Faculty Bibliography

Macrophage apoptosis in advanced atheromata, a key process in plaque necrosis, involves the combination of ER stress with other proapoptotic stimuli. We show here that oxidized phospholipids, oxidized LDL, saturated fatty acids (SFAs), and lipoprotein(a) trigger apoptosis in ER-stressed macrophages through a mechanism requiring both CD36 and Toll-like receptor 2 (TLR2). In vivo, macrophage apoptosis was induced in SFA-fed, ER-stressed wild-type but not Cd36(/) or Tlr2(/) mice. For atherosclerosis, we combined TLR2 deficiency with that of TLR4, which can also promote apoptosis in ER-stressed macrophages. Advanced lesions of fat-fed Ldlr(/) mice transplanted with Tlr4(/)Tlr2(/) bone marrow were markedly protected from macrophage apoptosis and plaque necrosis compared with WT -->Ldlr(/) lesions. These findings provide insight into how atherogenic lipoproteins trigger macrophage apoptosis in the setting of ER stress and how TLR activation might promote macrophage apoptosis and plaque necrosis in advanced atherosclerosis

Cholesterol metabolism is tightly regulated at the cellular level. Here we show that miR-33, an intronic microRNA (miRNA) located within the gene encoding sterol-regulatory element-binding factor-2 (SREBF-2), a transcriptional regulator of cholesterol synthesis, modulates the expression of genes involved in cellular cholesterol transport. In mouse and human cells, miR-33 inhibits the expression of the adenosine triphosphate-binding cassette (ABC) transporter, ABCA1, thereby attenuating cholesterol efflux to apolipoprotein A1. In mouse macrophages, miR-33 also targets ABCG1, reducing cholesterol efflux to nascent high-density lipoprotein (HDL). Lentiviral delivery of miR-33 to mice represses ABCA1 expression in the liver, reducing circulating HDL levels. Conversely, silencing of miR-33 in vivo increases hepatic expression of ABCA1 and plasma HDL levels. Thus, miR-33 appears to regulate both HDL biogenesis in the liver and cellular cholesterol efflux

Innate immunity is vital for protection from microbes and is mediated by humoral effectors, such as cytokines, and cellular immune defenses, including phagocytic cells (e.g., macrophages). After internalization by phagocytes, microbes are delivered into a phagosome, a complex intracellular organelle with a well-established and important role in microbial killing. However, the role of this organelle in cytokine responses and microbial sensing is less well defined. In this study, we assess the role of the phagosome in innate immune sensing and demonstrate the critical interdependence of phagocytosis and pattern recognition receptor signaling during response to the Gram-positive bacteria Staphylococcus aureus. We show that phagocytosis is essential to initiate an optimal MyD88-dependent response to Staphylococcus aureus. Prior to TLR-dependent cytokine production, bacteria must be engulfed and delivered into acidic phagosomes where acid-activated host enzymes digest the internalized bacteria to liberate otherwise cryptic bacterial-derived ligands that initiate responses from the vacuole. Importantly, in macrophages in which phagosome acidification is perturbed, the impaired response to S. aureus can be rescued by the addition of lysostaphin, a bacterial endopeptidase active at neutral pH that can substitute for the acid-activated host enzymes. Together, these observations delineate the interdependence of phagocytosis with pattern recognition receptor signaling and suggest that therapeutics to augment functions and signaling from the vacuole may be useful strategies to increase host responses to S. aureus

