Serum and cerebrospinal fluid immune mediators in children with autistic disorder: a longitudinal study

Autism spectrum disorder (ASD) is a lifelong neurodevelopmental disorder found in up to 1% of the US population [1], characterized by social communication deficits and restricted and repetitive behaviors [2]. Risk for ASD is highly associated with genetic factors [3], but current evidence suggests that neurobiological abnormalities in ASD are associated with changes in cytoarchitectural and neuronal organization that may be determined by the complex interplay of genetic, environmental, and immunological mechanisms [4–7]. Although ASD is not a classical immune-mediated disorder, there is increasing interest in examining the role of the immune system and inflammation in the development and persistence of the complex neurological and behavioral abnormalities associated with ASD [8–10].

Both innate and adaptive branches of the immune system are involved in critical mechanisms of brain development, neuronal and cortical organization, developmental and adaptive synaptic plasticity, and critical stages of brain function that determine neurological and behavioral activity into adulthood [11]. From fetal development to adulthood, the immune system and central nervous system (CNS) establish interactions which can influence both systemic immune responses (peripheral immune system) and local CNS immune function (neuroimmunity). Several lines of research have revealed abnormalities in the interactions of the immune system and CNS in ASD which involve disturbances in both adaptive and innate immunity [4, 6, 12, 13].

Evidence from the effects of maternal viral infections during pregnancy [14], an excess of autoimmune disorders in mothers of subjects with ASD or their families [15, 16], and the effects of environmental factors on the formation of the immune system [17] support the view that various types of disturbances of immune function play roles in the pathogenesis of ASD and the perpetuation of associated behavioral and neurological abnormalities. Studies of immune function in ASD frequently have focused on functional and quantitative studies of T cells and B cells, antibody production, and the presence of autoantibodies against neuronal epitopes. Studies of cellular immunity found abnormal function and number of T cells, as well as a lower percentage of CD4 T cells and a skewed CD4:CD8 T cell ratio [9]. However, these studies involved small samples, and more recent studies have demonstrated altered function in selected immune pathways and cell systems such as T regulatory cells and NK cells in subjects with ASD [18–20].

An emerging theme centers on the role of cytokines and chemokines, immune mediators which play important roles in pro-inflammatory or anti-inflammatory responses in the periphery and CNS. Cytokines and chemokines play important immune, homeostatic, and regulatory roles in the microenvironment of specific tissues or systems as well as circulating blood and immune cell populations [21–24]. Measures of circulating immune mediators or the rate of production by specific immune cell populations under specific conditions have been used for assessment of immune reactivity status including pro-inflammatory stages during disease. Although measurement of specific cytokines or chemokines lack specificity to define disease, their profiles and spectrum of expression may help to understand pathogenic mechanisms of disease in which the immune system may be involved [25–30]. Interestingly, cytokines and chemokines also may facilitate processes of immune-CNS interactions which modulate not only inflammatory responses within the CNS but also neuroimmune mechanisms associated with neuronal homeostasis, synaptic plasticity, and neuroglial function [31]. However, the literature is inconsistent in that no single marker is found to be abnormal consistently across studies. The majority of existing studies in ASD characterized cytokine and chemokine profiles in the plasma or serum [32], and each study finds a different profile of differences between children with ASD and children with developmental delay or typical development, most commonly elevations in ASD [33–36].

Fewer studies have examined the CNS directly, through the cerebrospinal fluid (CSF) or brain tissues; Vargas and colleagues [37] found increased levels of pro-inflammatory and modulatory cytokines, differentially expressed across the cortical regions in post-mortem samples from individuals with ASD. The goal of this study was to provide data testing the hypothesis that children with ASD differ in immune profile, especially in pro-inflammatory markers, from those without ASD. Given the importance of determining the role of immune-CNS interactions in ASD, the current study was designed to longitudinally assess immune mediators in children with ASD compared to children with typical development by focusing on the expression profile of cytokine and chemokine networks in the peripheral immune compartment (serum), to determine the pattern of expression of such immune mediators in the neuroimmune system compartment represented by CSF and to assess the correspondence between peripheral and neuroimmune system compartments.

