Autism Research and Treatment

Autism: Where Genetics Meets the Immune System

Guest Editors: Antonio M. Persico, Judy Van de Water, and Carlos A. Pardo Autism: Where Genetics Meets the Immune System Autism Research and Treatment Autism: Where Genetics Meets the Immune System

This is a special issue published in “Autism Research and Treatment.” All articles are open access articles distributed under the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. Editorial Board

Jean-Louis Adrien, France Mohammad Ghaziuddin, USA Klaus-Peter Ossenkopp, Canada M. Aman, USA Barry Gordon, USA Alan K. Percy, USA Elizabeth Aylward, USA H. Jyonouchi, USA Mikhail V. Pletnikov, USA R. F. Berman, USA Connie Kasari, USA Herbert Roeyers, Belgium Roberto Canitano, Italy Walter E. Kaufmann, USA John A. Sweeney, USA Manuel F. Casanova, USA Bryan King, USA Akio Wakabayashi, UK Jamel Chelly, France N. Kraus, USA Geraldine Dawson, USA Bennett L. Leventhal, USA Valsamma Eapen, Australia M.-Reza Mohammadi, Iran Contents

Autism: Where Genetics Meets the Immune System, Antonio M. Persico, Judy Van de Water, and Carlos A. Pardo Volume 2012, Article ID 486359, 2 pages

Is Autism a Member of a Family of Diseases Resulting from Genetic/Cultural Mismatches? Implications for Treatment and Prevention, Staci D. Bilbo, John P. Jones, and William Parker Volume 2012, Article ID 910946, 11 pages

Prenatal and Postnatal Epigenetic Programming: Implications for GI, Immune, and Neuronal Function in Autism, Mostafa I. Waly, Mady Hornig, Malav Trivedi, Nathaniel Hodgson, Radhika Kini, Akio Ohta, and Richard Deth Volume 2012, Article ID 190930, 13 pages

Decreased Levels of EGF in Plasma of Children with Autism Spectrum Disorder,CharityOnore, Judy Van de Water, and Paul Ashwood Volume 2012, Article ID 205362, 4 pages

HLA Immune Function Genes in Autism, Anthony R. Torres, Jonna B. Westover, and Allen J. Rosenspire Volume 2012, Article ID 959073, 13 pages

Intracellular and Extracellular Redox Status and Free Radical Generation in Primary Immune Cells from Children with Autism, Shannon Rose, Stepan Melnyk, Timothy A. Trusty, Oleksandra Pavliv, Lisa Seidel, Jingyun Li, Todd Nick, and S. Jill James Volume 2012, Article ID 986519, 10 pages

High Complement Factor I Activity in the Plasma of Children with Autism Spectrum Disorders, Naghi Momeni, Lars Brudin, Fatemeh Behnia, Berit Nordstrom,¨ Ali Yoseﬁ-Oudarji, Bengt Sivberg, Mohammad T. Joghataei, and Bengt L. Persson Volume 2012, Article ID 868576, 6 pages Hindawi Publishing Corporation Autism Research and Treatment Volume 2012, Article ID 486359, 2 pages doi:10.1155/2012/486359

Editorial Autism: Where Genetics Meets the Immune System

Antonio M. Persico,1, 2 Judy Van de Water,3 and Carlos A. Pardo4

1 Child and Adolescent Neuropsychiatry Unit, University Campus Bio-Medico, Via Alvaro del Portillo 21, 00128 Rome, Italy 2 Laboratory of Molecular Psychiatry and Neurogenetics, University Campus Bio-Medico, Via Alvaro del Portillo 21, 00128 Rome, Italy 3 Division of Rheumatology/Allergy and Clinical Immunology, UC Davis, 451 Health Science Drive, Suite 6510, GBSF, Davis, CA 95616, USA 4 Department of Neurology, Johns Hopkins University School of Medicine, 600 N. Wolfe Street, Baltimore, MD 21287, USA

Correspondence should be addressed to Antonio M. Persico, [email protected]

Received 24 July 2012; Accepted 24 July 2012 Copyright © 2012 Antonio M. Persico et al. This is an open access article distributed under the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.

Individuals with autism often display immune abnormalities toward microbial infections) or as a secondary event part in the form of altered cytokine profiles, autoantibodies, and of a broader immunomodulatory response aiming to blunt changesinimmunecellfunction.Aretheymere“smoke” an inflammatory process. F1 plasma levels suggest that this or real “fire”? Given what we now know about the cross- process could play a more relevant role in males and in small talk between the immune and nervous systems, one must ASD children. ask: are these immune abnormalities simply bystander The contribution by S. Rose et al. describes reduced effects of genetic/genomic variants directly responsible for glutathione-mediated redox/antioxidant capacity both inside abnormal neurodevelopment, or is there a sizable subgroup primary leukocytes and in the plasma of ASD children. Con- of patients with autism spectrum disorder (ASD), in which a sistently with this intracellular and extracellular imbalance of dysfunctional immune system plays a critical mediator role the glutathione redox status, intracellular production of free in the pathogenic chain of events leading to the disorder? radicals is also enhanced, especially in 30% of ASD cases. Is autism one of many genetic/genomic conditions, Down This and several previous studies provide converging evi- syndrome representing the best-known example, accompa- dence of excessive oxidative stress likely leading to abnormal nied by immune abnormalities not primarily responsible mitochondrial function in a similar percentage of autistic for changing the trajectory of neurodevelopment? Or could individuals. conceivably an unfortunate combination of genetic/genomic Along similar lines, M. I. Waly et al. direct their attention susceptibility and environmental factors converging onto the on the complex array of pathophysiological consequences same individual affect neurodevelopment through immune- produced by enhanced oxidative stress. In addition to mediated mechanisms, such as fetomaternal incompatibility, imbalanced glutathione redox status, reduced methionine autoimmunity, abnormal neuroinflammation, and so on? synthase activity is particularly interesting, as it results This special issue contains six contributions aimed at in lower availability of S-adenosyl-methionine and con- addressing different aspects of this question. sequently blunted DNA methylation. Curiously, a similar The paper by N. Momeni et al. documents significantly abnormality underlies autistic features also in two entirely elevated plasma levels of factor I among ASD children different contexts, namely, children exposed prenatally to compared to controls. Factor I is a plasma enzyme respon- valproic acid and in Rett syndrome girls, carrying MECP2 sible for degrading complement factor 3b, which in turn mutations. is the major opsonin in the complement system enabling The review by S. D. Bilbo et al. presents a stimulating phagocytosisofmicrobialagents.HigherfactorIlevelscan hypothesis, linking ASD to inflammatory, allergic, and be interpreted as either primary (i.e., conferring vulnerability autoimmune diseases. These “hyperimmune” disorders are 2 Autism Research and Treatment known to share steeply increasing incidence rates over the last decades, as well as significant loading in many families with children with autism. In the authors’ view, they also share an inappropriate activation of the immune system due to biome depletion in postindustrial societies, possibly suggesting preventive strategies based on biome reconstitution. The paper by C. Onore et al. reports an impressive threefold decrease in EGF plasma levels among small ASD children compared to controls. EGF is involved in wound healing in the skin, in the gastrointestinal, and respiratory systems, as well as in the central nervous system where this neurotrophic factor supports both adult subventricular zone and midbrain dopaminergic neurons, in addition to stem cell proliferation. A. R. Torres et al. review immune abnormalities in autism, focusing on HLA gene roles as potential contributors to autism pathogenesis through several distinct mechanisms. HLA genes or haplotypes have been found associated with several autoimmune diseases, but strong associations with autism have also been reported. These contributions, while not providing the ultimate answers, do add some new pieces to the autism puzzle, providing further support to the idea that immune abnormalities may play a pathophysiologically relevant role in a subgroup of families with autistic children. Future studies of the interaction of the immune and central nervous system are needed to clarify in more detail how neuroimmune mechanisms influence the enhancement of the neurodevelopmental disarray and disturbances of neurobehavioral trajectories determined by genetic and environmental factors. The heuristic potential of this line of investigation, both in terms of prevention and clinical therapeutics, ensures continued efforts until a more definitive answer can be given to our initial question, so crucial in today’s autism field. Antonio M. Persico Judy Van de Water Carlos A. Pardo Hindawi Publishing Corporation Autism Research and Treatment Volume 2012, Article ID 910946, 11 pages doi:10.1155/2012/910946

Review Article Is Autism a Member of a Family of Diseases Resulting from Genetic/Cultural Mismatches? Implications for Treatment and Prevention

StaciD.Bilbo,1 John P. Jones,2 and William Parker2

1 Systems and Integrative Neuroscience Group, Department of Psychology and Neuroscience, Duke University, Durham, NC 27710, USA 2 Department of Surgery, Duke University Medical Center, Durham, NC 27710, USA

Correspondence should be addressed to William Parker, [email protected]

Received 12 September 2011; Revised 18 January 2012; Accepted 10 April 2012

Academic Editor: Antonio M. Persico

Copyright © 2012 Staci D. Bilbo et al. This is an open access article distributed under the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.

Several lines of evidence support the view that autism is a typical member of a large family of immune-related, noninfectious, chronic diseases associated with postindustrial society. This family of diseases includes a wide range of inflammatory, allergic, and autoimmune diseases and results from consequences of genetic/culture mismatches which profoundly destabilize the immune system. Principle among these consequences is depletion of important components, particularly helminths, from the ecosystem of the human body, the human biome. Autism shares a wide range of features in common with this family of diseases, including the contribution of genetics/epigenetics, the identification of disease-inducing triggers, the apparent role of immunity in pathogenesis, high prevalence, complex etiologies and manifestations, and potentially some aspects of epidemiology. Fortunately, using available resources and technology, modern medicine has the potential to effectively reconstitute the human biome, thus treating or even avoiding altogether the consequences of genetic/cultural mismatches which underpin this entire family of disease. Thus, if indeed autism is an epidemic of postindustrial society associated with immune hypersensitivity, we can expect that the disease is readily preventable.

1. Introduction: Autism as a Member of a Large lupus, multiple sclerosis, hay fever, appendicitis, chronic Family of Postindustrial Epidemics fatigue syndrome, inﬂammatory bowel disease, asthma, Involving a Hyperimmune Response celiac disease, type 1 diabetes, Graves’ disease, some types of eczema, and a wide range of food allergies. Here, based In this paper, we outline a paradigm that points toward on a variety of evidence, we argue that autism is yet another autism as one disease among many other well-known member of this family of diseases, despite the slowness diseases which all share a common origin and, most of the medical community to recognize it as such. Since likely, a common prevention strategy [1]. These emerging, the pathogenesis of at least one form of autism has been noninfectious diseases are all epidemics of postindustrial directly linked to autoantibody production [2], and since culture, which are absent in antiquity in any culture, seven out of every eight cases of severe autism are associated and absent in present day developing cultures. Members with antineuronal antibodies in the serum [3], this idea of this family of disease are invariably associated with a has already been demonstrated, at least in part. Further, hyperimmune response and can have a very high preva- as described below, autism is associated with a wide range lence in postindustrial populations, with prevalence often of immune abnormalities. Even though some children with greater than 0.1% and sometimes greater than 1.0%. These autism appear to have “normal” immune systems, it is argued hyperimmune-associated diseases include a wide range of that adverse immune events early in development might lead allergic, autoimmune, and inﬂammatory diseases such as to an autistic phenotype. 2 Autism Research and Treatment

A primary consideration in this view of autism as a 4. Glial Cells Direct Normal hyperimmune-associated disease is the connection between Brain Development immunity and brain development in general, and between hyperimmune responses and autism in particular. Although Microglia are the resident macrophages of the central these connections have little bearing on the overall model nervous system, are associated with the pathogenesis of describing induction of disease by immune hypersensitivity many inflammatory diseases of the brain, and derive from in postindustrial society, the connections provide reason to primitive yolk sac macrophage precursors, which are of expect that the developing brain is sensitive to the same mesodermal origin and enter the neuroectoderm during postindustrial changes in the immune system which are embryogenesis [21]. Early in development, microglia are known to affect virtually all other organs of the human body highly mobile and primarily amoeboid, consistent with (e.g., kidneys, pancreas, epidermis, adult nervous tissues, their role in the phagocytosis of apoptotic cells [22]. The large and small bowel, cardiovascular system, thyroid, lungs, expression of many cytokines within the developing brain, andothers).Thenextfoursectionswillsummarizemuch including IL-1β and TGF-β, depends on the presence of of what is currently known regarding those very extensive amoeboid microglia [23]. Microglia transform into a highly connections. branched, ramified morphology by adulthood in most brain regions. This morphological transition occurs in parallel with neural cell genesis and migration, synaptogenesis, and 2. Pervasive Immune System synaptic pruning, suggesting functions for microglia in Abnormalities in Autism each of these processes, though these are just beginning to be explored [24–26]. Oligodendrocytes myelinate axons Immune system abnormalities exist throughout the body primarily during the postnatal period, and disruption of and brain of autistic children. These include evidence their function can be profoundly debilitating as in the of brain specific auto-antibodies, altered T, B, and NK case of periventricular leukomalacia leading to cerebral cell responses to antigen, altered cytokine production, an palsy [27]. Astrocytes mediate synapse formation within increased incidence of allergies and other autoimmune dis- the developing brain [28], in part via the secretion of orders, and functional changes in brain glial cells (microglia extracellular matrix proteins called thrombospondins (TSPs) and astrocytes) [4–11]. Microglia are the primary immuno- [29, 30]. Alterations in spine density via a putative TSP competent cells of the brain and rapidly respond to any mechanism are implicated in neurodevelopmental disorders infection, injury or other perturbation of homeostasis via such as Down’s and Rett Syndrome [31]. Notably, astrocyte a dynamic process of activation [12]. Notably, increased maturation marks the end of the perinatal synaptogenic microglial activation has been observed in several brain period when the brain is most plastic [32]. regions of autistic patients [13]. Once activated, glia produce a wide number of immune signaling molecules, including cytokines (e.g., interleukin [IL]-1β, tumor necrosis factor [TNF]-α), chemokines (e.g., monocyte chemoattractant 5. Long-Term Consequences of Immune protein [MCP]-1), and other inducible factors (e.g., nitric Activation during Early Development oxide), which may profoundly influence neural function Developmental or “fetal programming” is a growing field of [14]. science based on evidence that experiences during critical or sensitive periods of perinatal life may modulate or “program” 3. Immune System-Central Nervous the normal course of development, with the result that adult System Communication outcomes, including behavior, are significantly and often permanently altered [33]. Given the many ways in which the Beyond its traditional role in host defense and tissue repair, immune system is important for normal brain development, the immune system is now considered a diffuse sensory the capacity for immune-inducing events to influence the organ that works in concert with the endocrine, metabolic, long-term trajectory and function of these processes is likely and nervous systems to achieve and maintain homeostasis profound, perhaps more so than at any other stage of life throughout the body [15, 16]. In essence, the immune [34]. Notably, there is strong evidence that early-life infection system serves as an interface between the human body and can permanently alter stress reactivity, disease susceptibility, the environment, coordinating the response of the body and notably, vulnerability to cognitive and neuropsychiatric to the environment. Immunocompetent cells are located disorders, including Alzheimer’s disease, Parkinson’s disease, throughout every organ of the body, including the brain, schizophrenia, and autism [35–38]. One of the most dra- and regular communication occurs between the central matic and direct illustrations of this effect is the induction of nervous system and immune tissues during both health autism-like symptoms in mice by stimulation of the maternal and disease processes. Many excellent reviews have been immune system during pregnancy [39]. written on these topics [17–20]. Importantly, bidirectional Neuroinflammatory-induced alterations in neurogene- communication between the brain and immune system has sis, migration, axon growth, myelination, synaptogenesis, significant consequences for plasticity mechanisms within and dendritic spine density/function are all candidate mech- the brain, including cognition and emotion, which are anisms by which long-term changes in behavior might markedly altered in autism. result. Further, the long lifespan of microglia and their Autism Research and Treatment 3 pattern of central nervous system colonization during immune system. This effect is closely intertwined with an development suggest that these immune cells may have impact on metabolism and transport, as will be described particular significance in terms of neuroinflammatory effects later. These mismatches of postmodern culture involve some on developmental programming. Diverse early-life events of the most cherished attributes of postmodern culture, may have enduring consequences for the brain and behavior including the widespread use of toilets, water treatment facil- of organisms via an influence on these resident immune ities, indoor working environments, soaps and shampoos, cells, both by impacting their own intrinsic function as well modern medicine, and the loss of factors which demand as their interactions with ongoing developmental processes exercise for survival (e.g., human-eating predators, hardship [40, 41]. in finding food and procuring shelter for most of the population). These mismatches are not a target for therapy, because reversal of any of these mismatches is untenable. 6. Approaching Autism as Another Member of Thus, these mismatches can be described as belonging to the Family of Hyperimmune-Associated the “pretherapeutic zone” in the course of pathogenesis Diseases: Addressing the Underlying Causes (Figure 1). However, these mismatches lead directly to readily measurable consequences that, in turn, lead to disease The study of individual postindustrial associated diseases has if left untended. Fortunately, these direct consequences of often failed to enlighten researchers as to the underlying mismatches are readily treated or even avoided altogether. cause of diseases. While genetic factors associated with the Given that these consequences can be treated or avoided, epidemics of postindustrial society abound, these genetic this part of the pathogenesis of hyperimmune disease can factors have not changed over many generations, and thus be described as the “optimal therapeutic zone” (Figure 1). are not responsible for newly emerging epidemics of disease. In this paper we describe how mismatches between culture Almost paradoxically, even when specific triggers are iden- and genetics/biology have led to specific consequences which tified, these often cannot be said to cause newly emerging cause immune system hypersensitivity and/or destabiliza- epidemics. For example, peanuts and ragweed pollen are tion. All of these consequences are associated with a wide two well-known triggers for allergy, but these substances range of diseases that include allergic, autoimmune, and have been present in the environment for millennia without inflammatory diseases. triggering disease in humans. Similarly, viral infections and The single consequence of genetic/culture mismatches potentially some milk proteins [42] have been identified as that most profoundly impacts hyperimmune-associated dis- triggers for particular autoimmune diseases, but these factors ease has been described as a depletion of components have been present for much longer than the diseases they from the natural ecosystem of the human body, or simply trigger. Thus, as will be described in detail, it is not the “biome depletion” (Figure 1). The use of modern medicine, genetics of the disease which point toward a cure, and it is toilets, water treatment facilities, and cleaning agents in our generally not identification of the triggers for disease that culture has led to a substantial alteration or even loss of is most helpful. Rather, we suggest that the most effective living organisms that have coevolved with humans. Three and insightful approach toward defeating autism for future primary components of the biome which are potentially generations will be the neutralization of underlying factors in important have been affected [43]: (a) loss of interactions postindustrial culture which render postindustrial humans with soil bacteria, (b) alterations of the microbiome, the uniquely susceptible to disease, despite the presence of the normal bacteria that are associated with the human body, same genetics and many of the same triggers as found in and (c) loss of helminths, worms that typically inhabit preindustrial cultures. These factors have been identified the gut of mammalian species. Substantial evidence points as mismatches between normal human genetics and the toward all three of these factors as being important in environment of postindustrial culture. With that in mind, stabilization of the human immune system. Although not an examination of the factors in postindustrial culture which widely appreciated, several lines of evidence point very trigger the plagues of hyperimmune-associated diseases will strongly if not conclusively to the idea that helminths in be presented. particular are necessary for a stable immune system. This evidence has been reviewed extensively [1, 43]andcanbe very briefly summarized as follows. 7. Mismatches between Normal Human Genetics and Postindustrial Culture (i) Helminths are found almost ubiquitously in preindustrial humans [44] and in nondomesticated Leading to Hyperimmune Epidemics: mammals, including chimpanzees [45]. Even when Biome Depletion helminths cannot be found, the immune system shows signs of stimulation by helminths [46]. If indeed autism fits into the family of hyperimmune- associated diseases plaguing postindustrial culture, then the (ii) Helminths produce and secrete a number of pathogenesis or origins of these diseases merit detailed molecules which regulate the host immune system analysis in any consideration of autism. As shown in Figure 1, [47]. postindustrial culture has several mismatches with normal (iii) Addition of helminths to laboratory animals blocks human genetics (known as “evolutionary mismatches”) allergic, inflammatory, and autoimmune diseases which have a profoundly destabilizing effect on the human [47]. 4 Autism Research and Treatment

Postindustrial culture Indoor work, soap, Exercise not Pretherapeutic toilets, water treatment, shampoo, bathing essential for survival modern medicine zone

Optimal Biome Vitamin D Uncontrolled therapeutic depletion deﬁciency stress zone

Genetics, Immune hypersensitivity epigenetics, Alteration of and/or destabilization “triggers” transport and Allergy, Phase I/Phase II autoimmunity, metabolism chronic A wide range of Postoptimal inﬂammation Metabolic demands on therapeutic dysfunction, transport and zone toxicity issues, Phase I/Phase II oxidative stress metabolism Risk of cognitive dysfunction

Figure 1: The pathogenesis of epidemics of allergic, inflammatory, and autoimmune disease. Cultural mismatches with human biology (the pretherapeutic zone) have consequences that can be readily avoided (the optimal therapeutic zone). If biome depletion in particular is not avoided, the individual is left susceptible to a wide range of often complex immune-related diseases (the postoptimal therapeutic zone), sometimes associated with imbalances in metabolite transport and/or Phase I/Phase II metabolism. Other consequences such as vitamin D deficiency and uncontrolled stress may, in some but not all cases, add to the problem. Factors contributing to disease which are often naturally occurring and generally difficult or impossible to avoid are underlined and fit within the postoptimal therapeutic zone.

