Immune Responses in Autism

Extensive Microglial Activation
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Extensive chronic microglial activation (bottom) relative to a control group (top).
Suzuki K. et al. JAMA Psychiatry (2013)

Autism Spectrum Disorder is a multifactorial physical disorder making the abnormal behaviour associated with it different from typical frustration or tantrums. Individuals with the disease suffer from a widespread range of symptoms from sensory overload to gastrointestinal symptoms making every day life a challenge.

Prenatal and postnatal infections have each been hypothesized to lead to immune dysregulation occurring at critical time frames in development. This phenomenon has linked chronic inflammation (and neuroinflammation) with the behavioral abnormalities observed in those diagnosed with autism. Autistic children have shown increased levels in numerous growth factors and cytokines which are crucial for cell proliferation, differentiation and cell survival [1]. Anatomically; extensive microglial activation has been observed in the cerebellum as well as cortical areas of the brain[2].

Recent research regarding immune responses in autism has shifted the focus from being strictly neurological and to incorporate neuroimmunology discussing the gut and its secondary neurological effects[3]. While the relationships between immunology and the neurodevelopmental aspects of ASD show promising insight into the disorder – whether or not it is a cause or a corresponding effect of autism remains to be investigated.

1.0 Immune Dysregulation

Immune dysregulation is a critical phenomenon commonly observed in those diagnosed within the autism spectrum disorder. Both prenatal and postnatal infections are of crucial importance resulting in chronic activation of inflammatory response and potentially altering brain development. 

1.1 Prenatal Infection

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Effects of prenatal immune inflammation shown via an endotoxin (LPS) and its effects on the fetus
P. Boksa. (2010) Brain Behavior and Immunity. 24 (6)

Prenatal Stress as a result of infection has been shown to be of critical importance in numerous studies done with children diagnosed with autism. Several relationships were made between infections in the first or second trimester of pregnancy and diagnoses or development of autism in their child [4]. Whether the cause of maternal hospitalization was due to influenza or gastroentitis, prenatal systemic inflammatory responses are a growing concern for neurodevelopmental deficiency [4].
The placenta may be the culprit in transferring acute effects and large numbers of cytokines to the fetus resulting in placental dysfunction and apoptosis in the white matter of the brain[5]. Immune response triggering inflammation is thought to affect the placenta by increasing levels of TNFalpha activated by macrophages.
In addition; Interleukins 1 and 6 triggered via maternal immune response can also result in placental dysfunction inevitably altering the child’s social behavior. 
From a series of cell cultures and western blots, a study concluded the JAK2/STAT3 pathway was important for interleukin 6 activation [6]. The JAK2/STAT3 is crucial for CNS development and has been shown to also be important in neurodegenerative disorders such as Alzeihmers disease (see miRNA in Alzheimer's Disease). More importantly, once phosphorylation of the pathway was inhibited via flavonoids, abnormal social behavior was attenuated [6].

1.2 Chronic Inflammation

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Importance of balancing pro- and anti- inflammatory cytokines
Image from http://www.invitrogen.com/
BioProbes 67, June 2012.

Increased activation of both the innate and adaptive immune system has been associated with autism. Similar to the hypotheses presented regarding prenatal infection; high levels of numerous cytokines and chemokines (interleukin 8) have been reported in brain tissue of autistic patients post-natal. Cytokines such as: IL-6, IL-1β, TNF-α, have been reported to be consistently higher in the brain cortex when compared to relative controls [7]. Th1 has been shown to be the activated pathway as opposed to Th2 since no significant increases were observed in interleukins 2,5 and 10 [7]. Therefore; chronic localized neural activation specifically through the Th1 pathway is suggested to play a crucial role in the pathogenesis of the disorder [7]. One study has detected statistically significant increased concentrations of the cytokine TGFbeta in children diagnosed as autistic. Interestingly; children with developmental disabilities had very similar serum concentrations to the control group straightening the assumption of a more immune-mediated pathogenesis in autism [1]. Mast cells have also been discussed in relation to autism. Increased activation sensitivity may trigger allergy like symptoms[8]. While controversial; these symptoms were also shown to subside with use of flavonoids which inhibits granule release [8].

