autistic disorder and unrelated siblings. Int. J. Dev. Neurosci. 30,
121–127.
Masi, A., Breen, E.J., Alvares, G.A., Glozier, N., Hickie, I.B., Hunt, A., et al., 2017.
Cytokine levels and associations with symptom severity in male and female children
with autism spectrum disorder. Mol. Autism 8, 63.
Mazahery, H., Conlon, C.A., Beck, K.L., Mugridge, O., Kruger, M.C., Stonehouse, W., et al.,
2020. Inflammation (IL-1β) modifies the effect of vitamin d and omega-3 long chain
polyunsaturated fatty acids on core symptoms of autism spectrum disorder-an ex-
ploratory pilot study. Nutrients. 12 (3).
Saghazadeh, A., Ataeinia, B., Keynejad, K., Abdolalizadeh, A., Hirbod-Mobarakeh, A.,
Rezaei, N., 2019. Anti-inflammatory cytokines in autism spectrum disorders: a sys-
tematic review and meta-analysis. Cytokine. 123, 154740.
Schopler, E., Van Bourgondien, M.E., Wellman, G.J., 2010. Childhood Autism Rating
Scale. (2nd. CARS-2).
Spulber, S., Bartfai, T., Winblad, B., Schultzberg, M., 2011. Morphological and behavioral
changes induced by transgenic overexpression of interleukin-1ra in the brain. J.
Neurosci. Res. 89 (2), 142–152.
Stevens, H.E., Smith, K.M., Rash, B.G., Vaccarino, F.M., 2010. Neural stem cell regulation,
fibroblast growth factors, and the developmental origins of neuropsychiatric dis-
orders. Front. Neurosci. 4 (1), 1–14.
Suzuki, K., Matsuzaki, H., Iwata, K., Kameno, Y., Shimmura, C., Kawai, S., et al., 2011.
Plasma cytokine profiles in subjects with high-functioning autism spectrum disorders.
PLoS One 6, 1–6.
Theije, C.G.M., Wu, J., Koelink, P.J., Korte-Bouws, G.A.H., Borre, Y., Kas, M.J.H., et al.,
2014. Autistic-like behavioral and neurochemical changes in a mouse model of food
allergy. Behav. Brain Res. 261, 265–274.
Tsai, S.J., Hong, C.J., Liu, M.E., Hou, S.J., Yen, F.C., Hsieh, C.H., et al., 2010. Interleukin-
1 beta (C-511T) genetic polymorphism is associated with cognitive performance in
elderly males without dementia. Neurobiol. Aging 31, 1950–1955.