WO2009014863A2

WO2009014863A2 - Asc and pyrin-asc pyroptosomes and uses thereof

Info

Publication number: WO2009014863A2
Application number: PCT/US2008/068747
Authority: WO
Inventors: Emad S. Alnemri
Original assignee: Thomas Jefferson University
Priority date: 2007-06-29
Filing date: 2008-06-30
Publication date: 2009-01-29
Also published as: WO2009014863A9; WO2009014863A3

Abstract

The present invention relates to the field of immune responses and to inflammation. Embodiments of the invention relate to methods of: (1) isolating and detecting inflammasomes associated with pyroptosis-ASC pyroptosomes; (2) diagnosing inflammation; (3) detecting pathogen; (4) screening for anti-inflammatory agents; and (5) treatment of auto-inflammatary diseases and disorder.

Description

ASC AND PYRIN-ASC PYROPTOSOMES AND USES THEREOF

CROSS REFERENCE TO RELATED APPLICATION

[0001] This application claims benefit under 35 U.S.C. § 119(e) of the U.S. provisional applications No. 60/937,722 filed June 29, 2007 and No. 60/951,326 filed July 23, 2007, the contents of which are herein incorporated by reference in their entirety.

GOVERNMENT SUPPORT

[0002] This invention was made with Government support under Grant No.: AG14357-

09 awarded by the National Institutes of Health. The Government has certain rights in the invention.

FIELD OF THE INVENTION

[0003] The present invention relates to the field of immune responses and to inflammation. In particular, embodiments of the invention relate to methods for isolating and detecting inflammasomes associated with pyroptosis and uses thereof.

BACKGROUND OF INVENTION

[0004] Inflammation is a general term for the local accumulation of fluid, plasma proteins, and white blood cells that is initiated when a group of cells or an organism is put under stress, by physical injury such as DNA damages, infection, or a local immune response. This is also known as an inflammatory response. The cells that invade tissues undergoing inflammatory responses are often called inflammatory cells or an inflammatory infiltrate and help cells or organisms to improve their conditions as a response to the stress. Inflammation can lead to death of cells in the organ or affected tissue.

[0005] The inflammatory response is highly regulated. The inflammatory response is elicited upon exposure to foreign materials such as pathogens and pathogen-derived compounds. The inflammatory response should not be elicited by host-derived materials. However, deregulation of inflammation can occur, provoking inflammatory diseases. Inflammation entails four well-known symptoms, including redness, heat, tenderness/pain, and swelling that characterize so many common diseases and conditions. Chronic inflammatory diseases, such as rheumatoid arthritis, inflammatory bowel disease, systemic lupus erythematosus, multiple

11058275.3 1 sclerosis, and type 1 diabetes, affect almost half a billion of people. Many of these diseases are debilitating and are becoming increasingly common in our aging society.

[0006] One of the key factors that initiate inflammation is the cytokine IL-I (Dinarello,

2005), and IL-I is activated by the activation of immune cells such as macrophages. Macrophages and monocytes express a battery of plasma membrane associated Toll-like receptors (TLRs) and cytosolic Nod/NACHT-LRR (nod-like receptor-NLR) proteins that recognize a vast array of pathogen-associated molecular patterns (PAMPs) and non-pathogen- associated danger signals (Delbridge and O'Riordan M, 2006; Martinon and Tschopp, 2005; Ting et al., 2006). In response to these stimuli, macrophages and monocytes produce pro-IL-lβ and pro-IL-18 and activate their caspase-1. The active caspase-1 processes the inactive pro-IL- lβ and pro-IL-18 to produce the active cytokines IL- lβ and IL- 18, respectively, which are then released into the extracellular space. Active IL- lβ and IL- 18 are potent mediators of inflammation that stimulate fever, recruitment and activation of immune cells, and production of secondary cytokines (Delaleu and Bickel, 2004; Dinarello, 1998).

[0007] The activation of cytokines IL-lβ, IL-18, and caspase-1 occur via a cytosolic structure called the inflammasome (Drenth and van der Meer, 2006; Mariathasan, 2007; Ogura et al., 2006; Petrilli et al., 2005). An inflammasome is a multiprotein complex of more than 700 kDa that is responsible for the activation of caspase-1 and caspase-5 (Pertrilli V., et. al., 2002). Individual components of an inflammasome were found to include caspase-1, caspase-5, the ASC adaptor protein, and NALPl (Martinon et al., 2002). In the past few years a number of inflammasome complexes have been identified including ICE-protease activating factor (Ipaf), NaIp 1, Nalp2, cryopyrin/Nalp3, and pyrin inflammasomes (Agostini et al., 2004; Delbridge and O'Riordan M, 2006; Martinon and Tschopp, 2005; Poyet et al., 2001; Srinivasula et al., 2002; Ting et al., 2006; Yu et al., 2006). Ipaf and NaIp 1, both members of the NLR family, associate directly with caspase-1 via CARD-CARD interactions and promote its activation by inducing dimerization of the inactive monomeric pro-caspase-1 (Faustin et al., 2007; Poyet et al., 2001). On the other hand the NLR proteins Nalp2 and cryopyrin/Nalp3, and the TRIM-family member pyrin do not associate directly with pro-caspase-1 and require the adaptor protein ASC to recruit and activate caspase-1 (Agostini et al., 2004; Yu et al., 2006).

[0008] Although little is known about the mechanisms of activation of the inflammasomes, recent studies revealed that different inflammasomes are activated by different signals. Infection of macrophages with intracellular bacterial pathogens such as Salmonella typhimurium or cytosolic exposure to purified flagellin protein from these pathogens, have been

11058275.3 2 shown to specifically activate the Ipaf inflammasome (Amer et al., 2006; Franchi et al., 2006; Mariathasan et al., 2004; Miao et al., 2006), whereas exposure to anthrax lethal toxin or MDP have been shown to activate the NaIp 1 inflammasome (Boyden and Dietrich, 2006; Faustin et al., 2007). Interestingly, the cryopyrin inflammasome is activated by a broad range of signals including infection with intracellular bacteria Listeria monocytogenes and Staphylococcus aureus, TLR agonists plus potassium-depleting agents such as ATP, Nigericin or maitotoxin, danger signal monosodium urate (MSU), antiviral compounds R837 and R847, bacterial RNA and viral double- stranded RNA (Kanneganti et al., 2006a; Kanneganti et al., 2006b; Mariathasan et al., 2006; Martinon et al., 2006; Sutterwala et al., 2006). The mechanism by which these diverse signals converge to activate the cryopyrin inflammasome is not clear, but it is likely that they all activate a common physiological response that triggers cryopyrin inflammasome activation.

[0009] Apoptotic speck protein containing a caspase recruitment domain (CARD), ASC, is a 22 kDa adapter protein with an N-terminal pyrin domain (PYD) and a C-terminal CARD (Masumoto et al., 1999). It links the PYD-containing nod-like receptor (NLR) family member to procaspase-1, using its PYD to interact with the PYD of the NLRs and its CARD to interact with the CARD of procaspase-1 (Martinon et al., 2002; Srinivasula et al., 2002). Gene-targeted deletion of ASC in mice revealed that ASC is essential for activation of caspase- 1 and generation of mature IL-lbeta by almost all stimuli known to induce IL-lbeta generation, indicating that ASC is a key downstream effector of caspase- 1 activation (Kanneganti et al., 2006b; Mariathasan et al., 2004; Mariathasan et al., 2005; Mariathasan et al., 2006; Martinon et al., 2006; Sutterwala et al., 2006; Yamamoto et al., 2004). How ASC induces caspase-1 activation is not clear, but it is currently believed that PYD-containing NLR family members such as cryopyrin assemble an inflammasome complex with ASC which in turn recruits and activates caspase-1 (Martinon et al., 2002; Yu et al., 2006). Recently, it has been demonstrated that activation of caspase-1 not only leads to inflammation, but in certain instances causes an inflammatory form of cell death called pyroptosis (Cookson and Brennan, 2001; Fink and Cookson, 2005; Fink and Cookson, 2006; Swanson and Molofsky, 2005). Pyroptosis is a caspase-1 -dependent inflammatory form of cell death. Pyroptosis was initially observed in macrophages infected with the intracellular bacteria Salmonella typhimurium (Monack et al., 2001), but was later found to occur in response to infection with a number of other intracellular bacterial and viral pathogens (Hilbi et al., 1998; Johnston et al., 2005; KeIk et al., 2003; Mariathasan et al., 2005; Sansonetti et al., 2000; Sun et al., 2005; Thumbikat et al., 2005). An inflammatory form of cell death not caused by pathogenic infection, but similar to pyroptosis,

11058275.3 3 was also described in human THP-I macrophage cell line and murine macrophages in response to different stimuli including bacterial toxins, lipoproteins and LPS plus ATP (Aliprantis et al., 2001a; Aliprantis et al., 1999; Aliprantis et al., 2000; Perregaux and Gabel, 1994; Warny and Kelly, 1999). Since all these stimuli have been shown to activate caspase-1 in macrophages, it is likely that this infection-independent inflammatory form of cell death or pyroptosis is also dependent on caspase-1 activation. Little is known about the mechanism by which caspase-1 can be specifically activated to induce pyroptosis.

[0010] Host cell death due to pyroptosis resulting from direct pathogen infection and/or pathogen-derived pro-inflammatory compounds such as bacterial toxins, lipoproteins and LPS plus ATP can lead to impaired normal organ functions and also lead to associated signs and symptoms of diseases. Perhaps one of the most significant examples is neurodegenerative diseases such as Alzhemier's disease. It is well known that neurodegenerative diseases have an apoptotic component (Yuan and Yankner, 2000). Apoptotic phenotypes have been observed in neurons in age-related disorders such as Alzheimer's disease (Anderson et al., 1996; LeBlanc, 1996; Troncoso et al., 1996) and Parkinson's disease (Hartmann et al., 2000), and in rodent models of acute injury such as ischemic stroke (Chen et al., 1998; Namura et al., 1998).

[0011] Moreover, it is also known that excessive or uncontrolled IL-I production is harmful to the host and is therefore tightly controlled (Dinarello, 2005). Several proteins such as COP, PI- 9, Pyrin and ICEBERG are thought to regulate the inflammasome activity, through interference with either the recruitment or the activity of caspase-1. When the control is impaired, diseases are often manifested. For example, mutations in the gene coding for NALP3, CIASl, have been associated with several auto-inflammatory disorders such as Muckle-Wells syndrome, familial cold urticaria and CINCA (Chronic Infantile Neurological Cutaneous and Articular auto-inflammatory disease) (Martinon and Tschopp, 2004). These disorders are characterized by recurrent episodes of fever and serosal inflammation, due to increased production of IL-I .

[0012] Accordingly, there is a need in the art to identify and/or modulate the various protein/protein interactions of the apoptotic pathway(s) during pyroptosis, particularly proteins which modulate the activity of caspase proteins and the production of cytokines. A better understanding of the molecular components and their function in the apoptotic pathway(s) will provide the insight needed to develop novel molecular targets for treatment or intervention in diseases and disorders associated with inflammation induced cell death.

11058275.3 4 SUMMARY OF THE INVENTION

[0013] In macrophages, the early inflammatory response that eventually leads to apoptosis involves the formation of large supramolecular complexes comprising ASC dimers and procaspase-1. This supramolecular complex is called the ASC pyroptosome. Only a single complex is formed per macrophage and each complex is approximately 1-3 microns in size. The formation of the ASC pyroptosome is closely associated with caspase-1 activation and the related downstream inflammation events. Moreover, caspase-1 activation is dependent on the formation of the ASC pyroptosome. Therefore, the formation and/or presence of a ASC pyroptosome can function as an indicator of inflammation and pyroptosis. The large size of the ASC pyroptosome allows easy isolation and identification of the complex.

[0014] Accordingly, embodiments of the invention provide methods of isolating and detecting ASC pyroptosomes in a pellet of a sample, the method comprising centrifuging a sample to form a pellet and detecting the presence of ASC protein in said pellet. The sample can be macrophages from blood, whole blood, bone marrow, peritoneal fluid, or any bodily fluid. The cells in the whole blood, bone marrow, peritoneal fluid, or any bodily fluid can be lysed. The centrifugal force of more than 2000 x G and up to 100,000 x G is used to pellet the ASC pyroptosome from the lysate from the sample. The presence of the ASC protein in the pellet can be detected, for example, by immunochemistry.

[0015] Embodied in the invention is a method of diagnosing inflammation in an individual, the method comprising detecting an ASC pyroptosome in a sample from an individual, wherein a detectable presence of an ASC pyroptosome indicates that the individual is suffering from inflammation. The inflammation can be caused by an inflammatory disease or a pathogen infection. The sample from the individual macrophages can be from blood, whole blood, bone marrow, peritoneal fluid, or any bodily fluid. Whole cells for the collected sample can be used and an ASC pyroptosome can be detected, for example, by immunocytochemisty.

[0016] On one embodiment, the presence of an ASC pyroptosome in an individual is detected by a method comprising centrifuging a sample from an individual to form a pellet and detecting the presence of the ASC protein in the pellet after centrifugation. The cells in the whole blood, bone marrow, peritoneal fluid, or any bodily fluid can be lysed and centrifuged. The centrifugal force of more than 2000 x G and up to 100,000 x G is used to pellet the ASC pyroptosome from the lysate of the sample. The presence of the ASC protein in the ASC pyroptosome pellet can be detected, for example, by immunochemistry.

11058275.3 5 [0017] Embodied in the invention is a method of detecting the presence of a microbial pathogen, the method comprises the steps of: (a) contacting a reporter cell with a test sample suspected of containing a microbial pathogen; (b) detecting ASC pyroptosome; (c) determining that the test sample contains a microbial pathogen, wherein an ASC pyroptosome is detected.

[0018] In another embodiment, the invention encompasses a method for screening and identification of a compound that inhibits inflammation, the method comprises the steps of: (a) contacting a compound to be screened with a reporter cell in the presence of a pro-inflammatory stimulus; (b) detecting ASC pyroptosome; and (c) selecting the compound wherein ASC pyroptosome formation in the presence of the compound is not detectable or the amount of ASC pyroptosome formed is reduced compared to a reference amount.

[0019] In one embodiment, provided herein is a method for screening for a compound that inhibits the interaction of pyrin with PSTPIPl and/or activation of pyrin by PSTPIPl, the method comprising the steps of: (a) contacting a compound to be screened with reporter cells expressing pyrin, PSTPIPl, and ASC protein; (b) detecting ASC pyroptosome; and (c) selecting the compound wherein the percentage of cells having a ASC pyroptosome in the presence of the compound is reduced compared to a reference amount.

[0020] The presence of an ASC pyroptosome in the reporter cell can be detected by a method comprising centrifuging a lysate of the cell to form a pellet and detecting the presence of the ASC protein in the pellet after centrifugation. Centrifugal force of more than 2000 x G and up to 100,000 x G is used to pellet the ASC pyroptosome from the lysates. The presence of an ASC protein in the ASC pyroptosome pellet can be detected, for example, by immunochemistry. The reference amount of ASC pyroptosome is the amound formed in the absence of the test compound.

[0021] In one embodiment, the reporter cell is a macrophage. In another embodiment, the reporter cell is a non-macrophage.

[0022] In one embodiment, the reporter cell stably expresses ASC tagged with green fluorescent protein. In other embodiments, the ASC protein is fluorescently labeled.

[0023] In one embodiment, the presence of an ASC pyroptosome in reporter cell is detected by fluorescence.

[0024] In one embodiment, provided herein is a method for screening and identification of a compound that inhibits inflammation, the method comprises the steps: (a) contacting a

11058275.3 6 compound to be screened with a cell lysate; (b) detecting an amount of ASC pyroptosomes; (c) comparing the amount of ASC pyroptosome formed in the mixture with a reference amount; and (d) selecting the compound wherein no detectable ASC pyroptosome is formed in the presence of the compound or there is a reduced amount of ASC pyroptosome formed in the presence of the compound compared to the reference amount.

[0025] In one embodiment, the cell lysate is from a macrophage that is stably expressing an ASC-GFP fusion protein. In another embodiment, the cell lysate is from a non-macrophage that is stably expressing an ASC-GFP fusion protein.

[0026] In one embodiment, the cell lysate is SlOO lysate. In another embodiment, the cell lysate is crude lysate.

[0027] In one embodiment, the presence of an ASC pyroptosome in the reporter is detected by fluorescence.

[0028] Also encompassed in the invention is a method of determining the effectiveness of an anti-inflammatory treatment comprising: (a) obtaining a sample at one time point; (b) obtaining a sample at a second time point, the second time point being after the administration of an anti-inflammatory treatment; (c) detecting and/or analyzing the ASC pyroptosome in the samples; and (d) comparing the amount of ASC pyroptosome in each sample, wherein a decrease in the amount of ASC pyroptosome in the second time point sample provides an indication that treatment is effective.

[0029] In one embodiment, the invention provides a method of treatment of inflammation in a subject, the method comprising administering an effective amount of a BBox- peptide and a pharmaceutically acceptor carrier. The BBox peptide can be fused to other proteins or conjugated to polymer to enhance serum half life in vivo. The BBox peptide can also be fused to other protein or short tags to enhance protein expression and facilitate purification. Functional fragments, concervative amino acid substitutions variants and peptide mimics of the BBox-peptide are also included.

[0030] Encompassed in the invention is a diagnostic kit for the rapid detection of inflammation by detecting the presence of an ASC pyroptosomes in a sample. The kit comprise the components suitable to carry out separation of an ASC pyroptosome in a lysate of cells, the detection of the ASC protein in the ASC pyroptosome, and instructions to perform the rapid detection procedure. The sample can be whole blood, bone marrow, peritoneal fluid, or any

11058275.3 7 bodily fluid known in the art. The cells in the whole blood, bone marrow, peritoneal fluid, or any bodily fluid can be lysed by methods known in the art. The supramolecular ASC pyroptosome in a lysate of the sample can be separated by membrane filtration wherein the membrane has a pore size of less than 1 microns.

BRIEF DESCRIPTION OF THE DRAWINGS

[0031] Fig. IA. Western blot analysis of lysates from parental THP-I or stable THP-I-

ASC-GFP cells with anti-ASC antibody. Notice that the expression level of the ASC-GFP fusion protein (upper band) in the THP-I-ASC-GFP cells is comparable to the expression level of endogenous ASC (lower band).

[0032] Fig. IB. THP-I-ASC-GFP cells were pretreated with zVAD-FMK for 30 min and then treated with increasing amounts of crude LPS for the indicated periods of time. The percentages of cells containing ASC pyroptosomes were calculated as described under the "METHODS".

[0033] Fig. 1C. THP-I-ASC-GFP cells were treated as in D with LPS (1.0 μg/ml), MSU

(100 μg/ml), R837 (5.0 μg/ml) Pam3CSK4 (1.0 μg/ml) or FSL-I (1.0 μg/ml) for the indicated periods of time. The percentages of cells containing ASC pyroptosomes were determined as in Fig. IB.

[0034] Fig. ID. THP-I-ASC-GFP cells were preincubated with cycloheximide (10 μg/ml) for 30 minutes and then treated with LPS (1.0 μg/ml) for an additional Ih. The percentages of cells containing ASC pyroptosomes were determined as in Fig. IB.

[0035] Fig. 2C. Pyroptosome-induced pyroptotosis causes the release of intracellular

LDH. LDH release into the culture medium is shown as a percentage of LDH release by detergent.

[0036] Fig. 2D. Pyroptosome-induced pyroptotosis causes the release of IL- lβ. IL- lβ release into the culture media was determined by ELISA.

[0037] Fig. 3A. LPS (1.0 μg/ml) treatment of THP-I cells for 3h in the presence of z V AD-FMK lead to caspase-1 recruitment in ASC pyroptosomes. (L) lysates and (P) pellets of cell lysates.

11058275.3 [0038] Fig. 3B. LDH release into the culture medium by Bone marrow macrophages from WT or caspase-l-/-mice were treated with LPS (1.0 μg/ml) for 3h followed by ATP (4 mM) for 1 hour.

[0039] Fig. 3C. ASC pyroptosomes were isolated from WT or caspase-l-/-bone marrow macrophages after treatment with LPS plus ATP.

[0040] Fig. 4A. ASC pyroptosomes purified from LPS-stimulated THP-I-ASC-GFP cells were incubated with Flag-tagged WT or active site mutant (C/A) procaspase-1 together with pro-IL-lβat 37⁰C for 20, 40, or 60 minutes as indicated.

[0041] Fig. 4B. ASC pyroptosomes purified from in vitro assembled and purified ASC pyroptosomes from THP-I lysate.

[0042] Fig. 4C. Coomassie-stained SDS-polyacrylamide gel of a large preparation of purified ASC pyroptosomes from LPS-stimulated THP-I-ASC-GFP cells. Lane 1, endogenous ASC pyroptosomes purified from THP-I lysate. Lane 2, ASC pyroptosomes purified from THP- 1 -ASC-GFP lysate.

[0043] Fig. 4D. Western blots of purified in vitro assembled ASC pyroptosomes from

THP-I cell lysates (1st lanes), or purified ASC pyroptosomes from LPS-stimulated THP-I- ASC-GFP cells (2nd lanes) isolated in the presence of zVAD-FMK to trap the activated caspase- 1 on the pyroptosomes. The 3rd lane in the cryopyrin blot is a positive cryopyrin-containing lysates control from a stable 293 cells expressing cryopyrin.

[0044] Fig. 5A. The wild-type pyrin domain of ASC mediates formation of the ASC pyroptosome and activate procaspase-1.

[0045] Fig. 5B. The the ASC pyroptosome formed with wild-type pyrin ASC can be cross-linked by the cross-linking agent DSS.

[0046] Fig. 5C. A schematic illustration of the chimeric ASC-APAF which contains the

CARD of Apaf-1 at its C-terminus instead of its original CARD, and the C9-procaspase-l which contains the CARD of procaspase-9 at its N-terminus instead of its original CARD.

[0047] Fig. 5D. Autoradiography showing the effects of bacterially produced chimeric

ASC-APAF pyroptosomes or Apaf-1-591 on 35S-labeled procaspases-9 at 37⁰C for Ih.

11058275.3 [0048] Fig. 5E. Procaspase activation by bacterially produced chimeric ASC-APAF pyroptosomes were incubated with Flag-tagged C9-procaspase-l chimera (left panels) or WT procaspase- 1 (right panels) together with pro-IL-lβ at 4° C or 37⁰C for Ih as indicated.

[0049] Fig. 6A. ASC pyroptosome formation in vivo in THP-I-ASC-GFP cells treated with crude LPS (1 μg/ml) in the absence or presence of the indicated concentrations (mM) of KCl, or the potassium channel blocker TEA.

[0050] Fig. 6B. LDH release into the culture medium by THP-I cells were treated with crude LPS (1 μg/ml) in the absence or presence of the indicated concentrations of KCl.

[0051] Fig. 6C. IL-lβ release into the culture media by THP-I cells were treated with crude LPS (1 μg/ml) in the absence or presence of the indicated concentrations of KCl. IL-lβ was determined by ELISA.

[0052] Fig. 6D. ASC-GFP pyroptosomes formation in vivo by THP-I-ASC-GFP cells were treated with SAT (10 μg/ml) in the absence or presence of the of KCl (60 mM).

[0053] Fig. 6E. ASC-GFP pyroptosomes formation in vivo by THP-I-ASC-GFP cells were treated with digitonin in the absence or presence of the of KCl (60 mM).

[0054] Fig. 7A. Effects of potassium concentration inhibits ASC pyroptosomes formation from THP-I SlOO extracts.

[0055] Fig. 7B. Physiological potassium concentration inhibits caspase-1 activation from lysates from THP-I cells (10 μg/μl) incubated at 4⁰C or activated at 37⁰C in the presence of the indicated concentrations of KCl together with the cross-linking agent DSS (4 μM). The lysates were analyzed by western blotting with anti-caspase-1.

[0056] Fig. 7C. Physiological potassium concentration inhibits self-association of ASC dimers from lysates from THP-I cells (10 μg/μl) incubated at 4⁰C or activated at 37⁰C in the presence of the indicated concentrations of KCl together without the cross-linking agent DSS (4 μM). The lysates were analyzed by western blotting with anti-ASC antibodies.

[0057] Fig. 7D. Effects of potassium concentration on the activation of procaspases-1 mutant (C287A) by ASC pyroptosomes.

[0058] Fig. 7E. Effects of potassium concentration on the activation of WT procaspases-

1 by ASC pyroptosomes.

11058275.3 10 [0059] Fig. 8A. Effects of potassium concentration on the in vitro assembly of the ASC pyroptosome from purified recombinant ASC (10 ng/μl).

[0060] Fig. 8B. Assembled recombinant ASC pyroptosomes in the presence of 75 mM

KCl are composed of dimers.

[0061] Fig. 8C. Assembled recombinant ASC pyroptosomes in the presence of 75 mM

KCl can activate procaspases-1.

[0062] Fig. 9 A. Enhanced IL- lβ secreted by THP-I macrophages expression PAPA- associated PSTPIPl mutants as deteced by ELISA.

[0063] Fig. 9B. Enhanced IL-lβ secreted by THP-I macrophages expression PAPA- associated PSTPIPl mutants as deteced by Western blot.

[0064] Fig. 9C. Western blot analyses showing the effects on pyrin and pro-IL-lβ expression in THP-I cells infected with a GFP-encoding MSCV retrovirus for 24 h.

[0065] Fig. 9D. Western blot analyses showing the effects on pyrin and procaspase-lin

THP-I cells stable expressing the PSTPIP WT , A230T , or E250Q when infected by the MSCV retrovirus.

[0066] Fig. 9E. The secreted IL-lβ in the culture media of THP-I cells that are non- infected and infected by MSCV retrovirus expressing the PSTPIP WT , A230T , or E250Q, as measured using a human IL-lβ ELISA kit.

[0067] Fig. 9F. Effects of pyrin- specific (Pyr) siRNAs on the secretion of IL-lβ by mutant PSTPIPl A230T-expressing THP-I cells. Cells were transfected with control nonspecific (Con) or pyrin- specific (Pyr) siRNAs and then left untreated (Un-infected) or infected with a GFP-encoding MSCV retrovirus.

[0068] Fig. 1OA. Western blots showing that pyrin is required for activation of caspase-1 by PSTPIPl. Caspase-1 processing (top panels) and IL-lβ cleavage (bottom panel) were shown. The expression of PSTPIPl proteins in the transfected cells was determined by western blotting with anti-PSTPIPl antibody (middle panel).

[0069] Fig. 1OB. Western blots showing that PSTPIPl mutants potentiate caspase-1 processing in 293-ClAP cells. Note that WT PSTPIPl and PSTPIPl mutants induce caspase-1 activation only in the 293-ClAP, but not in the 293-ClAC cells.

11058275.3 11 [0070] Fig. 1OC. Western blots showing two independent stable cell clones of 293-Cl AP with different levels of pyrin (low or high) that were transfected with an empty vector or an A230T mutant PSTPIPl expression construct as indicated. The higher level of pyrin (4th lane) showed more caspase-1 activation in response to ectopic expression of PSTPIP 1-A230T mutant than cells with the lower level of pyrin (2nd lane).

[0071] Fig. 1 IA. PSTPIPl induces pyroptosome formation.

[0072] Fig. HB. PSTPIPl induces more pyroptosome formation in the presence of pyrin.

[0073] Fig. 11C. Western blots showing the interaction of pyrin with ASC in the presence or absence of WT or mutant PSTPIPl proteins.

[0074] Fig. 12A. Western blot showing that pyrin is a homotrimer.

[0075] Fig. 12B. Schematic representations of the domain structure of the full-length pyrin (FL) and the truncated pyrin mutants used Fig. 12C. PYD, pyrin domain; BB, B-Box; CC, coiled-coil; SPRY, domain in SPIa and Ryanodine receptor.

[0076] Fig. 12C. Western blots showing the bacterially-expressed T7-tagged truncated pyrin mutants that can form trimers.

[0077] Fig. 12D. Schematic representations of the three C-terminal-truncated pyrin mutants (1-580, 1-410 and 1-343) used in Fig. 12E.

[0078] Fig. 12E. Western blot analyses show that deletion of the coiled-coil or the coiled coil plu B-box domains impair both the basal and PSTPIPl -induced activation of pyrin.

[0079] Fig. 12F. ASC pyroptosomes formation in the presence of C-terminal-truncated pyrin mutants (1-580, 1-410 and 1-343).

[0080] Fig. 13A Schematic representations of the domain structure of pyrin and Trim5α, and the chimeric pyrin-Trim5αmutants used in Fig. 13B, C and F below.

[0081] Fig. 13B. Western blot analyses show that coiled-coil-mediated trimerization of pyrin is critical for its activity.

[0082] Fig. 13C. ASC pyroptosomes formation in the presence of chimeric pyrin-

Trim5αmutants.

11058275.3 12 [0083] Fig. 13D. Western blot analyses show the interaction of PSTPIPl with pyrin and

C-terminal-truncated pyrin mutants (1-580, 1-410 and 1-343).

[0084] Fig. 13E. Western blot analyses show the interaction of intact pyrin with WT

PSTPIPl and mutants PSTPIPl.

[0085] Fig. 13F. Western blot analyses show the interaction of full-length pyrin or the indicated pyrin-Trim5αchimeras with WT PSTPIPl and mutant PSTPIPl.

[0086] Fig. 13G. Western blot analyses show that the PYD of pyrin interacts with its B- box.

[0087] Fig. 14A. Western blot analyses show that colchicine inhibits processing of

Caspase-1.

[0088] Fig. 14B. Western blot analyses show that both colchicine and nocodazol inhibits

Caspase-1 processing.

[0089] Fig. 15. Schematic diagram showing mechanism of activation of pyrin by

PSTPIPl

[0090] Fig. 16 A. Schematic representation of the domain structure of PSTPIPl. The two

PAPA-associated mutations in the coiled-coil (CC) domain are indicated. Western blot analyses show that PSTPIPl is a homotrimer.

[0091] Fig. 16B. Western blot showing that formationof trimers by bacterially-produced

T7-tagged PSTPIPl or the truncated pyrin mutant LN-BB-CC.

[0092] Fig. 16C. Western blots showing that PSTPIPl monomers self-associated with each other to form multimers.

