WO2008131192A2 - Crystalline cholestrol and prevention of atherosclerosis - Google Patents

Abstract

The present invention discloses that innate immune responses to crystalline cholesterol plays a critical a role in directing the chronic inflammatory state associated with the development of atherosclerotic lesions. Hence, identifying the sensor system(s) that both detects and directs the inflammatory response to crystalline cholesterol will provide an opportunity to develop novel therapeutic agents for the treatment of atherosclerosis.

Description

INTERNATIONAL PATENT APPLICATION UNDER THE PATENT COOPERATION TREATY

To all whom it may concern :

Be it known that Christian W. SCHINDLER, Li SONG and Benvenuto PERNIS has invented certain new and useful improvements in CRYSTALLINE CHOLESTEROL AND PREVENTION OF ATHEROSCLEROSIS of which the following is a full, clear and exact description. CRYSTALLINE CHOLESTEROL AND PREVENTION OF ATHEROSCLEROSIS

[0001] This application claims the benefit of priority of U.S. Serial No. 60/912,541, filed April 18, 2007. The entire content and disclosure of the preceding applications is incorporated by reference into this application.

[0002] Throughout this application, various references or publications are cited. Disclosures of these references or publications in their entireties are hereby incorporated by reference into this application in order to more fully describe the state of the art to which this invention pertains.

FIELD OF THE INVENTION

[0003] The present invention relates to methods of inhibiting activation of innate immune responses induced by crystalline cholesterol, thereby preventing the development of atherosclerosis.

BACKGROUND OF THE INVENTION

[0004] Abbreviations used herein: CBD, Carbohydrate Binding Domains; LRR, Leucine Rich Repeat; NBD, Nucleotide Binding Domain; PAMP, Pathogen Associated Molecular Patterns; PRR, Pattern Recognition Receptors; TIR, TLR-IL-I Receptor; TLR, Toll Like Receptor.

[0005] The accumulation of lipid-laden macrophages in the subintimal space of blood vessels is a pathognomonic feature of atherosclerosis [10, 11, 29-33]. The cause for their accumulation, however, remains an active area of investigation. Intriguingly, many of the structural features (e.g., abundance of macrophages and the relative paucity of other leukocytes), as well as biochemical features (e.g., local secretion of inflammatory mediators) of atheromata are reminiscent of the chronic innate immune responses found in a subset of chronic human diseases, like osteoarthritis and silicosis [12, 18, 21]. The role cholesterol crystals plays in directly stimulating the chronic innate immune response that is characteristic of macrophages found in atherosclerotic lesions remains to be explored. Brief Overview of Innate Immunity

[0006] Microbes are widely believed to have been the driving force in the development of Pattern Recognition Receptors (PRRs) that specifically recognize and respond to conserved Pathogen Associated Molecular Patterns (PAMPs; reviewed in [2-7, 9, 34]). Intriguingly however, a number of the PRR sensing systems respond to host-derived factors. The role PRRs from the Toll Like Receptor (TLR), C-type lectin receptor, and Nucleotide Binding Domain (NBD) families play in stimulating innate immunity has become a growing area of investigation and will be briefly reviewed.

[0007] Transmembrane spanning TLRs feature an extra-cellular Leucine Rich Repeat (LRR) associated with PAMP sensing and an intracellular TLR-IL-I Receptor (TIR) domain important for signal transduction. The TIR domain transduces signals through MyD88 (except for TLR3, which employs TRIF), culminating in the activation of pro-inflammatory signaling pathways and downstream transcription factors (e.g., TKK----.->NFκB, TBKl =>TRFs, MAP kinases^ AP- 1/ATF ). Activated transcription factors (e.g., NFKB subunits ρ50, ρ65 and IicBζ, IRF-3, IRF -7, IRF -5 and AP- I, see [35-38]) bind to enhancer elements and direct the expression of inflammatory mediators, including cytokines (e.g., TNF, IL-I β, IL-6 &. type 1 IFNs [IFN-Is]X chcniokines (usually with delayed kinetics; e.g., CCl.2, CCl.4, CCL5, CXCLS, CXCL lO), as well as other inflammatory mediators. Reflecting a need to accumulate ligand in the appropriate compartment, the immune response stimulated by the subset of TLR receptors that sample endosomes (i.e., TLR3, TLR7, TLR8, TLR9) is more delayed [38, 39]. Card9, an NBD family member (see below), has recently been implicated in directing critical signals for these endosomal TLRs [40].

[0008] Transmembrane spanning, C-type lectin receptors feature one or more extra-cellular carbohydrate binding domains (CBD), and are often classified as scavenger receptors. As this name suggests, scavenger receptors are relatively promiscuous, usually directing the uptake of polyanionic molecules (reviewed in [9, 41]). Whether these receptors directly activate intracellular signaling pathways remains more controversial [9, 42-46]. C-type lectin receptors also play an important role in carbohydrate mediated cell-cell interactions; and in contrast to other important scavenger receptor families (e.g., Class A and Class B; [9]), a subset of these receptors (e.g., DAP- 12, Dectin-1) appear to transduce signals through immuno-receptor tyrosine activating motifs (ITAMs). The CBD of Dectin- 1 has recently been shown to exhibit specificity for zymosan/β-glucan, a characteristic carbohydrate component of fungal cell walls. Of note, a member of the NBD family, Card9, plays a critical role in directing Dectin-1 dependent activation of pro-inflammatory signaling pathways. This culminates, albeit with delayed kinetics, in the activation of a set of inflammatory mediators significantly overlapping with the ones activated by TLRs [34, 40]

As with the TLRs, cytosolic PRRs from the NBD family feature a carboxyl terminal LRR domain, as well as different amino terminal effector domains (e.g., Card, Pyrin and BIR; these proteins are also referred to Cards, Nalps, Naips & Nods; reviewed in [4, 47, 48]). These effector domains either direct a kinase dependent (e.g., Rip2) activation of a set of signaling pathways, analogous to those activated by TLRs, or activation of the "inflammasome". The inflammasome is a multi-protein complex (e.g., Nalpl, Nalp3, ASC, Cardinal, Ipaf, Caspasel & Caspase5) that directs that Caspasel dependent cleavage of pro-11 -I p, pro-lL-1 8 and pro-11 -33 into mature, secreted IL- lβ, IL- 18 and IL-33 [2, 16. 49-51 ]. Likely reflecting physical limitations on the interaction between ligand and receptor, Rip2 dependent activation of NFKB tends to proceed with slower kinetics than for surface expressed TLRs. This also corresponds with a subsequent delay in the expression of inflammatory mediators (e.g., TNF and IL-6). In contrast, NDB dependent activation of the inflammasome can occur rapidly, yielding secreted TT -I β (or TL-18 & IL-33), but this only occurs if sufficient substrate (i.e., pro-IL-l β, as well as pro-IL-18 & pro-IL- 33) is available for processing [1(_\ 50]. (Of note expression of pro-IL-lβ message appears to bε TNFKB dependent; [2, 16, 50, 52] } Caspasel activation has also been associated with enhanced cell death [2, 52-54]

It seems reasonable to speculate that the "pathogen" sensor domains found in PRRs (inc. scavengei receptors), which consist of a repetitive molecular structures, evolved to detect the conserved repetitive molecular patterns, PAMPs (e.g., LPS, Peptidoglycan, dsRNA, ssRNA, CpG-DNA motifs, flagcllin, etc , [2-9, 55]). Consistent with this, the LRR sensing domain is made up of 19-25 tandem copies of a 24-29 amino acid leucine rich repeat (LRR) motif (XLXXLXLXX or X XX XtFXXLX., where X any ammo acid; hydrophobic amino acid).

Crystals And Conserved Molecular Patterns

[0011] The best characterized bioaetive crystal is uric acid, which readily forms under physiological conditions and is the pathogenic agent υf gout [17 j. Recent studies have revealed thai the potent inflammatory response stimulated by this crystal is dependent on Nalp3, a member of the NDB family of innate cytosolic PRRs. In response to urate crystals, Nalp3 directs the inflammasomc dependent secretion of IL- I β, which in turn directs the expression of additional downstream inflammatory mediators (e.g., 7^"NF, IL-ό, fL-8). These are responsible for the recruitment and activation of neutrophils, a characteristic feature of acute inflammation associated with gυut [ 14, 50]. Similarly, the acute inflammatory response stimulated by calcium pyrophosphate dihydrate crystals, the etiologic agent of pseudo-gout, is dependent on Nalp3 [16]. Intriguingly, urate crystals have also been identified as the potent immune stimulating "adjuvant (i.e., danger signal)" released by dying cells [151. These observations suggest that urate crystals, and the intracellular receptors that recognize them, have evolved to play a critical role in regulating the innate response to dying cells [15]. However, prolonged activation can lead to a chronic and destructive inflammatory response.

[0012] The repetitive molecular pattern presented by urate crystals is also a common feature of other important biologically active crystals, raising the possibility that they may also be recognized by PRRs. For example, careful analysis of silica (SiO;), which directs the chronic inflammatory response associated with silicosis, has identified several distinct crystalline states. F.ach features a distinct arrangement of a basic tetrahedron, with a central Si atom [28]. Three crystalline forms, tridymite, cristobalite and quartz, exhibit potent immunostimulatory activity (and can cause devastating pulmonary inflammation), whereas stishovite and coesite crystals, as well as amorphous silica have little activity [12, 20, 21, 28, 56]. Intriguingly, these immune activating silica crystals present a distinct repeating pattern of the silanol (i.e., Si-OH) groups to the environment, which appears to be recognized by a component of the innate immune system [ iz, ^o j.

[0013] Hydroxy apatite crystals are also recognized for their ability to stimulate a chronic inflammatory state associated with disease J l S]. Moreover, the crystal structure of hydroxyapatite can be superimposed on that of cholesterol, accounting for the ability of cholesterol micro-crystals, found in numerous cellular and membrane compartments, to nucleate the formation of hydroxyapatite crystals [26J.