(iv) Addition of helminths to humans cures inflamma- infection, genetics or epigenetics) in the induction of tory bowel disease [48] and stops the progression of autism. multiple sclerosis [49]. 8. Mismatches between Normal Human (v) The effects of biome depletion on the immune system are not necessarily instantaneous because Genetics and Post-Industrial Culture long-term effects of immune interactions with the Leading to Hyperimmune Epidemics: biome can span decades or, through epigenetics, even Vitamin D Deficiency generations [1]. Nevertheless, the epidemiology of hyperimmune disease reveals an inverse correlation Based on the available information outlined above, it between helminths and disease. seems very likely that biome depletion is by far the most significant and most profoundly influential consequence (vi) Humans with helminths and rodents colonized with of genetic/cultural mismatches in postindustrial society. helminths have profoundly less reactive immune sys- However, other consequences which apparently exacerbate tems than do either without helminths [44, 50, 51]. the effects of biome depletion are evident. For example, (vii) A long coevolutionary history of helminths and ver- vitamin D deficiency has reached epidemic proportions in tebrates, in conjunction with the potent immunoreg- postindustrial society, is known to impact inflammation ulatory effects of the former, is consistent with the during early development [52], and, like biome depletion, idea that the human immune system is literally, has been linked to a spectrum of allergic, autoimmune, and physically dependent on helminths [1]. inflammatory diseases [53–55]. Further, epidemiology and other circumstantial evidence link vitamin D deficiency to We propose that biome depletion is changing the autism [56, 57]. Interestingly, the widely publicized asso- immune system at a population-wide level, which in ciation between rainfall and autism [58]canbeaccounted turn will undoubtedly impact the brain, particularly dur- for, perhaps more than in part, by decreased exposure to ing sensitive periods of development. Importantly, this sunshine in areas with high rainfall, and the subsequent modeldoesnotcompetewithanypriorhypothesesor impact of lower vitamin D levels. known risk factors for neuroinflammatory conditions, but Vitamin D deficiency is another consequence of rather complements them. For instance, biome deple- postindustrial culture’s mismatches with human biology tion may set the stage for exaggerated levels of neu- (Figure 1). Vitamin D production requires exposure of oils roinflammation, which interacts with other known risk on the body’s surface to sunlight. The resulting photochem- factors or triggers (psychological or metabolic stress, ical reaction and production of vitamin D is greatly reduced Autism Research and Treatment 5 by components of postindustrial culture that reduce expo- consequences of postindustrial culture can also be found sure to sunlight (e.g., indoor work environments, sunscreen). which are associated with a wide range of disease, but limits The association of vitamin D deficiency with a spectrum in space prevent a complete discussion. For example, chronic of hyperimmune-associated diseases places this consequence uncontrollable psychosocial stress is yet another factor that is of biology/culture mismatch alongside biome depletion as an generally associated with a range of hyperimmune-associated underlying agent destabilizing immune system function in diseases that include allergy, autoimmunity, and inflamma- postindustrial populations. However, vitamin D deficiency is tion [63–66]. Such stress derives from a complex range of an ancient problem, as indicated by the ancient occurrence biology/culture mismatches, including, for example, the loss of rickets, whereas epidemics of hyperimmune-associated of physical exercise [67–69] as a requirement for survival in diseases are more recent in nature. Thus, it seems likely postindustrial culture (Figure 1). As another example of con- that vitamin D deficiency is a contributor to hyperimmune- sequences of biology/culture mismatches which destabilizes associated disease, but not sufficient by itself to lead to immune function, deprivation from mother’s milk during disease. Further, the prevalence of vitamin D deficiency in infancy (not included in Figure 1) is associated with a wide postindustrial society is somewhat less than the prevalence range of allergic and autoimmune conditions [70–74]. This of biome depletion: although the prevalence of vitamin D genetic/culture mismatch, like other mismatches, involves an deficiency is substantial, with one study finding 24% of aspect of postindustrial society which should not be reversed individuals deficient (≤20 ng/mL) with an additional 34% (the survival of infants despite the unavailability of mother’s having levels that were insufficient (≤29 ng/mL) [59], the milk), and thus fits into the “pretherapeutic zone” of prevalence of biome depletion in postindustrial populations Figure 1. The extent to which factors such as uncontrollable is essentially 100%. Thus, it is expected that the impact of stress and lack of mother’s milk can contribute to disease vitamin D deficiency on immunity in postmodern culture in the presence of an otherwise normal biome remains to may be a matter of exacerbating the problems associated with be elucidated, although, as outlined above, biome depletion biome depletion more so than a significant problem when appears to be the single most important factor contributing considered in isolation. to the increased incidence of hyperimmune-associated dis- Some mechanisms by which vitamin D might stabilize eases. the immune system have been elucidated. Vitamin D acts as a signaling molecule to enhance immune cell function in the presence of infection and is important for maintaining the 10. Metabolism and Transport of Toxins and normal interface between the microbiome and the immune Hyperimmune-Associated Disease system [60]. However, the mechanisms by which vitamin D interacts within the body are complex, with the molecule The connection between metabolism and immunity is being intertwined either directly or indirectly in virtually important in a consideration of hyperimmune-associated all aspects of human biology. The active form of vitamin diseases. A variety of enzymes have been identified which are D binds and activates the Vitamin D receptor, altering the useful in the biotransformation of endogenous (produced expression of a variety of genes involved in such diverse by the body or the associated biome) as well as exogenous processes as cell growth and proliferation, bone remodeling, (pharmaceutical) substrates. This system of enzymes pro- and calcium homeostasis [61]. It has been estimated that vides the body with a means of processing a wide range vitamin D binds human DNA in more than 2,500 places of substances, some of which are toxins, and is profoundly and changes the expression of more than 200 genes [62]. affected by the immune system [75]. The metabolic portion Thus, while it is known that vitamin D is required for normal of this system is readily divided into two components: immune function, it is likely that vitamin D deficiency Phase I biotransformation includes the oxidative reactions could cause a general destabilization of the human biome of the microsomal cytochrome P450 monooxygenase system independent of the immune system, particularly during early that is expressed in the liver and gastrointestinal tract, development. Fortunately, regardless of the extent to which and to some extent in a variety of extrahepatic tissues. vitamin D deficiency destabilizes the human biome, dietary Phase II biotransformation includes conjugation reactions of supplements are readily available, and thus this consequence substrates with endogenous cofactors such as glucuronate, of culture/biology mismatch is easily avoided. sulphate, acetate, or glycine. In addition to Phase I and Phase II biotransformation, the transport of their substrates and byproducts is also an 9. Mismatches between Normal Human important component of this system. A variety of membrane Genetics and Postindustrial Culture spanning polypeptides have been identified in the liver, Leading to Hyperimmune Epidemics: gastrointestinal tract, kidney as well as other tissues in the Other Factors body, each responsible for the active trafficking of substrates across cell membranes. These transport proteins are in fact The above discussion points out two factors, primarily regulated by the same pathways that regulate the Phase I and biome depletion and secondarily vitamin D deficiency, II enzymes, and they can be thought of as being regulated in which generally destabilize the human immune system. tandem [76–80]. These factors can be identified by their association with Immune activation results in a downregulation of Phase a wide range of hyperimmune-associated diseases. Other I components [81, 82], transporters [83, 84] and potentially 6 Autism Research and Treatment

Phase II components in what might be viewed as a tradeoff Thus, if porcine whipworms do not work, it does not between immunity and metabolism. In time of duress, when suggest that the hypothesis is wrong. the immune system is geared to inflammation and defense, (v) This model does not suggest that helminths are the energy devoted to metabolism/transport may need to the only organisms involved in biome depletion. be temporarily diverted to host defense. This is apparently Normalization of the microbiome may also be a an effective strategy, but as a consequence, chronic immune ffi critical factor. In the face of modern practices in activation could result in metabolic di culties as outlined obstetrics and of widespread antibiotic use, this is an in Figure 1. Unfortunately, vitamin D deficiency is expected issue that must be considered. to make this problem worse, since vitamin D is required toupregulatebothPhaseIandPhaseIIcomponents[85] (vi) Pharmaceuticals may never prove adequate. The of metabolism. Further, accumulation of toxins as a result complexity of the immune system and the effects of of reduced Phase I, Phase II, or transport activity may biome depletion suggest that efforts to readjust the also stimulate the immune system, adding further to the system using drugs may be naive and overly opti- propensity for hyperimmune activity. mistic. Biome reconstitution may be the easiest and most straight forward way to bring the system into homeostasis. Similarly, metabolic factors may not 11. Implications of Autism as a Member of easily be compensated for by pharmaceuticals. Nor- the Family of Hyperimmune-Associated, malization and regulation rather than compensation Postindustrial Diseases and suppression are probably the better approaches. The implications of this model are several-fold and deserve (vii) Genetics may be relatively unimportant from a careful consideration. The consideration of some possible clinical perspective because it may not provide a assertions that are not supported by this model is probably means to prevent or treat disease. Triggers may also equally as important. be unimportant from a clinical perspective because (a) they may simply be unavoidable, and (b) even (i) This model indicates that prophylactic normalization if triggers for a particular disease can be identified ff of the biome will result in a profound decrease and avoided, such e orts may simply decrease the in the incidence of autism. Results with other prevalence of one disease at the expense of an hyperimmune-associated diseases have been promis- increased prevalence of another. ing, although a true normalization of the biome as a means of prevention has never been attempted for 12. The Required Approach any disease. Clinical trials are urgently needed to conclusively test a (ii) This model is consistent with the idea that a variety variety of questions. The approach needs to be extensive of factors affecting immunity and/or metabolism/ and systematic rather than timid and piecemeal. The tests transport may strongly influence the incidence of required will be on a large scale, although this scale pales autism. in comparison to the current amount of effort and energy (iii) There are several potential therapeutic approaches being poured into research on allergic, inflammatory, and that this model does not suggest which will necessar- autoimmune issues. This approach to medicine will not ily be successful. For example, this model does not likely be supported by any committee of experts containing predict that helminths will necessarily work as a cure members with vested interests in traditional immunological for autism. Whether helminths can be used to treat approaches (either in humans or in rodents) or in the autism will depend on the extent to which autism is pharmaceutical industry. Rather, this approach is much more reversible and the extent to which ongoing problems likely to be appreciated by those individuals with a broad with biome stability can be reversed. understanding of biology and whose primary interest is in improving health care and, ultimately, the health of the (iv) Although porcine whipworms are currently being ff population as a whole. With that in mind, the following used to treat the e ects of biome depletion in some questions need to be answered. studies, the model described in this paper does not predict that the porcine whipworm will prevent (i) Can autism be cured or effectively treated with autism, even prophylactically. (Although the use of biome reconstitution, or only prevented by biome porcine whipworms is currently underway for a few reconstitution? clinical studies, the use of porcine whipworms as a means of prophylaxis is probably not economically (ii) How important for children is the biome of the feasible because the costs of giving the necessary mother prior to and during pregnancy, and while biweekly doses would be prohibitive. This problem breastfeeding? (Does prevention require reconstitu- is not expected when using helminths adapted to tion of the mother’s biome?) humans, since one dose lasts for years.) It might, but (iii) Which helminths or combination of helminths is this potentially depends on the extent to which this both safe and effective? Although some concerns species recapitulates the presence of a normal biome. dealing with the use of helminths for therapy have Autism Research and Treatment 7

been expressed, these objections are easily dealt with P = 0.05) that autism is a consequence of evolutionary [1]. Several candidate species are well adapted to mismatches in postindustrial society and is consistent with humans, asymptomatic in low numbers, and thus are the idea that the parental immune status is important in obvious candidates for initial and immediate test- the development of autism. Further assessment of autism ing. Further, new technologies might be developed in preindustrial cultures may, however, prove unnecessary; which could improve on species that are naturally the easiest way to evaluate the idea that autism is related occurring. For example, irradiation of organisms to to biome depletion is probably to conduct the experiment. achieve sterility and eliminate the possibility of trans- Whether prophylactic biome reconstitution does or does mission, or cultivation of human-specific helminths not affect the incidence of autism will answer all questions in ultraclean and immunodeficient rodents or even conclusively, just as the effects of biome reconstitution in vitro might improve the utility of helminths for [49, 87] and vitamin D supplementation [88] on multiple widespread medical use. sclerosis, if confirmed by future studies, will effectively end (iv) What medical conditions such as a suppressed long-standing debates about the etiology of that disease. immune system, anemia, or coagulopathy might be The idea that autism is a result of evolutionary mis- contraindications for biome reconstitution? matches is predicated on the idea that autism often occurs in genetically “normal” individuals. Thus, if autism can (v) Does optimal biome reconstitution vary with human be attributed to genetic factors which dictate the presence genotype or other factors such as age, gender, and of disease even in individuals living in a preindustrial body size/composition? society, then biome depletion is probably not a factor in (vi) In addition to the restoration of helminths to the the pathogenesis of autism, and biome reconstitution will biome, what steps should be taken for restoration and probably not affect the incidence of autism. Certainly there maintenance of the microbiome? are cases where autism is associated with genetic mutations ff which are deleterious, such as fragile X syndrome, but at (vii) How will a reconstituted biome a ect other areas present most individuals with autism appear genetically of medicine, including aging, immunosuppression, normal [89]. However, it remains unknown what percentage infectious disease, cancer biology, and vaccine tech- of autism can be attributed directly to genetics, and the idea nology? that biome depletion and other evolutionary mismatches affect mutation rates is speculative and unexplored. Again, 13. Is Autism a Postindustrial, the most effective approach to resolving the debate regarding Hyperimmune-Associated Epidemic? the role of genetics may be to prophylactically reconstitute the biome of a sample population and await the results. The idea that autism is a result of evolutionary mismatches The epidemiology and the genetics of autism are is predicated on the idea that autism is epidemic in postin- inextricably woven together. It is anticipated that one of dustrial society. Thus, if the incidence of autism is the same two pictures will emerge. Either a postindustrial epidemic in preindustrial societies as it is in postindustrial societies, of autism with normal genetics will be identified, or then biome depletion is not a factor in the pathogenesis autism will be eventually defined as a “normal” part of of autism, and biome reconstitution will not affect the the human condition, potentially associated with certain incidence of autism. The epidemiology of autism has been genes or combinations of genes that predispose to disease hotly debated. Some changes in diagnostic criteria and regardless of the environment. In the former view, autism awareness of autism have certainly affected changes in the is due to an evolutionary mismatch, whereas in the latter reported incidence of autism over time. However, debates view, autism is due to evolution itself. It is of critical regarding the changing diagnosis of autism over the past few importance not to accept the latter view prematurely since decades have little bearing on the idea that biome depletion such acceptance may disable efforts at compensating for profoundly influences autism. Rather, the pertinent question evolutionary mismatches or discourage work on identifying is whether the prevalence of autism has changed over the postindustrial triggers for disease, with potentially tragic past 150 years as various cultures have transitioned from consequences. Fortunately, several lines of evidence argue pre to postindustrial over the course of several generations. against the idea that autism is a “normal” part of human Although it may be very difficult if not impossible to estimate biology. First, a general consideration of the nature of human the prevalence of autism prior to the industrial revolution evolution argues that evolution is not responsible for autism. in countries that are currently postindustrial, it might still The social interactions disrupted by autism are fundamental be possible to examine the prevalence of autism in some to survival of not only humans, but also a wide range of preindustrial societies. One very recent study by Schieve et mammalian and nonmammalian species, pointing to the al. is particularly informative in this regard: using a phone ancient evolutionary origins of those interactions. Given the survey technique to obtain data on 4,690 Hispanic children, intense selection pressures on early human populations, it a “striking heterogeneity” in the prevalence of autism seems unlikely that an error rate of 1% in social interactions spectrum disorder between US-born Hispanic children with would have developed and then persisted for many tens 2 US-born parents (autism spectrum disorder prevalence of thousands of years. However, the idea that autism was 2.39%) and otherwise similar children with 2 foreign- tolerated during human evolution because it is essentially a born parents (autism spectrum disorder prevalence 0.31%; tradeoff, an inherent “side-effect” of human brain function 8 Autism Research and Treatment that is required for survival, cannot be ruled out. A millions are affected by a wide range of allergic, inflam- second line of evidence pointing in favor of autism as a matory, and autoimmune disorders, and inflammation as a result of evolutionary mismatch rather than evolution is result of biome destabilization may play a key role in such the study, described above, showing that parental birth in common maladies as heart disease and dementia. With that a postindustrial culture seems to predispose offspring to in mind, the need for normalization of the biome cannot be autism [86]. Third, the observations (a) that the immune overemphasized, regardless of particular diseases that might system is profoundly destabilized by postindustrial culture be prevented by identifying and avoiding the environmental and (b) that brain development is closely tied with immune trigger. Thus, while it is hoped that a trigger that causes function, when considered together, argue strongly for the autism might be confidently identified in the near future, and idea that it is an evolutionary mismatch, not evolution, new cases of autism might be greatly reduced as a result, it is which is responsible for autism. Indeed, to the extent hypothesized that normalization of the human biome will be that human brain development is strongly tied to the necessary to avoid the full milieu of chronic immune-related immune system, it would be difficult to explain the idea diseases that plague postindustrial society. that postindustrial culture and the resulting consequences of evolutionary mismatches such as biome depletion do not profoundly affect brain development. Finally, the strong Acknowledgments association of autism with autoantibody production [3] The authors gratefully acknowledge the contribution of the and other immune abnormalities points toward the etiology Research Coordination Network in Ecological Immunology of autism as straight forward: another epidemic in the for facilitating discussion, and the Coalition for SafeMinds long list of hyperimmune associated epidemics caused by for providing the publication fee. The authors also thank biome depletion in postindustrial society. The fact that this Sharon L. Brenner, Zoie E. Holzknecht, and Susanne Meza- particular disease affects primarily the brain rather than Keuthen for helpful discussion. other organs or tissues undoubtedly leads to the tremendous complexity in the pathology associated with autism, but this complexity should not blind the medical community to the References simple roots that apparently underlie epidemics of all allergic and autoimmune diseases, or to the apparent solutions that [1] S. D. Bilbo, G. A. Wray, S. E. Perkins, and W. Parker, can currently be implemented to prevent those diseases [1]. “Reconstitution of the human biome as the most reasonable solution for epidemics of allergic and autoimmune diseases,” Medical Hypotheses, vol. 77, no. 4, pp. 494–504, 2011. 14. Summary/Conclusions [2]V.T.Ramaekers,N.Blau,J.M.Sequeira,M.C.Nassogne,and E. V. 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Research Article Prenatal and Postnatal Epigenetic Programming: Implications for GI, Immune, and Neuronal Function in Autism

Mostafa I. Waly,1 Mady Hornig,2 Malav Trivedi,3 Nathaniel Hodgson,3 Radhika Kini,3 Akio Ohta,3 and Richard Deth3

1 Department of Food Science and Nutrition, Sultan Qaboos University, Alkoudh 123, Muscat, Oman 2 Department of Epidemiology, Columbia University, New York, NY 10032, USA 3 Department of Pharmaceutical Sciences, Northeastern University, Boston, MA 02115, USA

Correspondence should be addressed to Richard Deth, [email protected]

Received 23 January 2012; Accepted 3 May 2012

Academic Editor: Antonio M. Persico

Copyright © 2012 Mostafa I. Waly et al. This is an open access article distributed under the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.

Although autism is first and foremost a disorder of the central nervous system, comorbid dysfunction of the gastrointestinal (GI) and immune systems is common, suggesting that all three systems may be affected by common molecular mechanisms. Substantial systemic deficits in the antioxidant glutathione and its precursor, cysteine, have been documented in autism in association with oxidative stress and impaired methylation. DNA and histone methylation provide epigenetic regulation of gene expression during prenatal and postnatal development. Prenatal epigenetic programming (PrEP) can be affected by the maternal metabolic and nutritional environment, whereas postnatal epigenetic programming (PEP) importantly depends upon nutritional support provided through the GI tract. Cysteine absorption from the GI tract is a crucial determinant of antioxidant capacity, and systemic deficits of glutathione and cysteine in autism are likely to reflect impaired cysteine absorption. Excitatory amino acid transporter 3 (EAAT3) provides cysteine uptake for GI epithelial, neuronal, and immune cells, and its activity is decreased during oxidative stress. Based upon these observations, we propose that neurodevelopmental, GI, and immune aspects of autism each reflect manifestations of inadequate antioxidant capacity, secondary to impaired cysteine uptake by the GI tract. Genetic and environmental factors that adversely affect antioxidant capacity can disrupt PrEP and/or PEP, increasing vulnerability to autism.