[0093] Fig. 16D. Western blots showing the size exclusion elution fractions from a

Superdex 200 FPLC column loaded with bacterially produced T7 -tagged PSTPIPl (WT and A230T). PSTPIPl are homotrimers and multimers.

[0094] Fig. 16E. Western blots showing the size exclusion elution fractions from a

Superdex 200 FPLC column loaded with bacterially produced T7 -tagged pyrin-LN-BB-CC. Pyrin-LN-BB-CC are homotrimers and multimers.

11058275.3 13 [0095] Fig. 17. Domain structures of human pyrin, human cryopyrin (h-Cryopyrin) and zebrafish cryopyrin (zf-Cryopyrin). The top diagram shows the domain structure of human pyrin and the regions that have been shown to interact with ASC, the cytoskeleton and PSTPIPl.

[0096] Fig. 18A. Western blots showing that the deletion of the B-box activates pyrin.

[0097] Fig. 18B. ASC ASC pyroptosomes formation in the presence of pyrin or pyrin-

ΔB-box (pyrin-deltaBB) and with WT or A230T PSTPIPl

[0098] Fig. 19A. Western blot analyses of pull down assay showing the strong binding of PSTPIPl WT/A230T heterotrimer to pyrin.

[0099] Fig. 19B. Western blot analyses of pull down assay showing the increased binding of the WT subunit(s) in the presence of A230-GST subunit(s).

[00100] Fig. 20. Western blot analyses of pull down assay showing the activated caspase-

1 cleaves pyrin. No pyrin cleavage occurs in lysates that do not contain ASC or caspase-1 (not shown).

[00101] Fig. 21A. A high throughput scheme for the identification and isolation of pyroptosome-assembly inhibitory molecules.

[00102] Fig. 21B. An example of proof of principle showing inhibition of pyroptosome formation by KCl. This assay was done in the 96- well plate format as illustrated in the scheme.

DETAILED DESCRIPTION OF THE INVENTION

[00103] Unless otherwise stated, the present invention was performed using standard procedures that are well known to one skilled in the art, for example, in Maniatis et al., Molecular Cloning: A Laboratory Manual, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y., USA (1982); Sambrook et al., Molecular Cloning: A Laboratory Manual (2 ed.), Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y., USA (1989); Davis et al., Basic Methods in Molecular Biology, Elsevier Science Publishing, Inc., New York, USA (1986); Methods in Enzymology: Guide to Molecular Cloning Techniques Vol.152, S. L. Berger and A. R. Kimmerl Eds., Academic Press Inc., San Diego, USA (1987); Current Protocols in Molecular Biology (CPMB) (Fred M. Ausubel, et al. ed., John Wiley and Sons, Inc.), Current Protocols in Protein Science (CPPS) (John E. Coligan, et. al., ed., John Wiley and Sons, Inc.); Current Protocols in Immunology (CPI) (John E. Coligan, et. al., ed. John Wiley and Sons, Inc.);

11058275.3 14 Current Protocols in Cell Biology (CPCB) (Juan S. Bonifacino et. al. ed., John Wiley and Sons, Inc.); Culture of Animal Cells: A Manual of Basic Technique by R. Ian Freshney, Publisher: Wiley-Liss; 5th edition (2005); and Animal Cell Culture Methods (Methods in Cell Biology, VoI 57, Jennie P. Mather and David Barnes editors, Academic Press, 1st edition, 1998), which are all incorporated by reference herein in their entireties.

[00104] Unless otherwise defined herein, scientific and technical terms used in connection with the present application shall have the meanings that are commonly understood by those of ordinary skill in the art. Further, unless otherwise required by context, singular terms shall include pluralities and plural terms shall include the singular.

[00105] Definitions of common terms in molecular biology are found in Benjamin Lewin,

Genes IX, published by Jones & Bartlett Publishing, 2007 (ISBN- 13: 9780763740634); Kendrew et al. (eds.), The Encyclopedia of Molecular Biology, published by Blackwell Science Ltd., 1994 (ISBN 0-632-02182-9); and Robert A. Meyers (ed.), Molecular Biology and Biotechnology: a Comprehensive Desk Reference, published by VCH Publishers, Inc., 1995 (ISBN 1-56081-569-8).

[00106] It should be understood that this invention is not limited to the particular methodology, protocols, and reagents, etc., described herein and as such may vary. The terminology used herein is for the purpose of describing particular embodiments only, and is not intended to limit the scope of the present invention, which is defined solely by the claims.

[00107] Other than in the operating examples, or where otherwise indicated, all numbers expressing quantities of ingredients or reaction conditions used herein should be understood as modified in all instances by the term "about." The term "about" when used in connection with percentages may mean +1%.

[00108] All patents and other publications identified are expressly incorporated herein by reference for the purpose of describing and disclosing, for example, the methodologies described in such publications that might be used in connection with the present invention. These publications are provided solely for their disclosure prior to the filing date of the present application. Nothing in this regard should be construed as an admission that the inventors are not entitled to antedate such disclosure by virtue of prior invention or for any other reason. All statements as to the date or representation as to the contents of these documents is based on the information available to the applicants and does not constitute any admission as to the correctness of the dates or contents of these documents.

11058275.3 15 [00109] The singular terms "a," "an," and "the" include plural referents unless context clearly indicates otherwise. Similarly, the word "or" is intended to include "and" unless the context clearly indicates otherwise. It is further to be understood that all base sizes or amino acid sizes, and all molecular weight or molecular mass values, given for nucleic acids or polypeptides are approximate, and are provided for description. Although methods and materials similar or equivalent to those described herein can be used in the practice or testing of this disclosure, suitable methods and materials are described below. The term "comprises" means "includes." The abbreviation, "e.g." is derived from the Latin exempli gratia, and is used herein to indicate a non-limiting example. Thus, the abbreviation "e.g." is synonymous with the term "for example."

[00110] Definitions

[00111] As used herein, an ASC pyroptosome refers to the 1-2 micron, large supramolecular complex formed in macrophages upon challenge with pro-inflammatory stimuli. Only one ASC pyroptosome is formed in each macrophage when the macrophage is challenged by pro -inflammatory stimuli. This large supramolecular complex can be separated and isolated from other cellular components, e. g. and ASC dimers in macrophages by a low speed centrifugation of not more than about 5000 x G. A centrifugal force of no less than 2000 x G and up to 100, 000 x G will essentially pellet the ASC pyroptosome while leaving other ASC containing complexes such as ASC dimers in the supernatant.

[00112] As used herein, the term "antibody-based binding moiety" or "antibody" includes immunoglobulin molecules and immunologically active determinants of immunoglobulin molecules, e.g., molecules that contain an antigen binding site which specifically binds (immunoreacts with) to ASC protein or fragments thereof. The term "antibody-based binding moiety" is intended to include whole antibodies, e.g., of any isotype (IgG, IgA, IgM, IgE, etc), and includes fragments thereof which are also specifically reactive with ASC protein and fragments thereof. Antibodies can be fragmented using conventional techniques. Thus, the term includes segments of proteolytically-cleaved or recombinantly-prepared portions of an antibody molecule that are capable of selectively reacting with a certain protein. Non limiting examples of such proteolytic and/or recombinant fragments include Fab, F(ab')2, Fab' , Fv, dAbs and single chain antibodies (scFv) containing a VL and VH domain joined by a peptide linker. The scFv's can be covalently or non-covalently linked to form antibodies having two or more binding sites. Thus, "antibody-based binding moiety" includes polyclonal, monoclonal, or other purified preparations of antibodies and recombinant antibodies. The term "antibody-based binding moiety" is further intended to include humanized antibodies, bispecific antibodies, and

11058275.3 16 chimeric molecules having at least one antigen binding determinant derived from an antibody molecule. In a preferred embodiment, the antibody-based binding moiety is detectably labeled.

[00113] "Labeled antibody", as used herein, includes antibodies that are labeled by a detectable means and include, but are not limited to, antibodies that are enzymatically, radioactively, fluorescently, and chemiluminescently labeled. Antibodies can also be labeled with a detectable tag, such as c-Myc, HA, VSV-G, HSV, FLAG, V5, or HIS. The detection and quantification of the ASC protein present in the pellet can be correlate to the intensity of the signal emitted from the detectably labeled antibody.

[00114] As used herein, a "subject" refers to a mammal, preferably a human. The term

"individual" and "subject" are used interchangeably.

[00115] As used herein, the term "therapeutically effective amount" means a dosage sufficient to reduce the effects and symptoms associated with the different inflammatory diseases and disorder. The ""therapeutically effective amount" should prevent further inflammations responses and/or alleviate the stmptoms.

[00116] The term "treatment" or "treating" means any therapeutic intervention in a subject, including: (i) prevention, that is, causing the clinical symptoms not to develop; (ii) inhibition, that is, arresting the development of clinical symptoms; and/or (iii) relief, that is, causing the regression of clinical symptoms.

[00117] As used herein, the term "pharmaceutical composition" refers to the active agent in combination with a pharmaceutically acceptable carrier of chemicals and compounds commonly used in the pharmaceutical industry.

[00118] As used herein, the term "conservative amino acid substitution" is one in which the amino acid residue is replaced with an amino acid residue having a side chain with a similar charge and size. Families of amino acid residues having side chains with similar charges have been defined in the art. These families include amino acids with basic side chains (e.g., lysine, arginine, histidine), acidic side chains (e.g., aspartic acid, glutamic acid), uncharged polar side chains (e.g., glycine, asparagine, glutamine, serine, threonine, tyrosine, cysteine), nonpolar side chains (e.g., alanine, valine, leucine, isoleucine, proline, phenylalanine, methionine, tryptophan), beta-branched side chains ( e.g., threonine, valine, isoleucine) and aromatic side chains (e.g., tyrosine, phenylalanine, tryptophan, histidine).

11058275.3 17 [00119] As used herein, the term "substantially similar" refer to peptides that differ from

SEQ. ID. No. 3, by having one or more conservative amino acid substitutions in their amino acid sequences and that peptides retain their binding to the PYD of pyrin and inhibitng ASC pyroptosome formation described herein and known in the art. "Substantially similar" BBox- peptide are functional variants of BBox-peptide.

[00120] As used herein, the term "fragment" refers to an amino acid sequence which is shorter than the original polypeptide encoded by the nucleic acid of ASC (SEQ. ID. No. 1; Genbank Accession No. BAA87339) or the coding sequence of the BBox- of pyrin, SEQ. ID. No. 3. A fragment is an incomplete or truncated ASC protein. The ASC protein is shortened or truncated in a "fragment." Examples of ASC fragments include fragments consisting of amino acids 1-150, amino acids 1-105, and amino acids 100-195. These fragments contain either the pyrin domain (PYR) and/or the caspase-recruiting domain (CARD).

[00121] As used herein, the term "inflammation" refers to any cellular processes that lead to the activation of caspase-1, or caspase-5, the production of cytokines IL-I and IL-8, and/or the related downstream cellular events resulting from the actions of the cytokines thus produced, for example, fever, fluid accumulation, swelling, abscess formation, and cell death. The term inflammation and pyroptosis are used interchangeably here.

[00122] As used herein, the term "vector" refers to a nucleic acid construct comprising the coding sequence of ASC (SEQ. ID. No. 1) (Genbank Accession No. BAA87339), the coding sequence of GFP (SEQ. ID. No. 2) (Genbank Accession No. E17099), SEQ. ID. No. 3, or small coding sequences thereof, wherein the nucleic acid construct is designed for the delivery into a host cell, transfer between different host cells, and/or for the expression of ASC-GFP fusion protein in cells. As used herein, a vector can be viral or non-viral.

[00123] As used herein, the term "nucleic acid" refers to DNA or RNA. The term encompasses sequences that include any of the known base analogs of DNA and RNA.

[00124] As used herein, the term "viral vector," refers to a nucleic acid vector construct that includes at least one element of viral origin and includes elements sufficient for or permissive of packaging into a viral vector particle. A viral vector can contain the coding sequence for a ASC-GFP fusion protein in place of non-essential viral genes. The vector and/or particle can be utilized for the purpose of transferring DNA, RNA or other nucleic acids into cells either in vitro or in vivo. Numerous forms of viral vectors are known in the art.

11058275.3 18 [00125] As used herein, the term "expression vector" refers to a vector that has the ability to incorporate and express heterologous DNA fragments in a foreign cell. An expression vector may comprise additional elements, for example, the expression vector may have two replication systems, thus allowing it to be maintained in two organisms, for example in human cells for expression and in a prokaryotic host for cloning and amplification.

[00126] As used herein, the term "microbial pathogen" refers to a microscopic living organism that can cause disease or illness. These include viruses, bacteria, protozoans, parasites, rickettsia, larval stages of insects, yeast, fungi, and helmiths. Examples include, but are not limited to Arcobacter species, Bacillus cereus, Campylobacter species, Clostridium botulinum, Clostridium perfringens, Cryptosporidium parvum, enteric viruses (eg. hepatitis A and Noroviruses), Escherichia coli O157:H7, Escherichia coli : non-0157 shiga toxin-producing (STEC), Giardia intestinalis, Listeria monocytogenes, Mycobacterium bovis, Norwalk-like viruses, Salmonella typhi, Salmonellae species: Non-typhoid, Shigella species, Staphylococcus aureus, Toxoplasma gondii, Vibrio cholerae, Vibrio parahaemolyticus, Vibrio vulnificus, and Yersinia enter ocolitica.

[00127] As used herein, a sample that is suspected of being contaminated with microbial pathogen include, but should not be construed to be limited to blood, sputum, feces, saliva, peritoneal fluid, synovial fluid, urine, cerebrospinal fluid, soil, water, rain, sewage, air, food, dust, and solid surface wipes.

[00128] As used herein, the term "inhibits" refers to the blocking, impeding and slowing of the formation of an ASC pyroptosome or inflammasome. It can also refer to the malformation of the pyroptosome such that the pyroptosome cannot or has a reduced capability to proteolytically activate caspase-1. It also refers to the increase degradation, turnover, and/or disassembly of an ASC pyroptosome or inflammasome complex. A reduced capability to proteolytically activate caspase-1 is less than or equal to 95% of an ASC pyroptosome activity relative to the activity in the absence of any compound to be tested. The Casp™ACE Assay System from Promega Inc. can be used to analyzed the proteolytically activation activity of the ASC pyroptosome. Other method are described in US Pat. Application 20070111934 and Yamamoto M, et. al., 2004, are all hereby explicitly incorporated by reference.

[00129] As used herein, the term "inhibitory compound" refers to any inorganic or organic compound, protein, peptide, peptidomimetic, siRNA and the like, synthetic compounds, small molecules and the like that can block, impede and/or slow the formation of an ASC

11058275.3 19 pyroptosome or inflammasome; cause the malformation of the pyroptosome such that the pyroptosome cannot or has a reduced capability to proteolytically activate caspase-1; and/or increase the rate of degradation, turnover, and/or disassembly of an ASC pyroptosome or inflammasome complex.

[00130] As used herein, the term "fusion protein" or "fusion polypeptide" refers to a protein created by joining two genes or two proteins / peptides together. In the laboratory, this is achieved through the creation of a fusion gene which is done through the removal of the stop codon from a DNA sequence of the first protein and then attaching the DNA sequence of the second protein in frame. The resulting DNA sequence will then be expressed by a cell as a single protein. In a fusion protein, the two proteins that will be joined together with a linker or spacer peptide added between the two protein. This linker or spacer peptide often contain protease cleavage site to facilitate the separation of the two proteins after expression and purification The making of fusion protein as a technique is commonly used for the identification and purification of proteins through the fusion of a GST protein, FLAG peptide or a hexa-his peptide.

[00131] Embodiments of the invention are based on the discovery of the ASC

Supramolecular Assembly Complex (ASC SMAC) that is formed in macrophages that have been challenged with pathogenic microbial products.

[00132] ASC stands for Apoptotic speck protein containing a caspase recruitment domain

(CARD), is a 22 kDa adapter protein with an N-terminal pyrin domain (PYD) and a C-terminal CARD (Masumoto et al., 1999). It links the PYD-containing nod-like receptor (NLR) family member to procaspase-1, using its PYD to interact with the PYD of the NLRs and its CARD to interact with the CARD of procaspase-1 (Martinon et al., 2002; Srinivasula et al., 2002). Other names for ASC are ASC, CARD5, MGC10332, TMS, TMSl. It is an adaptor protein. Gene- targeted deletion of ASC in mice revealed that ASC is essential for activation of caspase-1 and generation of mature IL-lbeta by almost all stimuli known to induce IL-lbeta generation, indicating that ASC is a key downstream effector of caspase-1 activation (Kanneganti et al., 2006b; Mariathasan et al., 2004; Mariathasan et al., 2005; Mariathasan et al., 2006; Martinon et al., 2006; Sutterwala et al., 2006; Yamamoto et al., 2004). How ASC induces caspase-1 activation is not clear, but it is currently believed that PYD-containing NLR family members such as cryopyrin assemble an inflammasome complex with ASC which in turn recruits and activates caspase-1 (Martinon et al., 2002; Yu et al., 2006). Recently, it has been demonstrated that activation of caspase-1 not only leads to inflammation, but in certain instances causes an inflammatory form of cell death called pyroptosis (Cookson and Brennan, 2001; Fink and

11058275.3 20 Cookson, 2005; Fink and Cookson, 2006; Swanson and Molofsky, 2005). Pyroptosis is a caspase-1 -dependent inflammatory form of cell death. Pyroptosis was initially observed in macrophages infected with the intracellular bacteria Salmonella typhimurium (Monack et al., 2001), but was later found to occur in response to infection with a number of other intracellular bacterial and viral pathogens (Hilbi et al., 1998; Johnston et al., 2005; KeIk et al., 2003; Mariathasan et al., 2005; Sansonetti et al., 2000; Sun et al., 2005; Thumbikat et al., 2005). An inflammatory form of cell death not caused by pathogenic infection, but similar to pyroptosis, was also described in human THP-I macrophage cell line and murine macrophages in response to different stimuli including bacterial toxins, lipoproteins and LPS plus ATP (Aliprantis et al., 2001a; Aliprantis et al., 1999; Aliprantis et al., 2000; Perregaux and Gabel, 1994; Warny and Kelly, 1999). Since all these stimuli have been recently shown to activate caspase-1 in macrophages, it is likely that this infection-independent inflammatory form of cell death or pyroptosis is also dependent on caspase-1 activation. Little is known about the mechanism by which caspase-1 can be specifically activated to induce pyroptosis.

[00133] The inventors found that the macrophages, in response to pro-inflammatory stimuli such as pathogenic infections and microbial products such as bacterial toxins, lipoproteins, LPS, and muramyl dipeptide (MDP), form a single ASC SMAC. The ASC SMAC is formed of ASC dimmers. Virtually all cellular ASC is recruited into this complex upon challenge with pro -inflammatory stimuli. Only one ASC SMAC is formed per challenged macrophage cell. The ASC SMAC formation precedes caspase-1 activation in these macrophages. The ASC SMAC formation is essential for the proteolytically activation of caspase-1 and this event is rapidly followed by caspase-1 -dependent inflammatory cell death (pyroptosis). As a result, this ASC SMAC that is formed preceding pyroptosis is termed an ASC pyroptosome. In the presence of pro-inflammatory stimuli, a single, large, supramolecular complex of the size of 1-3 microns forms in each challenged THP-I macrophage. Due to the supramolecular structure of the complex, a simple low speed centrifugation step of no more than 5000 x G is sufficient to pellet this complex in a preparation of cell lysate from cells undergoing an inflammatory response, thus permitting the rapid separation, isolation, and identification of an ASC pyroptosome.

[00134] Pyroptosis and other caspase-1 dependent inflammatory event depends on the activation of caspase-1. Inactive pro-caspase-1 is cleaved to active caspase-1. The active caspase-1 then cleaves the inactive pro-IL-lβ and pro-IL-18 to produce the active proinflammatory cytokines IL- lβ and IL- 18, respectively. Activation of caspase-1 occurs in

11058275.3 21 response to pathogenic infection or cellular stress and requires the assembly of intracellular molecular platforms, called inflammasomes (Drenth and van der Meer, 2006; Mariathasan, 2007; Ogura et al., 2006; Petrilli et al., 2005). A number of molecules involved in the assembly of different inflammasomes have been identified including ICE-protease activating factor (Ipaf), Nalpl, cryopyrin/Nalp3, pyrin and ASC (Agostini et al., 2004; Delbridge and O'Riordan M, 2006; Martinon and Tschopp, 2005; Poyet et al., 2001; Srinivasula et al., 2002; Ting et al., 2006; Yu et al., 2006). Ipaf and Nalpl associate directly with inactive monomeric procaspase-1 via CARD-CARD interactions and prompt its dimerization and activation (Faustin et al., 2007; Poyet et al., 2001). In contrast, cryopyrin/Nalp3, and pyrin do not associate directly with procaspase-1 and require the adaptor protein ASC to recruit and activate caspase-1 (Agostini et al., 2004; Yu et al., 2006).

[00135] The inflammasomes are critical elements of innate immunity and the host defense against pathogenic infections. The molecular mechanisms and signals that activate the different inflammasomes are poorly understood, but recent studies revealed that Ipaf is specifically activated by flagellin of intracellular pathogens such as Salmonella typhimurium and Legionella pneumophila, possibly via recognition of flagellin by its regulatory LRR domain (Amer et al., 2006; Franchi et al., 2006; Mariathasan et al., 2004; Miao et al., 2006). Although gene deletion studies in the mouse revealed that the Nalpl inflammasome is activated by anthrax lethal toxin (Boyden and Dietrich, 2006), a more recent study demonstrated that human Nalpl inflammasome can be activated in vitro by the microbial product, muramyl-dipeptide, and ribonucleoside triphosphates (Faustin et al., 2007).

[00136] Although caspase-1 plays a pivotal role in innate immunity and host response against pathogenic infections, deregulated activation of caspase-1 is reported to be responsible for a number of systemic autoinflammatory diseases in humans (Ting et al., 2006). These diseases represent a group of inherited disorders characterized by recurrent episodes of inflammation and fever without an apparent stimulus and of a major involvement of autoantibodies and autoreactive T cells (Galeazzi et al., 2006; Gumucio et al., 2002; Hull et al., 2003; McDermott and Aksentijevich, 2002; Stehlik and Reed, 2004). Mutations in basic and regulatory components of the inflammasome appear to be responsible for these autoinflammatory diseases. For example, mutations in the CIASl gene which encodes cryopyrin cause three autoinflammatory diseases; familial cold autoinflammatory syndrome (FCAS yfamilial cold urticaria (FCU), Muckle-Wells syndrome (MWS), and neonatal-onset multisystem inflammatory disease (NOMID )/Chronic infantile neurological cutaneous and

11058275.3 22 articular syndrome (CINCA) (Feldmann et al., 2002; Hoffman et al., 2001). Recent studies indicate that some of the disease-associated cryopyrin mutations increase self-self interactions and oligomerization of the mutant cryopyrin proteins thereby causing more oligomerization of ASC and activation of caspase-1 (Yu et al., 2006).

[00137] Mutations in the MEFV gene which encodes pyrin are associated with the most common autoinflammatory disease, Familial Mediterranean Fever (FMF) (Consortium, 1997a; Consortium, 1997b). Pyrin contains several domains including an N-terminal pyrin domain (PYD) followed by a long linker region, B-box, coiled-coil and SPRY domains. FMF-associated mutations have been observed in all of these regions with the highest percentage of mutations found in the SPRY domain (-44%) and the linker region between PYD and B-box (-38%). Pyrin interacts with ASC, and like cryopyrin, triggers ASC oligomerization, activation of procaspase-1 and IL-lβ processing (Yu et al., 2006). How the FMF-associated mutations lead to increased inflammation is not yet clear, but it is likely that these mutations alter the activity of pyrin leading to increased ASC oligomerization in FMF patients.

[00138] In the autoinflammatory PAPA syndrome, the inventors have found that pyrin is the receptor molecule for the cytoskeleton-organizing protein PSTPIPl and that the pyrin- PSTPIPl interaction enhances pyrin-dependent activation of ASC pyroptosome formation and the consequential caspase-1 activation and production of pro-inflammatory cytokine IL-lβ. In the disease state, the mutant PSTPIPl has increased binding affinity to pyrin and this increases the overall ASC pyroptosome formation and caspase-1 activation.

[00139] One embodiment of the invention provides a method of isolating and detecting

ASC pyroptosomes in a pellet of a sample. The method comprises centrifuging a sample to form a pellet and detecting the presence of the ASC protein in the pellet. The sample is subjected to a centrifugal force of more than 2000 x G and up to 100, 000 x G in order to pellet the ASC pyroptosome.

[00140] Various leukocytes are involved in the initiation and maintenance of inflammation. Generally speaking, acute inflammation is mediated by granulocytes or polymorphonuclear leucocytes, while chronic inflammation is mediated by mononuclear cells such as monocytes and macrophages. These cells can be further stimulated to maintain inflammation through the action of an adaptive cascade involving lymphocytes: T cells, B cells, and antibodies.

11058275.3 23 [00141] Accordingly, in one embodiment, the sample is a lysate of leukocytes. In one embodiment, the sample is a lysate of macrophages.

[00142] In another embodiment, the preferred sample is a lysate of macrophage cells. The macrophages are lysed and the lysate is subjected to centrifugation.

[00143] In one embodiment, the macrophages are obtained from a subject. In another embodiment, the leukocytes are obtained from a subject.

[00144] The macrophages can be harvested and isolated by various methods known in the art, for example from the circulating blood, by bronchoalveolar lavage, from the peritoneal cavity and bone marrow by syringe aspiration, and from the spleen.

[00145] The macrophages can be lysed by any method known in the art. For example, repeated drawing and aspiration of the suspension of macrophage cell through a hypodermic needle, ultrasound sonication, suspension in a hypotonic solution, freeze/thaw and homogenization using a tissue/cell grinder, using e.g., detergent-based reagents such as Poppers Protein Extraction Reagents that are commercially available.

[00146] In one embodiment, the macrophages are lysed by the following method. To the blood, peritoneal fluid or other fluid sample containing macrophages, equal volume of pre- warmed Hank's Buffered Salt Solution (HBSS) (full strength with carbonate: 0.137 M NaCl, 5.4 mM KCl, 0.25 mM Na₂HPO₄, 0.44 mM KH₂PO₄, 1.3 mM CaCl₂, 1.0 mM MgSO₄, 4.2 mM NaHCO₃) or other isotonic media is added and the mixture is centrifuged at no more than 1500 x G. The cells are spun gently to pellet them, and resuspended in about 1 ml of 0.83% ammonium chloride, pH 7. This lyses red blood cells. The remaining cells are incubated at room temp for 2-3 minutes, diluted with HBSS or media, and washed twice. The cells are resuspend in MEM with 10% FBS at a concentration of 10⁵/ml, and plated. After 24 hrs, the non-adherent cells are gently washed off. This procedure usually yields >95% pure macrophages by non-specific esterase staining. Other methods can be found in www.ubik.microbiol. washington.edu/protocols/bl3/MDMProtocol.pdf , M Knowles and D Hughes (1970), Mishra L,. et.al., (1995) and Current Protocols in Immunology (CPI).

[00147] Alternatively, the sample can be a lysate of whole blood, bone marrow, peritoneal fluid, or any bodily fluid known in the art. The cells in the whole blood, bone marrow, peritoneal fluid, or any bodily fluid can be lysed by methods known in the art.

11058275.3 24 [00148] With the ability to rapidly separate, isolate, and detect the supramolecular ASC pyroptosomes formed in macrophages that are elicited during an inflammatory response, the ASC pyroptosome can be used as an indicator of inflammation in an individual. The inflammation can be acute or chronic. The inflammatory response can be caspase-1, caspase-5, IL-I and/or IL- 18 dependent.

[00149] In one embodiment, the method described herein provides a method of diagnosing inflammation in an individual. The method comprises detecting an ASC pyroptosome in a sample from an individual, wherein the detectable presence of an ASC pyroptosome indicates that the individual is suffering from inflammation. A detectable presence of the ASC pyroptosome refers to a signal for the ASC protein in the ASC pyroptosome pellet that is at least 5% over that of the control immunochemistry signal obtained in the absence of an antibody against the ASC protein or fragments thereof or in the presence of a non-related, non- ASC binding antibody.

[00150] In one embodiment, in the method described herein, the presence of an ASC pyroptosome in an individual is detected by a method comprising centrifuging a sample from an individual to form a pellet and detecting the presence of the ASC protein in the pellet after centrifugation.

[00151] In one embodiment, the preferred sample from an individual is a lysate of macrophage cells. The macrophages are lysed, the lysate of macrophages is subjected to a centrifugal force of more than 2000 x G and up to 100, 000 x G, and the ASC protein in the pellet is detected by immmunochemistry as described herein. The macrophages can be harvested and isolated by various methods known in the art and the examples as described herein. The macrophages can be obtained from the circulating blood, bone marrow, spleen, peritoneal cavity, lungs, or any other bodily fluid from an individual suspected of suffering from inflammation or an individual susceptible to inflammation such as an individual diagnosed with arthritis, an autoimmunity disease described below, or have mutations in certain genes that make them more prone to inflammations (Fidder HH, et. al., 2003).

[00152] In another embodiment, immunocytochemistry can be used to diagnose inflammation in an individual. This method comprises detecting an ASC pyroptosome in a sample from an individual using immunocytochemistry. Whole, unlysed macrophages are harvested and isolated by various methods known in the art and the examples as described herein. The macrophages are fixed with fixatives (formaldehyde-PBS, acetone, methanol, or

11058275.3 25 ethanol) and permeated with detergent (Triton X-100 or Tween-20) that are well known in the art, and immunostained with antibodies against the ASC protein or fragments thereof. The bound anti-ASC antibodies can be visualized for examination under a microscope by using a secondary antibody against the anti-ASC antibodies. The secondary antibody is usually labeled with the fluorescent dye (eg. infrared dyes such as LI-COR IRDye^®680 and IRDye^®800cw, FITC, rhodamine, Texas Red, Cy5, Cy3, Alexa 568, and Alexa 488). Commercially available fluorescently or infrared labeled secondary antibodies can be purchased from Invitrogen- Molecular Probes and LI-COR. Methods of immunocytochemistry can be found in Current Protocols in Molecular Biology (CPMB), Current Protocols in Protein Sciences (CPPS), and Current Protocols in Immunology (CPI) described supra and they are incorporated herein by reference in their entirety.

