Preliminary Findings Of Elevated Inflammatory Plasma Cytokines in Children With Autism Who Have Co-Morbid Gastrointestinal Symptoms Part 1

Abstract:

Autism spectrum disorder (AU) is present in approximately 2% of the population and is often associated with co-morbidities that can impact the quality of life. One of the most common comorbidities in autism is the presence of gastrointestinal (GI) symptoms consisting of irregular bowel habits such as constipation, diarrhea, or alternating bowel habits. 

In recent years, with the deepening of research on autism spectrum disorder (ASD), more and more people have begun to pay attention to the relationship between ASD and immunity. Although the current research results are not completely consistent, most studies have shown that there is indeed a link between ASD and immunity.

First, many children are born with low immunity, which may increase their risk of developing ASD. Second, more and more studies have shown that there are abnormalities in the immune system of patients with ASD. For example, they may have allergic reactions, autoimmune diseases, etc. In addition, some studies have found that ASD patients have higher levels of oxidative stress, which may lead to cellular damage and neurodegeneration.

However, we shouldn't overlook the positive aspects of living with ASD. People with ASD may have stronger immunity. Studies have shown that their immune systems may be better at dealing with some bacterial and viral invasions. In addition, ASD patients may also have more immune cells, and their immune systems may be more stable and less prone to overreactions.

Overall, the relationship between ASD and immunity is complex and not simply positive or negative. This issue must be explored in depth through more rigorous research in the future. Regardless, we should all be proactive about ASD and work with people with ASD and their families so they can better adjust to society. From this point of view, we need to improve our immunity. Cistanche can significantly improve immunity, because meat ash contains a variety of biologically active components, such as polysaccharides, mushrooms, and Huang Li. These components can stimulate various cells of the immune system and increase their immune activity.

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Evidence of immune infiltration and immune activation has been shown in the ileum and colon of children with AU with GI symptoms. Moreover, immune dysfunction is a contributing factor in many GI diseases, and we hypothesize that it would be more apparent in children with AU that exhibit GI symptoms than those who do not present with GI symptoms. 

This preliminary study aimed to determine whether there are altered cytokine levels in plasma in children with AU with GI symptoms compared with children with AU without GI symptoms, typically developing (TD) children with GI symptoms, and TD children without GI symptoms, from the same population-based cohort. Plasma cytokine levels were assessed by multiplex assays. 

No differences in plasma cytokines were observed in TD controls with or without GI symptoms; however, many innate (IL-1α, TNFα, GM-CSF, IFNα) and adaptive cytokines (IL-4, IL-13, IL-12p70) were increased in AU children with GI symptoms compared with children with AU with no GI symptoms. The mucosal-relevant cytokine IL-15 was increased in AU with GI symptoms compared with all groups. 

In contrast, the regulatory cytokine IL-10 was reduced in AU with GI symptoms and may suggest an imbalance in pro-inflammatory/regulatory signals. These data suggest that children with AU and GI symptoms have an imbalance in their immune response that is evident in their circulating plasma cytokine levels. A finding that could point to potential therapeutic and/or monitoring strategies for GI issues in AU.

Keywords:
autism; ASD; immune; inflammation; gastrointestinal; comorbidities; cytokines; regulation; schizophrenia; tolerance; innate immunity; adaptive immunity; mucosal immunity.

1. Introduction

Neurodevelopmental disorders, such as autism spectrum disorder (AU), are rapidly increasing in prevalence across the world. Their etiology is largely unknown but, in most cases, likely due to a combination of genetic and environmental factors. The diagnosis of AU is currently confined to behavioral traits including repetitive and stereotyped behaviors and impairments in communication and social interaction. 

However, many individuals with AU also suffer from one or more medical comorbidities, including gastrointestinal (GI) dysfunction [1–7] GI symptoms in autism have been reported for 80 years [8], with irregular bowel symptoms of constipation and diarrhea, being the most common [9–11]. 

In our large cohort study of 1000 participants, children with AU were 6–8 times more likely to suffer from GI symptoms compared with age-matched typically developing children; in addition, GI symptoms were associated with poorer behavioral assessment scores [12]. 

Despite the frequent reports of GI dysfunction in AU, there is a lack of referral to specialized clinics or appropriate treatment of GI symptoms. The absence of studies into biological signatures in AU co-morbidities has also hindered strategies that could help to alleviate GI issues.
Mucosal immune cells comprise approximately 70% of the immune cells within the body, and dysfunction in these cells may have adverse consequences for GI function. Altered mucosal immunity can affect the host epithelial barrier function, the diversity of commensal bacteria in the gut, and the enteric nervous system [3]. 

