25-Hydroxycholesterol is a metabolite of cholesterol that is produced and secreted by macrophages in response to Toll-like receptor (TLR) activation. 25-hydroxycholesterol is a potent (EC₅₀≈65 nM) and selective suppressor of IgA production by B cells. In Vitro: The synthesis of 25-hydroxycholesterol is catalyzed by the enzyme cholesterol 25-hydroxylase, which uses cholesterol and molecular oxygen as substrates and NADPH as a cofactor. 25-hydroxycholesterol is a potent bioactive lipid in the innate and adaptive immune systems^[1].