B cell subsets are differentially regulated by desmosterol in an atherosclerotic environment
Abstract
Introduction: Atherosclerosis is an inflammatory disease of the large and medium size arteries that is characterized by deposition of oxidized lipids within the vessel. Inflammatory functions require proper metabolic reprograming to meet the increased energy demands of the cells. Cholesterol metabolism has recently been appreciated as a mechanism of regulation of such reprograming. Maintenance of cholesterol homeostasis is important in regulating potentially toxic levels of cholesterol accumulation. This process is likely dysregulated in a hyperlipidemic, atherosclerotic environment. Desmosterol, a precursor of cholesterol, serves as a negative regulator of macrophage functions. While it is known that different B cell subsets utilize divergent metabolic pathways, little is known about B cell subset specific cholesterol metabolism.
Methods: To understand the role of desmosterol in B cells, we generated a mouse model with overexpression of 24- dehydrocholesterol reductase (DHCR24), an enzyme that converts desmosterol into cholesterol (Dhcr24fl/fl mice were provided by Dr.Carlos Fernandez-Hernando), specifically in B cells using the cre/flox system crossed with an atherosclerotic mouse model (Dhcr24fl/flCd19cre/+Ldlr-/- mice). This overexpression of DHCR24 results in reduction of desmosterol specifically in B cells. B cell subsets were isolated through magnetic isolation or sorted using FACs Aria and purity checked using flow cytometry. Intracellular calcium flux and phosphorylation of SYK and BTK were assessed by flow cytometry to determine activation of B cells. Dhcr24fl/flCd19cre/+Ldlr-/- and control Dhcr24+/+Cd19cre/+Ldlr-/- mice were fed high fat diet for 12-16 wks.
Results: Reduced desmosterol levels in B cells increases B cell activation in response to BCR induced activation with increased calcium flux. This is accompanied by an increase in phosphorylation of SYK and BTK. Additionally, desmosterol regulates B cell subset distribution at homeostatic and atherosclerotic environments, suggesting a specific implication of desmosterol in functions of innate B cells including B1 and Marginal Zone B cells. Importantly, reduced desmosterol levels in B cells accelerated atherosclerosis in Dhcr24fl/flCd19cre/+Ldlr-/- in comparison with age-, and sex-matched control Dhcr24+/+Cd19cre/+Ldlr-/- mice. Desmosterol depletion also promoted B cell receptor-induced B cell activation in a subset specific manner. Genes important in cholesterol efflux and biosynthesis are regulated by desmosterol in B cells.
Conclusion: Thus, our data uncover a potential role of cholesterol metabolism intermediates in the regulation of B cell subset functions in health and diseases. These findings also highlight the critical function of desmosterol in atherogenesis by reducing inflammation via integration of B cell metabolism in the modulation of the humoral immune response.