Perinatal Arsenic Exposure-Induced Modulation of Pu.1/Csf1r Axis Leads to Microglial Proliferation in the Mouse Hippocampus

Earlier we showed that perinatal arsenic exposure induces microglial activation, proliferation and cognitive impairment in mouse pups. The present study is focused to identify the role of PU.1/CSF1R axis in arsenic-induced microglial proliferation. For in vivo study six to eight-week-old male and female BALB/c mice were used. For mating male and female mice were kept in the same cage at a ratio of (1:2). After mating pregnant females were divided into vehicle control and arsenic treatment group. Sodium arsenite was dissolved in reverse osmosis water and given to the treatment group. The treatment regimen continued from gestational day 5 (GD5) until the weaning period of pups around post-natal day 22 (PND22). Following the exposure regimen, pups were sacrificed for each of the western blot analysis, qRT-PCR and immunohistochemical staining. For in vitro studies, mouse micrglial cell line, BV2 was used. BV2 cells were treated with arsenic, L-methionine or siRNA as required in various combinations. Following in vitro treatment cells were harvested and used for western blot analysis or qRT-PCR. It was observed that perinatal arsenic exposure (0.38 mg/kg bd wt) from gestational day 5 till the sacrifice of the pups on PND 22 increased the microglial number. The expression of CSF1R was observed to be upregulated. The expression of CSF1R transcription factor PU.1 was also found to be increased in the arsenic-exposed group. The role of arsenic-induced PU.1 was confirmed by checking the expression of CSF1R following inhibiting PU.1 using siRNA in BV2 microglia. Reversal of PU.1 expression in the L-methionine-supplemented arsenic-exposed BV2 microglia indicates possible demethylation of the in silico predicted CpG island in the upstream regulatory region of the PU.1 promoter. Taken together, the study reveals that arsenic exposure increases microglial proliferation by modulating the PU.1/ CSF1R axis in mouse hippocampus.