Ionomycin – Stenabolic 0

Imbalance between the anti- and pro-inﬂammatory milieu in blood leukocytes of autistic children

a b s t r a c t
Accumulating evidence suggests an association between immune dysfunction and autism disorders in a signiﬁcant subset of children. In addition, an imbalance between pro- and anti-inﬂammatory path- ways has been proposed to play an important role in the pathogenesis of several neurodevelopmental disorders including autism; however, the role of anti-inﬂammatory molecules IL-27 and CTLA-4 and pro-inﬂammatory cytokines IL-21 and IL-22 has not previously been explored in autistic children. In the current study, we investigated the expression of IL-21, IL-22, IL-27, and CD152 (CTLA-4) following an in- vitro immunological challenge of peripheral blood mononuclear cells (PBMCs) from children with autism (AU) or typically-developing children (TD) with phorbol-12-myristate 13-acetate (PMA) and ionomycin. In our study, cells from children with AU had increased IL-21 and IL-22 and decreased CTLA-4 expres- sion on CD4+ T cells as compared with cells from the TD control. Similarly, AU cells showed decreased IL-27 production by CD14+ cells compared to that of TD control cells. These results were conﬁrmed by real-time PCR and western blot analyses. Our study shows dysregulation of the immune balance in cells from autistic children as depicted by enhanced pro-inﬂammatory cytokines, ‘IL-21/IL-22’ and decreased anti-inﬂammatory molecules, ‘IL-27/CTLA-4’. Thus, further study of this immune imbalance in autistic children is warranted in order to facilitate development of biomarkers and therapeutics.

1.Introduction
Autism is a neurodevelopmental disorder characterized by social shortfalls and stereotypical behavioral patterns and is usu- ally diagnosed in children around three years of age (Lord et al., 2000; American Psychiatric Association, 2015). Because there are no consistent biological markers for autism diagnosis is grounded on behavioral traits and developmental history (Le Couteur et al., 2008). The biological basis of autism is unclear but probably associates a relationship between genetic accountability and envi- ronmental exposures (Herbert, 2010). Furthermore, autoimmunity against components of the central nervous system (CNS) might have a role in the pathogenesis of autism (Enstrom et al., 2009; Al-Ayadhi and Mostafa, 2013). The most reliable treatment for the autism is behavioral therapy, which is most advanced beneﬁcial when initiated early in life (Dawson et al., 2012). Evidence of a role of immune system abnormalities in the pathogenesis of autism has recently been reported. Children with a diagnosis of autism present immune dysfunction at a young age (Ashwood et al., 2011; Abdallah et al., 2013; Ahmad et al., 2016). Moreover, immunological dysregu- lations in autisms are complex and may be associated to alterations in the prenatal immune environment, which could result in greater risk of autism (Goines et al., 2011).
Several hypotheses have been suggested that address the role of cytokines in the pathophysiology of autism (AL-Ayadhi, 2005; Ashwood et al., 2008). Correlations amongst the severity of behav- ioral impairments recognized as central diagnostic features of autism and cytokine levels have been described (Ashwood et al.,2008). Interleukin (IL)-21 cytokine secreted by CD4 T cell sub- sets, including natural killer (NK) and Th17 cells (Yi et al., 2010); however, the role of IL-21 in neuroinﬂammatory diseases is rea- sonably unknown. In neuromyelitis optica (NMO) and multiple sclerosis (MS), higher cerebrospinal ﬂuid IL-21 production and polymorphisms in the IL-21R gene involve IL-21 in the autoimmune pathogenesis of these diseases (Wu et al., 2012). Recently, IL-21 was revealed to be important for maintenance of local T cell responses in the CNS (Stumhofer et al., 2013). Furthermore, IL-21 optimizes T cell and humoral responses in the CNS (Phares et al., 2013). In addi- tion, it has been reported that IL-21 is highly upregulated in the injured mouse brain after cerebral ischemia and that IL-21 deﬁ- cient mice have smaller infarcts, better neurological function, and decreased lymphocyte accumulation (Clarkson et al., 2014). How- ever, the role of IL-21 in children with autism has not previously been studied.

