Paul Ashwood - US grants

DESCRIPTION (provided by applicant): This proposal is submitted in response to the following Title: Recovery Act Limited Competition: Research to Address the Heterogeneity in Autism Spectrum Disorders (R21). Request for Applications (RFA) Number: RFA-MH-09-172 Abstract: Though most studies have examined immune abnormalities in children with autism, it is also possible that an aberrant immune response in mothers during vulnerable, critical periods of neurodevelopment could produce neuronal injury in the fetal or neonatal brain, and produce long term neurological dysfunction characteristic of autism. We will examine several quantifiable biological signatures of immune cells in blood of mothers of children with autism: (1) RNA levels, (2) functional assays of white blood cells and (3) the presence of antibodies that are directed to fetal-brain proteins. We have preliminary data showing that a number of mothers of children with autism have RNA expression profiles in their peripheral blood that differ from mothers of control children. We have discovered that the function of Natural Killer (NK) cells in blood of a number of children with autism is impaired. We have also discovered that some mothers of children with autism have antibodies in their blood that are directed at fetal-brain proteins. Based upon these promising findings, we propose the following Specific Aims for this exploratory R21. Specific Aim #1. Determine whether there are subgroups of mothers of children with autism with different RNA expression profiles in their peripheral blood that differ from each other and differ from controls. Specific Aim #2: Examine Natural Killer (NK) cell function in mothers of children with autism;whether the NK functional changes are associated with changes of NK gene expression;and whether some of these mothers have autistic children with abnormal NK cell function. Specific Aim #3. Determine whether mothers of children with autism, who have antibodies that are directed at fetal-brain proteins and which are detectable in blood, have abnormal NK function and/or abnormal RNA expression profiles in blood that differ from other mothers of children with autism and differ from mothers of typically developing children. Hypotheses: We postulate that there is a subgroup of mothers with children with autism who has an immune abnormality detectable in peripheral blood that can be assessed using (1) RNA expression in the immune/ white blood cells (2) functional assays of white blood cells and (3) and by showing the presence of antibodies directed to fetal-brain proteins in blood. Significance and Impact. The ability to identify a subgroup of mothers of children with autism into a specific immune-related phenotype will improve the success of linkage and whole genome association studies to detect genes associated with this subgroup. The identification of biological signatures in mothers that are highly associated with having children with autism could eventually lead to the characterization of specific immune abnormalities in the mothers of children with autism and may shed light on the cause(s) of autism in this subgroup and potentially lead to prevention or treatment strategies prior to or during pregnancy. PUBLIC HEALTH RELEVANCE: There has been relatively little attention paid to the immune system of mothers of children with autism. The maternal immune system is important since a dysfunctional immune response during pregnancy might affect the fetal brain and result in autism. Thus, this proposal will focus on the immune system of women with children with autism compared to women of typically developing children.

Project Summary/Abstract: Autism Spectrum Disorders (ASD) are behaviorally defined by deficits in communication, social reciprocity, and repetitive stereotypic behaviors. While genetic and environmental factors are likely contributors to these disorders, little is known about the pathophysiology in ASD. Many in vitro studies, post-mortem brain studies and proteomic studies in plasma and cerebral spinal fluid have described the presence of increased immune activation in ASD, whilst clinical and epidemiological studies suggest that there is an increase in immune mediated conditions such as allergies, asthma and autoimmunity. Activation of these immune responses is more prominent in individuals with exacerbated behavioral impairments in ASD. We hypothesize that an underlying mechanism common to this diverse array of findings is the lack of immune control or regulation that can lead to immune activation and inflammation. Our published data and preliminary findings suggest a lack of production of immune regulatory cytokines such as transforming growth factor beta 1 (TGF?1) and interleukin (IL)-10. Decreases in these factors were associated with worse behavioral outcomes and more co-morbidities in ASD. In animal models with face and construct validity to ASD decreases in CD4+FoxP3+ regulatory T cells (Tregs) were observed. However, no studies have yet to address the functional cellular mechanisms of Tregs in ASD or their role in animal models of ASD. We will test the innovative hypothesis that a lack of cellular immune regulation by Tregs is a predictive risk factor for ASD diagnosis, and that targeting immune control mechanisms can alleviate behavioral abnormalities. Parallel clinical and preclinical experiments will be performed. The proposed studies will determine the Tregs cellular mechanisms of immune control in cord blood samples from children that later receive a diagnosis of ASD, and compare this to children with typical development (Aim #1). This proposal will directly assess specific cellular mechanisms for which there are novel therapeutic potential. One of these therapeutic approaches, adoptive transfer of Tregs, will be utilized to rescue the behavioral impairments present in a preclinical mouse model that exhibits many features with relevance to ASD (Aim #2). The proposal will directly assess the relationship of immune dysregulation in ASD and behavior abnormalities. If successful, this research will validate the transformative concept that ASD is, for some, a disorder due to defects in immune regulation and control by Tregs, and will validate a novel mechanism for one of the most visible public health concerns of our time.

