With support from Cure Autism Now, a study recently published in the Journal of Neuroimmunology has found that children with autism have a more active immune system. The research, led by Cynthia Molloy, MD,
also identified a potential mechanism for this immune dysregulation. The authors suggest that a cytokine called interleukin-10 (IL-10) could be a key part of the mechanism that leads to alterations in the adaptive immune response in individuals with autism. This new finding about the role of IL-10 provides another piece of the puzzle in understanding the complex nature of immune dysfunction in autism.
As early as the 1970's, immunological factors were identified in autism. Over time, a growing body of evidence has indicated a role of immune dysfunction in individuals with autism, but the exact nature is not fully clear, and no causal function has been established. One potent area of research has been the study of cytokines, chemicals in the body that serve as signaling molecules and play a crucial role in mediating specific types of immune responses. Cytokines are essential components of both the innate immune system (immune defense mechanisms that are the first line of defense against any kind of invading substance, and present from birth) and the adaptive immune system (immune defense mechanisms that develop in response to specific invading substances, built up as immunities to infection from diseases we have been exposed to over our lifetimes.) These important messengers control the strength, length, and direction of immune responses, and are essential in regulating the repair of tissue after injury. The many individual cytokines play different roles; some act as stimulators of immune system activation, while others provide inhibitory functions. Together, the various cytokines work in an intricately coordinated system, the success of which is dependent on their well-timed production by the various cell types of the immune system.
Interested in the impact of immune regulation on the development of autism, in 2003 Dr. Molloy received a pilot project grant from CAN. Dr. Molloy is an Assistant Professor of Pediatrics at the Center for Epidemiology and Biostatistics at Cincinnati Children's Hospital Medical Center, and is also the mother of a 13 year-old daughter with autism. While she began her career in pediatric emergency medicine, the emphasis of her work changed in 1999, when Dr. Molloy started a research fellowship in developmental disabilities at Cincinnati Children's Hospital Medical Center. She joined the faculty in 2003, where her research currently focuses on immune phenotypes and the contribution of genes on chromosome 21 to autism. Dr. Molloy highlights the benefits of teamwork at Cincinnati Children's Hospital, where she works closely with Marsha Wills-Karp, Ph.D. "I have been fortunate to collaborate with an exceptional immunobiologist to work on understanding the extent to which the immune system contributes to the pathogenesis of autism."
In this study, Dr. Molloy and her colleagues were interested in the levels of certain cytokines that are produced by a specific type of immune cell in the adaptive immune system, called helper T cells (T cells are a type of white blood cell). Helper T cells contribute to the immune response by promoting the production of other types of T and immune cells. The research team studied two types of helper T cells that work as a system: Th1 and Th2. Under normal circumstances, the Th1 and Th2 systems balance one another by inhibiting each other's activity. Each type of helper T cell produces different kinds of cytokines, with the T cell types defined by the cytokines they produce. These cytokines are termed interferons and interleukins, and the research group concentrated on a certain subset. Within the Th1 system, the dominant cytokine is interferon gamma (IFN-gamma), which is responsible primarily for reactions against viruses and intra-cellular microbes, and is pro-inflammatory. Among others, Th2 cells produce interleukins IL-4, IL-5, and IL-13. These interleukins are important for stimulating production of antibodies (immune proteins that identify specific foreign substances for destruction) and often have multiple functions. As part of the Th2 system, IL-4 and IL-13 are primarily anti-inflammatory (by inhibiting Th1 cells), but they also promote the growth and differentiation of other immune cells. IL-4 also has the very important role of producing the regulatory cytokine IL-10, which helps maintain the balance between the Th1- and Th2- produced cytokines.
Historically, the role of cytokines in the immune system dysregulation observed in studies of individuals with autism has not been conclusive, because different patterns of cytokine activation have been found. Some studies of the adaptive immune system in autistic individuals have shown that the cytokines of the Th1 cells are elevated, while other studies have found elevations in the cytokines of the Th2 system. Interestingly, a study of patient registries in Europe found that many individuals suffered from both allergies (generally Th2 driven) and autoimmune disorders (generally Th1 driven). Typically, autoimmune diseases and allergies are not seen together in an individual, because both Th systems are not usually overactive at the same time. One goal of Dr. Molloy's study was to determine if direct measures of the cytokine levels themselves (as opposed to measures of the allergic/autoimmune disorders produced by imbalances in these systems) would show the same simultaneous hyper-activation in individuals with autism.
To examine the adaptive immune system, Dr. Molloy's team measured cytokine production of children's immune cells in a cell culture, both at a baseline level and after stimulation by an allergen and a toxin. The team compared individual cytokine levels in blood samples from twenty children with autism and twenty unaffected controls matched on the basis of age, race, gender and date of study visit; this careful one-to-one matching was important for controlling some of the variability that has made previous studies of immune function in autism hard to interpret.