Cellular Biology of Cholesterol Metabolism

[0014] Apolipoproteins direct the formation of lipo-protein complexes, which transport significant concentrations of cholesterol, as well as other hydrophobic lipids, throughout the circulatory system [H]. These complexes are rich in phospholipids, which serve to prevent the spontaneous formation of cholesterol crystals. Elevated levels of cholesterol associated with the LDL apolipoprotein complex drives the formation of atherosclerotic plaques, which accumulate throughout life and cause significant human morbidity and mortality [10, 29, 32, 33]. Lipid- laden macrophages, referred to as foam cells (where the majority of the cholesterol is stored in the form of esterified cholesterol in lipid filled vesicle), represent the most prevalent and characteristic feature of atherosclerotic lesions. Moreover, the chronic inflammatory response associated with these foam cells has been ascribed a pivotal role in both lesion progression and plaque rupture, both with profound clinical consequences [33, 57, 58]. As macrophages do not express significant numbers of LDL receptors, oxidized-LDL has been credited with a central role in the formation of foam cells (reviewed in [10, 11, 29, 32, 33]). Although the process by which LDL is oxidized has not been fully elucidated, scavenger receptors (esp. SRAI/II & CD36) have been shown to play a pivotal role in transporting "modified" LDL (e.g., oxidized and/or acetylated) into the endosomal compartment of macrophages. Cholesterol, as well as free fatty acids are then recovered from the cholesterol rich LDL particles and begin to accumulate in the membranes of late endosomes/lysomes [22-24, 59]. These membranes are also rich in phospholipids, which serve to antagonize the spontaneous formation of cholesterol crystals. Normally, excess membrane cholesterol is removed through a process of esterification that is catalyzed by ER associated acyl-CoA cholesterol acyl transferase (ACAT). Esterified cholesterol subsequently accumulates in inert lipid droplets that are a characteristic feature of foam cells. However, over time this process becomes less efficient and membrane cholesterol levels begin to exceed the threshold for spontaneous crystallization, rapidly (i.e., within 12h) seeding the formation of plate-like cholesterol micro-crystals and eventually larger needle-like crystals [22-27].

[0015] Rising membrane cholesterol content has also been associated with activation of ER stress, reminiscent of the response to unfolded proteins [11, 46, 58]. Careful biochemical analysis of macrophages loaded with free cholesterol (i.e., through the addition of acetylated LDL and an ACAT blockade) has revealed a very delayed activation (i.e., peaking well after 6h) of pro-inflammatory signaling cascades (e.g., p38, Jnkl/2 & Erkl/2) and a corresponding very delayed expression TNF and IL-6 (i.e., peaking at 8-1Oh; [60]). Moreover, an associated increase in apoptosis (-10% of free cholesterol loaded macrophages) was found to be dependent on both p38 activation (via ER stress) and SRAI/II ligation (i.e., with Ac-LDL; [46]). Subsequent studies, have suggested that ligated SRAI/II transduces signals through an association with and activation of TLR4 [45]. However, the potential role fatty acids play in this process was not evaluated (fatty acids, an important component of LDL, have recently been shown to activate TLR4 [61]). [0016] TLRs have also been implicated in atherogenesis by several additional studies. Studies on TLR2 and TLR4 deficient mice have provided only modest evidence for a role in an innate response directed by lesional macrophages, but mice with MyD88[-/-] deficient macrophages exhibited a -50% reduction in atherosclerotic lesions size [62-66]. Surprisingly however, this reduction was largely associated with decreased expression of inflammatory chemokines, but not cytokines [63, 64]. Likewise, the variable changes in atherogenesis associated with mice deficient in their response to individual inflammatory cytokines (e.g., TNF, IL-I, IL-6 & IL-18) underscores a likely functional redundancy amongst these mediators [67-73]. In contrast, evidence of a critical role for chemokines (i.e., notably CCL2 and CX₃CLl) in the recruitment/accumulation of lesional macrophages remains compelling [74, 75].

Significance

[0017] Atherosclerosis is a remarkably pervasive disease, with over 85% of Americans in their

50s exhibiting pathological evidence of coronary involvement. Atherosclerotic lesions largely consist of cholesterol filled macrophages with inflammatory features. This includes elevated levels of cytokine (e.g., TNF, IL- lβ & IL-6) and chemokine (e.g., CCL2 and CX₃CLl) expression, as well as evidence of macrophage activation (e.g., increased ROS and MHC II expression). The efficacy of cholesterol lowering agents underscores the important role this lipid plays in disease pathogenesis. Yet, the mechanism by which cholesterol drives the development of an exuberant innate inflammatory response associated with these lesions has not been fully elucidated.

[0018] It is proposed that crystalline cholesterol, like other biologically active crystals, presents a surface to the environment that can effectively be recognized by components of the innate immune system, leading to chronic inflammatory state that defines atherosclerosis. Consistent with this model, cholesterol crystals are known to spontaneously form within macrophages during conditions of cholesterol accumulation. Moreover, this has been shown to correlate with their expression of inflammatory mediators (e.g., TNF, IL- lβ, IL-6, CCL2 & CX₃CLl). Identifying the mechanism by which these cholesterol crystals stimulate innate immunity in macrophages will not only provide important mechanistic insight into the pathogenesis of this disease, but will also provide an important opportunity to develop new therapeutic targets. Specifically, it is envisioned that it will be possible to develop therapeutics that block the ability of cellular sensor systems to respond to cholesterol crystals, thereby blocking inflammation and disease progression. SUMMARY OF THE INVENTION

[0019] The present invention provides a method of screening for a candidate compound capable of inhibiting macrophage-mediated inflammatory reaction during atherosclerosis, comprising the steps of contacting crystalline cholesterol with macrophages in the presence or absence of the candidate compound; and measuring secretion of one or more inflammatory mediators by the macrophages, wherein a reduced secretion of the inflammatory mediators in the presence of the candidate compound indicates the candidate compound is capable of inhibiting inflammatory reaction during atherosclerosis.

[0020] The present invention also provides a method of inhibiting macrophage-mediated inflammatory reaction during atherosclerosis, comprising the step of contacting macrophages with an agent that inhibits binding or uptake of crystalline cholesterol to the macrophages, thereby inhibiting macrophage-mediated inflammatory reaction during atherosclerosis.

[0021] The present invention also provides a method of inhibiting macrophage-mediated inflammatory reaction during atherosclerosis, comprising the step of contacting macrophages with an agent that inhibits receptor signaling upon binding of crystalline cholesterol to the macrophages, thereby inhibiting macrophage-mediated inflammatory reaction during atherosclerosis.

[0022] The present invention also provides a method of screening for a candidate compound capable of inhibiting crystallization of cholesterol, comprising the step of forming crystalline cholesterol in the presence or absence of the candidate compound, thereby identifying a candidate inhibitor. This method may further comprise the steps of (i) contacting crystalline cholesterol with macrophages in the presence or absence of the candidate inhibitor; and (ii) measuring secretion of one or more inflammatory mediators by the macrophages, wherein a reduced secretion of the inflammatory mediators in the presence of the candidate inhibitor indicates the candidate inhibitor is capable of inhibiting cholesterol induced inflammatory reaction.

[0023] The present invention also provides a method of predicting the risk of developing atherosclerosis in a subject, comprising the steps of: obtaining blood samples from the subject; examining levels of crystalline cholesterol in lipoprotein in the blood samples; and comparing the levels of crystalline cholesterol to that of a normal subject, wherein increased levels of crystalline cholesterol would indicate the subject has an increased risk of developing atherosclerosis.

BRIEF DESCRIPTION OF THE DRAWINGS

[0024] Figure 1 shows X-ray diffraction. Chemically matched samples of silica were analyzed by x-ray diffraction in the Laboratory of Mineralogy at the Institute of Industrial Medicine of the University of Milan.

[0025] Figure 2 shows dose response to crystalline silica and cholesterol. 1.5 x 10⁶ thioglycollate-elicited peritoneal macrophages, one day after harvest from C57B1/6J mice, were incubated with LPS (lμg/ml), tridymite (Trd), crystalline cholesterol for 2h as indicated. RNA was harvested, transcribed into cDNA, as previously reported [1] and evaluated by Q-PCR (Cyber Green; ABI), with specific primers for TNF, IL- lβ and β2-microglobulin. Relative cDNA expression (rel. exp.) was adjusted to the relative expression of the β2-microglobulin.

[0026] Figure 3 shows innate macrophage response to crystalline silica. 1.5 x 10⁶ thioglycollate-elicited peritoneal macrophages, one day after harvest from C57B1/6J mice, were incubated with LPS (1 μg/ml), tridymite (Trd), amorphous silica (Amr.) or quartz (Qtz), each at 0.4 mg/ml. Supernatants were collected and analyzed by ELISA (eBIOSCIENCES) for TNF, IL- lβ and IL-6, as indicated (Right panels). Cell pellets were collected for Q-PCR (Cyber Green; ABI), as outlined in Fig. 2. Again, the relative level of expression (rel. exp.) was adjusted with respect to a β2-microglobulin control and is presented both as a time course (Left panels) and in more detail as fold increase at 2 and 4 hour time points (Middle panels; note log scale).

[0027] Figure 4 shows innate macrophage response to crystalline cholesterol. Elicited peritoneal murine macrophages were prepared, stimulated and evaluated as outlined in the legend for Fig. 2. Crystallized cholesterol (Choi. Crystals) and amorphous cholesterol (Choi. Amorph) were added (1 mg/ml) as indicated.

[0028] Figure 5 shows cholesterol monohydrate crystal-induced inflammatory response is mediated by MyD88 transcription factor. Thioglycollate-elicited peritoneal macrophages from

C57B1/6J (B6) or MyD88-/- on a B6 background were incubated with 1 mg/ml of cholesterol monohydrate crystals from 0.5 to 24 hours in a RMPI- 1640 medium supplemented with 10% FCS and penicillin/streptomycin. Total RNA was harvested from the cell using Trizol (INVITROGEN) and 0.5 μg of total RNA were reverse transcribed and quantified by Q-PCR using 18S RNA as loading control (ABI BIOSYSTEMS) (top panel). The supernatants were collected at different time points and assayed for TNF production by an ELISA (eBIOSCIENCES) (middle panel). After 24 hours stimulation with cholesterol monohydrate crystals (CC, lmg/ml) or anhydrous cholesterol crystals (CA, lmg/ml), TNFα level was measured by ELISA in supernatants from peritoneal macrophages (bottom panel). Zymosan (100 μg/ml), which is a TLR2 stimulator, was used as a control.