[00153] In one embodiment, the methods described herein provides a method of diagnosing autoinflammation PAPA syndrome in an individual, the method comprises detecting an ASC pyroptosome in a sample from an individual suspected of suffering from autoinflammation PAPA syndrome.

[00154] Pyrin contains an N-terminal pyrin domain (PYD) followed by B-box, coiled-coil and SPRY domains. Pyrin is a cytosolic receptor for PSTPIPl. Pyrin forms homotrimers through homotypic interactions of its coiled-coil domain and is present in an inhibited state due to intramolecular interactions between its PYD and B-box. Ligation by PSTPIPl, which is also a homotrimer, unmasks the PYD of pyrin thereby allowing it to interact with ASC and facilitate its oligomerization into an active ASC pyroptosome.

[00155] Mutations in the cytoskeleton organizing protein PSTPIPl are associated with the dominantly-inherited autoinflammatory PAPA syndrome. Because of their high binding affinity to pyrin's B-box, PAPA-associated PSTPIPl mutants were found to be more effective than WT PSTPIPl in inducing pyrin activation and subsequent ASC pyroptosome formation and inflammation.

[00156] In one embodiment, the inflammation in the individual is caused by an inflammatory disease or a pathogen infection and/or exposure to pathogen toxins and pathogen- derived pro-inflammatory compounds such as bacterial toxins, lipoproteins, LPS, and muramyl dipeptide (MDP), Nigericin or maitotoxin, danger signal monosodium urate (MSU), antiviral compounds R837 and R847, bacterial RNA and viral double- stranded RNA.

11058275.3 26 [00157] In one embodiment, the methods described herein are suitable for detecting and diagnosing inflammation and/or cell death associated with infection by microbial pathogens e.g., Listeria monocytogenes, Mycobacterrium tuberculosis, Yersinia pseudotuberculosis, Chlamydia trachomatis, Pseudomonas aeruginosa, Streptococcus pneumoniae, Legionella pneumophilia, Haemophilus influenzae, Moraxella pneumoniae, Clostridium botulism, Clostridium difficile, Bordetella pertussis, Listeria monocytogenes, Neisseria meningitides, Haemophilus influenzae, Brucella species, Coxiella burnetii, Shigella species, Escherichia coli 0157 :H7, Mycoplasma pneumoniae, Mycoplasma tuberculosis, Mycoplasma αvzαm-intracellular complex, Mycoplasma gordonae, Mycoplasma kansaii, Staphylococci aurenus, Staphylococci epidermidis, Staphylococci saprophiticus, Staphylococci lugdunensis, Streptococcus pyogenes, Streptococcus agalactiae, Streptococcus pneumoniae, Enterococcus faecalis, Enterococcus faecium, Enterococcus gallinarum, Enterococcus casseliflavus, Klebsiella pneumoniae, Klebsiella oxytoca, Pseudomonas aeruginosa, Acinetobacter baumannii, Nocardia species, Salmonella species, Vibrio species, Yersinia.Salmonella Shigella flexneri, Escherichia coli, Straphylococcus aureus, and Candida albicans, and viruses such as Epstein-Barr virus (EBV, a member of the herpesvirus family), the hepatitis A, B, C, and D viruses, influenza viruses, chicken pox, avian flu viruses, smallpox, polio viruses and the like that infect humans and mammals.

[00158] In one embodiment, the inflammatory diseases include but are not limited to, rheumatoid arthritis, inflammatory bowel disease, pelvic inflammatory disease, ulcerative colitis, psoriasis, systemic lupus erythematosus, multiple sclerosis, and type 1 diabetes mellitus, multiple sclerosis, psoriasis, vaculitis, allergic inflammation such as allergic asthma, atopic dermiatitis, and contact hypersensitivity. Other examples of auto-immune-related diseases or disorders, include but should not be construed to be limited to, rheumatoid arthritis, multiple sclerosis (MS), systemic lupus erythematosus, Graves' disease (overactive thyroid), Hashimoto's thyroiditis (underactive thyroid), Type 1 diabetes mellitus, celiac disease, Crohn's disease and ulcerative colitis, Guillain-Barre syndrome, primary biliary sclerosis/ cirrhosis, sclerosing cholangitis, autoimmune hepatitis, Raynaud's phenomenon, scleroderma, Sjogren's syndrome, Goodpasture's syndrome, Wegener's granulomatosis, polymyalgia rheumatica, temporal arteritis / giant cell arteritis, chronic fatigue syndrome CFS), psoriasis, autoimmune Addison's Disease, ankylosing spondylitis, Acute disseminated encephalomyelitis, antiphospholipid antibody syndrome, aplastic anemia, idiopathic thrombocytopenic purpura, Myasthenia gravis, opsoclonus myoclonus syndrome, optic neuritis, Ord's thyroiditis, pemphigus, pernicious anaemia, polyarthritis in dogs, Reiter's syndrome, Takayasu's arteritis, warm autoimmune hemolytic anemia, Wegener's granulomatosis, fibromyalgia (FM),

11058275.3 27 autoinflammatory PAPA syndrome, Familial Mediaterranean Fever, familial cold autoinflammatory syndrome, Muckle-Wells syndrome, and the neonatal onset multisystem inflammatory disease.

[00159] The inventors has been found that human THP-I macrophages can become activated to elicit an inflammation response upon infection by intracellular pathogens as well as exposure to several pro -inflammatory stimuli (e. g. pathogen-derived compounds described herein). During this inflammation response, the macrophage forms a single supramolecular structure, the ASC pyroptosome. This formation of the ASC pyroptosome in response to pathogen infection and/or pathogen-derived pro -inflammatory stimuli can be used to screen for the presence of infectious, toxic pathogens, and/or pathogen toxins in the environment. This screening and detection of pathogens can be further simplified by using a macrophage cell line stably expressing an ASC-GFP fusion protein (GFP stands for green fluorescent protein). The ASC pyroptosome formed in such a macrophage cell line stably expressing an ASC-GFP fusion protein can be visualized directly in live cells by the GFP fluorescence, without the need to isolate the ASC pyroptosome from the lysate of macrophages by centrifugation and detection by immunochemistry.

[00160] Therefore, in one embodiment, a macrophage comprising a vector from which a

ASC-GFP fusion protein can be expressed is provided. The macrophage cell line will stably express an ASC-GFP fusion protein. Such a cell line can be made by any standard cell transfection methodology known in the art, using an ASC-GFP fusion protein expression vector. Recombinant molecular biology methods can be used to clone and fuse the coding sequences of ASC (SEQ. ID. No. 1) (Genbank Accession No.: BAA87339) with GFP (SEQ. ID. No. 2) (Genbank Accession No. E17099) as well as clone the fusion construct into an expression vector. The coding sequence of the GFP can come from Aequorea victoria, Aequorea macrodactyla or Renilla reniformis. The coding sequence of the GFP can also have point mutations that will change the emission spectrum and/or the stability of the protein in the cells, for example, increased resistance to quenching, degradation, and/or protein aggregation.

[00161] In other embodiments, the ASC can be fused to other proteins that fluoresces at different wavelengths. For example, the fluorescent protein can be red, yellow, cherry, plum, raspberry, strawberry, banana or cyan.

[00162] Molecular biology techniques that are well known to one skilled in the art can be used to construct the expression vector that expresses the aggregation-prone protein described

11058275.3 28 above. For example, PCR amplification and cloning. The making of the ASC-GFP vector and stably ASC-GFP expressing THl macrophage cell lines are fully described in Yu et. al. 2006, and it is hereby incorporated by reference in its entirety. Polymerase chain reaction primers can be designed and used to amplify the ASC cDNA and cloned the amplified cDNA directly into mammalian expression vectors carrying the fluorescent protein coding nucleic acid, e.g. pEGFP, pCMV-DsRed-Express, pmCherry, pmRaspberry, pmPlum, pmBanana, pmOrange, pmStrawberry, pDsRed, pZsYellowl, pAmCyan, pLVX- AcGFPl, pLVX-DsRed, pRetroQ- AcGFPl, pRetroQ-DsRed, pRetroX-IRES-ZsGreenl, and pRetroX-IRES-DsRedExpress from ClonTech Inc. Other expression vectors include viral vector such as adenovirus, adeno- associated virus, lentivirus, and retrovirus which have the added advangtage of inserting the heterologous ASC-fusion protein transgene into the host cell. Examples of viral vectors for the expression of fluorescent ASC protein are pLVX- AcGFPl, pLVX-DsRed, pRetroQ-AcGFPl, pRetroQ-DsRed, pRetroX-IRES-ZsGreenl, and pRetroX-IRES-DsRedExpress.

[00163] While not wishing to be bound by theory, stably transfected and expressing THl macrophage expresses ASC-GFP or ASC-fused with other fluorescent protein. When such cells are not challenged by pathogens or pathogen-derived products such as LPS that elicit an inflammatory response from the macrophage, the ASC proteins are found as dimers in the cytosol or lysate when such cells are lysed. Centrifugal force of 5000 x G and up to 100,000 x G for 30 min do not pellet the soluble ASC dimers from the lysate. However, in the presence of pathogens or pathogen-derived products, an inflammatory response is stimulated in these macrophages, and ASC pyroptosomes are formed. Such ASC pyroptosomes will be visible under fluorescence microscopy. Additionally, a centrifugal force of up to 5000 x G is sufficient to pellet the ASC pyroptosomes from the lysate of challenged marcophages. Therefore, in the absence of any pathogen or pathogen-derived products, no ASC protein will be detected in pellets obatined from the lysates of unchallenged macrophages nor will any fluroresent ASC pyroptosome be visible under fluorescence microscopy fro whole unchallenged macrophages.

[00164] Accordingly, in one embodiment, the invention described herein provides a method of detecting the presence of a microbial pathogen, the method comprises the steps of: (a) contacting a reporter cell with a test sample suspected of containing a microbial pathogen; (b) detecting an ASC pyroptosome; and (c) determining that the test sample contains a microbial pathogen, wherein an ASC pyroptosome is detected. A detectable presence of an ASC pyroptosome indicates that a microbial pathogen is present in the test sample. ASC pyroptosome

11058275.3 29 does not form in the absence of any inflammatory response stimulus such as pathogens or pathogen-derived products.

[00165] In one embodiment, the reporter cell is a macrophage. In another embodiment, the macrophage expresses ASC-GFP. In other embodiments, the macrophages express fluorescently labeled ASC.

[00166] In one embodiment, in the method described herein, the presence of an ASC pyroptosome in a macrophage is detected by a method comprising centrifuging the lysate of a macrophage to form a pellet and detecting the presence of the ASC protein in the pellet after centrifugation. The presence of the ASC pyroptosome is detected from the lysate of the macrophage pellet.

[00167] After the macrophage has been in contact with a test sample suspected of being contaminated with microbial pathogens for a period of time, e. g. 30 min at room temperature, the macrophages can be separated from the test sample and collected. The collected macrophage is lysed and the macrophage lysate is subjected to a centrifugal force of more than 2000 x G and up to 100, 000 x G, to sediment the supramolecular ASC pyroptosome in the macrophage lysate. The ASC protein in the ASC pyroptosome can be detected by immunochemistry, immunocytochemisty or fluorescence microscopy as described herein.

[00168] In one embodiment, when macrophage lysates are used, a detectable presence of an ASC pyroptosome refers to a immunochemistry signal for the ASC protein that is at least 5% over that of the control immunochemistry signal obtained with macrophages that are in the absence of any added test sample suspected of being contaminated with microbial pathogens. In one embodiment, the reference amount is the control immunochemistry signal obtained for the ASC protein when macrophages are in the absence of any added test sample suspected of being contaminated with microbial pathogens.

[00169] When whole, unlysed macrophages are used, a detectable presence of a ASC pyroptosome in a macrophage is a single aggregate of 1-3 microns that is visualized by immunocytochemistry and microscopy methods described herein and those that are known in the art. After the macrophage stably expressing the ASC-GFP fusion protein has been in contact with a test sample suspected of being contaminated with microbial pathogens, the macrophages can be separated from the test sample and collected. The collected macrophages can be prepared for immunocytochemistry. These collected macrophages can be fixed, permeated, and subjected to immunocytochemistry by methods well known in the art and by those described herein.

11058275.3 30 [00170] When whole, unlysed macrophages are used, a detectable presence of a ASC pyroptosome in a macrophage can be viewed by fluorescent microscopy using techniques well known in the art. Macrophages containing a single ASC pyroptosome are counted. The percentage of marcophages that have been exposed to the test sample and exhibit ASC pyroptosome aggregates intracellularly are determined. Similarly in the control where the macrophage have not exposed to the test sample, the percentage of these macrophages having ASC pyroptosome aggregate intracellularly are also determined. In one embodiment, the reference amount is the percentage of macrophages that are not exposed to any test sample, and having some ASC pyroptosome aggregate intracellularly. Unchallenged macrophages that have not been in contact with any sample suspected of containing a microbial pathogen should not form any ASC pyroptosome. In one embodiment, a detectable presence of an ASC pyroptosome refers to at least 5% of the macrophages in contact with a sample suspected of containing a microbial pathogen have an ASC pyroptosome aggregate intracellularly. In another embodiment, a detectable presence of an ASC pyroptosome refers to at least 5% more macrophages that are in contact with a sample suspected of containing a microbial pathogen have an ASC pyroptosome aggregate intracellularly compared to unchallenged control macrophages, i. e. the reference amount.

[00171] The formation of the ASC pyroptosome is essential for the downstream activation of caspase-1 and the production of cytokines such as IL-I and IL-18. Controlling the factors that are involved in the formation and destruction of the ASC pyroptosome will permit the development of new therapeutics for the treatment of pathological inflammatory responses. The macrophages stably expressing recombinant fluorescently labeled ASC fusion protein can used to screen libraries of compunds for therapeutic candidates that can prevent, slow, and/or inhibit the formation of the ASC pyroptosome. Therapeutic candidates can also cause the malformation of the pyroptosome such that the pyroptosome cannot or has a reduced capability to proteolytically activate caspase-1. It also refers to the increase degradation, turnover, and/or disassembly of an ASC pyroptosome or inflammasome complex. A reduced capability to proteolytically activate caspase-1 is less than or equal to 95% of an ASC pyroptosome activity relative to the activity in the absence of any compound to be tested. The Casp™ACE Assay System from Promega Inc. can be used to analyzed the proteolytically activation activity of the ASC pyroptosome. Other method are described in US Pat. Application 2007/0111934 and Yamamoto M, et. al., 2004, are all hereby explicitly incorporated by reference.

11058275.3 31 [00172] The ASC pyroptosome formation is initiated by self-association of the PYD of

ASC to form an ASC dimer, which subsequently oligomerizes with other ASC dimers to form the large pyroptosome. Therefore, identification small molecules that could interfere with self- association of the PYD of ASC, then these molecules can become potential anti-inflammatory candidates for treatment of inflammatory diseases caused by excessive activation of the ASC - caspase-1 pathway.

[00173] Therefore, encompassed in the methods described herein is a method for screening and identification of a compound that inhibits inflammation, the method comprises the steps of: (a) contacting a compound to be screened with a reporter cell in the presence of a proinflammatory stimulus; (b) detecting an ASC pyroptosome; and (c) selecting the compound wherein ASC pyroptosome formation in the presence of the compound is not detectable or an amount of ASC pyroptosome formed is reduced compared to a reference amount. The reduction indicates that the compound has an inhibitory effect on inflammation.

[00174] In one embodiment, the reporter cell is a macrophage. In another embodiment, the macrophage expresses ASC-GFP. In other embodiments, the macrophages express fluorescently labeled ASC.

[00175] In one embodiment, a presence of an ASC pyroptosome in the macrophage is detected by a method comprising centrifuging the lysate of the reporter cell to form a pellet and detecting the presence of the ASC protein in the pellet after centrifugation.

[00176] In one embodiment, a control screen is conducted in parallel wherein no compound is added to a reporter cell under indentical screening conditions, i. e. same buffers, reagents, temperature etc, etc. The reporter cell is in contact with a pro-inflammatory stimulus but not in contact with any compound being tested.

[00177] After the macrophage has been in contact with a compound for a period of time, e. g. 30 min at room temperature, and in the presence of a pro-inflammatory stimulus, e. g. E. coli LPS, the macrophages can be separated from the cpompound and collected. The ASC pyroptosome can be detected in the whole, unlysed macrophage or the in the lysate of the macrophage.

[00178] The collected macrophage is lysed and the macrophage lysate is subjected to a centrifugal force of more than 2000 x G and up to 100, 000 x G, to sediment the supramolecular

11058275.3 32 ASC pyroptosome in the macrophage lysate. The ASC protein in the pelleted ASC pyroptosome can be detected by immunochemistry or fluorescence as described herein.

[00179] When macrophage lysates are used and the ASC detection is by immunochemistry, the reference amount of ASC pyroptosome corresponds to the amount of ASC protein detected and quantified in the pelleted ASC pyroptosome obtained from the lysate of macrophage that was in contact with a pro-inflammatory stimulus but not in contact with the compound being tested, i. e. in the control screen conducted in parallel with the compound screen. A reduction in the amount of ASC pyroptosome formed in macrophages in contact with an inhibitory compound being tested compared with a reference amount of ASC pyroptosome is less than or equal to 95% to 0% of the reference amount of ASC pyroptosome, including all percentages between 95% and 0%, i.e. less than or equal to 95%, 80%, 70% , 20%, ,10

% , 5%, 2%....0% of the reference amount of ASC pyroptosome. When there is such a reduction, it indicates that the compound that is tested has an inhibitory effect on inflammation. It is envisioned that a compound can completely inhibit inflammation response elicited by the pro-inflammatory stimulus such that such that no detectable ASC protein is found in the pellet in the lysateof the reporter cell. No detectable ASC protein means an immunochemical signal obtained for the test compound that is equivalent to that of the control immunochemistry signal obtained in the absence of an antibody against the ASC protein or fragments thereof or in the presence of a non-related, non-ASC binding antibody.

[00180] The ASC pyroptosome can be detected in whole unlysed macrophage by immunocytochemisty or fluorescence microscopy. When whole, unlysed macrophages are used, the reference amount of ASC pyroptosome is the number of macrophages that is in contact with a pro-inflammatory stimulus but not in contact with the compound being tested, wherein in each macrophage has a single, intracellular ASC pyroptosome. A reduction in the number of macrophages possessing ASC pyroptosomes, when the cells are in contact with an inhibitory compound being tested, compared with a reference amount of ASC pyroptosome is less than or equal to 95% to 0% of the reference number of ASC pyroptosome-containing macrophages, including all percentages between 95% and 0%, i.e. less than or equal to 95%, 80%, 70% ,

20%, ,10 % , 5%, 2%....0% of the reference number of ASC pyroptosome-containing macrophages. Such a reduction indicates that the compound has an inhibitory effect on inflammation. When a cmpoung completely inhibit inflammation, no reporter cell will be observed to have an ASC pyroptosome.

11058275.3 33 [00181] In one embodiment, the method for the identification of a compound that inhibits inflammation comprises using a macrophage that is stably expressing ASC tagged with green fluorescent protein. A detectable presence of a ASC pyroptosome refers to a single GFP aggregate of 1-3 microns in a said macrophage viewed by fluorescent microscopy. A reference amount of ASC pyroptosome is the number of macrophages in contact with a pro-inflammatory stimulus but not in contact with said compound being tested, having a single, intracellular GFP aggregate of 1-3 microns in size. A reduction in the amount of GFP aggregates formed in macrophages in contact with an inhibitory compound being tested compared with a reference amount is less than or equal to 95% to 0% of the reference number of GFP aggregate containing macrophages, including all percentages between 95% and 0%, i.e. less than or equal to 95%,

80%, 70% , 20%, ,10 % , 5%, 2%....0% of the reference number of GFP aggregate

-containing macrophages. Such a reduction indicates that the compound has an inhibitory effect on inflammation.

[00182] After the macrophage stably expressing the ASC-GFP fusion protein has been in contact with a sample suspected of being contaminated with microbial pathogens, said macrophages can be separated from said sample and collected. The collected macrophages can be prepared and viewed by fluorescent microscopy using techniques known in the art.

[00183] In other embodiments, ASC protein fused to other fluorescrent proteins are envisioned herein. Methods for constructing recombinant vectors for various fluorescrently labeled ASC are well known in the art and are also described herein.

[00184] In one embodiment, the invention provides a method for screening and identification of a compound that inhibits inflammation, the method comprises the steps: (a) contacting a compound to be screened with a cell lysate; (b) detecting an amount of ASC pyroptosomes; (c) comparing the amount of ASC pyroptosome with a reference amount; and (d) selecting the compound wherein no detectable ASC pyroptosome is formed in the presence of the compound or there is a reduced amount of ASC pyroptosome formed in the presence of the compound compared to the reference amount. The reduced amount of ASC pyroptosome indicates that the compound has an inhibitory effect on inflammation.

[00185] In one embodiment, the cell lysate is from a macrophage that is stably expressing an ASC-GFP fusion protein. In another embodiment, the macrophage is stably expressing an ASC protein fused to a fluorescrent protein as described herein. Methods of constructing an

11058275.3 34 expression vector for a fluorescently labeled ASC are well known to one skilled in the art and are also described herein.

[00186] In one embodiment, the cell lysate is from a non-macrophage that is stably expressing an ASC-GFP fusion protein, PSTPIPl, and pyrin. In another embodiment, the ASC protein fused to a fluorescrent protein as described herein. In one embodiment, the non- macrophage cell is a HEK 239 cell as described herein.

[00187] In one embodiment, the cell lysate are clarified by centrifugation. The cells are pelleted by centrifugation (1000 X G for 10 min at 4°C) in a 50-ml centrifuge tube. The supernatant is carefully decanted or aspirated, lysed in 2.5 pack cell volume of ice-cold CHAPS buffer (20 mM Hepes-KOH, pH 7.5, 5 mM MgCl₂, 0.5 mM EGTA, 0.1 mM PMSF, 0.1 % CHAPS) and then centrifuged at (14, 000 rpm) -20, 000 x G for 8 min at 4°C to obtain crude lysates. The SlOO lysates were prepared from the crude lysates by centrifugation at 100,000 x G for 30 min at 4°C. The SlOO lysate is incubated with the compound to be screen at 37° C for 30- 40 min. The assembled ASC pyroptosomes formed is pelleted from the lysates by centrifugation at 1000 x G for 5 min at 4°C. The pellet contains the assembled ASC pyroptosome and is rinsed once in ice-cold CHAPS buffer. The ASC pyroptosome is pellet again as before and the fluorescence signal from is measured using a spectrophotometer.

[00188] In one embodiment, the cell lysate is the SlOO lysate. In another embodiment, the cell lysate is the crude lysate obtained after the centrifugation at 20, 000 x G for 8 min.

[00189] In one embodiment, the screening method is conducted in parallel with a control wherein the cell lysate is incubated at 37° C for 30-40 min in the absence of any added compound. The reference amount of ASC pyroptosome is the fluorescence signal measured for a pellet obtained from such a control, wherein the cell lysate has no added test compound. A reduction in the amount of ASC pyroptosome formed (in the form of ASC-GFP aggregates and measured by fluoresecence) in the presence of compound being tested compared with a reference amount is less than or equal to 95% to 2% of the reference fluorescence signal for the control, including all percentages between 95% and 2%, i.e. less than or equal to 95%, 80%,

70% , 20%, ,10 % , 5%, 2% of the reference fluorescence signal for the control.

Such a reduction indicates that the compound has an inhibitory effect on inflammation. It is envisioned that the test compound can completely inhibit the formation of any ASC pyroptosome. The fluorescence signal obatined in the presence of such compound will be

11058275.3 35 equivalent to the fluorescence signal of the cell lysis buffer or the lysate kept at 4°C. ASC pyroptosome do not form in cell lysates described herein at 4°C.

[00190] In another embodiment, the invention provides a method for screening for a compound that inhibits the interaction of pyrin with PSTPIPl and/or activation of pyrin by PSTPIPl. The compound can attenuate the the interaction of pyrin with PSTPIPl and/or activation of pyrin by PSTPIPl, in particular, the stronger interaction between pyrin and the mutant PSTPIPl proteins. Such inhibitory compound of pyrin and PSTPIPl interaction can be useful for the treatment of autoinflammatory PAP syndrome. The method comprises the steps of: (a) contacting a compound to be screened with a reporter cell expressing pyrin, PSTPIPl, and ASC protein; (b) detecting ASC pyroptosome; and (c) selecting the compound wherein ASC pyroptosome in the presence of the compound is not detectable or an amount of ASC pyroptosome is reduced compared to a reference amount. A reduction in the amount of ASC pyroptosome indicates that the compound has an inhibitory effect the interaction between pyrin and PSTPIPl and/or activation of pyrin by PSTPIPl.

[00191] In one embodiment, the reporter cells expresses mutant PSTPIPl. In another embodiment, the mutant PSTPIPl is A230T PSTPIPl.

[00192] In one embodiment, the reporter cells stably expresses ASC-GFP. Other fluorescently labeled ASC are also comtemplated.

[00193] In one embodiment, the reporter cell is a macrophage. For example, a ThI marcophage as described herein. In another embodiment, the reporter cell is a non-macrophage. For example, a HEK239 cell as described herein.

[00194] In one embodiment, a control screen is conducted in parallel wherein no compound is added to the reporter cell. The reference amount is the percentage of cells having a ASC pyroptosome is that obtained in this control screen. A reduction in the amount of ASC pyroptosome formed (in the form of ASC-GFP aggregates) in the presence of compound being tested compared with a reference amount is less than or equal to 95% to 2% of the reference fluorescence signal for the control, including all percentages between 95% and 0%, i.e. less than or equal to 95%, 80%, 70% , 20%, , 10 % , 5%, 2% of the reference fluorescence signal for the control.

[00195] Those compounds inhibiting ASC pyroptosome formation and activity, and those inhibiting pyrin and PSTPIPl interaction and pyrin activation are potential drugs susceptible to

11058275.3 36 be used in the treatment of inflammation diseases such as rheumatoid arthritis, inflammatory bowel disease, ulcerative colitis, psoriasis, systemic lupus erythematosus, multiple sclerosis, autoinflammatory PAPA syndrome, and type 1 diabetes. It is envisioned that all inflammation diseases and disorders that are associated caspase-1 activities can be treated with such drugs.

[00196] Compounds that can be screened according to the methods described herein include but are not limited to natural extracts of plants, animals or microorganisms, proteins, antibodies or small molecules. These compounds are screened either in a pure form or in mixtures with other compounds.

[00197] In one embodiment, compound libraries can be screened.

[00198] In one embodiment, the the sample is a lysate of leukocytes. In one embodiment, the sample is a lysate of macrophages.

[00199] In another embodiment, the preferred sample is a lysate of macrophage cells. The macrophages are lysed and the lysate is subjected to centrifugation.

[00200] The cells can be harvested and isolated by various methods known in the art, for example from the circulating blood, by bronchoalveolar lavage, from the peritoneal cavity and bone marrow by syringe aspiration, and from the spleen.

[00201] In one embodiment, the detecting and/or analyzing the ASC pyroptosome are performed according to the methods described herein.

[00202] In one embodiment, the anti-inflammation treatment include but are not limited to the non-steroidal anti-inflammatory drugs (NSAIDs - such as aspirin, ibuprofen or naproxen), corticosteroids (such as prednisone), anti-malarial medications (such as hydroxychloroquine), methotrexate, sulfasalazine, leflunomide, anti-TNF medications, cyclophosphamide and mycophenolate.

[00203] Encompassed in the invention is a diagnostic kit for the rapid detection of inflammation comprising detecting an ASC pyroptosomes in a sample. The kit comprise the components suitable for collecting a sample, reagents for lysing the cells, reagents for lysing the cells, reagents for lysing the cells, reagents for lysing the cells, for carry out separation of an ASC pyroptosome in a lysate of cells, the detection of the ASC protein in the ASC pyroptosome, and instructions to perform the rapid detection procedure. The sample can be whole blood, bone marrow, peritoneal fluid, or any bodily fluid known in the art. The cells in the whole blood,

11058275.3 37 bone marrow, peritoneal fluid, or any bodily fluid can be lysed by methods known in the art. The supramolecular AS pyroptosome can be separated by membrane filtration, wherein the membrane has a pore size of less than 1 microns. On the account of the large size of the ASC pyroptosome, the ASC pyroptosome will be retained on the membrane. The detection of the ASC protein in the ASC pyroptosome can be performed by immunochemistry methods described herein.

[00204] Also encompassed in the invention is a method of determining the effectiveness of an anti-inflammation treatment in an individual being treated, the method comprises: (a) obtaining a sample from the individual at one time point; (b) obtaining a sample from the individual at a second time point, the second time point is being after the administration of an anti-inflammation treatment and after the first time point; (c) detecting and analyzing the ASC pyroptosomes in the samples; and (d) comparing the ASC pyroptosome in the second sample with that in the first sample, wherein a decrease in the ASC pyroptosome in the second time point sample is indication that treatment is effective. A decrease in the amount of ASC pyroptosome in the second time point sample is less than or equal to 95% to 0% of the amount of ASC pyroptosome in the first time point sample, including all percentages between 95% and

0%, i.e. less than or equal to 95%, 80%, 70% , 20%, ,10 % , 5%, 2%....0% of the amount of ASC pyroptosomes in the first time point sample.

[00205] A sample can be whole blood, bone marrow, peritoneal fluid, or any bodily fluid known in the art. The cells in the whole blood, bone marrow, peritoneal fluid, or any bodily fluid can be lysed by methods known in the art.

[00206] In one preferred embodiment, the sample comprises macrophages harvested from whole blood, bone marrow, peritoneal fluid, or any bodily fluid known in the art. The macrophages can be lysed by methods well known in the art and also described herein. The amount of ASC pyroptosome is measured as the amount of ASC protein the pellet of ASC pyroptosome. Methods of determining the amount of ASC are also well known in the art and are also described herein.

[00207] As used herein, an anti-inflammation treatment aims to prevent or slow down

(lessen) an undesired physiological change or disorder, such as the development or progression of the inflammation. Beneficial or desired clinical results include, but are not limited to, alleviation of symptoms, diminishment of extent of disease, stabilized (i.e., not worsening) state of disease, delay or slowing of inflammation disease progression, amelioration or palliation of

11058275.3 38 the disease state, and remission (whether partial or total), whether detectable or undetectable. An anti-inflammation treatment can also mean prolonging survival as compared to expected survival if not receiving treatment. An anti-inflammation treatment can also completely suppress the inflammation response.