The present study describes a comprehensive longitudinal study of serum and CSF profiles of immune mediators and modulators in children with ASD. Three key points emerge from these data. First, we used rare, longitudinal CSF sampling to demonstrate striking differences in the expression of selected cytokines, immune-related growth factors, and chemokines in the CSF compartment compared to the circulating bloodstream compartment. These findings are consistent with data from other populations [51], which show that peripheral immune-related proteins do not mirror the neuroimmune and CNS microenvironment. Second, we used these unique longitudinal data to demonstrate the limitations of cross-sectional data. Although our analyses revealed few developmental trends in circulating peripheral immune-related proteins during childhood, we noted a striking lack of within-subject stability in most of the analytes. The low ICC estimates obtained in this study indicate that a single measurement is not a reliable longitudinal indicator of a child’s level of most of serum immune mediators and modulators we studied. Third, we found no evidence for major differences in the expression of circulating cytokines and chemokines between children with autism and typically developing controls. The results of our study do not support the hypothesis that an active systemic inflammatory process plays a role in the persistence of autistic disorder and suggest that previously observed increases in brain tissue cytokines and chemokines [37, 52] may reflect homeostatic non-inflammatory roles in response to CNS dysfunction [10].

An overview of basic immune parameters such as leukocyte count and profile of circulating immunoglobulins revealed no differences between AUT and TYP. In the peripheral immune system, as reflected by the circulating bloodstream, only proteins with both immune modulation and growth factor functions (EGF and sCD40L) were significantly elevated longitudinally in AUT compared to TYP. These differences occurred in the absence of within-subject stability, suggesting that while there was significant within-subject variability, the general range of values observed in AUT differed from that of TYP across timepoints.

Interestingly, proteins such as TNF-α, IL-6, and IFNγ and chemokines such as CCL2 and CXCL10, frequently associated with pro-inflammatory responses, did not differ between AUT and TYP. The group differences observed in two of the 39 analytes tested in this study, elevated circulating EGF and sCD40L, could be construed as suggesting that dysregulation of growth and modulatory pathways, rather than systemic inflammatory responses, may occur in autism. In the periphery, both EGF and sCD40L are carried by platelets and involved in mechanisms of tissue repair, vascular function, and immune modulation [53]. EGF is a growth factor that plays critical roles in the growth, proliferation, and differentiation of numerous cell types [54, 55], and is involved in several pathways of neuronal function and trophism [55, 56]. The results of the current study are consistent with other comparisons of serum EGF between children with and without autism. Using enzyme-linked immunosorbent assay (ELISA), one study documented significantly elevated serum EGF in 27 Turkish children with autism aged 2–11 years relative to age-matched typical controls [57]. A second report, using multiplex bead assay, found elevated serum EGF in 77 children and adolescents with autism (aged 5–15) compared to 19 healthy, slightly older, controls [35]. However, some studies have documented decreased EGF in the plasma of subjects with autism compared to controls [58–60]. These conflicting results may be related to the type of sample used for the assays, as serum assays reflect all growth factors released by platelets during the blood sampling processing for obtaining serum [53]. It is worth noting that in the current study, group differences remained when platelet count was added to the model, though platelet count was not available for all TYP participants.

Similar to EGF, the CD40 system plays a regulatory role in the immune and vascular systems. sCD40L modulates function of B cells including stimulation of activation-associated surface antigen, immunoglobulin isotype switching, immunoglobulin secretion, and lymphocyte memory generation. The interaction of sCD40L with its receptor, CD40, also plays important function in monocyte activation and dendritic cell maturation [61]. No data exist regarding sCD40L in ASD, though one relevant study documented decreasing levels of sCD40L in healthy individuals across neonatal, childhood, and adult age groups [62]. We found no changes in sCD40L levels across childhood, but high rates of out-of-range values may have obscured any developmental trend. The second study, comparing children with Prader-Willi syndrome to healthy siblings, found no significant difference between the groups in plasma sCD40L, though the trend was for higher values in the syndromic children [63]. There are several studies documenting elevated levels of sCD40L in adult psychiatric and health conditions, where a negative relationship between sCD40L and cognitive function has been demonstrated [64, 65].

Thus, the significance of the elevated blood circulating levels of EGF and sCD40L in our participants with autism remains unknown, especially given the high degree of within-subject variability in concentration. Future investigations could determine whether they influence the spectrum of CNS, neurobehavioral, and cognitive dysfunction in subjects with ASD, but the variable nature of these analytes dictates that this should be done only with extreme caution, in longitudinal samples, and with appropriate controls to demonstrate the specificity to ASD (rather than for general developmental disability, for example). One hypothesis is that genetically determined growth or immune-modulatory dysregulation, rather than active systemic inflammatory responses, is responsible for elevated EGF and sCD40L. Previous SNP analyses of growth factor genes revealed a haplotypic association of EGF with ASD [66], suggesting that genetic factors may cause elevated EGF levels. An alternative hypothesis is that growth pathway dysregulation during early development pre-established a context of elevated EGF. However, our findings of low stability over time of EGF (and most other analytes) necessitate caution in considering an immune endophenotype.