1.2.1 Microglial Activation

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Signalling cascade
Image taken from: Kaltschmidt Lab at Bielefeld

Chronic inflammation has been recognized as a factor in autism for decades. Inflammatory markers have been observed in the peripheral blood, as well as in the central nervous system [10]. Microglia are the immune-mediating cells of the central nervous system which bring about inflammation once activated via Protein Kinase-A (PKA). A cascade of following events results in the activation of NFkappaB. NFkappaB is a transcription factor which is responsible for mediating immune response and signaling for the production of cytokines and chemokines and is necessary for the physiological survival of a neuron. Postmortem studies on brain tissue slices showed significantly over activated microglia containing high levels of NFkappaB predominately in the orbitofrontal cortex of those diagnosed as autistic [10]. While the advantages of microglial activation are crucial for neuronal survival; extensive activation may lead to cytotoxicity.

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PET scan results indicating significantly activated brain areas in ASD
Suzuki K. et al. JAMA Psychiatry (2013)

A study at John Hopkins showed extensive astro- and microglial activation predominately in the white matter and the cerebellum of autistic case and has been suggested to be a cause of synaptic dysfunction [2]. High levels of transforming growth factor beta were found in overly active astrocytes, mostly in the cerebellum [2]. This study also showed high levels of both cytokines and chemokines present in overly active astrocytes located in the cerebellum [2].

A recent 2013 study studied PET (positron emission tomography) scans they obtained from adult autistic cases. Using a labile radioactive tracer they tagged for microglia in order to visualize a more widespread inflammation effect when compared to a control group of adults [12]. Primarily; they identified the cerebellum as being the most significantly different in terms of activation. They also identified other brain areas including: the midbrain, pons, fusiform gyri, and the anterior cingulate [12].

1.3 Animal Studies

Animal studies have observed the effects of injecting a pregnant rat with Lipopolysaccharide (LPS) to test for existence of oxidative stress in the fetus.  It confirmed previous assumptions and showed addition of N-acetylcysteine, Zinc or copper alleviated these effects [5]. Another study injected a single dose of the antibody Immunoglobin G (IgG) into a pregnant mouse affected sensory-motor and physical development while increasing anxiety in the offspring [9].

2.0 Gut Bacteria

Preview of a CBC documentary. The Nature of Things: The Autism Enigma

When little about pathogenesis of a disease is known, it is crucial to try and identify several biomarkers in order to understand the disorder. While the central nervous system is undoubtedly important for understanding the effects of autism, inflammation has also been noted in the peripheral blood monocytes of a specific subset of children diagnosed with autism [11]. This specific subset of autistic children showed chronic gastrointestinal symptoms which may, or may not, be disease specific.

Interestingly, transcription profiling of blood monocytes in both the mother and the child showed a genetic predisposition may be involved. While it is normal for autistic children to suffer from numerous comorbities at once, GI infections seems to co-occur the most frequently with cognitive deficits and abnormal behavior [11].

It has been suggested that improper digestion may be more prevalent in autistic children as opposed to controls groups. In addition; fecal samples were taken from autistic children and tested for levels of short chain fatty acid [13]. When compared to controls: abnormal fermentation and increased levels of ammonia were observed. [13] More specifically; the most predominant short chain fatty acid observed at significantly higher levels was propionic acid [13].

Another study has shown a large amount of sutterella wadsworthensis ,a gram negative bacteria, in the microbiota of autistic children who suffer from chronic GI abnormality [15]. Sutterella was not prevalent in those who suffered solely from gastrointestinal irregularities [15]. Behavioral difficulties in autistic children have been shown to intensify with severity of inflammation [11]. In addition, serotonin theory of autism also discusses the effects of gastrointestinal symptoms.

Normal TCA cycle and Altered Cycle
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Top: regular TCA cycle Bottom: Effect of propionic acid on TCA cycle inhibiting proximal portion
Frye, R.E., Melnyk, S., MacFabe, D.F. (2013)

2.1 Mitochondrial Disease and Propionic Acid

MacFabe et al published a study of propionic acid in 2007 and have followed up their research in 2013 asserting the notion "you are what you eat"[14]. There has also been some evidence to support the argument that a group of ASD cases may acquire mitochondrial disease (MD) allowing for further research to address the issue of inflammation in autistic children by focusing on a bacterium produced in the gut as a potential biomarker [3].

Propionic acid is an important metabolic short chain fatty acid that has many functions including neuronal development and is thought to trigger autistic-like characteristics in genetically susceptible groups. Not surprisingly; multiple physiological, behavioral, and metabolic effects similar to those observed in autistic patients were present in the rodents following treatment.

