The Neuroscience of Autism Spectrum Disorders || Immune Abnormalities and Autism Spectrum Disorders

C H A P T E R

2.9

Immune Abnormalities and AutismSpectrum Disorders

Majannie Eloi Akintude*, Luke Heuer*, Judy Van de Watery*University of California, Davis, CA, USA yDepartment of Internationl Medicine and the UC

Davis MIND Institute, Davis, CA, USA

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Historical Relationship of Immune Abnormalitieswith ASD
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Introduction to the Immune System
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Abnormalities in Innate Immunity in ASD
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Monocytes/Macrophages
235 Natural Killer Cells (NK) 236 Humoral Immunity 237
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Immunoglobins
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Complement
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23nce of Autism Spectrum Disorders.

org/10.1016/B978-0-12-391924-3.00016-8

Abnormalities in the Adaptive ImmuneResponse in ASD
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Cellular Immunity
239 T Lymphocytes (T cells) 239
B Lymphocytes (B cells)
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Cytokines and Chemokines
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Autoimmunity
242 Auto-Antibodies e Children 242 Auto-Antibodies e Maternal 243
Conclusions
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HISTORICAL RELATIONSHIP OFIMMUNE ABNORMALITIES WITH ASD

Autism spectrum disorders (ASD) are characterizedby difficulties in social interactions, impairment inverbal and/or nonverbal communication, and in thedevelopment of stereotyped behaviors and restrictedinterest (Lord et al., 2000; APA, 2000). Over the pasttwenty-five years the prevalence of ASD has increasedrapidly for a variety of reasons (see Chapter 1.1). TheCenter for Disease and Control (CDC) estimates that 1to 110 live births in the United States is diagnosedwith ASD ((CDC), 2006; Boyle et al., 2011; Investigatorsand (CDC), 2009). The increased prevalence of ASD issuggested to be the result of increased awareness,more effective diagnosis and classification approaches,as well as due to truly higher incidence rates (Hertz-Picciotto and Delwiche, 2009). Autism is typically

diagnosed between the ages of 2–3 years, and hasa male to female ratio of 4:1. Currently, the factorsleading to autism are not completely known and thereis no biological marker common to all forms of thesyndrome. Because of the diversity in phenotypes andthe differences in the severity of the symptoms amongindividuals with ASD, it is a referred to as a spectrumdisorder which is presumably caused by different etiol-ogies (Altevogt et al., 2008). Genetic and environmentalfactors are both suggested as having a role in the etiologyof ASD. Genetic studies on individuals diagnosed withASD have demonstrated that whilemany different genesare implicated in ASD, there is extreme genetic andgenomic heterogeneity [see Chapters 2.1–2.4, also(Zhao et al., 2007)]. In addition, there is now a growingbody of research suggesting that abnormalities in theimmune system may also be a contributing factor inthe etiology of ASD (Ashwood et al., 2006).

Copyright � 2013 Elsevier Inc. All rights reserved.

http://dx.doi.org/10.1016/B978-0-12-391924-3.00016-8

2.9. IMMUNE ABNORMALITIES AND AUTISM SPECTRUM DISORDERS234

As autism is a spectrum disorder, a better under-standing of the immune abnormalities noted in theseindividuals is critical, since immune profiling could beused in the future to identify subpopulations of ASDpatients. Investigation of the immune response in chil-dren with autism is a new and growing research fieldthat has developed tremendously over the past 30 years.Despite a somewhat rocky start, numerous recentstudies now provide us with strong evidence indicatingimmune abnormalities in children with autism, andunderstanding these abnormalities might make avail-able new tools for enhanced diagnosis and treatmentof ASD.

First described in 1977, investigators demonstratedthat children with autism had a decrease in theirimmune response after immune challenge (Stubbs andCrawford, 1977). Over the next 30 years, multiplestudies continued to describe immune anomalies inindividuals with ASD. There were early issues withpoorly controlled studies, but as the field has matured,investigators have consistently demonstrated immuneabnormalities in children with ASD, as well as in imme-diate family members. Some of the most compellingrecent research is the presence of neuroinflammation,skewed cytokine profiles, altered lymphocyte activation,elevated circulating monocytes and autoimmunephenomena in children with autism and their mothers(Ashwood et al., 2006; Braunschweig et al., 2008;Enstrom et al., 2009b; Heuer et al., 2008; Vargas et al.,2005). Research to understand the direct relationshipbetween immune dysfunction and autism continues,and such investigations could lead to a better under-standing of the interplay between the immune andnervous systems. The following chapter will attempt todescribe the current state of immune-related researchin autism, and to explain how, through this research,there is the potential for a better understanding of thisenigmatic disorder.

INTRODUCTION TO THE IMMUNESYSTEM

To understand the potential role of the immunesystem relative to ASD, one must first be introduced tothe major players involved in the immune system. Theimmune system as the body’s defense mechanismagainst invaders is a very complex structure known tointeract with many other systems in the body, includingthe endocrine and nervous systems (Carpentier andPalmer, 2009; Deverman and Patterson, 2009; McAllisterand Van de Water, 2009). Upon invasion or infectionwith a pathogen, the immune system reacts by removalof the agent and develops immunological memory toprevent reoccurrence of infection, which is a major

2. ETIOLOGY OF AUTISM

strength of the immune response. The development ofimmunological memory helps the host by establishinga mechanism to quickly remove invaders in subsequentinfections. Immune cell memory relies upon an intricateweb of interaction between multiple immune factorsand cell subtypes. A proper immunological responsemay only be achieved when all the immunologicalfactors are well balanced, and thus in a homeostaticstate. However, in disease states such as autoimmunity,the immune systemmay respond to ‘self-protein’, seeingit as the foreign antigen, thus creating a chronic activa-tion or inflammatory atmosphere (Murphy, 2011). Inother cases, the immune systemmay be unable to mounta proper immune response due to the depletion ordysfunction of the immune cells, such as in the case ofHIV/AIDS. Overall, the immune system is solelyresponsible for the host’s defense and is controlled andmaintained by the delicate interplay between severalimmunological factors that can be simply divided intotwo arms; the innate immune and the adaptive immuneresponse.

The innate immune response is a first-line defensesystem and is non-specific in its response to pathogens.These cells recognize non-specific pathogens by genericreceptors on innate immune cells. This mechanismprovides the host with a generic way to quickly identifycertain component patterns derived from bacterial andviral particles that activate particular receptors whenmounting an immune response (Murphy, 2011). Theseresponses subsequently lead to the activation of themore specific adaptive immune response that requirespresentation of processed antigens to other immunecells. Major players in the innate immune responseinclude the monocyte/macrophage lineage, which playsa significant role in phagocytosis, the NK cells which areimportant in viral infections and tumor destruction,immunoglobins and the complement molecules, whichboth play a role in the clearance of pathogens.