Project Summary/Abstract: Autism spectrum disorders (ASD) are a set of pervasive, highly prevalent lifelong disorders for which pharmacological interventions are not readily available. While genetic factors are likely contributors to these disorders, heritability estimates indicate strong environmental contributions. Of particular interest is the link between fetal neurodevelopment and the activation of the maternal immune system during gestation. Epidemiological reports suggest a strong association between periods of maternal immune activation and an increased risk of having a child with ASD, including immune conditions such as allergies and asthma. Unique immune cascades representative of asthma and allergy responses have been detected in amniotic fluid, newborn bloodspots and maternal mid-gestation serum samples of mothers whose child was later diagnosed with ASD. Acute exacerbations are common in pregnant asthmatic women with as many 35% suffering attacks during pregnancy. In addition, air pollution, a major exacerbating factor in allergic asthma, has been separately linked with an increased risk for ASD. However, little is known about the consequences of maternal allergic asthma (MAA) responses, air pollution, or their combined effects on fetal development. In this exploratory proposal, we will create a novel model in which to test the innovative hypothesis that the particulate matter (PM) fraction of air pollution and MAA are causally linked, and in combination lead to exacerbated ASD-relevant behaviors in offspring of PM + MAA exposed dams. The proposed studies will examine the priming effects of PM sampled from the Sacramento region in combination with MAA on ASD-relevant behaviors (Aim #1). We will test the hypothesis that the combined effects of PM + MAA exposures during gestation lead to changes in immune function and microglia activation in the fetus that disrupt neurodevelopment. The proposed studies will examine whether behavioral outcomes of PM + MAA exposures are IL-4 dependent (Aim #2). If successful, this research will validate the transformative concept that ASD is, for some, a disorder due to the direct effects of common environmental contaminants, and will identify a novel mechanism for one of the most visible public health concerns of our time.

