Recruitment of inflammatory cells plays the key pathogenic role in all inflammatory and autoimmune diseases. Inflammation is characterized by the local tissue expression of chemokines, a large group of chemotactic cytokines, which play an important pathogenic role in inflammatory diseases by enhancement of leukocyte recruitment and activation at inflammatory sites [1–4].

CCR4 is a receptor that binds two 8 kDa CC chemokines, macrophage-derived chemokine (MDC/CCL22) and thymus and activation-regulated chemokine (TARC/CCL17) [5]. MDC is synthesized by cells of macrophage lineage, while TARC is expressed in the thymus. The gene encoding both chemokines is clustered on chromosome 16q13 [6–8]. CCR4 ligands, MDC and TARC, act on their CCR4 receptors to enhance the recruitment and activation of T helper (Th) 2 cells with a subsequent production of type 2 cytokines that include interleukin-4 (IL-4), IL-5, IL-9 and IL-13 [9, 10]. The Th2-associated chemokines, MDC and TARC, were the highest at birth and then decreased with age [11]. CCR4 ligands have an important pathogenic role in inflammatory conditions such as allergy and some autoimmune diseases [12–18]. Chemokines and their receptors have been implicated as functional mediators of immunopathology of autoimmune neuroinflammatory diseases [19, 20].

The presence of autoantibodies to neural tissues/antigens in autism [21–28] and the increase in the frequency of autoimmune disorders among autistic families [29–33] suggest that autoimmunity may play an important role in the pathogenesis of autism [21].

This study aimed to measure serum levels of CCR4 ligands in relation to the degree of the severity of autism in a group of autistic children.

Autistic children had significantly higher serum levels of MDC (median (IQR) = 1289.5 (1079) pg/ml) than healthy controls (median (IQR) = 190 (295) pg/ml), P <0.001 (Figure 1). Similarly, autistic children had significantly higher serum levels of TARC (median (IQR) = 880.5 (1206) pg/ml) than healthy children (median (IQR) = 137.25 (173) pg/ml), P <0.001, (Figure 2).

There were significant positive correlations between serum levels of MDC and TARC in autistic children, P < 0.001 (Figure 3).

Serum levels of both MDC and TARC had no significant correlations with the age of the children with autism (P = 0.87 and P = 0.47), respectively. In addition, there was no significant difference between male and female autistic children in serum levels of both MDC (P >0.05) and TARC (P >0.05).

All study subjects had normal weight (body mass index (BMI) was between the 5th and less than the 85th percentiles based on age and sex). There was no significant difference between the BMI of healthy children and autistic children (P >0.05). In addition, there were no significant correlations between the BMI and serum levels of both MDC (P >0.05) and TARC (P >0.05) in children with autism.

CCR4 ligands direct trafficking and recruitment of Th2 cells [9, 10] and they were reported to play an important role in some autoimmune diseases [15–18]. In the present study, autistic children had significantly higher serum levels of MDC and TARC than healthy controls (P <0.001 and P <0.001, respectively). A previous study reported increased MDC and TARC in the anterior cingulate gyrus of autistic patients [36]. Proinflammatory chemokines, such as TARC and monocyte chemotactic protein-1 (MCP-1) along with proinflammatory cytokines were reported to be elevated in the brains of some individuals with autism.

The transport or synthesis of cytokines in the brain may contribute to neuroinflammation and possible neurotransmitter imbalances in autism [21]. Abnormal immune responses, as assessed by multiplex analysis of cytokines and chemokines, may serve as one of the biological trait markers for autism [37]. Active neuroinflammatory process in the cerebral cortex, white matter, and notably in cerebellum of autistic patients was reported. Immunocytochemical studies showed marked activation of microglia and astroglia, and cytokine profiling indicated that macrophage chemoattractant protein-1 and tumor growth factor-beta1, derived from neuroglia, were the most prevalent cytokines in brain tissues. Cerebrospinal fluid (CSF) showed a unique proinflammatory profile of cytokines, including a marked increase in MCP-1 [36].

Elevated levels of chemokines have been detected in the brain and CSF of individuals with autism. Few studies have examined chemokine levels in the plasma of children with autism, but these studies did not correlate the plasma levels of chemokines with the disease severity. Elevated plasma MCP-1, RANTES and eotaxin in some autistic children and their association with more impaired behaviors may have etiological significance. Chemokines and their receptors might provide unique targets for future therapies in autism [38]. Another study reported decreased plasma levels of chemokines involved in hematopoiesis and the Th cell immune system in the children with autism compared with unrelated siblings without autism. The investigators of this study recommended further studies to confirm immunological disturbances influencing hematopoiesis and antibody production in the children with autism [39]. An increased risk for infantile autism with elevated MCP-1 in amniotic fluid was recently reported. Elevated levels of MCP-1 may play an indirect role in the pathophysiology of autism [40].

