Paul Ashwood

The immune response in autism: a new frontier for autism research

Autism spectrum disorders (ASD) are part of a broad spectrum of neurodevelopmental disorders known as pervasive developmental disorders, which occur in childhood. They are characterized by impairments in social interaction, verbal and nonverbal communication and the presence of restricted and repetitive stereotyped behaviors. At the present time, the etiology of ASD is largely unknown, but genetic, environmental, immunological, and neurological factors are thought to play a role in the development of ASD. Recently, increasing research has focused on the connections between the immune system and the nervous system, including its possible role in the development of ASD. These neuroimmune interactions begin early during embryogenesis and persist throughout an individual’s lifetime, with successful neurodevelopment contingent upon a normal balanced immune response. Immune aberrations consistent with a dysregulated immune response, which so far, have been reported in autistic children, include abnormal or skewed T helper cell type 1 (TH1)/TH2 cytokine profiles, decreased lymphocyte numbers, decreased T cell mitogen response, and the imbalance of serum immunoglobulin levels. In addition, autism has been linked with autoimmunity and an association with immune‐based genes including human leukocyte antigen (HLA)‐DRB1 and complement C4 alleles described. There is potential that such aberrant immune activity during vulnerable and critical periods of neurodevelopment could participate in the generation of neurological dysfunction characteristic of ASD. This review will examine the status of the research linking the immune response with ASD.

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

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

The role of immune dysfunction in the pathophysiology of autism

Autism spectrum disorders (ASD) are a complex group of neurodevelopmental disorders encompassing impairments in communication, social interactions and restricted stereotypical behaviors. Although a link between altered immune responses and ASD was first recognized nearly 40 years ago, only recently has new evidence started to shed light on the complex multifaceted relationship between immune dysfunction and behavior in ASD. Neurobiological research in ASD has highlighted pathways involved in neural development, synapse plasticity, structural brain abnormalities, cognition and behavior. At the same time, several lines of evidence point to altered immune dysfunction in ASD that directly impacts some or all these neurological processes. Extensive alterations in immune function have now been described in both children and adults with ASD, including ongoing inflammation in brain specimens, elevated pro-inflammatory cytokine profiles in the CSF and blood, increased presence of brain-specific auto-antibodies and altered immune cell function. Furthermore, these dysfunctional immune responses are associated with increased impairments in behaviors characteristic of core features of ASD, in particular, deficits in social interactions and communication. This accumulating evidence suggests that immune processes play a key role in the pathophysiology of ASD. This review will discuss the current state of our knowledge of immune dysfunction in ASD, how these findings may impact on underlying neuro-immune mechanisms and implicate potential areas where the manipulation of the immune response could have an impact on behavior and immunity in ASD.

Consensus Paper: Pathological Role of the Cerebellum in Autism

There has been significant advancement in various aspects of scientific knowledge concerning the role of cerebellum in the etiopathogenesis of autism. In the current consensus paper, we will observe the diversity of opinions regarding the involvement of this important site in the pathology of autism. Recent emergent findings in literature related to cerebellar involvement in autism are discussed, including: cerebellar pathology, cerebellar imaging and symptom expression in autism, cerebellar genetics, cerebellar immune function, oxidative stress and mitochondrial dysfunction, GABAergic and glutamatergic systems, cholinergic, dopaminergic, serotonergic, and oxytocin-related changes in autism, motor control and cognitive deficits, cerebellar coordination of movements and cognition, gene–environment interactions, therapeutics in autism, and relevant animal models of autism. Points of consensus include presence of abnormal cerebellar anatomy, abnormal neurotransmitter systems, oxidative stress, cerebellar motor and cognitive deficits, and neuroinflammation in subjects with autism. Undefined areas or areas requiring further investigation include lack of treatment options for core symptoms of autism, vermal hypoplasia, and other vermal abnormalities as a consistent feature of autism, mechanisms underlying cerebellar contributions to cognition, and unknown mechanisms underlying neuroinflammation.

Small intestinal enteropathy with epithelial IgG and complement deposition in children with regressive autism.