The adaptive immune system is a highly specializedgroup of cells that respond specifically to eliminate orprevent reoccurrence of pathogens by immunologicalmemory. Its strength arises from its ability to recognizeand remember specific pathogens (Murphy, 2011). Themajor cell types of the adaptive immune system includethe B and T lymphocytes. B-lymphocytes, or B cells,differentiate into plasma cells to secrete antibodiesupon stimulation and activation, an extremely impor-tant component of the humoral immune response.T lymphocytes, or T cells, are the major effector cells ofthe adaptive immune system and are responsible forthe elimination of infectious agents (Murphy, 2011).Mediators of both the innate and adaptive arms of theimmune system exist in the form of cytokines and che-mokines. Cytokines are released from immune cellsand signal to other cells for activation, replication, and

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Xenobiotics and Infections that Influence the

Immune Response and Neurodevelopment

Innate Immune Response Adaptive Immune Response

Toll-like Receptors (TLR)

Complement

B cells (Plasma Cell)

T cells

Toxicants

Viruses

Monocytes/Macrophage

B cells

Altered NK Cells

•Decreased NK cells•Decreased cytotoxic activity

Altered Complement Protein

•C4B protein•C4B null allele

NK cells

Altered TLR Signaling

•TLR2•TLR4•TLR9

IL- 6

TNF- α

IL-1 β

Altered Cytokine Profile

Altered T cell subsets

•Decreased •CD8+ •CD4+•CD95+


Increased Plasma

Chemokines

•MCP-1•Rantes•Eotaxin

Increased Plasma Cytokines

•IL-6•TNF- α•IFN- γ

Decreased Ig

•IgM•IgGIncreased Ig

•IgG2•IgG4

Auto-antibodies:

•Fetal brain proteins (maternal•Cerebellum•Thalamus

Dendritic Cells

Bacteria

FIGURE 2.9.1 Overview of immune abnormalities in children with autism. Research has shown children with autism have variousimpairments in both the innate and adaptive immune response. In the innate immune response, research has shown the presence of alteredcomplement protein levels, reduced NK cell activity, and changes in both the Toll-like Receptor (TLR) signaling pathways in monocytes, as wellas cytokine and chemokine dysreguation. For the adaptive immune response, effector cells such as Tcells have also been shown to have cytokineand chemokine dysregulation. Decreased IgG and IgM, as well as auto-antibodies brain proteins have been described in children with autism,and maternal auto-antibodies to fetal brain proteins are highly specific for ASD. Xenobiotic exposure and/or infections are also suggested toinfluence the immune response in children with autism. Taken together, alterations in the immune response are strongly indicated in childrenwith autism.

ABNORMALITIES IN INNATE IMMUNITY IN ASD 235

killing. Chemokines are involved in directing theimmune cells to migrate to sites of infection (Murphy,2011). Each of the above-described systems has beenimplicated to play a role in the pathogenesis of autism.They are discussed in further detail in the followingsections, and summarized in Figure 2.9.1.

ABNORMALITIES IN INNATEIMMUNITY IN ASD

Various studies have indicated differences in theinnate immune response between children with ASDand unaffected individuals. As mentioned earlier, theinnate immune system is the host’s first line of defense.


The differences described in innate immunity in chil-dren with ASD compared to unaffected individualsinclude changes in monocytes/macrophages andnatural killer (NK) cell activation and cell numbers(Enstrom et al., 2009b; 2010; Onore et al., 2011). Comple-ment levels have also been reported to be altered in ASD(Mostafa and Shehab, 2010;Warren et al., 1991). An over-view of the innate immune abnormalities found in chil-dren with ASD is given in the next section.

Monocytes/Macrophages

Monocytes are major players in the innate immuneresponse. Upon infection, peripheral blood monocytesdifferentiate into macrophages in the tissue and travel

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to the site of infection. As the most prominent effectorcells in the innate immune response, macrophages areinvolved in identifying pathogens, phagocytosingthem, and activating the adaptive immune responsethrough antigen presentation via the MHC (major histo-compatibility complex) (Murphy, 2011). Macrophagesexpress TLRs, which broadly recognize commonPAMPS on viruses and bacteria. Altered monocyte/macropaghe numbers and activation levels have beenindicated in children with autism. A study that exam-ined the absolute leukocyte counts in age- and gender-matched healthy children compared to those with ASDshowed a significantly higher monocyte count in thelatter group, with no difference in the absolute leukocytecounts (Sweeten et al., 2003b). This is an interestingfinding since high bloodmonocyte counts are often asso-ciated with autoimmune disorders and some infectiousdiseases (Sweeten et al., 2003b). This study indicatedthat the immune system of children with autism mightoperate at a higher level of activation compared tohealthy children. Thus, this increased activation maylead to alterations in neurodevelopment.

In an attempt to examine the function and activationof monocytes in a well-characterized group of childrenwith autism, Enstrom et al. examined monocytesignaling via TLR-2, TLR-4, and TLR-9. Monocyteswere cultured and activated with mitogens specific foreach signaling pathway and resulted in altered TLRsignaling in ASD compared to controls. TLR-2 activatedmonocytes demonstrated an increase in pro-inflamma-tory cytokines, IL-1b, IL-6 and TNF-a, while TLR-4 acti-vation also showed an increase in IL-1 b (Enstrom et al.,2010). TLR-9 activation resulted in a decrease in IL-1b,IL-6, GMCSF and TNF-a. These findings are intriguingas pro-inflammatory cytokines are thought to affect neu-rodevelopment (Aly et al., 2006; Doherty, 2007). There-fore, the innate immune system might have an impacton the function and development of the nervous system,which may lead to changes in behavioral outcomes.These results also indicated that children with autismhave a dysfunction in monocyte signaling that maylead to long-term problems in their response to infec-tion. Therefore, it is important to continue the investiga-tions of monocyte function and cytokine expression inchildren with ASD.

Several reports demonstrate disturbances in gastroin-testinal activity in children with autism (Ashwood andWakefield, 2006; Maenner et al. 2011; Wasilewska et al.,2009). One study examining monocyte activation levelsin the blood of children with autism with persistentgastrointestinal abnormalities indicated that these chil-dren had innate immune abnormalities (Ashwoodet al., 2003). Monocytes were isolated from the childrenand activated via mitogen stimulation. The results sug-gested that children with gastrointestinal problems in


conjunction with ASD produced lower amounts of thepro-inflammatory cytokines IL-6, IL-1b, IL-12, IL-23and the counter-regulatory cytokine IL-10 when mono-cytes were stimulated (Jyonouchi et al., 2011). Thisimpaired signaling was in response to Toll-like receptoragonists for TLR2/6 and TLR 7/8, which are intracel-lular receptors for ssRNA. The authors suggested thatimpaired innate signaling might be an indication of anincreased vulnerability to common microbial infectionsin children with autism who have gastrointestinal prob-lems. Overall, the authors concluded from this researchthat this sub-group of children with ASD and gastroin-testinal problems may be more vulnerable to commonmicrobial infections and that classical monocytes mayplay a role in disease pathogenesis in children withASD and gastrointestinal co-morbidities (Jyonouchiet al., 2011).

As much research is still needed in the area of mono-ctye activity in children with ASD, it would beintriguing to consider abnormalities in monocytesignaling and activation as a potential biomarker foraltered immune function in a sub-group of childrenwith autism.