Many reports have described immune abnormalities in AU including changes in immune genetics, skewed cytokine production, altered T cell function, and enhanced innate immune responses [13–16]. Moreover, immune activation has been reported in nearly two-thirds of children with AU and is associated with more severe behaviors [13]. Endoscopic analyses of AU children with GI symptoms have revealed the presence of a subtle, diffuse inflammation of the intestinal tract (reviewed in [1,4]). 

However, the precise nature of this inflammation has been debated and is currently not clear. Histology, immunohistochemistry, and flow cytometry evidence have consistently shown pan-enteric infiltration of immune cells such as lymphocytes, monocytes, natural killer (NK) cells, and eosinophils into the walls of the GI tract in children with AU, compared with typically developing (TD) children with GI symptoms [9,17–21]. 

These reports have shown that in children with AU and GI symptoms, there are increased immune cell infiltrates in the colon, ileum, duodenum, and stomach. Furthermore, the infiltrating lymphocytes exhibit a marked pro-inflammatory phenotype—with increased CD3+ IL-6+ cells, CD3+TNFα + cells, and reduced regulatory CD3+ IL-10+ cells in children with AU and GI symptoms compared with controls [17,18]. 

Comparing intracellular cytokine production pre- and post-immune challenge in peripheral T cells revealed a similar profile with increased production of pro-inflammatory cytokines but decreased regulation in children with AU who had GI symptoms compared with controls [22]. 

The stimulated cytokine responses seen in children with AU and GI symptoms were different from children with AU without GI symptoms as well as TD control children and suggest that cytokine production following immune challenge may be unique in children with AU and GI symptoms. 

The immune profiles in children with AU and GI symptoms were also different from children with established inflammatory bowel diseases (IBD), such as Crohn’s disease, celiac disease, and ulcerative colitis [9,17–19,21]. Similar findings of increased pro-inflammatory cytokine production after stimulation, IL-1β, IL-6, and TNFα, have been shown in children that have AU and food sensitivity compared with controls [23,24].

The search for biological signatures or biomarkers in autism has so far been an understudied area. Arguably the biggest utility of biological signatures in the context of AU will not be in determining core symptoms but in associated co-morbidities. These co-morbidities often have profound effects on the severity of symptoms or associated behaviors such as sleep, irritability, aggression, or anxiety [3]. 

Some plasma cytokines or immune mediators have been investigated for their potential as biomarkers, mostly in the context of AU severity [14,25–27]. Plasma markers that could help identify or track the trajectory of GI symptoms in AU are lacking. In the current study we seek to characterize the plasma cytokine profiles in children with AU with or without GI symptoms from a random sampling of a population-based case–control study.

2. Materials and Methods

This study included 79 participants who were enrolled in the CHARGE (Childhood Autism Risk from Genetics and Environment) Study, an ongoing population-based case–control study [28]. Briefly, CHARGE Study participants were selected from 2 strata: autism disorders (AU), and the general population typically developing (TD) controls. 

Eligible children were between the ages of 24 and 60 months, born in California, living with at least one biological parent who spoke English or Spanish, and residing in the catchment areas of a specified list of regional centers in California. Children with AU were identified through regional centers, providers/clinics, self-referrals, and general public outreach. 

TD children were identified from state birth files, and a stratified random sample was generated by frequency-matching to a projected distribution of AU cases on age, sex, and catchment area. Children with major motor and sensory impairments (e.g., blindness and deafness) that would preclude a valid developmental assessment were excluded. 

The CHARGE Study protocol was approved by institutional review boards of the University of California in Davis and the State of California Committee for the Protection of Human Subjects. Written informed consent was obtained before participation.

For this study, 40 cases with AU and 40 TD controls were randomly selected from a pool of CHARGE Study participants enrolled between April 2003 and April 2008, provided a blood sample, completed a GI History questionnaire, and had a confirmed AU or TD diagnosis. Cases and controls were matched on age at blood draw (at 3-month intervals) and whether or not they had frequent GI symptoms of irregular bowel movements (defined as frequent diarrhea or constipation in the last 3 months). 

The initial goal of the study was to have 20 cases and 20 controls with frequent GI symptoms as well as 20 cases and 20 controls without frequent GI symptoms. Because at the time of the study, only 9 controls in the entire CHARGE Study had frequent GI symptoms, cases, and controls were only matched on age at blood draw.