1. Introduction As all cells possess the same DNA, their differentiation into various cell types reflects stable suppression of some Neurological and behavioral symptoms implicate abnormal genes and activation of others, accomplished in large part brain development as a core pathophysiological feature of by epigenetic regulation. Such regulation involves reversible autism, but increasing evidence also indicates immune [1–8] modifications of both DNA nucleotides and histone proteins and gastrointestinal (GI) abnormalities in a significant subset [13]. These modifications or epigenetic marks favor or [1, 8–12]. This triad of dysfunctional systems provides not disfavor formation of nucleosomes, DNA/histone complexes only a more complete description of autism and autism spec- that maintain genes in a compacted, inactive state (hete- trum disorders (ASDs), but also an important opportunity rochromatin). Methylation of DNA is the most fundamental to consider the mechanisms which could result in the shared involvement of these three particular systems, especially epigenetic mark, enabling binding of proteins containing in the context of development. Recent elucidation of the methylDNA binding domains, such as methyl CpG binding central role of epigenetic regulation of gene expression in protein 2 (MeCP2). Proteins with methylDNA binding development presents a molecular framework within which domains recruit other proteins, including those capable of prenatal and postnatal maturation of neuronal, immune, and modifying histones at their tail regions [14]. Multiple sites GI systems can be viewed. and forms of histone modification (e.g., methylation, acety- 2 Autism Research and Treatment lation, and phosphorylation) make this a highly complex 2. Materials and Methods mode of regulation, affected by diverse signaling pathways + that adjust gene expression to local cellular metabolic 2.1. Purification of Regulatory T Cells. To purify CD4 CD25+ regulatory T cells, spleen cells and lymph node conditions [15]. Whereas certain epigenetic marks are to cells from C57BL/6 mice were combined as a cell source. some degree reversible, others are not readily reversed and, The cells were labeled with FITC-conjugated anti-CD24 once in place, these may be sustained for an entire lifespan and anti-CD8 mAbs and with anti-FITC microbeads. After and/or be transmitted across generations through germline the removal of CD8+ T cells and CD24-expressing B cells modifications [16]. Accordingly, epigenetic marking or pro- using an AutoMACS separator (Miltenyi Biotec, Auburn, gramming early in development has the potential to exert CA), regulatory T cells were purified by positive selection of long-lasting effects [17]. CD25+ cells using PE-conjugated anti-CD25 mAb and anti- There is considerable evidence that epigenetic pro- PE microbeads. All antibodies except for anti-CD25 mAb gramming is highly sensitive to changes in the cellular were from BD Biosciences (San Jose, CA). Other materials environment, as broadly defined [18]. Indeed, it appears that were from Miltenyi Biotec. Purity of CD4+ CD25+ cells was epigenetic regulation is a widely utilized adaptive mechanism higher than 95 %. to allow cells to maintain a favorable metabolic status under different conditions, including differential exposure to physiological substances (e.g., hormones, neurotransmitters, 2.2. RNA Isolation. RNA was isolated using the RNAqueous- or growth-regulation factors), xenobiotics (e.g., pollutants, 4PCR kit (Ambion, Naugatuck, CT), then treated with toxic chemicals), or even infectious agents (e.g., bacteria or DNase to eliminate contaminating genomic DNA, and viruses, fungi or parasites) [19]. Epigenetic regulation may quantified using an ND-1000 NanoDrop spectrophotometer. facilitate adaptation to changes in the cellular environment through stable alterations of cellular phenotype, potentially 2.3. cDNA Synthesis. cDNA synthesis was performed using resulting in progressive differentiation and maturation dur- the First strand cDNA synthesis (Roche, Nutley, NJ). 1 μg ing fetal and possibly postnatal development [20]. RNA was used as template, along with 1 mM dNTP mix, During prenatal development, nutritional support to 60 uM random hexamer primers and dH20 to make a final μ ◦ the fetus is provided via the transplacental circulation volume of 13 L. Samples were denatured at 65 C for 5 min- μ as a function of the available maternal nutriture, with utes and then placed on ice. Transcriptor RT (20 units/ L), μ metabolic consequences for both the mother and developing Protector RNase inhibitor (40 U/ L), 5x Transcriptor Reverse Transcriptase Reaction Buffer, and dH 0inafinalvolume child. During postnatal development, nutritional support is 2 of 7 μL were used to bring the final volume to 20 μL. This provided by oral food intake into the GI tract: breast-milk- ◦ was followed by incubation at 25 C for 10 min followed by or cow-milk-based formula at first, followed by introduction ◦ 30 min incubation at 55 C. The reverse transcriptase enzyme of other foods, usually in a controlled, gradual manner, but was inhibited by incubating at 85◦C for 5 min and then the provision of adequate nutritional resources to support cooling on ice. further development of the newborn is not assured. Thus, the prenatal/postnatal transition is a critical juncture in metabolic adaptation. This transition is particularly linked 2.4. Primers. Primers were designed using the Invitrogen OligoPerfect Designer to have approximately 50–60% GC to aerobic metabolism, as the delivery of oxygen via newborn content, an annealing temperature of 60◦C, and a length lungs and its ultimate rate of utilization must be balanced by of 20 bases. Glyceraldehyde phosphate dehydrogenase the available antioxidant capacity of body tissues. Epigenetic ff (GAPDH) expression was used for normalization. Primers regulation o ers an opportunity for adaptation during this for EAAT3 (designated EAAC1 in mice) and GAPDH were metabolic transition, allowing the level of ongoing aerobic EAAT3-Forward: 5-TTACAGCCACCGCTGCCAGC-3; metabolism in each organ system, tissue, and cell type to EAAT3-Reverse: 5-GGCAGCCCCACAGCACTCAG-3; be maintained in homeostatic equilibrium with antioxidant GAPDH-Forward: 5-TCTCCACACCTATGGTGCAA-3; metabolism. GAPDH-Reverse: 5-CAAGAAACAGGGGAGCTGAG-3. We use the term postnatal epigenetic programming (PEP) to describe the ongoing adaptive changes in gene 2.5. qRT-PCR Analysis. qRT-PCR was performed on dupli- expression occurring in response to the transition from fetal cate samples using the LightCycler 480 from Roche. 5 μLof to postnatal metabolism, as distinct from prenatal epigenetic cDNA was used for qRTPCR, along with 10 μM sense and programming (PrEP), at term that recognizes the dynamic antisense primers, 10 μL SYBR Green I Master from Roche, occurrence of similar changes which occur in utero. While anddH20inafinalvolumeof20μL. The following thermal it is clear that autism can result from a variety of genetic parameters were used: incubation for 5 min at 95◦C, followed and environmental factors, by focusing on factors affecting by 45 cycles of 95◦C for 10 sec, 60◦C for 20 sec and 72◦Cfor antioxidant and methylation status in the developing GI 30 sec, and lastly a single cycle of 95◦Cfor5sec,1minat65◦C tract, immune system, and brain, we hope to illuminate the and 97◦C for the melting curve. No template controls were pathological mechanisms underlying ASDs and other related run on each plate and dissociation curves were generated to disorders. determine any nonspecific products. Data was normalized to Autism Research and Treatment 3

GAPDH, and the ΔCt second derivative method was used for of intracellular redox status. The relatively high concentra- analysis of EAAT3 expression. tion of GSH within the cell imposes a pervasive influence on essentially all aspects of metabolism. Although all cell types and tissues rely upon the same core antioxidant mechanisms, 2.6. MS Assay. Brain cortex samples were obtained from variations on this central theme are recognized. For example, thimerosal-treated or untreated C57BL/6 and SJL/J mice, as the intracellular level of GSH in liver hepatocytes is close to per the previously published protocol [21]. MS activity in 10 mM [25], whereas in neurons it is 0.2 mM, some 50-fold mouse cortex was determined by measuring incorporation lower [26]. of radiolabel from [methyl-14C]methyltetrahydrofolate into As the rate of ROS formation must be quantitatively methionine, as previously described [22]. Enzyme assays balanced by the available antioxidant capacity, a multitude were performed under anaerobic conditions by bubbling of mechanisms have evolved to restrict aerobic metabolism nitrogen gas through stoppered vials for 1 hour at 37◦Cand when antioxidant status is low and vice versa. One prominent terminated by heating at 98◦C for 2 min after which samples example involves glutathionylation of cysteine residues in were cooled on ice. Radiolabeled methionine was separated complex I of the mitochondrial electron transport chain, from unreacted radiolabeled methylfolate by passing the which limits the flow of electrons, thereby restricting down- reaction mixture through an anion exchange column. The stream ROS formation [27]. In glutathionylation, GSSG, column was washed with 2 mL of water and the aqueous the oxidized form of glutathione, reacts with a reduced samples were collected and counted. Reported values are cysteine, resulting in GSH release and modification of the normalized to protein content and corrected for the counts protein in proportion to the prevailing redox state [28]. This observed in control assays in which sample enzyme was regulatory mechanism not only controls ROS formation but omitted. also provides an auxiliary pathway for glutathione reductase activity and production of GSH. Additional intracellular 2.7. GSH Measurement. A 100 μLaliquotofcortexsuper- control over the balance between ROS and antioxidant natant was incubated with 2 μL of monochlorobimane reservoirs is achieved by the capacity for reversal of the glu- (25 mmol/L) and 2 μL of glutathione-S-transferase reagent tathionylation of complex I by the action of glutaredoxin 2, was added, as provided by a commercial kit (Biovision, in conjunction with the selenoprotein thioredoxin reductase, Mountain View, CA). After a 30 min incubation at 37◦C, using NADPH-derived electrons [27]. fluorescence was read at 380 nm excitation and 460 nm More than ten studies have reported significantly lower emission. GSH content was determined by comparison with plasma levels of GSH in autism, accompanied by lower values from a standard curve using freshly prepared GSH. levels of cysteine [29–39]. Among these studies, the average decrease in GSH was 37%, representing a substantial reduction in antioxidant capacity. Several studies also reported a 2.8. Statistical Methods. Statistical analysis was carried out significant decrease in the GSH to GSSG ratio [8–20, 22–36], using Graph Pad Prism version 5.01. Data was expressed indicative of oxidative stress, consistent with other reports as mean ± standard error of the mean (SEM). Student’s ff of increased biomarkers of oxidative stress [29, 32, 40]. t-test was conducted to determine the di erences between One consequence of persistent oxidative stress, an increase individual groups. in glutathionylation of complex I of the mitochondrial electron transport chain, provides a potential explanation 3. Results and Discussion for the mitochondrial dysfunction frequently reported in autism [7]. Genetic factors affecting mitochondrial function 3.1. Antioxidant Capacity and Aerobic Metabolism. Aerobic may interact with oxidative stress. For example, SLC25A12, metabolism is unavoidably accompanied by the risk of the gene encoding the Ca2+-dependent aspartate-glutamate oxidative damage, most notably caused by reactive oxygen carrier isoform 1 (AGC1), has been identified as a putative species (ROS) generated as by-products of mitochondrial autism susceptibility gene [41]. AGC1 facilitates the supply oxidative phosphorylation and ATP production. Increased of NADH for oxidative phosphorylation, and elevated Ca2+ metabolic activity requires higher rates of ATP production levels will, therefore, increase ROS formation via increased and increased ROS formation. Maintenance of redox equilib- AGC1 activity. Higher Ca2+-dependent AGC1 transport rates rium requires inactivation of ROS before these by-products were demonstrated in autistic subjects [41], and it has been can oxidize cellular components, as well as repair of oxidized proposed that dyregulated Ca2+ levels may be a broadly biomolecules (proteins, lipids, DNA, and RNA). Catalase, influential factor in causing autism [42]. superoxide dismutase and the selenoprotein glutathione peroxidase inactivate ROS, and a wide array of redox-active proteins and enzymes participate in the repair of oxidized 3.2. Intestinal Absorption of Cysteine. Starting from birth, molecules and the maintenance of thiols in their reduced the systemic availability of cysteine to support GSH synthesis state, with glucose-derived NADPH serving as the source of is dependent upon absorption of the food-derived sulfur- reducing electrons [23, 24]. containing amino acids cysteine and methionine from the As elegantly reviewed by Schafer and Buettner [25], the GI tract (Figure 1). Adequate GI uptake of selenium is equilibrium between reduced and oxidized forms of glu- also critical for maintaining GSH in its reduced state. tathione (GSH and GSSG, resp.) is the primary determinant Intestinal epithelial cells express different transporters on 4 Autism Research and Treatment

GI tract Blood Brain

Astrocytes GI Blood epithelial cells brain barrier Cystine GSH GSH GSH EAAT3

Cysteine Cysteine Cysteine Cysteine EAAT3 Methionine Cystine Cystine

GSH Cysteine Dietary protein Neurons

Cysteine Cystine Liver

Figure 1: Absorption and systemic distribution of dietary cysteine and methionine. GI epithelial cells take up cysteine and methionine, with EAAT3 being particularly important for cysteine uptake in the distal ileum. A portion of methionine is converted to cysteine via the methionine cycle and transsulfuration of homocysteine. Absorbed cysteine is taken up by the liver, which releases oxidized cystine. Cystine, but not cysteine, is able to traverse the blood brain barrier and is taken up by astroctyes, which convert it to GSH. Cysteine from astrocyte- derived GSH is available for EAAT3-mediated neuronal uptake, supporting neuronal GSH synthesis. their luminal brush border surface that are critical for proportion of active transporters in the plasma membrane. uptake of sulfur and selenium-containing amino acids. These Oxidative stress inhibits EAAT3 transport activity [47], and amino acids are then subject to intracellular metabolism several cysteine residues have been identified as being critical before being ultimately released on the contralateral surface, for this mode of regulation, raising the possibility that either in their free form or incorporated into proteins or they might be targets for glutathionylation. Oxidative stress peptides, including GSH. Availability of transported cysteine affecting gut epithelium would, therefore, decrease EAAT3- ff or selenium can, therefore, a ect the GSH levels and redox dependent absorption of cysteine, with the local and systemic status of the epithelial cells themselves, as well as that of consequences of lower GSH levels. the adjacent cells located within the intestinal wall, including Cells express a multitude of adaptive mechanisms that immune cells. allow them to maintain homeostatic redox equilibrium, Among amino acid transporters in GI epithelial cells, of which the Keap1/Nrf2 system is a prominent example. EAAT3 (excitatory amino acid transporter 3, EAAC1) is Nrf2 (nuclear factor erythroid 2-like 2) is a transcription selective for cysteine and was initially cloned from GI factor protein with a high turnover rate, that is, tightly epithelial cells [43]. Subsequent studies revealed that EAAT3 bound to Keap-1 (Kelch-like ECH-associated protein 1); is most prominently expressed in the small intestine, espe- however, in response to a more oxidative redox state, Nrf2 cially in the terminal ileum [43, 44], a prominent site of is released from Keap-1 and moves to the nucleus where it inflammation in subjects with autism [45]. The highest levels promotes expression of a large number of genes via binding were found in crypt cells and lower villus regions, the locus to antioxidant response element (ARE) sites [48]. It has of multipotent stem cells that sustain the epithelial lining of been recently demonstrated that Keap1/Nrf2 mediates up- + + the gut [44]. EAAT3 is a member of a family of Na -andH - regulation of EAAT3 in response to oxidative stress [49]. dependent amino acid transporters named for their ability to Similar to autism, GI disorders such as celiac disease transport glutamate and aspartate, but EAAT3 is unique in its and inflammatory bowel disease have an autoimmune preference for cysteine. EAAT3 exists in equilibrium between component [50], and diets excluding gluten and/or casein the endoplasmic reticulum and the plasma membrane, (i.e., GF/CF diet) are frequently beneficial in both conditions. and its transport activity is regulated by the PI3 kinase Prompted by the recognition that digestion of both gluten signaling pathway, similar to insulin regulation of glucose and casein yields peptides with opiate activity [51, 52], we transporters [46]. In resting cells, about 75% of EAAT3 recently examined the activity of these peptides on EAAT3- resides inside the cell, where it is inactive, but activation mediated cysteine uptake and redox status in human GI of either PI3 kinase or protein kinase C increases the epithelial and neuronal cells and found that they significantly Autism Research and Treatment 5

Astrocyte Oxidative stress or Cystine Cysteine gluten/casein Growth Cysteine Cysteinylglycine GSH GSH factors (−) EAAT3 Neuron (+) GSSG GSH PI3-kinase γ-glutamylcysteine

Cysteine Partially blocked in neuronal cells Cystathionine Adenosine Adenosine D4SAH D4HCY HCY SAH (−) MethylTHF MethylTHF > Phospholipid Methionine 200 methylation synthase Methylation reactions THF THF e.g. D4SAM D4MET MET SAM DNA methylation PP+Pi ATP ATP PP+Pi