This study is the first to investigate serum levels of CCR4 ligands in relation to the degree of the severity of autism, which was assessed by using CARS. In the present work, children with severe autism had significantly higher serum levels of MDC than patients with mild to moderate autism (P <0.001 and P = 0.01, respectively). There were significant positive correlations between the values of CARS and serum levels of both MDC (P <0.001) and TARC (P <0.001) in autistic children. This may indicate that the extent of the elevation of CCR4 ligands was closely linked to the degree of the severity of autism. However, it is not easy to determine whether this increase is a mere consequence of autism or has a pathogenic role in the disease. Further research is warranted to determine the pathogenic role of CCR4 ligands and their relation to serum levels of brain-specific autoantibodies in autistic children.

CCR4 ligands have many cellular sources including monocyte-derived dendritic cells, keratinocytes and bronchial epithelial cells [41–43]. MDC is also produced by natural killer cells and macrophages [44]. Early signals elicited during innate immune response, such as Th2-type cytokines and bacterial products, induce the rapid secretion of CCR4 [45, 46]. The current study revealed significant positive correlations between serum MDC and serum TARC levels in autistic children (P <0.001). This could be explained by the fact that MDC is produced concomitantly with TARC during immune inflammation. Both chemokines are ligands to the same receptor, which is characterized on Th2 cells [9] resulting in the activation and long-term recruitment of Th2 cells [47] and subsequently in the pathomechanism of some autoimmune diseases, including autism [15–17].

Both Th2 chemoattractants, MDC and TARC, were reported to be elevated and play a role in some autoimmune diseases such as systemic lupus erythematosus [15] and systemic sclerosis [16]. In addition, MDC levels were reported to be elevated in the CSF of female patients with multiple sclerosis (MS). MDC was suggested to play a role in the development of MS possibly by influencing the intracerebral recruitment of Th2 cells which express CCR4 receptors [17]. Furthermore, MDC plays a role in experimental autoimmune encephalomyelitis (EAE), the animal model of MS. MDC/CCL22 gene is a part of a chemokine cluster, which includes also TARC/CCL17. The frequency of the C/T and C/A single nucleotide polymorphisms (SNPs) in the promoter and coding sequence of CCL22 as well as the C/T SNP in the promoter of CCL17 were determined in 370 patients with MS compared with 380 controls. A trend toward a decreased allelic frequency of the A allele of the CCL22 C/A SNP as well as of the T allele of the CCL17 C/T SNP was found in patients compared with controls [19]. Similar genetic studies of MDC/CCL22 gene are recommended in children with autism.

The immunomodulatory effects of CCR 4 ligands may act not only through modulation of Th cell function, but also through decreasing the induction of CD4 + CD25high regulatory T cells (T-regs) and the promotion of the differentiation of Th17 cells [48]. Th17 cells have been implicated in the pathogenesis of autoimmune diseases, which is supported by recent clinical trials using anti-IL-17 in the treatment of these diseases. Th17 cells are characterized by a strong IL-17-producing capacity. IL-17 family cytokines control the inflammatory responses by triggering the secretion of proinflammatory cytokines and chemokines [49, 50]. In addition, T-regs play an important role in the establishment of the immunological self-tolerance and thereby, prevent autoimmunity as T-regs suppress Th17 cells [51]. One study reported deficiency of T-regs in 73.3% of autistic children [32]. Another studies reported significantly higher plasma concentrations of IL-17 [37, 52] and some of Th2 cytokines, such as IL-5 and IL-13, in autistic children compared to healthy matched controls [37, 38].

Previous studies reported the possible pathogenic role of Th2 cells, Th17 cells and deficiency of T-regs in the induction of autoimmunity in a subgroup of patients with autism [32, 35, 36, 50]. The results of this study may indicate the possible contributing role of CCR4 ligands in the induction of autoimmunity in autism, possibly as a result of long-term recruitment of Th2 cells, suppression of the induction of T-regs and enhancement of the differentiation of Th17 cells. However, this is an initial report that cannot prove the pathogenic role of CCR4 ligands in autism, but rather raises additional questions. So, these data should be treated with caution until further investigations are performed. Studies should be conducted to investigate the relationship between serum levels of CCR4 ligands and both T-regs and Th17 cells in children with autism.

Decreasing chemokine receptor binding may be an important potential therapeutic target in allergy and immunology [4]. This can be performed by using small molecule chemokine receptors antagonists or by using blocking antibodies [53]. Thus, studies concerning the effect of CCR4-ligand antagonism on amelioration of autistic manifestations in children are mandatory.

Serum levels of CCR4 ligands were elevated in autistic children and they were significantly correlated to the degree of the severity of autism. However, further research is warranted to determine the pathogenic role of CCR4 ligands in autism and to shed light on the therapeutic role of CCR4-ligand antagonism in autistic children.