Natural Killer Cells (NK)

NK cells are a unique subset of lympocytes that walka fine line between the innate immune system and theadaptive immune system.NKcells only represent a smallnumber of total lymphocytes in peripheral blood and donot express the traditional specific receptors as their Tand B lymphocyte counterparts (Schleinitz et al., 2010).NK cells have been shown to play a role in immunityagainst parasitic and viral infections, cancer, and havea suggested role in autoimmunity. These cells are foundin abundance in both the human liver and uterus (Schlei-nitz et al., 2010), and are themost prominent lymphocytesubset found in the uterus during pregnancy. Further,research has shown a link between low numbers of NKcells and miscarriage recurrence (Tang et al., 2011).Therefore researchers have explored the link betweenpregnancy, autoimmunity, and NK cell function asa possible mechanism for tolerance induction. An imbal-ance between inhibitory and activating NK cells hasbeen implicated in several autoimmune disorders, suchas systemic lupus erythematosus (SLE), multiple scle-rosis, diabetes, rheumatoid arthritis (RA), and the neuro-developmental disorder, autism (Enstrom et al., 2009b;Schleinitz et al., 2010).

Over two decades ago Warren et al., (1987) describeda decrease in NK cell function in children with autism. Amore recent study examined mRNA expression inperipheral blood of children with ASD and found anincrease in expression of NK cell associated genes(Gregg et al., 2008). In another study, which examined

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ABNORMALITIES IN INNATE IMMUNITY IN ASD 237

a large population of autism subjects, lower NK cellactivity was found in about 45% of a subset of childrenwith ASD (Vojdani et al., 2008). In an expansion of thestudy by Gregg et al. (2008), Enstrom et al. (2009b),examined NK cell activity in children with ASD togetherwith age-matched typically developing controls, andlooked for changes in activation and killing. Usingmicroarray analysis, the researchers profiled NK geneexpression and a performed functional analysis on NKcells isolated from peripheral blood. Results showedthat after stimulation, there was a decrease in NK cellcytotoxic activity compared to age-matched controls.In addition, there was an up-regulation in RNA expres-sion of NK cell receptors and effector molecules(Enstrom et al., 2009b).

There has been one report of an infection model forautism using mice that implicates an alteration in NKcell numbers (Hsiao and Patterson, 2011). In a rodentmodel of maternal immune activation (MIA), whichmimics viral infection using a synthetic double-strandedRNA (poly(I:C)) and leads to autism-related behaviorsin offspring, examination of the placentas followinginfection revealed an increase in uterine NK cells (Hsiaoand Patterson, 2011). This is an important finding, as analteration in the NK cells located in the placenta duringfetal development might have a negative impact on neu-rodevelopment. Thus, it will be interesting to furtherinvestigate NK cell activity in the placenta and uterusduring pregnancy. Abnormalities in NK cells, such asdescribed above, may represent a susceptibility factorin ASD, and may predispose to the development ofautoimmunity and/or adverse neuroimmune interac-tions during critical periods of development.

Research findings highlighted in this section suggestthat abnormalities in the innate immune response areprevalent in children with ASD. As the innate immunesystem is thebody’sfirst lineofdefense, it is critical to fullyunderstandwhy childrenwith ASD have such abnormal-ities. As a fully functional innate immune response isnecessary for the adaptive immune response to occur,abnormalities in the innate response will affect the subse-quent adaptive immune response, which may result inchildren with ASD having increased adaptive immuneabnormalities – as described in the next section.

Humoral Immunity

Immunoglobins

In addition to cell-mediated immunity, the humoralimmune response makes up the other functional armof the immune system. Humoral immunity involvesthe substances found in the humors or body fluid, andis primary comprised of antibodies and components ofthe complement system. Antibodies are secreted by Bcells, which receive co-stimulation from CD4þ helper


T cells as well as antigen presenting cells, such as thedendritic cells. Antibodies or immunoglobulins (Ig) are‘Y’-shaped glycoproteins that are found in the bloodand tissue. In humans, there are four major types of anti-bodies; IgMwhich is produced first, IgA, which is foundin mucosal tissue, IgE, which plays a role in the allergyresponse, and IgG which makes up the bulk of immuno-globulin in the circulation and is also known to cross theplacenta during gestation. Each Ig has a different biolog-ical function and property for a specific immunedefense. Ig acts by priming macrophages for phagocy-tosis and neutralization of foreign objects such asbacteria and viruses (Murphy, 2011). It may also causeagglutination and precipitation of antigen-antibodycomplexes and stimulation of the complement pathway.

Several studies have examined circulating immuno-globulin levels in autism. One such study showeddecreased levels of IgM and IgG in plasma from childrenwith autism, which correlated with more aberrantbehaviors (Heuer et al., 2008). In another study,increased levels of the atypical isotypes, IgG2 andIgG4 were reported in the plasma of children withautism (Croonenberghs et al., 2002b; Enstrom et al.,2009a). Immunoglobulin levels are the result of thecomplex interaction between antigen presenting cells,T cells and B cells. In addition, Ig levels are extremelylow at birth and take up to 10 years to reach adult levels.As such, these levels may be a useful measure of normaldevelopment of the humoral immune system.

The finding that children with ASD have lower over-all levels that correlate with behavioral symptoms mightindicate a common factor that affects the development ofboth the immune and nervous systems. Identification ofthis common defect would be instrumental in definingat least one pathological pathway in ASD.

Complement

The complement pathway plays a critical role in boththe innate and adaptive immune response. Composed ofthree different distinct pathways including classical,mannose binding lectin (MBL), and alternative(Murphy, 2011), complement is involved in the lysisand removal of infectious organisms from the blood,and is suggested to be involved in cellular apoptosis inthe brain (Corbett et al., 2007). The complement pathwayactivates over 25 proteins and protein fragments that canopsonize pathogens, induce inflammation, and formantibody-antigen complexes.

Research has proved that an association existsbetween complement levels and some autoimmunedisorders (Mostafa and Shehab, 2010; Samano et al.,2004). Since recent studies have indicated an autoim-mune phenomenon in autism, investigators have soughtto identify a potential role for the complement systemin autism; the complement component C4 has been

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studied most extensively. Complement component C4commences the initial step of activation in the classicalpathway of complement activation and humoral defenseand is determined by two pairs of allotypes, C4A andC4B (Samano et al., 2004; Warren et al., 1994). Theplasma C4 protein is composed of the protein productsof the C4A and C4B genes, which are localized inthe middle of the class III region of the MHC molecule(Martinez et al., 2001). Deficiency in the C4 allotypehas been associated with several autoimmune-associ-ated diseases such as SLE, and has also been linkedwith autism (Onore et al., 2011; Samano et al., 2004).Research has shown that individuals with autism hada significant increase in the frequency of the null alleleat the C4B locus compared to controls (Warren et al.,1991). A more recent study also linked the C4B nullallele to autism and a family history of autoimmunity(Mostafa and Shehab, 2010). Results indicated that thepresence of the C4B null allele was significantly higherin children with autism (37%) compared to controls(8%) (Mostafa and Shehab, 2010). In addition, theincreased presence of the C4B null allele had a signifi-cantly higher association with autism, and a familyhistory of autoimmunity (Mostafa and Shehab, 2010;Sweeten et al., 2008; Warren et al., 1995). Taken together,these studies indicate a possible link between the C4Bnull allele and autoimmunity in association with autism.