Project Summary/Abstract: Autism spectrum disorders (ASD) are behaviorally defined and affects 1 in 68 children in the United States; however, little is known about its etiology and pathophysiology. Interestingly, over half of children with Autism Spectrum Disorders (ASD) have co-morbid gastrointestinal (GI) symptoms. GI problems occur 6-8 times more frequently in ASD than in typically developing (TD) children and are associated with more exacerbated behavioral impairments. Although Very little is known about the pathophysiological pathways underlying GI problems in ASD. Increased immune activation, pro-inflammatory cytokine production and autoantibodies directed to gut epithelium have been reported in children with ASD and GI symptoms. Regulatory T cells (Tregs) are key mediators of peripheral tolerance that maintain their lineage commitment and function through epigenetic regulation and prevent inappropriate mucosal inflammation in response to bacteria and other luminal antigens/components. We and others have previously demonstrated decreased blood levels of the immunosuppressive and Tregs-associated cytokines transforming growth factor (TGF)?1 and interleukin (IL)-10, fewer putative Tregs and alterations in epigenetic mechanisms in children with ASD. Our new preliminary data shows that these immune regulatory deficits are more severe in children with ASD and persistent GI symptoms. In animal models with face and construct validity to ASD, defects in GI barrier integrity and decreases in Tregs were observed. However, no studies have yet addressed the functional cellular mechanisms of Tregs in ASD or preclinical models of ASD. We will test the innovative hypothesis that deficits in the ability to control immune responses by Tregs are an underlying pathophysiological mechanism in children with ASD who experience GI co-morbidities. This is an important area of investigation since therapeutic targeting of immune control mechanisms might improve GI barrier integrity and alleviate behavioral abnormalities. Parallel clinical and preclinical experiments will be performed to investigate this hypothesis. The proposed studies will determine Treg cellular function (Aim #1) and epigenetic mechanisms controlling Treg commitment and stability (Aim #2) in both children with ASD and TD controls with and without GI symptoms. This proposal will directly assess specific cellular mechanisms with potential for novel therapies. One of these therapeutic approaches, adoptive transfer of Tregs, will be utilized to rescue GI barrier integrity and behavioral impairments present in a preclinical mouse model that exhibits many ASD-relevant features (Aim #3). If successful, this research will validate the transformative concept that ASD is, for some, a disorder due to defects in immune regulation and control by Tregs, and will validate a novel mechanism for one of the most visible public health concerns of our time.

Project Summary/Abstract: Autism spectrum disorders (ASD) are pervasive, highly prevalent lifelong disorders for which pharmacological interventions are not readily available. While genetic factors are likely contributors to these disorders, heritability estimates indicate strong environmental contributions. Of particular interest is the link between fetal gestation and the activation of the maternal immune system during critical periods of development. Epidemiological reports suggest a strong association between periods of maternal immune activation and an increased risk of having a child with ASD including immune conditions such as allergies and asthma. Unique immune cascades representative of asthma and allergy responses have been detected in amniotic fluid and maternal serum mid-pregnancy of mothers whose child was later diagnosed with ASD. Acute exacerbations are common in pregnant asthmatic women with as many 45% suffering attacks during pregnancy. However, little is known about the consequences of maternal allergic asthma (MAA) responses on fetal development. We have described the first set of preclinical studies to test the hypothesis that MAA induced during gestation imparts alterations in brain neurobiology and functional behavioral outcomes in the offspring that have a high degree of face validity for ASD symptoms. Pregnancy is a time when epigenetic changes help a static genome adapt to the maternal environment, so that if the maternal immune system is overly activated, the fetal immune system will also be over activated at the expense of brain development. The proposed studies will examine whether MAA alters epigenetic landscape of microglia, alters microglia activation and impacts early brain development (Aim #1). We will test the hypothesis that MAA exposures during gestation leads to a susceptibility to further challenges or stressors in juvenile offspring that can impact inflammation in the offspring, including neuroinflammation and elevated microglia activation, neuropathology and behavioral outcomes (Aim #2). If successful, this research will validate the transformative concept that ASD is, for some, a disorder due to the direct effects of aberrant immune activation and will identify a novel mechanism for one of the most visible public health concerns of our time.

