Symptom or cause? Autism and immune dysfunction

There are tantalising clues that anomalies in immune function may be related to autism spectrum disorders. In unpicking this connection, one lab needed a new approach to the immune assay… 

Autism Spectrum Disorders (ASD) are neurodevelopmental diseases characterised by difficulty with social interaction, delayed and sometimes deficient language skills, and repetitive behaviors. The behavioural effects of ASD have the potential to drastically impact the lives of patients. Currently, there are no reliable biomarkers for diagnosing ASD. Rather, diagnosis relies on behavioural observation and assessment. Similarly, pharmacological treatments are limited; behavioural therapy is the most widely used and effective treatment, and has the biggest influence on patient outcomes when begun early in life. With as many as 1 in 100 UK citizens living with ASD, according to estimates from the Health and Social Care Information Centre¹, research into its causes that could lead to diagnostics and treatment is vital.

Our laboratory at the University of California, Davis is studying one potential contributor to neurodevelopmental changes found in ASD – an altered immune system. The brains of individuals with ASD seem to develop differently than the brains than those without ASD, including “over-connections” in some areas and weak links in others, as well as a lack of coordination between some regions². Similarly, the immune systems of individuals with ASD seem to respond differently than those individuals not on the spectrum. The connection between ASD and immune system anomalies was reported as early as 1976³. The idea that the immune and neural systems communicate is not unheard of; research of autoimmune disorders, such as systemic lupus erythematosus (SLE), points to abnormal immune responses impacting cognitive and behavioural patterns?.

The immunological changes experienced by individuals with ASD have been heavily investigated over the past decade?,?. Altered cytokine profiles, incomplete immune response activation, and increased incidence of autoimmune disease are just the tip of the iceberg. We are particularly interested in examining the altered cytokine profiles of children with ASD and their families.

Cytokines are small proteins important to cell signalling, especially at the initiation and amplification of an immune response.

Our research on ASD examines not only connections to immunology, but also the relationship between specific anomalies and severity of behavioural symptoms. There are many questions of interest to our group: Are immune abnormalities directly related to development and behaviour or just coincidental? When do these anomalies in immune function begin? Could these immune profiles be predictive of ASD and, even more powerfully, subtypes of ASD?

At our lab, we focus on two areas of the immune system’s influence on neurodevelopment: Firstly, the role of the maternal immune system, including circulating maternal autoantibodies and cytokine dysregulation, during pregnancy and secondly, the immune systems of the children with ASD at birth and during early life.

We have observed that approximately 20% percent of mothers of children with ASD have autoantibodies against fetal brain proteins?.

Furthermore, the presence of maternal autoantibodies seems to correlate with behaviour patterns, such as language scores, irritability levels, and stereotypic behavior, of the children?. In children with ASD, we seek to understand the relationship between their immune profile, defined by the cytokines present and their expression levels, and the severity of their disorder. Identifying biomarker(s) that are predictive of behavioural outcomes has the potential to lead to more targeted treatment methods.

[caption id="attachment_52430" align="alignnone" width="540"] Finding an assay to detect multiple cytokines at minute levels, is a focus of growing importance[/caption]

During an immune response, dozens of cytokines are involved, necessitating assaying several different cytokines to create a reliable immunologic snapshot. Traditionally, cytokines have been analysed using enzyme-linked immunosorbent assays (ELISAs), which recognize the analyte of interest using antibodies. ELISAs, however, only allow for the analysis of a single analyte at a time. Adding to this challenge, the available samples are typically extremely limited; it is often only possible to retrieve microlitre volumes of usable sample from either the dried blood spots collected from newborns, or blood draws from children. We frequently only have one shot to analyse these samples. There simply would not be enough sample to do ELISAs on all of the cytokines of interest. Therefore, finding an assay capable of reliably analysing several cytokines in a single reaction is crucial, often at low levels.

To this end, our study of immune dysregulation in ASD has been greatly aided by multiplexed immunoassays analysed using the Bio-Plex system (Bio-Rad Laboratories) with Luminex xMap technology. Much like ELISAs, the technology takes advantage of antibody recognition of the desired analytes. However, in a Bio-Plex assay the antibodies for each cytokine are linked to color-coded beads. The different beads are then mixed into a single reaction, and each bead emits at a unique wavelength when excited by a laser, allowing for the detection of the various analytes bound to them. In addition to detection, the assays can also be used to determine the concentration of the cytokines present. For quantitation, a fluorescently labeled control antibody specific for analyte bound to the bead-linked antibody is used. Exciting this labelled antibody with a laser specific to the fluorescent compound allows measurement of how much of that antibody is present. We can then compare the sample’s fluorescence levels to the fluorescence of known standards to calculate the analyte concentrations.

Because the developing immune system is immature, cytokine production in children is lower than in adults.

This decreased production requires sensitive assays if cytokine levels are to be reliably quantitated. Assays with insufficient sensitivity would be unable to detect cytokines that are present at these lower levels. For this reason, we use high-sensitivity (low limit of detection) Bio-Plex Precision Pro Human Cytokine kits. Luminex technology has been especially valuable for the analysis of cytokines in neonatal blood spots, as well as samples from very young children. It was because of the ability to measure several analytes simultaneously at high-sensitivity that we have had success identifying several cytokines that are correlated to severity of ASD symptoms?.

Further research is required to determine whether the link between ASD and immune anomalies is causal or coincidental. However, the observed link between cytokine levels and ASD severity raises an intriguing new avenue for immune-based diagnostics. Our research, and similar studies by other investigators, will facilitate a better understanding of the biological contributions to the development of ASD, potentially leading to the new therapeutic strategies.

Author

Judy Van de Water is Professor at the Department of Internal Medicine at the University of California, Davis Center for Children’s Environmental Health.

References

1. Health & Social Care Information Centre. Available online. 2. Shriber, L. Autism: A Neurological and Sensory Based Perspective. In: JH Stone, M Blouin, editors. International Encyclopedia of Rehabilitation. 2010. Available online. 3. Stubbs, EG. Autistic children exhibit undetectable hemagglutination-inhibition antibody titers despite previous rubella vaccination. J Autism Child Schizophr. 1976. 6(3): p. 269. 4. Diamond, B. et al. Immunity and acquired alterations in cognition and emotion: lessons from SLE. Adv Immunol. 2006. 89: p. 289. 5. Goines, P and Van De Water, J. The Immune System’s Role in the Biology of Autism. Curr Opin Neurol. 2010. 23(3): p. 111. 6. Ashwood, P, Willis, S and Van De Water, J. The Immune Response in Autism: A New Frontier for Autism Research. J Leukoc Biol. 2006. 80(1): p. 1. 7. Braunschweig D, et al. Autism-specific maternal autoantibodies recognize critical proteins in developing brain. Transl Psychiatry. 2013. 3:e277. 8. Krakowiak, P. et al. Neonatal Cytokine Profiles Associated with Autism Spectrum Disorder. Biol Psychiatry. 2015.epub ahead of print.