Research findings have also indicated that C4B nullalleles have an increased frequency in children withautism independent of autoimmunity (Odell et al.,2005; Sweeten et al., 2003a). Investigators discoveredthat an increased frequency of the C4B null allele insubjects with the human leukocyte antigen class IIIC4BQ0 had an increased risk of developing autism(Odell et al., 2005). In this study, the researchers exam-ined families with and without ASD. An estimated42.4% of subjects with autism were carriers comparedto only 14.5% of controls. The investigators concludedthat this genetic trait leads to a relative risk of 4.33 ofdeveloping autism (Odell et al., 2005). In another study,researchers examined the association of the C4B nullallele with a common mutation in CYP21A2, a genelocated 3Kb downstream of C4 that encodes for anenzyme important in the synthesis of cortisol and themaintenance of proper androgen levels (Sweeten et al.,2003a). This association was investigated due to concur-rent deletions of the C4B null allele and the CYP21A2gene (Carroll et al., 1985). However, no associationbetween the C4B null allele and CYP21A2 genetic muta-tion was noted for autism (Sweeten et al., 2008).

Other, early, studies describing the levels of theC4B protein in serum from children with autismcompared to control subjects indicated that activatedT lymphocytes were inversely correlated with decreasedplasma levels of C4B protein (Warren et al., 1995).


Another study by the same group indicated a decreasein C4B protein concentration in the plasma of autisticpatients compared to that of normal subjects (Warrenet al., 1994). A more recent case-control proteomic study,which examined serum from children with autismcompared to typical control subjects, found a differentialexpression of proteins in apolipoprotein, complementfactor H related protein (FHRI), complement C1q, andfibronectin 1 (FN1). In addition, complement depositionhas been indicated in both brain and gastrointestinaltissue from children with autism (Torrente et al., 2002;Vargas et al., 2005).

Studies using brain tissue taken from individualswith ASD are limited, due to the rare availability of thesetissue samples from young children. However, in oneseminal study, Vargas and colleagues examined post-mortem brain tissue samples from children with autism.Using immunocytochemistry, the investigators founddeposition of complement proteins in the cerebellumthat was absent in control brains (Vargas et al., 2005).The complement complexes were located in the Purkinjecells as well as the microglia, which are the macrophage-like cells of the nervous system. The presence of comple-ment protein in these tissue sections suggests thatcomplement activation might play a role in tissuedestruction in the brains of these individuals. Of greatinterest, the complement deposits were not found to beco-localized with immunoglobulin in the brain. Thus,there may be an alternative role for complement in thebrain, especially when found near microglia. A role forC1q in axonal pruning has been described in the devel-oping brain, and perhaps the increased C1q levels in theautistic brain represent an attempt at repair or remodel-ing (Amler et al., 2006).

In addition to looking for complement components inthe brain, researchers have also examined duodenalbiopsies from children with regressive autism andcompared them to children with various autoimmunedisorders such as celiac disease, cerebral palsy, mentalretardation, as well as to normal controls. Using immu-nohistochemistry, an increase in the deposition of thecomplement protein C1q was found on the epithelialsurface, co-localized with IgG in biopsy samples fromchildren with regressive autism (Torrente et al., 2002).The presence of C1q deposits co-localized with IgGsuggests immune activation in gut lesions in childrenwith regressive autism. This provides further evidenceof possible immune-mediated pathology in autism.

Evidence of changes in blood complement compo-nent levels, now noted by several groups, as well astissue deposition of C1q, strengthens the notion thatcomplement may in some way be associated with path-ological changes in a subgroup of individuals withautism. However, additional studies are needed todetermine the exact mechanism, if any, of the

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ABNORMALITIES IN THE ADAPTIVE IMMUNE RESPONSE IN ASD 239

relationship between components of the complementsystem and the development of autism.

ABNORMALITIES IN THE ADAPTIVEIMMUNE RESPONSE IN ASD

In addition to abnormalities in the innate arm of theimmune system, abnormalities in the cells and compo-nents that comprise the adaptive immune system havebeen noted in some individuals with autism. We willhighlight the major findings in this section.

Cellular Immunity

T Lymphocytes (T cells)

T lymphocytes, or T cells, play a major role in cell-mediated immunity as effector cells of the adaptiveimmune response. They are distinguished from otherlymphocytes, such as B cells, by the T cell receptor(TCR) on the cell surface. T cells can further be dividedby the expression of TCR accessory proteins such asCD4 and CD8 on the cell surface. CD4 positive cellsare helper Tcells (Th) that become activated upon recog-nition of a specific peptide in conjunction with MHCclass II molecules located on antigen-presenting cells.Once activated, the cells differentiate into specific Thsubtypes depending on what cytokines are secreted bythe antigen presenting cell. Each subtype has a specificrole in directing the immune system according to thetype of pathogen encountered. T helper-1 cells protectagainst intracellular pathogens, T helper-2 cells protectagainst multi-cellular parasitic pathogens, and Thelper-17 cells protect against extra-cellular bacteria atmucosal surfaces. In addition, there are CD4þ subsetslike Th0, Th3, and Tregs that protect against excessiveinflammation during infection, or the perpetuation ofautoimmunity. These different subsets of the T helperlineage secrete different cytokines to facilitate differentactions within the immune response, and are usuallydefined by both their cell surface molecule expressionand the cytokines they secrete. The CD8-positive T cells,or cytotoxic Tcells (CTL), play an important role in viralimmunity and cancer defense. These cells are presentedwith antigen via MHC class I molecules, which arepresent on almost every cell type within the body,including the neurons of the brain (Murphy, 2011;Needleman et al., 2010).

Over the course of autism research, several studieshave indicated abnormalities in T cell immunity in chil-dren with autism compared to healthy controls. In addi-tion to the original Stubbs and Crawford study in 1977that examined decreased lympocytes counts in childrenwith ASD, a subsequent study, which examined a group


of children aged 7–15 with autism, cultured lympho-cytes challenged with PHA displayed a decrease in thepercentages of T helper cells following stimulation,and a lower suppressor cell ratio as determined byflow cytometry (Denney et al., 1996). In a more recentpaper, utilizing a well-characterized population of chil-dren with autism compared to age-matched controls,peripheral blood mononuclear cells (PBMCs) were iso-lated and challenged with PHA or tetanus, and cytokineprofiles from the cellular response were determined.Children with autism exhibited an increase in GM-CSF, TNF-a and IL-13 expression and a decrease inIL-12p40 expression. Of great interest, cytokine expres-sion was associated with altered behaviors in childrenwith autism. For example, an increase in T helper-1 cyto-kines was associated with greater impairments in corefeatures of autism and aberrant behaviors (Ashwoodet al., 2011b). As for cellular markers on T cells, therewas a significant decrease in the expression of CD3,CD4, and CD8 T cells, which may partially explainthe altered cytokine profiles in children with autism(Ashwood et al., 2011b).

