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Sensitization To A Milk Allergen Induces Differential Behavioral And Neurological Responses In Allergic C57BL/6J And BALB/cJ Mice

Smith, N;

Studies have highlighted an association between food allergies and neuropsychiatric disorders such as anxiety, depression, and attention deficit hyperactivity disorder. Though the precise mechanism behind this relationship is unknown, it marks a potential novel therapeutic strategy outside of traditional neuropharmacological intervention. Food allergy is a heterogeneous disorder of the immune system characterized by an immune response that occurs reproducibly to a given food. Chronic allergen exposure in patients with mild or non-anaphylactic food allergies may cause or exacerbate psychiatric conditions. However, the variability in food allergies due to factors like genetic background introduces selection bias for individuals with overt anaphylactic reactions. To elucidate the framework of a mechanism by which peripheral non-anaphylactic food allergies cause behavioral changes, we sensitized mice to the cow’s milk allergen ?-lactoglobulin (BLG). This dissertation consists of three studies to characterize the behavioral effects of mild cow’s milk allergy (CMA) and the underlying mechanism. The first study was to profile the behavioral symptoms of mice following sensitization to BLG and their underlying brain pathology. The second builds upon the findings of the first, to investigate changes in the brain using RNA sequencing and further histological analysis to understand what is happening in the brains of allergic mice. The third study compares two strains of mice with different genetic backgrounds and their responses to allergic sensitization. We evaluate the difference in their clinical symptoms, behavior, microbiomes, and released immune mediators in response to allergic challenge to investigate how genetic predisposition influences the effects we observed in our model. In study 1, male C57BL/6J mice sensitized to BLG presented increased anxietylike behavior in the elevated-zero maze and grooming behavior tests and depression-like behavior during the tail-suspension test 24-48 hrs following allergic challenge. Since the mice sensitized to BLG were confirmed to be allergic based upon the abundance of BLGspecific IgE and IgG1, we histologically analyzed their intestines as the primary site of insult for CMA. In the ileum region of the small intestine, we observed a decrease in the tight junction protein occludin in BLG sensitized mice. A decrease in the abundance of occludin is common in intestinal inflammation and often leads to dysfunction of normal gut barrier function. Knowing male mice sensitized to BLG had a behavioral change, gut pathology, and were confirmed allergic, we then looked for evidence of brain pathology. We evaluated the status of astrocytes within the brain by staining for their structural protein glial fibrillary acidic protein (GFAP). We found that astrocytes in the substantia nigra were hypertrophic, and there was evidence of perivascular glial scarring which is common in neuroinflammatory conditions. The perivascular scarring also coincided with increased abundance of the proinflammatory cytokine, tumor necrosis factor-alpha (TNF?), which astrocytes both respond to and produce when activated. Together these data suggest CMA leads to anxiety and depression-like behavior in male mice and that the astrocyte response and signaling of cytokines like TNF? are involved in these behavioral changes. In the second study, we investigate region transcriptional profiles of BLG sensitized C57BL/6J male mice. Across the multiple brain regions, using ingenuity pathway analysis, we found pathways involved in inflammatory signaling, neuronal signaling, cell structural pathways, and disease states differentially activated in BLG sensitized mice. We validated some of our findings histologically using myelin basic protein (MBP) and IgG as targets based upon our previous glial findings and pathways involved in other glial cells and blood-brain barrier integrity. We observed no evidence of differential myelination through MBP in the brains of BLG sensitized mice but increased extravascular IgG in the brain parenchyma. High amounts of IgG staining within the brain implies impairment of normal blood-brain barrier function, which coincides with our previous astrocyte data. In study 3, we compared the C57BL/6J and BALB/cJ strains because of their genetic differences in the allergy response. The differences were apparent when observing the overt anaphylactic response to BLG challenge; only the BALB/cJ mice showed significant clinical symptoms. However, both strains produced BLG-specific IgE in response to treatment, but only the BALB/cJ strain produced IgG antibodies. Despite the described differences, males of both strains demonstrated similar anxiety-like behavior though the changes were more pronounced in C57BL/6J mice. Knowing the differences in immune responses observed in these strains, we quantified the cytokines released into the plasma, finding increases in a Th2 cytokines interleukin (IL)-10, -13, and -21, in addition to various chemokines in male C57BL/6J mice, but no increases in male BALB/cJ mice. Based on the observed differences, we wanted to investigate the impact BLG-sensitization had on the microbiome. Both strains were found to have distinct profiles, and BLG-sensitization led to strain-specific changes in the microbiome. Despite vastly different microbiome profiles, when we performed brain-specific pathway analysis of the microbiomes, the two strains had similar activation states of serotonergic, dopaminergic, addiction pathways, and various neurodegenerative diseases.