High levels of propionic acid is thought to alter regular tricarboxylic acid cycle activity by increasing levels of succinyl-CoA on the distal side of the pathway and inhibiting the production of succinyl-CoA from the proximal portion starting with acetyl-CoA (visualized by grey arrows in fig. 3). This alteration effectively shuts off one half of the TCA cycle and cuts down NADH production leading to numerous defects in metabolic functions [13]

Theoretically; metabolism and the production of propionic acid following digestion were to result neural inflammation as well as a decrease in the tri-peptide glutathione. In order to test this theory; experimental animal models were used to further understand the effects of MD in the pathogenesis of autism by intracerebroventricularly treating rodents with propionic acid [3]. Results showed a significant similarity in the pathogenesis and biomarkers found in both autistic individuals with MD as well as the PPA rodent models.


Bibliography
1. Ashwood, P., Enstrom, A., Krakowiak, P., Hertz-Picciotto, I., Hansen, R. L., Croen, L. A., Ozonoff, S., & Pessa, I. N. (2008). Decreased transforming growth factor beta1 in autism: A potential link between immune dysregulation and impairment in clinical behavioral outcomes.Journal of Neuroimmunology, 204, 149-153.
2. Vargas, D. L., Nascimbene, C., Krishan, C., Zimmerman, A. W., & Pardo, C. A. (2005). Neuroglial activation and neuroinflammation in the brain of patients with autism. Annals of Neurology, 57(1), 67-81.
3. Frye, R.E., Melnyk, S., MacFabe, D.F. (2013) Unique acyl-carnitine profiles are potential biomarkers for acquired mitochondrial disease in autism spectrum disorder. Translational Psychiatry, 3(1)
4. Atladóttir H.O., Thorsen P., Østergaard L., et al. (2010). Maternal Infection Requiring Hospitalization During Pregnancy and Autism Spectrum Disorders.
5. Boksa, P. (2010). Effects of prenatal infection on brain development and behavior: A review of findings from animal models.
6. Parker-Athill E., Luo D., Bailey, A. (2009) Flavonoids, a prenatal prophylaxis via targeting JAK2/STAT3 signaling to oppose IL-6/MIA associated autismJournal of Neuroimmunology. 217 (1-2), pg. 20-27
7. Xiaohong L., Chauhana, A. et al. Elevated immune response in the brain of autistic patients Journal of Neuroimmunology.
8. Theoharides, T.C., Angelidou A., et al. (2012) Mast cell activation and autism BBA - Molecular Basis of Disease. 1822 (1), pg. 34-41 
9. Braunschweig D., Golub M.S., (2012). Maternal autism-associated IgG antibodies delay development and produce anxiety in a mouse gestational transfer model Journal of Neuroimmunology. 252 (1-2), pg. 56-65 
10. Young, A.M, Campbell, E., Lynch, S., Suckling, J., Powis, S.J., (2011)  Aberrant NF-KappaB Expression in Autism Spectrum Condition: A Mechanism for Neuroinflammation. Frontiers in Psychiatry. 2:27
11. Jynouchi, H., Gange, L. Streck, D.L., Touruner, G.A. (2011).  Children with autism spectrum disorders (ASD) who exhibit chronic gastrointestinal (GI) symptoms and marked fluctuation of behavioral symptoms exhibit distinct innate immune abnormalities and transcriptional profiles of peripheral blood (PB) monocytes. Journal of Neuroimmunology. 238 (73-80)
12. Suzuki, K., Sugihara, G. et al. (2013). Microglial Activation in Young Adults With Autism Spectrum Disorder. JAMA Psychiatry. 70(1). 49-58.
13. Wang L., Christophersen C.T., Sorich, M.J., Gerber, J.P., Angley, M.T., Conlon, M.A. (2012). Elevated fecal short chain fatty acid and ammonia concentrations in children with autism spectrum disorder. Dig Dis Sci. 57(8):2096-102
14. MacFabe D.F., Cain D.P., Rodriguez-Capote K., et al (2007) Neurobiological effects of intraventricular propionic acid in rats: possible role of short chain fatty acids on the pathogenesis and characteristics of autism spectrum disorders. Behav Brain Res. 10;176(1):149-69.
15. Williams, B.L., Hornig M., Parekh T., and Lipkin, W.I. (2012) Application of Novel PCR-Based Methods for Detection, Quantitation, and Phylogenetic Characterization of Sutterella Species in Intestinal Biopsy Samples from Children with Autism and Gastrointestinal Disturbances. mBio vol. 3 no. 1

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