Cholesterol accumulation and inflammation in skin and skin-draining lymph nodes in ApoE-KO mice lacking murine ApoA-I

Background. Skin is one the largest organs in the body and serves as a major barrier to infection, water loss, and abrasion. Maintenance of the integrity of the skin requires the synthesis of large amounts of lipids. For this reason, skin is a very active site of cholesterol synthesis and contains a substantial fraction of apoA-I. Lymph nodes are positioned at strategic sites surveying the diverse tissues of the body. Cells, antigens, and lipids drained from the tissues reach the lymph nodes with the afferent lymph. Previous experimental findings demonstrate that diet-induced hypercholesterolemia, in atherosclerosis-prone mice lacking apoA-I, leads to a massive accumulation of cholesterol in skin and skin-draining lymph nodes. Aim. To determine how different apoA-I levels could affect skin and skin-draining lymph nodes morphology and composition of athero-prone mice in normocholesterolemic conditions. Methods. C57Bl/6 (WT), apoE-KO (EKO), apoA-I-deficient/apoE-KO (dKO) or dKO overexpressing human apoA-I (hA-I) mice were fed a chow diet for 30 weeks. At sacrifice, plasma cholesterol concentration and distribution among lipoproteins were evaluated. Skin biopsies were harvested and processed for light microscopy and transmission electron microscopy. Axillary/inguinal skin-draining lymph nodes, mesenteric gut-draining lymph nodes and spleen were histologically characterized. Results. Plasma total cholesterol concentration in dKO mice was comparable with that of WT mice and 3-fold lower than the concentration observed in EKO and hA-I mice. Cholesterol in WT mice was almost exclusively confined to the HDL fraction whereas in EKO mice an elevated cholesterol accumulation in VLDL/LDL and low HDL levels were observed. In dKO mice, HDL particles were absent and cholesterol accumulation in the VLDL/LDL fractions was lower than that found in EKO mice. hA-I mice were characterized by a less prominent presence of VLDL/LDL, but a larger HDL cholesterol peak than that found in EKO mice. Skin morphology of EKO and hA-I mice was comparable with that of WT mice. Conversely, dKO mouse skin was characterized by increased dermal thickness, large accumulation of cholesterol and massive accumulation of foam cells and lymphocytes. Additionally, electron microscopy highlighted the presence of cholesterol crystals both in the extracellular milieu and within foamy macrophages only in dKO mouse skin. Compared to all the other experimental groups, dKO mice had enlarged axillary and inguinal skin-draining lymph nodes, characterized by the accumulation of foamy macrophages, increased cholesterol and lipid deposition, together with the presence of cholesterol crystals surrounded by granulomatous reactions, and dilated sinuses. To evaluate whether the impairment observed in axillary and inguinal lymph nodes was due to a systemic inflammation, the gut-draining mesenteric lymph nodes and spleen were evaluated. In this case no differences in both size and histology were observed among the mouse lines. Conclusions. Our study demonstrates that HDL depletion in apoEKO mice fed a low-fat/low-cholesterol diet leads to cholesterol levels comparable with those of WT mice. This finding notwithstanding, an aberrant accumulation of cholesterol with a concomitant infiltration of foamy macrophages and lymphocytes in the skin and in axillary and inguinal lymph nodes was observed. We can conclude that apoA-I deficiency itself, in the absence of hyperlipidemia, causes cholesterol overload and associated inflammation in skin and skin-draining lymph nodes.