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Elevated plasma cytokines in autism spectrum disorders provide evidence of immune dysfunction and are associated with impaired behavioral outcome

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Abstract

Autism spectrum disorders (ASD) are characterized by impairment in social interactions, communication deficits, and restricted repetitive interests and behaviors. A potential role for immune dysfunction has been suggested in ASD. To test this hypothesis, we investigated evidence of differential cytokine release in plasma samples obtained from 2 to 5 year-old children with ASD compared with age-matched typically developing (TD) children and children with developmental disabilities other than autism (DD). Participants were recruited as part of the population based case-control CHARGE (Childhood Autism Risks from Genetics and Environment) study and included: 97 participants with a confirmed diagnosis of ASD using standard assessments (DSM IV criteria and ADOS, ADI-R), 87 confirmed TD controls, and 39 confirmed DD controls. Plasma was isolated and cytokine production was assessed by multiplex Luminex™ analysis. Observations indicate significant increases in plasma levels of a number of cytokines, including IL-1β, IL-6, IL-8 and IL-12p40 in the ASD group compared with TD controls (p < 0.04). Moreover, when the ASD group was separated based on the onset of symptoms, it was noted that the increased cytokine levels were predominantly in children who had a regressive form of ASD. In addition, increasing cytokine levels were associated with more impaired communication and aberrant behaviors. In conclusion, using larger number of participants than previous studies, we report significantly shifted cytokine profiles in ASD. These findings suggest that ongoing inflammatory responses may be linked to disturbances in behavior and require confirmation in larger replication studies. The characterization of immunological parameters in ASD has important implications for diagnosis, and should be considered when designing therapeutic strategies to treat core symptoms and behavioral impairments of ASD.

Introduction

Autism Spectrum Disorders (ASD) are a heterogeneous group of neurodevelopmental disorders characterized by severe impairments in social interaction and communication, and restricted, stereotyped interests that manifest in early childhood (American Psychiatric Association, 2000). Recent epidemiologic data suggest that approximately 1% of children are diagnosed with an ASD (MMWR, 2009). In the majority of cases, the etiology of ASD is not known and likely involves complex interactions between genetic, epigenetic and environmental factors. Recent papers have described links between genes that encode for immune-related proteins and ASD, suggesting that abnormalities in the immune system may influence aspects of brain development and synaptic functions that negatively impact clinical outcomes relevant to ASD (reviewed in Enstrom et al., 2009a). Taken together with well-established reports of cytokine mediated influences on neuronal function, differentiation, migration, proliferation, and behavioral impairments in animal models, there is an emerging view of synergistic relationships between immune dysfunction and genetic predisposition that contribute to a subset of ASD cases.

Altered immune responses in individuals with ASD have been reported for nearly 40 years including the presence of self-reactive antibodies to brain and CNS proteins (Cabanlit et al., 2007, Connolly et al., 2006, Silva et al., 2004, Todd et al., 1988, Vojdani et al., 2002, Wills et al., 2009) and evidence for increased neuroinflammation in brain and CNS specimens obtained from individuals with ASD (Vargas et al., 2005, Li et al., 2009, Garbett et al., 2008). Several studies have shown peripheral immune abnormalities in children with ASD including abnormal or skewed T helper cell cytokine profiles (Ashwood and Wakefield, 2006, Gupta et al., 1998), decreased lymphocyte numbers (Ashwood et al., 2003), altered T cell responses to mitogens and recall responses (Molloy et al., 2006), an imbalance of serum immunoglobulin levels (Enstrom et al., 2009b), NK cell activation (Enstrom et al., 2009c), increased monocyte responses (Enstrom et al., 2010) and increased levels of complement components (Corbett et al., 2007). Taken together, these findings support alterations in the immune responses in a significant proportion of children with ASD.

Few studies have addressed if cytokine patterns differ in the sera or plasma in ASD (Ashwood et al., 2008a, Ashwood et al., 2008b, Croonenberghs et al., 2002, Enstrom et al., 2008a, Okada et al., 2007, Singh et al., 1991, Singh, 1996, Sweeten et al., 2004, Zimmerman et al., 2005) and of these only three have attempted to evaluate whether cytokine levels are associated with core deficits of ASD or impairments in associated behaviors and/or onset patterns of ASD (Ashwood et al., 2008a, Ashwood et al., 2008b, Enstrom et al., 2008a, Okada et al., 2007). A subset of these studies have demonstrated increased levels of cytokines that can induce inflammation in ASD, such as IFNγ or IL-12, (Sweeten et al., 2004, Singh, 1996), or a decreased production of cytokines that negatively regulate inflammation, such as TGFβ1 (Ashwood et al., 2008a, Ashwood et al., 2008b, Okada et al., 2007). However, varied experimental designs, diagnostic criteria, ages of the probands and control populations (including the comparison of children with ASD with adult controls) have confounded interpretation of the results. Moreover, small sample sizes and the use of non-clinically assessed siblings of children with ASD as controls have dampened the ability to detect differences in cytokine patterns, thwarting scientific progress towards a consensus regarding whether particular cytokine patterns reflect an inherent immune dysfunction in ASD.

Based on the abnormal immune dysfunction observed in ASD, we herein evaluated a comprehensive panel of inflammatory cytokines associated with general immune activation in plasma samples from a large series of well-characterized participants enrolled in a population based case-control study. To better define the immune status of children with ASD, cytokine levels were assessed in ASD children 24–60 months and compared with typically developing children and children with developmental disabilities other than ASD who were of the same age. In addition, cytokine profiles in children with ASD were investigated for any associations with clinical behavioral outcomes.

Section snippets

Subjects

Participants in the study were enrolled in the CHARGE (Childhood Autism Risks from Genetics and Environment) study conducted at the UC Davis M.I.N.D. Institute (Hertz-Picciotto et al., 2006). The study protocols including recruitment and behavioral assessments have been described in detail (Ashwood et al., 2008a, Ashwood et al., 2008b, Hertz-Picciotto et al., 2006, Enstrom et al., 2009b). In brief, after clinical evaluations, participants were placed in one of three groups: (1) diagnosed with

Results

Levels of IL-6 and IL-12p40 were significantly higher in children with ASD compared with TD and DD controls (Table 1). After adjusting for child’s age and gender, levels of IL-6 were approximately twofold higher in plasma collected from ASD children (median 20.3; interquartile range 9.5–43.5 pg/mL) when compared with TD (11.8; 3–29.2 pg/mL; p = 0.01), and elevated nearly seven fold compared with DD controls (3; 3–18.8 pg/mL; p = 0.03). Levels of IL-12p40 were significantly higher in plasma sampled

Discussion

Although the etiology and pathogenesis of ASD remain unclear, it is suggested that there may be an association with immune dysfunction (Ashwood et al., in press, Enstrom et al., 2009a). Marked neuroinflammation and microglia cell activation is one of the most prominent ASD characteristics (Vargas et al., 2005), along with increased circulating autoantibodies to brain or CNS tissue (Wills et al., 2009). Further, altered T cell activation (Ashwood et al., 2004, Ashwood and Wakefield, 2006, Molloy

Acknowledgments

This study was funded by the NIEHS Children’s Center Grant (2 P01 ES011269), US EPA STAR program Grant (R833292 and R829388), NIEHS CHARGE study (R01ES015359) Autism Speaks Foundation, Peter Emch Foundation, The Boler Company Foundation and the Johnson Foundation. We would like to thank and the staff of both the UC Davis M.I.N.D Institute and the CHARGE study, and Christina Kwong and Angela Tarver for their technical support. The commitment of the families who took part in these studies is

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