[00208] Since the BBox of pyrin is an inhibitory domain, it can be envisioned that a soluble BBox can bind to activated pyrin via the available PYD of pyrin and prevent pyrin from engaging the ASC dimmers, and consequently the formation of the ASC pyroptosomes. Accordingly, in one embodiment, the invention provides a method of treatment of inflammation in a subject, the method comprising administering an effective amount of a BBox-peptide and a pharmaceutically acceptor carrier.

[00209] In one embodiment, the BBox-peptide is derived from Pyrin (Genbank Accession

No. AAB70557). In one embodiment, the BBox-peptide comprises the sequence CKRHLKQVQLLFCEDHDEPICLICSLSQEHQGHRVRPI (SEQ. ID. NO. 3). Conservative amino acid substitution of the BBox-peptide and smaller functional fragments thereof are also envisioned. The functional fragments the BBox-peptide and substantially similar fragments can inhibit ASC pyroptosome formation and caspase-1 activation as described herein. In yet another embodiment, the BBox-peptide comprises at least 10 amino acid residues of SEQ. ID. No. 3. In another embodiment, the BBox-peptide is a peptidomimetic of the original the BBox-peptide of pyrin (SEQ. ID. No. 3).

[00210] The BBox peptide comprises the B-Box-type zinc finger motif, which also known as the zinc binding domain (CHC3H2). This motif is often present in combination with other motifs, such as RING zinc finger, NHL motif, coiled-coil or RFP domain in functionally unrelated proteins, most likely mediating protein-protein interaction.

[00211] In one embodiment, the BBox-peptide is a fused to another protein or a portion thereof or conjugated to a polymer, thus forming a fusion protein or a conjugated protein. This produces an isolated chimeric BBox containing protein comprising a first portion and a second portion. The first portion can be the BBox-peptide (SEQ. ID. No. 3), a substantially similar BBox-pepide having one or more conservative amino acid substitution, a functional fragment of SEQ. ID. No. 3, or a peptide mimic thereof. The second portion can be but is not limited to serum transferrin or portions thereof, albumin, transthyretin, Fc of IgG (See G. M. Subramanian, (2007), Nature Biotechnology 25, 1411 - 141), and polymers such as polyethylene glycol for the purpose of enhancing the serum half life. The suitable polymers include, for example,

11058275.3 39 polyethylene glycol (PEG), polyvinyl pyrrolidone, polyvinyl alcohol, polyamino acids, divinylether maleic anhydride, N-(2-Hydroxypropyl)-methacrylamide, dextran, dextran derivatives including dextran sulfate, polypropylene glycol, polyoxyethylated polyol, heparin, heparin fragments, polysaccharides, cellulose and cellulose derivatives, including methylcellulose and carboxymethyl cellulose, starch and starch derivatives, polyalkylene glycol and derivatives thereof, copolymers of polyalkylene glycols and derivatives thereof, polyvinyl ethyl ethers, and α,β-Poly[(2-hydroxyethyl)-DL-aspartamide, and the like, or mixtures thereof. A polymer may or may not have its own biological activity. The polymers can be covalently or non-covalently conjugated to the first portion. Methods of conjugation for increasing serum half life and for radiotherapy are known in the art, for example, in U. S. Pat. Nos.: 5,180,816, 6,423,685, 6,884,780, and 7,022,673, which are hereby incorporated by reference in their entirety. The fusion or conjugation as described served to enhanced the serum half life of the BBox-peptide in vivo.

[00212] In another embodiment, the second portion can be but is not limited to with other proteins or short amino acid residues for the purposes of facilitating protein expression and purification, e.g. thioredoxin, glutathione-S-synthetase (GST), FLAG and six histidine tags.

[00213] In one embodiment, the BBox-peptide, peptide mimics, chimeric fusion protein or conservative amino acid substitution variant thereof include modification within the sequence, such as, modification by terminal-NH2 acylation, e.g., acetylation, or thioglycolic acid amidation, by terminal-carboxylamidation, e.g., with ammonia, methylamine, and the like terminal modifications. Terminal modifications are useful, and is well known, to reduce susceptibility to proteinase digestion, and therefore serve to prolong half life of the polypeptides in solutions, particularly biological fluids where proteases may be present.

[00214] In one embodiment, the method of treatment is administered in conjunction with other anti-inflammation treatment. Examples of the anti-inflammation treatment include but are not limited to the non-steroidal anti-inflammatory drugs (NSAIDs - such as aspirin, ibuprofen or naproxen), corticosteroids (such as prednisone), anti-malarial medications (such as hydroxychloroquine), methotrexate, sulfasalazine, leflunomide, anti-TNF medications, cyclophosphamide and mycophenolate.

[00215] In one embodiment, the inflammation is due to autoinflammatory diseases.

[00216] Compound librarires and HST screening

11058275.3 40 [00217] Commercially available combinatorial small molecule drug libraries can be screened for such inhibitors using assay methods well known in the art and those described herein. For example, libraries from Vitas-M Lab and Biomol International, Inc. A comprehensive list of compound libraries can be found at http://www.broad.harvard.edu/chembio/platform/screening/compound_libraries/index.htm. Other chemical compound libraries such as those from of 10,000 compounds and 86,000 compounds from NIH Roadmap, Molecular Libraries Screening Centers Network (MLSCN) can be screened successfully with the developed HTS assay. A chemical library or compound library is a collection of stored chemicals usually used ultimately in high-throughput screening or industrial manufacture. The chemical library can consist in simple terms of a series of stored chemicals. Each chemical has associated information stored in some kind of database with information such as the chemical structure, purity, quantity, and physiochemical characteristics of the compound.

[00218] In one embodiment, the screening method is a high-throughput screening. High- throughput screening (HTS) is a method for scientific experimentation that uses robotics, data processing and control software, liquid handling devices, and sensitive detectors. High- Throughput Screening or HTS allows a researcher to quickly conduct millions of biochemical, genetic or pharmacological tests. High-Throughput Screening are well known to one skilled in the art, for example, those described in U. S. Pat. Nos. 5,976,813, 6,472,144, 6,692,856, 6,824,982 , and 7, 091,048, and these are hereby incorporated by reference in their entirety.

[00219] HTS uses automation to run a screen of an assay against a library of candidate compounds. An assay is a test for specific activity: usually inhibition or stimulation of a biochemical or biological mechanism. Typical HTS screening libraries or "decks" can contain from 100,000 to more than 2,000,000 compounds (circa 2008).

[00220] The key labware or testing vessel of HTS is the microtiter plate: a small container, usually disposable and made of plastic, that features a grid of small, open divots called wells. Modern (circa 2008) microplates for HTS generally have either 384, 1536, or 3456 wells. These are all multiples of 96, reflecting the original 96 well microplate with 8 x 12 9mm spaced wells. Most of the wells contain experimentally useful matter, often an aqueous solution of dimethyl sulfoxide (DMSO) and some other chemical compound, the latter of which is different for each well across the plate. (The other wells may be empty, intended for use as optional experimental controls.)

11058275.3 41 [00221] To prepare for an assay, the researcher fills each well of the plate with some biological entity that he or she wishes to conduct the experiment upon, such as a protein, some cells, or an animal embryo. After some incubation time has passed to allow the biological matter to absorb, bind to, or otherwise react (or fail to react) with the compounds in the wells, measurements are taken across all the plate's wells, either manually or by a machine. Manual measurements are often necessary when the researcher is using microscopy to (for example) seek changes or defects in embryonic development caused by the wells' compounds, looking for effects that a computer could not easily determine by itself. Otherwise, a specialized automated analysis machine can run a number of experiments on the wells (such as shining polarized light on them and measuring reflectivity, which can be an indication of protein binding). In this case, the machine outputs the result of each experiment as a grid of numeric values, with each number mapping to the value obtained from a single well. A high-capacity analysis machine can measure dozens of plates in the space of a few minutes like this, generating thousands of experimental data points very quickly.

[00222] The compound or group of compounds being selected by the method according to the invention are then used in the development of drugs to be used in the treatment of conditions where control of inflammation is needed, such as the diseases listed above.

[00223] Detection of ASC protein.

[00224] The ASC protein in the pelleted ASC pyroptosome can be detected by any method known in the art. The ASC pyroptosome in the pellet can be dissociated and solubilized with detergents and heat. Preferably the detection method is an immunochemical method involving the binding of the ASC protein with an antibody-based binding moiety that specifically binds to ASC or a fragment of an ASC protein. Formation of the antibody- protein complex is then detected by a variety of methods known in the art.

[00225] In one preferred embodiment, the antibody-based binding moiety is detectably labeled by linking the antibody to an enzyme. The enzyme, in turn, when exposed to it's substrate, will react with the substrate in such a manner as to produce a chemical moiety which can be detected, for example, by spectrophotometric, fluorometric or by visual means. Enzymes which can be used to detectably label the antibodies of the present invention include, but are not limited to, malate dehydrogenase, staphylococcal nuclease, delta- V- steroid isomerase, yeast alcohol dehydrogenase, alpha-glycerophosphate dehydrogenase, triose phosphate isomerase, horseradish peroxidase, alkaline phosphatase, asparaginase, glucose oxidase, beta-galactosidase,

11058275.3 42 ribonuclease, urease, catalase, glucose- VI-phosphate dehydrogenase, glucoamylase and acetylcholinesterase.

[00226] Detection can also be accomplished using any of a variety of other immunoassays. For example, by radioactively labeling an antibody, it is possible to detect the antibody through the use of radioimmune assays. The radioactive isotope can be detected by such means as the use of a gamma counter or a scintillation counter or by audioradiography. Isotopes which are particularly useful for the purpose of the present invention are ³H, ¹³¹1, ³⁵S, ¹⁴C, and preferably ¹²⁵I.

[00227] It is also possible to label an antibody with a fluorescent compound. When the fluorescently labeled antibody is exposed to light of the proper wavelength, its presence can then be detected due to fluorescence. Among the most commonly used fluorescent labeling compounds are CYE dyes, fluorescein isothiocyanate, rhodamine, phycoerytherin, phycocyanin, allophycocyanin, o-phthaldehyde and fluorescamine.

[00228] An antibody can also be detectably labeled using fluorescence emitting metals such as 152Eu, or others of the lanthanide series. These metals can be attached to the antibody using such metal chelating groups as diethylenetriaminepentaacetic acid (DTPA) or ethylenediaminetetraacetic acid (EDTA).

[00229] An antibody also can be detectably labeled by coupling it to a chemiluminescent compound. The presence of the chemiluminescent-antibody is then determined by detecting the presence of luminescence that arises during the course of a chemical reaction. Examples of particularly useful chemiluminescent labeling compounds are luminol, luciferin, isoluminol, theromatic acridinium ester, imidazole, acridinium salt and oxalate ester.

[00230] The ASC protein in the pellet can be detected by immunoassays, such as enzyme linked immunoabsorbant assay (ELISA), radioimmunoassay (RIA), Immunoradiometric assay (IRMA), Western blotting, immunocytochemistry or immunohistochemistry, each of which are described in more detail below. Immunoassays such as ELISA or RIA, which can be extremely rapid, are more generally preferred.

[00231] The most common enzyme immunoassay is the "Enzyme-Linked Immunosorbent

Assay (ELISA)." ELISA is a technique for detecting and measuring the concentration of an antigen using a labeled (e.g. enzyme linked) form of the antibody. There are different forms of ELISA, which are well known to those skilled in the art. The standard techniques known in the

11058275.3 43 art for ELISA are described in "Methods in Immunodiagnosis", 2nd Edition, Rose and Bigazzi, eds. John Wiley & Sons, 1980; Campbell et al., "Methods and Immunology", W. A. Benjamin, Inc., 1964; and Oellerich, M. 1984, J. Clin. Chem. Clin. Biochem., 22:895-904.

[00232] In a "sandwich ELISA", an antibody (e.g. anti-ASC) is linked to a solid phase

(i.e. a microtiter plate) and exposed to a pellet sample containing ASC protein. The solid phase is then washed to remove unbound ASC protein. A labeled antibody (e.g. enzyme linked) is then bound to the bound- ASC protein forming an antibody- antigen- antibody sandwich. Examples of enzymes that can be linked to the antibody are alkaline phosphatase, horseradish peroxidase, luciferase, urease, and β-galactosidase. The enzyme linked antibody reacts with a substrate to generate a colored reaction product that can be measured.

[00233] In a "competitive ELISA", antibody is incubated with a pellet sample containing the ASC protein. The antigen- antibody mixture is then contacted with a solid phase (e.g. a microtiter plate) that is coated with ASC protein. The more ASC protein is present in the pellet sample, the less free antibody that will be available to bind to the solid phase ASC protein. A labeled (e.g., enzyme linked) secondary antibody is then added to the solid phase to determine the amount of primary antibody bound to the solid phase.

[00234] In an "immunohistochemistry assay" a sample pellet can be fixed on glass slides and treated with anti-ASC antibodies. The antibodies can then be visualized by any of a number of methods to determine the presence of the ASC protein. Examples of methods used to visualize antibodies are, for example, through enzymes linked to the antibodies (e.g., luciferase, alkaline phosphatase, horseradish peroxidase, or beta-galactosidase), or chemical methods (e.g., DAB/Substrate chromagen). The sample is then analyzed microscopically, most preferably by light microscopy of a sample stained with a stain that is detected in the visible spectrum, using any of a variety of such staining methods and reagents known to those skilled in the art.

[00235] Alternatively, "Radioimmunoassays" can be employed. A radioimmunoassay is a technique for detecting and measuring the concentration of an antigen using a labeled (e.g.. radioactively or fluorescently labeled) form of the antigen. Examples of radioactive labels for antigens include ³H, ¹⁴C, and ¹²⁵I. The concentration of the ASC protein in the pellet sample is measured by having the ASC protein in the pellet compete with the labeled (e.g. radioactively) ASC protein for binding to an antibody specific for the ASC protein. To ensure competitive binding between the labeled ASC protein and the unlabeled ASC protein, the labeled ASC protein is present in a concentration sufficient to saturate the binding sites of the antibody. The

11058275.3 44 higher the concentration of ASC protein in the pellet, the lower the concentration of labeled ASC protein that will bind to the antibody.

[00236] In a radioimmunoassay, to determine the concentration of labeled antigen bound to antibody, the antigen-antibody complex must be separated from the free antigen. One method for separating the antigen- antibody complex from the free antigen is by precipitating the antigen-antibody complex with an anti-isotype antiserum. Another method for separating the antigen-antibody complex from the free antigen is by precipitating the antigen- antibody complex with formalin-killed S. aureus. Yet another method for separating the antigen- antibody complex from the free antigen is by performing a "solid-phase radioimmunoassay" where the antibody is linked (e.g., covalently) to Sepharose beads, polystyrene wells, polyvinylchloride wells, or microtiter wells. By comparing the concentration of labeled antigen bound to antibody to a standard curve based on samples having a known concentration of antigen, the concentration of antigen in the biological sample can be determined.

[00237] An "Immunoradiometric assay" (IRMA) is an immunoassay in which the antibody reagent is radioactively labeled. An IRMA requires the production of a multivalent antigen conjugate, by techniques such as conjugation to a protein e.g., rabbit serum albumin (RSA). The multivalent antigen conjugate must have at least 2 antigen residues per molecule and the antigen residues must be of sufficient distance apart to allow binding by at least two antibodies to the antigen. For example, in an IRMA the multivalent antigen conjugate can be attached to a solid surface such as a plastic sphere. Unlabeled "sample" antigen and antibody to antigen which is radioactively labeled are added to a test tube containing the multivalent antigen conjugate coated sphere. The antigen in the sample competes with the multivalent antigen conjugate for antigen antibody binding sites. After an appropriate incubation period, the unbound reactants are removed by washing and the amount of radioactivity on the solid phase is determined. The amount of bound radioactive antibody is inversely proportional to the concentration of antigen in the sample.

[00238] Other techniques can be used to detect ASC protein in the pellet sample. One such technique is Western blotting (Towbin et at., Proc. Nat. Acad. Sci. 76:4350 (1979)), wherein the ASC pyroptosome in the pellet can be dissociated with detergents and heat, and run on an SDS-PAGE gel before being transferred to a solid support, such as a nitrocellulose filter. Detectably labeled anti- enzyme antibodies can then be used to assess enzyme levels, where the intensity of the signal from the detectable label corresponds to the amount of enzyme present. Levels can be quantified, for example by densitometry.

11058275.3 45 [00239] In one embodiment, the ASC protein in the pellet sample can be detected by mass spectrometry such as MALDI/TOF (time-of-flight), SELDI/TOF, liquid chromatography-mass spectrometry (LC-MS), gas chromatography-mass spectrometry (GC-MS), high performance liquid chromatography-mass spectrometry (HPLC-MS), capillary electrophoresis-mass spectrometry, nuclear magnetic resonance spectrometry, or tandem mass spectrometry (e.g., MS/MS, MS/MS/MS, ESI-MS/MS, etc.). See for example, U.S. Patent Application Nos: 20030199001, 20030134304, 20030077616, which are herein incorporated by reference.

[00240] Mass spectrometry methods are well known in the art and have been used to quantify and/or identify biomolecules, such as proteins (see, e.g., Li et al. (2000) Tibtech 18:151-160; Rowley et al. (2000) Methods 20: 383-397; and Kuster and Mann (1998) Curr. Opin. Structural Biol. 8: 393-400). Further, mass spectrometric techniques have been developed that permit at least partial de novo sequencing of isolated proteins. Chait et al., Science 262:89- 92 (1993); Keough et al., Proc. Natl. Acad. Sci. USA. 96:7131-6 (1999); reviewed in Bergman, EXS 88:133-44 (2000).

[00241] In certain embodiments, a gas phase ion spectrophotometer is used. In other embodiments, laser-desorption/ionization mass spectrometry is used to analyze the sample. Modern laser desorption/ionization mass spectrometry ("LDI-MS") can be practiced in two main variations: matrix assisted laser desorption/ionization ("MALDI") mass spectrometry and surface-enhanced laser desorption/ionization ("SELDI"). In MALDI, the analyte is mixed with a solution containing a matrix, and a drop of the liquid is placed on the surface of a substrate. The matrix solution then co-crystallizes with the biological molecules. The substrate is inserted into the mass spectrometer. Laser energy is directed to the substrate surface where it desorbs and ionizes the biological molecules without significantly fragmenting them. See, e.g., U.S. Pat. No. 5,118,937 (Hillenkamp et al.), and U.S. Pat. No. 5,045,694 (Beavis & Chait).

[00242] In SELDI, the substrate surface is modified so that it is an active participant in the desorption process. In one variant, the surface is derivatized with adsorbent and/or capture reagents that selectively bind the protein of interest. In another variant, the surface is derivatized with energy absorbing molecules that are not desorbed when struck with the laser. In another variant, the surface is derivatized with molecules that bind the protein of interest and that contain a photolytic bond that is broken upon application of the laser. In each of these methods, the derivatizing agent generally is localized to a specific location on the substrate surface where the sample is applied. See, e.g., U.S. Pat. No. 5,719,060 and WO 98/59361. The two methods can

11058275.3 46 be combined by, for example, using a SELDI affinity surface to capture an analyte and adding matrix-containing liquid to the captured analyte to provide the energy absorbing material.

[00243] For additional information regarding mass spectrometers, see, e.g., Principles of

Instrumental Analysis, 3rd edition., Skoog, Saunders College Publishing, Philadelphia, 1985; and Kirk-Othmer Encyclopedia of Chemical Technology, 4.sup.th ed. Vol. 15 (John Wiley & Sons, New York 1995), pp. 1071-1094.

[00244] The detection of ASC protein in the pellet will typically depend on the detection of signal intensity. This, in turn, can reflect the quantity and character of the ASC protein bound to the substrate. For example, in certain embodiments, the signal strength of peak values from spectra of a first sample and a second sample can be compared (e.g., visually, by computer analysis etc.), to determine the relative amounts of particular biomolecules. Software programs such as the Biomarker Wizard program (Ciphergen Biosystems, Inc., Fremont, Calif.) can be used to aid in analyzing mass spectra. The mass spectrometers and their techniques are well known to those of skill in the art.

[00245] In on embodiment, the detection of ASC protein in the pellet is accomplished using antibodies against the ASC protein. The antibodies for use in the methods described herein can be obtained from a commercial source such as Millipore/Chemicon polyclonal antibody product No. AB3607. The antibodies can be polyclonal or monoclonal antibodies. Alternatively, antibodies can be raised against the ASC protein (Genbank Accession No. BAA87339) or fragments thereof by one of skill in the art. Methods for the production of antibodies are disclosed in PCT publication WO 97/40072 or U.S. Application. No. 2002/0182702, which are herein incorporated by reference. The processes of immunization to elicit antibody production in a mammal, the generation of hybridomas to produce monoclonal antibodies, and the purification of antibodies may be performed by described in "Current Protocols in Immunology" (CPI) (John Wiley and Sons, Inc.) and Antibodies: A Laboratory Manual (Ed Harlow and David Lane editors, Cold Spring Harbor Laboratory Press 1988) which are both incorporated by reference herein in their entireties.

[00246] The detection of ASC protein in the pellet is considered positive when the immunoassay signal is at least 5% over that of the control immunoassay signal in the absence of an antibody against the ASC protein or fragments thereof or in the presence of a non-related, non-ASC binding antibody. When the ASC protein in the pellet is detected by mass

11058275.3 47 spectrometry, a positive result refers to distinct peak corresponding to the mass/charge of ASC and its proteolytic fragments.

[00247] Synthesis of the BBox-peptide and chimeric BBox containing protein.

[00248] BBox-peptide, variants that are substantially similar, peptide mimetics thereof and fusion proteins thereof can also be synthesized and purified by molecular methods that are well known in the art. Preferably molecular biology methods and recombinant heterologous protein expression systems be used. For example, recombinant protein may be expressed in bacteria, mammal, insects, yeast, or plant cells.

[00249] Standard techniques known to those of skill in the art can be used to introduce mutations in the nucleotide sequence encoding the BBox-peptide, including, for example, site- directed mutagenesis and PCR-mediated mutagenesis which result in amino acid substitutions. Preferably, the variants encode less than 10 amino acid substitutions, less than 5 amino acid substitutions, less than 4 amino acid substitutions, less than 3 amino acid substitutions, or less than 2 amino acid substitutions relative to the BBox-peptide of SEQ. ID. No. 3.

[00250] Alternatively, mutations can be introduced randomly along all or part of the coding sequence of the BBox-peptide (SEQ. ID. No. 4), such as by saturation mutagenesis, error-prone PCR, shuffling, oligonucleotide-directed mutagenesis, recursive ensemble mutagenesis, exponential ensemble mutagenesis, site-specific mutagenesis, gene reassembly, GSSM and any combination thereof. The resultant mutants can be screened for the ability to inhibit ASC pyroptosome formation as described herein.

[00251] The introduced mutations can be silent or neutral missense mutations, i.e., have no, or little, effect on the BBox-peptide inhibitory activity with regards to ASC formation. These types of mutations can be useful to optimize codon usage, or improve recombinant BBox- peptide or chimeric fusion protein expression and production. Alternatively, non-neutral missense mutations can alter the BBox-peptide inhibitory activity, such as enhancing the inhibitory activity. One of skill in the art would be able to design and test mutant molecules for desired properties such as no alteration to the BBox-peptide inhibitory activity. Following mutagenesis, the encoded protein can routinely be expressed and the functional and/or biological activity of the encoded protein, (e.g., ability to inhibit ASC pyroptosome formation) can be determined using techniques described herein or by routinely modifying techniques known in the art.

11058275.3 48 [00252] In one embodiment, the coding nucleic sequence for the BBox-peptide of pyrin is: 5'-

TGTAAGCGCCACCTGAAGCAGGTCCAGCTGCTCTTCTGTGAGGATCACGATGAGCC CATCTGCCTCATCTGCAGTCTGAGTCAGGAGCACCAAGGCCACCGGGTGCGCCCCA TT -3'(SEQ. ID. No. 4). The coding nucleic sequence can be cloned into a general purpose cloning vector such as pUC19, pBR322 , pBluescript vectors (Stratagene Inc.) or pCR TOPO® from Invitrogen Inc. In the example below, the cDNA is subcloned into the vector pDNR-dual. The resultant recombinant vector carrying coding nucleic sequence can then be used for further molecular biological manipulations such as site-directed mutagenesis or can be subcloned into protein expression vectors or viral vectors for protein synthesis in a variety of protein expression systems using host cells selected from the group consisting of mammalian cell lines, insect cell lines, yeast, and plant cells. In the example below, Cre recombinase to move the cDNA's into pCMVneo for expression.

[00253] Examples of other expression vectors and host cells are the pET vectors

(Novagen), pGEX vectors (Amersham Pharmacia), and pMAL vectors (New England labs. Inc.) for protein expression in E. coli host cell such as BL21, BL21(DE3) and AD494(DE3)pLysS, Rosetta (DE3), and Origami(DE3) (Novagen); the strong CMV promoter-based pcDNA3.1 (Invitrogen) and pCIneo vectors (Promega) for expression in mammalian cell lines such as CHO, COS, HEK-293, Jurkat, and MCF-7; replication incompetent adenoviral vector vectors pAdeno X, pAd5F35, pLP-Adeno-X-CMV (Clontech), pAd/CMV/V5-DEST, pAd-DEST vector (Invitrogen) for adeno virus-mediated gene transfer and expression in mammalian cells; pLNCX2, pLXSN, and pLAPSN retrovirus vectors for use with the Retro-X™ system from Clontech for retroviral-mediated gene transfer and expression in mammalian cells; pLenti4/V5- DEST™, pLenti6/V5-DEST™, and pLenti6.2/V5-GW/lacZ (Invitrogen) for lentivirus-mediated gene transfer and expression in mammalian cells; adenovirus-associated virus expression vectors such as pAA V-MCS, pAAV-IRES-hrGFP, and pAAV-RC vector (Stratagene) for adeno- associated virus-mediated gene transfer and expression in mammalian cells; BACpakό baculovirus (Clontech) and pFastBac™ HT (Invitrogen) for the expression in Spodopera frugiperda 9 (Sf9) and SfIl insect cell lines; pMT/BiP/V5-His (Invitrogen) for the expression in Drosophila Schneider S2 cells; Pichia expression vectors pPICZα, pPICZ, pFLDα and pFLD (Invitrogen) for expression in Pichia pastoris and vectors pMETα and pMET for expression in P. methanolica; pYES2/GS and pYDl (Invitrogen) vectors for expression in yeast Saccharomyces cerevisiae. Recent advances in the large scale expression heterologous proteins in Chlamydomonas reinhardtii are described by Griesbeck C. et. al. 2006 MoI. Biotechnol.

11058275.3 49 34:213-33 and Fuhrmann M. 2004, Methods MoI Med. 94:191-5. Foreign heterologous coding sequences are inserted into the genome of the nucleus, chloroplast and mitochondria by homologous recombination. The chloroplast expression vector p64 carrying the most versatile chloroplast selectable marker aminoglycoside adenyl transferase (aadA), which confer resistance to spectinomycin or streptomycin, can be used to express foreign protein in the chloroplast. Biolistic gene gun method is used to introduce the vector in the algae. Upon its entry into chloroplasts, the foreign DNA is released from the gene gun particles and integrates into the chloroplast genome through homologous recombination.

[00254] Specific site-directed mutagenesis of coding nucleic sequence in a vector can be used to create specific amino acid mutations and substitutions. Site-directed mutagenesis can be carried out using the QuikChange® site-directed mutagenesis kit from Stratagene according to manufacture's instructions or any method known in the art.

[00255] Envisioned in the methods described herein are chimeric BBox containing protein can be fused to transferrin, IgG, or albumin, to name a few, to enhance serum half life and pharmacokinetics in the individual being treated. BBox-peptide or chimeric BBox containing protein can also be fused to a tag protein such as tandem histidine residues(6xHis), GST, myc, thioredoxin first 105 amino acids or HA tag for the purification and/or enhance solubility of the expressed recombinant protein in heterologous system. Enzymatic digestion with serine proteases such as thrombin and enterokinase cleave and release the BBox-peptide or BBox containing protein from the histidine or myc tag, releasing the recombinant BBox-peptide or the chimeric BBox containing protein from the affinity resin while the histidine-tags and myc-tags are left attached to the affinity resin. Other reasons for tagging the BBox-peptide or chimeric BBox containing protein include monitoring the distribution of the protein over time in the individual.

[00256] BBox peptides, variants, and peptidomimetics.

[00257] BBox-peptide, variants that are substantially similar and peptidomimetics thereof can be chemically synthesized and purified by biochemical methods that are well known in the art such as solid phase peptide synthesis using t-Boc (tert-butyloxycarbonyl) or FMOC (9- flourenylmethloxycarbonyl) protection group described in "Peptide synthesis and applications" in Methods in molecular biology Vol. 298, Ed. by John Howl and "Chemistry of Peptide Synthesis" by N. Leo Benoiton, 2005, CRC Press, (ISBN-13: 978-1574444544) and "Chemical Approaches to the Synthesis of Peptides and Proteins" by P. Lloyd- Williams, et. al., 1997, CRC-

11058275.3 50 Press, (ISBN-13: 978-0849391422). Solid phase peptide synthesis, developed by R. B. Merrifield, 1963, J. Am. Chem. Soc. 85 (14): 2149-2154, was a major breakthrough allowing for the chemical synthesis of peptides and small proteins. An insoluble polymer support (resin) is used to anchor the peptide chain as each additional alpha-amino acid is attached. This polymer support is constructed of 20-50 μm diameter particles which are chemically inert to the reagents and solvents used in solid phase peptide synthesis. These particles swell extensively in solvents, which makes the linker arms more accessible.

[00258] Organic linkers attached to the polymer support activate the resin sites and strengthen the bond between the (-amino acid and the polymer support. Chloromethyl linkers, which were developed first, have been found to be unsatisfactory for longer peptides due to a decrease in step yields. The PAM (phenylacetamidomethyl) resin, because of the electron withdrawing power of the acid amide group on the phenylene ring, provides a much more stable bond than the classical resin. Another alternative resin for peptides under typical peptide synthesis conditions is the Wang resin. This resin is generally used with the FMOC labile protecting group.