BACKGROUND & AIMS: Nonalcoholic steatohepatitis (NASH) is a disorder that consists of steatosis and hepatic inflammation. It is not known why only some people with steatosis develop NASH. Recently, we identified dietary cholesterol as a factor that directly leads to hepatic inflammation and hepatic foam cell formation. We propose a mechanism by which Kupffer cells (KCs) take up modified cholesterol-rich lipoproteins via scavenger receptors (SRs). KCs thereby accumulate cholesterol, become activated, and may then trigger an inflammatory reaction. Scavenging of modified lipoproteins mainly depends on CD36 and macrophage scavenger receptor 1. METHODS: To evaluate the involvement of SR-mediated uptake of modified lipoproteins by KCs in the development of diet-induced NASH, female low-density lipoprotein receptor-deficient (Ldlr(-/-)) mice were lethally irradiated and transplanted with bone marrow from Msr1(+/+)/Cd36(+/+)or Msr1(-/-)/Cd36(-/-) mice and fed a Western diet. RESULTS: Macrophage and neutrophil infiltration revealed that hepatic inflammation was substantially reduced by approximately 30% in Msr1(-/-)/Cd36(-/-)-transplanted mice compared with control mice. Consistent with this, the expression levels of well-known inflammatory mediators were reduced. Apoptotis and fibrosis were less pronounced in Msr1(-/-)/Cd36(-/-)-transplanted mice, in addition to the protective phenotype of natural antibodies against oxidized low-density lipoprotein in the plasma. Surprisingly, the effect on hepatic inflammation was independent of foam cell formation. CONCLUSIONS: Targeted inactivation of SR pathways reduces the hepatic inflammation and tissue destruction associated with NASH, independent of hepatic foam cell formation

The inflammatory nature of atherosclerosis is well established but the agent(s) that incite inflammation in the artery wall remain largely unknown. Germ-free animals are susceptible to atherosclerosis, suggesting that endogenous substances initiate the inflammation. Mature atherosclerotic lesions contain macroscopic deposits of cholesterol crystals in the necrotic core, but their appearance late in atherogenesis had been thought to disqualify them as primary inflammatory stimuli. However, using a new microscopic technique, we revealed that minute cholesterol crystals are present in early diet-induced atherosclerotic lesions and that their appearance in mice coincides with the first appearance of inflammatory cells. Other crystalline substances can induce inflammation by stimulating the caspase-1-activating NLRP3 (NALP3 or cryopyrin) inflammasome, which results in cleavage and secretion of interleukin (IL)-1 family cytokines. Here we show that cholesterol crystals activate the NLRP3 inflammasome in phagocytes in vitro in a process that involves phagolysosomal damage. Similarly, when injected intraperitoneally, cholesterol crystals induce acute inflammation, which is impaired in mice deficient in components of the NLRP3 inflammasome, cathepsin B, cathepsin L or IL-1 molecules. Moreover, when mice deficient in low-density lipoprotein receptor (LDLR) were bone-marrow transplanted with NLRP3-deficient, ASC (also known as PYCARD)-deficient or IL-1alpha/beta-deficient bone marrow and fed on a high-cholesterol diet, they had markedly decreased early atherosclerosis and inflammasome-dependent IL-18 levels. Minimally modified LDL can lead to cholesterol crystallization concomitant with NLRP3 inflammasome priming and activation in macrophages. Although there is the possibility that oxidized LDL activates the NLRP3 inflammasome in vivo, our results demonstrate that crystalline cholesterol acts as an endogenous danger signal and its deposition in arteries or elsewhere is an early cause rather than a late consequence of inflammation. These findings provide new insights into the pathogenesis of atherosclerosis and indicate new potential molecular targets for the therapy of this disease

In atherosclerosis and Alzheimer's disease, deposition of the altered self components oxidized low-density lipoprotein (LDL) and amyloid-beta triggers a protracted sterile inflammatory response. Although chronic stimulation of the innate immune system is believed to underlie the pathology of these diseases, the molecular mechanisms of activation remain unclear. Here we show that oxidized LDL and amyloid-beta trigger inflammatory signaling through a heterodimer of Toll-like receptors 4 and 6. Assembly of this newly identified heterodimer is regulated by signals from the scavenger receptor CD36, a common receptor for these disparate ligands. Our results identify CD36-TLR4-TLR6 activation as a common molecular mechanism by which atherogenic lipids and amyloid-beta stimulate sterile inflammation and suggest a new model of TLR heterodimerization triggered by coreceptor signaling events

