Over the last several decades, cholesterol has become a household word, familiar to most people because the levels and type of cholesterol in their bodies are indicators of cardiovascular health. Although cholesterol is an indispensable lipid in vertebrate physiology, its imbalance has long been associated with heart disease, and cholesterol crystals are a major constituent of atherosclerotic lesions. However, whether cholesterol induces the lesions or is an inert and harmless bystander has been disputed. In 2010, a research team led by Dr. Eicke Latz of the Department of Infectious Diseases and Immunology, University of Massachusetts Medical School, showed that, at least in mice, cholesterol crystals are one of the crucial inflammatory stimuli in atherogenesis (Duewell et al. 2010). Their results suggest that therapies designed to either degrade these crystals or increase the concentration of cholesterol-sequestering high density lipoprotein (HDL) may help alleviate cardiovascular disease.
Because cholesterol crystals are soluble in the organic solvents used in histology, only large crystals had been identifiable in tissue samples prepared from atherosclerotic lesions. In fact, they were identifiable only from so-called cholesterol "crystal clefts," which outline the space the crystals occupy before they dissolve. Because the clefts are generally seen only in advanced lesions, many researchers thought they form late in atherosclerosis. However, because atherosclerosis is so intimately linked to cholesterol levels, the research team suspected the crystals form much earlier.
To investigate, they fed atherosclerosis-prone apolipoprotein E (Apoe)-deficient mice (B6.129P2-Apoetm1Unc/J, 002052) a high-cholesterol diet. Two weeks later, they examined very early atherosclerotic lesions in the animals' aortic sinuses with a combination of laser reflection and fluorescence confocal microscopy. Not only did they find many small crystals, but immune cells, such as macrophages, were being recruited to the sites of crystal deposition. As long as the mice were fed the high-cholesterol diet, crystal deposition and immune-cell recruitment increased steadily. Cholesterol crystal clefts were not visible histologically for another six weeks. The authors also found that, in human atherosclerotic lesions, small crystals are abundant in areas rich in immune cells. These results clearly indicate that cholesterol crystals form and probably induce the recruitment of immune cells very early in atherogenesis.
By performing a series of human and mouse cell culture experiments, the authors found that cholesterol crystals activate the NLR family, pyrin domain containing 3 (NLRP3) inflammasome. This inflammasome is a multi-protein complex consisting of caspase 1 (CASP1, an enzyme that cleaves other proteins, such as precursors of the IL1 family cytokines interleukin 1β (IL1β) and IL18, into their biologically active forms), PYCARD (also known as ASC, an adaptor protein that helps assemble large signaling complexes in the inflammatory and apoptotic signaling pathways and recruits CASP1 into the inflammasome), and NLRP3, an intracellular receptor with multiple protein interaction domains, that comprises the bulk of the inflammasome scaffold and recruits ASC (Cassela et al. 2009). The team found that cholesterol crystals activate the NLRP3 inflammasome (indicated by the CASP1-dependent release of IL1β and/or IL18) in phagocytes in a process that involves phagolysosomal damage: the crystals induce lysosomes to rupture and spill their proteolytic contents into the cytosol. Some of the cholesterol crystals may be formed from cholesteryl esters supplied by oxidized low density lipoprotein (LDL). In contrast, the inflammasome is either not or only mildly triggered in crystal-treated macrophages from Nlrp3 -, Pycard-, cathespin B (Ctsb)-, and cathespin L (Ctsl)-deficient mice.
The authors confirmed their in vitro results by conducting a series of in vivo experiments using genetically altered mice. They found that whereas cholesterol crystals injected into the peritoneum induce a robust recruitment of neutrophils in wild-type C57BL/6J (000664) or B6129F2/J (101045) controls, they induce a lower recruitment in Nlrp3-, Ctsb-, Ctsl-, interleukin 1 (Il1a/b)-, and interleukin 1 receptor, type 1 (Il1r1)-deficient (B6.129S7-Il1r1tm1Imx/J, 003245) mice, demonstrating that cholesterol-induced inflammation is either very low or absent when NLRP3 inflammasome components or lysosomal proteases are missing or the IL1 signaling pathway is disrupted.
To test whether the NLRP3 inflammasome is involved in the chronic inflammation that underlies atherogenesis, the authors tested whether the absence of NLRP3, ASC, or IL1 cytokines modulates atherogenesis in LDL receptor (Ldlr)-deficient JAX® Mice strain B6.129S7-Ldlrtm1Her/J (002207), a model of hypercholesterolemia. They reconstituted lethally irradiated Ldlr-deficient mice with bone marrow from wild-type, Nlrp3-, Pycard-, or II1a/b-deficient mice and fed them a high-cholesterol diet for eight weeks. At the end of the eight weeks, the blood cholesterol levels among these groups were comparable, and all except those reconstituted with wild-type bone marrow had significantly reduced levels of plasma IL18, an atherosclerosis biomarker whose secretion depends on inflammasomes. Additionally, mice reconstituted with Nlrp3- or II1a/b-deficient bone marrow were markedly less prone to atherosclerosis. These data indicate that activation of the NLRP3 inflammasome by bone marrow-derived cells is a major contributor to diet-induced atherosclerosis in mice.
The findings by the Latz research team strongly suggest that cholesterol crystals are not inert. Rather, they appear to be key stimuli for atherosclerosis-associated inflammation. Indeed, the inflammatory responses they apparently induce are similar to those induced by crystalline uric acid in gout or silica and asbestos in occupational diseases such as silicosis and asbestosis respectively. The immune system seems incapable of degrading these substances. Likewise, cholesterol is not degraded by immune cells. It is removed from peripheral tissues and packaged into HDL, which carries it to the liver for disposal. Thus, clearing cholesterol crystals may depend on the availability of HDL, the low concentration of which significantly increases the risk of atherosclerosis. No wonder pharmacological methods for increasing HDL's concentration are being actively pursued. The findings reported by this research team suggest that novel therapies capable of reducing cholesterol crystals or blocking the NLRP3 inflammasome pathway might also stop or slow down atherogenesis.
Cassela SL, Jolyb S, Sutterwalab FS. 2009. The NLRP3 inflammasome: a sensor of immune danger signals. Seminars in Immunology 21:194-8.
Duewell P, Kono H, Rayner KJ, Sirois CM, Vladimer G, Bauernfeind FG, Abela GS, Franchi L, Nu–ez G, Schnurr M, Espevik T, Lien E, Fitzgerald KA, Rock KL, Moore KJ, Wright SD, Hornung V, Latz E. 2010. NLRP3 inflammasomes are required for atherogenesis and activated by cholesterol crystals. Nature 464:1357-61.