[00259] A labile group protects the alpha-amino group of the amino acid. This group should be easily removed after each coupling reaction so that the next alpha-amino protected amino acid may be added. Typical labile protecting groups include t-Boc and FMOC t-Boc is a very satisfactory labile group which is stable at room temperature and easily removed with dilute solutions of trifluoroacetic acid (TFA) and dichloromethane. FMOC is a base labile protecting group which is easily removed by concentrated solutions of amines (usually 20-55% piperidine in N-methylpyrrolidone). When using FMOC alpha-amino acids, an acid labile (or base stable) resin, such as an ether resin, is desired.

[00260] The stable blocking group protects the reactive functional group of an amino acid and prevents formation of complicated secondary chains. This blocking group must remain attached throughout the synthesis and may be removed after completion of synthesis. When choosing a stable blocking group, the labile protecting group and the cleavage procedure to be used should be considered.

[00261] After generation of the resin bound synthetic peptide, the stable blocking groups are removed and the peptide is cleaved from the resin to produce a "free" peptide. In general, the stable blocking groups and organic linkers are labile to strong acids such as TFA. After the peptide is cleaved from the resin, the resin is washed away and the peptide is extracted with

11058275.3 51 ether to remove unwanted materials such as the scavengers used in the cleavage reaction. The peptide is then frozen and lyophilized to produce the solid peptide. This is then characterized by HPLC and MALDI before being used. In addition, the peptide should be purified by HPLC to higher purity before use.

[00262] Commercial peptide synthesizing machines are available for solid phase peptide synthesis. For example, the Advanced Chemtech Model 396 Multiple Peptide Synthesizer and an Applied Biosystems Model 432A Peptide synthesizer. There are commercial companies that make custom synthetic peptide to order, e.g. Abbiotec, Abgent, AnaSpec Global Peptide Services, LLC. Invitrogen and rPeptide, LLC.

[00263] Designing peptide mimetics.

[00264] Methods of designing peptide mimetics and screening of functional peptide mimetics are well known in the art. One basic method of designing a molecule which mimics a known protein or peptide, is first to identifies the active region(s) of the known protein (for example in the case of an antibody- antigen interaction one identifies which region(s) of the antibody enable binding to the antigen), and then searches for a mimetic which emulates the active region. Since the active region of the known protein is relatively small, it is hoped that a mimetic will be found which is much smaller (e.g. in molecular weight) than the protein, and correspondingly easier and cheaper to synthesis. Such a mimetic could be used as a convenient substitute for the protein, as an agent for interacting with the target molecule.

[00265] For example, Reineke et al. (1999, Nature Biotechnology, 17;271-275) designed a mimic molecule which mimics a binding site of the interleukin-10 protein using a large library of short peptides were synthesized, each of which corresponded to a short section of interleukin 10. The binding of each of these peptides to the target (in this case an antibody against interleukin-10) was then tested individually by an assay technique, to identify potentially relevant peptides. Phage display libraries of peptides and alanine scanning method can be used.

[00266] Other methods for designing peptide mimetic to a particular peptide or protein include The Chemical Computing Group's Molecular Operating Environment" (M. O. E.) software, European Patent EP1206494, the SuperMimic program by Andrean Goede et. al. 2006 BMC Bioinformatics, 7:11; and MIMETIC program by W. Campbell et. al.,2002, Microbiology and Immunology 46:211-215. The SuperMimic program is designed to identify compounds that mimic parts of a protein, or positions in proteins that are suitable for inserting mimetics. The application provides libraries that contain peptidomimetic building blocks on the one hand and

11058275.3 52 protein structures on the other. The search for promising peptidomimetic linkers for a given peptide is based on the superposition of the peptide with several conformers of the mimetic. New synthetic elements or proteins can be imported and used for searching. The MIMETIC computer program, which generates a series of peptides for interaction with a target peptide sequence is taught by W. Campbell et. al.,2002. In depth discussion of the topic is reviewed in "Peptide Mimetic Design with the Aid of Computational Chemistry" by James R. Damewood Jr. in Reviews in Computational Chemistry Reviews in Computational Chemistry, Jan 2007, Volume 9 Book Series: Reviews in Computational Chemistry, Editor(s): Kenny B. Lipkowitz, Donald B. BoydPrint ISBN: 9780471186397 ISBN: 9780470125861 Published by John Wiley &Sons, Inc.; and in T. Tselios, et. al., Amino Acids, 14: 333-341, 1998.

[00267] Methods for preparing libraries containing diverse populations of peptides, peptoids and peptidomimetics are well known in the art and various libraries are commercially available (see, for example, Ecker and Crooke, Biotechnology 13:351-360 (1995), and Blondelle et al., Trends Anal. Chem. 14:83-92 (1995), and the references cited therein, each of which is incorporated herein by reference; see, also, Goodman and Ro, Peptidomimetics for Drug Design, in "Burger's Medicinal Chemistry and Drug Discovery" Vol. 1 (ed. M. E. Wolff; John Wiley & Sons 1995), pages 803-861, and Gordon et al., J. Med. Chem. 37:1385-1401 (1994), each of which is incorporated herein by reference). One skilled in the art understands that a peptide can be produced in vitro directly or can be expressed from a nucleic acid, which can be produced in vitro. Methods of synthetic peptide and nucleic acid chemistry are well known in the art.

[00268] A library of peptide molecules also can be produced, for example, by constructing a cDNA expression library from mRNA collected from a tissue of interest. Methods for producing such libraries are well known in the art (see, for example, Sambrook et al., Molecular Cloning: A laboratory manual (Cold Spring Harbor Laboratory Press 1989), which is incorporated herein by reference). Preferably, a peptide encoded by the cDNA is expressed on the surface of a cell or a virus containing the cDNA.

[00269] Therapeutic compositions and administration

[00270] In one embodiment, the methods described herein comprise administering a pharmaceutical composition comprising a BBox peptide and a pharmaceutically acceptable carrier.

[00271] All dosage forms of the pharmaceutical composition, along with methods for their preparation, are well known in the pharmaceutical and cosmetic art (see HARRY'S

11058275.3 53 COSMETICOLOGY (Chemical Publishing, 7th ed. 1982); REMINGTON'S PHARMACEUTICAL SCIENCES (Mack Publishing Co., 18th ed. 1990)). Other desirable ingredients for use in such preparations include preservatives, co-solvents, viscosity building agents, carriers, etc. The carrier itself or a component dissolved in the carrier may have palliative or therapeutic properties of its own, including moisturizing, cleansing, or anti- inflammatory/anti-itching properties. Penetration enhancers may, for example, be surface active agents; certain organic solvents, such as di-methylsulfoxide and other sulfoxides, dimethyl- acetamide and pyrrolidone; certain amides of heterocyclic amines, glycols (e.g. propylene glycol) propylene carbonate; oleic acid; alkyl amines and derivatives; various cationic, anionic, nonionic, and amphoteric surface active agents; and the like.

[00272] In one embodiment, dosage forms include pharmaceutically acceptable carriers that are inherently nontoxic and nontherapeutic. Examples of such carriers include ion exchangers, alumina, aluminum stearate, lecithin, serum proteins, such as human serum albumin, buffer substances such as phosphates, glycine, sorbic acid, potassium sorbate, partial glyceride mixtures of saturated vegetable fatty acids, water, salts, or electrolytes such as protamine sulfate, disodium hydrogen phosphate, potassium hydrogen phosphate, sodium chloride, zinc salts, colloidal silica, magnesium trisilicate, polyvinyl pyrrolidone, cellulose- based substances, and polyethylene glycol. For all administrations, conventional depot forms are suitably used. Such forms include, for example, microcapsules, nano-capsules, liposomes, plasters, inhalation forms, nose sprays, sublingual tablets, and sustained release preparations. For examples of sustained release compositions, see U.S. Pat. No. 3,773,919, 3,887,699, EP 58,481A, EP 158,277A, Canadian Patent No. 1176565, U. Sidman et al., Biopolymers 22:547 (1983) and R. Langer et al., Chem. Tech. 12:98 (1982). Biologies such as antibodies and proteins will usually be formulated at a concentration of about 0.1 mg/ml to 100 mg/ml and the viral vector that carry the gene for expressing the biologies in vivo should be in the range of 106 to 1 x 1014 viral vector particles per application per patient.

[00273] In one embodiment, other ingredients can be added to pharmaceutical formulations, including antioxidants, e.g., ascorbic acid; low molecular weight (less than about ten residues) polypeptides, e.g., polyarginine or tripeptides; proteins, such as serum albumin, gelatin, or immunoglobulins; hydrophilic polymers such as polyvinylpyrrolidone; amino acids, such as glycine, glutamic acid, aspartic acid, or arginine; monosaccharides, disaccharides, and other carbohydrates including cellulose or its derivatives, glucose, mannose, or dextrins; chelating agents such as EDTA; and sugar alcohols such as mannitol or sorbitol.

11058275.3 54 [00274] In one embodiment, the pharmaceutical formulation to be used for therapeutic administration must be sterile. Sterility is readily accomplished by filtration through sterile filtration membranes (e.g., 0.2 micron membranes). The BBox peptide or chimeric BBox contain protein ordinarily can be stored in lyophilized form or as an aqueous solution if it is highly stable to thermal and oxidative denaturation. The pH of the BBox peptide or chimeric BBox contain protein preparations typically will be about from 6 to 8, although higher or lower pH values may also be appropriate in certain instances.

[00275] The pharmaceutical compositions described herein can also be administered systemically in a pharmaceutical formulation. The preferred formulation is also sterile saline or Lactated Ringer's solution. Lactated Ringer's solution is a solution that is isotonic with blood and intended for intravenous administration. Systemic routes include but are limited to oral, parenteral, nasal inhalation, intratracheal, intrathecal, intracranial, and intrarectal. The pharmaceutical formulation is preferably a sterile saline or lactated Ringer's solution. For therapeutic applications, the preparations described herein are administered to a mammal, preferably a human, in a pharmaceutically acceptable dosage form, including those that may be administered to a human intervenously as a bolus or by continuous infusion over a period of time, by intramuscular, intraperitoneal, intracerebrospinal, subcutaneous, intra-arterial, intrasynovial, intrathecal, oral, topical, or inhalation routes. A preferred embodiment is the nasal inhalation of a BBox peptide or chimeric BBox contain protein formulated for used in a nebulizer. Viral vectors encoding a BBox peptide or chimeric BBox contain protein can be formulated for use with a nebulizer. For these uses, additional conventional pharmaceutical preparations such as tablets, granules, powders, capsules, and sprays may be preferentially required. In such formulations further conventional additives such as binding- agents, wetting agents, propellants, lubricants, and stabilizers may also be required. In one embodiment, the therapeutic compositions described herein are formulated in a cationic liposome formulation such as those described for intratracheal gene therapy treatment of early lung cancer (Zou Y. et. al., Cancer Gene Ther. 2000 May;7(5):683-96). The liposome formulations are especially suitable for aerosol use for delivery to the lungs of patients. Vector DNA and/or virus can be entrapped in 'stabilized plasmid- lipid particles' (SPLP) containing the fusogenic lipid dioleoylphosphatidylethanolamine (DOPE), low levels (5-10 mol%) of cationic lipid, and stabilized by a polyethyleneglycol (PEG) coating (Zhang Y. P. et. al. Gene Ther. 1999, 6:1438- 47). Other techniques in formulating expression vectors and virus as therapeutics are found in "DNA-Pharmaceuticals: Formulation and Delivery in Gene Therapy, DNA Vaccination and Immunotherapy" by Martin Schleef (Editor) December 2005, Wiley Publisher, and "Plasmids

11058275.3 55 for Therapy and Vaccination" by Martin Schleef (Editor) May 2001, are incorporated herein as reference. In one embodiment, the dosage for viral vectors is 106 to 1014 viral vector particles per application per patient.

[00276] The route of administration, dosage form, and the effective amount vary according to the potency of the BBox peptide or chimeric BBox contain protein, and expression vectors and viral vectors used the gene therapy, and their physicochemical characteristics. The selection of proper dosage is well within the skill of an ordinarily skilled physician.

[00277] Gene Therapy

[00278] In one embodiment, a BBox peptide or chimeric BBox contain protein is administered to an individual by any one of several gene therapy techniques known to those of skill in the art. In general, gene therapy can be accomplished by either direct transformation of target cells within the mammalian subject (in vivo gene therapy) or transformation of cells in vitro and subsequent implantation of the transformed cells into the mammalian subject (ex vivo gene therapy). A viral vector carries the nucleic acid encoding the transgene under a tissue specific regulatory element. The tissue specific regulatory element allows the expression of the transgene in the target cells, for example, the lung epithelial cells.

[00279] The principles of gene therapy are disclosed by Oldham, R. K. (In: Principles of

Biotherapy, Raven Press, N.Y., 1987), and similar texts. Disclosures of the methods and uses for gene therapy are provided by Boggs, S. S. (Int. J. Cell Clon. 8:80-96 (1990)); Karson, E. M. (Biol. Reprod. 42:39-49 (1990)); Ledley, F. D., In: Biotechnology, A Comprehensive Treatise, volume 7B, Gene Technology, VCH Publishers, Inc. NY, pp 399-458 (1989)), all of which references are incorporated herein by reference.

[00280] The nucleic acid encoding the transgene of interest can be introduced into the somatic cells of an animal (particularly mammals including humans) in order to provide a treatment for non-allergen induced asthma. Most preferably, viral or retroviral vectors are employed for as the transfer vehicle this purpose. A suitable vehicle for gene therapy will not promote an immune response to the transgene described herein. The gene therapy virus can be in the form of an adenovirus, adeno-associated virus or lentivirus.

[00281] The term "vector", as used herein, refers to a nucleic acid construct designed for delivery to a host cell or transfer between different host cells.

11058275.3 56 [00282] As used herein, a "retroviral vector" refers to an expression vector that comprises a nucleotide sequence that encodes a transgene and that further comprises nucleotide sequences necessary for packaging of the vector. Preferably, the retroviral transfer vector also comprises the necessary sequences for expressing the transgene in cells.

[00283] Retroviral vectors are a common mode of delivery and in this context are retroviruses from which all viral genes have been removed or altered so that no viral proteins are made in cells infected with the vector. Viral replication functions are provided by the use of retrovirus "packaging" cells that produce all of the viral proteins but that do not produce infectious virus.

[00284] Introduction of the retroviral vector DNA into packaging cells results in production of virions that carry vector RNA and can infect target cells, but such that no further virus spread occurs after infection. To distinguish this process from a natural virus infection where the virus continues to replicate and spread, the term transduction rather than infection is often used.

[00285] Recombinant lenti virus can be used for the delivery and expression of a gene of interest in either dividing and non-dividing mammalian cells. The HIV-I based lentivirus can effectively transduce a broader host range than the Moloney Leukemia Virus (MoMLV)-base retroviral systems. Preparation of the recombinant lentivirus can be achieved using the pLenti4/V5-DEST™, pLenti6/V5-DEST™ or pLenti vectors together with ViraPower™ Lentiviral Expression systems from Invitrogen.

[00286] Examples of use of lentiviral vectors for gene therapy for inherited disorders and various types of cancer, and these references are hereby incorporated by reference (Klein, C. and Baum, C. (2004). Hematol. J., 5, 103-111; Zufferey, R et. al. (1997). Nat. Biotechnol., 15, 871- 875; Morizono, K. et. al. (2005). Nat.Med., 11, 346-352; Di Domenico, C. et. al. (2005), Hum.Gene Ther., 16, 81-90; Kim, E. Y., et. al., (2004). Biochem. Biophys. Res. Comm., 318, 381-390).

[00287] Non-retroviral vectors also have been used in genetic therapy. One such alternative is the adenovirus (Rosenfeld, M. A., et al., Cell 68:143155 (1992); Jaffe, H. A. et al., Nature Genetics 1:372-378 (1992); Lemarchand, P. et al., Proc. Natl. Acad. Sci. USA 89:6482- 6486 (1992)). Major advantages of adenovirus vectors are their potential to carry large segments of DNA (36 Kb genome), a very high titer (1011 /ml), ability to infect non-replicating cells, and suitability for infecting tissues in situ, especially in the lung. The most striking use of this vector

11058275.3 57 so far is to deliver a human cystic fibrosis transmembrane conductance regulator (CFTR) gene by intratracheal instillation to airway epithelium in cotton rats (Rosenfeld, M. A., et al., Cell 63:143-155 (1992)). Similarly, herpes viruses may also prove valuable for human gene therapy (Wolfe, J. H. et al., Nature Genetics 1:379-384 (1992)). Of course, any other suitable viral vector may be used for genetic therapy with the present invention.

[00288] U.S. Patent No. 6,531,456 provides methods for the successful transfer of a gene into a solid tumor cell using recombinant AAV virions. Generally, the method described in U.S. Patent No. 6,531,456 allows for the direct, in vivo injection of recombinant AAV virions into tumor cell masses, e.g., by intra-tumoral injection. The invention also provides for the simultaneous delivery of a second gene using the recombinant AAV virions, wherein the second gene is capable of providing an ancillary therapeutic effect when expressed within the transduced cell. U.S. Patent No. 6,531,456 is hereby incorporated by reference.

[00289] The viron used for gene therapy can be any viron known in the art including but not limited to those derived from adenovirus, adeno-associated virus (AAV), retrovirus, and lenti virus. Recombinant viruses provide a versatile system for gene expression studies and therapeutic applications.

[00290] The recombinant AAV virions described above, including the DNA of interest, can be produced using standard methodology, known to those of skill in the art. The methods generally involve the steps of (1) introducing an AAV vector into a host cell; (2) introducing an AAV helper construct into the host cell, where the helper construct includes AAV coding regions capable of being expressed in the host cell to complement AAV helper functions missing from the AAV vector; (3) introducing one or more helper viruses and/or accessory function vectors into the host cell, wherein the helper virus and/or accessory function vectors provide accessory functions capable of supporting efficient recombinant AAV ("rAAV") virion production in the host cell; and (4) culturing the host cell to produce rAAV virions. The AAV vector, AAV helper construct and the helper virus or accessory function vector(s) can be introduced into the host cell either simultaneously or serially, using standard transfection techniques. Using rAAV vectors, genes can be delivered into a wide range of host cells including many different human and non-human cell lines or tissues. Because AAV is nonpathogenic and does not illicit an immune response, a multitude of pre-clinical studies have reported excellent safety profiles. rAAVs are capable of transducing a broad range of cell types and transduction is not dependent on active host cell division. High titers, > 10⁸ viral particle/ml,

11058275.3 58 are easily obtained in the supernatant and 10¹¹ -10¹² viral particle/ml with further concentration. The transgene is integrated into the host genome so expression is long term and stable.

[00291] A simplified system for generating recombinant adenoviruses is presented by He

TC. et. al. Proc. Natl. Acad. Sci. USA 95:2509-2514, 1998. The gene of interest is first cloned into a shuttle vector, e.g. pAdTrack-CMV. The resultant plasmid is linearized by digesting with restriction endonuclease Pme I, and subsequently cotransformed into E. coli. BJ5183 cells with an adenoviral backbone plasmid, e.g. pAdEasy-1 of Stratagene's AdEasy™ Adenoviral Vector System. Recombinant adenovirus vectors are selected for kanamycin resistance, and recombination confirmed by restriction endonuclease analyses. Finally, the linearized recombinant plasmid is transfected into adenovirus packaging cell lines, for example HEK 293 cells(El -transformed human embryonic kidney cells) or 911 (El -transformed human embryonic retinal cells) (Human Gene Therapy 7:215-222, 1996). Recombinant adenovirus are generated within the HEK 293 cells.

[00292] The use of alternative AAV serotypes other than AAV-2 (Davidson et al (2000),

Proc. Natl. Acad. Sci. USA 97(7)3428-32; Passini et al (2003), J. Virol. 77(12):7034-40) has demonstrated different cell tropisms and increased transduction capabilities. With respect to brain cancers, the development of novel injection techniques into the brain, specifically convection enhanced delivery (CED; Bobo et al (1994), Proc. Natl. Acad. Sci. USA 91(6):2076- 80; Nguyen et al (2001), Neuroreport 12(9): 1961-4), has significantly enhanced the ability to transduce large areas of the brain with an AAV vector.

[00293] Large scale preparation of AAV vectors is made by a three -plasmid cotransfection of a packaging cell line: AAV vector carrying a DNA coding sequence of interest, AAV RC vector containing AAV rep and cap genes, and adenovirus helper plasmid pDF6, into 50 x 150 mm plates of subconfluent 293 cells. Cells are harvested three days after transfection, and viruses are released by three freeze-thaw cycles or by sonication.

[00294] AAV vectors are then purified by two different methods depending on the serotype of the vector. AA V2 vector is purified by the single- step gravity- flow column purification method based on its affinity for heparin (Auricchio, A., et. al., 2001, Human Gene therapy 12;71-6; Summerford, C. and R. Samulski, 1998, J. Virol. 72:1438-45; Summerford, C. and R. Samulski, 1999, Nat. Med. 5: 587-88). AAV2/1 and AAV2/5 vectors are currently purified by three sequential CsCl gradients.

11058275.3 59 [00295] Pharmaceutical compositions used in the methods described herein can be delivered systemically via in vivo gene therapy. A variety of methods have been developed to accomplish in vivo transformation including mechanical means (e.g, direct injection of nucleic acid into target cells or particle bombardment), recombinant viruses, liposomes, and receptor- mediated endocytosis (RME) (for reviews, see Chang et al. 1994 Gastroenterol. 106:1076-84; Morsy et al. 1993 JAMA 270:2338-45; and Ledley 1992 J. Pediatr. Gastroenterol. Nutr. 14:328- 37).

[00296] Another gene transfer method for use in humans is the transfer of plasmid DNA in liposomes directly to human cells in situ (Nabel, E. G., et al., Science 249:1285-1288 (1990)). Plasmid DNA should be easy to certify for use in human gene therapy because, unlike retroviral vectors, it can be purified to homogeneity. In addition to liposome-mediated DNA transfer, several other physical DNA transfer methods, such as those targeting the DNA to receptors on cells by conjugating the plasmid DNA to proteins, have shown promise in human gene therapy (Wu, G. Y., et al., J. Biol. Chem. 266:14338-14342 (1991); Curiel, D. T., et al., Proc. Natl. Acad. Sci. USA, 88:8850-8854 (1991)).

[00297] For gene therapy viruses, the dosage ranges from 10⁶ to 10 ¹⁴ particles per application. Alternatively the biolistic gene gun method of delivery may be used. The gene gun is a device for injecting cells with genetic information, originally designed for plant transformation. The payload is an elemental particle of a heavy metal coated with plasmid DNA. This technique is often simply referred to as biolistics. Another instrument that uses biolistics technology is the PDS- 1000/He particle delivery system. The proteins, expression vector, and/or gene therapy virus can be coated on minute gold particles, and these coated particles are "shot" into biological tissues such as hemangiomas and melanoma under high pressure. An example of the gene gun-based method is described for DNA based vaccination of cattle by Loehr B. I. et. al. J. Virol. 2000, 74:6077-86.

[00298] This invention is further illustrated by the following examples which should not be construed as limiting. The contents of all references cited throughout this application, as well as the figures and table are incorporated herein by reference.

[00299] It should be understood that this invention is not limited to the particular methodology, protocols, and reagents, etc., described herein and as such may vary. The terminology used herein is for the purpose of describing particular embodiments only, and is not intended to limit the scope of the present invention, which is defined solely by the claims.

11058275.3 60 [00300] Other than in the operating examples, or where otherwise indicated, all numbers expressing quantities of ingredients or reaction conditions used herein should be understood as modified in all instances by the term "about." The term "about" when used in connection with percentages may mean +1%.

[00301] All patents and other publications identified are expressly incorporated herein by reference for the purpose of describing and disclosing, for example, the methodologies described in such publications that might be used in connection with the present invention. These publications are provided solely for their disclosure prior to the filing date of the present application. Nothing in this regard should be construed as an admission that the inventors are not entitled to antedate such disclosure by virtue of prior invention or for any other reason. All statements as to the date or representation as to the contents of these documents is based on the information available to the applicants and does not constitute any admission as to the correctness of the dates or contents of these documents.

EXAMPLES

[00302] Materials and methods

[00303] Generation of Stable THP-I-ASC-GFP Cell Line.

[00304] The monocytic cell line THPl was cultured in RPMI 1640 supplemented with 10 mM N-(2-hydroxyethyl) piperazine-N'-(2-ethanesulfonic acid), 1 mM sodium pyruvate, 55 μM β-mercaptoethanol, 10 % fetal bovine serum and 200 μg»ml-l penicillin and 100 μg»ml-l streptomycin sulfate. Stable THP-I cells expressing ASC-GFP fusion protein were generated by retroviral gene transfer with recombinant MSCV expression vectors. The amphotropic packaging cell line Phoenix (G. P. Nolan's laboratory, Stanford University Medical Center, Stanford, CA) was transfected pMSCVpuro- ASC-EGFP-Nl plasmid using the LipofectAMINE transfection method. Forty-eight hours after transfection, the GFP expressing cells were sorted three times over a period of 4 weeks by flow cytometry until more than 95% of the cells is stable GFP positive. To infect THP-I cells, the stable Phoenix cells were seeded in THP-I culture medium for 24 h and then the culture supernatants containing retroviral particles were collected and filtered through 0.45 μm membrane. THPl cells (1 x 10⁶ cells/well) were then centrifuged in 6-well plate for 60 min at 2500 rpm at 32⁰C in the presence of 3 ml of retrovirus-enriched culture supernatant supplemented with 4 μg/ml of polybrene (Sigma). Plates were placed back in a CO₂ incubator at 37⁰C for 2 h. Fresh THP-I medium was then added and the cells were allowed to recover for 24 h. The cells were subjected to another cycle of infection and then

11058275.3 61 allowed to recover for 72h before sorting by flow cytometry. The GFP expressing cells were sorted three times over a period of 4 weeks until more than 95% of the cells is stable GFP positive. The stable expression of ASC-GFP was verified by western blotting and functional assays.

[00305] Generation of Stable HEK293T Cell Lines.

[00306] HEK293T cells were cultured in DMEM/F-12 supplemented with 10% fetal bovine serum, 200 μg»ml-l penicillin and 100 μg»ml-l streptomycin sulfate. 293-caspase-l, and 293-cells have been described before (Yu et al., 2006). To generate the 293-caspase-l -ASC- EGFP-Nl (293-Cl-ASC-GFP) and 293-caspase-l-EGFP-Nl (293-Cl-GFP) stable cell lines, the parental 293-caspase-l cells were transfected with pEGFP-Nl-ASC (Yu et al., 2006) or pEGFP- Nl plasmids respectively. The stable GFP expressing cells were then sorted three times over a period of 4 weeks by flow cytometry until more than 95% of the cells were GFP positive.

[00307] Live-cell Bioimaging.

[00308] THP-I-ASC-GFP cells were seeded in 35 mm cover glass bottom culture dishes and then primed with PMA (0.5 μM) for 3h and allowed to attach for 24 h. Time-lapse imaging was performed on an LSM 510 META Confocal Microscope System (Carl Zeiss) equipped with a temperature and Cθ₂-controlled sample chamber for live-cell imaging. The GFP protein were excited with the 488 nm Argon laser. The nuclear Hoecht 33342 stain was excited with the 405 nm diode laser. The mitotracker red was excited with the 543 nm He/Ne Laser 1. Ten minutes after crude LPS (5 μg/ml) stimulation, images from the GFP, Hoecht and mitotracker fluorescence signals were recorded simultaneously every 17.5 seconds for an additional 30 minutes. Static bioimaging was done with wide-field fluorescence microscope or LSM 510 META Confocal Microscope.

[00309] ASC pyroptosome quantitation in live cells.

[00310] THP-I-ASC-GFP cells were seeded in 12- well plates and then primed with PMA

(0.5 μM) for 3h. After priming the PMA containing medium was replaced with fresh medium and the cells were allowed to attach to the plates for 24 h. The cells were pretreated with zVAD- FMK (50 μM) for 30 min to prevent cell death, and then treated with different agents and observed by fluorescent microscopy at different periods of time after treatment. The number of cells containing ASC-GFP pyroptosomes were counted in several fields. The percentage of cells

11058275.3 62 with ASC pyroptosomes was calculated by dividing the number of cells with ASC pyroptosomes over the total cells counted.

[00311] Treatment of THP-I cells and assay of LDH and IL-lβ Secretion.

[00312] PMA primed THP-I cells were treated with ultrapure LPS (100 ng/ml) for 1 h to induce the synthesis of pro-IL-lβand then treated with different agents for different periods of time. The culture supernatants were collected at different time points and assayed for LDH with the CytoTox96 LDH-release kit (promega), and IL-lβ by enzyme-linked immunosorbent assay (ELISA) (R&D systems, Minneapolis, MN, USA), as described by the manufacturer's protocols.

[00313] Purification of ASC pyroptosomes from LPS-stimulated macrophages and chemical cross-linking.

[00314] THP-I cells were treated with PMA and seeded in 10 cm dishes. Three hours after PMA treatment the medium containing PMA was replaced with fresh medium and the cells were allowed to attach to the plates overnight. Next day cells were preincubated with zVAD- FMK (50 μM) for 30 min and then treated with LPS (5 μg/ml) for 3h. Cells were harvested and then lysed in buffer A (20 mM Hepes-KOH, pH7.5, 10 mM KCl, 1.5 mM MgCl₂, 1 mM EDTA, 1 mM EGTA, 320 mM sucrose). The cell lysate was centrifuged in 1.5 ml Eppendorff tubes at 1500 rpm to remove the bulk nuclei and the resulting supernatant was diluted 2X with buffer A and then filtered using a 5 micron filter to remove any remaining nuclei. The supernatant was diluted with one volume CHAPS buffer (20 mM Hepes-KOH, pH 7.5, 5 mM MgCl₂, 0.5 mM EGTA, 0.1 mM PMSF, 0.1 % CHAPS) and then centrifuged at 5000 rpm to pellet the ASC pyroptosomes. The crude pellet was resuspended in CHAPS buffer, and either was chemically cross-linked using the non-cleavable Disuccinimidyl suberate (DSS) cross linker (4 mM) for 30 minutes, or subjected to further purification. To further purify the ASC pyroptosomes, the crude 5000 rpm pellet from above was resuspended in CHAPS buffer, layered over a 40% percoll cushion and centrifuged at 14000 rpm in an Eppendorff table top centrifuge. The pelleted ASC pyroptosomes at the bottom of the 40% percoll cushion were then washed in CHAPs buffer and centrifuged again at 12,000 rpm. The purified ASC pyroptosomes were resuspended in CHAPS buffer and used for different assays. ASC pyroptosomes were isolated from bone marrow derived mouse macrophages after stimulation with LPS (3 h) followed by ATP (1 h) in the absence of zVAD-FMK and then cross-linked as described above.