The cardiovascular complications of obesity have prompted interest in dietary interventions to reduce weight, including low-carbohydrate diets that are generally high in protein and fat. However, little is known about the long-term effects of these diets on vascular health. We examined the cardiovascular effects of a low-carbohydrate, high-protein diet (LCHP) in the ApoE(-/-) mouse model of atherosclerosis and in a model of ischemia-induced neovascularization. Mice on a LCHP were compared with mice maintained on either the standard chow diet (SC) or the Western diet (WD) which contains comparable fat and cholesterol to the LCHP. LCHP-fed mice developed more aortic atherosclerosis and had an impaired ability to generate new vessels in response to tissue ischemia. These changes were not explained by alterations in serum cholesterol, inflammatory mediators or infiltrates, or oxidative stress. The LCHP diet substantially reduced the number of bone marrow and peripheral blood endothelial progenitor cells (EPCs), a marker of vascular regenerative capacity. EPCs from mice on a LCHP diet also manifest lower levels of activated (phosphorylated) Akt, a serine-threonine kinase important in EPC mobilization, proliferation, and survival. Taken together, these data demonstrate that in animal models LCHP diets have adverse vascular effects not reflected in serum markers and that nonlipid macronutrients can modulate vascular progenitor cells and pathophysiology

Lymphatic vessels play a key role in maintaining tissue-fluid homeostasis, immune surveillance and metastasis. The hyaluronan receptor, LYVE-1, is widely used as a molecular marker for adult and embryonic lymphatic endothelium, but its physiological functions have not yet been established in vivo. In agreement with a recent report, LYVE-1(-/-) mice, which are healthy and fertile, do not display any defects related to congenital abnormalities of the lymphatic system. One hypothesis for the absence of a phenotype in LYVE-1 null mice is that other hyaluronan receptors, such as CD44, may compensate for LYVE-1. To test this hypothesis, we created LYVE-1/CD44 double knockout mice with appropriate littermate controls. Lymphatic vessel structure and function, as determined by histological methods and intravital microscopy, show that LYVE-1(-/-), CD44(-/-) and LYVE-1(-/-)/CD44(-/-) mice are indistinguishable from wild-type mice under normal conditions. Furthermore, resolution of carrageenan-induced paw edema is comparable in all genotypes. However, LYVE-1(-/-)/CD44(-/-) mice exhibit increased edema formation in a carrageenan-induced paw inflammation model compared to wild-type mice, but not to LYVE(-/-) or CD44(-/-) mice. These data suggest that LYVE-1 and CD44 are not required for the formation or function of lymphatics, but do not rule out a role for LYVE-1 in inflammation

Receptors involved in innate immunity to fungal pathogens have not been fully elucidated. We show that the Caenorhabditis elegans receptors CED-1 and C03F11.3, and their mammalian orthologues, the scavenger receptors SCARF1 and CD36, mediate host defense against two prototypic fungal pathogens, Cryptococcus neoformans and Candida albicans. CED-1 and C03F11.1 mediated antimicrobial peptide production and were necessary for nematode survival after C. neoformans infection. SCARF1 and CD36 mediated cytokine production and were required for macrophage binding to C. neoformans, and control of the infection in mice. Binding of these pathogens to SCARF1 and CD36 was beta-glucan dependent. Thus, CED-1/SCARF1 and C03F11.3/CD36 are beta-glucan binding receptors and define an evolutionarily conserved pathway for the innate sensing of fungal pathogens

OBJECTIVE: The scavenger receptors SR-A and CD36 have been implicated in macrophage foam cell formation during atherogenesis and in the regulation of inflammatory signaling pathways, including those leading to lesional macrophage apoptosis and plaque necrosis. To test the impact of deleting these receptors, we generated Apoe(-/-) mice lacking both SR-A and CD36 and fed them a Western diet for 12 weeks. METHODS AND RESULTS: We analyzed atheroma in mice, assessing lesion size, foam cell formation, inflammatory gene expression, apoptosis, and necrotic core formation. Aortic root atherosclerosis in Apoe(-/-)Cd36(-/-)Msr1(-/-) mice, as assessed by morphometry, electron microscopy, and immunohistochemistry, showed no decrease in lesion area or in vivo foam cell formation when compared to Apoe(-/-) mice. However, Apoe(-/-)Cd36(-/-)Msr1(-/-) lesions showed reduced expression of inflammatory genes and morphological analysis revealed a approximately 30% decrease in macrophage apoptosis and a striking approximately 50% decrease in plaque necrosis in aortic root lesions of these mice. CONCLUSIONS: Although targeted deletion of SR-A and CD36 does not abrogate macrophage foam cell formation or substantially reduce atherosclerotic lesion area in Apoe(-/-) mice, loss of these pathways does reduce progression to more advanced necrotic lesions. These data suggest that targeted inhibition of these pathways in vivo may reduce lesional inflammation and promote plaque stability