Dopamine

Figure 2: Redox and methylation pathways in neurons. The amino acid cysteine is rate-limiting for glutathione (GSH) synthesis and it is provided either by uptake from astrocyte-derived cysteine or by transsulfuration of homocysteine (HCY). The methionine cycle of methylation (lower right) depends upon both dietary methionine (MET) and remethylation of HCY by methionine synthase. Since formation of HCY from S-adenosylhomocysteine (SAH) is reversible and SAH inhibits methylation, decreased methionine synthase activity (e.g., caused by oxidative stress) both augments GSH synthesis and inhibits methylation reactions. Thus, redox status and methylation activity are closely linked. The D4 dopamine receptor carries out a cycle of dopamine-stimulated phospholipid methylation which is completely dependent upon methionine synthase and is therefore also sensitive to oxidative stress. Growth factors promote EAAT3 activity by increasing its location on the cell surface. EAAT3 activity is inhibited by oxidative stress and by food-derived opiate peptides. inhibited uptake [53]. Their action was associated with oxidizing conditions) are able to exert epigenetic effects a decrease in GSH levels and the SAM/SAH ratio, along via their influence on MS and the SAM/SAH ratio. Recog- with alteration in the expression of redox-related genes. nizing this metabolic relationship, we previously proposed Important differences, however, were observed between pep- a “redox/methylation hypothesis” of autism, linking this tides derived from bovine versus human casein, which may neurodevelopmental disorder to oxidative stress induced by contribute to the well-recognized benefits of breastfeeding. xenobiotic exposures [55], and now we apply this perspective to epigenetic programming of GI and immune function. 3.3. Postnatal Epigenetic Regulation (PEP). Methylation of During oxidative stress associated with GI inflammation, DNA and histones is fundamental to epigenetic regulation impaired EAAT3 activity will decrease availability of cys- of gene expression, providing factors affecting methylation teine for GSH synthesis, leading to increased ROS levels, capacity with an opportunity to affect development. S- diminished MS activity, a decrease in the SAM/SAH ratio adenosylmethionine (SAM) is the donor of methyl groups and a global decrease in methylation capacity, altering the to more than 200 methyltransferase reactions, including pattern of DNA and histone methylation in epithelial cells. DNA and histone methyltransferases [54], whereas its de- The distribution of EAAT3 suggests that this inflammation- methylated form, S-adenosylhomocysteine (SAH), inhibits induced epigenetic response will be most prominent in crypt methylation by virtue of its competition with SAM. The stem cells of the terminal ileum, where it can influence both SAM to SAH ratio determines methylation capacity for the proliferation and the functionality of the GI epithelium. all methylation reactions, providing an exceptionally broad Notably, the terminal ileum is also a critical location for folate influence over cellular metabolism. and vitamin B12 absorption [56, 57]. The SAM/SAH ratio is responsive to the activity of Postnatal infant nutrition is most commonly provided by methionine synthase (MS), the folate, and vitamin B12- either human breast milk or bovine milk. Human breast milk dependent enzyme that converts homocysteine (HCY) to is comparatively rich in sulfur-containing amino acids, and methionine (MET) as part of the methionine cycle of colostrum contains 2.5-fold higher levels versus mature milk methylation (Figure 2). Its vitamin B12 (cobalamin) cofactor [58]. A comparison of plasma cysteine levels in 12-week- renders MS activity highly sensitive to oxidative stress; old infants found significantly higher levels for breast-fed changes in cellular redox status (shifts toward reducing or versus formula-fed infants [59]. Thus, breast feeding appears 6 Autism Research and Treatment to provide a superior supply of the essential raw materials for GI tract can be matched to the ongoing level of metabolic synthesis of the antioxidant GSH. As this source of cysteine activity. In other words, the supply of antioxidant must be is made available, EAAT3 and the factors that modulate its matched to the demand for antioxidant, and the rate of activityserveascriticalregulatorsoftheextenttowhich postnatal growth and development must be restricted so as cysteine may be absorbed. to not exceed the supply provided by the GI tract. Breast milk contains a number of different growth fac- As a developmental disorder in which the availability tors, including insulin-like growth factor-1 (IGF-1), epider- of GSH is reported to be reduced by almost 40%, autism mal growth factor (EGF), vascular endothelial growth factor can be viewed as a syndrome resulting from an imbalance (VEGF), hepatocyte growth factor (HGF), and transform- between antioxidant supply and demand—essentially, a state ing growth factor-β1andβ2(TGF-β1/β2), among others of oxidative stress that interrupts the normal epigenetically [60, 61]. These factors activate PI3 kinase and mitogen- based program of development. In the absence of a large- activated protein (MAP) kinase signaling pathways to achieve scale prospective study, it is not possible to determine their growth-promoting effects, which are associated with whether the autism-associated GSH deficit is present from increased metabolic activity and its attendant increase in birth or begin during postnatal development. The former ROS production. We recently demonstrated the ability case would likely reflect a significant maternal deficit in of IGF-1 and other PI3 kinase-activating growth factors antioxidant resources, and the finding that plasma levels to stimulate EAAT3-mediated cysteine uptake in cultured of GSH and the SAM/SAH ratio are significantly decreased human neuronal cells, accompanied by an increase in GSH in mothers of autistic children [36] is consistent with levels, an increased SAM/SAH ratio, and increased DNA such a possibility. Under the latter scenario, the imbalance methylation [62]. HGF, acting via the MAP kinase pathway, might develop in a progressive manner during postnatal increases expression of the two enzymes which convert development if escalating metabolic activity and ROS pro- cysteine to GSH [63]. Thus, growth factors promote GSH duction outstripped the ability of the GI tract to provide synthesis via both increased cysteine uptake and transcrip- sufficient antioxidant, and inhibition of EAAT3-mediated tional effects, corresponding to short-term and longer-term cysteine uptake in response to developing oxidative stress effects, respectively. These observations imply that breast- would also exacerbate this self-reinforcing cycle. Postnatal milk-derived growth factors can stimulate cysteine uptake exposure to agents which interfere with antioxidant capacity, and increase GSH in the GI epithelium, coincident with including mercury, lead, pesticides, and other xenobiotics, the initiation of postnatal digestion. The extent to which could shift the redox balance sufficiently to initiate develop- antioxidant resources (i.e., cysteine and GSH) are made mental regression secondary to impaired methylation and its available to the rest of the body depends upon the efficiency epigenetic consequences. of this process. Microbes within the GI tract compete for nutrient Human breast milk is also rich in selenium, and breast- resources in a normally symbiotic manner, but dysbiosis fed infants have higher selenium levels than formula-fed can intervene when the normal array of organisms is infants [64]. A significant proportion (up to 30%) of breast displaced or skewed. GI dysfunction, which is common in milk selenium is in the form of glutathione peroxidase (GPx), autism [8–12], may be associated with a failure of the GI which is hydrolyzed to selenocysteine during digestion. epithelium to absorb antioxidant nutrients, such as cysteine The importance of GPx activity in breast milk may be to or selenocysteine. Increased availability of these critical combat oxidation, especially of vulnerable omega-3 fatty antioxidant nutrients for intestinal microbes may encourage acids. However, GPx appears to also represent an important the growth of previously restricted microorganisms, while reservoir for delivery of selenium to the developing infant. As diminishing the growth of others. Once established, the a structural analog of cysteine, breast-milk-derived seleno- dysbiotic microbial population will decrease availability of cysteine can also be transported by EAAT3 and contributes antioxidant nutrients for absorption by further competing to the antioxidant capacity and redox status [65]. with the host, perpetuating the antioxidant deficit. As MS activity and the SAM/SAH ratio are responsive The importance of PEP varies by cell and tissue types, to redox status, it is reasonable to propose that the post- depending upon the extent of the developmental matu- natal transition to independent nutrition is associated with ration of specific cells and organs at birth. Delaying the epigenetic responses and that growth factor stimulation of development of selected neural networks (e.g., cortex and cysteine uptake is a key event in this transition, which we hippocampus) until after birth allows their programming call postnatal epigenetic programming or PEP.Although PEP to be guided by experience, in contrast to other networks is an ongoing body-wide process affecting the development (e.g., brainstem respiratory centers) which must function of all tissues, but we specifically propose that the GI immediately upon birth. Indeed, unique features of human tract plays a uniquely important role as the source of brain allow PEP to be dynamically exploited throughout the nutritional support for metabolism, and that availability of lifespan, providing the capacity for memory [66]. In a similar antioxidant resources is the critical determinant of the level manner, maturation of the immune system is restrained of metabolic activity that may be achieved without cellular until birth so as to optimize its response to environmental damage. Epigenetic regulation of gene expression provides a exposures, while at the same time minimizing its poten- molecular mechanism by which the functional state of the tial for autoimmunity. While this perspective encompasses Autism Research and Treatment 7 the triad of neuronal, immune, and GI symptom domains in reliance on and retention of selenoproteins are an insufficient autism, the prominence of each component may vary among means of maintaining antioxidant defenses under certain individuals, reflecting, in part, genetic differences in the conditions, such as exposure to mercury. Selenoproteins vulnerability to oxidative stress and impaired methylation. are exquisitely sensitive to inhibition by mercury, and any mercury that penetrates the brain will interfere with redox 3.4. Redox Signaling in Human Brain. As previously noted, signaling and disrupt normal epigenetic regulation [70, 71]. cysteine availability is limited for GSH synthesis. However, Very tight binding of mercury by selenoproteins, especially the level of cysteine in cerebrospinal fluid (CSF) is only to selenoprotein P, contributes to the long-term retention one tenth the level in blood [67], meaning that the raw of mercury in the brain and its accumulation across the material for making antioxidants is much scarcer in the lifespan, and it has been proposed that selenoprotein P,which brain. Indeed, the GSH level in neurons is the lowest contains ten selenocysteine residues, has evolved to serve a reported for any cell type [25], despite the fact that the specific role to protect against mercury toxicity [72]. brain utilizes oxygen at a ten times higher rate than other In addition to conversion of HCY to MET, MS carries tissues. As illustrated in Figure 2, neurons obtain their out a second reaction, providing methyl groups to the D4 cysteine from neighboring astrocytes, which release GSH dopamine receptor; the D4 receptor, when stimulated by that is subsequently broken down to cysteine, which is then dopamine, subsequently transfers these methyl groups to available for uptake by neurons, depending upon the level membrane phospholipids—a unique activity carried out of EAAT3 activity, which is controlled by growth factors. by the D4 dopamine receptor [73]. Genetic variants of Growth factors utilize redox status to regulate neuronal the D4 receptor (such as the 7-repeat variant) have been function via the intermediate involvement of MS, a form linked to novelty-seeking behavior and are important risk of “redox signaling.” By allowing more cysteine into cells factors for attention-deficit/hyperactivity disorder (ADHD) and increasing GSH synthesis, growth factors increase MS [74]. Although the role of dopamine-stimulated phospho- activity and increase DNA methylation. These increases in lipid methylation remains incompletely understood, we first MS activity and DNA methylation lead to epigenetic changes proposed that it facilitates attention by modulating the in gene expression as well as many other metabolic effects frequency of neural networks, promoting their synchro- through >200 methylation reactions, all affected by the nization [75]; others subsequently confirmed dopamine- SAM/SAH ratio. The increase in antioxidant level also allows stimulated phospholipid methylation to be critically involved cells to safely increase their oxidative metabolism (that is, in neuronal synchronization during attention [76]. As D4 their mitochondrial activity); the resultant increase in ATP receptor-mediated phospholipid methylation is absolutely supports a higher level of neuronal activity. In this way, redox dependent upon MS activity, it is thus also dependent on signaling not only regulates gene expression but also controls EAAT3, raising the possibility that ADHD might involve a the level of neuronal function. decrease in MS activity secondary to oxidative stress. A second brain-specific feature that augments redox signaling is a brain-specific limitation in the level of trans- 3.5. Redox and the Immune Response. In the past several sulfuration activity, which restricts conversion of HCY to years, a previously unappreciated role for redox regulation cysteine [68]. This unique feature contributes to the low of the immune response has been elucidated, involving GSH level in neurons, making them more responsive to extrusion of GSH from activated antigen presenting cells growth factor-induced EAAT3 activation. Together with the (APCs, e.g., dendritic cells, macrophages, or B cells), fol- limited availability of extracellular cysteine, this makes epi- lowed by release of its cysteine, which is then taken up by T genetic regulation exceptionally dynamic. Along with other cells [77] This relationship is analogous to the relationship methylation-dependent consequences of redox signaling, between astrocytes and neurons illustrated in Figure 2. the long-term nature of epigenetic effects endows neurons Uptake of this cysteine by na¨ıve CD4+ CD25− effector T with the ability to create “metabolic memories” that are cells (Teff) leads to their activation and proliferation, in coordinated with experience via the input of sensory systems. association with increased GSH synthesis and the epigenetic This view is consistent with recent studies identifying the consequences thereof. Activated Teff cells release cytokines central role of epigenetic changes in learning and memory that attract other cells and promote inflammation, as well as formation [66, 69]. Compromised function of these redox- stimulate B cell activation and antibody production (Helper dependent systems could contribute to the types of cognitive T cells). However, the extent of Teff cell activation is limited function deficits and neurodevelopmental delays that are by CD4+ CD25+ Foxp3+ regulatory T cells (Treg) that com- primary to autism. pete with Teff cells for available extracellular cysteine, thereby The restricted availability of GSH in the brain is partly suppressing immune and autoimmune responses [77]. compensated for by the presence of alternate mechanisms Recognizing this newly described mode of T cell regu- for sustaining antioxidant capacity, including an increased lation, we hypothesized that EAAT3 might be involved in dependence upon selenoproteins. The brain has developed cysteine uptake. To evaluate this possibility, we used qRT- the capacity to retain selenium under conditions of scarcity, PCR to determine whether EAAT3 was expressed in murine even when other tissues become depleted. Nonetheless, T cells and compared the level of expression in T cell subsets, 8 Autism Research and Treatment

2 40

30 ∗ 1.5 20 ∗∗ ∗∗ g/mg protein) μ

GSH ( 10 1

0 C57BL6/J SJL/J Thimerosal C57BL6/J 0.5 Thimerosal SJL/J

EAAT3 expression normalized to GAPDH normalized to expression EAAT3 Figure 4: GSH levels in frontal cortex of SJL/J and C57BL6/J mice with or without thimerosal treatment. GSH levels were significantly lower (P<0.05) in cortex of SJL/J autoimmune-prone mice compared to C57BL6/J strain mice. GSH levels were not affected 0 by thimerosal treatment. UNSEP CD4+ T cells CD8+ T cells B cells (a) expression by immune cells provides a mechanism for them to take up cysteine in a competitive manner, and higher EAAT3 expression in Treg cells may underlie the ability of ∗ this cell subset to limit the immune response and restrict autoimmunity. 4 The SJL/J strain of mice has been widely employed as a model for autoimmunity, but these mice also exhibit deficits in GSH, similar to those reported in autistic children. In SJL/J mice, the deficit is due to mutations in the enzymes which synthesize GSH; factors underlying GSH deficits in autism are as yet unknown. In a previous study, we showed that the thiol-reactive compound thimerosal caused 2 growth impairment, as well as neurodevelopmental and neurochemical effects in SJL/J mice, but not in C57BL6/J mice, including upregulated brain levels of EAAT3 [21]. To EAAT3 expression normalized to GAPDH normalized to expression EAAT3 further characterize the role of redox and methylation status in autoimmunity, we measured both GSH levels and MS activity in the liver and brain of control and thimerosal- 0 treated SJL/J mice, as compared to C57BL6/J mice. GSH Treg CD4+ levels were significantly lower in both liver and brain of SJL/J (b) as compared with the levels found in C57BL6/J mice, but they did not differ as a function of thimerosal treatment Figure 3: Expression of EAAT3 in murine lymphocyte subsets. in either strain (Figure 4). Methionine synthase activity Spleen and lymph node-derived lymphocytes were separated by was also significantly lower in SJL/J versus C57BL6/J mice, magnetic cell sorting, followed by qRT-PCR analysis for EAAT3 ff (EAAC1) expression. (a) EAAT3 expression was significantly higher but not di erent in thimerosal-treated animals (Figure 5). (P<0.05) in CD4+ T cells as compared to unseparated lymphocytes These results confirm the occurrence of low GSH in the (UNSEP), CD8+ T cells, or B cells. (b) EAAT3 expression was autoimmune-prone SJL/J strain, along with the novel finding significantly higher (P<0.05) in FoxP3+ Treg cells. that MS activity is reduced, although neonatal exposure to thimerosal did not alter these features. Expression of EAAT3 was significantly lower in CD4+ T-cells from SJL/J mice separated on the basis of surface epitopes by magnetic cell versus C57BL6/J mice, suggesting that autoimmunity is asso- sorting. As illustrated in Figure 3(a), EAAT3 expression was ciated with impaired capacity for cysteine uptake (Figure 6). detected in unseparated cells, as well as in CD4+ and CD8+ T Together these findings provide support for the roles of cells and B cell populations, with the highest expression level oxidative stress and impaired methylation in autoimmunity. in CD4+ cells. Further analysis showed that the expression Abnormal immune function, including, but not limited of EAAT3 by Treg cells is almost two-fold higher than to, increased autoimmunity, has been extensively docu- that in unseparated CD4+ cells (Figure 3(b)). Thus, EAAT3 mented in autism. Of particular relevance are autoantibodies Autism Research and Treatment 9

20 2

∗

1.5 10 ∗∗ ∗∗

5 ∗ ∗∗ 1 MS activity (pmol/min/mg protein)

0 expression EAAT3 MethylB12 HydroxoB12 0.5 C57BL6/J SJL/J Thimerosal C57BL6/J Thimerosal SJL/J ∗ Figure 5: Methionine synthase activity in cortex of SJl/J and C57BL6/J mice with or without thimerosal treatment. Methionine 0 synthase activity was measured in the presence of either methylco- C57 SJL balamin (MethylB12) or hydroxocobalamin (HydroxoB12). Activity was significantly lower in SJL/J autoimmune-prone mice compared C57BL6/J to the C57BL6/J strain, irrespective of thimerosal treatment. SJL/J Activity was higher in the presence of methylcobalamin. ∗P<0.02 compared to C57BL6/J group, ∗∗P<0.001 compared to C57BL6/J Figure 6: Expression of EAAT3 in CD4+ T-cells from C57BL6/J group. andSJL/Jmice.Spleenandlymphnode-derivedlymphocyteswere separated by FACS analysis cell sorting, followed by qRT-PCR analysis for EAAT3 (EAAC1) expression. EAAT3 expression was significantly higher in CD4+ T-cells from C57BL6/J mice versus targeting brain and neuronal proteins [2, 3, 5]aswellas autoimmune-prone SJL/J mice (P<0.05). autoantibodies targeting the folate receptor [78–80]. The latter were initially linked to the relatively rare cerebral folate deficiency syndrome [78] but recently were found to 4. Conclusions be present in a high proportion (75%) of autistic subjects [80], and a casein-free diet greatly reduced the circulating The prominence of neurological, GI, and immune symptoms level of anti-folate receptor antibodies [79]. The number in autism suggests a shared mechanism of dysregulation that of circulating Treg cells was reported to be significantly may relate to the significant deficits in systemic reservoirs lower in autistic subjects, with 73% showing abnormally low of antioxidant GSH reported in this neurodevelopmental levels [4]. The Treg cell decrease was more prominent in disorder. Impaired GI absorption of cysteine, the essential the more severe cases, as well as among individuals with GSH precursor, is proposed as a crucial factor in the allergic manifestations such as asthma and atopic dermatitis. pathogenesis of this triad of symptoms, wherein reduced Although autistic subjects with low Treg cells did not cysteine availability leads to a condition of local and sys- manifest clinical or laboratory-based signs of autoimmunity temic oxidative stress, and subsequent disruption of normal (arthritis or arthralgia, elevated erythrocyte sedimentation epigenetic regulation of gene expression. In some cases, the rate, or leucopenia), the frequency of autoimmunity was extent of impaired cysteine absorption may be severe enough higher in their families. The same researchers found ele- to result in overt GI inflammation, while in other cases vated markers of oxidative stress (increased plasma F2- the restriction may only alter immune and/or neurological isoprostane and/or decreased GPx activity) in 89% of autistic development and function. Awareness of the redox-based subjects and evidence of antineuronal antibodies in 55%, linkage between GI, brain, and immune systems serves to leading them to suggest a link between oxidative stress illuminate a number of diseases whose origins may reflect and autoimmunity [5]. Notably, expression of Foxp3, the the critically important roles of PrEP and PEP. Indeed, transcription factor which defines Treg cells, is epigenetically adaptive epigenetic responses to changes in redox status are regulated, with increased expression when DNA methylation likely to play a critical role in diseases arising across the is decreased [81], and Treg cell levels are elevated in lifespan, especially those that can be traced to environmental male, but not female, SJL mice [82]. As illustrated in exposures that interfere with antioxidant homeostasis. Figure 7, these observations suggest that autoimmunity in autism may be related to inflammation in the distal ileum, Acknowledgment where impaired activity of EAAT3 in Treg cells can lead to formation of autoantibodies directed against the folate This report was supported in part by a research grant from receptor. SafeMinds to R Deth. 10 Autism Research and Treatment

TerminalTerminalalal partparpaa t of ileocolicileoiil colic c artearteryr CecaCecalal brbbranchesan IleaIleall branchesbrb

Ileum Terminal ileum

Cecum AAppendicularpp ararteryte

Vermiformf process

GI Anti-FR-Ab Body lumen

APC Epigenetic regulation Growth factors (+) + FR CD4 Redox status Opiate peptides (−) Na¨ıve T-cell GSH GSH GSSG GSH CYS CYS Redox CYS status Se-CYS Treg CYS Se-proteins Se-CYS cell EAAT3 Mucosal immune cells GI epithelial cell Figure 7: Cysteine and selenocysteine uptake in the terminal ileum regulates autoimmunity. EAAT3 transports diet-derived cysteine (CYS) and selenocysteine (Sel-CYS) into GI epithelial cells in the terminal ileum. Within the cell, cysteine is directed to GSH synthesis and selenocysteine is incorporated into selenoproteins, which maintain GSH in its reduced state. EAAT3 activity is regulated by multiple factors, including intracellular redox status, Nrf2-dependent transcription, growth-factor-dependent translocation to the cell surface, and the inhibitory eﬀects of casein and gluten-derived opiate peptides. Systemic availability of cysteine and GSH depends upon these terminal ileum events, as does the local mucosal environment. Immune cells in the terminal ileum can be a source of auto-antibodies, including anti-folate receptor (FR) antibodies, which are commonly present in subjects with autism [80]. Under oxidative stress conditions, EAAT3- mediated uptake of cysteine by Treg cells is impaired, limiting their suppression of na¨ıve CD4+ T cells, and increasing autoimmune responses to antigens present locally, such as the folate receptor.

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cerebral folate deﬁciency syndrome,” Developmental Medicine and Child Neurology, vol. 50, no. 5, pp. 346–352, 2008. [80] R. E. Frye, J. M. Sequeira, E. V. Quadros, S. J. James, and D. A. Rossignol, “Cerebral folate receptor autoantibodies in autism spectrum disorder,” Molecular Psychiatry. In press. [81] J. K. Polansky, L. Schreiber, C. Thelemann et al., “Methylation matters: Binding of Ets-1 to the demethylated Foxp3 gene contributes to the stabilization of Foxp3 expression in regulatory T cells,” Journal of Molecular Medicine, vol. 88, no. 10, pp. 1029– 1040, 2010. [82] S. Hussain, S. J. Kirwin, and S. A. Stohlman, “Increased T regulatory cells lead to development of Th2 immune response in male SJL mice,” Autoimmunity, vol. 44, no. 3, pp. 219–228, 2011. Hindawi Publishing Corporation Autism Research and Treatment Volume 2012, Article ID 205362, 4 pages doi:10.1155/2012/205362

Research Article Decreased Levels of EGF in Plasma of Children with Autism Spectrum Disorder

Charity Onore,1, 2 Judy Van de Water,2, 3 and Paul Ashwood1, 2

1 Department of Medical Microbiology and Immunology, University of California, Davis, USA 2 The M.I.N.D. Institute, University of California, Davis, Sacramento, CA 95817, USA 3 Division of Rheumatology, Allergy and Clinical Immunology, University of California, Davis, USA

Correspondence should be addressed to Paul Ashwood, [email protected]

Received 14 September 2011; Revised 3 November 2011; Accepted 16 December 2011

Academic Editor: Antonio M. Persico

Copyright © 2012 Charity Onore et al. This is an open access article distributed under the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.

Autism Spectrum Disorder (ASD) is a neurodevelopmental disorder estimated to affect 1 in 110 children in the U.S., yet the pathology of this disorder is not fully understood. Abnormal levels of several growth factors have been demonstrated in adults with ASD, including epidermal growth factor (EGF) and hepatocyte growth factor (HGF). Both of these growth factors serve important roles in neurodevelopment and immune function. In this study, concentrations of EGF and HGF were assessed in the plasma of 49 children with ASD aged 2–4 years old, and 31 typically developing controls of a similar age as part of the Autism Phenome Project (APP). Levels of EGF were significantly reduced in the ASD group compared to typically developing controls (P = 0.003). There were no significant differences in HGF levels in young children with ASD and typically developing controls. EGF plays an important role in regulating neural growth, proliferation, differentiation and migration, and reduced levels of this molecule may negatively impact neurodevelopment in young children with ASD.