[0029] Figure 6 shows among different forms of cholesterol crystals, only cholesterol monohydrate crystals stimulate macrophages. Thioglycollate-elicited peritoneal macrophages from a wild type C57B1/6J mouse were incubated with cholesterol (chol., SIGMA), cholesteryl acetate (chol. acetate), cholestanol, cholestanol monohydrate (cholestanol-H2O), cholestane, cholesterol monohydrate (chol.H2O), or anhydrous cholesterol (chol. anhydrate) at lmg/ml for 24 hours. TNFα and IL-I β levels were determined by ELISA. LPS (100 ng/ml) was used as a control.

[0030] Figure 7 shows cholesterol monohydrate crystal-induced TNF production can be inhibited by a chemical. Thioglycollate-elicited peritoneal macrophages from a wild type C57B1/6J mouse were treated with either N-(quinolin-8-yl)benzenesulfonamides (a potential ubiquitin E3 ligase inhibitor; 5 μM) or DMSO for 30 min and then incubated with cholesterol monodydrate crystal (CC, lmg/ml) or LPS (100 ng/ml) for 4 hours. TΝF and 18S RΝA was quantified by Q-PCR.

[0031] Figure 8 shows cholesterol monohydrate crystals- and silica crystals-induced macrophage cell death depends on inflammasome proteins. Mouse bone marrow-derived macrophages from wild type C57B1/6J (B6) mice, ASC-/- mice, CIAS (Νalp 3-/-) mice, or Monarch (Νalp 12-/-) mice were incubated with crystalline silica (Tridymite, 0.4mg/ml), noncrystalline amorphous silica (0.4mg/ml), or cholesterol monohydrate crystals (Crystalline chol.) for 8 hours. Apoptotic cell and necrotic cells were determined by staining the cells with Annexin V-FITC and propidium iodide (PI) (APO-AF kit, Sigma). Apoptotic cells were positive for Annexin V while necrotic cells were positive for both Annexin V and PI. Cells growing in a serum free RPMI- 1640 media were used as a control. The number of positive cells were counted and averaged from 3 random IOOX fields. Total number of cells per field ranged from 600 to 1000. DETAILED DESCRIPTION OF THE INVENTION

[0032] The central role cholesterol plays in atherosclerosis is well documented and has led to the development of important therapeutic agents. Within lesions, cholesterol chronically accumulates in macrophages, leading to the characteristic formation of foam cells. Understanding why macrophages abnormally accumulate in the intima and the role cholesterol plays in this process remains an important area of investigation.

[0033] Reflecting on the important role macrophages play in the innate response to microbial infections may provide insight into their pathological role in the development of atherosclerotic lesions. In healthy organisms macrophages migrate to and reside in virtually all tissues, serving both to eliminate apoptotic cells and as sentinels against potential microbial infections. The recent identification of pattern recognition receptors (PRRs) from the Toll like receptor (TLR) family, as well as other families (non-TLR), has provided important insight into how macrophages detect the presence of microbial pathogens [2-9]. Upon recognizing a molecular pattern associated with a given pathogen, these PRR sensors activate intracellular signaling pathways, which culminate in the activation of an innate inflammatory response. This includes up-regulation of receptors involved in endocytosis (e.g., scavenger receptors), antimicrobial enzymes, and the secretion of inflammatory mediators, which in turn direct the recruitment and activation of additional immune cells. In most cases, the offending agent is eliminated and homeostasis reestablished. However, in some cases the accumulation of macrophages becomes chronic, culminating in the modification and/or destruction of local tissues, as is the case during the development of atherosclerotic lesions, pulmonary fibrosis and infection with clever pathogens like Mycobacterium tuberculosis [10-13].

[0034] Intriguingly, monosodium urate crystals, which form spontaneously when cells die, have recently been identified as a potent adjuvant that acutely activates the innate immune system through Nalp3, a member of the non-TLR PRR/sensor family [14-16]. Crystalline silica and hydroxyapatite are also known to stimulate innate immune responses, but the mechanism(s) have not been elucidated. In contrast to the more acute inflammatory response to urate, these crystals stimulate development of a chronic, macrophage dependent, inflammatory response [12, 17-21]. Preliminary studies have determined that crystalline cholesterol, which is also known to form spontaneously within cholesterol-loaded macrophages [22-26], effectively stimulates the expression of inflammatory mediators. Intriguingly, a chemically matched sample of amorphous cholesterol is functionally inert. Notably, each of these crystals presents a distinct pattern of -OH groups to the environment [12, 27, 28], raising the possibility that they may also be recognized by pattern recognition sensors, which in turn direct the development of a chronic inflammatory state.

[0035] The present invention explores the hypothesis that the innate response to crystalline cholesterol plays a critical a role in directing the chronic inflammatory state associated with the development of atherosclerotic lesions. Identification of the sensor system(s) that both detects and directs the inflammatory response to crystalline cholesterol will not only provide important insight into the pathogenesis of atherosclerotic plaques, but also provide an opportunity to develop novel therapeutic agents.

[0036] The present invention provides a method of screening for a candidate compound capable of inhibiting macrophage-mediated inflammatory reaction during atherosclerosis, comprising the steps of contacting crystalline cholesterol with macrophages in the presence or absence of the candidate compound; and measuring secretion of one or more inflammatory mediators by the macrophages, wherein a reduced secretion of the inflammatory mediators in the presence of the candidate compound indicates the candidate compound is capable of inhibiting inflammatory reaction during atherosclerosis. In general, the macrophages can be human or murine macrophages. Examples of macrophages include, but are not limited to, peritoneal macrophages, bone marrow derived macrophages, donor derived peripheral blood macrophages, and macrophage cell lines (e.g., RAW and THP-I cells). In one embodiment, the crystalline cholesterol has a size of about 1-15 μM. In another embodiment, the crystalline cholesterol is cholesterol monohydrate. Examples of inflammatory mediators include, but are not limited to, TNF and IL- lβ. In another embodiment, the method comprises the steps of contacting crystalline cholesterol with macrophages in the presence or absence of the candidate compound; contacting the macrophages with an agent that stimulates Pattern Recognition Receptors that recognize conserved Pathogen Associated Molecular Patterns, and measuring secretion of one or more inflammatory mediators by the macrophages, wherein a reduced secretion of the inflammatory mediators in the presence of the candidate compound indicates the candidate compound is capable of inhibiting inflammatory reaction during atherosclerosis. Examples of Pattern Recognition Receptors are well known in the art.

[0037] The present invention also comprises a composition comprising a candidate compound identified by the screening method described above. In another embodiment, there is also provided uses of such candidate compound as a medicament for inhibiting macrophage- mediated inflammatory reaction during atherosclerosis.

[0038] The present invention also provides a method of inhibiting macrophage-mediated inflammatory reaction during atherosclerosis, comprising the step of contacting macrophages with an agent that inhibits binding or uptake of crystalline cholesterol to the macrophages, thereby inhibiting macrophage-mediated inflammatory reaction during atherosclerosis. In one embodiment, the agent inhibits phagocytosis by the macrophages. Examples of such inhibitors include, but are not limited to, nocodazole, cytocholasin D, latrunculin A, and colchicine.

[0039] Alternatively, the agent inhibits binding of crystalline cholesterol to scavenger receptors. Examples of scavenger receptors include, but are not limited to, Scavenger Receptor A-I/II, MARCO, CD36, Mannose Receptor, and Dectin-1. In another embodiment, the agent inhibits binding of crystalline cholesterol to Pattern Recognition Receptor (PRR) from Toll Like Receptor (TLR) family or non-TLR family. Examples of Pattern Recognition Receptors include, but are not limited to, members of the Nucleotide Binding Domain (NBD) family, Nalp3, ASC, and members of the CATERPILLER family (see Table 1; Ting J. et al, Ann Rev. Immuno. (2004) 23:387-414). In one embodiment, the agent inhibits binding of crystalline cholesterol by competitive binding to the receptors described above. For example, it has been reported that phosphorothioate (PS)-modified deoxyribose homopolymers acted as Toll Like Receptor 9 and Toll Like Receptor 7 antagonists. They displayed high affinity to both TLRs and did not activate on their own, but they competitively inhibited ligand-TLR interaction and activation [122].

[0040] The present invention also provides a method of inhibiting macrophage-mediated inflammatory reaction during atherosclerosis, comprising the step of contacting macrophages with an agent that inhibits receptor signaling upon binding of crystalline cholesterol to the macrophages, thereby inhibiting macrophage-mediated inflammatory reaction during atherosclerosis. In one embodiment, the agent inhibits protein synthesis. Examples of protein synthesis inhibitors include, but are not limited to, Puromycin and Cycloheximide. In another embodiment, the agent inhibits signaling through Pattern Recognition Receptor (PRR) from Toll Like Receptor (TLR) family. For example, targets of signaling molecules include IRAK- l/IRAK-4, NIK, Tak-1, NFKB, p38, Erkl/2, and Jnkl/2. These are targets that can be inhibited by standard and commercially available inhibitors. In yet another embodiment, the agent inhibits signaling through Pattern Recognition Receptor (PRR) from non-Toll Like Receptor (TLR) family. For example, targets of signaling molecules include Rip2, Card9, ASC, Ipaf, NaIp 1, and Nalp3. These are the targets that will be inhibited by RNAi approaches as described herein. In another embodiment, the agent is a nucleotide analog that targets Nucleotide Binding Domains. In yet another embodiment, the agent is an E3-ligase inhibitor.

[0041] In another embodiment, there is also provided an agent for use as a medicament for inhibiting macrophage-mediated inflammatory reaction during atherosclerosis, wherein the agent inhibits binding or uptake of crystalline cholesterol to the macrophages, or inhibits receptor signaling upon binding of crystalline cholesterol to the macrophages. Examples of agents or targets for inhibiting the binding of crystalline cholesterol to macrophages or the subsequent receptor signaling have been discussed above.

