Conceived and designed the experiments: KS MT TS NM. Performed the experiments: HM KI YK CS. Analyzed the data: KJT YI KN. Wrote the paper: KS NM. Obtained informed consent from participants: SK YY TW KT ST KM.
The authors have declared that no competing interests exist.
Accumulating evidence suggests that dysregulation of the immune system is involved in the pathophysiology of autism spectrum disorders (ASD). The aim of the study was to explore immunological markers in peripheral plasma samples from non-medicated subjects with high-functioning ASD.
A multiplex assay for cytokines and chemokines was applied to plasma samples from male subjects with high-functioning ASD (n = 28) and matched controls (n = 28). Among a total of 48 analytes examined, the plasma concentrations of IL-1β, IL-1RA, IL-5, IL-8, IL-12(p70), IL-13, IL-17 and GRO-α were significantly higher in subjects with ASD compared with the corresponding values of matched controls after correction for multiple comparisons.
The results suggest that abnormal immune responses as assessed by multiplex
analysis of cytokines may serve as one of the biological
Autism spectrum disorders (ASD) are a group of neurodevelopmental disorders
characterized by pervasive abnormalities in social interaction and communication,
and repetitive and restricted behavioral patterns and interests. ASD include
autistic disorder, Asperger's disorder and pervasive developmental disorder,
not otherwise specified
Accumulating evidence suggests that dysregulation of the immune system may be
implicated in the pathophysiology of ASD
Recent advances in multiplex technologies have enabled measurement of multiple
analytes simultaneously. Multiplexing provides data on a large number of analytes,
even when the sample volumes are limited
The characteristics of all participants are summarized in
Characteristic | Mean (SD) [Range] | |
Control,
|
ASD,
|
|
Age, years | 12.3 (2.3) |
12.1 (3.3) |
Full IQ | 101.5 (11.5) [82–124] | 99.6 (18.6) [72–136] |
Height, cm | 149.6 (12.5) [121.4–172.8] | 147.1 (17.0) [110.0–175.0] |
Weight, kg | 40.4 (10.6) [24.0–62.0] | 40.4 (13.2) [17.5–72.4] |
BMI, kg/m2 | 17.7 (2.4) [14.4–25.3] | 18.1 (2.6) [13.9–24.2] |
Scores on Autism Diagnostic Interview-Revised | ||
Domain A (social) | N/A | 19.9 (5.2) |
Domain BV (communication) | N/A | 13.7 (4.0) |
Domain C (stereotype) | N/A | 5.6 (2.2) |
Domain D (age of onset) | N/A | 3.2 (1.1) |
Abbreviations: ASD, autism spectrum disorder; IQ, intelligence quotient; BMI, body-mass index; and N/A, not applicable.
The comparison of cytokine and chemokine detection is summarized in
Control group (n = 28) | ASD group (n = 28) | FDR- corrected |
||||
Analytes | mean | SD | mean | SD | ||
Group I | ||||||