1. Introduction [10–12], which may be influenced by atypical growth factor activity. Growth factor dysregulation may contribute to ASD Autism Spectrum Disorder (ASD) is a developmental disor- pathology by directly affecting CNS development, and/or by der characterized by impairments in social interaction and augmenting immune function. communication, and the presence of restricted behaviors or Epidermalgrowthfactor(EGF)andhepatocytegrowth interests [1]. According to the most current CDC estimate, factor (HGF) are both involved in the growth and prolifera- ASD affects 1 in 110 children in the US [2], yet the patho- tion of several cell types, including neurons and glia of the physiology of the disorder is largely unknown. Recently, sev- CNS. EGF is present at high levels in the central nervous eral growth factors have been found to be dysregulated in a system (CNS) and plays a critical role in controlling pro- substantial proportion of adults with ASD [3]. liferation and differentiation of nervous tissue during neu- In the central nervous system, growth factors can regulate rogenesis [13, 14]. In addition to the function of EGF as a the processes of neuronal growth, differentiation, and pro- CNS growth factor, it is a central factor in promoting wound liferation, as well as regulating neuronal survival, neuronal healing. EGF is expressed at sites of injury and inhibits the migration, and the formation or elimination of synapses [3]. activity of nitric oxide synthase, preventing inflammation In addition to their central function in regulating neuro- [4, 15]. EGF deficiency results in several neurological, development, current literature has also illustrated the dual gastrointestinal, dermal, and pulmonary abnormalities in nature of many growth factors as immune modulators and animal models [16]. An increased frequency of EGF single highlighted their involvement in crosstalk between the im- nucleotide polymorphisms has been reported in ASD, as well mune system and the central nervous system (CNS) [4–7]. as lower plasma EGF levels in adults with autism [17, 18]. Many studies suggest the presence of aberrant immune HGF also plays a dual role as a neurological growth factor activity in ASD, in the CNS [8, 9] and in the periphery and an immune modulator. HGF can modulate immune 2 Autism Research and Treatment responses by signaling through the MET receptor on antigen was measured on a Wallac Victor3 multilabel-plate reader presenting cells, which results in a tolerogenic phenotype (PerkinElmer, Boston, MA) at 450 nm. with reduced proinflammatory cytokine production and cellular activity [5]. HGF is also essential for normal neu- 2.3. Statistical Analysis. Statistical analysis to compare levels rodevelopment, and disruption of HGF signaling results in of growth factors between ASD and TD groups was con- complex alterations in GABAergic neuron development in ducted with unpaired Student’s t-test. All analyses were con- the forebrain of animal models [19]. Lower levels of HGF ducted with GraphPad Prism statistical software (GraphPad in sera of autistic adults have been described [20], as well Software Inc., San Diego, CA). as decreased expression of the HGF receptor in postmortem brain samples [21]. To determine if there exists a differential profile for 3. Results peripheral blood growth factor levels in ASD, we analyzed plasma EGF and HGF in well-characterized young children Plasma levels of EGF were approximately 3-fold lower in . ± . aged 2–4 years old with a diagnosis of ASD and unrelated the ASD group (23 1 6 2 pg/mL) compared with the TD . ± . P = . typically developing children who were frequency-matched group (76 9 20 0pg/mL)( 0 003). Plasma levels of HGF ff for age. In addition, levels of plasma EGF and HGF were were not statistically di erent between children with ASD . ± . . ± . investigated for any associations with clinical behavioral and (423 5 20 9 pg/mL) and TD controls (436 6 19 2pg/mL) developmental outcomes. (Figure 1).

2. Methods and Materials 4. Discussion In this study, we investigated the levels of EGF and HGF, 2.1. Subjects and Behavioral Assessments. Eighty study par- two growth factors involved in neurodevelopment. Previous ticipants aged between 2–4 years of age were recruited as studies have found decreased levels of EGF in high function- part of the APP [22]. Participants consisted of 49 children ing adults with autism [18], but no studies have looked at with ASD (median age of 2.88 years, interquartile range children with ASD who are close to the onset of the disorder. 2.66–3.41 years, 42 males) and 31 typically developing (TD) We found that levels of plasma EGF were significantly children (median age of 2.96 years, interquartile range reduced in young children with ASD as compared with 2.85–3.27, 20 males). Diagnostic instruments included the similarly age-matched typically developing control children. Autism Diagnostic Observation Schedule-Generic (ADOS- This finding is consistent with that seen in adults with high G) [23] and the Autism Diagnostic Interview-Revised (ADI- functioning autism and may suggest that a deficiency in EGF R) [24]. All diagnostic assessments were conducted or is persistent throughout the time course of ASD. directly observed by trained, licensed clinical psychologists Current research has demonstrated that EGF is involved who specialize in autism and had been trained according in growth, differentiation, and maintenance of several tissues to research standards for these tools. Inclusion criteria for including the CNS and the gastrointestinal tract (GI) [14]. ASD were taken from the diagnostic definition of ASD Notably, abnormalities of both the CNS and the GI have been in young children formulated and agreed upon by the reported in ASD [26].IntheCNS,EGFservesasapotent Collaborative Programs of Excellence in Autism. Inclusion neurotrophic factor, and in vivo studies have demonstrated criteria for TD controls included developmental scores the effect of EGF in promoting proliferation, differentiation, within two standard deviations of the mean on all subscales survival, and migration of multipotent neural progenitor of the MSEL. Exclusion criteria for TD controls included cells and the differentiation of these cells into astrocytes and a diagnosis of mental retardation, pervasive developmental neurons [27]. In this paper, we were limited to investigating disorder or specific language impairment, or any known EGF in plasma and not in cerebral spinal fluid. However, developmental, neurological, or behavioral problems. TD EGF rapidly transports across the blood-brain barrier [28], children were screened and excluded for autism with the suggesting peripheral EGF levels could be representative of Social Communication Questionnaire (scores > 11) (SCQ— levels in the CNS that may impact neurodevelopment. Lifetime Edition) [25]. In the GI tract, EGF is necessary for normal development of the intestinal mucosa, and mice deficient in EGF receptor 2.2. Measurement of EGF. Peripheral blood was collected in suffer from symptoms similar to those of necrotizing ente- acid-citrate-dextrose Vacutainers (BD Biosciences, San Jose, rocolitis, with gradual destruction of villi, become severely CA). The plasma fraction was immediately harvested by malnourished, and typically die before postnatal day 8 centrifugationandstoredasaliquotsat−80◦C until the date [16]. EGF promotes wound healing in animal models of of assay. Plasma levels of EGF were determined by Human ulcerative colitis [29, 30]. Although GI symptoms affect a EGF enzyme-linked immunosorbant assay (ELISA) kit (R&D large proportion of children with ASD, the exact nature and Systems, Minneapolis, MN). Plasma levels of HGF were extent of GI inflammation in ASD are still controversial [31– determined by Human HGF enzyme-linked immunosorbant 34]. Interestingly, preliminary investigations in this study assay (ELISA) kit (R&D Systems). The assays were performed show that EGF levels are associated with increased bloating according to the protocols provided by the manufacturer, in children with ASD, as well as with raw, standard, and and all samples were assayed in duplicate. Optical density percentile rank scores on the Peabody picture vocabulary Autism Research and Treatment 3

P = 0.0032 500 100

400

300

HGF (pg/mL) 200 EGF (pg/mL)

25 100

0 0 ASD Control ASD Control

ASD ASD Control Control (a) (b)

Figure 1: Plasma levels of EGF and HGF. Levels of EGF and HGF in peripheral blood plasma from ASD participants and similarly aged typically developing controls as measured by ELISA (data is shown as mean and SEM). EGF levels are significantly lower in ASD subjects in comparison to typically developing controls (P = 0.003). HGF levels did not differ significantly between the two groups. Significance was determined by two-tailed unpaired Student’s t-test.

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Van Der Kooy, “In vivo growth factor expansion Hindawi Publishing Corporation Autism Research and Treatment Volume 2012, Article ID 959073, 13 pages doi:10.1155/2012/959073

Review Article HLA Immune Function Genes in Autism

Anthony R. Torres,1 Jonna B. Westover,1 and Allen J. Rosenspire2

1 Center for Persons with Disabilities, Utah State University, 6804 Old Main Hill, Logan, UT 84322, USA 2 Department of Immunology and Microbiology, School of Medicine, Wayne State University, 221 Lande Building, Detroit, MI 48201, USA

Correspondence should be addressed to Anthony R. Torres, [email protected]

Received 10 October 2011; Accepted 11 November 2011

Academic Editor: Judy Van de Water

Copyright © 2012 Anthony R. Torres et al. This is an open access article distributed under the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.

The human leukocyte antigen (HLA) genes on chromosome 6 are instrumental in many innate and adaptive immune responses. The HLA genes/haplotypes can also be involved in immune dysfunction and autoimmune diseases. It is now becoming apparent that many of the non-antigen-presenting HLA genes make signiﬁcant contributions to autoimmune diseases. Interestingly, it has been reported that autism subjects often have associations with HLA genes/haplotypes, suggesting an underlying dysregulation of the immune system mediated by HLA genes. Genetic studies have only succeeded in identifying autism-causing genes in a small number of subjects suggesting that the genome has not been adequately interrogated. Close examination of the HLA region in autism has been relatively ignored, largely due to extraordinary genetic complexity. It is our proposition that genetic polymorphisms in the HLA region, especially in the non-antigen-presenting regions, may be important in the etiology of autism in certain subjects.

1. Autism the prenatal period or infancy stage of life. Exposure to mercury, maternal viral infections, autoimmune disorders, Leo Kanner first described autism in 1943 [1] after finding 11 and the inheritance of certain gene combinations have been children with common symptoms of obsessiveness, stereo- implicated in the etiology. Unfortunately, none of these areas typy, and echolalia at Johns Hopkins University. Autism have given clear answers as to the etiology. Fortunately, remained an esoteric disorder for several decades until psychologists have made significant strides in treating chil- physicians and parents connected these symptoms with an dren and it appears the earlier behavioral treatment starts, increasing number of patients. It is important to note that the better the outcome. Nevertheless, medical researchers the diagnostic criteria have been modified over the years continue to search for the cause(s) of ASD. This paper to include a broader category of symptoms, thus increasing discusses a possible role for the immune system, and in the number of children diagnosed with the disorder, now particular immune function genes in the human leukocyte referred to as Autism Spectrum Disorder (ASD) [2]. Current- antigen region (HLA), as a research area that should be more ly, the Centers for Disease Control and Prevention (CDC) closely investigated. states that the incidence of ASD is 1 out of 110 children in the United States [3]. The severity of ASD varies greatly with the most severe forms, much like Kanner autism, displaying 2. Infections language regression, seizures, and lower IQ. Altevogt et al. [4] have suggested that autism, or more properly ASD, is not One of the first areas of interest in the 1960s and 1970s was a single disorder, but a collection of similar disorders each the search for an infectious agent that might be involved in with different characteristics and perhaps etiologies. the etiology of ASD [5]. During this time, there were many Even after several decades of research, there is much case reports in the literature that suggested an association debate around the world on the etiology of ASD. It is clear between congenital rubella infection and resultant autistic that ASD results from abnormal brain development in either behaviors. However, after decades of research no definite role 2 Autism Research and Treatment for infectious agents in autism etiology has been confirmed. study of ASD. These newer approaches have identified CNV On the other hand, these endeavors have led to observations mutation differences in genes involved in neuronal cell that perhaps the immune system was involved in autism, and adhesion and ubiquitin degradation as being associated with evidence continues to mount that immune abnormalities are ASD [18]; however, these results have yet to be replicated by indeed associated with ASD. other researchers. The neurexin-1 gene has been associated with a variety of developmental disorders including ASD [19]. The neurexin- 3. Familial Studies 1 gene (2p16.3) is one of the largest genes in the human Studies indicating familial clustering and the increases of genome with 24 exons in 1.1 Mb. With two independent promotors there can be over 1,000 neurexin isoforms gener- ASD in twins have been interpreted by many as an indication ff of genetic predisposition. Twin studies show the concordance ated from the 24 exons in di erent cells or tissue. Another rates of monozygotic twins at 36–96%, whereas dizygotic gene of interest is the contactin-associated protein-like 2 twins are 0–24% concordant, resulting in an estimated herit- (CNTNAP2) gene that was shown to be associated in ability of autism at >90% [6–8]. Additionally, family studies Old-Order Amish subjects with intractable epilepsy and have shown autism to have familial aggregation with 3–8% of ASD [20].Threeothergroupshavenowconfirmedthe subsequently born siblings either being autistic or showing involvement of CNTNAP2 in ASD [21–23]. some form of pervasive developmental disorder (PDD) [9, Both the neurexin-1 and the CNTNAP2 genes are in- 10]. This is a 3- to 8-fold increase in risk for siblings over the volved in synaptic function. Although these approaches have general population. A more recent study gave an estimate of shown a strong association of certain genes with ASD, 18.7% sibling recurrence risk for ASD, a 20-fold increase over only a small percentage of subjects with ASD have these the general population [11]. It is important to note, however, mutations. For example, the neurexin-1gene is found in only that family data should be looked at with great caution, as about 0.5% of autism cases and 0.2% of controls. The 90% individuals living in the same household will have similar missing inheritance may be largely due to marked genetic ff exposures to microorganisms and environmental chemicals. heterogeneity, suggesting that di erent ASD phenotypes Taking this into account, a recent paper based on data from should be examined separately [24–26]. Recent genetic re- twin pairs estimates the genetic heritability for ASD to be search has also associated numerous immune function genes 14–67% [12]. However, this idea is somewhat controversial with autism [27–30]. A large study that examined SNP data as many in the research community continue to feel that the from several genomewide scans on 3,130 subjects with schiz- results from family studies are indicative of a strong genetic ophrenia found that the 5 most significant SNP markers are etiology [13, 14]. found across the HLA region [31]. It appears that some of missing inheritance, at least in schizophrenia, was uncovered in the HLA region and we suggest a similar finding will be 4. Genetics confirmed/uncovered in autism. Many of the early genetic studies involved the examination of microsatellites throughout the human genome in an attempt 5. Immune Abnormalities in Autism to find genomic regions that would associate with autism. Overall, this approach was not very fruitful and researchers It is no surprise to see immune gene associations in ASD, as quickly started to examine single-nucleotide polymorphisms numerous researchers have reported immune abnormalities (SNPs) as techniques advanced [15]. With this approach in autism for over 20 years. It has become increasingly obvi- many researchers proposed that multiple candidate genes ous that inflammatory processes are associated with autism. were associated with ASD. Unfortunately, studies proposing Blood levels of the inflammatory cytokines IL-6, INF-γ,and candidate genes were often contradictory and proved to be TNF-α were shown to be elevated in autistic individuals com- unreliable [15]. One of the most interesting genetic findings pared to controls [32, 33]. Later, seminal work by Vargas et in ASD is the association of autism with the MET receptor al. [34] utilized direct morphological analysis and immuno- tyrosine kinase gene located on chromosome 7q31 as MET histologic techniques to show an active neuroinflammatory signaling participates in gastrointestinal repair, immune process in the cerebral cortex, white matter, and in particular function, neocortical, and cerebellar growth [16]. It is impor- the cerebellum of ASD patients that was dependent on tant to mention that the autism MET associations have activation of microglia and astroglia. Cytokine profiling been replicated by other research groups [17]. This autism demonstrated that neuroinflammation was accompanied by association showed a relative risk of 2.27 which is much upregulation of the macrophage chemoattractant protein lower than the relative risk for HLA gene/allele associations (MCP-1) and TGF-beta in brain tissue, and that MCP-1 was discussed below. also upregulated in cerebral spinal fluid [34]. More recent It is very reasonable to believe that deletions or dupli- work has directly demonstrated that aside from blood, IL- cations of genetic regions, which can cause lower or higher 6, TNF-α,andINF-γ are elevated in ASD brains, along with levels of gene expression, could produce pathological phe- the other inflammatory cytokines GM-CSF and IL-8 [35]. notypes. Consequently, newer approaches examining copy Most recently, it has been reported that the important number variation (CNV) and microarray analyses of 500,000 inflammatory-associated transcription factor, nuclear factor SNPs or more have been in vogue for several years in the kappa-light-chain-enhancer of activated B cells (NF-κB) is Autism Research and Treatment 3

Table 1: A list of proteins against which autoantibodies have been fairly recently, it was believed that most immature lym- found in the serum of subjects with autism. phocytes recognizing self-antigens (autoimmune repertoire) were normally neutralized by virtue of central tolerance Protein Reference before they could mature, and that peripheral tolerance Transglutaminase 2 [39] would insure the removal of any self-reactive lymphocyte 45 and 62 kDa proteins in cerebellum [40, 41] escaping central tolerance. Peripheral tolerance refers to the Voltage dependent anion channel (VDAC) [42] process whereby mature self-reactive lymphocytes which Hexokinase-1 [42] have escaped central tolerance are eliminated, largely by Mitochondrial protein [43] CD95-mediated apoptosis [62]. Thus responses to foreign Antimitochondrial DNA auto-antibodies [43] antigens were viewed as normal, while anti-self responses Nuclear proteins [44] were considered necessarily pathologic. However, with the 52 kDa protein in hypothalamus and thalamus [45] realization that many self-reactive lymphocytes survive cen- 43–48 kDa protein in the hypothalamus [45] tral and peripheral tolerance, this view has had to be modified [49]. Limited immune responses to self-antigens Folate receptor [46] (autoimmunity) are now understood to be normal and not Brain-derived neuro trophic factor [47] necessarily pathologic [63]. HSP90 [48] Upon stimulation of the system with a pathogen, cognate Myelin basic protein (MBP) [38, 49] lymphocyte clones representative of the antiforeign reper- Myelin-associated glycoprotein (MAG) [50] toire normally expand and mature, providing a protective Myelin oligodendrocyte glycoprotein (MOG) [50] immune response. On the other hand, it is the expansion and Neuron-axon filament protein [51] maturation of those clones representative of the autoimmune Glial fibrillary acidic protein [51] repertoire that leads to autoimmune disease. In other words, the developing immune system can be characterized as bal- ancing production between antiforeign (protective) and anti- self (autoimmune) repertoires. While a beneficial function of upregulated in both blood [36] and brain tissue [37]of naturally occurring, low level autoimmune antibodies, also autistic individuals. Other immune abnormalities such as referred to as natural antibodies (NAs), remains a matter of autoantibodies started to be reported in 1993 [38](Table 1). debate, it appears as if the repertoire of NA is reflective of Autoantibodies to myelin basic protein have been noted the susceptibility to develop specific autoimmune diseases by at least a couple of researchers [38, 52]. An increase [64, 65]. Because central tolerance of T cells depends to a in autoantibody reactivity has been reported against other large extent upon the strength of the TCR interaction with brain proteins in ASD including nerve growth factor [53], an autoantigen or an HLA class I or II complex [59], the brain endothelium [54], cerebellar proteins [40], and sero- NA repertoire will to a large extent depend upon the HLA tonin 5-HT receptors [55] and transglutaminase-2, a protein haplotype, with some haplotypes favoring autoantibodies important in synaptic stabilization [39]. Croonenberghs et targeting one antigen over another. For instance, in the case al. [56] noted a significant increase in gamma globulin of ASD we have found an association with low level antibody especially of the IgG2 and IgG4 subclasses in children with responses to tissue transglutaminase, and that this response autism over a control population. Autoantibodies to several appears linked to the HLA-DR3/DQ2 and DR7/DQ2 hap- uncharacterized brain-specific proteins have been reported lotypes [39]. It is likely that the other autoantibodies noted in the plasma of autistic individuals. In particular, western above as being associated with ASD may be linked to different blot analysis has shown the presence of IgG autoantibodies haplotypes. targeting a protein of approximately 52 kDA located in the Aside from autoantibodies and altered cytokine levels hypothalamus and thalamus of adult brain [45]. Other there appear to be other immune abnormalities associated autoantibodies targeting three brain proteins of 43–48 kDA with ASD. It was noted that there were decreased numbers located in the hypothalamus have also been observed in the of T-lymphocytes and an altered ratio of suppressor T- serum of autistic individuals [45]. Autoantibodies targeting lymphocytes to helper T-lymphocytes [66, 67]andalteredT- cerebellar proteins of 45 and 62 kDA have been associated lymphocytes responses in children with autism [68]. Warren with ASD [40]. These autoantibodies may be specific to et al. [69]reportedthatsubjectswithautismhadreduced cerebellar Golgi cells, which are GABAergic interneurons NK cell killing in the standard K562 target cell cytotoxicity [41]. Autoantibodies reactive to human brain proteins in the assay. This observation of decreased NK cell killing has been 36–39 and 61 kDA range have also been found in the sera of repeated by at least a couple of other research teams [28, 70]. mothers of autistic children. [57, 58]. One research group [71] observed that monocyte counts and It is important to note that while autoantibodies associ- neopterin levels were increased in autistic children compared ated with ASD may be biomarkers, they may not necessarily to gender and age-matched healthy controls suggesting that be pathologic in and of themselves. Central tolerance refers the immune system was overactivated in the ASD group. to the process whereby immature lymphocytes are negatively Another elegant set of experiments involved the stimulation selected based on the ability of their antigen receptors (the of cultured monocytes with several toll-like-receptor (TLR) B cell receptor or BCR for B cells and the T-cell receptor ligands. The monocytes from subjects with ASD had signif- or TCR for T cells) to recognize self-antigens [59–61]. Until icant increases or decreases in proinflammatory cytokines 4 Autism Research and Treatment

Table 2: Genes and alleles in the HLA region.