IL-22 is known as one of the signature cytokines of Th17 cells, and has been implicated as a key pathogenic cytokine in encephalomyelitis (Liang et al., 2006). It also plays an important role in multiple sclerosis by promoting leukocyte inﬁltration into the brain (Kebir et al., 2007). Th17 cells are deﬁned by the sig- nature transcription factor retinoic acid receptor-related orphan receptor gamma T (RORγT) and have been implicated in autism through both genetic associations and elevated levels of IL-17 (Durant et al., 2010; Ciofani et al., 2012; Suzuki et al., 2011; Al- Ayadhi and Mostafa, 2012). IL-22-secreting Th17 cells have also been shown to be involved in inﬂammatory diseases, such as pso- riasis, asthma, and inﬂammatory bowel disease (Eyerich et al., 2010), and their upregulation often correlates with disease activity (Zenewicz, 2011). However, the presence of IL-22 secreting CD4+ T cells has not been explored in autistic children.IL-27 suppresses immune responses by inhibiting the devel- opment of Th17 cells (Batten et al., 2006a) while inducing IL-10 production in a Stat1 and Stat3-dependent manner (Iwasaki et al., 2015). Moreover, IL-27 RA-deﬁcient mice exhibit exacerbated experimental allergic encephalomyelitis (EAE), which suggests that IL-27 signaling plays an important role in inﬂammatory conditions (Fitzgerald et al., 2007).Cytotoxic T lymphocyte associated gene-4 (CTLA-4) is consti- tutively expressed on T regulatory cells and is a key molecule required for their function (Takahashi et al., 2000; Wing et al., 2008). Mice that show a Treg speciﬁc deﬁciency in CTLA-4 develop severe autoimmune diseases and Treg cells from these mice dis- play decreased suppressive capacity (Wing et al., 2008). Further, CTLA-4 contributes to the maintenance of peripheral tolerance and prevention of autoimmune responses (Hurwitz et al., 2002), and astrocytes have been shown to induce Th cell energy via CTLA- 4 (Gimsa et al., 2004). Nonetheless, to our knowledge there are no studies that have evaluated the anti-inﬂammatory/suppressive functions of IL-27 and CTLA-4 in autism.Keeping these observations in mind, our aim in the present study
was to investigate the pro- and anti-inﬂammatory milieu in periph- eral blood mononuclear cells (PBMCs) from autistic children. To this end, we show increased expression of pro-inﬂammatory cytokines, IL-21/IL-22, and decreased expression of CTLA-4/IL-27 by CD14+ or CD4+ T cells in peripheral blood mononuclear cells (PBMCs) from AU children.

2.Materials and methods
2.1.Participants
This cross-sectional study was conducted using cells from 50 children, who had classic-onset autism, over a period of 6 months from the beginning of June 2015 to the end of November 2015.The autistic group included 50 children (40 males and 10 females) enrolled from the Autism Research and Treatment Center, College of Medicine, King Saud University, Saudi Arabia, Riyadh. Sub- jects were fulﬁlling the standards for the ‘diagnosis of autism’ according to the 5th edition of the Diagnostic and Statistical Man- ual of Mental Disorders (The American Psychiatric Association), (American Psychiatric Association, 2015). Their ages ranged among 3 and 11 years (mean SD = 7.69 2.26 years). Children included in this study had no related neurological diseases such as (tuberous sclerosis and cerebral palsy) or metabolic disorders e.g. (phenylke- tonuria), as these associated comorbidities with autism may impact results. Also, the involved subjects were not getting any medica- tions.The control group included 45 age and sex matched healthy children (10 females and 35 males), which were the healthy older siblings of the healthy infants who attend the College of Medicine, Well Baby Clinic, King Khalid University Hospital, King Saud Univer- sity, Riyadh, KSA, for routine follow-up of their growth parameters. The control children were not associated to the children with autism, and demonstrated no clinical outcomes suggestive of neuropsychiatric or immunological disorders. Their ages ranged between 3 and 11 years (mean SD = 7.76 2.45 years). The King Saud University, College Ethical Committee, Riyadh, KSA, approved this work. In addition, an informed written agreement of participa- tion in this study was signed by the parents or the legal guardians of the studied subjects.