11058275.3 63 [00315] Most of the centifugation are performed in a Eppendorff table top centrifuge uses a 8.5 cm radius centrifugation rotor, except for the ultracentrifugation and the initial harvesting of the macrophages.

[00316] In vitro assembly and purification of ASC pyroptosomes from THP-I cell lysates.

[00317] THP-I cell pellets were lysed in CHAPS buffer and then centrifuged at 14,000 rpm to obtain crude lysates. SlOO lysates were prepared from the crude lysates by centrifugation at 100,000 x G for 30 min. The lysis of the macrophages and the various centrifugation are conducted at 4°C. Buffers are chilled before use. The lysate is allowed to warm up to 37°C for the AS pyroptosome formation assay. ASC pyroptosomes were in vitro assembled by incubation of SlOO lysates at 37° C for 30 min. The assembled ASC pyroptosomes were pelleted from the lysates by centrifugation at 5000 rpm. The pellets were resuspended in CHAPS buffer, layered over a 40% percoll cushion and centrifuged at 14,000 rpm. The pelleted ASC pyroptosomes at the bottom of the 40% percoll cushion were then washed in CHAPs buffer and centrifuged again at 12,000 rpm. The purified ASC pyroptosomes were resuspended in CHAPS buffer and used for different assays.

[00318] In vitro caspase-1 activation and IL-lβ cleavage Assays. Flag-tagged procaspase-

1 (WT and C/A) and untagged pro-IL-lβ were isolated from 293-caspase-l or 293-pro-IL-lβ stable cell lines.

[00319] Purified ASC pyroptosomes were incubated with procaspase-1 together with pro-

IL-lβ in CHAPS buffer for different periods of time at 37° C. The reaction mixtures were then fractionated by SDS-PAGE and analyzed by western blotting with anti-Flag and anti-IL-lβ antibodies.

[00320] Expression and purification of recombinant ASC from bacteria. WT ASC or chimeric ASC proteins were expressed in bacteria with N-terminal His-tag using the pET-28a vector. ASC was purified by standard procedures using Talon affinity beads.

[00321] Generation of Antibodies. The anti-human caspase-1 polyclonal antibody was raised in rabbits against a bacterially produced protease domain (p30) of human caspase-1 (amino acids 120-402). The anti-IL-lβ monoclonal antibody (32D) was obtained from the NCI preclinical repository, Biological resource branch. Anti human and mouse ASC antibodies were obtained from J. Sagara (Japan). Anti-human cryopyrin monoclonal antibody (anti-Nalpy-3b) was obtained from Alexis.

11058275.3 64 [00322] Retrovirus Infection of THP-I and Assay of IL- lβ Secretion and Caspase-1

Activation

[00323] THP-I cells were infected with culture supernatants containing retroviral particles produced in Phoenix cells as described in the supplementary information. 24 h after infection, the culture supernatants were collected and assayed for IL- lβ by enzyme-linked immunosorbent assay (ELISA) (R&D systems, Minneapolis, MN, USA). In some experiments, THP-I cells were transfected with the pyrin specific HS_MEFV_2_HP siRNA (Qiagen) using Amaxa Nucleofector™ (Amaxa, Cologne, Germany) method according to the manufacturer' s protocol. 48h after transfection, the cells were infected with MSCVgfp retrovirus for 24h and then the culture supernatants were collected and assayed for IL-lβby ELISA. In some experiments the cell pellets were collected, lysed and analyzed by western blotting with anti- human caspase-1, pyrin, IL- lβ, or PSTPIPl antibodies.

[00324] Caspase-1 Processing and IL-lβCleavage Assays in HEK293 Cells

[00325] Confocal and Fluorescence Microscopy

[00326] The 293-ASC-EGFP-Nl cells were seeded on cover slips or in 6-well plates and then transfected with empty vector or pyrin or cryopyrin expression constructs together with or without PSTPIPl expression plasmids. The transfections were done using Lipofectamine PLUS- reagent (Invitrogen) according to the manufacturer's instructions. In some experiments 293- caspase-1 -ASC-EGFP-Nl cells or 293-ClP-ASC-EGFP-Nl cells were seeded in 6-well plates and then transfected with empty vector or different PSTPIPl plasmids as above. After 24-48 h of transfection cells were stained with DAPI. Cells on cover slips were observed using a Zeiss LSM 510 Meta confocal microscope, while cells in 6-well plates were observed with a fluorescent microscope.

[00327] Full-length cDNAs and Expression Constructs

[00328] The mammalian expression plasmids for full-length human pyrin (pcDNA-pyrin- myc-His), cryopyrin (pcDNA-cryopyrin-Flag), pro-IL-lβ (pcDNA-pro-IL-lβ) and ASC (pMSCVpuro-ASC) were described previously (Yu et al., 2006). Full-length cDNAs for WT and the disease-associated mutants of human PSTPIPl, A230T and E250Q were generated by PCR using the IMAGE clone 4180398 (Genbank accession # BC008602) from Invitrogen as a template. For expression studies in 293T cells, each PSTPIPl cDNA was cloned into the mammalian expression vector pcDNA3 with C-terminal T7 or Flag epitope tags. For expression

11058275.3 65 studies in THP-I cells, each PSTPIPl cDNA was cloned without tags into the Hpa I site of a modified retrovirus expression vector, pMSCVgfp, in which the puromycin selection marker was replaced with GFP. cDNAs for the C-terminal truncated pyrin 1-580, pyrin 1-410 and pyrin 1-343 were generated by PCR using the pcDNA-pyrin-myc-His plasmid (Yu et al., 2006) as a template. The cDNAs were then cloned into the Nde I/Xho I sites of the bacterial expression vector pET-21a (+) in-frame with the vector's C-terminal His6 tag to generate pET-21a-pyrinl- 580, pET-21a-pyrinl-410 and pET-21a-pyrin 1-343 plasmids, or the Nhe I/Xho I sites of the mammalian expression vector pcDNA3.1-myc-His (-) B (Invitrogen) in-frame with the vector's C-terminal myc-His tag to generate the pcDNA-pyrinl-580-myc-His, pcDNA-pyrinl-410-myc- His and pcDNA-pyrinl-343-myc-His plasmids. The full-length pyrin in pET-21a (pET-21a- pyrin-myc-His) was generated by removing the 3' Sac Il/Xho I Fragment from pET-21a-pyrinl- 580 and replacing it with the Sac IU AfI II fragment from pc-DNA-pyrine-myc-His plasmid after blunting the Xho I and AfI II ends. cDNAs for the truncated pyrin mutants pyrin-LN-BB-CC, pyrin-LN-BB and pyrin-LN, or the pyrin-Trim5α chimeras PT-CC-SPRY and PT-BB-CC- SPRY were generated by PCR using pcDNA-pyrin-myc-His plasmid and pcDNA-Trim5α as a template. The cDNAs were then cloned into appropriate sites of the bacterial expression plasmid pET21a or mammalian expression plasmid pcDNA3.1 -myc-His (-) B. pMSCVpuro-ASC-EGFP- Nl plasmid was generated by excising the ASC-GFP fusion cDNA from the pEGFP-Nl-ASC construct (Yu et al., 2006) with BgI II and Not I and inserting it in the BgI II and Hpa I sites of pMSCVpuro after blunting the Not I end. pMSCVgfp-ASC was generated from pMSCVpuro- ASC by excising the puromycin selectable marker and replacing it with GFP. The nucleotide sequences of all constructs were confirmed by automated sequencing.

[00329] Generation of Antibodies

[00330] The anti-pyrin polyclonal antibody was raised in rabbits against the N-terminal

343 amino acids of pyrin. Pyrinl-343 was produced in bacteria with a C-terminal His6 tag to facilitate purification using the pET-21a-pyrinl-343 plasmid. The anti-PSTPIPl polyclonal antibody was raised in rabbits against the full-length protein. Full-length PSTPIPl was produced in bacteria with a C-terminal His6 tag using the pET-2 Ia-PSTPIPl plasmid. The human anti- caspase-1 antibody was a kind gift from Dr. Douglas Miller (Merck). The anti-IL-lβ monoclonal antibody (32D) was obtained from the NCI preclinical repository, Biological resource branch.

[00331] Generation of Stable THP-I Cell Lines

11058275.3 66 [00332] The monocytic cell line THP-I was cultured in RPMI 1640 supplemented with

10 mM N-(2-hydroxyethyl) piperazine-N'-(2-esthanesulfonic acid), 1 mM sodium pyruvate, 55 μM β-mercaptoethanol, 10% fetal bovine serum and 200 μg-ml-1 streptomycin sulfate. The amphotropic packaging cell line Phoenix (G.P. Nolan's laboratory, Stanford University medical Center, Stanford, CA) was transfected with the empty vector pMSCVgfp or pMSCVgfp- PSTPIPl (WT, A230T, E250Q) or pMSCVpuro- ASC-EGFP-Nl vectors using the LipofectAMINE transfection method. Forty-eight hours after transfection, the GFP expressing cells were sorted three times over a period of 4 weeks by flow cytometry until more than 95% of the cells were GFP positive. To infect THP-I cells, the stable Phoenix cells were seeded in THP- 1 culture medium for 24 h and culture supernatants containing retroviral particles were collected and filtered through 0.45 μm membrane. THP-I cells (1 x 10⁶ cells/well) were then centrifuged in 6-well plates for 60 min at 2500 rpm at 32⁰C in the presence of 3 ml of retrovirus-enriched culture supernatant supplemented with 4 μg/ml of polybrene (Sigma). Plates were placed back in a CO₂ incubator at 32⁰C for 2 h. Fresh THP-I medium was then added and the cells were allowed to recover for 24 h. The cells were subjected to another cycle of infection and then allowed to recover for 72 h before sorting by flow cytometry. The GFP-expressing cells were sorted three times over a period of 4 weeks until more than 95% of the cells were stable GFP positive. The stable expression of the various transgenes was verified by western blotting.

[00333] Generation of Stable HEK293T Cell Lines

[00334] HEK293T cells were cultured in DMEM/F-12 supplemented with 10% fetal bovine serum, 200 μg-ml-1 penicillin and 100 μg-ml-1 streptomycin sulfate. 293-caspase-l, 293- caspase-1-ASC, 293-ASC and 293-ASC-EGFP-N! cells have been described before (Yu et al., 2006). To generate the 293-caspase-l -ASC-pyrin (293-ClAP) and 293-caspase-l-ASC- cryopyrin (293-ClAC) stable cell lines, the parental 293-caspase-l -ASC cells were transfected with pcDNA-pyrin-myc-i-His or pcDNA-cryopyrin-Flag plasmid together with a construct containing the hygromycin selectable marker (pMSCVhygro) using LipofectAMINE (Invitrogen). After a few weeks of selection in hygromycin containing media stable 293-ClAP and 293-ClAC clones were isolated and characterized. The expression of pyrin and cryopyrin in the selected clones was verified by western blot analysis. To generate the 293-caspase-l -pyrin stable cells, the parental 293-caspase-l were transfected with pcDNa-pyrin-myc-His and pMSCVhygro and stable cell clones were selected as described above. The 293-C1P-ASC- EGFP-Nl stable cell line was generated from the parental 293-caspase-l -pyrin by transfection with pMSCVpuro- ASC-EGFP-Nl constructs. The stable GFP expressing cells were then sorted

11058275.3 67 three times over a period of 4 weeks by flow cytometry until more than 95% of the cells were GFP positive. The 293-caspase-l-ASC-EGFP-Nl was generated from the 293-caspase-l cells by transfection with pMSCVpuro- ASC-EGFP-Nl followed by sorting by flow cytometry as described above.

[00335] Immunoprecipitation and Pull-down Assays

[00336] 293T cells were transfected with pcDNa-pyrin-myc-His plasmid together with empty vector or pcDNA3-PSTPIP-l-Flag expression plasmids encoding WT, A230T or E250Q PSTPIPl variants in 100 mm dishes and 24 h after transfection the transfected cells were lysed by syringing (20X) in an IP buffer (20 mm Hepes, pH 7.4, 10 mM KCl, 1.5 mM MgCl₂, 1 mM EDTA, 0.1 mM PMSF, 2 μg/ml leupeptin, 1 mM Na₃VO₄, 5 mM NaF) and centrifuged at 16,000 x G for 10 min. The pyrin-PSTPIPl complexes were immunoprecipitated from the lysates with an anti-pyrin antibody and immobilized on protein G-sepharose beads. The bead- bound proteins were then fractionated by SDS-PAGE and immunoblotted with anti-pyrin or anti-Flag antibodies.

[00337] In vitro pull-down assays were performed with bacterially expressed GST-tagged

PYD of pyrin. GST or GST-PYD were isolated from bacterial lysates by gluthathione-affinity purification on glutathione-agarose beads. The bead-bound proteins were then incubated for 2 h at 4⁰C with in vitro translated 35S-methione-labeled mutant pyrin proteins. After incubation the complexes were fractionated by SDS-PAGE and detected by autoradiography.

[00338] Example 1. LPS induces formation of an ASC supramolecular assembly in THP-

1 macrophages.

[00339] Crude LPS preparations from gram-negative bacteria or bacterial lipoproteins have been shown to induce caspase-1 activation and a pyroptosis-like cell death in human THP- 1 macrophages (Aliprantis et al., 2001a; Aliprantis et al., 2001b; Aliprantis et al., 1999; Aliprantis et al., 2000). However, the mechanism by which caspase-1 is activated in these cells, and the role of ASC were not determined. As a first step to characterize the role of ASC in this model of inflammatory cell death, a THP-I macrophage cell line that stably expresses physiological amounts of an ASC-GFP fusion protein was generated (data not shown, Fig. IA). Live PMA primed THP-I-ASC-GFP cells were left untreated or treated with LPS (1 μg/ml) or MSU (100 μg/ml) for 60 min and observed by fluorescence microscopy (2OX magnification). PMA primed THP-I-ASC-GFP cells seeded on glass cover slips were treated with crude LPS for 60 min and then fixed and stained with DAPI. Cells were then observed and photographed

11058275.3 68 by fluorescence confocal microscopy (63X magnification). There was uniformly distributed green fluorescent ASC-GFP in one cell that did not form ASC pyroptosome and the large perinuclear green fluorescent ASC pyroptosomes formed in three other cells. The data indicate that pro-inflammatory stimuli induce formation of ASC pyroptosome in THP-I cells.

[00340] In unstimulated cells, ASC-GFP was evenly distributed in the cytoplasm and nucleus. However, after stimulation with crude E. coli LPS for 1 h, the entire ASC-GFP fluorescence accumulated as distinct bright clusters or oligomers in the cytoplasm of -30 % of the treated cells (data not shown). These oligomers are large and measured about 2 μm in diameter and only one oligomer is formed in each cell (data not shown). These ASC oligomers were designated pyroptosomes, since all cells containing these oligomers showed morphological and biochemical characteristics of pyroptosis (see below).

[00341] Quantitative analysis of the number of cells with ASC pyroptosomes revealed that the number of cells containing these pyroptosomes increased in a time and dose-dependent fashion after stimulation with LPS (Fig. IB). At moderate LPS concentrations, more than 90% of pyroptosome formation occurred within the first 2.5 h after drug treatments. Formation of the ASC pyroptosomes did not require new protein synthesis as pre-incubation of cells with the protein synthesis inhibitor cycloheximide did not inhibit their formation in response to LPS treatment (Fig. ID). Since the cytotoxic effect of crude LPS preparations is attributed to contamination with bacterial lipoproteins (Aliprantis et al., 2001a), synthetic bacterial lipopeptide analogs Pam3CSK4 and FSL-I were tested too, which have been shown to induce cell death in THP-I cells (Aliprantis et al., 2001a), to ascertain if they could also induce formation of the ASC pyroptosome. Treatment of the THP-I-ASC-GFP cells with these lipopeptides also induced a time-dependent formation of ASC pyroptosomes (Fig. 1C). These results thus indicate that bacterial lipoproteins in crude LPS preparations are likely responsible for induction of the ASC pyroptosome. Consistent with this conclusion, stimulation of the THP- 1 -ASC-GFP cells with ultrapure LPS induced very little ASC pyroptosomes, compared to crude LPS (data not shown). In addition, treatment of these cells with MDP, another common contaminant of LPS, did not induce ASC pyroptosome formation (data not shown).

[00342] LPS and bacterial Pam3CSK4 or FSL-I lipopeptides have been shown recently to induce caspase-1 activation and IL- lβ secretion from mouse bone marrow macrophages independent of the cryopyrin inflammasome (Kanneganti et al., 2006b). To investigate if signals that activate the cryopyrin inflammasome could also induce the formation of the ASC pyroptosome, THP-I-ASC-GFP cells were treated with the danger signal MSU or the antiviral

11058275.3 69 compound R837, both of which require cryopyrin to activate caspase-1 (Kanneganti et al., 2006b; Martinon et al., 2006). As shown in Fig. 1C, MSU and R837 were also able to induce ASC pyroptosome formation in a dose and time dependent manners. Combined, these results indicate that formation of the ASC pyroptosome can be induced by both cryopyrin and non- cryopyrin-dependent signals.

[00343] Example 2. Formation of the ASC pyroptosome is rapid and precedes cell death.

[00344] To observe the dynamics of assembly of the ASC pyroptosome, time-lapse confocal imaging on LPS-stimulated THP-I-ASC-GFP cells was performed. Formation of the ASC pyroptosome causes cell death in THP-I cells. Time series confocal images (63X magnification) of crude LPS-stimulated THP-ASC-GFP cells in the absence or presence of z V AD-FMK (70 μM). Time-lapse movies showing the assembly of the ASC pyroptosome in the absence or presence of zV AD-FMK were recorded.These experiments revealed that the entire process from the start until the complete assembly of the whole ASC pyroptosome in a single cell takes less than 3 minutes. Within seconds after ASC pyroptosome formation the plasma membrane ruptures releasing cellular contents and membrane vesicles in the culture supernatants. The plasma membrane then appears to rapidly reseal and starts to swell to form a balloon shaped vesicle around the nucleus, which also undergoes dramatic rounding and condensation. The mitochondria also lose membrane potential and release their contents as evidenced by the concurrent loss of the mitochondrial vital stain mitotracker. These phenotypic features were also observed in the parental THP-I macrophages after treatment with LPS (data not shown), which indicates that oligomerization of ASC causes cell death in THP-I macrophages.

[00345] To determine whether the LPS-induced pyroptosome formation and cell death is dependent on caspase activation, cells were pretreated with the pancaspase inhibitor zVAD- FMK and then stimulated with LPS. Pretreatment with zV AD-FMK blocked all the morphological features of cell death but did not affect ASC pyroptosome formation in response to LPS treatment (data not shown). These data indicate that LPS-induced cell death is caspase- dependent. In contrast, pyroptosome formation per se is not dependent on caspase activation. The morphological features of this form of cell death are clearly a combination of apoptosis (condensation of the nucleus and loss of mitochondrial membrane integrity) and oncosis (plasma membrane swelling). Since this form of cell death is associated with caspase-1 activation and secretion of inflammatory cytokines (see below), it is refered to as pyroptosis (Fink and Cookson, 2005). Plasma membrane swelling and rupture triggered by formation of the ASC

11058275.3 70 pyroptosome might be a mechanism by which dying macrophages release their cellular contents of inflammatory cytokines such as IL- lβ. Indeed, stimulation of the parental THP-I cells with different proinflammatory agents induced concomitant cell death as measured by LDH release and IL-lβ release in the culture media (Fig. 2A and B). PMA-primed parental THPl or THP-I- ASC-GFP cells were pretreated for Ih with ultrapure LPS to induce pro-IL-lβ and then treated with crude LPS (1.0 μg/ml), R837 (10 μg/ml) or Pam3CSK4 (0.5 μg/ml) for the indicated periods of time (Fig. 2A and B). Similar results were obtained with THP-I-ASC-GFP cells (data not shown). Collectively, our data indicate that pro-inflammatory stimuli cause a pyroptotic cell death in THP-I macrophages by inducing the formation of an ASC pyroptosome. This form of cell death is responsible for the release of the mature IL-lβ cytokine from activated macrophages.

[00346] Example 3. Caspase-1 is the apical caspase in the macrophage pyroptotic cell death pathway.

[00347] The rapid activation of pyroptotic cell death after formation of the ASC pyroptosome and the ability of the pan caspase inhibitor to block this form of cell death but not the formation of the ASC pyroptosome, indicates that the ASC pyroptosome is responsible for activation of a cellular caspase that causes cell death. Considering that ASC can only associate with procaspase-1 (Srinivasula et al., 2002), it is likely that activation of caspase-1 by the ASC pyroptosome is responsible for the pyroptotic cell death observed in THPl cells. To test this hypothesis, a biochemical procedure was devised to isolate the ASC pyroptosome from the parental THP-I macrophages after LPS stimulation. Since the diameter of the ASC-GFP pyroptosome in the THP-I-ASC-GFP cells is ~2 μm, we estimated that the diameter of the untagged endogenous ASC pyroptosome in the parental THP-I cells could be in the range of ~ 1 μm. Therefore, it is possible to separate the ASC pyroptosome from nuclei by a combination of membrane filtration through a 5 micron filter to remove nuclei, and centrifugation at relatively low speed (5000 rpm; -2200 x G) to pellet the ASC pyroptosomes. This procedure was performed on control and LPS-stimulated THP-I cells that were previously preincubated with z V AD-FMK to prevent cell death and trap the apical caspase on the ASC pyroptosome. The resulting cell-free pellets were then subjected to chemical cross-linking with the non-cleavable protein cross-linking agent Disuccinimidyl suberate (DSS) to determine the oligomeric state of ASC in the control and LPS-stimulated cells.

[00348] The ASC pyroptosomes present in the lysates (L) were pelleted (P) by centrifugation at 5000 rpm as described under "METHODS". The lysates (L) and pellets (P)

11058275.3 71 were then fractionated by SDS-PAGE and western blotted with anti-ASC (upper panel) or anti- caspase-1 (lower panel) antibodies. The pellets in 3rd and 6th lanes were incubated with DSS for 30 min before SDS-PAGE. As shown in Fig. 3A upper panel, ASC pyroptosomes were predominantly present in the pellets from the LPS -stimulated cells (5th lane) compared to the control unstimulated cells (2nd lane), which contained very small amount of ASC. Chemical cross-linking of the isolated pellets yielded cross-linked ASC oligomers in the pellets from the LPS-treated cells (6th lane), but not from the control untreated cells (3rd lane). The ASC dimer was the major cross-linked species, indicating that the ASC pyroptosome is composed of oligomerized ASC dimers. The ASC pyroptosomes were associated with unprocessed and processed caspase-1 p35 fragment as determined by western blotting with a specific caspase-1 antibody (Fig. 3A, lower panel), indicating that the ASC pyroptosomes recruit and activate caspase-1 after LPS-stimulation of THP-I cells.

[00349] To provide additional evidence that caspase-1 is the apical caspase in the pyroptotic cell death pathway triggered by formation of the ASC pyroptosome, stable HEK293 cells expressing physiological amounts of ASC-GFP protein alone (293-ASC-GFP cells), or ASC-GFP and procaspase-1 together (293-Cl-ASC-GFP)were generated. The 293 stable cell lines were left untreated or treated with PMA to induce formation of ASC pyroptosomes. Cells were then observed by confocal microscopy (63X magnification). The pyroptotic phenotype induced by ASC pyroptosome in the 293-Cl-ASC-GFP cells, which express both procaspase-1 and ASC-GFP, but not in the 293-ASC-GFP, which express only ASC-GFP, or 293-Cl-GFP, which express only procaspase-1 and GFP. Stimulation with PMA induced formation of ASC pyroptosomes in both cell lines (data not shown). However, only cells expressing both ASC- GFP and procaspase-1 (293-Cl-ASC-GFP) showed morphological features of pyroptosis. The 293-ASC-GFP cells, which do not express caspase-1 remained viable even after formation of the ASC pyroptosome (data not shown). Control HEK293 cells, stably transfected procaspase-1 and GFP alone (293-Cl-GFP), but no ASC, was also not sensitive to PMA-induced pyroptosis. These results indicate that caspase-1 activation by the ASC pyroptosome induces pyroptosis not only in macrophages but also in other cell lines like HEK293 cells, in presence of caspase-1.

[00350] To further investigate the role of caspase-1 in the pyroptotic process, bone marrow macrophages was isolated from WT and caspase-1 -/-mice and stimulated them with LPS for 3 h followed by ATP for Ih. Unlike human THP-I cells, stimulation of primary mouse macrophages with LPS alone does not induce robust caspase-1 activation and requires an ATP stimulus to activate caspase-1 (Mariathasan et al., 2006). Stimulation with LPS plus ATP

11058275.3 72 induced cell death in WT macrophages but not in caspase-1 -/-cells as revealed by LDH release (Fig. 3B). Confocal micrographs of the bone marrow macrophages from WT or caspase-1-/- mice treated with LPS plus ATP was observed. The pyroptotic phenotype in the LPS plus ATP- treated WT, but not the caspase-1 -/- cells. The morphological features of cell death in these cells resembled THP-I cell death, with nuclear condensation and plasma membrane swelling (data not shown), indicating that these cells die by pyroptosis, which is mediated by caspase-1 activation. As with LPS-stimulated THP-I cells, LPS plus ATP- stimulated WT and caspase-1- deficient mouse macrophages contained ASC pyroptosomes, as revealed by chemical-cross linking (Fig. 3C, 6th and 12th lanes). These, results indicate that stimulation with LPS plus ATP induces formation of ASC pyroptosomes in both WT and casp-1 -/-macrophages. However, formation of ASC pyroptosome alone does not induce cell death, but requires the presence of procaspase-1 to produce active caspase-1, which in turn processes its physiological substrate pro-IL-lβ and cause cell death to release the active IL- lβ cytokine from the macrophages. Thus, caspase-1 is the apical caspase in pyroptotosis and inflammation.

[00351] Example 4. The ASC pyroptosome is a potent caspase-1 activation platform.

[00352] To provide direct biochemical evidence that the ASC pyroptosome is indeed capable of activating procaspase-1, ASC pyroptosomes was purified from LPS-stimulated THP- 1 cells. The purified ASC pyroptosomes were then incubated with recombinant WT or active site mutant (C285A) procaspase-1. ASC pyroptosomes purified from LPS-stimulated THP-I- ASC-GFP cells were incubated with Flag-tagged WT or active site mutant (C/A) procaspase-1 together with pro-IL-lβat 37⁰C for 20, 40, or 60 minutes as indicated. The reaction products were then analyzed by SDS-PAGE and western blotting with anti-Flag (upper panel) or anti-IL- lβ (lower panel) antibodies. As shown in Fig. 4A, purified ASC pyroptosomes induced activation of the WT caspase-1, but not the C285A caspase-1 mutant. Only WT caspase-1 (1st to 4th lanes), but not active site mutant caspase-1 (C/A) (5th-8th lanes), can be activated by ASC pyroptosomes. The asterisks indicate a non-specific band. The activated WT caspase-1 was able to process pro-IL-lβ to the mature IL- lβ cytokine, indicating that purified ASC pyroptosome is responsible for activating caspase-1 and generation of mature IL- lβ during pyroptosis. The kinetics of assembly of the ASC pyroptosome in LPS-stimulated THP-I-ASC-GFP cells and the fact that only one pyroptosome per cell is formed indicates that the assembly process is driven by self-association of the ASC monomers to form dimers, which then oligomerize to form a large ASC pyroptosome. To test if this process can be recapitulated in vitro by incubation of lysates from THP-I cells at 37⁰C, cell lysates from THP-I-ASC-GFP and the parental THP-I

11058275.3 73 cells were prepared in a hypotonic buffer. The resulting lysates were incubated on ice or at 37⁰C for 30 min in the presence of rhodamine-tagged zVAD-FMK (red-zVAD) to label and trap activated caspase-1 on the ASC pyroptosome. The lysates were then centrifuged at 5000 rpm to pellet the ASC pyroptosomes, and the pellets containing ASC pyroptosomes were further purified by centrifugation over a 40% percoll cushion. Examination of the purified pellets by fluorescent confocal microscopy revealed that incubation at 37⁰C induced formation of ASC pyroptosomes in these lysates (data not shown). Confocal micrographs (6X magnification) of ASC-GFP pyroptosomes purified from THP-I-ASC-GFP cell lysates, or endogenous ASC pyroptosomes from THP-I cell lysates. The ASC-pyroptosomes were isolated in the presence of rhodamine-labeled zVAD-fmk to label the associated caspase-1. Highly organized star-shaped structure of the ASC pyroptosome. These ASC pyroptosomes bound red-zVAD-FMK indicating that they contain activated caspase-1. The shape of these ASC pyroptosomes were similar to those formed in vivo in LPS-stimulated THP-ASC-GFP cells, but were larger in size and measured ~3 μm in diameter. The in vitro assembled ASC pyroptosomes from both THP-I and THP-I-ASC-GFP cells have star- shaped crystal-like (quasicrystal) structure, indicating that they are not merely aggregates of ASC. Interestingly, the ASC-GFP pyroptosomes have sharper spikes than the ASC pyroptosomes, possibly because of the presence of GFP at the C-terminus of ASC. No ASC pyroptosomes were seen in lysates incubated on ice (not shown). These results indicate that ASC pyroptosomes can form spontaneously in cell free lysates by mere incubation at 37⁰C. These results also explain previous observations which showed that caspase-1 is spontaneously activated in hypotonic THP-I lysates after incubation at 37⁰C (Martinon et al., 2002).

[00353] To determine whether the in vitro assembled ASC pyroptosomes could activate caspase-1, purified ASC pyroptosomes from 37°C-activated THP-I lysates were incubated with recombinant WT or active site mutant (C285A) procaspase-1. As expected, the purified ASC pyroptosomes activated WT procaspase-1, but not the active site mutant (C285A) procaspase-1 (Fig. 4B, upper panel). The active WT caspase-1 was able to process pro-IL-lβ to the mature IL- lβ cytokine (Fig. 4B, lower panel). Combined, these results indicate that the ASC pyroptosome is indeed responsible for activation of caspase-1.