IL-1β | 1.1 | 0.8 | 1.7 | 0.8 | −2.616 | *0.049 |
IL-1RA | 85.4 | 49.2 | 135.0 | 43.4 | −4.002 | *0.003 |
IL-2 | BDR | BDR | - | - | ||
IL-4 | 2.1 | 0.9 | 2.7 | 0.9 | −2.487 | 0.06 |
IL-5 | 2.8 | 1.4 | 3.8 | 1.3 | −2.906 | *0.033 |
IL-6 | 5.9 | 3.2 | 6.8 | 2.4 | −1.207 | 0.37 |
IL-7 | 10.8 | 3.1 | 12.8 | 3.4 | −2.201 | 0.09 |
IL-8 | 8.7 | 3.7 | 11.6 | 2.5 | −3.391 | *0.014 |
IL-9 | 13.0 | 10.1 | 14.8 | 9.9 | −0.672 | 0.68 |
IL-10 | 2.5 | 1.8 | 2.9 | 1.4 | −1.039 | 0.45 |
IL-12 (p70) | 21.3 | 12.6 | 38.1 | 13.7 | −4.784 | *0.001 |
IL-13 | 11.8 | 5.2 | 16.3 | 5.0 | −3.329 | *0.011 |
IL-15 | BDR | BDR | - | - | ||
IL-17 | 7.2 | 4.8 | 17.7 | 11.9 | −4.287 | *0.002 |
Eotaxin | 86.7 | 50.8 | 107.6 | 35.3 | −1.789 | 0.16 |
Basic FGF | BDR | BDR | - | - | ||
G-CSF | 4.8 | 3.4 | 6.9 | 3.2 | −2.368 | 0.07 |
GM-CSF | BDR | BDR | - | - | ||
IFN-γ | 80.0 | 46.4 | 107.2 | 49.6 | −2.123 | 0.10 |
IP-10 | 1912.2 | 3202.5 | 1075.1 | 322.0 | 1.376 | 0.33 |
MCP-1 | 26.4 | 17.3 | 28.2 | 13.9 | −0.419 | 0.83 |
MIP-1α | 6.5 | 2.6 | 6.7 | 2.8 | −0.227 | 0.91 |
MIP-1β | 125.0 | 45.3 | 159.8 | 57.1 | −2.527 | 0.06 |
PDGF-BB | 11053.2 | 3023.3 | 12465.3 | 1548.4 | −2.200 | 0.09 |
RANTES | 6303.5 | 809.6 | 6103.5 | 598.4 | 1.051 | 0.46 |
TNF-α | 8.6 | 9.1 | 18.0 | 19.6 | −2.316 | 0.08 |
VEGF | 74.3 | 65.6 | 124.9 | 75.4 | −2.682 | 0.05 |
Group II | ||||||
CTACK | 555.8 | 138.7 | 606.1 | 145.8 | −1.324 | 0.33 |
GRO-α | 60.5 | 38.3 | 99.0 | 47.4 | −3.347 | *0.013 |
HGF | 213.1 | 83.5 | 266.0 | 66.9 | −2.619 | 0.06 |
IFN-α2 | 38.4 | 11.1 | 38.1 | 8.2 | 0.150 | 0.92 |
IL-1α | 0.5 | 0.4 | 0.6 | 0.4 | −0.990 | 0.47 |
IL-2Rα | 59.6 | 20.0 | 56.3 | 22.2 | 0.579 | 0.76 |
IL-3 | 17.1 | 16.6 | 17.3 | 9.9 | −0.051 | 0.96 |
IL-12 (p40) | 43.4 | 23.2 | 56.2 | 26.8 | −1.907 | 0.15 |
IL-16 | 210.8 | 90.0 | 220.6 | 73.1 | −0.447 | 0.83 |
IL-18 | 60.3 | 24.3 | 61.3 | 17.2 | −0.171 | 0.93 |
LIF | BDR | BDR | - | - | ||
MCP-3 | 7.2 | 3.4 | 5.8 | 3.7 | 1.443 | 0.30 |
M-CSF | 10.7 | 7.2 | 13.2 | 7.6 | −1.259 | 0.35 |
MIF | 78.3 | 31.3 | 81.0 | 28.1 | −0.333 | 0.88 |
MIG | 415.2 | 270.8 | 471.3 | 520.5 | −0.505 | 0.80 |
β-NGF | 3.1 | 1.9 | 3.1 | 1.0 | 0.059 | 0.98 |
SCF | 144.0 | 24.3 | 150.3 | 39.3 | −0.718 | 0.68 |
SCGF-β | 29762.4 | 6331.6 | 32684.0 | 5613.8 | −1.827 | 0.16 |
SDF-1α | 162.7 | 55.7 | 180.8 | 43.6 | −1.347 | 0.33 |
TNF-β | 3.4 | 4.6 | 3.1 | 3.4 | 0.285 | 0.88 |
TRAIL | 160.7 | 58.9 | 133.9 | 48.9 | 1.851 | 0.16 |
Concentrations of analytes are shown in [pg/ml]. Note the
statistically significant difference between the two groups
(*
The results represent the % concentration relative to the mean concentration of each analyte in the control group (dashed line in red). Data are expressed as the mean plus standard error of the mean.