A major finding of the present study is the lack of correspondence in the profiles of circulating immune-related modulators in the blood and CSF of participants with ASD. This incongruence highlights essential differences in the CNS/neuroimmune and peripheral immune system environments [67, 68]. Importantly, EGF and sCD40L, the two serum analytes on which AUT and TYP differed, had extremely low fraction of intrathecal production rates, indicating that peripheral activity had little bearing on central presence of these analytes. The relatively high levels and increased fraction of intrathecal production of immune mediators such as FLT3L, IL-15, CX3CL1, CXCL8, and CCL2 likely reflect the specific production of these mediators by neuroglia or neuronal cell populations in the CNS environment. The CSF profile of these immune mediators likely reflects a homeostatic role, as most of them have selective function on microglia and the neuroglia-neuronal interactions required for maintaining CNS homeostasis. For example, FLT3L promotes cell differentiation, proliferation, and survival and influence in the function of microglia cells in the CNS. Similarly, CX3CL1 and CCL2, chemokines that facilitate migration of monocytes to areas of injury, are critical for monocytes physiology and migration and play critical roles in the homeostatic function of microglia [69–71]. Interestingly, IL-15, a widespread expressed cytokine which is involved in multiple signaling pathways in the CNS of neurogenesis and neuroplasticity, is also a potent microglia modulator [72–74]. These observations suggest that the CNS environment in our population of subjects with ASD was enhanced in favor of microglia function rather than other adaptive neuroinflammatory responses, perhaps in response to a persistent stage of developmental synaptic plasticity or neurodevelopmental processes in the brain of these subjects [75].

Although this study focused on the assessment of immune mediators in the blood and CSF compartments, these results do not totally preclude the possibility that systemic immune factors or inflammation may influence brain development and alter neurobiological trajectories and subsequent long-term neurobehavioral changes. Such effects may only be observable when the immune markers are derived from specific microenvironment niches, or when they result from specific immune cell populations in response to exogenous challenges (e.g., isolated PBMC [76]). In those cases, the immune response is highly influenced by genetic determinants of the host, but its relevance to pathogenesis of autism remains uncertain. Regardless, the within-subject stability should be considered in future explorations of non-CSF compartment individual markers or profiles of these markers, as the high degree of variability of most analytes necessitates caution in interpreting single observations as an indicator of an enduring endophenotype. In addition, although CSF analyses permit a more direct window into the physiological status of the CNS than do peripheral measures, even they are not capable of providing information about neuroimmune responses occurring in selected areas of the brain, as has been demonstrated in previous studies of brain tissues [37].

One limitation of this study was the lack of comparison data for the CSF, as ethical constraints on lumbar punctures for healthy children precluded a comparison group in the present study. Little is known about potential differences between children and adults in CSF concentrations of immune mediators; still, comparison to adult samples may be helpful for context. AUT CSF concentrations of the six immune mediators with the highest percent transfer were similar to that of the healthy adults and more dissimilar to adults with active neuroinflammatory disorders, analyzed in the same lab (Additional file 1: Figure S1). A second limitation of this study, which we protected against by correcting for the false detection rate, was the large number of comparisons which may lead to spurious results. A third limitation was the detection limits of the assays, and high rates of out-of-range values in several cases required us to model the proportion out-of-range rather than the mean value. It is worth noting that a difference in rate of out-of-range values is similar to, but not the same as, a difference in mean values. This type of dichotomization does reduce the power of a statistical test to detect relationships, and assays with different sensitivities may have produced different results. Finally, although the majority of participants contributed at least two serum samples to the longitudinal analysis, it is possible that the degree of data missingness may have influenced the results.

In conclusion, this is the first longitudinal study of the profile of serum immune mediators in children with ASD compared to controls. The large sample size and repeated measures design of this study are significant strengths, as the power to detect differences between groups is increased when error variance due to individual differences is reduced. Further, we explicitly modeled the potential confounding effects of age in this sample, which spanned from 2–8 years, and reduced the risk spurious results due to age-related differences in immune profiles. We also utilized groups matched on sex, and controlled for sex in our statistical model, given some data suggesting that males and females respond differently to in utero inflammation [77]. Finally, this is the only longitudinal study of chemokines and cytokines in the CSF of children with ASD. These data provide important information about the lack of relationship between central and peripheral immune markers, signaling that caution should be taken when interpreting the available studies implicating current immune dysfunction in the phenomenology of ASD, as few have included direct measures of CNS status.