Oral sensitization to whey proteins induces age- and sex-dependent behavioral abnormality and neuroinflammatory responses in a mouse model of food allergy: a potential role of mast cells.

BACKGROUND: Growing evidence has strengthened the association of food allergy with neuropsychiatric symptoms such as depression, anxiety, and autism. However, underlying mechanisms by which peripheral allergic responses lead to behavioral dysfunction are yet to be determined. Allergen-activated mast cells may serve as mediators by releasing histamine and other inflammatory factors that could adversely affect brain function. We hypothesized that eliciting food allergy in experimental animals would result in behavioral changes accompanied by mast cell accumulation in the brain. Our hypothesis was tested in a mouse model of milk allergy using bovine milk whey proteins (WP) as the allergen. METHODS: Male and female C57BL/6 mice at 4 weeks (young) and 10 months (old) of age underwent 5-week WP sensitization with weekly intragastric administration of 20 mg WP and 10 µg cholera toxin as an adjuvant. Age-matched sham animals were given the vehicle containing only the adjuvant. All animals were orally challenged with 50 mg WP in week 6 and their intrinsic digging behavior was assessed the next day. Animals were sacrificed 3 days after the challenge, and WP-specific serum IgE, intestinal and brain mast cells, glial activation, and epigenetic DNA modification in the brain were examined. RESULTS: WP-sensitized males showed significantly less digging activity than the sham males in both age groups while no apparent difference was observed in females. Mast cells and their activities were evident in the intestines in an age- and sex-dependent manner. Brain mast cells were predominantly located in the region between the lateral midbrain and medial hippocampus, and their number increased in the WP-sensitized young, but not old, male brains. Noticeable differences in for 5-hydroxymethylcytosine immunoreactivity were observed in WP mice of both age groups in the amygdala, suggesting epigenetic regulation. Increased microglial Iba1 immunoreactivity and perivascular astrocytes hypertrophy were also observed in the WP-sensitized old male mice. CONCLUSIONS: Our results demonstrated that food allergy induced behavioral abnormality, increases in the number of mast cells, epigenetic DNA modification in the brain, microgliosis, and astrocyte hypertrophy in a sex- and age-dependent manner, providing a potential mechanism by which peripheral allergic responses evoke behavioral dysfunction.

Region-specific regulation of central histaminergic H3 receptor expression in a mouse model of cow's milk allergy.

Central histaminergic H3 receptor (H3R) has been extensively investigated as a potential therapeutic target for various neurological and neurodegenerative disorders. Despite promising results in preclinical rodent models, clinical trials have not provided conclusive evidence for the benefit of H3R antagonists to alleviate cognitive and behavioral symptoms of these disorders. Inconsistent pharmacological efficacies may arise from aberrant changes in H3R over time during disease development. Because H3R is involved in feedback inhibition of histamine synthesis and secretion, the expression of the autoreceptor may also be reciprocally regulated by altered histamine levels in a pathological condition. Thus, we investigated H3R expression in a mouse model of cow's milk allergy, a condition associated with increased histamine levels. Mice were sensitized to bovine whey proteins (WP) over 5 weeks and H3R protein and transcript levels were examined in the brain. Substantially increased H3R immunoreactivity was observed in various brain regions of WP-sensitized mice compared to sham mice. Elevated H3R expression was also found in the thalamic/hypothalamic region. The expression of histaminergic H1, but not H2, receptor subtype was also increased in this and the midbrain regions. Unlike the brain, all three histaminergic receptors were increased in the small intestine. These results indicated that the central histaminergic receptors were altered in WP-sensitized mice in a subtype- and region-specific manner, which likely contributed to behavioral changes we observed in these mice. Our study also suggests that altered levels of H3R could be considered during a pharmacological intervention of a neurological disease.

Astrogliosis Associated With Behavioral Abnormality in a Non-anaphylactic Mouse Model of Cow's Milk Allergy.

Etiology of neuropsychiatric disorders is complex, involving multiple factors that can affect the type and severity of symptoms. Although precise causes are far from being identified, allergy or other forms of hypersensitivity to dietary ingredients have been implicated in triggering or worsening of behavioral and emotional symptoms, especially in patients suffering from depression, anxiety, attention-deficit hyperactivity, and/or autism. Among such ingredients, cow's milk, along with wheat gluten, is commonly suspected. However, the contributory role of cow's milk in these disorders has not been elucidated due to insufficient pathophysiological evidence. In the present study, we therefore investigated neuroinflammatory changes that are associated with behavioral abnormality using a non-anaphylactic mouse model of cow's milk allergy (CMA). Male and female C57BL/6J mice were subjected to a 5-week oral sensitization procedure without or with a major milk allergen, beta-lactoglobulin (BLG). All mice were then later challenged with BLG, and their anxiety- and depression-associated behaviors were subsequently assessed during the 6th and 7th weeks. We found that BLG-sensitized male mice exhibited significantly increased anxiety- and depression-like behavior, although they did not display anaphylactic reactions when challenged with BLG. Female behavior was not noticeably affected by BLG sensitization. Upon examination of the small intestines, reduced immunoreactivity to occludin was detected in the ileal mucosa of BLG-sensitized mice although the transcriptional expression of this tight-junction protein was not significantly altered when measured by quantitative RT-PCR. On the other hand, the expression of tumor necrosis factor alpha (TNFα) in the ileal mucosa was significantly elevated in BLG-sensitized mice, suggesting the sensitization had resulted in intestinal inflammation. Inflammatory responses were also detected in the brain of BLG-sensitized mice, determined by the hypertrophic morphology of GFAP-immunoreactive astrocytes. These reactive astrocytes were particularly evident near the blood vessels in the midbrain region, resembling the perivascular barrier previously reported by others in experimental autoimmune encephalitis (EAE) mouse models. Interestingly, increased levels of COX-2 and TNFα were also found in this region. Taken together, our results demonstrated that BLG sensitization elicits inflammatory responses in the intestine and brain without overt anaphylactic signs of milk allergy, signifying food allergy as a potential pathogenic factor of neuropsychiatric disorders.