Project Summary/Abstract: Many in vitro studies, post-mortem brain studies, and proteomic studies in plasma and cerebral spinal fluid have described the presence of increased immune activation in ASD, whilst clinical and epidemiological studies suggest that there is an increase in immune mediated conditions such as atopy and autoimmunity. Activation of these immune responses is more prominent in individuals with exacerbated behavioral impairments in ASD. Despite recent advances, there is a large gap in our knowledge regarding pathophysiological pathways in ASD. We hypothesize that an underlying mechanism common to this diverse array of findings is the lack of immune control or regulation that can lead to immune activation and inflammation. Regulatory T cells (Tregs) are key mediators of immune tolerance that maintain their lineage commitment and function through epigenetic regulation, and restrain inappropriate inflammation. We and others have previously demonstrated decreased frequencies of Tregs-and immunosuppressive cytokines including transforming growth factor (TGF)?1 and interleukin (IL)-10.Our new preliminary data shows that these immune regulatory deficits are associated with more severe behavioral phenotypes. However, no studies have yet to address the functional cellular mechanisms of Tregs in ASD. We will test the innovative hypothesis that a lack of cellular immune regulation is an early predictive risk factor for ASD, and that it endures in children with ASD who have received a diagnosis. Considering that impairments in social interaction and anxiety are key features of ASD, the amygdala has been extensively implicated in ASD pathophysiology. We will also address the absence of immune regulation in the amygdala of individuals with ASD and matched controls over development. This is an important area of investigation since therapeutic targeting of immune control mechanisms might improve immune function, address abnormal amygdala development and alleviate behavioral abnormalities. The proposed studies will determine immune regulation, Treg cellular function and epigenetic mechanisms controlling Tregs commitment and stability (Aim #1) in children with ASD and typically developing (TD) controls. In two prospective, population based cohorts we will examine immune regulatory mechanisms in cord blood samples from children that later receive a diagnosis of ASD, or TD (Aim #2). The proposal will directly assess the relationship of predictive and longitudinal measures of immune dysregulation and behavior abnormalities in ASD. We will examine amygdala growth over development into adulthood and immune regulation in human samples from ASD and TD controls (Aim #3). If successful, this research will validate the transformative concept that ASD is, for some, a disorder due to defects in immune regulation and will validate a novel mechanism for one of the most visible public health concerns of our time. .

Project Summary/Abstract: Autism spectrum disorders (ASD) are behaviorally defined and affects 1 in 68 children in the United States; however, little is known about its etiology and pathophysiology. Interestingly, over half of children with Autism Spectrum Disorders (ASD) have co-morbid gastrointestinal (GI) symptoms. GI problems occur 6-8 times more frequently in ASD than in typically developing (TD) children and are associated with more exacerbated behavioral impairments. Although Very little is known about the pathophysiological pathways underlying GI problems in ASD. Increased immune activation, pro-inflammatory cytokine production and autoantibodies directed to gut epithelium have been reported in children with ASD and GI symptoms. Regulatory T cells (Tregs) are key mediators of peripheral tolerance that maintain their lineage commitment and function through epigenetic regulation and prevent inappropriate mucosal inflammation in response to bacteria and other luminal antigens/components. We and others have previously demonstrated decreased blood levels of the immunosuppressive and Tregs-associated cytokines transforming growth factor (TGF)?1 and interleukin (IL)-10, fewer putative Tregs and alterations in epigenetic mechanisms in children with ASD. Our new preliminary data shows that these immune regulatory deficits are more severe in children with ASD and persistent GI symptoms. In animal models with face and construct validity to ASD, defects in GI barrier integrity and decreases in Tregs were observed. However, no studies have yet addressed the functional cellular mechanisms of Tregs in ASD or preclinical models of ASD. We will test the innovative hypothesis that deficits in the ability to control immune responses by Tregs are an underlying pathophysiological mechanism in children with ASD who experience GI co-morbidities. This is an important area of investigation since therapeutic targeting of immune control mechanisms might improve GI barrier integrity and alleviate behavioral abnormalities. Parallel clinical and preclinical experiments will be performed to investigate this hypothesis. The proposed studies will determine Treg cellular function (Aim #1) and epigenetic mechanisms controlling Treg commitment and stability (Aim #2) in both children with ASD and TD controls with and without GI symptoms. This proposal will directly assess specific cellular mechanisms with potential for novel therapies. One of these therapeutic approaches, adoptive transfer of Tregs, will be utilized to rescue GI barrier integrity and behavioral impairments present in a preclinical mouse model that exhibits many ASD-relevant features (Aim #3). If successful, this research will validate the transformative concept that ASD is, for some, a disorder due to defects in immune regulation and control by Tregs, and will validate a novel mechanism for one of the most visible public health concerns of our time.