At baseline, the researchers found that immune cells of children with autism produced higher levels of both the Th1 and Th2 cytokines, including IFN-gamma and IL-4, -5, -13, than the cells cultured from the control group. In contrast, in the experiment using stimulation by an allergen or toxin, there was no difference between cases and controls, indicating that the cells in both groups were equally capable of producing the cytokines and generating an immune response.
These findings demonstrate that, in children with autism, both the Th1 and Th2 cytokines are more highly activated in the immune system's resting state, indicating potential underlying hypersensitivity to exposures in the general environment. Dr. Molloy's study shows that immune dysregulation is found in the adaptive immune system, as has been previously shown for the innate immune system, confirming that children with autism exhibit hyper-sensitivity in both innate and adaptive systems. Dr. Molloy's research has found increases in both pro- and anti- inflammatory cytokines in the Th1 and Th2 system which is indicative of dysregulation in the two systems. Instead of focusing on the exact role of the anti- or pro- inflammatory cytokines, the study highlights the importance of balanced regulation between these two systems in the adaptive immune system.
In an intriguing twist, although baseline levels of almost all the cytokines measured were higher in children with autism than in control individuals, Dr. Molloy found an exception in the relatively lower levels of the critical regulatory cytokine, IL-10, in individuals with autism. If both Th1 and Th2 cells are just generally overactive in individuals with autism, elevated IL-10 production would have been predicted as well. Dr. Molloy explains that "it is unusual to see both the Th1 and Th2 arms of the adaptive immune response so active at the same time; it is even more unusual to see this increased activation without a proportional increase in the regulatory cytokine IL-10, which is involved in Th1 and Th2 system regulation." Although previous research has shown that IL-10 regulates the Th1 and Th2 systems, the exact mechanisms contributing to the balance within the two systems is currently not known. Dr. Molloy proposes that "many of the paradoxical findings that have been reported about immune responses in autism could possibly be explained by the general dysfunction of IL-10." The finding that IL-10 levels were not elevated in individuals with autism, even when the levels of both Th1 and Th2 cytokines were elevated, suggests that the immune response dysfunction seen in autism may be a problem with regulating the cytokine system. Dr. Molloy hypothesizes that "children with autism may not be able to down-regulate their Th1 and Th2 systems" either because of a dysfunction in the production of IL-10 or because of a dysfunction with the activity of IL-10 itself.
Dr. Molloy's research contributes a crucial piece of information to the ability to determine how these cytokines function within the complex interactions of an adaptive immune system response. Further study of IL-10 is needed to determine how it contributes to the balance between the Th1 and Th2 systems. Therefore, Dr. Molloy plans to follow-up her CAN-funded project with a study that investigates the function of IL-10, as well as Transforming Growth Factor (TGF ), another regulatory cytokine shown to mediate the balance of the Th1 and Th2 systems through unknown mechanisms.
Dr. Molloy's research is complemented by other CAN-funded projects which are currently examining cytokine activity. A 2006 CAN pilot project by Paul Ashwood, Ph.D. from the University of California, Davis will examine plasma cytokine levels in autistic individuals, in order to develop an immunological marker that can be used as a diagnostic bio-marker. In his study, Dr. Ashwood will measure several types of cytokines and compare their levels of production in typically developing children and in autistic children who have different clinical and behavioral symptoms. Dr. Ashwood's study may help define phenotypic subgroups within the spectrum and explain why we see different patterns of cytokine elevation in different studies. In addition, several other CAN-funded researchers are studying how elevated cytokine levels in the blood may affect brain development. Lisa Boulanger, Ph.D. of the University of California, San Diego is examining the effects of cytokines on the expression of immune genes in the brain, and CAN Scientific Advisory Board member Paul Patterson, Ph.D. of the California Institute of Technology is examining how a maternal infection during pregnancy may alter cytokine levels and contribute to development of autism.
Cytokines, and the immune system overall, may play a very important role in the development of autism. These cellular and molecular studies are vital, both for identifying the function of cytokines in autistic individuals and for understanding how the cells that produce these cytokines may play a factor in immune dysfunction. A better understanding of underlying biology can inform studies examining how these structures contribute to the immune impairments that are observed in individuals with autism. Dr. Molloy emphasizes this point, "If the cells that contribute to the dysregulation can be identified, then from this, models can be developed that identify how these cells work in the active immune system to give rise to the dysregulation in autism." Such studies are also critical to the development of treatments. By documenting specific changes in the immune system and when they might occur, these researchers studying the immune system can provide the foundation for the development of preventive measures and treatments that can target the dysfunctions in the underlying biology.
Reference: Molloy, C., Morrow, A., Meinzen-Derr, J., Schleifer, K., Dienger, K., Manning-Courtney, P., Altaye, M., & Wills-Karp, M. (2006). Elevated cytokine levels in children with autism spectrum disorder. Journal of Neuroimmunology, 172, 198-205.