The Fas receptor (FasR), also referred to as the cellsurface marker CD95, has also been associated withASD. The Fas receptor serves as a death receptor, whichupon ligation with Fas ligand, initiates programmedcells death or apoptosis. Within the immune system,the Fas receptor is involved with maintaining immuno-logical tolerance (Stranges et al., 2007). This is likelyachieved by limiting lymphocyte proliferation andattenuation of CD95 signalingmay play a role in autoim-munity (Stranges et al., 2007). CD4þ T lymphocytesfrom children with autism showed a decreased expres-sion of CD95. This suggests that children with autismmight have difficulty in regulating the cellular immuneresponse (Stranges et al., 2007).

Regulatory T cells play an important role in immuno-logical self-tolerance and the prevention of autoimmu-nity. T regulatory cells are identified by expression ofCD4 and CD25 on their cell surface, as wells as the intra-cellular expression of the transcription factor FOXP3. Astudy examining regulatory cells indicated that childrenwith autismhad a significantly lower frequency ofCD4þCD25þ T regulatory cells compared to controls (Mostafaet al., 2010). Results also indicated lower Tregulatory cellfrequencies in children with autism that had allergicmanifestations and a family history of autoimmunity.Thus, the decreased frequency of T regulatory cells inchildren with autism may be a contributing factor toautoimmunity in a subgroup of autistic subjects (Mostafaet al., 2010).

T cells are critical components of the adaptiveimmune response. Thus, abnormalities in this cell typemay lead to disruption in other aspects of the adaptiveimmune response. It is therefore important to fully

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comprehend the abnormalities found in both T cellnumbers and function, in order to better understandthe mechanisms of immune dysfunction in ASD.

B Lymphocytes (B cells)

B cells also serve an important role in the adaptiveimmune response, specifically the humoral immuneresponse. B cells recognize invading pathogens throughthe B cell receptor, a membrane bound version of immu-noglobulin. This recognition triggers phagocytosis,degradation of the pathogen, and presentation ofpeptide fragments through MHC II molecule to helperT cells. Depending on the type of T cell help provided,B cells can proliferate and class switch to the appropriateimmunoglobulin for fighting the particular pathogenencountered (IgG, IgA, IgE, or remain IgM). Of this pop-ulation of activated B cells, some will differentiate intoplasma cells that migrate to the bone marrow andsecrete large quantities of antigen-specific immunoglob-ulin, or they will become long-lived memory B cells thatserve as a primer for a secondary response if the samepathogen is encountered a second time.

Despite the preponderance of studies examiningimmunoglobulin levels in children with autism, veryfew studies have investigated either B cell function orperipheral prevalence. One study examined the totallymphocyte counts using flow cytometry. Results indi-cated a decrease in circulating CD20þ B cells in childrenwith autism compared to healthy controls and siblings(Yonk et al., 1990). A more recent study has demon-strated a 20% increase in the number of B cells pervolume of blood in children with autism (Ashwoodet al., 2011a). In addition, of the cellular activationmarkers measured, CD38 was significantly higher inchildren with autism compared to controls.

While these studies indicate a possible change in theB cell population of the immune system, further studiesare needed to address the relationship between thesefindings and the etiology of autism. Later in this chapterwe will describe the autoimmune phenomena of auto-antibodies directed against brain proteins in childrenwith ASD, as well as antibodies to fetal brain proteinsin the blood of their mothers. It will be important to fullyunderstand the role of the humoral and innate immunesystems in the production of these auto-antibodies.


Cytokines and chemokines play a very important rolein intercellular communication through autocrine, para-crine, and/or endocrine action. While the term cyto-kine/chemokine has come to describe most smallextracellular signaling molecules, which are used innearly every tissue including the nervous system, theterm has its origins in the immune system. Historically,as well as in modern immune nomenclature, the


cytokines expressed by cells of the immune system areused not only to distinguish the cells phenotypically,but to describe their function as well. For example, Thelper-1 cells are promoted by the presence of IL-12and IFN-g, and in turn secrete IFN-g, a major player inthe inflammatory response and critical for defenseagainst intracellular bacteria. T helper-2 cells arepromoted by IL-4 and secrete IL-5, IL4, and IL-13, majorplayers in defense against multi-cellular parasites andthe development of allergies. T helper-17 cells secreteIL-17 and IL-23, and regulate bacterial invasion atmucosal surfaces, while regulatory T cells secrete IL-10and TGF-b to promote tolerance to self-proteins, aswell as regulate the inflammatory response. Elevatedcytokines in plasma or following cellular activationmay be indicative of a disease state or the presence ofan inflammatory process. Therefore, measuring cyto-kines as well as chemokine levels is an excellent indi-cator of immune status. As such, they may serve in thefuture as potential biomarkers to identify immunedysfunction in children with autism.

There are a plethora of studies examining the levels ofcytokines and chemokines in the plasma of individualswith autism (Table 2.9.1). Subjects with autism havebeen reported to have a significant increase in IFN-gand IL-1RA with a trend towards increased IL-6 andTNF-a (Croonenberghs et al., 2002a). More recently,one study indicated an increase in IL-1RA as well asIL-1b, IL-5, IL-8, IL-12(p70), IL-13, IL-17 and Gro-alphain the plasma of ASD subjects compared to age-matchedcontrols (Suzuki et al., 2011). Another study demon-strated an increase in IL-1b, IL-6, IL-8 and IL-12(p40)in subjects with autism, which was associated with chil-dren that had a regressive form of ASD and impairedcommunication and aberrant behaviors (Ashwoodet al., 2011b). Elevated chemokine levels have alsobeen indicated in children with autism. A recent studydescribed an increase in plasma levels of MCP-1, Rantes,and eotaxin, which were all associated with higher aber-rant behavioral scores and more impaired developmentand adaptive function (Ashwood et al., 2011c). Whilemost of the cytokines reported to be increased in ASDare of a pro-inflammatory nature, the levels of regula-tory cytokines have also been investigated. Results indi-cated that children with autism have decreased plasmalevels of TGF-b compared to age-matched, typicallydeveloping controls and demonstrate a correlationbetween lower TGF-b cytokine levels and adaptivebehavioral symptoms (Ashwood et al., 2008a; Okadaet al., 2007).

Plasma cytokine levels provide informationregarding the current immune status of the individual.By understanding which cytokine is elevated providesinformation about what cell type or cellular processmight be abnormal. An over-activated immune system,

SPECTRUM DISORDERS

TABLE 2.9.1 Comprehensive Studies of Cytokine and Chemokine Profiles in Autism

Source Study description Reference

Plasma Elevated levels of IL-1, IL-6, IL-8 and IL-12p40. Associated with regression (Ashwood et al., 2011b)

Increase in chemokine MCP-1, Rantes and Eotaxin levels in ASD subjectscompared to age-matched typically developing controls. An association betweenincreased chemokines levels with aberrant behaviors.

(Ashwood et al., 2011c)

In male ASD subjects, an increase in cytokines IL-1b, IL-1RA, IL-5, IL-8, IL-12(p70),IL-13, IL-17 and GRO-a.