2.2.Study measurements
The clinical assessment of autistic subjects was established on clinical history taken from neuropsychiatric assessment, care- givers, and clinical examination. In addition, the disease severity was evaluated by using the Childhood Autism Rating Scale (CARS) (Schopler et al., 1986), which rates the child on a scale from one to four in each of ﬁfteen areas (relating to people; visual, listening and emotional responses; non-verbal communication; imitation; body and object uses; fear or nervousness; verbal communication; adaptation to change).

2.3.Antibodies and chemicals
The antibodies and chemicals, used in this work were purchased from the following companies. Primary and secondary antibodies against IL-21 and IL-22 used for Western blotting were obtained from Santa Cruz Biotechnology, Inc, USA. Heparin, PMA, and Ionomycin were obtained from Sigma-Aldrich, USA. Fluoroisoth- iocyanate (FITC) labeled CD4 and CD14 anti-human monoclonal antibodies, and phycoerythrin (PE) labeled IL-22, IL-27 and CD152 (CTLA-4) and allophycocyanin (APC) labeled anti-IL-21 anti-human monoclonal antibodies, GolgiStop, FcR blocking reagent, Brefeldin A, RBC lysing, permeabilizing and ﬁxation buffers were purchased from (BD Biosciences and BioLegend, USA and Miltenyi Biotech, Germany). The primers were used to evaluate gene expression were obtained from (Genscript, Piscataway, USA). The SYBR® Green PCR master mix and high capacity cDNA reverse transcription kit were obtained from (Applied Bio Systems, Paisley, UK). TRIzol was obtained from (Life Technologies, Grand Island, USA).

2.4Isolation of peripheral blood mononuclear cells (PBMCs)
Blood was collected in an acid-citrate-dextrose Vacutainer tubes (BD Biosciences, USA) and handled for ﬂowcytometry, protein and gene expression analyses. Blood was mixed 1:1 with Hank’s Bal- anced Salts-Solution (Gibco, Gaithersburg, USA), and the diluted peripheral blood was then sensibly layered over a Ficoll-Paque(Sigma-Aldrich, USA) and centrifuged at 400 g for 50 min at 25 ◦C (Careaga et al., 2014).

2.5.Intracellular staining and ﬂow cytometric determinations
Peripheral Blood Mononuclear Cells for ﬂow cytometry was incubated with GolgiStop immediately after addition of phorbol- 12-myristate 13-acetate (PMA)/ionomycin (10 µg/ml) for 4 h at 37 ◦C. The expression of cell surface receptors, and cytokines were assessed by intracellular staining with the following antibodies: Fluorescein isothiocyanate (FITC)-conjugated anti-CD4 and anti- CD14. PBMCs were directly pipetted into a FACS tubes containing 20 µl of monoclonal antibodies against CD4, CD14 and CD152 cell surface antigens. After washing with wash buffer the 20 µl, anti-IL- 21, anti-IL-22 and IL-27 antibodies were added and incubation for 30 min. The lymphocytes were ﬁrst gated by their physical prop- erties (Ahmad et al., 2015). All the measurements were done on a Beckman Coulter ﬂow cytometer with CXP software application.

2.6.RNA isolation and real-time quantitative RT-PCR analysis
Total RNA was extracted from PBMCs using Trizol reagent (Life Technologies, Grand Island, USA) as previously reported (Sthoeger et al., 2013). High-capacity cDNA reverse transcription kit (Applied Biosystems, USA) for initiating cDNA synthesis according to the manufacturer’s instructions. The primers used in these assays were selected from PubMed and other databases, GAPDH expression served as an internal
5r-GGGATGACACCTGATTGGGG-3r. The RT-PCR data were ana- lyzed using the relative and comparative gene expression (i.e.,
∆∆CT) method. The data were presented as the fold change in gene expression normalized to housekeeping gene (GAPDH) (Abd-Allah et al., 2014).

