Chronic exposure to aflatoxin B1 increases hippocampal microglial pyroptosis and vulnerability to stress in mice

Background: Chronic aflatoxin B1 (AFB1) exposure may increase the risk of multiple neuropsychiatric disorders. Stress is considered one of the main contributors to major depressive disorder. Whether and how chronic AFB1 exposure affects vulnerability to stress is unclear.

Methods: Mice were exposed for three weeks to AFB1 (100 µg/kg/d) and/or chronic mild stress (CMS). The vulnerability behaviors in response to stress were assessed in the forced swimming test (FST), sucrose preference test (SPT), and tail suspension test (TST). Microglial pyroptosis was investigated using immunofluorescence, enzyme-linked immunosorbent assays, and western blot assay in the hippocampus of mice. Hippocampal neurogenesis and the effects of AFB1-treated microglia on proliferation and differentiation of neural stem/precursor cells (NSPCs) were assessed via immunofluorescence in the hippocampus of mice.

Results: Mice exposed to CMS in the presence of AFB1 exhibited markedly greater vulnerability to stress than mice treated with CMS or AFB1 alone, as indicated by reduced sucrose preference and longer immobility time in the forced swimming test. Chronic aflatoxin B1 exposure resulted in changes in the microglial morphology and increase in TUNEL⁺ microglia and GSDMD⁺ microglia in the hippocampal dentate gyrus. When mice were exposed to both CMS and AFB1, pyroptosis-related molecules (such as NLRP3, caspase-1, GSDMD-N, and interleukin-1β) were significantly upregulated in the hippocampus. These molecules were also significantly enhanced by AFB1 in primary microglial cultures. AFB1-treated mice showed decrease in the numbers of BrdU⁺, BrdU-DCX⁺, and BrdU-NeuN⁺ cells in the hippocampal dentate gyrus, as well as the percentages of BrdU⁺ cells that were NeuN⁺ in the presence or absence of CMS when compared with vehicle-treated mice. The combination of AFB1 and CMS exacerbated these effects to an even greater extent. The number of DCX⁺ cells correlated negatively with the percentage of ameboid microglia, TUNEL⁺ microglia and GSDMD⁺ microglia in the hippocampal dentate gyrus. AFB1-treated microglia suppressed the proliferation and neuronal differentiation of NSPCs in vitro.

Conclusion: Chronic AFB1 exposure induces microglial pyroptosis, promoting an adverse neurogenic microenvironment that impairs hippocampal neurogenesis, which may render mice more vulnerable to stress.