[0042] The present invention also provides a method of screening for a candidate compound capable of inhibiting crystallization of cholesterol, comprising the step of forming crystalline cholesterol in the presence or absence of the candidate compound, thereby identifying a candidate inhibitor. Methods of forming cholesterol crystals have been described herein. Examples of candidate compounds include, but are not limited to, lecithin, compound 48/80, and polyvinyl pyridine N-oxide (PVPNO).

[0043] The present invention also provides a method of predicting the chance of developing atherosclerosis in a subject, comprising the steps of: obtaining blood samples from the subject; examining levels of crystalline cholesterol in lipoprotein in the blood samples; and comparing the levels of crystalline cholesterol to that of a normal subject, wherein increased levels of crystalline cholesterol would indicate the subject has an increased chance of developing atherosclerosis.

[0044] The invention being generally described, will be more readily understood by reference to the following examples which are included merely for purposes of illustration of certain aspects and embodiments of the present invention, and are not intended to limit the invention.

EXAMPLE 1

Silica Crystals And Macrophage Activation

[0045] Studies highlighting an important role for IFN-γ in promoting atherosclerosis led to an effort to understand how a misdirected immune response contributed to atherogenesis [76]. Subsequent efforts revealed that neither lymphocytes [77] nor IL-6 [67] play a pivotal role in the development of murine atheromata, and shifted the focus towards macrophages and innate immunity. Recent studies showing potent innate immune response stimulated by crystalline, but not amorphous silica (see below), prompted the inventors to explore the potential role crystalline cholesterol my play in driving activation of innate immunity during atherogenesis.

[0046] Studies on the debilitating diseases associated with silica exposure have identified at least three crystalline forms, tridymite, cristobalitc and quartz, as important in disease pathogenesis [12, 20, 21, 28]. To explore the potential of crystalline silica in directly stimulating macrophages, a pure preparation of the most active crystal, tridymite, was made from quartz through a standard sintering protocol and then milled to an average size of 0.5 - 1 μm in the Laboratory of Mineralogy at the Institute of Industrial Medicine of the University of Milan [78]. An identical sample of melted quartz was rapidly cooled, but milled to the same size, and represents a chemically matched sample of amorphous silica. An x-ray diffraction analysis of these crystal preparations, as well as the quartz starting material (Sigma Aldrich), revealed the corresponding characteristic diffraction patterns (see Fig. 1). This analysis also confirmed a >99% purity for each sample. The inflammatory activity of these silica preparations was evaluated by layering them on thioglycollate elicited peritoneal macrophages one day after harvest. Quantitative PCR (Q-PCR) revealed a robust and dose dependent increase in the levels of TNF and IL- lβ transcripts, with peak activity at 0.8 mg/ml (Fig. 2). A dose of 0.4 mg/ml of silica was selected for subsequent studies.

[0047] Next, kinetic studies comparing the ability of tridymite and amorphous silica to induce TNF and IL- lβ expression were carried out. As anticipated, stimulation with LPS revealed a rapid and robust induction of TNF, IL- lβ and IL-6 expression. Tridymite also stimulated a robust, albeit relatively delayed increase in the levels of TNF, IL- lβ and IL-6 transcripts (but not IFN-I, as the case with LPS; [79]; data not shown). Transcription of TNF and IL- lβ peaked at about 2 hours, whereas only a modest level of IL-6 expression was evident at 4 hours (Fig. 3), peaking at about 8 hours (not shown). This is consistent with the need to induce IκBζ expression and a potential requirement for Card9 [37, 40]. In stark contrast, amorphous silica failed to stimulate the induction of TNF and IL- lβ transcripts (see Fig. 3). A corresponding robust accumulation of TNF and IL- lβ protein was also detected in the supernatants of tridymite stimulated macrophage cultures (see Fig. 3). Intriguingly, tridymite stimulated TNF protein production preceded that of IL- lβ, suggesting IL- lβ production may be secondary to TNF. Over time, tridymite crystals also induced significant macrophage death (>75 % in 12 hrs), as determined by diminished uptake of fluorescein acetate (not shown; [80-82]). Similar results were obtained with day 7 cultures of murine Bone Marrow-derived murine Macrophages (BMMs; not shown). These observations support the hypothesis that silica crystals present a repetitive molecular pattern that is effectively recognized by components of the innate immune system, culminating in the robust expression of inflammatory cytokines.

Cholesterol Crystals Activate Macrophages

[0048] Next, the possibility that cholesterol crystals stimulate an analogous innate immune response was explored. Cholesterol crystals (plate-like and predominately 15-20 μm in diameter; [83]; not shown) were prepared by slowly adding water to ethanol dissolved free cholesterol, as previously reported [22, 27, 83]. Redissolving these crystals in ethanol and rapidly evaporating the solvent served to generate a chemically matched mixture of amorphous and anhydrous cholesterol crystals which are functionally distinct from cholesterol monohydrate crystals. These control cholesterol preparations are referred to as either amorphous or anhydrous, but the latter term is more accurate. Stimulation of murine macrophages with crystalline cholesterol led to a robust and dose dependent induction of both TNF and IL- lβ transcripts (see Figs. 2 & 4). There was however, no corresponding increase in the level of IL-6 transcripts, even at 8 hours (not shown). Likewise, there was no induction of IFN-I expression (not shown). Analogous to what had been observed with silica, chemically identical amorphous/anhydrous cholesterol (i.e., prepared from the same starting material) was inactive, indicating that contaminating LPS cannot account for the response to crystalline cholesterol. Intriguingly, cholesterol induced TNF and IL- lβ transcripts were expressed with delayed kinetics with respect to tridymite (i.e., peaking at 4h vs. 2h; see Fig. 4) and LPS (i.e., with significant TNF induction by 0.5h; not shown), further excluding the possibility that LPS mediated this response. Again, this correlated with a robust accumulation of TNF and IL- lβ protein, albeit with a corresponding delay in kinetics (Fig. 4). Analogous to what had been observed with silica, TNF secretion preceded that of IL-I β, contrasting the response observed with urate crystals [16]. This not only indicates that activation of the inflammasome is delayed, but raises the possibility that pro-IL-lβ expression and secretion is TNF dependent. In contrast to silica, there was little cell death at 12 hours (not shown). These observations highlight significant differences in the innate response to crystalline cholesterol versus that of silica and uric acid. The nature of this response will be explored below. Summary

[0049] Specific forms of crystalline urate, silica, cholesterol and hydroxyapatite stimulate distinct innate immune responses in macrophages. Urate crystals (presumably small; [15]), known to cause gout, rapidly induce IL- lβ production, which in turn drives TNF and chemokine expression [17]. This is associated with robust neutrophil recruitment and an acute inflammatory response [14, 17]. In contrast, small silica crystals drive a more chronic inflammatory process [12, 20]. Consistent with this, these crystals induce a different pattern of cytokine expression (i.e., TNF preceding IL- lβ and IL-6; see Fig. 2). Although the potential role crystal size plays in the innate response to crystalline silica has not been rigorously evaluated, preliminary studies reveal that small hydroxyapatite crystals stimulated a similarly rapid, albeit less potent pattern of TNF and IL- lβ expression. Likewise, hydroxyapatite crystals are associated with a chronic inflammatory response [18]. In contrast, large cholesterol crystals stimulate the robust, but delayed innate response where TNF expression precedes that of IL-I β. Moreover, cholesterol crystals failed to stimulate IL-6 expression.

[0050] It is hypothesized that during atherosclerosis, the capacity of macrophages to store cholesterol in an inert form (i.e., esterified cholesterol) becomes overwhelmed ("cholesterol overload"). Free cholesterol then begins to accumulate within endosomes and their membranes. This quickly leads to saturating cholesterol concentrations, whereupon micro- and then macro- cholesterol crystals form [22-27]. Preliminary data suggest these crystals are recognized by components of the innate immune system, leading to activation of pro-inflammatory signaling cascades and the secretion of inflammatory mediators. In the setting of this chronic innate immune response, atherosclerotic plaques grow in size and complexity. Preliminary data on cytokine expression are consistent with published studies on "cholesterol loaded macrophages" (e.g., Ac-LDL + ACAT blockade), which reveal that: (1) cholesterol crystals form within 12 hrs of cholesterol loading [22-24, 59]; (2) this correlates with the activation of pro-inflammatory signals and the secretion of inflammatory mediators ([46, 60]; see also Fig. 4).

EXAMPLE 2 Size of Cholesterol Crystals And Immune Responses

[0051] Like other metabolites, the ability of free cholesterol to accumulate at concentrations that exceed the threshold for spontaneous crystallization presents the immune system with a unique challenge. Moreover, recent elegant biochemical studies have demonstrated that crystalline cholesterol rapidly forms within the endocytic compartments of cholesterol loaded macrophages. These cholesterol crystals are however at least initially considerably smaller than the crystals employed in preliminary studies, which revealed a robust, albeit delayed activation of an innate immune response. The large size of cholesterol crystals (i.e., too big to be rapidly phagocytosed) raises the question as to whether these crystals accurately model the immune response stimulated by the smaller crystals that form within foam cells. It will be important to address this issue to fully evaluate some of the studies proposed below.

[0052] It is hypothesized that the relatively delayed immune response (i.e., with respect to tridymite or LPS) stimulated by the ectopic addition of cholesterol crystals can be attributed to their large size. Specifically, the large cholesterol crystals must either be remodeled to enter the cell, or cholesterol specific PRRs must be redistributed to the cell surface to become activated. A corollary to this hypothesis is that smaller ectopic crystals, which can be phagocytosed, will more accurately model the innate response to crystals that form within foam cells. Specifically, they will more rapidly stimulate the expression of inflammatory mediators, analogous to the kinetics observed with tridymite and hydroxyapatite.