(Suzuki et al., 2011)

Increase in leptin levels in ASD subjects compared to age-matched controls. (Ashwood et al., 2008b)

Increase in macrophage migration inhibitory factor (MIF) in ASD subjectscompared to age-matched controls.

(Grigorenko et al., 2008)

Decrease in TGF-b in subjects with ASD compared to controls. (Ashwood et al., 2008a;Okada et al., 2007)

Increase in IL-12 and IFN-g in ASD subjects compared to age-matched controls. (Singh, 1996)

Cytokine and chemokineexpression in cell cultures

In isolated PBMCs stimulated with PHA, increase in GM-CSF, TNF-a and IL-13.A decrease in IL-12(p40) in ASD subjects compared to age-matched controls.

(Ashwood et al., 2011d)

Stimulation of various TLR with ligands on monocytes isolated from subjects withASD compared to age-matched controls. Increase in IL-1b, IL-6, TNF-a, withstimulation of TLR2. Increase in IL-1b, with stimulation of TLR4. Decrease inIL-1b, IL-6, GMCSF, TNF-a with TLR9.

(Enstrom et al., 2010)

Increase in IFN-g in NK cells from subjects with ASD. (Enstrom et al., 2009b)

Increased production of cytokines from Th1 and Th2 cytokines in ASD subjectscompared to age-matched controls.

(Molloy et al., 2006)

Increase in IL-12 and TNF-a in ASD subject with GI symptoms. (Jyonouchi et al., 2005)

Increase in IFN-g and TNF-a in isolated PBMCs from ASD subjects compared toage-matched controls stimulated with LPS.

(Jyonouchi et al., 2002)

Unstimulated whole blood cultures from subjects with ASD compared to age-matched controls had increased production of IFN-g and IL-1RAwith a trendtowards higher IL-6 and TNF-a.

(Croonenberghs et al.,2002a)

PBMCs isolated from ASD subjects unstimulated produced higher levels ofTNF-a, IL-1b, and IL-6 compared to controls. PBMCs stimulated with LPS, PHAand tetanus produced increase levels of IL-12 and IL-1b.

(Jyonouchi et al., 2002)

ABNORMALITIES IN THE ADAPTIVE IMMUNE RESPONSE IN ASD 241

resulting in elevated cytokine and chemokine levels,may promote cellular processes to occur that are dysre-gulated, and may go on unchecked. In other words,elevated cytokine and chemokine levels might havea negative effect on other biological processes in thedeveloping body, such as the nervous system (McAllisterandVandeWater, 2009). Understandingwhich cytokinesand chemokines are elevated in plasma could provideclues that aid in the understanding of immune dysfunc-tion in children with autism and how these might relateto changes in neurodevelopment.

As mentioned above, plasma cytokine levels pro-vide information on the subjects’ current immunestatus at the time of blood draw. However, in a humanpopulation, it is difficult to measure the response toimmune challenge following infection. Through the use


of cell culture systems, peripheral immune cells may beisolated from subjects to measure the in vitro immuneresponse to global cell stimulation molecules (mitogens)as well as recall antigens such as vaccine proteins. Byexposing immune cells to mitogenic challenge, investiga-tors have shown that subjects with autism exhibit anincrease in IL-13/IL-10 and IFN-g/IL-10 ratios comparedto control subjects (Molloy et al., 2006). The authorsconcluded that autism subjects have increased activationof both Th1 and Th2 cytokines with a predominance ofTh2 (Molloy et al., 2006).

In a more recent study, in vitro stimulation of PBMCswith PHA (a T mitogen) and tetanus toxoid producedan increase in GM-CSF, TNF-a, and IL-13, and a decreasein IL-12(p40). The investigators interpreted this asa possible skewing toward a T helper-2 cytokine profile

SPECTRUM DISORDERS


in children with ASD and away from a T helper-1 cyto-kine profile. A major strength of this research study wasthat the investigators coupled behavioral analysis withcytokine profiles. An association was found betweenthe proinflammatory cytokines, TNF-a and IL-8, the Thelper-1 cytokine, IFN-g, and the severity of certaincore behavior measurements (Ashwood et al., 2011b).From these results, the authors suggest that T helper-1skewing was associated with a more impaired behavior,and the T helper-2 cytokine response was associatedwith improved levels of adaptive function in childrenwith autism.

Although the above-described results indicatechanges in the immune response in individuals withautism, these studies provide only a snapshot of theimmune system at a particular time point. To fully eval-uate the immune response in childrenwith ASD, a longi-tudinal study using repeated cytokine measurementswould be necessary. Finally, while it is not entirely clearexactly how cytokine levels might influence behavior,a better understanding of the mechanisms leading toimmune dysfunction would provide information thatcould result in potential therapeutic intervention.Thus, it remains important to grasp the full extent ofthe relationship between cytokine markers of immunedysfunction and behavior in children with autism.

AUTOIMMUNITY

Auto-Antibodies – Children

Several investigators have proposed an autoimmune-based etiology for a subset of children with autism. Theprincipal evidence in support of this hypothesis hasbeen the discovery of auto-antibodies directed againstvarious brain components (Connolly et al., 2006;Connolly et al., 1999; Goines et al., 2011; Wills et al.,2007; 2009; 2011). While still poorly understood, theseauto-antibodies have the potential to be pathologicallysignificant. Auto-antibodies may interfere with neuro-development by acting as a ligand for a target such asa receptor, by blocking receptor function, or by inducingcellular damage and/or activation of neuronal cells(Diamond et al., 2009). While it has been proposed thatthese auto-antibodies may have pathological conse-quences, they could simply be an epiphenomenonresulting from tissue destruction. Numerous studieshave indicated that children with autism have anti-bodies against serotonin receptors, heat shock proteins,glial filament proteins and myelin basic protein, andother proteins with significant neurological relevance(Evers et al., 2002; Mostafa and AL-ayadhi, 2011a; Singhet al., 1997a, b; 1993; Vojdani et al., 2002). Although thefindings in these studies are interesting, many lacked


proper controls and have some shortcomings. Forexample, the Singh et al. study involved subjects havinglarge age (Singh et al., 1997b), from 4 to 30 years in thosewith autism, and from 5 to 48 years in healthy controls.Such studies should be viewed with caution, as theimmune system of a child differs vastly from that ofan adult. In addition, some of these studies did nothave a verified diagnosis for the children with autism.Despite these possible issues, several recent reports,with proper control groups and diagnostic methods,continue to report the presence of auto-antibodies inplasma of children with autism.