2.7.Total cellular protein extraction from PBMCs for Western blotting
The total proteins were isolated from PBMCs as previously described method (Chen et al., 2007). Total protein concentra- tions were measured by the Lowry method (Seevaratnam et al., 2009). Brieﬂy, 20–40 µg of total protein from each sample was sep- arated by 10% SDS-polyacrylamide gel electrophoresis (PAGE) and electrophoretically transferred onto a PVDF membrane (Bio-Rad Laboratories, Inc, UK). Membrane were blocked overnight at 4 ◦C, followed by incubation with primary antibodies against IL-21 or IL-22 (Santa Cruz Biotechnology, USA), followed by incubation for 1 h with peroxidase-conjugated secondary antibodies at 25 ◦C. The IL-21 and IL-22 bands were visualized using the luminata Forte Western HRP substrate (Millipore, Billerica, USA) and quantiﬁed relative to β-actin.

2.8.Statistical analyses
The data are expressed as mean standard deviation (SD). Com- parison between two groups was conducted by Student’s t-test. The level of statistical signiﬁcance was set at p < 0.05 for differences between the groups. All the statistical analyses were performed using the Graph Pad Prism statistical package. 3.Results 3.1.Altered cell surface receptor expression and cytokine induction in cells from autistic children The characteristics of the study subjects are revealed in Table 1. The frequencies of autoimmune diseases among families of chil- dren with autism were signiﬁcantly higher than healthy controls. Recently, IL-21 was displayed to be important for maintenance of T cell responses in the CNS (Stumhofer et al., 2013). We wanted to explore whether changes in IL-21 cytokine production were also associated with the neurodevelopment of autism. Fig. 1A demon- strates that there is a signiﬁcant increase in the number of IL-21+ cells in samples from children with AU compared to those from the TD controls. Further, we observed an increased number of CD4+ T cells secreting IL-21 in the samples from children with AU (Fig. 1A). We next assessed IL-21 mRNA expression in PBMCs and found that cells from children with AU exhibited an upregulation of IL-21 expression compared to the TD control (Fig. 1B). In addition, west- ern blot analysis conﬁrmed the strong induction of IL-21 protein expression in PBMCs from AU children as compared to those from TD children (Fig. 1C). Therefore, these ﬁndings suggest that there may be a link between IL-21 expression and the pathogenesis of autism.Previous studies have indicated that IL-22 plays a role in the regulation of autoimmune neuroinﬂammatory diseases; however, the role of this cytokine has not previously been studied in autistic disorders. We found that there was a signiﬁcant increase in the percentage of IL-22+ and CD4+IL-22+ cells after PMA/ionomycin stimulation of cells from children with AU compared to the TD control cells (Fig. 2A). Moreover, PBMCs from children with AU exhibited a signiﬁcant increase in IL-22 mRNA expression as determined by quantitative RT-PCR analysis (Fig. 2B). We fur- ther characterized IL-22 protein expression levels by western blot analysis of PBMCs from children with AU and found a signiﬁcant upregulation of IL-22 as compared to the TD control (Fig. 2C). Overall, this data suggests that IL-22 may contribute to the neu- roinﬂammatory processes of autism. Anti-inﬂammatory signaling was also explored in the samples from autistic children as these pathways could be activated to counteract the pro-inﬂammatory pathways. For this purpose, we assessed the expression of IL-27 and CD152 (CTLA-4) in samples from children with AU and in TD controls. Our results indicated that IL-27 production was signiﬁcantly decreased in cells from chil- dren with AU compared to TD controls (Fig. 3A). Fig. 1. A Flowcytometric analysis of intracellular IL-21 cytokine production by all cells and speciﬁcally by CD4+ T cells in the PBMCs (peripheral blood mononuclear cells) from typically-developing children (TD) and children with autism (AU). (B) Quantitative RT-PCR of IL-21 gene expression in the PBMC. (C) Western blot analysis of IL-21 protein expression in the PBMCs. (D) Dot plots show events from PBMCs taken from one representative AU patient and from one control patient. Statistical analyses were performed using Student’s t-test. All data in ﬁgures are presented as the