[0053] Studies outlined in this example will exploit three distinct but complementary approaches to test the hypothesis that the delayed response to crystalline cholesterol can be attributed to the large size of crystal preparations. The first approach will directly compare the innate response between large and small cholesterol crystals. The second approach will explore the potential role scavenger receptors play in the response to cholesterol crystals. These receptors, which are already widely recognized for a role in atherogenesis, should exhibit a strong preference for small crystals. The final approach will evaluate an alternate hypothesis, that the delayed response to cholesterol can be attributed to an initial need to synthesize components of this sensor/response pathway, as is the case in several other innate immune response pathways [16, 89]. This approach will also explore the potential effect membranes have on the innate response to cholesterol crystals (in vivo, cholesterol crystals are initially seeded in membranes; [24]). Crystalline silica will serve as an important control for each of these studies.

Does The Large Size of Cholesterol Crystals Delay Recognition?

[0054] Preliminary studies reveal that the innate response to crystalline cholesterol is delayed in comparison to tridymite, which is phagocytosed [81, 82], and LPS which is detected on the cell surface. The hypothesis that the large size of cholesterol crystals impedes uptake, thereby delaying the subsequent innate response (i.e., cytokine expression) will be tested. These studies will facilitate characterization of the cholesterol sensor/response pathway by determining whether these crystals are recognized at the cell surface (like LPS) or internally.

[0055] The initial experimental strategy will entail comparing the kinetics of the innate response to small (i.e., < 1 μm) vs. large cholesterol crystals, in a manner analogous to the studies outlined in Figure 4. Small cholesterol crystals will be generated by two methods. First, one can employ the approach that was exploited to generate small silica crystals (i.e., grinding larger crystals in a motorized, jeweled mortar and pestle at the Laboratory of Mineralogy at the Institute of Industrial Medicine of the University of Milan; [78, 80]). Second, crystalline cholesterol will be prepared under conditions that favor smaller crystals (i.e., more rapid addition of water). Smaller crystals will be selected by fractionation through a porous membrane. The size and homogeneity of these crystals will be evaluated both microscopically and by light scattering (part of a Dept of Biochemistry core facility).

[0056] Once small crystals have been prepared, their capacity to stimulate macrophage expression of TNF and IL- lβ transcripts will be compared to that of the larger standard cholesterol crystals exploited in preliminary studies (see Figs. 2 & 4). Similarly sized amorphous cholesterol and tridymite, as well as LPS, will serve as important controls. In addition, crystal uptake will be monitored by side scatter, microscopic methods or labeling techniques, as previously reported [22, 23, 25, 82, 83].

[0057] It is anticipated that smaller cholesterol crystals will be taken up robustly and therefore stimulate a more rapid induction of TNF and IL-I β mRNA (i.e. analogous to the pattern observed with tridymite and type I hydroxyapatite). This outcome will suggest that the delayed response to larger crystals is secondary to "slow" uptake. If this is the case, then subsequent studies will confirm a role for phagocytosis, with a panel of well characterized inhibitors (e.g., nocodazole, cytocholasin D, latrunculin A and finally colchicine, which is known to block the response to urate crystals [16, 90]). Additional studies will evaluate the potential role scavenger receptors play in this process. This outcome will also suggest that the response to large crystals is likely to be delayed either because the crystal must be remodeled to gain entry into the cell, or the cholesterol specific sensor/PRR must be redirected to the cell surface. These possibilities will be explored as outlined below or in future studies. [0058] Another possibility is that the current crystal preparations are "contaminated" with a significant number of small crystals (i.e., too small to be readily visualized), which are in turn responsible for the induction of TNF and IL- lβ expression. This possibility is not favored because: 1) macrophages can be observed rapidly attaching to large cholesterol crystals (not shown; see also [83]); and 2) "contamination" with small crystals should just reduce the amplitude of the response and not affect kinetics. None-the-less, the proposed experiments will test these less likely possibilities.

[0059] If on the other hand, large and small cholesterol crystals elicit the same delayed response, then subsequent studies will determine whether this delay (i.e., vs tridymite) reflects a specific need for new protein synthesis and/or use of a unique scavenger receptor. Both of these outcomes will provide valuable insight into the nature of the unique sensor system that recognizes and directs the response to cholesterol crystals.

Do Scavenger Receptors Account For Kinetic Differences In The Response To Cholesterol Crystals?

[0060] Studies on crystalline silica have determined that scavenger receptors play an essential role in crystal uptake, as is the case for many other "bioactive complexes" (e.g., microbes, apoptotic cells & oxidized/modified apoplipoprotein complexes; [9, 10, 81, 82]). At least two scavenger receptors, SRAI/III and CD36, have also been linked to atherogenesis through their capacity to promote macrophage dependent uptake of modified apolipoprotein complexes [10]. More recent studies have even raised the possibility that in addition to uptake, these receptors may transduce signals that promote atherosclerosis [8, 42-46]. It is therefore, important to determine whether scavenger receptors contribute to the innate response to cholesterol crystals, especially if studies outlined herein determine that small crystals are potent activators of this response. Specifically, the hypothesis that scavenger receptors play an important role in the innate response to cholesterol crystals will be tested. Specifically, these studies will explore whether three scavenger receptors, previously implicated in silica and cholesterol uptake (i.e., Scavenger Receptor A-MI [SRA-I/II], MARCO & CD36 [81, 82]), as well as the Mannose Receptor (MR), play an obligate role in the innate response to cholesterol crystals. Studies on a potential role of another "scavenger" receptor, Dectin-1, are also outlined herein. It is important to note that scavenger receptors are unlikely to contribute to the innate response to cholesterol crystals that spontaneously form within foam cells, which will be explored below. [0061] The experimental strategy will entail evaluating the ability of the most effective cholesterol crystals (presumably small crystals, but large crystals will be tested as well) to induce cytokine expression in available SRA-I/II, MARCO, CD36 and MR knockout macrophages, as detailed in Figures 3 & 4. Macrophages from corresponding wild type mice (i.e., 129 and C57B16/J), as well as tridymite, amorphous cholesterol and LPS will serve as important controls. Although the initial screen will not directly focus on genetic differences in scavenger receptor expression, subsequent follow up studies will rigorously evaluate these potential differences [82]. Side scatter, which effectively monitors tridymite uptake [82], will be explored as a potential assay to evaluate cholesterol crystal uptake. Alternatively, cholesterol crystal uptake will be monitored microscopically or with a radioactive or fluorescent tag [22, 23, 25, 83].

[0062] It is anticipated that SRA-I/II and CD36, which have previously been implicated in cholesterol uptake (albeit modified LDL; [10, 11, 33]), will be found to play an important role in the uptake of cholesterol crystals (i.e., as measured by cytokine expression). This is further expected to contrast the prevalent role MARCO plays in uptake and subsequent response to silica crystals [82]. These results will suggest that these scavenger receptors deliver these crystals to a compartment within the cell where they are then recognized by PRR/sensor systems from the innate immune system, which will be explored below.

[0063] If SRA-I/II and CD36 are found to be important in the response to cholesterol crystals, then subsequent studies will determine whether a need to up regulate scavenger receptor expression accounts for the delayed response (i.e., many scavenger receptors function as acute phase response genes; [9]). Results will be confirmed in available CD36-SRAI/III double knockout macrophages [82].

[0064] Alternatively, these studies may determined that the MR and potentially MARCO play an important role in both the response to cholesterol and silica; but this outcome will not provide insight into kinetic differences between these two crystals.

[0065] Another potential outcome is that none of these four scavenger receptors will be found to play an important role in the innate response to cholesterol crystals. This will indicate that either an alternative scavenger receptor(s) [9], or no scavenger receptor is involved in response to/uptake of cholesterol crystals. This outcome will suggest that the model system discussed herein more closely reflect what happens in foam cells, where crystal form spontaneously within macrophages. One additional scavenger-like receptor, Dectin-1 will be explored below.

[0066] There are two additional outcomes worth considering. First, scavenger receptors may take up and direct cholesterol (and/or silica) crystals to a "noninflammatory intracellular compartment", in other words receptor knockout macrophages will exhibit a more rapid and/or robust response to crystals, as has recently been suggested for silica and inhaled oxidants [81, 91]. This intriguing outcome will support a hypothesis that scavenger receptors play an important anti-inflammatory role, which becomes overwhelmed during chronic disease or with excessive ligand exposure (e.g., during atherosclerosis). Second, scavenger receptors may directly participate in the activation of innate response signals. However, this possibility appears less likely, as kinetic studies reveal a very delayed induction of signaling pathways and cytokine expression in macrophages stimulated with modified LDL or other scavenger receptor ligands (e.g. peaking after 8h; see Background; [46, 60]). This possibility will, however, be explored if one or more scavenger receptor knockout macrophages fail to respond to cholesterol crystals, and/or there is no evidence that PRR sensor systems direct the innate response to cholesterol crystals. Subsequent/follow-up studies will entail evaluating the response of mutant scavenger receptors (i.e., missing critical cytosolic domains) ectopically expressed in corresponding null macrophages, an approach routinely exploited to study IFN receptor dependent signaling (Zhao et al, 2007). Additional follow up studies will explore the possibility that scavenger receptors transduce signals through association with innate receptors, as recently suggested [45].

Does The Need For New Protein Synthesis Account For The Delayed Response To Cholesterol Crystals?

[0067] The rapid and direct induction of target gene expression is a characteristic feature of the innate response triggered by surface expressed TLRs (e.g., the initial response to LPS or PaIn₃CSK₄ does not require new protein synthesis; [38, 65]). This contrasts a modestly delayed response directed by endocytic TLRs (TLR3, TLR7/8 & TLR9) and members of the NBD family of cytosolic sensors, where a full response requires ligand uptake, processing and in several cases the expression of new genes [6, 7, 38, 39, 47]. This includes the response to urate crystals [14-16]. This approach will test the alternate hypothesis that the delayed response to cholesterol crystals can be attributed to the need to synthesize one or more critical components of the response pathway. Induced genes may include a receptor(s) (i.e., both for uptake or signaling), signaling molecules and/or components of a cytosolic sensor/response system. Note, a delayed secretion of IL- lβ can be attributed to a need to first express pro-IL-lβ, likely an NFKB dependent response [47].