We then examined the correlations between plasma levels of IL-1β, IL-1RA,
IL-5, IL-12(p70), IL-13, IL-17 and GRO-α and clinical variables in the
subjects with ASD. There were no statistically significant correlations between
the plasma levels of analytes and clinical variables, including age, weight,
height, BMI, IQ (full, verbal and performance) and severities in autistic
symptoms as assessed by the ADI-R. When correlation coefficients were evaluated
among the 7 analytes that showed significant elevation in ASD, there were
significant correlations between IL-1β and IL-1RA (Pearson's
In the present study, plasma levels of IL-1β, IL-1RA, IL-5, IL-8, IL-12(p70),
IL-13, IL-17 and GRO-α in the high-functioning male subjects with ASD were
significantly higher than those of carefully matched control subjects. Our
participants with ASD showed no signs or symptoms implying inflammatory diseases and
were similar in parameters of obesity, including BMI, to controls. Thus, it is
likely that the elevations in plasma levels of those analytes were significantly
associated with the diagnosis of ASD. These results are in line with the studies
mentioned above, which reported altered immune responses in individuals with ASD
IL-1β is a pro-inflammatory cytokine produced by various sources, including
monocytes, macrophages, dendritic cells, neutrophil leukocytes and endothelial
cells
IL-5 is mainly produced by T helper 2 (Th2) cells and mast cells, and belongs to
the Th2 cytokine family
IL-12(p70) is a heterodimeric cytokine that consists of two subunits, p35 and p40
Outside the Th1/Th2 cell paradigm, a distinct T helper cell subset that produces
IL-17 has recently been discovered, and is known as the Th17 cell subset
Both IL-8 and GRO-α are chemokines produced by macrophages and other cell
types, such as epithelial and endothelial cells. These chemokines have
chemotactic activity on neutrophils and play important roles in the innate
immune response. Previous studies have examined IL-8 levels in plasma or serum,
and found either increases
There were limitations in the present study. The small sample size renders the data presented here preliminary, and a larger study with more subjects with ASD will be necessary. However, recruitment for the current study was limited to a group of high-functioning subjects with ASD, none of whom were given psychotropic drugs. Therefore, our data are free from possible confounding factors and thus reflect a certain common immunological pathology among people with ASD.
Twenty-eight male subjects with ASD and 28 healthy male controls participated in
this study. All the participants were Japanese, born and living in restricted
areas of central Japan, including Aichi, Gifu, and Shizuoka prefectures. Based
on interviews and available information, including hospital records, diagnoses
of ASD were made by an experienced child psychiatrist (TS) based on the
DSM-IV-TR criteria
Fasting blood samples from all the participants were obtained between 11:00 and noon by venipuncture and collected into EDTA-containing tubes. Immediately after the sampling, samples were centrifuged for 10 min at 4°C, divided into 200-µl of aliquots, and stored at −80°C until use. The mean time interval for preparation of plasma from blood samples was 4.5 min (3 to 6 min). Multiplex kits for measuring cytokines and chemokines were purchased from Bio-Rad (Bio-Plex Pro Human Cytokine Group I [27-plex] and Group II [21-plex] panels; Bio-Rad, Hercules, CA). The kits were used per the manufacturer's instructions. Plasma samples were diluted using the appropriate sample diluents provided in each kit in accordance with the manufacturer's instructions. Concentrations (pg/ml) of different analytes in the plasma samples were determined by using the standard curves generated in the multiplex assays. Each standard curve was generated using eight points of concentrations, and a nonlinear least squares minimization algorithm was used for the curve fitting by the five-parameter logistic equation and to determine the high and low limits of detection. Data points for analytes that were occasionally above or below the detection range were discarded.
Comparisons of concentrations of analytes between subjects with ASD and controls
were made by an unpaired
The authors thank Mses Tae Takahashi, Erina Sakamoto, and Mika Oyaizu for their excellent technical assistance.