HLA genes Number HLA alleles Number HLA class I genes 6 HLA class I alleles 2215 HLA class II genes 12 HLA class II alleles 986 HLA class I-like genes 2 HLA class I-like alleles 94 Non-HLA genes 112 Total genes 132 HLA class I genes A/B/C/E/F/G HLA class I-like genes MICA/MICB HLA class II genes DRA/DRB/DQ/DP/DM/DO depending on the particular TLR ligand added to the cell cul- specialization, central nervous system (CNS) development ture media [72]. [80–84], and even neurological disorders [85]. The genes One large-scale study found that the frequency of of the HLA region are shown in Figure 1. Shiina et al. autoimmune disorders in the families with autistic chil- [86, 87] have published two excellent reviews on the HLA dren was found to be higher than in control subjects, super-locus. Not only is there extraordinary complexity in especially mothers of autistic children [73]. Another group the HLA genes, there are extensive haplotype-related tran- demonstrated that autoimmune diseases were increased scriptional differences [88]. It has been shown that genetic significantly in families with ASD compared with those of mechanisms outside of the non-antigen-binding HLA genes healthy control subjects [74], suggesting a link between the in the ancestral haplotype 8.1 (also referred to as COX) are disorders. Croen et al. [75] showed that maternal psoriasis associated with susceptibility to many autoimmune diseases diagnosed around the time of pregnancy is significantly [89]. It is our proposition that HLA genes/proteins should associated with a subsequent diagnosis of autism in the child. be more carefully examined due to increasing evidence of Additionally, they showed a 2-fold increase in risk for a child autoimmune type associations in autism. having ASD if the mother was diagnosed and with asthma or allergies during pregnancy. An association between a family history of type 1 diabetes mellitus (T1DM) and infantile 7. HLA Associations in ASD autism as well as a significant association between maternal It was suggested over 30 years ago by Stubbs and Magenis histories of either rheumatoid arthritis (RA) or celiac disease [90] that the HLA region might be important in autism. ´ and ASDs was noted by Atladottir et al. [76]. Warren et al. [91] first reported that the HLA ancestral haplotype 44.1 (B44-SC30-DR4) was associated with autism 6. Autism HLA Genetics with a relative risk of 7.9. That result was confirmed in a separate case/control population [92]. Interestingly, the HLA is the name for the major histocompatibility complex individual components of AH 44.1 (A2-B44-SC30-DR4) (MHC) in humans and HLA and MHC are often used include a deleted C4B gene and DRβ1∗0401, both of which interchangeably in the literature. The HLA region on chro- have been shown independently to be significantly associated 6 mosome 6p21 (about 4 × 10 bp) is of major interest in basic with ASD [93, 94]. Examination with different genetic research as well as medicine as genes/proteins in this region markers than those used by Warren suggested that certain are involved in many biological processes such as histocom- HLA haplotypes are associated with autism in Sardinian and patibility, inflammation, ligands for immune cell receptors, Italian families [95, 96]. and the complement cascade. The HLA region has 20 typical Warren et al. [97] reported that the shared epitope- HLA genes and 112 nontypical HLA genes (Table 2) that are binding pocket (DRβ1∗0401, ∗0404, and ∗0101) in the third inherited together as frozen blocks of DNA called ancestral hypervariable region of DRβ1 has a strong association with or extended haplotypes. Complete DNA sequences have autism. A relative risk of 19.8 for autism was reported for been published for 8 of the more common ancestral hap- subjects with one of the two extended HLA haplotypes. Both lotypes in an effort to expedite basic and disease research of these haplotypes have many allelic similarities especially [77]. It should be mentioned that smaller haplotypes can the DRβ1∗0401. also be constructed for genes that are linked. HLA genes AH 44.1 (HLA-A2, Cw5, B44, Bf∗S, C2∗C, C4A3, also play a role in reproduction, pregnancy maintenance, C4BQ0, DRβ1∗0401, DQB1∗0301). mate selection, and even kin recognition [78, 79]andhave AH 62.1 (HLA-A2, Cw3, B15, Bf∗S, C2∗C, C4A3, C4B3, been associated with over 100 diseases/disorders including DRβ1∗0401, DQB1∗0302). autism. The proteins encoded by HLA genes are ligands, The shared epitope has been associated with several receptors, cytokines, signaling factors, heat shock proteins, autoimmune diseases such as rheumatoid arthritis, psoriatic transcription regulators, and so forth. Current research is arthritis, and systemic lupus erythematosus [98]. Torres et increasingly demonstrating a role for HLA proteins in neural al. [93] confirmed the association of the HLA-DR4 allele and cell interactions, synaptic function, cerebral hemispheric also found that the DR13 and DR14 alleles occurred less Autism Research and Treatment 5

Telomeric side Chr. 6 Telomere block κ Short arm block α Class II region Centromere Class I region Class III region Class I region Long arm Extended class II region Extended

Centromeric side block β Telomere

Figure 1: Gene map of the human leukocyte antigen (HLA) region. The major histocompatibility complex (MHC) gene map corresponds to the genomic coordinates of 29677984 (GABBR I) to 33485635 (KIFC1) in the human genome build 36.3 of the National Center for Biotechnology Information (NCBI) map viewer. The regions separated by arrows show the HLA subregions such as extended class I, classical class I, class III, classical class II, and extended class II regions from telomere (left and top side) to centromere (right and bottom side). White, gray, striped and black boxes show expressed genes, gene candidates, noncoding genes and pseudogenes, respectively. The location of the alpha, beta, and kappa blocks containing the cluster of duplicated HLA class I genes in the class I region are indicated. (Reprinted with permission from the Journal of Human Genetics [87].) often in subjects with autism, suggesting a possible protective three additional research groups. Lee et al. [99] demonstrated mechanism. Interestingly, the DR13 allele was inherited less that boys with autism and their mothers had a significantly frequently than expected from the mothers. Associations higher frequency of DR4 than normal control subjects (odds with autism and the DR4 allele have since been confirmed in ratios 4.20 and 5.54, resp.), suggesting that a maternal-fetal 6 Autism Research and Treatment immune interaction could be involved in autism. Johnson In the CNS, TNF-α is made in microglia cells, astrocytes, and et al. [100] reported significant transmission disequilibrium neurons [141]. In the normal state, TNF-α drives acute and for HLA-DR4 (odds ratio 4.67) from maternal grandparents chronic inflammatory responses that leads to the removal of to mothers of children with autism which also suggests a injurious stimuli and the restoration of homeostasis. TNF- maternal-fetal interaction for HLA-DR4. It has been recently α is necessary for neural cell differentiation and neuron shown in Han Chinese that the HLA-DRβ1allelefrequencies maturation and there is evidence that it is critical in the including DR4 are different in subjects with autism versus normal brain for proper synaptic function. control subjects [101]. There is extensive research that implicates TNF-α as a key Itwasreported20yearsagothatsubjectswithautism mediator in disease progression, inflammation, blood-brain- had a significant increase in the C4B null allele (C4B barrier deterioration, and even cell death [142]. Elevated lev- gene deletion) compared to control subjects [91]. After this els are present in numerous neurological disorders including observation, it was also noted that subjects with autism had multiple sclerosis, Alzheimer’s disease, Parkinson’s disease, a significant deficiency in the plasma C4B protein [102]. ischemia, traumatic brain injury, and as mentioned above, These initial findings of an increase in the deletion of the ASD [141]. It is unclear if TNF-α contributes to the disease C4B gene was supported by examining a new population of state or the higher concentrations limit neuronal injury. subjects with autism [94]. Mostafa and Shehab [103]have There are three adjacent genes: lymphotoxin alpha and beta recently reported a significant increase in the deletion of the (LTA and LTB) and leukocyte-specific transcript-1 (LST1) C4B gene in the Egyptian population. They also reported an in the TNF-α block. LTA and LTB are proinflammatory increased risk when there was a family history of autoim- cytokines like TNF-α, and LST1 plays a role in inflammatory mune diseases in the autism population. Descriptions of and infectious diseases. It has been suggested that vaccine- several non-antigen-binding HLA genes that are associated induced immunity changes with certain haplotypes in these with autoimmune diseases are discussed below and listed genes [143]. Two other genes important in immune function in Table 3. are adjacent to the TNF-α block. Nuclear factor of kappa light polypeptide gene enhancer in B-cells inhibitor-like 1(NF- κBIL1) on the telomeric side of TNA-α is an inhibitor of NF- 8. Non-Antigen-Binding HLA Genes in Class I κB, an important pleiotropic immune system transcription factor that is upregulated in ASD [144, 145]. In addition, NF- Although MICA/MICB genes are structurally similar to clas- κ sical antigen-binding HLA class I genes, they encode proteins BIL1 has been associated with several autoimmune diseases that interact with different T-cell receptors in response to (Table 3). The natural cytotoxicity trigger receptor three stress as infection or neoplastic transformation. The two (NCR3) gene on the centromeric side encodes proteins that genes are located on the centromeric end of the class I region are ligands for activating receptors on NK cells that recognize near HLA-B at the border of the class III region (Figure 1) tumor cells [146]. Typical HLA-B and -C proteins are also [87]. MICA/MICB proteins are ligands for NKG2D recep- ligands for KIR receptors on NK cells. tors on NK cell and gamma/delta T-cell receptors. Adap- There are numerous heat shock proteins (HSPs), also tive immunity involves three lymphocyte populations (B referred to as chaperones, that assist the folding of newly cells, alpha/beta T cells, and gamma/delta T cells). Gam- synthesized proteins as well as those that have been unfolded ma/delta T cells represent a small population of T cells that or denatured [147]. HSPs are also important in cell protective possess a T-cell receptor that is distinct from the typical functions and in inhibiting the apoptosis cascade. They are alpha/beta T cell. They are concentrated in the intestinal mu- named by their molecular weights (HSP100, HSP90, HSP70, cosa and appear to have a prominent role in recognizing HSP60, and smaller HSPs). Although the proteins appear small bacterial phosphoantigens and undefined antigens in normal cellular functions, they are induced to higher presented by MICA/MICB proteins. Gamma/delta T cells levels in trauma, epilepsy, neurodegenerative diseases, and have potent cytotoxic activity and have been considered a other injuries. An underlying feature among AD, Parkinson’s link between innate and adaptive immunity. Polymorphisms disease, spinocerebellar ataxia, and other neurodegenerative in MICA/MICB genes have been associated with T1DM, AD, diseases is the accumulation of misfolded proteins and and SLE independent of DRβ1 alleles. There are several genes HSPs are being studied because of their role in folding associated with psoriasis (PSORSI locus). It is unclear how and refolding proteins [147]. There is intense interest in the risk is spread among these genes as they are closely linked. HSPs as they appear to protect neurons [148]andonemust Two genes RNF39 and TRIM39 in the class I region have been remember that postmitotic neurons are unable to dilute associated with Behçet’s disease. misfolded or aggregated proteins through division. There are three HSP70 genes (HSPA1L, HSPA1A, and 9. Non-Antigen-Binding HLA Genes in Class III HSPA1B) in the class III HLA region that are adjacent but separate genes. HSP70 proteins have been demonstrated to Tumor necrosis factor-alpha (TNF-α) is a proinflammatory, stimulate IL-6 and TNF-α production, activate microglial multifunctional cytokine that plays important roles in cell cells, and stimulate phagocytosis [148]. HSP70 proteins are physiology. It is synthesized in numerous cells including also important in autoimmunity by enhancing antigen pre- macrophages, NK cells, T cells, mast cells, osteoblasts, gran- sentation in both HLA class I and HLA class II systems [147, ulocytes, smooth muscle cells, fibroblasts, and keratinocytes. 149]. Peptides that are associated with HSP70 at the time of Autism Research and Treatment 7

Table 3: Non-classical HLA genes associated with autoimmune diseases.

Gene HGNC gene number Autoimmune disease References Extended class I OR2H2 8253 SLE [104] RNF39 18064 Behçet’s disease [105] TRIM39 10065 Behçet’s disease [105] Class I PSORS1 locus 9573 Systemic sclerosis, Psoriasis [106–111] MICA 7090 T1DM, AD, SLE [112–114] MICB 7091 SLE [104] BAT1–BAT5 13917–21 Alzheimer’s, AIDS [115, 116] NFKBIL1 7800 Sjogren’s¨ syndrome, SLE, RA [117, 118] Alzheimer’s, Psoriasis, Autoimmune TNF Block 11892 [115, 119–121] hepatitis, Sarcoidosis AIF1 352 T1DM [122] Class III HSP70 genes 5232–4 MS [123] Complement genes 1248, 1324, 1323 SLE, myasthenia gravis, T1DM [124–126] SKIV2L 10898 SLE [127] ATF6B (CREBL1) 2349 SLE [104] NOTCH4 7884 Systemic sclerosis [128] C6orf10 13922 SLE [104] BTNL2 1142 Ulcerative colitis, Sarcoidosis [129, 130] TAP2 44 Psoriasis [131] PSMB8 9545 Hypersensitivity pneumonitis [132] Psoriasis [133] Class II TAP1 43 Vitiligo [134] PSMB9 9546 Psoriasis, Vitiligo [133, 134] Psoriasis, Antiphospholipid HLA-DM 4934, 4935 [131, 135–138] auto-antibodies, RA, SLE, T1DM HLA-DO 4936, 4937 common variable immunodeficiency [139] HSD17B8 3554 SLE [104] Class II Extended DAXX 2681 MS [140] Abbreviations: (HGNC) HUGO Gene Nomenclature Committee (http://www.genenames.org/); (SLE) systemic lupus erythematosus; (AIDS) acquired immune deficiency syndrome; (T1DM) type 1 diabetes mellitus; (AD) Addison’s disease; (RA) rheumatoid arthritis; (MS) multiple sclerosis, PSORS1 psoriasis locus genes (CDSN, PSORS1C1, PSORS1C2, CCHCR1, POU5F1, PSORS1C3), TNF Block genes (LTA, TNF, LTB, LST1), HSP70 genes (HSPA1L, HSPA1A, HSPA1B), Complement genes (C2, C4B, C4A).

T-cell presentation have been shown to be more antigenic HLA proteins. DM stabilizes and edits the peptide repertoire and therefore involved in autoimmunity [149, 150]. presented by DQ proteins by catalyzing CLIP release. The Another gene that is independently associated with associations of DQ2 with T1DM and celiac disease have been autoimmune diseases outside of non-antigen-binding HLA known for several decades; however, the biochemistry behind alleles is allograft inflammatory factor-1 (AIF1). The SNP these associations has not been elucidated. It is now known rs2269475 C > T in AIF1 has been associated with RA. that DQ2 is a poor substrate for DM and it has been proposed The T allele was significantly higher in the RA patients and that antigen presentation in the thymus and periphery can be ff there was no significant linkage disequilibria between the a ectedbyimpairedDQ-DMinteractionssoastopromote AIF1 SNP and DRβ1 alleles. Anticyclic citrullinated peptide autoimmune disease [152]. HLA-DO is another nonclassical antibodies commonly used to monitor RA were significantly class II HLA protein that is involved in the loading of peptides increased in carriers with the T allele [151] but not the C to HLA-DR proteins by modulating the function of HLA- allele. DM [153]. There are two genes in the class II region that encode proteins involved in the transport of antigen from the cytoplasm 10. Non-Antigen-Binding HLA Genes in Class II to the endoplasmic reticulum for binding to class I HLA proteins: transporter associated with antigen-processing 1 Although HLA-DM and -DQ proteins have structures like and 2 (TAP1 and TAP2). Several research groups have classical antigen-binding HLA proteins, they work in the reported associations of TAP2 genes with SLE independent of cytoplasm and not at the cell surface antigen-presenting classical HLA alleles [154]. This interaction is very interesting 8 Autism Research and Treatment as it means that class II genes can affect class I antigen References binding. [1] L. Kanner, “Autistic disturbances of affective contact,” The There are two other genes in the extended class II Nervous Child, vol. 2, no. 2, pp. 217–250, 1943. region that have been associated with autoimmune dis- [2] F. R. Volkmar, M. State, and A. Klin, “Autism and autism eases (Table 3). Hydroxysteroid 17-beta dehydrogenase 8 spectrum disorders: diagnostic issues for the coming decade,” β (HSD17 8) is important in regulating the concentrations of Journal of Child Psychology and Psychiatry and Allied Disci- active estrogens and androgens and high levels of estrogen plines, vol. 50, no. 1-2, pp. 108–115, 2009. are well known to be associated with SLE and other autoim- [3] Centers for Disease Control and Prevention (CDC), “Preva- mune diseases. 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Research Article Intracellular and Extracellular Redox Status and Free Radical Generation in Primary Immune Cells from Children with Autism

Shannon Rose, Stepan Melnyk, Timothy A. Trusty, Oleksandra Pavliv, Lisa Seidel, Jingyun Li, Todd Nick, and S. Jill James

Department of Pediatrics, Arkansas Children’s Hospital Research Institute, University of Arkansas for Medical Sciences, Little Rock, AR 72202, USA

Correspondence should be addressed to S. Jill James, [email protected]

Received 30 July 2011; Revised 12 August 2011; Accepted 12 September 2011

Academic Editor: Antonio M. Persico

Copyright © 2012 Shannon Rose et al. This is an open access article distributed under the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.

The modulation of the redox microenvironment is an important regulator of immune cell activation and proliferation. To investigate immune cell redox status in autism we quantified the intracellular glutathione redox couple (GSH/GSSG) in resting peripheral blood mononuclear cells (PBMCs), activated monocytes and CD4 T cells and the extracellular cysteine/cystine redox couple in the plasma from 43 children with autism and 41 age-matched control children. Resting PBMCs and activated monocytes from children with autism exhibited significantly higher oxidized glutathione (GSSG) and percent oxidized glutathione equivalents and decreased glutathione redox status (GSH/GSSG). In activated CD4 T cells from children with autism, the percent oxidized glutathione equivalents were similarly increased, and GSH and GSH/GSSG were decreased. In the plasma, both glutathione and cysteine redox ratios were decreased in autistic compared to control children. Consistent with decreased intracellular and extracellular redox status, generation of free radicals was significantly elevated in lymphocytes from the autistic children. These data indicate primary immune cells from autistic children have a more oxidized intracellular and extracellular microenvironment and a deficit in glutathione-mediated redox/antioxidant capacity compared to control children. These results suggest that the loss of glutathione redox homeostasis and chronic oxidative stress may contribute to immune dysregulation in autism.