[0068] The experimental strategy will entail evaluating the innate response (i.e., cytokine transcription; see Figs. 3 & 4) of macrophages to the most effective preparation of cholesterol crystals. Test macrophages will be pretreated with puromycin (1, 10 and 25 μM) Ih prior to stimulation with cholesterol crystals. The IFN-α dependent induction of IRF-I, ISG-15 and Mx- 1, which are effectively blocked by 5 μM puromycin (Song and Schindler; unpublished observation), will serve as important controls. Additional controls will include stimulation with amorphous cholesterol, LPS and tridymite.

[0069] It is anticipated that in contrast to LPS, puromycin will block the innate response to cholesterol crystals (i.e., TNF and IL- lβ transcription). The response to tridymite may also be puromycin sensitive if MARCO or pro-ILlβ expression must first be induced [2, 82].

[0070] If, as anticipated, the response to cholesterol is puromycin dependent, then it can be concluded that important components of the uptake and/or sensor pathway must be induced prior to an effective innate response to cholesterol crystals. This outcome will prompt an attempt to circumvent a potential block in uptake by coating small cholesterol crystals in lipids as outlined in the following paragraph. This important observation will also prompt analysis of RNA expression profile (i.e., an AFFYMETRIX gene array at the Columbia University Cancer Center Genomics Core) to identify candidate genes in the cholesterol-response pathway. This observation will also provide an important opportunity to carry out a future ectopic retroviral cDNA library expression-complementation screen (i.e., macrophages will be infected with a retroviral cDNA library recently developed with the Goff laboratory and then screened for their ability to rapidly induce TNF/IL-lβ expression in response to cholesterol crystals; [92]).

[0071] If on the other hand the innate response to cholesterol crystals is found to be independent of protein synthesis, then effort can be focused on two models: 1) the delayed response to cholesterol crystals can be attributed to a slow but obligate accumulation of cholesterol crystals within the cell (e.g., scavenger receptor dependent); 2). the need to redistribute components of the PRR/sensor/response pathway to the cell surface (i.e., where the crystals are) account for the delay in response. Evidence of a puromycin resistant response will also support the model that cholesterol crystals initially nucleate within endosomal compartments during "cholesterol overload" [22-24, 59], thereby triggering an innate immune response. Additional studies to support this model will entail determining whether the response to small cholesterol crystals can be enhanced (i.e., with respect to magnitude and/or kinetics) by exploiting liposomes (e.g., Lipofectamine & FuGene), which should facilitate a more rapid accumulation of these crystals in endosomal compartments, as is the case with TLR3 ligands [93-95].

EXAMPLE 3 Recognition of Cholesterol Crystals And Innate PRR/Sensor System

[0072] Kinetic and functional differences in the innate response to LPS, tridymite and cholesterol crystals raise the possibility that these crystals are sensed by distinct PRRs (see Figs. 3-4). Notably, PRRs from the TLR and NOD-LRR family activate distinctive sets of intracellular signaling pathways, including IKK^>NFκB, IRF3/7^>IFN-I, IRF5^>TNF, IκBζ^>IL-6, Card9^>MEK3/6^>p38 & Card9^>MEK2/3^>Jnkl/2 [7, 37, 38, 40, 96-99]. This approach will test the hypothesis that cholesterol crystals activate a pattern of pro-inflammatory signaling pathways that is distinct from those activated by tridymite and/or LPS. Studies in this approach will also expand on the "inflammatory signature" of cholesterol crystals by evaluating the expression of additional important inflammatory mediators (e.g., CCL2, CCL4, CCL5, CXCL8 & CX₃CLl; [37, 74, 96, 100]). (Note, although arguably descriptive, the identification of signaling pathways activated by PRRs is considered both essential and informative by those studying these sensor pathways [89, 94, 96, 100]. Moreover, these studies can be carried out quite quickly.)

[0073] These studies will initially evaluate the ability of cholesterol crystals to stimulate the activation of NFKB, p38, Erkl/2 and Jnkl/2. The expression of several chemokines (by Q-PCR; see above) will be included to further define the signature of this response. Activation of IRF3 will not be evaluated since neither crystal induces IFN-I expression (not shown; [6]). Activation of IRF5 will also not initially be evaluated in the absence of a reliable assay [36]. The experimental approach will entail stimulating murine macrophages with the most potent preparations of cholesterol crystals. Extracts will be prepared and evaluated for activation kinetics (t = 15 min, 30 min, Ih, 2h, 4h, 6h and 16h) of: NFKB (by EMSA or immunoblotting for IκBα & IκBζ; [23]); p38; Jnkl/2 and Erkl/2 (all by phospho-immunoblotting; [60, 99, 101]). Stimulation with amorphous cholesterol, tridymite, amorphous silica, LPS and MDP (Muramyl Dipeplidc [MDPj, a Nod2 ligand) will serve as important controls [5, 38, 98].

[0074] As is the case for all characterized TLR and NOD-LRR ligands, it is anticipated that cholesterol crystals will exhibit distinct patterns/profiles of NFKB, p38, Erkl/2 and Jnkl/2 activation, as well as differing in the set of chemokines it induces [99]. If this is true, then the most informative assays will be exploited in identification of the sensor components/PRR that directs the innate response to these crystals, as outlined herein. Additional follow up studies will exploit pharmacological inhibitors (e.g., Erkl/2-PD98059 or upstream MEKl /MEK2-U0126; p38-SB20219; JNK-SP600125) to confirm the potentially critical role these inflammatory pathways play in the innate response to cholesterol crystals, as previously reported [46, 60, 99, 101]. The activation of NFKB will be inhibited through the ectopic expression of a dominant interfering mutant recently tested in vivo successfully [102]. Again, the comparison to tridymite, LPS and MDP, as well as amorphous preparations of cholesterol and silica will be informative.

[0075] If no differences are observed in the activation of the initial set of inflammatory response pathways, then an additional effort will be made to evaluate the potentially important roles of other signaling pathways/components. This will include: IRF5 (i.e., through lentiviral mediated RNAi knockdown); PI3Ki-Akt activation (i.e., through phospho-immunoblotting for evidence of Akt activation and LY294002 directed PBKi blockade; [101]); and NFAT (i.e., through EMSA and cyclosporin blockade [103, 104]). It will be important to recognize that cholesterol crystals, as well as the other ligands, may activate more than one inflammatory response pathway (e.g., those stimulated by endocytosis and those stimulated by PRR activation; [45]).

Do TLRs Recognize And Initiate The Inflammatory Response To Cholesterol Crystals? [0076] Recent reports implicating TLR2, TLR4 and MyD88 in the development of atherosclerotic lesions in mice raises the possibility that TLRs contribute to the innate response to cholesterol [45, 61-65]. Moreover, gene targeting studies have underscored the essential role MyD88 plays in transducing signals for the TLR family of PRRs, several of which exclusively sample the endosomal compartment [7]. TRIF on the other hand only directs activation of the IRF3-IFN-I axis (i.e., for TLR3 and TLR4), which is induced by LPS, but not crystalline cholesterol or silica (data not shown). Therefore, the focus of the studies will be on MyD88 dependent TLR responses. Specifically, the hypothesis that cholesterol crystals fail to stimulate TNF and IL-I β expression in MyD88[-/-] macrophages can be tested.

[0077] The experimental strategy entails evaluating cytokine expression in available MyD88[- /-] macrophages after stimulation with the most effective preparation of cholesterol crystals (see Fig. 4). Macrophages from C57B1/6J mice will serve as wild type controls. Additional controls will include tridymite, amorphous preparations of cholesterol, as well as LPS (a TLR4 ligand) and PaIn₃CSK₄ (a TLR2 ligand; [38]).

[0078] It is anticipated that MyD88[-/-] macrophages will exhibit distinct defects in response to stimulation with LPS, PaIn₃CSK₄, tridymite and cholesterol crystals, as previously reported for LPS and PaIn₃CSK₄ [34, 36, 38, 65, 105]. If, as expected, cholesterol crystals are not able to stimulate TNF and IL- lβ expression in MyD88[-/-] macrophages, then whether specific TLRs function as the PRR for these crystals will be examined. Initial follow-up studies will exploit available knockout mice to evaluate the potential role two MyD88 dependent TLRs, TLR2 and TLR4, play in the innate response to crystalline cholesterol. These two TLRs have been selected, because they are well characterized in macrophages and functionally pleiotropic. Their functional pleiotropism entails their ability to respond to multiple distinct ligands, which has been attributed to their capacity to associate with additional receptor chains (which may require protein synthesis; [38, 105, 106]). Moreover, TLR2 ligands exacerbate atherosclerosis [65]. If these studies determine that neither TLR2 (which can dimerize with TLRl or TRL6) nor TLR4 contribute to the response to cholesterol crystals, but MyD88 is essential, then future studies will evaluate a potential role for TLR7, 8 & 9, currently ascribed specificity for nucleic acid ligands. The outcome of these studies will also need to be considered in context of the results from studies outlined above.

Do Non-TLR Innate Receptors Direct The Inflammatory Response To Cholesterol Crystals?

[0079] The relatively delayed kinetics of NFKB activation and cytokine expression stimulated by the NBD family of non-TLR sensors identifies them as appealing candidates for the PRR involved in the response to cholesterol crystals [3, 4, 6, 98, 100, 108-111]. Intriguingly, non- TLR sensors/PRR proteins include a growing number of functionally and structurally diverse members [3, 4, 111]. Even though many of these have not yet been knocked-out, recent advances in RNAi technology have greatly facilitated the ease with which candidate sensors/PRRs (or associated components) can be genetically evaluated. Specifically, RNAi can be used to test the hypothesis that non-TLR sensor systems direct the innate response to cholesterol crystals.