We have reported lymphocytic colitis in children with regressive autism, with epithelial damage prominent. We now compare duodenal biopsies in 25 children with regressive autism to 11 with coeliac disease, five with cerebral palsy and mental retardation and 18 histologically normal controls. Immunohistochemistry was performed for lymphocyte and epithelial lineage and functional markers. We determined the density of intraepithelial and lamina propria lymphocyte populations, and studied mucosal immunoglobulin and complement C1q localisation. Standard histopathology showed increased enterocyte and Paneth cell numbers in the autistic children. Immunohistochemistry demonstrated increased lymphocyte infiltration in both epithelium and lamina propria with upregulated crypt cell proliferation, compared to normal and cerebral palsy controls. Intraepithelial lymphocytes and lamina propria plasma cells were lower than in coeliac disease, but lamina propria T cell populations were higher and crypt proliferation similar. Most strikingly, IgG deposition was seen on the basolateral epithelial surface in 23/25 autistic children, co-localising with complement C1q. This was not seen in the other conditions. These findings demonstrate a novel form of enteropathy in autistic children, in which increases in mucosal lymphocyte density and crypt cell proliferation occur with epithelial IgG deposition. The features are suggestive of an autoimmune lesion.

Intestinal Lymphocyte Populations in Children with Regressive Autism: Evidence for Extensive Mucosal Immunopathology

Inflammatory intestinal pathology has been reported in children with regressive autism (affected children). Detailed analysis of intestinal biopsies in these children indicates a novel lymphocytic enterocolitis with autoimmune features; however, links with cognitive function remain unclear. To characterize further, the nature and extent of this disease we examined the mucosal infiltrate using flow cytometry. Duodenal, ileal, and colonic biopsies were obtained from 52 affected children, 25 histologically normal, and 54 histologically inflamed, developmentally normal controls. Epithelial and lamina propria lymphocyte populations were isolated and examined by multicolor flow cytometry. Adjacent biopsies were assessed by semiquantitative histopathology. At all sites, CD3+ and CD3+CD8+ IEL as well as CD3+ LPL were significantly increased in affected children compared with developmentally normal noninflamed control groups (p<0.01) reaching levels similar to inflamed controls. In addition, two populations—CD3+CD4+ IEL and LP CD19+ B cells—were significantly increased in affected children compared with both noninflamed and inflamed control groups including IBD, at all sites examined (p<0.01). Histologically there was a prominent mucosal eosinophil infiltrate in affected children that was significantly lower in those on a gluten- and casein-free diet, although lymphocyte populations were not influenced by diet.The data provide further evidence of a pan-enteric mucosal immunopathology in children with regressive autism that is apparently distinct from other inflammatory bowel diseases.

Stereotypies and hyperactivity in rhesus monkeys exposed to IgG from mothers of children with autism

Autism together with Asperger syndrome and pervasive developmental disorder not otherwise specified form a spectrum of conditions (autism spectrum disorders or ASD) that is characterized by disturbances in social behavior, impaired communication and the presence of stereotyped behaviors or circumscribed interests. Recent estimates indicate a prevalence of ASD of 1 per 150 (Kuehn, 2007). The cause(s) of most cases of ASD are unknown but there is an emerging consensus that ASD have multiple etiologies. One proposed cause of ASD is exposure of the fetal brain to maternal autoantibodies during pregnancy [Dalton, P., Deacon, R., Blamire, A., Pike, M., McKinlay, I., Stein, J., Styles, P., Vincent, A., 2003. Maternal neuronal antibodies associated with autism and a language disorder. Ann. Neurol. 53, 533-537]. To provide evidence for this hypothesis, four rhesus monkeys were exposed prenatally to human IgG collected from mothers of multiple children diagnosed with ASD. Four control rhesus monkeys were exposed to human IgG collected from mothers of multiple typically developing children. Five additional monkeys were untreated controls. Monkeys were observed in a variety of behavioral paradigms involving unique social situations. Behaviors were scored by trained observers and overall activity was monitored with actimeters. Rhesus monkeys gestationally exposed to IgG class antibodies from mothers of children with ASD consistently demonstrated increased whole-body stereotypies across multiple testing paradigms. These monkeys were also hyperactive compared to controls. Treatment with IgG purified from mothers of typically developing children did not induce stereotypical or hyperactive behaviors. These findings support the potential for an autoimmune etiology in a subgroup of patients with neurodevelopmental disorders. This research raises the prospect of prenatal evaluation for neurodevelopmental risk factors and the potential for preventative therapeutics.