It has been suggested that the presence of auto-antibodies in children with autismmight be a phenotypeof a subgroup of ASD (Careaga et al., 2010). Perhapsthese auto-antibodies are a consequence of a previousdamaging events, which may have occurred duringbrain development (Careaga et al., 2010). Some of themost compelling work to date has been the characteriza-tion of auto-antibodies directed against adult brainproteins using western blot analysis and immunohisto-chemistry in brain tissue sections (Cabanlit et al., 2007;Goines et al., 2011; Rossi et al., 2011; Wills et al., 2011).For example, a large well-characterized study usingwestern blot analysis found that the blood of a subsetof children with autism contains antibodies directedagainst human hypothalamus and thalamus (Cabanlitet al., 2007). This study revealed that 13/63 (21%) ofsubjects with ASD possessed antibodies that demon-strated specific reactivity to a cerebellar protein withan apparent molecular weight of approximately45 kDa compared with only 1/63 (2%) of the typicallydeveloping controls. In a follow-up study, cerebellum-specific antibodies were also assessed by immunohisto-chemical (IHC) staining of sections from macue monkeycerebellum. Intense immunoreactivity to what wasdeterminedmorphologically to be Golgi cells of the cere-bellum was noted for 7/34 (21%) of subjects with ASD,compared with 0/23 of the typically-developingcontrols. Furthermore, there was a strong associationbetween the presence of antibodies reactive to the45kDa protein by western blot with positive immunohis-tochemical staining of cerebellar Golgi cells in the ASDgroup, but not controls. More recently, a study by Willset al. established that the auto-antibodies are immunore-active with GABAergic neurons, which are distributedthroughout a number of brain regions (Wills et al., 2011).

In a subsequent study, further characterization ofthe behavioral phenotypes associated with these auto-antibodies showed that auto-antibodies specific fora 45kDa cerebellar protein in children were associatedwith a diagnosis of autism, while auto-antibodiesdirected towards a 62kDa protein were associated withthe broader diagnosis of ASD (Goines et al., 2011). Ofgreat interest, children with auto-antibodies directed

SPECTRUM DISORDERS

AUTOIMMUNITY 243

against the 45kDa protein demonstrated more severescores on the ABC (Aberrant Behaviors Checklist) Leth-argy subscale, the ABC Stereotypy subscale, MSEL(Mullen Scales of Early Learning) test, as well as theVABS (Vineland Adaptive Behavioral Scale), in compar-ison to children without auto-antibody reactivity(Goines et al., 2011). Children with reactivity to the62kDa protein demonstrated impaired scores on theABC Inappropriate Speech subscale. Therefore, childrenwith reactivity to proteins at 62kDa and 45kDa hadlower adaptive and cognitive function, as well asincreased aberrant behaviors when compared to chil-dren without these antibodies (Goines et al., 2011).

Collectively, this research indicates that auto-antibodies specific to brain proteins in children are asso-ciated with lower adaptive and cognitive function aswell as core behavioral deficits associated with autism.The exact mechanism of these auto-antibodies has notbeen determined. The authors suggest that the differ-ence in these reactivity patterns could result from thetarget proteins being involved in alternate pathways,each having a different biological outcome with respectto behavior. Currently, studies to determine the identityof these antigens are underway. Their identification willprovide clues to which specific brain pathways are tar-geted, and how these pathways relate to the behavioralchanges noted in children with autism.

The question remains regarding the pathologicalsignificance of these auto-antibodies. Under normalhealthy conditions, antibodies are too large to cross theblood-brain barrier (BBB) and enter the brain. However,in various disease states and with certain infections, theBBB may become permeable and large molecules suchas antibodies may cross this barrier (Diamond et al.,2009). Such states include hypertension, infections, toxinstress, and the presence of bacterial toxins such as per-tussin toxin (Kuang et al., 2004; Kugler et al., 2007).Once the antibody enters the brain, it may act as a ligandfor a receptor, or block receptor function. In addition,they may be able to fix complement, which can laterlead to immune activation and tissue destruction(Diamond et al., 2009). One example of an autoimmunedisorder in which auto-antibodies are able to cross theBBB barrier and lead to tissue destruction is the case ofsystemic lupus erythematosis (SLE) with associatedcognitive and neuropsychiatric symptoms. Researchhas demonstrated that auto-antibodies cross the BBBand act as the NMDA receptor for glutamate throughmolecular mimicry (Kowal et al., 2004). This phenom-enon was also proven in an animal model in whichthe auto-antibodies were administered to mice,leading cognitive impairment and neuronal cell death(Huerta et al., 2006). This is an excellent example ofa neurodevelopmental disorder in which auto-anti-bodies are known to lead to brain destruction.


Studies have also indicated that children with autismhave antibodies directed against transglutaminase.Rosenspire et al. have recently reported that:

1) A sub-population of ASD children (~25–30%) expressa significantly higher than expected autoimmuneresponse directed to transglutaminase II (anti-TG2),and

2) That expression of high levels of anti-TG2 aresignificantly linked in to the (HLA)-DR3, Q2 andDR7, DQ2 haplotypes (Rosenspire et al., 2011).

TG2 is a multifunctional enzyme of about 80 kDaperhaps best known for its ability to catalyze the cova-lent cross linking of a g-glutaminyl residue ofa protein/peptide substrate to an ε-lysl residue ofa protein/peptide co-substrate (De Vivo et al., 2009).TG2 is important for normal neural development, astransglutaminase activity typically increases duringearly development, especially in the cerebellar cortex(Ruan and Johnson, 2007). During development, TGactivity is associated with enhanced neurite outgrowth(Maccioni and Seeds, 1986; Mahoney et al., 1996), andthe stabilization of CNS synapses through TG2 medi-ated formation of protease resistance cross-linkingbetween synaptic proteins (Festoff et al., 2001). Due tothe critical role of TG in neurodevelopment, it will beimportant to determine the pathological significance ofthese auto-antibodies in the etiology of autism.

Gangliosides have recently become another protein ofinterest in autism. These are sialylated glycosphingoli-pids located on the outer leaflets of the plasmamembrane found in all vertebrates. Ganglioside proteinsare abundant in the nervous system and are involved inneurotransmission, and as such play an important rolein neural function (Ariga et al., 2008; Zitman et al.,2010). Ganglioside MI is the most abundant gangliosidein the neural membrane. In a recent study, children withautism had a significantly higher frequency of serumanti-ganglioside MI antibodies as measured by ELISA(Mostafa and AL-ayadhi, 2011b).

While there are several intriguing clues regardingauto-antibodies and their potential role in autism, westill have a long way to go with respect to proving theirpathological significance, and in some cases, identifyingthe target antigens. However, there may come a timewhen, at least for a sub-group of individuals, we mayhave strong biomarkers of autism risk, and the potentialfor future therapeutic intervention.