[0080] Non-TLR sensors, and associated signaling pathways that will initially be targeted and evaluated for their response to cholesterol crystals will include: Rip2 (which directs critical signals for Nodi and Nod2, as well as potentially additional PRRs; [112]); Card9 (which transduces signals for Dectin-1, as well as intracellular PRRs [34, 40]); ASC (a critical regulator for several bacterial PAMPs, may also be important in initiating necrosis, and is an important component in bacterial and ATP/P2X/K⁺ dependent activation of the inflammasome; [16, 50, 52, 113]); Ipaf (a critical regulator for several bacterial PAMPs and down stream activation of the inflammasome [50, 52, 114]); and NaIp 1 & Nalp3 (which are components of the inflammasome responsible for IL- lβ secretion; may also direct secondary TNF expression; [2, 16, 48, 50]). Of note, PRRs from the non-TLR family that sense viral RNA can be excluded since cholesterol crystals do not induce IFN-I expression (data not shown; [6, 7]).

[0081] The initial experimental strategy will entail evaluating crystalline cholesterol dependent cytokine expression in macrophages knocked down (by RNAi) for six candidate sensor components. Briefly, each candidate molecule will be targeted by three cDNA-specific shRNAi sequences cloned into pSuper. The single most effective shRNAi vector, as determined by immunoblotting and/or RNA expression analysis, will be selected for cholesterol studies. Knockdown macrophages, collected by FACS sorting, will be stimulated with the most effective preparation of crystalline cholesterol and evaluated for expression of TNF, IL- lβ and chemokines. Additional controls will include macrophages stimulated with: tridymite; amorphous cholesterol; LPS; and when appropriate urate crystals (for Nalp3 knockdown; [16]); β-glucan/zymosan (for Card9 knockdown; [34]); MDP (for Rip2 and Card9 knockdown; [4, 111]); flagellin (for Ipaf knockdown; [115]); and ATP (for ASC knockdown; [52]). Studies, in order of priority for targeting will be, Rip2, Card9, ASC, Ipaf, Nalp3 and NaIp 1. Positive results will initially be confirmed by RNAi resistant cDNA re-expression analysis. Subsequently, macrophages from the corresponding knockout mice, most of which are available [16, 34, 50, 52, 111 , 115], will be employed to confirm the phenotype.

[0082] It is anticipated that one or more of these knockdowns will impede the cholesterol dependent expression of TNF and IL- lβ, as well as chemokines. Moreover, loss of one or more NBD sensors involved in inflammasome activity (e.g., Ipaf, ASC, Nalp3 and Nalpl; [2-4]) is likely to impede crystalline cholesterol dependent IL- lβ secretion. This may however have no affect on TNF transcription (or IL- lβ transcription) and TNF secretion, suggesting this defect represents the loss of a generic innate response (i.e., one activated by several up stream pathways). This outcome will provide limited insight into which sensor system is responsible for the crystalline cholesterol dependent induction of TNF (or IL- lβ) transcripts.

[0083] Positive results (i.e., loss in more than just IL- lβ secretion) will direct attention to each corresponding upstream PRRs as follows. For example, if Rip2 is found to be important, then attention will be focused on whether Nodi and/or Nod2 play an important role in recognizing and responding to crystalline cholesterol. This will entail studying the response in Nodi and Nod2 knockout BMMs [108, 111]. MDP and GlcNAc-MurNAc will serve as important positive controls for these studies [4, 112, 116]. Additionally, comparative stimulations with PaIn₃CSK₄ (a TLR2 ligand) and PGN (a TLR2 & Nod2 ligand) will be important in exploring the potential antagonistic relationship between Nod2 and TLR2 in directing the biological response to crystalline cholesterol [98, 107]. If these subsequent studies support a role for Nodi or Nod2 in this innate response, then future studies will exploit knockout macrophages to carry out: Nod over expression studies; cholesterol dose response studies; and Nod domain mapping analysis to develop additional insight into how Nodi and/or Nod2 direct the innate response to crystalline cholesterol (studies will be modeled on those characterizing the Nod2 dependent response to MDP and GI bacteria; [4, 40, 98, 108, 111, 116]). In addition, Rip2/LDL-r and Nod/LDL-r double knockout mice will be generated in the future to evaluate their capacity to develop atherosclerosis.

[0084] If Card9 is identified as important to the cholesterol response, then attention will focus on Dectin 1 and the handful of intracellular PRR/sensors shown to signal through Card9 (e.g., Nod2, TLR3 & TLR7; [34, 40]). Specifically, knockout macrophages will be evaluated for their ability to respond to crystalline cholesterol. The order of priority for PRR/sensor evaluation will be Dectin 1 [117, 118], Nod2 (see above) and TLR7. It is anticipated that evidence for an important role for Card9 in the cholesterol response will correlate with data demonstrating a similarly important role for p38 and JNK in this response (i.e., Card9 is an important regulator of p38 and JNK; [40]). If these studies support a role for one of these receptors in this innate response to crystalline cholesterol, then future studies will exploit knockout macrophages to carry out domain mapping analysis in an effort to develop additional insight into how these receptors sense cholesterol. In addition, Card9/LDL-r and subsequently Dectin 1/LDL-r (or TLR7/LDL-r) double knockout mice will be generated to evaluate their capacity to develop atherosclerosis.

[0085] Very few if any NDB proteins appear to function upstream of Ipaf. Therefore, evidence implicating Ipaf will focus attention on Ipaf as the cholesterol sensor. Initial studies will entail evaluating the cholesterol dependent response in Ipaf knockout macrophages [52, 115]. It will be important to rigorously establish whether Ipaf directs the cholesterol dependent induction of TNF expression, in addition to its likely role in regulating the inflammasome (i.e., IL- lβ secretion). If studies support a role for Ipaf in the innate response to crystalline cholesterol, then future studies will exploit knockout macrophages to carry out domain mapping analysis in an effort to develop additional insight into how Ipaf senses cholesterol. In addition, Ipaf/LDL-r double knockout mice will be generated and evaluated for their capacity to develop atherosclerosis.

[0086] ASC appears to serve a more pleiotropic role in innate immunity, with multiple upstream inputs. If both knockdown and knockout studies implicate ASC in the cholesterol dependent induction of TNF expression, then future studies will determine whether ASC is directly involved in sensing cholesterol, or is downstream from another NDB family member (e.g., Monarch/Nalpll or PYPAFi, PYPAF4, etc.; [3J). A similar directed approach will be exploited to evaluate NaIp 1 and Nalp3, currently believed to integrate upstream signals into activation of the inflammasome [2].

[0087] If none of the candidate PRRs/PRR-components are involved, then future studies will entail establishing an ectopic cDNA expression screen to select for genes that are able to confer, on a normally unresponsive cell (e.g., CHO cells), the ability to rapidly induce the expression of TNF and IL- lβ transcripts (or specific reporters; [96, 119]) upon stimulation with cholesterol crystals. These studies will be guided by results from the gene array proposed herein and will require many controls. Although this approach is likely to identify unrelated regulators of TNF and IL- lβ expression, it was very successful in discovering several components of a newly elucidated cytosolic viral PRR/sensing system [6, 7, 89]. EXAMPLE 4 Cholesterol Monohydrate Induces MyD88-Dependent Expression of TNF

[0088] Figure 5 shows that cholesterol monohydrate crystals, but not anhydrous crystals, induced MyD88-dependent expression of TNF. This strongly suggests that TLRs are involved in both TNF and IL- lβ expression (note even though IL- lβ is secreted by the inflammasome, an NFkB dependent signal is required to induce production of the pro-ILlβ precursor). This either implies that TLR receptors, likely sampling the endosomal compartment (e.g., TLR3, TLR7, TLR8, TLR9 and likely TLRl 2), play a direct role in sampling cholesterol crystals, or that they sample another PAMP associated with cholesterol crystals (i.e., a contaminant) that synergize with cholesterol monohydrate, but not anhydrous crystals, to promote acute inflammation. It is speculated that contaminating LPS (or another PAMP) may be responsible for the MyD88- dependent component of this response. Of note, there is a long and storied history of the role infections play in promoting exacerbations in atherosclerosis. Thus, cholesterol crystals could provide one "hit" and a concomitant infection (e.g., stimulating TLRs) the second "hit". This is also the strategy the immune system exploits to induce a full inflammatory rather than a tolerant response stimulated by a single effector.

[0089] Figure 6 shows that cholesterol monohydrate crystals are much more effective than any other cholesterol crystal (e.g. cholesterol acetate; cholestenol; cholestanol acetate; cholestan; or anhydrous cholesterol) in inducing both TNF and IL- lβ secretion. These data argue for specificity and raise the possibility that cholesterol analogs might be able to impede the formation of cholesterol crystals, yet not impair vital activities, like membrane formation/regulation. These cholesterol analogs might include plant sterols.

[0090] Figure 7 shows that an E3-ligase inhibitor, which is one of the N-(quinolin-8- yl)benzenesulfonamides identified for its ability to block NFkB activity [121], blocks cholesterol monohydrate crystal-dependent induction of TNF expression. These data suggest a concrete pharmaceutical agent/target. Accordingly, agents that block inflammation (e.g., NFkB activity, Caspasel activity, etc) may be valuable in blocking the inflammatory response to cholesterol monohydrate and silica crystals.

[0091] Figure 8 shows cholesterol monohydrate crystals-induced macrophage cell death depends on inflammasome proteins. Macrophages were taken from wild-type, ASC-/-, CIAS (NaIp 3-/-), or Monarch (NaIp 12-/-) mice. Cholesterol monohydrate and silica crystals, as well as the "non-crystalline" control agents can induce macrophage cell death. Necrotic cell death is considered pro-inflammatory, whereas apoptotic death is anti-inflammatory. Cell death was measured by an in situ annexin/PI stain, after which cells were scored for apoptosis and necrosis (typically 20-50 cells out of a field of 600-800 macrophages on a cover slip, i.e. 5-19 %). As a control, cells were serum starved to induce apoptosis. Tridymite induced much less necrotic death in ASC, CIAS, or Monarch null macrophages. This is consistent with the notion that the inflammasome (which requires ASC, CIAS and potentially monarch) may promote necrotic death. The results for cholesterol crystals were more interesting. ASC and monarch null macrophages revealed an increase in apoptotic cell death, raising the possibility that if necrotic cell death is not available, the next best thing, apoptotic death, is chosen. The CIAS null macrophages revealed increased necrosis. These results are consistent with the idea that the NBDs are involved in the innate response to both crystalline silica and cholesterol.

