Elevated immune response in the brain of autistic patients

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Abstract

This study determined immune activities in the brain of ASD patients and matched normal subjects by examining cytokines in the brain tissue. Our results showed that proinflammatory cytokines (TNF-α, IL-6 and GM-CSF), Th1 cytokine (IFN-γ) and chemokine (IL-8) were significantly increased in the brains of ASD patients compared with the controls. However the Th2 cytokines (IL-4, IL-5 and IL-10) showed no significant difference. The Th1/Th2 ratio was also significantly increased in ASD patients. Conclusion: ASD patients displayed an increased innate and adaptive immune response through the Th1 pathway, suggesting that localized brain inflammation and autoimmune disorder may be involved in the pathogenesis of ASD.

Introduction

Autistic spectrum disorders (ASD) are complex, pervasive developmental disorders of childhood characterized by impairments in social interaction, deficits in verbal and non-verbal communication, and restricted repetitive and stereotyped patterns of behavior and interests (DSMIV criteria, American Psychiatric Association, 1994). The prevalence of ASD is currently estimated to be 6–7 per 1000, affecting 4 times more boys than girls (Rice, 2007). Susceptibility to ASD is clearly attributable to genetic factors (Folstein and Rosen-Sheidley, 2001), but the etiology of the disorder is unknown, and no biomarkers have yet been identified as characteristic of ASD. Recent reports suggest that a combination of environmental or perhaps in utero risk factors, autoimmune risk factors and localized inflammation of the central nervous system may contribute to the pathogenesis of ASD (Nelson et al., 2001, Vargas et al., 2005, Zimmerman et al., 2005, Chez et al., 2007). Jyonouchi et al., 2001, Jyonouchi et al., 2002 demonstrated that elevated levels of the proinflammatory cytokines tumor necrosis factor (TNF)-α and IL-1β were produced by peripheral blood mononuclear cells from children with ASD. Singh (1996) reported increased plasma levels of the Th1 cytokines IL-12 and IFN-γ, and Croonenberghs et al. (2002) found increased levels of IFN- γ in the supernatant of whole blood cultures from children with ASD. In addition, Molloy et al. (2006) reported that children with ASD had increased activation of both Th2 and Th1 arms of the adaptive immune response in blood mononuclear cells, with a Th2 predominance, and without the compensatory increase in the regulatory cytokine IL-10. In both peripheral blood and intestinal mucosa, Ashwood and Wakefield (2006) found that CD3 + TNF-α and CD3 + IFNγ were increased and CD3 + IL-10 were markedly decreased in ASD children with GI symptoms. However, it is difficult to interpret these findings with respect to the pathogenesis of autism since it is not clear that the immune findings in peripheral blood mononuclear cells in autistic children correlate with immune-mediated pathology within the central nervous system. Recently, one study investigated the nature of inflammatory responses in the brain of autistic patients by examining 79 proteins including cytokines, chemokines and growth factors using protein array methods (Vargas et al., 2005). In this study, Vargas et al. demonstrated that transforming growth factor (TGF)-β1, derived from neuroglia, was significantly increased in the middle frontal gyrus (MFG) of autistic patients, while macrophage chemoattractant protein (MCP)-1, IL-6 and IL-10 were increased in the anterior cingulated gyrus (ACG). In addition, using protein array approach, Vargas et al. (2005) also found that MCP-1, IL-6, IL-8 and IFNγ were significantly increased in the cerebrospinal fluid (CSF). One of the advantage of protein array is the ability to detect a whole category of proteins in a very efficient way using very small quantity of tissues. But it is also less specific comparing with enzyme-linked immunosorbent assay (ELISA). (Simpson, 2003). To further investigate whether immune-mediated mechanisms are involved in the pathogenesis of autism and gain a clearer picture of cytokine activities in the brain of autistic patients, we carried out the study to determine the activities of a set of cytokines including the proinflammatory cytokines (IL-6, IL-1β, TNF-α, GM-CSF), Th1 cytokines (IL-2, IFN-γ), Th2 cytokines (IL-4, IL-5, IL-10) and chemokine (IL-8) in the brain of ASD patients using Multiplex Bead Immunoassays. We focused on using the brain tissue other than blood and serum samples, so that the results can directly reflect the immune response in the brain. The reason we chosen the above set of cytokines to be measured is because that they represent cytokines from different category and therefore both innate and adaptive immune responses can be determined. Our results showed that TNF-α, IL-6, GM-CSF, IFN-γ and IL-8 were significantly increased in the brains of ASD patients compared with the control subjects. Th1/Th2 ratio was also significantly elevated in ASD patients. In addition, we found that the regulatory IL-10 was not compensatory increased in response to the Th1 cytokines increase in ASD patients.

Section snippets

Study material

Frozen human brain tissue (frontal cerebral cortex) of 8 autistic patients and 8 age and gender matched normal subjects were obtained from the NICHD Brain and Tissue Bank for Developmental Disorders. Donors with autism fit the diagnostic criteria of the Diagnostic and Statistical Manual—IV, as confirmed by the Autism Diagnostic Interview—Revised. This study was approved by the Institutional Review Board of the NY State Institute of Basic Research and the subjects' information were summarized in

Results

The mean age of autistic group and the control group is 12.8 years and 12.5 years respectively. PMI (post-mortem index) is the time elapsed until brain tissue obtainment post-mortem. The mean values of PMI in autistic group and the control group were 23 ± 11 and 18 ± 5 respectively. There was no significant differences between the two groups (P > 0.05). In addition, there was no significant regressional relationship found between the length of PMI and the values of cytokines in these samples. We

Discussion

Although the pathogenesis of ASD is not well understood, recent studies have suggested that localized inflammation of the central nervous system may contribute to the pathogenesis of ASD. A number of studies have shown that TNFα, IFNγ, IL-1β and IL-12 were increased in the peripheral blood of ASD patients (Zimmerman et al., 2005, Molloy et al., 2006, Ashwood and Wakefield, 2006). TNFα was also shown to be increased in the cerebral spinal fluid of autistic patients (Vargas et al., 2005; Chez,

Acknowledgements

This work was supported by the NYS office of Mental Retardation and Developmental Disabilities, and by grants to A. Chauhan (PI) from Autism Speaks, Autism Research Institute, Alexander and Bo MacInnis.

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