[00354] To determine the composition of the ASC pyroptosome, large preparations of purified pyroptosomes from either THP-I (Fig. 4C, lane 1) or THP-ASC-GFP (Fig. 4C, lane 2) cells were subjected to SDS-PAGE and Coomassie staining. Notice the presence of endogenous ASC (lane 2, middle band) in the ASC-GFP pyroptosome preparation. The ASC-GFP (lane 2,

11058275.3 74 top band) migrates slightly above the 45 kDa marker. The bottom band in lanes 1 and 2 is an endogenous short isoform of ASC. The pyroptosome preparation from THP-I cells contained two distinct bands migrating as 19 and 25 kDa species (Fig. 4C, lane 1), which were identified by western blotting and mass spectroscopy to be full length ASC (25 kDa band) and a short isoform of ASC (19 kDa band). Similarly, the pyroptosome preparation from THP-I-ASC-GFP cells contained the 25 kDa and 19 kDa ASC species together with the ASC-GFP fusion protein which migrates as a -50 kDa band (Fig. 4C, lane T). No high molecular weight species in the range of 90-110 kDa corresponding to cryopyrin or other related family members were detected in these preparations. Very faint bands ranging in sizes between 30 to 50 kDa were also detected in these preparations. These bands correspond to proform and processed caspase-1 as determined by western blotting and mass spectroscopy, which detected only ASC and caspase-1 (Fig. 4D, left panels), but not cryopyrin (Fig. 4D, right panel), in these preparations. The blots were probed with anti-ASC (upper panel, exposure time 15 sec), anti-caspase-1 (lower panel, exposure time 30 sec) or anti-cryopyrin (right panel, exposure time 30 min) antibodies. The 3rd lane in the cryopyrin blot is a positive cryopyrin-containing lysates control from a stable 293 cells expressing cryopyrin. These results demonstrate clearly that the ASC pyroptosome is composed largely of oligomerized ASC dimers and a small amount of caspase-1. The absence of cryopyrin or other NLRs from the purified pyropotosome can not be attributed to dissociation of these proteins during purification, since the pyroptosome purification process, which is a gentle few step process did not dissociate caspase-1 from the pyroptosome. Furthermore, quantitative estimates of the cellular concentrations of ASC (-1.4 μg/mg total cytosolic proteins) and cryopyrin (-15 pg/mg total cytosolic proteins) in THP-I as measured by quantitative immunoblot analysis with specific ASC and cryopyrin antibodies revealed that the molar ratio of ASC to cryopyrin in THP-I cells is -500 to 1. Therefore, it is unlikely that the endogenous ASC pyroptosome would contain equimolar amounts of ASC and cryopyrin, since almost all cellular ASC assembles into one pyroptosome in each stimulated THP-I cell. Collectively, these data support the ASC pyroptosome as the molecular platform responsible for recruitment and activation of caspase-1 during inflammation and pyroptosis.

[00355] Example 5. The ASC pyroptosome is formed by self-association of the pyrin domain of ASC.

[00356] ASC contains two domains, an N-terminal pyrin domain (PYD) and a C-terminal

CARD domain. The N-terminal PYD has been shown to mediate self-association of ASC (Moriya et al., 2005). To determine if the PYD of ASC is responsible for formation of the ASC

11058275.3 75 pyroptosome, a point mutation (K26A) was introduced in the PYD of ASC, which has been previously shown to prevent self-association of the isolated PYD of ASC (Moriya et al., 2005). The WT and mutant ASC proteins were then stably expressed in 293-caspase-l cells. Lysates from stable 293T cells (10 μg/μl) expressing Flag-tagged procaspase-1 and either WT or K26A mutant ASC were activated by incubation at 37⁰C or left at 4⁰C for Ih as indicated. The lysates were then analyzed by SDS-PAGE and western blotted with anti-Flag (upper panel) or anti-ASC (lower panel) antibodies. Incubation of lysates from the WT ASC expressing cells, but not the K26A ASC mutant at 37⁰C induced caspase-1 activation (Fig. 5 A, 2nd lane). [00357] The lysates were also incubated at 4⁰C or 37⁰C for 30 min with the cross-linking agent DSS as indicated. The lysates were then analyzed by SDS-PAGE and western blotted with anti-ASC antibody. Chemical cross-linking of lysates from these cells revealed that only the WT ASC but not the K26A mutant, was able to form ASC pyroptosomes (Fig. 5B, 2nd lane). To provide additional evidence that the PYD is responsible for assembly of the ASC pyroptosome, whereas the CARD domain is needed to recruit procaspase-1, a chimeric ASC molecule which contains ASC PYD (residues 1-100) followed by the CARD domain of Apaf-1 was generated (Fig. 5C), the CARD domain of Apaf-1 interacts with the CARD domain of procaspase-9. This chimeric protein was expressed and purified from bacteria and then assembeled into pyroptosomes and were then analyzed by SDS-PAGE and. Both ASC-APAF and Apaf-1 -591 can activate procaspase-9. Incubation of the chimeric ASC pyroptosomes with procaspase-9 resulted in activation of procaspase-9 (Fig. 5D, 3rd lane). The reaction products were also analyzed by SDS-PAGE and western blotting with anti-Flag (upper panels) or anti-IL-lβ (lower panels) antibodies. As expected the chimeric ASC pyroptosome was able to activate a chimeric procaspase-1, which contains the CARD domain of procaspase-9 instead of its original CARD, but not WT procaspase-1 (Fig. 5E, left panels). Together, these results indicate that ASC forms pyroptosomes by self-association of its PYD, whereas the CARD domain recruits and activates procaspase-1.

[00358] Example 6. The assembly of the ASC pyroptosome is mediated by potassium depletion.

[00359] Unrelated and diverse agents such as LPS, MSU, R837, Pam3CSK4 and FSL-I can induce the formation of the ASC pyroptosome. Despite their distinct mechanisms of action these agents might stimulate a common downstream event that triggers formation of the ASC pyroptosome. One possibility is that these agents alter the physiological milieu of the cell, which is sensed either by a molecule upstream of ASC (i, e., cryopyrin) or by ASC itself, triggering ASC oligomerization. Potassium efflux is a common event induced by a broad range of stimuli

11058275.3 76 and has been shown to play an important role in cell death, caspase activation and IL- lβprocessing (Cain et al., 2001; Mariathasan et al., 2006; Perregaux and Gabel, 1994; Walev et al., 1995; Warny and Kelly, 1999). Given that depletion of intracellular potassium by bacterial toxins has been shown to induce pyroptotic cell death and IL- lβ release from THP-I cells (Warny and Kelly, 1999), could intracellular potassium depletion induces formation of the ASC pyroptosome in macrophages? To answer this question, the effect of inhibiting potassium efflux by increasing the extracellular potassium concentration to 30 or 60 mM was examined. Potassium depletion triggers formation of the ASC pyroptosome in vivo in THP-I-ASC-GFP cells were treated with crude LPS (1 μg/ml) in the absence or presence of the indicated concentrations (mM) of KCl, or the potassium channel blocker TEA for 2h. As shown in Fig. 6A, inhibition of potassium efflux by high extracellular potassium concentrations inhibited LPS- induced formation of the ASC pyroptosome in these cells. High extracellular potassium also blocked pyroptotosis and IL- lβ secretion from THP-I cells as measured by LDH release and ELISA, respectively (Fig. 6B and C). Moreover, high extracellular potassium inhibited Pam3CSK4 and R837-induced ASC pyroptosome formation, pyroptosis and IL-lβsecretion from THP-I cells (data not shown). The potassium channel blocker tetraethylammonium (TEA) has been shown to inhibit IL-lβsecretion from human monocytes (Walev et al., 1995).

[00360] To provide additional evidence that the formation of ASC pyroptosome is dependent on intracellular potassium depletion, the THP-I-ASC-GFP cells were incubated in the presence TEA. As expected, inhibition of potassium efflux by TEA also decreased LPS-induced ASC pyroptosome formation at a concentration as low as 2 mM (Fig. 6A). To provide more direct evidence that potassium depletion induces formation of the ASC pyroptosome in THP-I- ASC-GFP cells we stimulated these cells with Staphylococcus aureus alpha-toxin (SAT). SAT selectively permeabilizes the plasma membrane for monovalent ions and has been previously shown to dramatically decrease intracellular potassium concentrations to below 50 mM in THP- 1 cells (Warny and Kelly, 1999). Treatment of THP-I-ASC-GFP cells with SAT induced ASC pyroptosome formation in more than 65 % of the cells (Fig. 6D). Consistent with the critical role of potassium, treatment of THP-I-ASC-GFP with SAT in the presence of 60 mM extracellular potassium completely inhibited ASC pyroptosome formation. To rule out the possibility that other bacterial contaminants in the Staphylococcus aureus alpha-toxin (SAT) preparation is responsible for activation of the ASC pyroptosome, the effect of digitonin, a mild non-ionic detergent used to permeabilize plasma membrane, on THP-I-ASC-GFP cells was examined (Fig. 6E). Treatment of these cells with low concentration of digitonin (10 μg/ml) induced robust formation of the ASC pyroptosome (Fig. 6E). The effect of digitonin was also inhibited

11058275.3 77 by high extracellular potassium (Fig. 6E). Together, the results indicate that depletion of intracellular potassium by different agents with distinct mechanisms of actions triggers formation of the ASC pyroptosome.

[00361] Example 7. Subphvsiological concentrations of potassium induce ASC oligomerization and enhances recruitment and activation of procaspase-1 by the ASC pyroptosome.

[00362] How does low intracellular potassium concentration induce formation of the ASC pyroptosome? Based on our observations that the PYD of ASC mediates its self-association to form the ASC pyroptosome (Fig. 5), it is possible that low potassium concentrations favor self- association of the ASC PYD. To test this possibility, the effect of different concentrations of potassium on self-assembly of the ASC pyroptosome in vitro was examined. THP-I SlOO extracts were stimulated by incubation at 37⁰C in the presence of increasing concentrations of potassium for 30 minutes and the assembled ASC pyroptosomes were isolated by low speed centrifugation. ASC pyroptosomes were assembled in vitro by incubation of THP-I SlOO extracts (10 μg/μl) at 37⁰C in the presence of increasing concentrations of KCl. The reaction mixtures were centrifuged at 5000 rpm, and the resulting pellets, which contain the assembled pyroptosomes, and the remaining supernatants were then fractionated by SDS-PAGE followed by western blotting with anti-ASC or anti-caspase-1 antibodies. The ASC blot (Fig. 7A, 1st panel from the top, pellet) was exposed for 2 min. The caspase-1 blot (2nd panel from the top, pellet) was exposed for 3h to detect caspase-1. The supernatant blots (3rd and forth panels from the top) were exposed for 2 min. As anticipated, no ASC pyroptosomes were found at high or near physiological potassium concentration (5th and 6th lanes). In contrast, ASC pyroptosomes were found at potassium concentrations below 120 mM with maximum amount at 30 mM (1st to 4th lanes). This experiment demonstrates that indeed, low potassium concentrations favor the assembly of ASC pyroptosomes, whereas physiological concentrations of potassium (150 mM) block assembly in vitro (Fig. 7A, upper panel).

[00363] To further study the effect of potassium on ASC oligomerization and caspase-1 activation, THP-I SlOO lysates was incubated at 37° C in the presence of increasing potassium concentrations and then the status of caspase-1 in the lysates was determined by western blotting (Fig. 7B). Consistent with the above findings, the results of this experiment show clearly that physiological concentrations of potassium inhibit in vitro caspase-1 activation. Chemical cross- linking of the THP-I lysates with DSS during incubation at 37⁰C revealed that the potassium- mediated inhibition of caspase-1 activation is caused by a direct inhibitory effect of potassium

11058275.3 78 on self-oligomerization of ASC (Fig. 7C). Potassium depletion might not only be important for ASC oligomerization, but it could also be important for recruitment and activation of procaspase-1 by the ASC pyroptosome.

[00364] To examine the effect of varying potassium concentrations on the recruitment of procaspase-1 to the ASC pyroptosome, an active site mutant procaspase-1 C285A was incubated with purified pre-formed ASC pyroptosomes in the presence of different potassium concentrations. Purified preformed ASC pyroptosomes were incubated with inactive procaspases-1 mutant (C287A) in the presence of the indicated potassium concentrations at 37⁰C for Ih. The ASC pyroptosomes were then pelleted by low speed centrifugation washed three times and then fractionated by SDS-PAGE followed by western blotting with anti-caspase- 1 (top panel) or anti-ASC (bottom panel) antibodies. As shown in Fig. 7D, increasing potassium concentrations inhibited recruitment of procaspase-1 to the pyroptosome in a dose dependent manner with maximum inhibition observed at 150 mM KCl. Similarly, Purified preformed ASC pyroptosomes were incubated with with WT procaspases-1 in the presence of the indicated potassium concentrations at 37⁰C for Ih. The ASC pyroptosomes were then pelleted by low speed centrifugation washed three times and then fractionated by SDS-PAGE followed by western blotting with anti-caspase-1 (top panel) or anti-ASC (bottom panel) antibodies. Consistent with the previous results with procaspases-1 mutant (C287A), increasing potassium concentrations also inhibited in a dose-dependent manner, the activation of WT procaspase-1 by preformed ASC pyroptosomes (Fig. 7E). The total reaction mixtures in Fig 7E were fractionated by SDS-PAGE followed by western blotting with anti-caspase-1 (top panel) or anti-ASC (bottom panel) antibodies. Together, these results indicate that potassium depletion plays a critical role in macrophage pyroptosis by inducing the formation of the ASC pyroptosome and subsequently enhancing the recruitment and activation of caspase-1. Thus, in the absence of a potassium efflux, procaspase-1 cannot be activated.

[00365] Example 8. In vitro assembly of the ASC pyroptosome using purified recombinant ASC.

[00366] The next question that was addressed was whether the assembly of ASC pyroptosome could be mediated by an indirect effect of potassium depletion on a molecule upstream of ASC (i,e., cryopyrin), which facilitates ASC oligomerization, or is a direct effect on ASC itself. Bacterially expressed ASC purified to complete homogeneity in a buffer containing physiological concentration of potassium (150 mM). In this buffer, ASC was completely soluble. Purified recombinant ASC (10 ng/μl) was incubated at 37⁰C or at 4⁰C in the presence of

11058275.3 79 decreasing concentrations of KCl as indicated. The samples were centrifuged at 5000 rpm, and the resulting pellets (upper panel), which contain the assembled ASC pyroptosomes, and the remaining supernatants (lower panel) were then fractionated by SDS-PAGE followed by Coomassie staining. As shown in Fig. 8A, incubation at 37⁰C in subphysiological concentrations of potassium induced the formation of ASC pyroptosomes which pelleted at 5000 rpm (4th lane). No ASC pyroptosomes were found in the sample that was incubated in 75 mM KCl- containing buffer at 4⁰C, (Fig. 8A, 5th lane) indicating that heat treatment is also an important factor in ASC oligomerization. The reconstituted ASC pyroptosomes have the same shape as the ASC pyroptosomes derived from THP-I cells (not shown). Chemical cross-linking of the assembled ASC pyroptosomes with DSS, demonstrated that they are composed of oligomerized ASC dimers similar to those isolated from THP-I cells (Fig. 8B). Increasing amounts of the assembled recombinant ASC pyroptosomes from A above were incubated with procaspases-1 for Ih at 37⁰C, and then fractionated by SDS-PAGE followed by western blotting with anti- caspase-1 (top panel) or anti-ASC (bottom panel) antibodies. The reconstituted recombinant ASC pyroptosomes were able to activate procaspase-1 in a dose dependent manner (Fig. 8C), indicating that these pyroptosomes are functional. Collectively, these results indicate that subphysiological concentrations of potassium could directly induce ASC oligomerization in the absence of other cellular proteins.

[00367] Example 9. Expression of PAPA-associated PSTPIPl mutants in THP-I induces caspase-1 activation.

[00368] PAPA syndrome, like FMF, is associated with increased generation of IL- lβ and is responsive to treatment with the IL-I receptor antagonist anakinra (Chae et al., 2006; Dierselhuis et al., 2005). The mutant PSTPIPl proteins were tested to evaluate whether the mutant protein attributed to an excessive activation of caspase-1. The effect of retrovirus- mediated transient expression of the disease-associated PSTPIPl mutant proteins in THP-I monocytes on caspase-1 activation and IL- lβ generation were studied. THP-I cells were infected with an empty MSCV retroviral vector, or MSCV retroviruses encoding the indicated WT or mutant PSTPIPl proteins as described under "Materials and methods". The culture supernatants were collected and assayed for IL- lβ by ELISA. Additionally, the cells were lysed and analyzed by western blotting with anti-human caspase-1 (Fig. 9B, upper panel) or PSTPIPl (Fig. 9B, lower panel) antibodies. As shown in Fig. 9A and B, expression of the A230T or E250Q PSTPIPl mutants in THP-I cells using a retroviral vector resulted in substantially more secretion of IL-lβ compared with expression of WT PSTPIPl or an empty vector control. This

11058275.3 80 increased IL- lβ secretion was a result of robust caspase-1 activation by the A230T and E250Q PSTPIPl mutants compared with the WT PSTPIPl or empty vector control, which induced relatively little caspase-1 activation. These results show for the first time that expression of the PAPA-associated PSTPIPl mutants in a human monocytic cell line induces activation of caspase-1, which then processes pro-IL-lβ into the mature IL-lβ cytokine.

[00369] Example 10. Retroviral infection of THP-I induces pyrin and enhances caspase-1 activation by the mutant PSTPIPl proteins.

[00370] While studying the effect of retrovirus-mediated expression of PSTPIPl variants in THP-I cells, it was noticed that retroviral infection, even with an empty retroviral vector, causes a dramatic increase in the expression of the endogenous pyrin and pro-IL-lβ proteins in the infected cells 16-24 h after infection. THP-I cells were infected with a GFP-encoding MSCV retrovirus for 24 h. The cells were then lysed and their lysates analyzed by western blotting with anti-pyrin (upper panel), anti-pro-IL-lβ (middle panel) or anti-β-actin (lower panel) antibodies (Fig. 9C). Therefore, it was possible that pyrin might play an important role in the robust caspase-1 activation and IL-lβ generation by the autoinflammatory PSTPIPl mutants, especially since pyrin has been shown to interact with PSTPIPl (Shoham et al., 2003), and also to induce caspase-1 activation (Yu et al., 2006). The effects of retroviral infection on caspase-1 activation in THP-I cells that stably express WT PSTPIPl or the two PAPA-associated PSTPIPl mutants A230T or E250Q were examined. Stable THP-I cells expressing an empty vector (1st and 5th lanes), or WT (2nd and 6th lanes), A230T (3rd and 7th lanes) or E250Q (4th and 8th lanes) PSTPIPl proteins were left untreated (1st to 4th lanes) or infected with a GFP- expressing MSCV retrovirus for 24 h. The cells were then lysed and the resulting lysates were western blotted with anti-caspase-1 (upper panel), anti-PSTPIPl (middle panel) or anti-pyrin (lower panel) specific antibodies, or the secreted IL-lβ in the culture media of these infected cells was measured using a human IL-lβ ELISA kit. As shown in Fig. 9D, the expression of endogenous pyrin was substantially increased in the retrovirus-infected cells (5th to 8th lanes) compared with the un-infected cells (1st to 4th lanes). The appearance of the caspase-1 p20 band in the mutant PSTPIPl -expressing THP-I cells (7th and 8th lanes, upper panel) indicating caspase-1 activation. There was also more caspase-1 activation (Fig. 9D, 5th to 8th lanes) and IL- lβ generation (Fig. 9E, 5th to 8th columns) in the infected cells compared to un-infected cells. Significantly, retroviral infection caused more caspase-1 activation in the mutant PSTPIPl- expressing cells (7th and 8th lanes) compared to the empty vector (5th lane) or the WT PSTPIPl-expressing cells (6th lane). The infection also caused more IL-lβ secretion from the

11058275.3 81 mutant PSTPIPl -expressing cells (Fig. 9E, 7th and 8th columns) compared to the empty vector (5th column) or the WT PSTPIPl -expressing cells (6th column). Combined, these results reveal that pyrin is induced by retroviral infection, and its induction is associated with increased caspase-1 activation and IL- lβ generation in cells expressing the PAPA-associated PSTPIPl mutants.

[00371] Example 11. Pyrin is important for IL- lβ generation by the mutant-PSTPIPl proteins in THP-I cells.

[00372] After establishing that retroviral infection causes induction of pyrin and more activation of caspase-1 in the mutant PSTPIPl -expressing cells, the effect of siRNA-mediated knockdown of pyrin on retro virus-induced IL- lβ generation in the stable PSTPIPl A230T- expressing cells were examined. These cells were transiently transfected with a control or a pyrin- specific siRNA, and 48 h after transfection the cells were infected with a GFP-encoding retrovirus for an additional 24 h. Mutant PSTPIPl A230T-expressing THP-I cells were transfected with control non-specific (Con) or pyrin-specific (Pyr) siRNAs and the cells were then left untreated (Un-infected) or infected with a GFP-encoding MSCV retrovirus as described under "Materials and methods". The secreted IL-lβin the culture media of these cells was measured. Pyrin expression in these cells was assayed by western blotting with anti-pyrin antibody (upper panel). As shown in Fig. 9F, knocking-down pyrin significantly reduced retrovirus-induced IL- lβ ecretion from these cells. Taken together, the results indicate that pyrin plays an important role in the activation of caspase-1 by the auto-inflammatory PSTPIPl mutants, since its induction increases caspase-1 activation and IL- lβ generation and its knockdown has an opposite effect.

[00373] Example 12. Pyrin is necessary for PSTPIPl -induced caspase-1 activation.

[00374] To investigate in more detail the role of pyrin in the mechanism of caspase-1 activation by the auto-inflammatory PSTPIPl mutants, a HEK-293 cell-based reconstitution system was used. HEK293 cells do not normally express any detectable amounts of PSTPIPl, pyrin, caspase-1 or the adaptor protein ASC (Yu et al., 2006), which makes it an ideal system to reconstitute the PSTPIPl -pyrin complex to study how PSTPIPl interacts with pyrin to induce caspase-1 activation. Therefore, stable HEK293T cell lines (293-ClAP) that express physiological levels of procaspase-1, ASC and pyrin, were generated. Other cell lines that stably express procaspase-1, ASC and cryopyrin (293-ClAC), procaspase-1 and pyrin (293-caspl- pyrin) or procaspase-1 and ASC (293-casp-l-ASC) were also produced to use as controls. Next,

11058275.3 82 these cell lines were transfected with WT PSTPIPl or PAPA-associated PSTPIPl mutants and assayed the activation of caspase-1 by western blot analysis and IL-lβ processing. 293-caspase- 1-ASC, 293-caspase-l -pyrin or 293-ClAP cells were transfected with an empty vector or the indicated PSTPIPl expression constructs. 24 h after transfection caspase-1 processing (top panels) and IL-lβ cleavage (bottom panel) were assayed as in "Materials and methods". The expression of PSTPIPl proteins in the transfected cells was determined by western blotting with anti-PSTPIPl antibody (middle panel). As shown in Fig. 1OA right panels, expression of WT PSTPIPl or PAPA-associated mutants in 293-ClAP cell line, which expresses procaspase-1, ASC and pyrin, resulted in caspase-1 activation and IL-lβ processing. As observed in THP-I cells, the PAPA-associated mutants induced more caspase-1 activation and IL-lβ processing than the WT PSTPIPl protein (3rd and 4th lanes). In contrast, expression of these PSTPIPl proteins in the 293-casp-l-ASC cells that express procaspase-1 and ASC without pyrin, did not induce caspase-1 activation (Fig. 1OA, left panels), indicating that pyrin is required for PSTPIPl -induced caspase-1 activation. Similarly, expression of the PSTPIPl proteins in the 293-caspl -pyrin cell line that expresses pyrin and procaspase-1 without ASC also did not induce caspase-1 activation (Fig. 1OA, middle panels), indicating that ASC is also required in addition to pyrin for PSTPIPl to induce caspase-1 activation.

[00375] To further demonstrate that pyrin is specifically required for PSTPIPl -induced caspase-1 activation, the effect of expression of PSTPIPl variants on caspase-1 activation was compared in the pyrin-expressing 293-ClAP and the cryopyrin-expressing 293-ClAC cell lines. PSTPIPl mutants potentiate caspase-1 processing in 293-ClAP cells. 293-ClAP (293-caspase- 1-ASC-pyrin) or 293-ClAC (293-caspase-l -ASC-cryopyrin) cells were transfected with an empty vector or the indicated PSTPIPl constructs. After transfection, caspase-1 and IL- lβprocessing were assayed as in A. Note that WT PSTPIPl and PSTPIPl mutants induce caspase-1 activation only in the 293-ClAP, but not in the 293-ClAC cells. In contrast to 293- ClAP, ectopic expression of WT or the PAPA-associated PSTPIPl mutants in the cryopyrin- expressing 293-ClAC cells did not induce caspase-1 activation or IL-lβ processing (Fig. 10B). These results indicate that pyrin, but not cryopyrin, is required for PSTPIPl to induce caspase-1 activation and that the disease-associated PSTPIPl mutants are more potent than the WT PSTPIPl in inducing pyrin-dependent caspase-1 activation. Consistent with the above results, the ability of PSTPIPl to activate caspase-1 was dependent on the level of pyrin in the cell. Two independent stable cell clones of 293-ClAP with different levels of pyrin (low or high) were transfected with an empty vector or an A230T mutant PSTPIPl expression construct as indicated. After transfection, caspase-1 processing (top panels) was assayed as in A. Note that

11058275.3 83 the cells with the higher level of pyrin (4th lane) showed more caspase-1 activation in response to ectopic expression of PSTPIP 1-A230T mutant than cells with the lower level of pyrin (2nd lane).When the PSTPIPl A230T mutant protein was expressed in two stable 293-ClAP cell lines having different levels of pyrin, it activated more caspase-1 in the higher pyrin-containing cells, than in the lower pyrin-containing cells (Fig. 10C). Similar results were obtained with WT and E250Q mutant PSTPIPl (data not shown). Taken together, the results demonstrate that pyrin is absolutely required for caspase-1 activation by PSTPIPl and that the ability of PSTPIPl to activate caspase-1 is enhanced at higher pyrin concentrations. These data indicate that Pyrin is required for activation of caspase-1 by PSTPIPl.

[00376] Example 13. PSTPIPl induces formation of the ASC pyroptosome in a pyrin- dependent manner.

[00377] Diverse pro -inflammatory stimuli trigger the assembly of an ASC pyroptosome in monocytes and macrophages by inducing ASC dimerization (Fernandes-Alnemri et al., 2007). ASC pyroptosome assembly can be observed in live cells using a THP-I cell line (THP-I-ASC- GFP cells) that stably expresses an ASC-GFP fusion protein. Considering that ASC is also important for PSTPIPl -induced caspase-1 activation, the effect of retrovirus-mediated ectopic expression of the A230T PSTPIPl mutant was examined on ASC-GFP in the THP-I-ASC-GFP cells. THP-I-ASC-GFP cells were left untreated (none) or infected with an empty MSCV (Vec) or PSTPIP l-A230T-encoding (A230T) retroviral vectors. The percentages of cells containing ASC pyroptosomes were determined as described under the "Materials and methods". As shown in Fig. HA, infection with an empty retroviral vector induced small amount of pyroptosome formation in these cells. In contrast, infection with a retrovirus encoding the A230T PSTPIPl mutant induced substantially more ASC pyroptosome formation compared to the empty vector control. These results indicate that PSTPIPl induces caspase-1 activation by triggering the formation of the ASC pyroptosome in THP-I monocytes.

[00378] To reconstitute the signaling pathway that leads to the formation of the ASC pyroptosome in response to PSTPIPl, a 293T-based ASC pyroptosome assembly assay similar to the THP-1-based assay was used. 293-ASC-EGFP-Nl cells were transfected with an empty vector (1st to 4th columns) or plasmids encoding pyrin (5th to 8th columns) or cryopyrin (9th to 12th columns) together with an empty vector (1st, 5th, 9th columns), wildtype PSTPIPl plasmid (2nd, 6th , 10th columns), A230T PSTPIPl mutant plasmid (3rd, 7th, 11th columns) or E250Q PSTPIPl mutant plasmid (4th, 8th, 12th columns) as indicated. 28 h after transfection, the cells were observed by fluorescence microscopy and the percentages of cells containing ASC-GFP

11058275.3 84 pyroptosomes were calculated. As shown in Fig. 1 IB, expression of the WT PSTPIPl or the PAPA-associated PSTPIPl mutants with pyrin in a stable 293-ASC-EGFP-Nl cell line, which expresses an ASC-GFP fusion protein, induced substantially more ASC pyroptosome formation than expression of pyrin alone. Consistent with the caspase-1 activation results (Fig. 10), the PSTPIPl mutants induced more ASC pyroptosomes than WT PSTPIPl in these cells (Fig. HB). No significant change in ASC pyroptosome formation was observed when PSTPIPl proteins were expressed without pyrin, or when co-expressed with cryopyrin, indicating that pyrin is specifically required for PSTPIPl -induced ASC pyroptosome formation (Fig. HB).

[00379] To provide additional evidence on the critical role of pyrin in PSTPIPl -induced

ASC pyroptosome formation, the effect of ectopic expression of WT or mutant PSTPIPl proteins was examined on ASC pyroptosome formation in a HEK293 cell line that expresses caspase-1, pyrin and an ASC-GFP fusion protein (designated 293-ClP- ASC-EGFP-Nl cells) and a control HEK293 cell line that expresses only caspase-1 and ASC-GFP without pyrin (designated 293-Cl-ASC-EGFP-Nl cells). 293-ASC-EGFP-Nl cells were seeded on slide cover slips and then transfected with empty vector or the indicated expression constructs. 24 h after transfection the cells were fixed and stained with DAPI and then observed by fluorescence confocal microscopy. Stable 293-caspase-l-ASC-EGFP-Nl cells, which do not express pyrin, or 293-ClP-ASC-EGFP-Nl cells which express pyrin were transfected with empty vector (EV) or the indicated PSTPIPl expression constructs (WT, A230T, and E250Q). 24 h after transfection the cells were observed and photographed by fluorescence microscopy. Notice the extensive formation of ASC pyroptosomes by mutant PSTPIPl proteins only in the pyrin-expressing cells.