[0092] As discussed above, there are two major classes of pattern recognition receptors implicated in the expression of inflammatory cytokines, the TLRs and NBDs. The studies presented in this disclosure focused on TLR-dependent pathways and a handful of NBDs, because both of these families play a prominent role in cytokine expression. The TLRs and several NBDs (e.g. Nodi, Nod2, Card9, Cardl l) appear to be important in TNF and IL-6 expression/secretion, whereas other NBDs (e.g., Ipaf, ASC, NaIp 1 and Nalp3) are important in IL- lβ secretion. Several recent studies have exploited Nalp3 knockout macrophages to underscore the critical role this NBD plays in the innate response to uric acid and pyrophosphate crystals. Recent data also indicated that Nalp3 may play a critical role in the innate response to silica crystals, at least for IL- lβ secretion.

[0093] The data presented herein indicate that both cholesterol monohydrate and silica crystals induce the secretion of IL- lβ as well as TNF. The IL- lβ response is pathognomonic of inflammasome (i.e., Caspasel plus either Nalp3 or Nalpl) activity. Thus, it is reasonable to speculate that components of the inflammasome are involved in the innate responses to cholesterol monohydrate and silica crystal. There is emerging data suggesting that inflammasome activity and the regulation of cholesterol metabolism may be intimately related.

This is not surprising since the inflammasome promotes the secretions of exosomes (small membrane surrounded vesicles), which necessitates having extra lipids available. It has been known for a number of years that the cholesterol sensor Scap plays a critical role in regulating the cleavage of SREPB, a family of transcription factors that direct the expression of genes that increase cellular cholesterol and lipid stores (e.g., by inducing the expression of the LDL-R and HMGCoA reductase - both targets of cholesterol lowering drugs). In low cholesterol states Scap allows membrane tethered SREBP to move to the Golgi apparatus, whereupon they come into contact with two Golgi-associated proteases (SlPand S2P). SREBP is freed from its membrane tether and can induce the expression of target genes. Recent studies from Gisou van der Goot revealed that Caspasel, from the inflammasome, can cleave SREBP, thereby promoting expression of genes important in cholesterol accumulation (see above). Thus, the inflammasome, which is associated with exosome secretion, also induces expression of genes that promote cholesterol accumulation. Thus, crystalline cholesterol could initiate a positive feedback cycle that promotes cholesterol uptake and inflammation. One could therefore speculate that a number of cholesterol lowering agents or anti-inflammatory agents might server or break this vicious cycle.

[0094] It is believed that full innate response to cholesterol monohydrate and silica crystals (as illustrated in all of the figures) requires a second stimulus. This is not surprising, since most studies on urate and pyrophosphate crystals, as well as toxin dependent IL- lβ secretion, entail a prior treatment with ATP or even in LPS. The preparations of cholesterol used herein are possibly contaminated with small amounts of LPS (and potentially other PAMPs as well). In theory, one could make preparations of cholesterol from cows' brain and eliminate this contaminant. It is believed that the contaminating LPS provides a second necessary signal, much like the ATP used by groups studying the inflammasome.

[0095] The data presented herein suggest that MyD88-dependent TLRs and several NBDs (e.g., Nalp3, ASC and possibly monarch) are involved in the innate inflammatory response to crystalline cholesterol monohydrate. Thus, drugs that block these pathways (e.g., inhibitors of IRAKs, RIPs, TRAFs Tak-1, possibly the IKK complex) would be potential candidates for blocking inflammation associated with atherosclerosis.

[0096] Regarding the NBDs, it is reasonable to speculate that Caspase 1 or 11 (in humans this is Caspase 5 and potentially Caspase 4; see [120]) are potential targets. Since NBDs require nucleotides to become activated (i.e., they catalyze dimerization through a Walker ATP binding motif), nucleotide analogs that specifically target the NBDs like Nalp3 or Monarch (ASC does not have this domain) might block this response. Similarly, small drugs that block the assembly of the multi-component inflammasome or promote the association of inhibitory subunit might work in blocking cholesterol monohydrate crystal-induced inflammation. [0097] Drugs that block cholesterol crystallization might include amphipathic amines like imipramine or phospholipids like lecithin, sphingomyelin or phosphotidyl choline. It is reasoned from the fact that structures that accumulate high concentrations of cholesterol, like membranes and Apolipoproteins, prevent crystallization by intercalating the cholesterol between phospholipids. Screening assays for agents that impede cholesterol crystallization may be set up to test compounds like phospholipids. For human application, these amines or phospholipids can be safely taken as food supplements.

[0098] Another way nature avoids cholesterol crystallization is by storing cholesterol esters in lipid droplets. Thus, if the function of cholesterol esterase is blocked, increasing amounts of cholesterol would be stored in this "inert" form impeding the formation of crystals. A potential drawback is that foam cells are macrophages loaded with cholesterol ester droplets. They accumulate in atherosclerotic lesions. Many people feel these cells contribute to the disease process. Overwhelming a macrophage with this stored cholesterol could lead to a backup of normal trafficking, actually promoting cholesterol accumulation in the membrane (because the cell is so full of these droplets there is no place to store more), and leading to cholesterol crystallization.

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Claims

What is claimed is:

1. A method of screening for a candidate compound capable of inhibiting macrophage- mediated inflammatory reaction during atherosclerosis, comprising the steps of a) contacting crystalline cholesterol with macrophages in the presence or absence of the candidate compound; and b) measuring secretion of one or more inflammatory mediators by the macrophages, wherein a reduced secretion of the inflammatory mediators in the presence of the candidate compound indicates the candidate compound is capable of inhibiting inflammatory reaction during atherosclerosis.

2. The method of claim 1, wherein the macrophages are selected from the group consisting of peritoneal macrophages, bone marrow-derived macrophages, peripheral blood-derived macrophages, and macrophage cell lines.

3. The method of claim 1, wherein the crystalline cholesterol has a size of about 1-15 μM.

4. The method of claim 1, wherein the crystalline cholesterol is cholesterol monohydrate crystals.

5. The method of claim 1, wherein the inflammatory mediator is TNF or IL- lβ.

6. The method of claim 1, wherein in step (a) the macrophages are further contacted with an agent that stimulates Pattern Recognition Receptors that recognize conserved Pathogen Associated Molecular Patterns.

7. A composition comprising the candidate compound identified by the method of any one of claims 1-6.

8. A candidate compound identified by the method of any one of claims 1-6 for use as a medicament for inhibiting macrophage-mediated inflammatory reaction during atherosclerosis.

9. A method of inhibiting macrophage-mediated inflammatory reaction during atherosclerosis, comprising the step of contacting macrophages with an agent that inhibits binding or uptake of crystalline cholesterol to the macrophages, thereby inhibiting macrophage-mediated inflammatory reaction during atherosclerosis.

10. The method of claim 9, wherein the agent inhibits phagocytosis by the macrophages, .

11. The method of claim 9 or 10, wherein the agent is selected from the group consisting of nocodazole, cytocholasin D, latrunculin A, and colchicine.

12. The method of claim 9, wherein the agent inhibits binding of crystalline cholesterol to scavenger receptors, to Pattern Recognition Receptors (PRR) of Toll Like Receptor

(TLR) family or to receptors of non-TLR family.

13. The method of claim 12, wherein the non-TLR family is Nucleotide Binding Domain (NBD) family.

14. The method of claim 12, wherein the receptors are selected from the group consisting of Scavenger Receptor A-I/II, MARCO, CD36, Mannose Receptor, and Dectin-1, Nalp3, ASC, and members of the CATERPILLER family.

15. The method of any one of claims 9-14, wherein the macrophages are further contacted with an agent that inhibits binding to or signaling from Pattern Recognition Receptors that recognize conserved Pathogen Associated Molecular Patterns.

16. A method of inhibiting macrophage-mediated inflammatory reaction during atherosclerosis, comprising the step of contacting macrophages with an agent that inhibits receptor signaling upon binding of crystalline cholesterol to the macrophages, thereby inhibiting macrophage-mediated inflammatory reaction during atherosclerosis.

17. The method of claim 16, wherein the agent inhibits protein synthesis.

18. The method of claim 17, wherein the agent is Puromycin or Cycloheximide.

19. The method of claim 16, wherein the agent inhibits signaling through Pattern Recognition Receptors (PRR) of Toll Like Receptor (TLR) family or receptors of non- TLR family.

20. The method of claim 16, wherein the agent inhibits signaling through a molecule selected from the group consisting of IRAK-I /IRAK-4, Nik, Tak-1, MyD88, NFKB, p38, Erkl/2, Jnkl/2, RIPs, TRAFs, IKK complex, Rip2, Card9, ASC, Ipaf, Nalpl, and Nalp3.

21. The method of claim 16, wherein the agent is a nucleotide analog that targets Nucleotide Binding Domains.

22. The method of claim 16, wherein the agent is an E3-ligase inhibitor.

23. The method of any one of claims 9-22, wherein the crystalline cholesterol is cholesterol monohydrate crystals.

24. An agent for use as a medicament for inhibiting macrophage-mediated inflammatory reaction during atherosclerosis, wherein the agent inhibits binding or uptake of crystalline cholesterol to the macrophages, or inhibits receptor signaling upon binding of crystalline cholesterol to the macrophages.

25. A method of screening for a candidate compound capable of inhibiting crystallization of cholesterol, comprising the step of forming crystalline cholesterol in the presence or absence of the candidate compound, thereby identifying a candidate inhibitor.

26. The method of claim 25, wherein the candidate compound is selected from the group consisting of lecithin, compound 48/80, and polyvinyl pyridine N-oxide (PVPNO).

27. A method of predicting the risk of developing atherosclerosis in a subject, comprising the steps of: a) obtaining blood samples from the subject; b) examining levels of crystalline cholesterol in lipoprotein in the blood samples; and c) comparing the levels of crystalline cholesterol to that of a normal subject, wherein increased levels of crystalline cholesterol would indicate the subject has an increased risk of developing atherosclerosis.

28. The method of claim 27, wherein the crystalline cholesterol is cholesterol monohydrate crystals.