Mice, bone marrow transplantation, immunization, and diet

Niemann-Pick type C1^nih mutant (Npc1 ^mut) mice (a kind gift from Prof. Dr. Lieberman from University of Michigan Medical School) were backcrossed into a C57BL/6 background for more than 10 generations. Npc1 ^mut and Ldlr ^−/− mice were housed under standard conditions and had access to food and water ad libitum. Experiments were performed according to Dutch regulations and approved by the Committee for Animal Welfare of Maastricht University.

To generate myeloid Npc1 ^mut deficient Ldlr ^−/− mice, bone marrow transplantations were performed. Twenty-two week-old female Ldlr ^−/− mice received antibiotic water containing neomycin (100 mg/l; Gibco, Breda, the Netherlands) and 6*10⁴ U/l polymycin B sulphate (Gibco, Breda, the Netherlands) one week before and four weeks after irradiation. One day before and on the day of the transplantation, Ldlr ^−/− mice were lethally irradiated with 6 Gray of γ-radiation, thus receiving 12 Gray in total. Lethally irradiated Ldlr ^−/− mice were then injected with 1*10⁷ bone marrow cells donated from either Npc1 ^mut mice or wildtype littermate controls (Npc1 ^wt). In order to fully ensure bone marrow replacement, mice had a nine week recovery period. After nine weeks of recovery, transplanted (-tp) mice received a high-fed, high-cholesterol (HFC) diet, containing 21% butter and 0.2% cholesterol (diet 1635; Scientific Animal Food and Engineering, Villemoissonsur-Orge, France) for 12 weeks. Five weeks after bone marrow transplantation, mice were divided into three groups. One group received the equivalent of 10⁸ colony-forming units of heat-killed R36A (unencapsulated Streptococcus pneumoniae) emulsified in 200 µl sterile 0.9% NaCl for the primary subcutaneous immunization. Subsequently, two intraperitoneal booster immunizations were administered every two weeks. The two other control groups received an 0.9% NaCl injection only. From the start of the diet, intraperitoneal booster immunizations were administered every three weeks. An overall overview of the experimental set-up is depicted in Supplementary Fig. ^S4.

Liver tissue was isolated and snap-frozen in liquid nitrogen and stored at −80 °C or fixed in 4% formaldehyde/PBS. The biochemical determination of plasma cholesterol and liver triglyceride levels, electron microscopy, RNA isolation, complementary DNA synthesis, quantitative polymerase chain reaction and auto-antibody titers against anti-oxLDL IgM antibodies are described extensively^4,5,43–46. Liver cholesterol levels were quantified as described previously⁴⁷.