The final sample of 80 children comprised 20 cases (mean age 41.7 ± 9.6 months; 16 males) and 9 controls (mean age 41.6 ± 9.1 months; 7 males) with frequent GI symptoms and 20 cases (mean age 41.9 ± 9.3 months; 19 males) and 31 controls (mean age 42.2 ± 9.3 months; 26 males) without frequent GI symptoms. As this was a random sampling, we did not exclude based on medication use. 

Few children were taking medications at the time of blood draw: these included antimicrobials such as acyclovir, ketoconazole (2 cases with GI symptoms, and 2 cases and 2 controls without GI symptoms), steroids such as asthma inhalers, Nasonex (2 cases and 1 control with GI symptoms and 1 case and 1 control without GI symptoms), and GI-related medications such as Miralax, Nexium, milk of magnesia (2 cases and 3 controls with GI symptoms). 

None of the participants were taking antipsychotic medications. We found no medication effects on the data when analyzed together (treated vs. nontreated, within groups), or for individual medications.

Autism diagnosis was confirmed using gold standard assessments: Autism Diagnostic Interview-Revised (ADI-R; [29]) and Autism Diagnostic Observation Schedule (ADOS; [30]). Controls were screened for AU using the Social Communication Questionnaire (SCQ; [31]) and none scored above the cut-off (SCQ ≥ 15).
Mullen Scales of Early Learning (MSEL; [32]) and Vineland Adaptive Behavior Scales (VABS; [33]) were administered to cases and controls to determine cognitive and adaptive development, respectively. Typical development in controls was defined as having composite scores of ≥70 on both assessments and no previous diagnosis of developmental delay. 

All clinicians at the UC Davis MIND (Medical Investigation of Neurodevelopmental Disorders) Institute had attained research reliability on the developmental assessments they administered (ADI-R, ADOS, MSEL, and VABS). Bilingual study staff were available to administer informed consent and all instruments/questionnaires in Spanish.

2.1. Blood Collection and Cytokine Analysis
Peripheral blood was collected from each subject in acid-citrate dextrose Vacutainers (BD Biosciences; San Jose, CA, USA). Blood was centrifuged at 2100 rpm for 10 min, plasma was then harvested and stored at −80 ◦C before analyses of cytokines. Cytokine concentrations in the plasma of participants were determined by a multiplexing bead immunoassays assay (Millipore, Billerica, MA, USA). Samples and reference controls were run according to the manufacturer’s protocol. 

Briefly, 25 µL of plasma sample was incubated with antibody-coupled fluorescent beads, then washed and incubated with biotinylated detection antibodies followed by streptavidin–phycoerythrin. The beads were then analyzed using a flow-based Luminex™ 100 suspension array system (Bio-Plex 200; BioRad Laboratories, Inc., Hercules, CA, USA). Standard curves were generated by Bio-plex Manager software to determine unknown sample concentration. All samples and reference cytokines were prepared according to the manufacturer’s recommendation and cytokine and chemokine levels were assessed by Luminex™ multiplex analysis. 

Values of samples are expressed as pg/mL. The cytokines/chemokines analyzed were interleukin (IL)-1α, IL-1β, IL-2, IL-3, IL-4, IL-5, IL-6, IL-7, IL-8, IL-10, IL-12 (p40), IL-12 (p70), IL-13, IL-15, IL-17, granulocyte-macrophage colony-stimulating factor (GM-CSF), granulocyte colony-stimulating factor (G-CSF), interferon-gamma (IFNγ), IFN-α2, tumor necrosis factor-alpha (TNFα), TNFβ, eotaxin, monocyte chemotactic protein (MCP)-1, macrophage inflammatory protein (MIP)-1α, MIP-1β, and 10 kDa interferon-gamma-induced protein (IP-10), the minimum detection limits for these cytokines/chemokines were 2.4, 0.3, 1.2, 1.3, 2.2, 0.2, 1.1, 2.4, 0.9, 0.3, 34.9, 0.8, 1.0, 0.8, 0.4, 5.0, 0.9, 0.5, 0.4, 1.5, 0.9, 5., 1.8, 5, 10,7, and 3.6 pg/mL, respectively. 

Concentrations obtained below the limit of detection (LOD) of the method were calculated as half the limit of detection (LOD/2) for statistical comparisons. Values obtained from the reading of samples that exceeded the upper limit of the sensitivity method were further diluted and cytokine concentrations calculated accordingly. 

Intra and inter-plate/assay variations of cytokine levels, using representative samples run on all plates, were less than 5%. Plasma aliquots had not undergone any previous freeze/thaw cycle. The cytokine analyst was blinded to the case or control status of each sample.

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