1. Introduction (GSH) and several of its metabolic precursors, an increase in oxidized glutathione disulfide (GSSG), and a decrease in Autism is a behaviorally defined neurodevelopmental disor- glutathione redox ratio (GSH/GSSG) in case-control evalua- der that usually presents in early childhood and is charac- tions of plasma and lymphoblastoid cell lines derived from terized by significant impairments in social interaction and children with autism [13–16]. Recently, several interactive communication and by abnormal repetitive hyper-focused polymorphisms in enzymes regulating glutathione synthesis behaviors. The prevalence of autism spectrum disorders has were found to be more prevalent in children with autism increased more than 10-fold in the last two decades, now suggesting that the glutathione deficit and predisposition to affecting one in 110 US children, yet the etiology of these oxidative stress may be genetically based in some children disorders remains elusive [1]. Glutathione depletion and oxi- [17]. dative stress have been implicated in the pathology of numer- Oxidative stress occurs when cellular antioxidant defense ous neurobehavioral disorders including schizophrenia [2], mechanisms fail to counterbalance endogenous ROS produc- bipolar disorder [3], and Alzheimer’s disease [4]. Accumu- tion and/or exogenous prooxidant environmental exposures. lating evidence suggests that redox imbalance and oxidative Glutathione (γ-L-glutamyl-L-cysteinylglycine) is a tripeptide stress may also contribute to autism pathophysiology. Mul- that functions as the major intracellular antioxidant and tiple biomarkers of oxidative stress have been identified in redox buffer against macromolecular oxidative damage. The blood samples from children with autism [5–12]. Our group glutathione thiol/disulfide redox couple (GSH/GSSG) is the has reported a decrease in concentrations of glutathione predominant mechanism for maintaining the intracellular 2 Autism Research and Treatment microenvironment in a highly reduced state that is essential with autism have been shown to have abnormal responses for antioxidant/detoxification capacity, redox enzyme regula- to stimulation, we also elected to challenge the PBMCs with tion, cell cycle progression, and transcription of antioxidant immune activators known to promote oxidative stress and response elements (ARE) [18–23]. Subtle variation in the measure the resulting intracellular glutathione redox status relative concentrations of reduced and oxidized glutathione in activated isolated monocytes and T cells. provides a dynamic redox signaling mechanism that regulates these vital cellular processes [24–27]. For example, in both CNS precursor cells and na¨ıve immune cells, intracel- 2. Subjects and Methods lular glutathione redox status is the primary determinant 2.1. Participants. This investigation was conducted on a modulating the cellular decision to undergo cell cycle arrest, subset of children from the autism IMAGE (Integrated differentiation, or proliferation [27]. A reducing intracellular Metabolic and Genomic Endeavor) study at Arkansas Chil- environment is required for proliferation, while a more dren’s Hospital Research Institute (ACHRI) that has recruit- oxidized microenvironment favors cell cycle arrest and dif- ed over 162 case and control families to date. The IMAGE ferentiation. A chronic deficit in the GSH/GSSG redox ratio cohort for this study consisted of 43 children diagnosed is considered to be a reliable indicator of oxidative stress and with autistic disorder and 41 unaffected control children increased vulnerability to oxidative damage from prooxidant (16 of which were unaffected siblings). The autism case environmental exposures [28, 29]. families were recruited locally after referral to the University In the extracellular plasma compartment, the cysteine/ of Arkansas for Medical Sciences (UAMS), Dennis Devel- cystine (thiol/disulfide) redox couple independently provides opmental Center and diagnosed by trained developmental the ambient redox environment for circulating immune cells. pediatricians. Children aged 3 to 10 with a diagnosis of The ambient extracellular cysteine/cystine redox potential autistic disorder as defined by the Diagnostic and Statistical has been shown to be more oxidized than the intracellular Manual of Mental Disorders, Fourth Edition (DSM-IV 299.0), GSH/GSSG redox potential and is independently regulated the Autism Diagnostic Observation Schedule (ADOS), [30]. Dynamic shifts in the plasma cysteine/cystine redox and/or the Childhood Autism Rating Scales (CARS >30) potential alter the redox status of cysteine moieties in cell were enrolled. Children diagnosed with other conditions on surface proteins to induce conformational changes in protein the autism spectrum or rare genetic diseases associated with structure that can reversibly alter function [31, 32]. For ex- symptoms of autism were excluded from the study. Children ample, under oxidizing extracellular conditions, redox-sensi- with chronic seizure disorders, recent infection, and high- tive cysteine residues in the catalytic core of protein tyrosine dose vitamin or mineral supplements exceeding the RDA phosphatases become oxidized and reversibly inactivate were also excluded because these conditions are potential enzyme activity depending on the ambient cysteine/cystine confounders that could affect redox status. Unaffected sib- redox potential [31, 33, 34]. Extracellular cysteine/cystine lings and unrelated, neurotypical children aged 3 to 10 with redox status is emerging as an important new signal trans- no medical history of behavioral or neurologic abnormalities duction mechanism that can induce posttranslational alter- by parent report made up the comparison group. The ations in downstream redox-sensitive proteins including a protocol was approved by the Institutional Review Board at variety of enzymes, transcription factors, receptors, adhesion UAMS, and all parents signed informed consent. molecules, and membrane signaling proteins resulting in the dynamic modulation of their activity and function [32, 35, 36]. 2.2. Materials. Culture flasks, plates, and pipettes were ob- Recent studies have revealed numerous immunologic tained from Corning Life Sciences (Lowell, Mass, USA). abnormalities among children with autism including alter- RPMI 1640, penicillin/streptomycin, Dulbecco’s phosphate- ff ations in immune cell proportions [37–40] and shifts in bu ered saline (PBS), fetal bovine serum (FBS), and glutamine were purchased from Life Technologies (Carlsbad, helper T-cell subpopulations after mitogenic stimulation [41, 42]. Peripheral blood mononuclear cells (PBMCs) from Calif, USA). Carboxy-H2DCFDA (6-carboxy-2 ,7 -dichlo- individuals with autism have been shown to produce higher rodihydrofluorescein diacetate, diacetoxymethyl ester) was levels of proinflammatory cytokines and abnormal levels of obtained from Molecular Probes (Carlsbad, Calif, USA). regulatory cytokines compared to control PBMCs at baseline Human Monocyte Isolation Kit II and Human CD4 T and upon mitogenic stimulation [43–46]. Taken together, cell Isolation Kit II were purchased from Miltenyi Biotec the immunological studies suggest a role for a dysregulated (Bergisch-Gladbach, Germany). Histopaque-1077 and all immune system in autism that potentially could be related to other chemicals were obtained from Sigma-Aldrich (St. a deficit in glutathione-mediated antioxidant capacity and an Louis, Mo, USA). oxidized microenvironment in immune cells. To investigate this possibility, we examined whether primary immune cells 2.3. Isolation of PBMCs and Stimulation of Monocytes and (PBMCs) from children with autism exhibit decreased in- CD4 T Cells. Fasting blood samples (≤20 mL) were collected tracellular glutathione redox capacity compared to PBMCs before 9:00 AM into EDTA-Vacutainer tubes and immedi- from age-matched control children and whether a more ately chilled on ice before centrifuging at 1300 ×gfor10min oxidized intracellular and extracellular microenvironment at 4◦C. Aliquots of plasma were stored at −80◦C in cryostat is associated with increased production of oxidizing intra- tubes until extraction and HPLC quantification. PBMCs cellular free radicals. Because immune cells from children were isolated by centrifugation over Histopaque-1077. Red Autism Research and Treatment 3 blood cells were lysed using a brief (15 s) incubation with Table 1: Demographics of study population. 1 mL ice-cold water. Approximately, 30 × 106 PBMCs were resuspended in RPMI 1640 medium (supplemented with Case children Control children n = 43 n = 41 10% FBS, 1% penicillin/streptomycin, and 2 mM glutamine) at a density of 106 cells/mL. Note that because we were unable Age; mean (SD) 5.42 (1.98) 6.16 (2.29) n to obtain 20 mL blood volume from every child, it was not Male; (%) 36 (84) 20 (49) possible to isolate and analyze monocytes and CD4 T cells White; n (%) 38 (88.4) 31 (75.6) for all participants. For monocyte stimulation, PBMCs were Asian; n (%) 2 (4.65) 0 (0) treated with 0.1 μg/mL lipopolysaccharide (LPS); for T-cell African American; n (%) 2 (4.65) 8 (19.5) stimulation, PBMCs were treated with 10 ng/mL phorbol 12- Hispanic; n (%) 1 (2.3) 2 (4.9) μ myristate 13-acetate (PMA) and 1 g/mL ionomycin. Cells OTC multivitamin use; n (%) ◦ 17 (39.5) 8 (19.5) were placed in a humidified 5% CO2 incubator at 37 C for 4 hr. Stimulated monocytes and CD4 T cells were then isolated by negative selection using magnetic cell labeling as fluorescence normalized to DCF fluorescence of a standard described by the manufacturer (Miltenyi Biotec, Bergisch- PBMC preparation. As an internal control, the standard Gladbach, Germany). Using flow cytometry, we determined PBMC preparation was isolated from a 100 mL blood sample ≥ that 75% of isolated monocytes are positive for CD14 and from an unaffected healthy adult volunteer, aliquoted and ≥ that 87% of isolated CD4 T cells are positive for CD4. For frozen at −180◦C in 90% FBS/10% DMSO. An aliquot of HPLC quantification of GSH and GSSG, approximately 2 × 6 the standard PBMC preparation was stained and analyzed 10 unstimulated (resting) PBMCs, stimulated monocytes, with each subject sample. Evaluation of oxidizing free radical or stimulated CD4 T cells were pelleted, snap frozen on dry production was possible only in those case and unrelated − ◦ ice,andstoredat 80 C. control samples for which sufficient (∼20 mL) blood volume was obtained. 2.4. Cell Extraction and HPLC Quantification of Intracellular Glutathione and Plasma Cysteine Redox Status. The storage 2.6. Statistical Analysis. Within the control group, 16 of the ◦ interval at −80 C before extraction was consistently between 41 unaffected control children were case siblings. There were 1-2 weeks after blood draw and cell isolation to minimize 27 additional case children without a sibling and 25 addi- potential metabolite interconversion. The methodological tional unrelated control children comprising the total case- details for intracellular and extracellular GSH extraction control cohort of 84 children. To down-weight the impact of and HPLC elution and electrochemical detection have been outliers, three metabolites observations were curtailed at the described previously [15, 16], and metabolite detection does extremes of the distributions for PBMC GSH, PBMC GSSG, not require derivatization. Although most GSSG is present and Monocytes GSH/GSSG (see footnote in Table 2). The as a mixed disulfide with other thiols including cysteine, sibling data are correlated resulting in a combined sample our measurements detect only the free GSSG in plasma. of correlated and uncorrelated data; thus, the assumption of Glutathione and cysteine concentrations were calculated all data being independent is not satisfied for the standard from peak areas of standard calibration curves using HPLC two-sample t-test. To make use of all data from dependent software. Intracellular results are expressed as nanomoles and independent observations, we used the corrected Z-test per milligram of protein using the BCA Protein Assay Kit proposed by Looney and Jones [47]. This statistical approach (Pierce, Rockford, Ill, USA), and plasma results are expressed provides adequate control of Type 1 errors and has more as micromoles per liter. power than a standard Student’s t-test. Because the DCF data compared cases and unrelated controls (without siblings) 2.5. Measurement of Intracellular Free Radicals. Carboxy- the standard Student’s t-test was used with significance set H2DCFDA (DCF) is a membrane-permeable ROS/RNS-sen- at 0.05. Nonparametric intercorrelations (Spearman correla- sitive probe that remains nonfluorescent until oxidized by tion coefficients) between age and gender and the 7 outcome intracellular free radicals. The intensity of DCF fluorescence variables, GSH, GSSG, GSH/GSSG, % oxidized glutathione, is directly proportional to the level of free radical oxidation. cysteine, cystine, and cysteine/cystine were determined with Approximately, 106 PBMCs were resuspended in 1 mL RPMI the significance level set at 0.05. Data was analyzed using SAS 1640 medium supplemented with 10% FBS, 1% penicil- 9.2 software (SAS Institute Inc, Cary, NC, USA). lin/streptomycin, and 2 mM glutamine and stained in the ◦ dark for 20 min with 1 μMDCFat37C. Stained cells were 3. Results washed and resuspended in PBS and analyzed immediately onaPartecCyFlowflowcytometer(Gorlitz,¨ Germany) using 3.1. Demographics of Study Population. Table 1 lists the 488 nm excitation wavelength with 530/30 nm (FL1) emis- demographics of the study population. The only major sion filter. For each analysis, the fluorescence properties of differences between cases and controls are that the control 10000 cells were collected, and the data were analyzed using group was composed of a greater proportion of females and the FCS Express software (De Novo Software, Los Angeles, African Americans, whereas the case group had a greater pro- Calif, USA). Intracellular free radical levels are expressed as portion of Asian subjects. Over-the-counter multivitamin median fluorescence intensity (MFI) of subject sample DCF supplement use was higher among cases (39.5%) compared 4 Autism Research and Treatment

Table 2: Intracellular glutathione redox status in resting PBMCs and activated monocytes and CD4 T cells.

Case children Control children Corrected Z-test Metabolite Diﬀerence n Mean ± SD n Mean ± SD P value (95% CI) Resting PBMCs GSH (nmol/mg protein) 43 25.45 ± 8.16 41 23.35 ± 6.38 2.09 (−1.09, 5.29) 0.19 GSSG (nmol/mg protein) 43 0.90 ± 0.3410.66 ± 0.23 0.24 (0.13, 0.35) <0.001 GSH/GSSG 43 29.58 ± 9.04 41 37.58 ± 10.89 −7.99 (−12.51, −3.48) <0.001 Oxidized GSH (%) 43 0.07 ± 0.02 41 0.05 ± 0.01 0.02 (0.0075, 0.024) <0.001 Activated monocytes GSH (nmol/mg protein) 18 7.73 ± 3.16 20 8.55 ± 2.5 −0.82 (−2.02, 0.38) 0.18 GSSG (nmol/mg protein) 18 0.62 ± 0.24 20 0.47 ± 0.17 0.14 (0.03, 0.25) 0.01 GSH/GSSG 18 13.31 ± 7.26 20 19.30 ± 6.35 −5.98 (−9.99, −1.97) 0.003 Oxidized GSH (%) 18 0.14 ± 0.05 20 0.10 ± 0.03 0.04 (0.02, 0.07) <0.001 Activated CD4 T cells GSH (nmol/mg protein) 18 6.82 ± 3.01910.16 ± 3.74 −3.33 (−5.24, −1.42) <0.001 GSSG (nmol/mg protein) 18 0.68 ± 0.29 19 0.63 ± 0.24 0.05 (−0.11, 0.22) 0.51 GSH/GSSG 18 10.47 ± 4.19 19 17.49 ± 6.95 −7.02 (−10.17, −3.87) <0.001 Oxidized GSH (%) 18 0.17 ± 0.05 19 0.11 ± 0.05 0.05 (0.03, 0.08) <0.001 GSH: glutathione; GSSG: oxidized glutathione disulﬁde; oxidized GSH: (%)2GSSG/(GSH+2GSSG); curtailment: PBMC GSH >45 set = 45 (n = 1); PBMC GSSG >1.75 set = 1.75 (n = 1); Monocytes GSH/GSSG >35 set = 35 (n = 1).

to controls (19.5%); however, the glutathione redox status of the outcome measures. The protein content per 106 cells was statistically unaffected by vitamin use (data not shown). did not differ between case and control children (data not shown). 3.2. Decreased Intracellular Glutathione Redox Status in Autism. Table 2 presents the relative intracellular concen- 3.3. Decreased Extracellular Glutathione and Cysteine Redox trations of GSH, GSSG, the glutathione redox ratio, and Status in Autism. Table 3 presents the relative concentrations the percentage of oxidized glutathione equivalents in resting of GSH, GSSG, GSH/GSSG, % oxidized GSH, cysteine, (unstim-ulated) PBMCs and in isolated stimulated mon- cystine, and the cysteine/cystine redox ratio in the extracel- ocytes and CD4 T cells from children with autism and lular plasma compartment. Children with autism exhibited age-matched control children. The percent oxidized gluta- a significantly decreased extracellular concentration of GSH thione is expressed in absolute glutathione equivalents as (∼21%) and GSH/GSSG (∼54%) and increased concentra- 2GSSG/(GSH+2GSSG). Relative to controls, the intracellular tion of GSSG and the percent oxidized glutathione (52% concentration of GSSG and the percent oxidized glutathione and 82%, resp., P<0.001). Figures 1(a) and 1(b) com- were significantly increased (∼40%), and the GSH/GSSG pare GSH/GSSG and % oxidized glutathione equivalents, ratio decreased (∼21%) in PBMCs from children with respectively, in plasma, T cells, and monocytes from case and autism (P<0.001). After stimulation with LPS, mono- control children and graphically demonstrates the consistent cytes from children with autism also exhibited significantly decrease in both extracellular and intracellular glutathione decreased GSH/GSSG (∼31%, P = 0.003), increased GSSG redox status among the case children. concentration (∼32%, P = 0.01), and 40% higher percent The dynamic balance between the reduced and oxidized oxidized glutathione (P<0.001). In mitogen-stimulated forms of glutathione can also be expressed as the redox CD4 T cells from children with autism, the intracellular GSH potential or reducing power of the GSH/GSSG redox couple concentration was ∼33% lower, the GSH/GSSG was ∼40% (Eh) and can be calculated from the Nernst equation, Eh = lower (P<0.001), and the percent oxidized glutathione E0 + RT/nF ln[disulfide]/([thiol 1] ∗ [thiol 2]), where E0 was ∼55% higher than in stimulated CD4 T cells from is the standard potential for the glutathione redox couple control children (<0.001). As expected, activation with LPS (−264 mV), R is the gas constant (8.314 J/◦Kmol), T is the and PMA both resulted in decreased intracellular GSH absolute temperature of analytical measurement (25◦C = levels and GSH/GSSG in isolated monocytes and CD4 T 298◦K), n is 2 for the number of electrons transferred, cells compared to resting (unstimulated) PBMCs. Upon and F is Faraday’s constant (96,485 coulomb/mol) [48]. stimulation, there was a greater decrease in intracellular The calculated Eh value for the GSH pool in the children GSH and GSH/GSSG in both CD4 T cells and monocytes with autism is −116 mV, which is 12 mV more oxidized from children with autism compared to control children. than in the control children, with an Eh value of −128 mV Neither age nor gender was significantly correlated with any (Table 3). Autism Research and Treatment 5

Table 3: Extracellular (plasma) glutathione and cysteine redox status.

Case children Control children Corrected Z-test Metabolite Diﬀerence n Mean ± SD n Mean ± SD P value (95% CI) Plasma GSH (μM) 38 1.58 ± 0.23 41 1.99 ± 0.22 −0.41 (−0.50, −0.31) <0.001 GSSG (μM) 38 0.20 ± 0.06 41 0.13 ± 0.04 0.07 (0.05, 0.09) <0.001 GSH/GSSG 38 8.24 ± 2.20 41 17.14 ± 5.54 −8.73 (−10.52, −6.94) <0.001 Oxidized GSH (%) 38 0.20 ± 0.05 41 0.11 ± 0.03 0.09 (0.07, 0.10) <0.001 Eh for GSH −116 mV −128 mV Cysteine (μM) 41 21.7 ± 4.88 41 21.43 ± 4.08 0.13 (−1.88, 2.14) 0.90 Cystine (μM) 41 29.2 ± 10.64119.26 ± 4.8 9.73 (6.25, 13.2) <0.001 Cysteine/Cystine 41 0.79 ± 0.18 41 1.14 ± 0.18 −0.33 (−0.41, −0.26) <0.001 Eh for Cysteine −106 mV −111 mV 2 GSH: glutathione; GSSG: oxidized glutathione disulﬁde; Eh: steady-state redox potential; Eh for GSH: −264 mV +(30 mV) ∗ log([GSSG]/[GSH] ); Eh for cysteine: −250 mV + (30 mV) ∗ log([CySSCy]/[Cys]2).

30 0.3 ∗ 25 0.25 ∗ ∗ ∗ 20 0.2 ∗ 15 0.15 ∗

GSH/GSSG 10 0.1

5 Oxidized GSH (%) 0.05

0 0 Plasma Activated monocytes Activated T cells Plasma Activated monocytes Activated T cells

Control Control Case Case (a) (b)

Figure 1: Intracellular and extracellular glutathione redox imbalance in autism. (a) presents the GSH/GSSG in plasma, isolated activated monocytes, and CD4 T cells from case and control children; (b) presents the % oxidized glutathione equivalents. Both extracellular and intracellular glutathione redox status are consistently signiﬁcantly decreased among the case children (∗P<0.01).

The concentration of cystine, the oxidized form of and unaffected control children (normalized to MFI of the cysteine, was significantly elevated (∼52%), while the cys- standard PBMC preparation). Gated lymphocytes from chil- teine/cystine redox ratio was significantly decreased (∼31%) dren with autism exhibited a significantly higher mean level in plasma from children with autism (P<0.001). The Eh of intracellular free radicals compared to lymphocytes from value for the cysteine pool can also be calculated from the control children (P<0.05). No differences in free radical Nernst equation (see above) where the E0 for cysteine is equal production were observed in gated monocytes from case and to −250 mV [30]. The calculated Eh value for the cysteine control children. Intracellular free radical production was pool in children with autism is −106 mV, or 5 mV more not correlated with age or gender in this cohort. oxidized than the control Eh value of −111 mV (Table 3). 4. Discussion 3.4. Elevated Free Radical Production in Autism. The level of intracellular free radicals was measured in available resting Oxidative stress is generally defined as an imbalance between PBMCs from children with autism (n = 15) and unaffected oxidant production and endogenous antioxidant defense control children (n = 16) using DCF, an ROS/RNS-sensitive mechanisms and can be clinically defined in humans by a fluorescent probe. Monocytes and lymphocytes were gated decrease in the redox status of GSH/GSSG and cysteine/cys- based on light scatter properties (size and density) and ana- tine thiol/disulfide redox couples [49].Therelativeequi- lyzed separately. Figure 2 presents the median fluorescence librium between reduced and oxidized sulfhydryl groups intensity (MFI) of lymphocytes from children with autism defines the ambient redox state. Low glutathione redox status 6 Autism Research and Treatment has been associated with the pathophysiology of several neu- 1.5 ∗ robehavioral disorders including schizophrenia [2, 50], bipolar disorder [3], alcoholism [51], HIV [52], and Alzheimer’s disease [53]. This is the first study to evaluate intracellular glutathione-mediated antioxidant/redox capacity in primary 1 cells from children with autism as well as the extracellular plasma cysteine/cystine redox status. Because these two redox systems are compartmentalized and independently regulated, evaluation of both redox couples provides a complete 0.5 picture of the primary immune cell microenvironment in Intracellular free radicals children with autism. Supporting and extending our previ- (normalized to standard) ous findings of decreased plasma and lymphoblastoid cell GSH/GSSG, we now report that both primary immune cell 0 GSH/GSSG and plasma cysteine/cystine redox couples are Control Case similarly compromised resulting in a more oxidized immune Figure 2: Intracellular Free Radicals are Elevated in Lymphocytes cell microenvironment in children with autism compared to from Children with Autism. Intracellular free radicals were mea- control children. sured in freshly isolated PBMC from children with autism and Recent evidence supports the notion that subtle fluctu- unaffected control children using 1 uM DCF. Presented is median ations in ambient redox status may provide an important fluorescent intensity (MFI) of the gated lymphocyte population regulatory mechanism that can dynamically modulate im- from subject samples normalized to MFI of a standard PBMC mune cell function. Activation and proliferation of T cells preparation also treated with 1 uM DCF and analyzed with each require a reducing intracellular microenvironment, whereas subject sample. Lymphocytes from children with autism exhibited a significantly higher mean level of intracellular free radicals than a more oxidized environment promotes cell cycle arrest and controls (P = 0.04). Control median (95% CI) = 0.576 (0.551– blunted responsiveness to immune stimulation [54–57]. For 0.640); case median (95% CI) = 0.689 (0.561–1.086). example, a mechanism involving extracellular redox modulation by regulatory T cells (Tregs) was recently elucidated by Yan et al. [35]. Tregs were shown to inhibit the release of cysteine into the immune synapse between dendritic cells to be associated with decreased proliferative response after and na¨ıve T cells, which effectively reduces GSH levels in T mitogen stimulation in children with autism [58]. cells by eliminating the rate-limiting amino acid for GSH The more oxidized GSH/GSSG redox status in plasma synthesis. A high ratio of reduced to oxidized glutathione is and primary immune cells in children with autism (Figure 1) required for cell cycle progression from G1 to S phase and may offer a mechanistic explanation for the abnormal adap- induction of the T-cell proliferative response [55]. Thus, the tive immune response previously reported in these children. more oxidized GSH/GSSG redox state of the intracellular When intracellular oxidative stress exceeds glutathione redox glutathione pool in PBMCs and in activated CD4 T cells capacity, cells export GSSG into the plasma as a mechanism observed in children with autism (Table 2) would suggest to restore internal redox homeostasis [49, 63]. The elevated a hyporesponsive phenotype that is less conducive to T-cell GSSG concentrations in PBMCs (Table 2) suggest that activation and proliferation. Consistent with this hypothesis, the GSSG export mechanism and intracellular antioxidant several recent studies have documented abnormalities in capacity were not sufficient to maintain intracellular redox the adaptive immune response in children with autism homeostasis and that redox imbalance was chronic in these [44, 58]. children. The association between a more oxidized immune A glutathione deficit in T cells has been shown to nega- cell microenvironment and an abnormal adaptive immune tively affect the adaptive immune response and T-cell pro- response warrants continued investigation especially in light liferation by reducing IL-2 receptor turnover and IL-2- of the potential reversibility of immune dysfunction with dependent DNA synthesis [59, 60]. In monocytes, an oxi- targeted treatment to restore redox homeostasis [15]. dized intracellular environment has been shown to alter the The calculated Eh values for the extracellular GSH and cytokine profile and skew the Th1 and Th2 balance [61, 62]. cysteine pools (Table 3) in our control population differ Studies in mice have demonstrated that the intracellular GSH somewhat from previously published values. In adults, the content of antigen presenting cells (APCs) reversibly alters plasma glutathione Eh is more reduced at around −137 mV, the Th1 and Th2 cytokine response pattern [61]. Specifically, and the plasma cysteine redox couple is more oxidized at a GSH deficit reduced Th1-associated IFN-γ production and −80 mV [30, 48]. These discrepancies may reflect method- exaggerated Th2-associated IL-4 production. Restoration of ological differences in sample preparation in that our electro- GSH restored the Th1 cytokine response and normalized chemical detection does not require derivatization for detec- the Th2 response. Consistent with these observations, two tion. It is also possible that children (age 3–10 years) may independent studies have reported that helper T-cell sub- have less reducing capacity than previously reported in adults populations in PBMCs from children with autism are shifted (age 25–35 years) [48]. Nonetheless, our calculated Eh values towards T helper 2 (Th2) dominance [41, 42]. Further, a are consistent with previous reports that plasma cysteine Eh decrease in T-cell IL-2 receptor expression has been reported (−111 mV) is more oxidized than that of GSH (−128 mv). Autism Research and Treatment 7

