25-hydroxycholesterol mediates brain cytokine production and neutrophil infiltration in a mouse model of LPS-induced neuroinflammation.

Neuroinflammation has been implicated in the pathogenesis of several neurologic and psychiatric disorders. Microglia are key drivers of neuroinflammation and in response to different inflammatory stimuli overexpress a proinflammatory signature of genes. Among these, Ch25h is a gene overexpressed in brain tissue from Alzheimer's disease as well as various mouse models of neuroinflammation. Ch25h encodes cholesterol 25-hydroxylase, an enzyme that hydroxylates cholesterol to form 25-hydroxycholesterol (25HC). 25HC, an immune-oxysterol primarily produced by activated microglia, is further metabolized to 7?,25-dihydroxycholesterol, which is a potent chemoattractant for leukocytes. We have also previously shown that 25HC increases production and secretion of the proinflammatory cytokine, IL-1?, by mouse microglia treated with lipopolysaccharide (LPS). In the present study, wildtype (WT) and Ch25h-knockout (CKO) mice were peripherally administered LPS to induce an inflammatory state in the brain. In LPS-treated WT mice, Ch25h expression and 25HC levels increased in brain relative to vehicle-treated WT mice. Interestingly, 25HC levels were significantly higher in LPS-treated WT female compared to male mice. Activation of microglia and astrocytes in response to systemic LPS was suppressed in CKO mice relative to WT mice. Proinflammatory cytokine production and intra-parenchymal infiltration of neutrophils strongly correlated with brain 25HC levels and were significantly lower in CKO compared to WT mice. Overall, our results show that 25HC mediates a sex-specific proinflammatory response in the brain characterized by activation of both microglia and astrocytes but also by neutrophil migration into the brain parenchyma presumably due to 7?,25-diHC produced from 25HC. Overall design: Wild-type (C57BL/6J) and ch25h-/- (CKO) mice from the Jackson Laboratory were used in this study. At 23-26 weeks of age, mice were intraperitoneally (i.p.) administered LPS (2 mg/kg per dose) or vehicle (PBS) twice with a 24-hours interval. Animals were perfused 24 hours after the last injection. Left hemisphere was fixed in 4% paraformaldehyde for immunostaining. Hippocampus from the right hemisphere was flash frozen and stored at -80°C until RNA isolation using Zymo Research Quick-RNA MiniPrep Plus kit (Cat. #: R1058).