Differential monocyte responses to TLR ligands in children with autism spectrum disorders

Autism spectrum disorders (ASD) are characterized by impairment in social interactions, communication deficits, and restricted repetitive interests and behaviors. Recent evidence has suggested that impairments of innate immunity may play an important role in ASD. To test this hypothesis, we isolated peripheral blood monocytes from 17 children with ASD and 16 age-matched typically developing (TD) controls and stimulated these cell cultures in vitro with distinct toll-like receptors (TLR) ligands: TLR 2 (lipoteichoic acid; LTA), TLR 3 (poly I:C), TLR 4 (lipopolysaccharide; LPS), TLR 5 (flagellin), and TLR 9 (CpG-B). Supernatants were harvested from the cell cultures and pro-inflammatory cytokine responses for IL-1beta, IL-6, IL-8, TNFalpha, MCP-1, and GM-CSF were determined by multiplex Luminex analysis. After in vitro challenge with TLR ligands, differential cytokine responses were observed in monocyte cultures from children with ASD compared with TD control children. In particular, there was a marked increase in pro-inflammatory IL-1beta, IL-6, and TNFalpha responses following TLR 2, and IL-1beta response following TLR 4 stimulation in monocyte cultures from children with ASD (p<0.04). Conversely, following TLR 9 stimulation there was a decrease in IL-1beta, IL-6, GM-CSF, and TNFalpha responses in monocyte cell cultures from children with ASD compared with controls (p<0.05). These data indicate that, monocyte cultures from children with ASD are more responsive to signaling via select TLRs. As monocytes are key regulators of the immune response, dysfunction in the response of these cells could result in long-term immune alterations in children with ASD that may lead to the development of adverse neuroimmune interactions and could play a role in the pathophysiology observed in ASD.

Immune activation of peripheral blood and mucosal CD3+ lymphocyte cytokine profiles in children with autism and gastrointestinal symptoms.

Gastrointestinal pathology, characterized by lymphoid nodular hyperplasia and entero-colitis, has been demonstrated in a cohort of children with autistic spectrum disorder (ASD). Systemic and intestinal mucosal immune dysregulation was assessed in ASD children with gastrointestinal (GI) symptoms (n = 18), and typically developing controls (n = 27), including non-inflamed controls (NIC) and inflamed GI control children with Crohns disease (CD), by analysis of intracellular cytokines in CD3+ lymphocytes. In both peripheral blood and mucosa, CD3+ TNFalpha+ and CD3+ IFNgamma+ were increased in ASD children compared with NIC (p < 0.004) and reached levels similar to CD. In contrast, peripheral and mucosal CD3+ IL-10+ were markedly lower in ASD children with GI symptoms compared with both NIC and CD controls (p < 0.02). In addition, mucosal CD3+ IL-4+ cells were increased (p < 0.007) in ASD compared with NIC. There is a unique pattern of peripheral blood and mucosal CD3+ lymphocytes intracellular cytokines, which is consistent with significant immune dysregulation, in this ASD cohort.

Spontaneous Mucosal Lymphocyte Cytokine Profiles in Children with Autism and Gastrointestinal Symptoms: Mucosal Immune Activation and Reduced Counter Regulatory Interleukin-10

A lymphocytic enterocolitis has been reported in a cohort of children with autistic spectrum disorder (ASD) and gastrointestinal (GI) symptoms. This study tested the hypothesis that dysregulated intestinal mucosal immunity with enhanced pro-inflammatory cytokine production is present in these ASD children. Comparison was made with developmentally normal children with, and without, mucosal inflammation. Duodenal and colonic biopsies were obtained from 21 ASD children, and 65 developmentally normal paediatric controls, of which 38 had signs of histological inflammation. Detection of CD3+ lymphocyte staining for spontaneous intracellular TNFα, IL-2, IL-4, IFNγ, and IL-10, was performed by multicolor flow cytometry. Duodenal and colonic mucosal CD3+ lymphocyte counts were elevated in ASD children compared with noninflamed controls (p<0.03). In the duodenum, the proportion of lamina propria (LP) and epithelial CD3+TNFα+ cells in ASD children was significantly greater compared with noninflamed controls (p<0.002) but not coeliac disease controls. In addition, LP and epithelial CD3+IL-2+ and CD3+IFNγ+, and epithelial CD3+IL-4+ cells were more numerous in ASD children than in noninflamed controls (p<0.04). In contrast, CD3+IL-10+ cells were fewer in ASD children than in noninflamed controls (p<0.05). In the colon, LP CD3+TNFα+ and CD3+IFNγ+ were more frequent in ASD children than in noninflamed controls (p<0.01). In contrast with Crohn’s disease and non-Crohn’s colitis, LP and epithelial CD3+IL-10+ cells were fewer in ASD children than in nondisease controls (p<0.01). There was a significantly greater proportion of CD3+TNFα+ cells in colonic mucosa in those ASD children who had no dietary exclusion compared with those on a gluten and/or casein free diet (p<0.05). There is a consistent profile of CD3+ lymphocyte cytokines in the small and large intestinal mucosa of these ASD children, involving increased pro-inflammatory and decreased regulatory activities. The data provide further evidence of a diffuse mucosal immunopathology in some ASD children and the potential for benefit of dietary and immunomodulatory therapies.