Mean intracellular free radical production was higher in cysteine/cystine-dependent signaling for mitochondrial ROS primary lymphocytes from children with autism relative to production is not yet clear; the authors provide evidence of lymphocytes from age-matched control children (Figure 2) a possible link to changes in the redox state of cytoskeletal and was driven by a subset of 5 (33.3%) children whose proteins that could be functionally linked to the mitochon- lymphocytes exhibited especially high levels of free radicals. drial membrane. Other studies have demonstrated that an Mitochondria are the primary producers and targets of oxidized plasma cysteine/cystine redox potential is associated intracellular free radicals, and mitochondrial dysfunction has with proinflammatory conditions [78, 79]andcanbemod- been postulated to be a contributing factor in the pathogen- ulated by diet [80, 81]. These observations support the pos- esis of autism and numerous other neurological disorders sibility that the oxidized plasma cysteine/cystine in children [64–67]. In a lymphoblastoid cell model, we previously dem- with autism may be functionally related to the increase in onstrated that the GSH/GSSG redox ratio in mitochondria lymphocyte free radical production observed and contribute was significantly lower in autism compared to control cells to immune cell abnormalities in these children. and was associated with a significantly lower mitochondrial In summary, we show for the first time that both the membrane potential after nitrosative stress [16]. It is well extracellular and intracellular immune cell compartments established that mitochondria are highly concentrated in are more oxidized in children with autism compared to age- presynaptic terminals and that loss of redox control can neg- matched unaffected control children. Randomized clinical atively affect the efficiency of neurotransmission and synap- trials will be needed to determine whether treatment to tic plasticity [68, 69]. Similarly, mitochondrial localization normalize plasma and intracellular redox status will improve and redox signaling at the immunological synapse between immune cell function and possibly the health and behavior lymphocytes and antigen presenting cells are required for in children with autism. immune activation, and excessive ROS can interrupt these signaling pathways [70–72]. A recent study of mitochondrial Conflict of Interests defects in lymphocytes from children with autism found decreased complex I activity and overreplication of and de- The authors declare no conflict of interests. letions in mitochondrial DNA compared to control lymphocytes [73]. Based on this evidence, it is plausible to hy- Acknowledgments pothesize that mitochondria may be the source of the increased levels of lymphocyte free radicals observed in the The authors would like to express their gratitude to the subset of autistic children presented in Figure 2. Consistent families in Arkansas affected by autism whose participa- with this hypothesis, a recent meta-analysis estimated that tion made this study possible. They also acknowledge the mitochondria dysfunction may affect up to 30% of children invaluable help of the nurses and clinicians at the Dennis with autism [64]. Based on this evidence, further study of Developmental Center for referral and evaluation. This mitochondrial function and redox status in lymphocytes research was supported, in part, with funding from the from children with autism is warranted. National Institute of Child Health and Development (RO1 Relevant to our observations, two recent papers have HD051873; SJJ), the Department of Defense (AS073218P1; revealed that an oxidized extracellular cysteine/cystine redox SJJ), and by grants from the Arkansas Children’s Hospital status can initiate a redox signaling cascade that stimulates and Arkansas Biosciences Institute (SJJ). intracellular mitochondrial ROS production as a mechanism to initiate an inflammatory immune response [74, 75]. The signal transduction from the extracellular to intracellular References compartments occurs through oxidative modification of [1] CDC, “Prevalence of autism spectrum disorders—autism and redox-reactive cysteines on cell surface proteins. 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Clinical Study High Complement Factor I Activity in the Plasma of Children with Autism Spectrum Disorders

Naghi Momeni,1 Lars Brudin,2 Fatemeh Behnia,3 Berit Nordstrom,¨ 4 Ali Yoseﬁ-Oudarji,5 Bengt Sivberg,4 Mohammad T. Joghataei,5 and Bengt L. Persson1

1 School of Natural Sciences, Linnaeus University, 39182 Kalmar, Sweden 2 Department of Clinical Physiology, Kalmar County Hospital, 39185 Kalmar, Sweden 3 Department of Occupational Therapy, University of Social Welfare and Rehabilitation Sciences, Tehran, Iran 4 Department of Health Sciences, Autism Research, Faculty of Medicine, Lund University, Box 157, 22100 Lund, Sweden 5 Cellular and Molecular Research Centre, Tehran University of Medical Sciences (TUMS), Tehran, Iran

Correspondence should be addressed to Bengt Sivberg, [email protected]

Received 17 June 2011; Revised 22 August 2011; Accepted 22 August 2011

Academic Editor: Judy Van de Water

Copyright © 2012 Naghi Momeni et al. This is an open access article distributed under the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.

Autism spectrum disorders (ASDs) are neurodevelopmental and behavioural syndromes affecting social orientation, behaviour, and communication that can be classified as developmental disorders. ASD is also associated with immune system abnormality. Im- mune system abnormalities may be caused partly by complement system factor I deficiency. Complement factor I is a serine protease present in human plasma that is involved in the degradation of complement protein C3b, which is a major opsonin of the complement system. Deficiency in factor I activity is associated with an increased incidence of infections in humans. In this paper, we show that the mean level of factor I activity in the ASD group is significantly higher than in the control group of typically developed and healthy children, suggesting that high activity of complement factor I might have an impact on the development of ASD.

1. Introduction The complement system comprises a group of proteins which, when activated, provide one of the first lines of Autism spectrum disorders (ASDs) are characterized by im- defence by promoting lysis and the removal of invading pairments in social interaction, communication, and repeti- microbes. Activation of the complement system in response tive or restricted patterns of interests, or behaviours, and are to an infection or foreign antigen is achieved via three com- classified as developmental disorders in DSM-IV [1]. ASD is plement pathways, the classical pathway, which is activated about 4-5 times more prevalent in boys than in girls. The by antigen-antibody complexes, the lectin pathway, which is ratio is estimated to range from 5.5 : 1.4 to 16.8 : 4.0 [2]. activated by the interaction of microbial carbohydrates with Recent research clearly indicates that the underlying causes mannose-binding proteins in the plasma and tissue fluids, of autism are neurobiological disorders and combinations of ff and the alternative complement pathway, which is activated di erent factors, such as environmental and genetic factors, by C3b binding to microbial surfaces and to antibody mole- and abnormality in the communication between neurons, cules. All of the three pathways converge with the activation probably associated with an abnormal set of neuropeptides of the central C3 component. This leads to a final common in the brain [3–9]. The symptoms of ASD have been linked with elevated pathway with assembly of the C5–C9 components to form plasma levels of serotonin [10, 11] and opioid [12], abnormal a cell surface transmembrane pore (membrane attack com- levels of melatonin [13], altered levels of activity of the serine plex) [22, 23]. It has been shown by comparison with healthy protease prolyl endopeptidase [14], and infectious and im- control children that several differentially expressed proteins munological factors such as abnormalities of T cells, B cells, are related to the complement system in children with ASD natural killer (NK) cells, and of the complement system [15– [22]. The alternative pathway consists of six proteins: C3, fac- 21]. tor B, factor D, factor H, factor I, and properdin. The plasma 2 Autism Research and Treatment glycoprotein factor I (C3b/C4b inactivator) is a serine pro- of Pediatrics in its Embargo from 2007 [35]. The control tease that acts as a regulator of the complement C3 cascade. group consisted of typically developed and healthy children Factor I has a molecular weight of about 88 kDa, consists of showing no signs of neurodevelopmental disorders who were two disulfide-linked polypeptide chains (50 kDa and 38 kDa, recruited from the same area as the children with ASD. resp.), and is synthesized as a single-chain precursor in the Children who had any kind of infection/infectious disease liver [24, 25]. Factor I cleaves C3b and C4b in a reaction, within two weeks prior to the time of examination were wherefIisdependentonvariouscofactors,suchasfactor excluded from this study. H, C4b-binding protein CR1 and membrane cofactor protein The study was approved (MT/1247) by the ethics com- (MCP) [26]. Factor I-mediated cleavage of the α chain of C3b mittee of the Iran University of Medical Sciences, Tehran. liberates 3 fragments with molecular weights of 68 kDa, 43 kDa, and 2 kDa. Degradation of C3b by fI abrogates the 2.2. Procedure action of this protein in the C3 pathway [27]. Complement C3b is the major opsonin of the complement system which 2.2.1. Blood Sample Collection. Blood samples were collected facilitates the phagocytosis process by coating antigens (each by a paediatric nurse, and those from the children diagnosed of the phagocytes expresses a complement receptor such as with autism were collected under the supervision of a child CR1, CR3, or CR4 that binds C3b, C4b, or C3bi) [28, 29]. psychiatrist with special training in the field of childhood Factor I deficiency can be conferred by a C3 deficiency, since psychosis. Venous blood was collected into 3 mL EDTA this also increases susceptibility to pyogenic infections by tubes (Vacutainer System; Becton-Dickinson Inc., Plymouth, UK), and plasma was separated immediately thereafter by Neisseria meningitides, Haemophilus influenza, and Strep- ◦ tococcus pneumonia and increases the incidence of immune centrifugation at 1,300 g for 10 min at 4 C. Thereafter, an μ complex diseases due to impaired complement-mediated inhibitors cocktail (30 L per 1 mL plasma) was added to the function [30]. Immune system abnormalities have been asso- resultant plasma sample (cocktail inhibitor solution: 2.0 M μ ciated with autism [15–20], and it has been suggested that Tris, 0.9 M Na-EDTA, 0.2 M Benzamidine, 92 M E-64, and 48 μM Pepstatin; Sigma, St. Louis, Mich, USA). The plasma children with ASD might have an increased incidence of bac- ◦ terial inflammation [31]. Immunological aspects of the early was stored at −80 C. onset of autism have recently highlighted the fact that immune dysfunction may occur in some children with autism 2.2.2. Assay. Methods based on the hydrolysis of fluorogenic [31, 32]. substrates have previously been described by Tsiftoglou and Having previously discovered altered levels of the serine Sim [36] and Gupta et al. [15]. The following assay procedure was found to be optimal for assaying fI activity in the protease prolyl endopeptidase in children with ASD [14], the μ μ ff aim of this study was to investigate if an association exists plasma. 20 L of plasma was incubated with 80 Lofbu er (100 mM phosphate buffer, pH 7.5, containing 1 mM EDTA, between serine protease fI deficiency and the development of ◦ ASD. 1 mM DTT and 1 mM sodium azide) for 10 min at 37 Cto reach thermal equilibrium. 100 μL of substrate solution (200 μM Boc-Asp(OBz)-Pro-Arg-7-amino-4-methylcoumarin; Bachem, Bubendorf, Switzerland) in 25 mM phosphate 2. Materials and Methods buffer, pH 7.4, was then added, and the mixture was incubated at 37◦C for 60 min (see Figure 1). The reaction was 2.1. Participants. Thirty children with ASD and thirty typical inhibited by the addition of 1 mL of 1.5 M acetic acid, and the control children participated in this study. The ASD group release of 7-amino-4-methylcoumarin was measured by comprised 23 boys and 7 girls with a mean age of 4.5 years spectrofluorometer (Hitachi-f 2000; λ : 360 nm; λ : (age range 3–9 years). The control group comprised 13 boys ex em 440 nm; slit width: 2.5). All measurements were carried out and 17 girls, mean age 6.0 years (age range 3–12 years), randomized and in duplicate. Background fluorescence in (Table 1). the assay was monitored by the use of plasma in the absence Children in the ASD group were recruited from the Au- of substrate and was subtracted from values obtained in the tism Rehabilitation Centre at the University of Social Welfare presence of substrate. and Rehabilitation Sciences in Tehran, Iran. After having obtained informed consent from the parents, blood samples 2.3. Data Analysis and Statistics. Plasma fI activity was log- were collected. All children with ASD were examined by cli- normally distributed, and logarithmic values were, therefore, nical specialists on autism. A child psychiatrist and a child used when analysing differences between the ASD group and neurologist or child psychologist examined all of the chil- the control group. To adjust for age (dichotomized using the dren. All consultants agreed on the diagnosis of autism ac- median value, 5 years) and gender, factorial ANOVA was cording to the DSM-IV criteria [1]. However, diagnostic pro- used. A P value < 0.05 was considered statistically significant. cedures applied in Europe and in the US/Canada using the Statistica 8.0 (StatSoft c , Tulsa, Okla, USA) was used. Intra- autism diagnostic observation schedule (ADOS) [33] and the and interassay variability of the plasma fI activity was expres- Autism Diagnostic Interview—Revised [34]werenotusedin sed as the standard error of a single determination (Smethod), the diagnostic process applied in Iran. This shortcoming was using the formula first proposed by Dahlberg [37] compensated for by the extensive clinical experience by the √ d 2 child neurologist/child psychiatrist who was familiar with the S = i method n ,(1) core behaviours in autism stated by the American Academy (2 ) Autism Research and Treatment 3

Table 1: Age/y, gender, and medication of the participants.

Parameter ASD (n = 30) Controls (n = 30) P value∗ Mean (SD) 4.8 (1.7) 6.1 (2.3) Median (range) 4.5 (3–9) 6 (3–12) 0.033 Age ≤5 years 21 14 >5 years 9 16 0.115 Males 23 13 Gender Females 7 17 0.017 No speciﬁc medication 8 30 — Medication Risperdal alone or in combination 18 0 — Ritalin alone or in combination 4 0 — ∗ Diﬀerence between ASD and controls. Mann-Whitney U-test for age and Fisher’s exact test for age category and gender.

Factor I activity and substrate stability medication with Risperdal to reduce hyperactivity and vio- 3000 lent behavior, and a few were under medication with Ritalin to improve attention (Table 1). It would have been ethically questionable to discontinue medication with the purpose 2500 of controlling the experimental design. We have correlated the data shown in Figure 2(a) with the type of medication 2000 the children in the ASD group were receiving. Although we did not see any clear correlation between medication and distribution for the scatter plot data, it cannot be excluded 1500 that some differences in the pattern may be influenced by medication, as has been previously discussed [22]. The values were statistically significantly higher in the 1000 children with ASD, and there was a weak association with Fluorescence intensity unit gender. No statistically significant differences were found, however, between the age groups. 500 The methodological intra-assay error was small, 0.5%. The interassay methodological error was 13%. We found a significantly higher complement factor I enzyme activity 0 123in children with ASD compared to the control group of Time (hours) around the same age. This is, as far as we know, the first report regarding dysfunction of fI activity in children with Figure 1: Fluorescence intensity of release of 7-amino-4-methyl ASD. Although not statistically significant, males tended to coumarin as a function of plasma incubation period (mean ± SD; exhibit higher fI activity than females, and the difference 1h(974 ± 44.2), 2h (1995 ± 45.2) and 3 h (2374 ± 1.2)). between the control group and the ASD group was more convincing amongst the younger children, as shown in Figure 2. Due to fI’s role as a regulating factor in the comple– ment system pathway, an fI abnormality could play a role where di is the difference between the i : th paired measure- in the onset of ASD. A defect in this pathway makes the- ment and n is the number of differences. The Smethod was ex- individual more vulnerable to various inflammations. Some pressed as the coefficient of variation (%). reports [21, 24–26] provide increasing evidence of a connection between immunological abnormality and human disease. ASD numbers among the types of disorders that 3. Results are associated with immune system abnormalities [28, 38]. Our results are consistent with a recent proteomic study of There was significantly higher activity of plasma fI in the serum from ASD children, where a significant differential children with ASD (geometric mean (95% confidence limit): expression was shown for proteins related to the complement 523 (154–1776) when compared with the control group: 361 system [22]. (135–967; ANOVA P = 0.015, adjusted for age and gender; Figure 2). No statistically significant interactions were found with 4. Discussion regard to gender and age, and no significant associations were found between fI activity and age or gender (ANOVA; P = The aetiology of ASD is still largely unknown despite the fact 0.25 for gender and 0.42 for the two age groups, Figure 2). In that many factors such as genetic, environmental, immuno- the ASD group, some children with severe autism were under logical, and neurological aspects are thought to influence 4 Autism Research and Treatment

3000

Age ≤ 5 years Age > 5years 7 2000

6.6 1000 Males

Fluorescence intensity Males 6.2

0 Females Control ASD 5.8

Participants log (ﬂuorescence intensity) Females

Non medicated Combination (Risperdal and Ritalin) 5.4 Risperdal Control ASD Control ASD Others Ritalin Participants Participants (a) (b)

Figure 2: (a) Complement factor I activity in EDTA plasma from children with ASD (n = 30) and healthy control children (n = 30). A scatter plot of factor I activity for each individual is shown. Samples from ASD children who were not under medication at the time of the investigation, those under medication with Risperdal, Ritalin, a combination thereof, or other medications such as antipsychotics (thiorida- zine) or anticonvulsants (fenobarbital and sodium valproate) are shown. (b) Mean values and standard errors of the complement factor I activity in EDTA plasma are shown for the different age groups and genders for both children with ASD and the healthy control group. In the ASD group; age ≤ 5 years: males (n = 17), females (n = 4) and age > 5 years: males (n = 6), females (n = 3). In the healthy control group; age ≤ 5 years: males (n = 4), females (n = 10) and age > 5 years: males (n = 9), females (n = 7). the development of ASD [39]. A dysregulated immune res- a functioning C3b protein. C3b is degraded by fI, and abnor- ponse has been reported among children with ASD. Pro- mal fI activity might cause an abnormal and uncontrolled tein products of immune activation, such as cytokines, can degradation of C3b protein, resulting in the loss of the be linked to core features of ASD, such as difficulties to reg- phagocytosis function for this particular protein (a function ulate affect, sleep, nutritional uptake, and can also affect which partly protects the body from invasion of foreign behaviour and social communication [39]. This study high- organisms). Altered levels of other serine protease activities, lights an elevated level of factor I that may contribute to a such as that of proline endopeptidase (PEP), have also been dysregulation of the immune response associated with the found in a group of children with ASD when compared to a complement system. Recent research proposes an extensive control group [14]. The results of the present study, together communication between the immune and nervous systems, with our previous findings on altered levels of PEP activity, affecting both the development of the central nervous system may indicate a connection between the onset of ASD and [32] in the promotion of health as well as disease. Early serine protease dysfunction. The higher plasma fI activity damage during critical periods in neurodevelopment of the observed in the male group as compared to that of the foetus has in a mouse model study been shown to affect female group (Figure 2) is paralleled by a higher occurrence cognitive development. It is reasonable to suggest that of ASD in male children. Also, the higher plasma fI activity in complement factor I might contribute to ASD, while changes children younger than six years of age may indicate that the in the complement system may predispose the mother or inflammatory process is more active in younger ages or that foetus to infections during development, possibly causing we may be dealing with two subgroups of children with ASD resultant abnormalities in brain development [22]. Also, the with different onsets of the disease. frequency of autoimmune diseases has been reported to be significantly higher in families with a child with ASD [40] 5. Conclusions than in families with children with typical development. The pathophysiology of ASD is poorly understood. Chil- The preliminary findings of this study together with our dren with ASD are prone to recurrent viral and bacterial in- previous report [14] suggest that there may be an associa- flammations. There are also some reports of immune sys- tion between abnormal serine protease activity and the deve- tem abnormalities in children with ASD [15–20]. An associa- lopment of ASD. Further research is needed, however, to tion between ASD and immune system abnormalities toge- establish a possible role of serine proteases in the aetiology ther with the vulnerability of the ASD group with regard to of ASD. inflammatory processes may indicate an impaired mechanism in this system. It is known that phagocytosis is an Conflict of Interests important part of the body’s defence mechanism. This mechanism requires an active complement system and The authors declare that they have no conflict of interest. Autism Research and Treatment 5

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