Auto-Antibodies – Maternal

In addition to the presence of auto-antibodies in chil-dren with autism, the presence of auto-antibodies hasalso been indicated in some mothers of children withautism. The presence of maternal antibodies in autism

SPECTRUM DISORDERS


were first described by Dalton et al., and were thought toaffect behavior in a rodent model (Dalton et al., 2003).Since then, several studies have expanded upon thisearlier work to more fully characterize these antibodies.For example, in one large well-characterized populationstudy, plasma from a subset of mothers of children withautism demonstrated antibody reactivity against fetaland adult brain proteins for two bands at 37 and73 kDa in about 12% of mothers (Braunschweig et al.,2008). This reactivity was not found in plasma frommothers of typically-developing children. In anexpanded study of the same population, Braunschweig,et al. profiled fetal-brain reactive auto-antibodies for anadditional 338 mothers, including 215 mothers of chil-dren diagnosed with an ASD, and 123 mothers of typi-cally developing children (Braunschweig et al., 2011).Highly significant associations between the presence ofIgG reactivity to fetal brain proteins at 37 kDa and73kDa and a childhood diagnosis of full autism (AU)(p¼ 0.0005) was maintained in this follow up study.Interestingly, the 37/73 kDa band pattern was associatedwith lower scores on measures of expressive language(p¼ 0.005). Additionally, the authors reported pairedreactivity to proteins at 39 kDa and 73 kDa, which corre-lated strongly with a broader diagnosis of ASD(p¼ 0.0007); this pattern was significantly associatedwith increased irritability on the Aberrant BehavioralChecklist (p¼ 0.05). Further characterization of thematernal antibody targets revealed conserved expres-sion of both antigen pairs in late-gestation Rhesus brainas well as variable presence in human fetal CNS andnon-CNS tissues. This study provides strong evidencefor more than one pattern of reactivity to fetal brainproteins by maternal antibodies associated with ASDs,and such reactivity appears to have a relationship withcertain behaviors characteristic of ASD.

Other research groups have also demonstrated serumauto-antibody reactivity to brain proteins. In a smallerstudy using only 11 mothers, Zimmerman andcolleagues tested serum reactivity to prenatal, postnataland adult rat brain using western blot analysis frommothers of children with autism (Zimmerman et al.,2007). Auto-antibody reactivity to fetal brain proteinfrom mothers of children with autism was present for2 to 18 years after birth. In another slightly larger study,using 100 mothers of children with autism and 100mothers of typically developing controls, serum auto-antibody reactivity was tested via western blot analysisagainst rodent embryonic brain tissue and human fetalbrain tissue (Singer et al., 2008). A predominant bandingpattern was observed in samples from mothers ofchildren with autism at 36kDa and 39kDa. Althoughthe identity of the target antigens remains under inves-tigation in the above studies, the researchers suggestthat the presence of such IgG antibodies in the blood


during gestation might cross the BBB potentially leadingto alterations in fetal brain development.

In order to determine whether these auto-antibodiesdo indeed have pathological significance, variousanimal model studies have been conducted. A pilotnon-human primate study was conducted, whereinRhesus monkey dams were exposed during pregnancyto IgG from mothers with fetal brain-specific antibodies,or to IgG from control mothers with no reactivity. Theoffspring of these monkeys were evaluated for specificsocial, communication, and stereotypic behaviors thatare characteristic of autism. The results indicated thatthe monkeys exposed to IgG from the mothers of chil-dren with autism showed increases in stereotypicaland hyperactive behaviors (Martin et al., 2008). Anotherstudy used a mouse model to determine the possiblemechanism of the auto-antibodies from mothers of chil-dren with autism. In this model, pregnant mice wereadministered with IgG from mothers of children withautism, and from typical controls, at embroyonic days13 through 18. Behavioral testing demonstrated thatmice exposed to IgG from mothers of children withautism displayed anxiety-like behaviors and alterationsin sociability (Singer et al., 2009) providing an additionalexample of behavioral consequences in offspringexposed to these antibodies. This is an extremelyintriguing finding, as the presence of these auto-anti-bodies in the serum of mothers might be used asa biomarker to determine susceptibility to having a childwith autism. In addition, once the target antigens arefully characterized, therapeutic interventions could bedeveloped to prevent a subset of autism.

One must also consider the risk factors that lead tothe presence of maternal antibodies to fetal brainproteins. One such risk factor is the genetic suscepti-bility to the production of auto-antibodies througha polymorphism in the tyrosine kinase MET receptor(Campbell et al., 2006; 2007; 2008; Eagleson et al.,2011). In an initial population case-controlled study ofover 500 autism families, researchers found a geneticassociation between the MET ‘C’ allele in the promoterregion of the MET gene. A relative risk of 2.27 was fromautism diagnosis associated with a CC genotype anda risk of only 1.67 associated for the CG genotypecompared to the GG genotype (Campbell et al., 2006).MET signaling is involved in neurodevelopmentand gastrointestinal repair (Campbell et al., 2006) anda polymorphism affecting cell surface expression ofthis receptor could have long-lasting effects on neuro-development. MET also plays an important role in theimmune system as a negative regulator, through controlof the level of activation of antigen-presenting cellssuch as dendritic cells, macrophages, and B cells (Beil-mann et al., 1997; 2000; Okunishi et al., 2005). Thus,alteration in this receptor may lead to immune

SPECTRUM DISORDERS

REFERENCES 245

abnormalities, such as a lack of immune regulation,thereby setting the stage for auto-antibody production.A very strong association between the presence of theMET ‘C’ allele and maternal auto-antibodies to fetalbrain proteins was in fact noted in a recent study byHeuer et al. (2011). In a sample of 365 mothers,including 202 mothers of children with ASD, the func-tional MET promoter variant rs1858830 C allele wasstrongly associated with presence of an ASD-specific37 kD þ 73 kD band pattern of maternal auto-antibodiesto fetal brain proteins (P¼ 0.003). To determine themechanism of this genetic association, MET proteinand cytokine production were measured in freshlyprepared peripheral blood mononuclear cells from 76mothers of ASD and TD children. The MET rs1858830C allele was significantly associated with MET proteinexpression (P¼ 0.025). Moreover, decreased expressionof the regulatory cytokine IL-10 was associated withboth the MET gene C allele (P¼ 0.001) and reducedMET protein levels (P¼ 0.002) (Heuer et al., 2011).These results indicate genetic distinction amongmothers who produce ASD-associated antibodies tofetal brain proteins, and suggest a potential mechanismfor how a genetically determined decrease in METprotein production may lead to a reduction in immuneregulation. Thus, this study was the first to describea genetic susceptibility factor associated with autism,and a functional outcome of that factor. Identificationof a pre-disposing genetic factor such as MET shouldcontribute greatly towards the understanding of themechanisms involved in a subset of ASD cases.

CONCLUSIONS

In conclusion, there is a great deal of convincingevidence suggesting that abnormalities of the immunesystem are frequently found in children with autism.We have described herein several studies supportingthe concept that children with autism have dysfunctionin both the innate and adaptive immune systems. Theseabnormalities include alterations in monocyte activity,complement levels, NK cell number and function,immune cellular signaling patterns, and auto-antibodiesfound in both the children as well as their mothers.Much progress has been made in the field of immunityand autism since the first paper describing immuneabnormalities in autism was published in 1977 (Stubbsand Crawford, 1977). ASD is still a serious healthconcern, and the numbers of affected children haveexpanded dramatically in recent years. Therefore, it isessential to understand the exact mechanisms behindthese various immune abnormalities and how theymight serve as a biological marker of neuroimmuneanomalies. It is also critical to determine how the various


immune biomarkers associate with behavioral outcome.It is through this research that we might finally elucidatedelete some of the etiological mechanisms that lead tothis enigmatic disorder.

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The Neuroscience of Autism Spectrum Disorders || Immune Abnormalities and Autism Spectrum Disorders