mean ± standard deviation (SD) of children with autism (n = 50) and control group (n = 45). The level of statistical signiﬁcance was set at *p < 0.05 compared to TD controls (Fig. 3A), and IL-27 mRNA expression was also lower in the AU samples (Fig. 3B).Fig. 4A demonstrates that there was a signiﬁcant decrease in the number of CTLA-4+ cells in the samples from children with AU compared with the TD controls. Further, we observed a decrease in the number of CD4+ and CTLA-4+ double positive cells in the children with AU as compared to TD control (Fig. 4A). Therefore, these results provide evidence of the dysregulation of anti-inﬂammatory/immune suppressive signals, such as IL-27 and CTLA-4, in autistic children. Fig. 2. A Flowcytometric analysis of intracellular IL-22 cytokine production by all cells and speciﬁcally by CD4+ T cells in the PBMCs from typically-developing children (TD) and children with autism (AU). (B) Quantitative RT-PCR of IL-22 gene expression in the PBMC. (C) Western blot analysis of IL-22 protein expression in the PBMCs. (D) Dot plots show events from PBMCs taken from one representative AU patient and from one control patient. Statistical analyses were performed using Student’s t-test. All data in ﬁgures are presented as the mean ± standard deviation (SD) of children with autism (n = 50) and control group (n = 45). The level of statistical signiﬁcance was set at *p < 0.05. 4.Discussion Autism is a range of complex neurodevelopmental condi- tions principally characterized by dysfunctions linked to mental development (Abrahams and Geschwind, 2008). Immune system dysfunction and overexpression of proinﬂammatory mediators have been connected with several neuropsychiatric disorders, such as schizophrenia and depression, as well as neurodegenerative dis- eases including Parkinson’s and Alzheimer’s (Borsini et al., 2015). Recent studies have revealed that there are more than 1000 highly Fig. 3. A Flowcytometric analysis of intracellular IL-27 cytokine production by all cells and speciﬁcally by CD14+ T cells in the PBMCs from typically-developing children (TD) and children with autism (AU). (B) Quantitative RT-PCR of IL-27 gene expression in the PBMC. (C) Dot plots show events from PBMCs taken from one representative AU patient and from one control patient. Statistical analyses were performed using Student’s t-test. All data in ﬁgures are presented as the mean ± standard deviation (SD) of children with autism (n = 50) and control group (n = 45). The level of statistical signiﬁcance was set at *p < 0.05 interconnected genes expressed mainly in the brain, which are likely involved in autism (Moreno-Ramos et al., 2015). There are also various reports of peripheral immune abnormalities in autis- tic individuals (Onore et al., 2012). Immune abnormalities reliable with a dysregulated immune response that have thus far been reported in autistic children include abnormal or skewed Th1 and Th2 cytokine proﬁles, reduced lymphocyte numbers, and the imbal- ance of serum immunoglobulin levels (Al-Ayadhi and Mostafa, 2012; Ashwood et al., 2006).Inﬂammation plays a key role in many neurological diseases as well as in neurodegenerative diseases characterized by cognitive and behavioral impairments. There are several reports of cytokine imbalance in autism and these imbalances could have a pathogenic role or might be markers of underlying genetic and environmen- tal inﬂuences (Goines et al., 2011). While cytokines act primarily as mediators of immunological activity, they also have signiﬁcant interactions with the CNS (Goines et al., 2011). In fact, it has been suggested that the synthesis or transport of cytokines in the brain may contribute to neuroinﬂammation and possibly to neurotrans- mitter imbalances in autism (Cohly and Panja, 2005). Furthermore, the presence of IL-21+ cells in human brain tissue during differ- ent neuroinﬂammatory conditions was recently reported (Tzartos et al., 2011). Our study shows that IL-21 expression is greatly increased in the PBMCs of children with autism. Since IL-21 is a pleiotropic pro-inﬂammatory cytokine, its upregulation may lead to activation of inﬂammatory pathways. Moreover, IL-21 is known to be involved in the differentiation of Th17 cells, which have been implicated as a major player in neurodevelopmental disorders (Geri et al., 2011). The upregulation of IL-21 in autism may play a role in the modulation of behaviors and core features of autism. Therefore, IL-21 may signify a link between immune and neuronal dysfunc- tion in autism. Previously, children with autism were shown to have signiﬁcantly higher serum IL-17A levels than healthy controls (Al-Ayadhi and Mostafa, 2012; Mandal et al., 2011).IL-22, a strong proinﬂammatory cytokine, is initiate in many settings of acute and chronic inﬂammation, and is known to affect the hypothalamic function (Chen et al., 2015). In addition, IL-22 is expressed in the brain and spinal cord (Levillayer et al., 2007), and has been reported to affect the integrity of the blood brain barrier allowing lymphocyte ingress into the CNS. We found increased IL-22 expression in the PBMCs of children with autism. This increase may be the result of increased IL-21, as it is known to lead to enhanced proliferation/activation of Th17 cells. There- fore, our results suggest that induction of IL-21/IL-22 in PBMCs may be associated with the pathogenesis of autistic disorder, and could signify a link between immune and neuronal dysfunction in Fig. 4. A Flowcytometric analysis of CTLA-4 production by all cells and speciﬁcally by CD4+ T cells in the PBMCs from typically-developing children (TD) and children with autism (AU). (B) Dot plots show events from PBMCs taken from one representative AU patient and from one control patient. Statistical analyses were performed using Student’s t-test. All data in ﬁgures are presented as the mean ± standard deviation (SD) of children with autism (n = 50) and control group (n = 45). The level of statistical signiﬁcance was set at *p < 0.05. autism. However further investigations are required to delineate IL-21/IL-22 related downstream cellular signaling.CTLA-4 and IL-27 play important roles in immunological sig- naling through modulation of T cell activation. Further, increasing evidence suggests that neuroinﬂammation is modulated by the interaction between T cells and antigen-presenting cells, such as microglial cells, in the CNS (Ziv and Schwartz, 2008). Our ﬁndings show a marked decrease in CTLA-4 and IL-27 expression in cells from children with autism compared with controls. CTLA-4 plays an important role in regulating peripheral T cell tolerance and is recognized one of the most effective negative regulators of T cell activation (Santos et al., 2014). Moreover, CTLA-4 deﬁcient mice develop a severe lymphoproliferative disorder (Takahashi et al., 2000), and decreased CTLA-4 expression has been reported in the frontotemporal lobe of dementia patients (Santos et al., 2014). Deﬁ- ciency in IL-27 has also been reported to lead to aggravation of multiple sclerosis in a mouse model (Batten et al., 2006b). More- over, in this model of multiple sclerosis, IL-27 RA-deﬁcient mice exhibit an increase in the frequency of CNS-inﬁltrating IFN-γ+ and IL-17+CD4+ T cells (Mascanfroni et al., 2013). Together these resultssuggest that peripheral immune suppression may be associated with autism in children and further study is warranted. Our study focused on cytokines/biomolecules with pro- and anti-inﬂammatory nature in PBMCs. Moreover, our study did not explore cause and effect relationship of study parameters with the disease outcome. The cytokines/biomolecules investigated in this study function through different mechanisms which could be through JAK-STAT or co-receptor interaction signaling. This is an interesting area for future research. Some of the downstream pathways of these cytokines have already been elucidated in our recent publication (Ahmad et al., 2016). Our future work is directed at further delineating speciﬁc cytokine induced cellular signaling in different immune cells such as CD4+/CD8+ T cells or mono- cytes/neutrophils in autistic children. 5.Conclusions Overall, our results indicate enhanced peripheral inﬂamma- tion and suppressed anti-inﬂammatory pathways in PBMCs from autistic children, which may play a role in the modulating the development and progression of this disorder. In this study, we provide the evidence of the immune dysfunction in autism with implications for the identiﬁcation Ionomycin of possible novel biomarkers or therapeutic targets, and therefore, our ﬁndings further the under- standing of this neurodevelopmental disorder.