[00380] It was observed as in the 293-ASC-EGFP-Nl cells, the ASC-GFP in the 293-

C IP-ASC-EGFP-Nl cells was evenly distributed in the entire cytoplasm and nucleus, indicating that stable co-expression of pyrin and caspase-1 together with ASC-GFP does not affect its distribution. Consistent with the above results, the PAPA-associated PSTPIPl mutants induced dramatic ASC pyroptosome formation in the pyrin-expressing 293-ClP- ASC-EGFP-Nl cells (data not shown). In contrast, these PSTPIPl mutants were not able to induce ASC pyroptosome formation in the control HEK293-C1-ASC-EGFP-N1 cells, which do not express pyrin (data not shown). These results underscore the essential role of pyrin in mutant PSTPIP-I -induced ASC oligomerization. The results also indicate that the engagement of pyrin by the mutant PSTPIPl proteins generates the molecular signal necessary for ASC oligomerization.

[00381] To determine how engagement of pyrin by mutant PSTPIPl induces more ASC oligomerization, the interaction of pyrin with ASC in the presence or absence of PSTPIPl was

11058275.3 85 examined. 293-ASC cells were transfected with empty vector or pcDNA-pyrin-myc-His plasmid together with constructs for the indicated WT or mutant PSTPIPl proteins. 24 h after transfection, the cell lysates were immunoprecipitated with an anti-pyrin antibody and protein G-sepharose beads. The bead-bound proteins were then fractionated by SDS-PAGE and immunoblotted with the appropriate antibodies to detect ASC or pyrin. As shown in Fig. HC, the interaction between pyrin and ASC was enhanced by co-expression of pyrin with WT PSTPIPl and further enhanced by co-expression with the disease-associated PSTPIPl mutants. These results indicate that PSTPIPl induces ASC oligomerization by increasing the interaction of pyrin with ASC.

[00382] Example 14. Pyrin is a homotrimer.

[00383] The ability of pyrin to induce ASC oligomerization suggests that pyrin itself is an oligomer or it oligomerizes before it engages ASC. To examine the first possibility, chemical cross-linking analyses with ethylene glycol bis (succinimidylsuccinate) (EGS) was performed to determine the oligomeric state of full length pyrin. Lysates from 293T cells transfected with full length pyrin plasmid (left panel), THP-I cell lysates containing endogenous pyrin (middle panel), or purified bacterially-produced full length pyrin (right panel) were cross-linked with the indicated concentrations of EGS. Pyrin was then immunoprecipitated with a pyrin- specific antibody and fractionated by SDS-PAGE followed by western blotting with anti-pyrin antibody. As shown in Fig. 12A, treatment of full-length pyrin from 293T or THP-I cells or purified from bacteria with low concentrations of EGS produced a major cross-linked species with apparent molecular mass of -300 kDa. Since monomeric pyrin has an apparent molecular mass of -100 kDa in SDS-PAGE, this indicates that native pyrin is a homotrimer. These results were confirmed by gel-filtration on Superdex 200, which also revealed that the native form of pyrin is indeed a homotrimer (Fig. 16E).

[00384] Example 15. The coiled-coil domain of pyrin mediates its homotrimerization.

[00385] Human pyrin contains four distinct domains; the N-terminal PYD (residues 1-92) followed by the B-box (BB) domain (residues 370-412), the coiled-coil (CC) domain (residues 420-582) and the PRY-SPRY domain (residues 597-781) (Fig. 12B, and Fig. 17). Shown in Fig. 17 are the domain structures of pyrin and related proteins. The top diagram shows the domain structure of human pyrin and the regions that have been shown to interact with ASC, the cytoskeleton and PSTPIPl. By analogy to Trim5α, the PRY-SPRY domain of pyrin might interact with pathogen-associated molecules. The numbers in parenthesis represent the numbers

11058275.3 86 of FMF-associated mutations identified in these domains. The bottom panel shows the domain structures of human cryopyrin (h-Cryopyrin) and zebrafish cryopyrin (zf-Cryopyrin). To sense pathogens human cryopyrin contains a C-terminal LRR domain whereas the zebrafish cryopyrin contains both LRR and PRY-SPRY domains. Between the PYD and B-box, pyrin contains a 278 amino acid long linker region with no homology to any known domains.

[00386] The PYD of pyrin is required for pyrin-induced ASC oligomerization since PYD mutations that abolish its interaction with ASC or deletion of the PYD of pyrin inhibit pyrin- induced ASC oligomerization ((Yu et al., 2006) and data not shown). To identify the exact region in pyrin that mediates its homotrimerization, the oligomeric state of a truncated pyrin mutant lacking the PRY-SPRY domain (1-580) was determined. Bacterially-expressed TV- tagged truncated pyrin mutants were cross-linked with EGS and then fractionated by SDS- PAGE followed by western blotting with anti-T7 antibody. As shown in Fig. 12C, left panel, treatment of pyrin 1-580, which has an apparent molecular mass of -75 kDa in SDS-PAGE, with low concentrations of EGS yielded a major cross-linked species with apparent molecular mass of -220 kDa corresponding to a trimeric form of pyrin. This indicates that deletion of the SPRY-PRY domain of pyrin does not affect the trimeric state of pyrin. Next, the N-terminal PYD and the C-terminal SPRY domain were deleted and determine the oligomeric state of this truncated pyrin mutant (designated pyrin-LN-BB-CC) (Fig. 12B). Treatment of this truncated pyrin mutant with low concentrations of EGS also yielded a distinct trimeric species (Fig. 12C, middle panel, 2nd and 3rd lanes). This indicates that the remaining linker region, B-box or coiled-coil domain mediates pyrin homotrimerization. To test this possibility, two additional truncated pyrin mutants were generated; one containing the linker region and the B-box (pyrin- LN-BB) and the other containing only the linker region (pyrin-LN). In contrast to the pyrin-LN- BB-CC, the pyrin-LN-BB and pyrin-LN mutants which lack the coiled-coil domain were no longer able to form any higher molecular mass cross-linked species and migrated in SDS-PAGE as monomers (Fig. 12C, middle panel, 4th to 6th lanes; right panel 1st to 3rd lanes). Collectively, these results indicate that pyrin is a homotrimer and that its trimeric state is maintained by self-association of its coiled-coil domain. These findings are consistent with previous structural predictions that the coiled-coil domains have the ability to form dimers and trimers (Kovacs et al., 2002; Lumb and Kim, 1995; Lupas, 1996). In addition, the coiled-coil domains have been implicated in self dimerization, trimerization and formation of high molecular weight oligomeric complexes in the pyrin-related Trim family of proteins (Javanbakht et al., 2006; Mische et al., 2005; Peng et al., 2000).

11058275.3 87 [00387] Example 16. Homotrimerization of pyrin is important for its ability to induce

ASC oligomerization and caspase-1 activation.

[00388] To determine if homotrimerization of pyrin is critical for its activity, the effect of different deletions that remove the PRY-SPRY domain, PRY-SPRY plus coiled-coil domains, or PRY-SPRY, coiled-coil plus B-box domains (Fig. 12D) on the ability of pyrin to induce ASC pyroptosome formation and caspase-1 activation were examined in the 293-ASC-EGFP-Nl and 293-caspase-l-ASC cells, respectively. 293-caspase-l-ASC cells were transfected with an empty vector (1st lane), or the indicated pyrin expression constructs together with an empty vector (2nd, 5th, 8th, 11th lanes), wildtype PSTPIPl plasmid (Fig. 12D, 3rd, 6th, 9th, 12th lanes) or A230T PSTPIPl mutant plasmid (Fig. 12D, 4th, 7th, 10th, 13th lanes) as indicated. 28 h after transfection, the cells were lysed in hypotonic CHAPS buffer and the resulting cell lysates were western blotted with the anti-Flag (caspase-1) antibody (Fig. 12D, upper panel), anti-PSTPIPl antibody (middle panel), or anti-pyrin antibody (Fig. 12D, lower panel). The decrease in pyrin expression (FL and 1-580) in the presence of PSTPIPl is due to cell death and cleavage of pyrin by the activated caspase-1. The data show that caspase-1 cleaves pyrin into smaller fragments (see Fig. 20). Activated caspase-1 cleaves pyrin. Lysates from stable 293-ClAP cells (10 μg/μl), which express Flag-procaspase-1, ASC and pyrin were activated by incubation at 37⁰C or left at 4⁰C for the indicated times. The lysates were then analyzed by SDS-PAGE and western blotted with anti-Flag (upper panel) or ani-pyrin (lower panel) antibodies. Incubation at 37⁰C activates the ASC pyroptosome which in turn activates caspase-1 (Fernandes-Alnemri et al., 2007). Notice the cleavage of pyrin by the activated caspase-1. No pyrin cleavage occurs in lysates that do not contain ASC or caspase-1 (not shown).

[00389] Additionally, 293-ASC-EGFP-Nl cells were transfected with an empty vector

(1st column) or plasmids encoding full-length pyrin (Fig. 12F, 2nd to 4th columns), pyrin 1-580 (Fig. 12F, 5th to 7th columns), pyrin 1-410 (Fig. 12F, 8th to 10th columns) or pyrin 1-343 (Fig. 12F, 11th to 13th columns) together with an empty vector (Fig. 12F, 2nd, 5th, 8th, 11th columns), wild type PSTPIPl plasmid (Fig. 12F, 3rd, 6th, 9th, 12th columns) or A230T PSTPIPl mutant plasmid (Fig. 12F, 4th, 7th, 10th, 13th columns) as indicated. 24 h after transfection, the cells were observed by fluorescence microscopy and the percentages of cells containing ASC-GFP ASC pyroptosomes were calculated.

[00390] As shown in Fig. 12E and F, deletion of the PRY-SPRY domain of pyrin did not affect the basal or PSTPIPl -induced activities of pyrin, as similar amounts of ASC pyroptosome formation and caspase-1 activation was observed with pyrinl-580 compared with the full-length

11058275.3 88 protein. In contrast, further deletion of the coiled-coil or the coiled-coil plus B-box domains impaired both the basal and PSTPIPl -induced activities of pyrin. These results indicate that the coiled-coil domain of pyrin is critical for its basal and PSTPIPl -induced activities, whereas the PRY-SPRY domain is not essential for either activity. Considering that deletion of the coiled- coil domain inactivates pyrin by abrogating its homotrimerization, we asked whether restoring homotrimerization with a homologous domain from the related family member Trim5α (Javanbakht et al., 2006; Mische et al., 2005) could restore pyrin activity. The coiled-coil and SPRY domains of pyrin were deleted and replaced with the homologous domains from Trim5α (Fig. 13A, 3rd diagram from top). 293-caspase-l-ASC cells were transfected with an empty vector (Fig. 13B, 1st lane), or expression constructs for pyrin or the indicated chimeric pyrin- Trim5α mutants together with an empty vector (Fig. 13B, 2nd, 4th, 6th lanes) or A230T PSTPIPl mutant plasmid (Fig. 13B, 3rd, 5th, 7th lanes) as indicated. 28 h after transfection, cell lysates were western blotted with the anti-Flag (Fig. 13B, caspase-1) antibody (Fig. 13B, upper panel), anti-pyrin antibody (Fig. 13B, middle panel) or anti-PSTPIPl antibody (Fig. 13B, lower panel). The decrease in pyrin levels in 3rd, 5th, 6th and 7th lanes is due to cell death and cleavage of pyrin by the activated caspase-1. Additionally, 293-ASC-EGFP-Nl cells were transfected with an empty vector (Fig. 13C, 1st column), or expression constructs for pyrin or the indicated chimeric pyrin-Trim5αmutants together with an empty vector (Fig. 13C, 2nd, 4th, 6th columns) or A230T PSTPIPl mutant plasmid (Fig. 13C, 3rd , 5th , 7th columns) as indicated. 24 h after transfection the cells were observed by fluorescence microscopy and the percentages of cells containing ASC-GFP ASC pyroptosomes were calculated as described under the "Materials and methods". The resulting PT-CC-SPRY chimeric protein, which contains the first 410 residues of pyrin followed by the coiled-coil and SPRY domains of Trim5α exhibited similar low basal activity as the WT pyrin protein, and induced similar amounts of ASC pyroptosome formation and caspase-1 activation (Fig. 13B and C). Furthermore, like the WT pyrin, the activity of the chimeric protein was also enhanced by co- expression with the A230T mutant PSTPIPl, indicating that the first 410 amino acids of pyrin contains all the necessary elements required for regulation by PSTPIPl. Together, these results indicate that coiled-coil-mediated trimerization of pyrin is critical for its activity.

[00391] Example 17. PSTPIPl activates pyrin by binding to its B-box.

[00392] The ability of PSTPIPl to enhance the activity of pyrin suggests that PSTPIPl might interact with a regulatory domain in pyrin to modulate its activity. To map the PSTPIPl- interaction domain in pyrin, co-immunoprecipitation experiments with WT PSTPIPl or the

11058275.3 89 A230T PSTPIPl mutant, and full-length pyrin or the truncated pyrin mutants were performed. HEK293 cells were transfected with empty vector, or expression constructs for full-length (FL) pyrin or the indicated pyrin truncated mutants together with pcDNA-PSTPIPl-Flag plasmids encoding WT (Fig. 13D, left panels) or mutant A230T PSTPIPl (Fig. 13D, right panels) as indicated. Lysates from these cells were immunoprecipitated (IP) with anti-pyrin antibody and immunoblotted with anti-Flag antibody to detect PSTPIPl (Fig. 12D, 1st panel from top) or anti- pyrin antibody (Fig. 13D, 3rd panel from top). The total lysates were also immunoblotted with anti-Flag antibody (Fig. 13D, 2nd panel from top) or anti-pyrin antibody (Fig. 13D, 4th panel from top). Both WT and the A230T mutant PSTPIPl interacted with full length, pyrin 1-580 and pyrin 1-410, but not with the B-box-truncated pyrin 1-343 mutant (Fig. 13D), indicating that the B-box is required for this interaction. Interestingly, the disease-associated PSTPIPl mutants, A230T and E250Q, exhibited substantially more binding to pyrin compared with the WT PSTPIPl (Fig. 13E). HEK293 cells were transfected with empty vector, or an expression construct for full-length pyrin together with pcDNA-PSTPIPl-Flag plasmids encoding WT or mutant PSTPIPl A230T and E250Q as indicated. Lysates from these cells were immunoprecipitated (IP) with anti-pyrin antibody and immunoblotted with anti-Flag antibody to detect PSTPIPl (Fig. 13E, 1st panel from top) or anti-pyrin antibody (Fig. 13E, 2nd panel from top). The total lysates were also immunoblotted with anti-Flag antibody Fig. 13E, 3rd panel from top) or anti-pyrin antibody (Fig. 13E, 4th panel from top). These results indicate that PSTPIPl interacts with the B-box of pyrin and that the disease-associated PSTPIPl mutants exhibit more binding to pyrin than the WT PSTPIPl. These results also explain why the disease- associated PSTPIPl mutants induce more robust activation of pyrin compared with the WT PSTPIPl protein.

[00393] Example 18. The B-box of pyrin is an inhibitory domain.

[00394] The above results demonstrate that WT pyrin and the pyrin-Trim5α chimera

PTCC-SPRY are not fully active without binding of PSTPIPl to their B-box. This indicates that in the unoccupied state the B-box might exert an inhibitory effect on the PYD of pyrin thereby preventing it from engaging ASC. To examine these possibilities, the pyrin B-box in the PT-CC- SPRY chimera was substituted with the homologous B-box from Trim5α (Fig. 13 A, 4th diagram from top). Unlike WT pyrin or the PT-CC-SPRY chimera, the new chimera (PT-BB-CC-SPRY), which contains the first 363 residues of pyrin followed by the B-box, coiled-coil and SPRY domains of Trim5α could not bind to PSTPIPl (Fig. 13F), indicating that the B-box of Trim5α does not interact with PSTPIPl. HEK293 cells were transfected with empty vector, or

11058275.3 90 expression constructs for full-length pyrin or the indicated pyrin-Trim5α chimeras together with pcDNA-PSTPIPl-Flag plasmid encoding mutant PSTPIPl A230T as indicated. Lysates from these cells were immunoprecipitated (IP) with anti-pyrin antibody and immunoblotted with anti- Flag antibody to detect PSTPIPl (Fig. 13F, 1st panel from top) or anti-pyrin antibody (Fig. 13F, 3rd panel from top). The total lysates were also immunoblotted with anti-Flag antibody (Fig. 13F, 2nd panel from top) or anti-pyrin antibody (Fig. 13F, 4th panel from top). Significantly, the basal activity of the new PT-BB-CC-SPRY chimera was substantially higher than that of the WT pyrin or the PT-CC-SPRY chimera (Fig. 13B, 6th and 7th lanes; Fig. 13C, 6th and 7th columns). The basal activity of the new chimera was comparable to the PSTPIPl -induced activity of WT pyrin and the PT-CC-SPRY chimera, and was not enhanced by co-expression with PSTPIPl. These results indicate that the B-box of pyrin indeed functions as an inhibitory domain to maintain pyrin in an inactive conformation, and that binding of PSTPIPl to the B-box or substituting it with the B-box of Trim5α relieves its inhibitory activity resulting in activation of pyrin. Consistent with this, deletion of the B-box of pyrin generates a constitutively active pyrin that does not require PSTPIPl to induce robust ASC oligomerization and caspase-1 activation (Fig. 18). 293-caspase-l-ASC cells were transfected with an empty vector (Fig. 18A, 1st lane), or expression constructs for pyrin or pyrin-ΔB-box (pyrin-ΔBB) together with an empty vector (Fig. 18A, 2nd and 4th lanes) or A230T PSTPIPl mutant plasmid (Fig. 18A, 3rd and 5th lanes) as indicated. 28 h after transfection, the cells were lysed in hypotonic CHAPS buffer and the resulting cell lysates were western blotted with the anti-Flag (caspase-1) antibody (upper panel) or anti-pyrin antibody (lower panel). 293-ASC-EGFP-Nl cells were transfected with an empty vector (Fig. 18B, 1st column), or expression constructs for pyrin or pyrin-ΔB-box (pyrin-deltaBB) together with an empty vector (Fig. 18B, 2nd and 4th columns) or A230T PSTPIPl mutant plasmid (Fig. 18B, 3rd and 5th columns) as indicated. 24 h after transfection the cells were observed by fluorescence microscopy and the percentages of cells containing ASC-GFP pyroptosomes were calculated as described under the "Materials and methods".

[00395] However, the B -box-deleted pyrin mutant was slightly less active than the

PSTPIPl-activated WT pyrin, perhaps because the B-box is important for proper folding and trimerization of pyrin. Indeed, an intact B-box is required for efficient oligomerization of the ret finger protein, which is also a member of the Trim family (Cao et al., 1997). The B-box might inhibit the activity of pyrin by binding and sequestering the PYD thereby preventing it from engaging ASC. To test this possibility, the interaction of the isolated PYD of pyrin with truncated pyrin-LN-BB-CC mutant which lacks the PYD and SPRY domain, was measured. The PYD of pyrin interacts with its B-box. GST or GST-PYD fusion protein (pyrin PYD residues 1-

11058275.3 91 100) were incubated with ³⁵S-labeled pyrin-LN-BB-CC (Fig. 13G, left panels) or pyrin-LN- ΔBB-CC (right panels) in the absence (Fig. 13G, 2nd and 3rd lanes) or the presence of dIAP as a non-specific control (Fig. 13G, 4th lane) or PSTPIPl A230T (Fig. 13G, 5th lane). The bound proteins were fractionated by SDS-PAGE and detected by autoradiography (Fig. 13G, top panels). The corresponding immobilized GST and GST-PYD proteins are shown in the lower panels. As shown in Fig. 13G, left panel, the isolated bacterially expressed PYD was able to interact with this mutant (3rd lane). This interaction was substantially reduced by PSTPIPl (5th lane) indicating that it is mediated by the B-box, since PSTPIPl binds to the B-box of pyrin. Indeed, deletion of the B-box from this mutant (pyrin-LN-ΔBB-CC) substantially reduced this interaction (Fig. 13G, right panel). Collectively, these results indicate that the B-box inhibits the activity of pyrin by sequestering the PYD and that binding of PSTPIPl to the B-box or deletion of the B-box unmasks the PYD resulting in activation of pyrin.

[00396] Example 19. Colchicine inhibits pyrin activity.

[00397] FMF is highly responsive to treatment with the microtubule-disrupting agent colchicine (Dinarello et al., 1974; Margolis and Wilson, 1977; Zemer et al., 1986; Zemer et al., 1974) and the colchicine's responsiveness is an important diagnostic tool for FMF. Furthermore, low doses of colchicine have been shown to be effective in the treatment of pyoderma gangrenosum (Kontochristopoulos et al., 2004), a condition similar to PAPA syndrome. These observations indicate that the cytoskeleton is an important element in the pyrin inflammatory pathway. Considering these observations and that PSTPIPl and pyrin are associated with the cytoskeleton, the disruption of the cytoskeleton by colchicine could inhibit activation of caspase- 1 by pyrin. 293-ClAP cells were transfected with an empty vector (Fig. 14A, 1st to 3rd lanes) or a construct encoding the A230T PSTPIPl mutant (Fig. 14A, 4th to 6th lanes). 16h after transfection, the cells were left untreated (none) or treated with the indicated concentrations of colchicine. 6h after treatment, the cells were lysed and the lysates were analyzed by western blotting with anti-caspase-1 antibody to detect caspase-1 processing. Consistent with the therapeutic benefit of colchicine in FMF, colchicine completely inhibited processing of caspase- 1 in response to ectopic expression of PSTPIPl (Fig. 14A). Similar results were obtained with nocodazol, another microtubule-disrupting agent (Fig. 14B). 293-ClAP cells were transfected with an empty vector (Fig. 14B, 1st lane) or a construct encoding the E250Q PSTPIPl mutant Fig. 14B, 2nd to 4th lanes). 16h after transfection the cells were left untreated (none) or treated with colchicine or nocodazol. Caspase-1 processing was assayed as in Fig. 14A. These results thus provide further support for the critical role of pyrin as a pro-inflammatory molecule, and

11058275.3 92 show for the first time that the pyrin-dependent caspase-1 activation process is a target for the microtubule-disrupting agents like colchicine and nocodazol.

[00398] The model illustrated in Fig. 15 demonstrates how PAPA-associated PSTPIPl mutants induces potent ASC pyroptosome assembly by way of pyrin, as experimentally demonstrated by chemical cross-linking and gel filtration analyses, both pyrin and PSTPIPl preexist as homotrimers (Fig. 12 and Fig. 16). PSTPIPl is a homotrimer. Fig. 16A (Upper diagram) shows the schematic representation of the domain structure of PSTPIPl. The two PAPA-associated mutations in the coiled-coil (CC) domain are indicated. Lower panels, Lysates containing full-length PSTPIPl (WT and A230T) expressed in 293T cells (left panel) or endogenous PSTPIPl from THP-I cells (right panel) was cross-linked with the indicated EGS concentrations. PSTPIPl was then immunoprecipitated with a PSTPIPl specific antibody and fractionated by SDS-PAGE followed by western blotting with PSTPIPl antibody. Notice the presence of trimeric PSTPIPl in the 2nd, 3rd, 5th and 6th lanes of left panel, and 2nd and 3rd lanes of right panel. Fig. 16B. The indicated bacterially-produced T7-tagged PSTPIPl (left panel) or the truncated pyrin mutant LN-BB-CC were cross-linked with EGS, immunoprecipitated with T7-agarose and then fractionated by SDS-PAGE followed by western blotting with anti-T7 antibody. Fig. 16C shows that 293T cells were transfected with the indicated PSTPIPl constructs. Lysates were then immunoprecipitated (IP) with anti-Flag and then fractionated by SDS-PAGE followed by western blotting with anti-T7 antibody (1st panel from top) or anti-Flag antibody (3rd panel from top). Lysates were also western blotting with anti-T7 antibody (2nd panel from top) or anti-Flag antibody (4th panel from top). Notice the association of the Flag-tagged PSTPIPl with the T7-tagged PSTPIPl in the 3rd lane, 1st panel from top, indicating that the PSTPIPl monomers self-associated with each other to form multimers. In Fig. 16D and E, bacterially produced T7-tagged PSTPIPl (WT and A230T) and pyrin-LN-BB-CC were fractionated on Superdex 200 FPLC column in phosphate buffered saline pH 7.0, containing 0.5% NP40. The indicated fractions (0.5 ml) were then western blotted with anti-T7 antibody. The basic units of both pyrin and PSTPIPl are homotrimers, which further oligomerize to form large multimers.

[00399] In the unbound conformation, the PYD of pyrin is masked by direct interactions with its B-box, preventing recruitment of ASC to the PYD of pyrin. PSTPIPl homotrimer binds to the pyrin homotrimer via a direct interaction with the B-box. Binding of PSTPIPl to pyrin results in unmasking of the PYD of pyrin, which now becomes free to interact with the PYD of ASC. The close proximity of ASC monomers on the surface of the pyrin homotrimer induces

11058275.3 93 ASC oligomerization, which we believe is an important initial step in the nucleation and subsequent assembly of the ASC pyroptosome. The PYD of pyrin is critical for ASC oligomerization, since point mutations in the PYD of pyrin that abrogate its interaction with the PYD of ASC also abrogate ASC oligomerization and caspase-1 activation by pyrin (Yu et al., 2006).

[00400] The region required for optimal binding to PSTPIPl is present within the B-box of pyrin. This region is not only important for PSTPIPl binding but also important for auto- inhibition of pyrin, since substitution with a homologous region from Trim5α or deletion of this region resulted in constitutive activation of pyrin. These important observations explain why pyrin has a low basal activity, although it is a homotrimer. Pyrin preexists in an autoinhibited homotrimeric state, and binding of PSTPIPl to its B-box transforms it into the active conformation. Homotrimerization of pyrin is mediated by its coiled-coil domain. This domain is a protein-protein interaction domain that has been shown to mediate oligomerization and formation of high molecular weight oligomeric complexes in the pyrin-related Trim family of proteins (Javanbakht et al., 2006; Meroni and Diez-Roux, 2005; Mische et al., 2005; Peng et al., 2000; Reymond et al., 2001). Like pyrin, PSTPIPl contains a coiled-coil domain, which likely mediates its homotrimerization (Fig. 16A).

[00401] The disease-associated PSTPIPl mutations are clearly gain-of-function mutations because PAPA syndrome is a dominantly inherited disease. The model described herein shows this enhanced association of PSTPIPl mutants with pyrin B-box could induce constitutive activation of pyrin thereby leading to more ASC oligomerization, and subsequently more caspase-1 activation. Binding of PSTPIPl WT/A230T heterotrimer to pyrin. (A) Wildtype (WT), A230T, or a 1:1 mixture of WT and A230T expression plasmids were in vitro transcribed/translated using TNT® coupled reticulocyte lysates system (Promega) in the presence of 35S-methionine. 10 μl of each translation mixture was then incubated at 4⁰C for 1.5 h with TALON™-agarose-bound GST-His6 (Fig. 19A, 4th and 5th lanes) or pyrin l-580-His6 (Fig. 19A, 6th to 8th lanes) in a 100 μl reaction as indicated. The bead-bound complexes were washed several times and then boiled in 30 μl of sample buffer. Each sample was loaded onto a 12.5% SDS polyacrylamide gel and separated by electrophoresis. The gel was dried and exposed to x-ray film. Lanes 1 to 3 show the input of each translation mixture (1 μl). Notice that unlike WT PSTPIPl (Fig. 19A, 6th lane), the PSTPIPl WT/A230T heterotrimer (Fig. 19A, 8th lane) exhibits strong binding to pyrin, explaining why mutant PSTPIPl behave dominantly.This is indeed supported by immunoprecipitation experiments, which revealed that pyrin exhibits

11058275.3 94 enhanced association with ASC in the presence of the disease-associated PSTPIPl mutants. Moreover, pyrin induced more ASC pyroptosome assembly in the presence of the PSTPIPl mutants compared to WT PSTPIPl. It was also observed that a heterotrimer of WT and A230T mutant PSTPIPl exhibit strong binding to pyrin comparable to binding of a homotrimer of mutant A230T to pyrin (Fig. 19A). Untagged wildtype (WT) PSTPIPl (lane 1), GST-tagged A230T (A230T-GST) PSTPIPl (lane 2), or a 1:1 mixture of WT and A230T-GST (Fig. 19B, lane 3) expression plasmids were in vitro transcribed/translated using TNT® coupled reticulocyte lysates system. The translation mixtures were bound to TALON™-agarose-bound pyrin 1-58O-His6 (Fig. 19B, 4th to 6th lanes) and analyzed as described in A above. The GST- tagged A230T was used to differentiate between the binding of WT and A230 subunits. Notice the increased binding of the WT subunit(s) (Fig. 19B, 6th lane) in the presence of A230-GST subunit(s).The WT PSTPIPl subunit(s) in the heterotrimer exhibited significantly more binding than the WT subunits in the WT homotrimer (Fig. 19B, compare 4th and 6th lanes). These results explain why mutations of PSTPIPl behave dominantly.

[00402] Example 20. High-throughput screen (HTS) to identify small molecule inhibitors of ASC pyroptosome formation.

[00403] Incubation of THP-I lysates containing a GFP-tagged ASC at 37⁰C for 30 min triggers formation of 2-3 pm-large pyroptosomes (Fig. 5A). These pyroptosomes can be separated easily from solution by simple low speed centrifugation at -3000 rpm (-900 X G). The GFP -fluorescence signal associated with the pelleted pyroptosomes is measured using a specrofluorometer. If the pyroptosome assembly is performed in the presence of a pyroptosome inhibitory molecule, the pyroptosome will not assemble, and therefore, there will be no GFP signal in that pellet. To screen large libraries of small molecules for potential pyroptosome inhibitory molecules, the in vitro pyroptosome assemble assay can be adapted to allow for high throughput screening, using 96-well plates (Fig. 21A). In this format, each compound in a library of small molecules is pipetted into a seperate well in a 96-well plate and then a fixed amount of THP-I lysates containing ASC-GFP is added to each well. The plate is then incubated at 37⁰C for 30 min. After incubation the ASC-GFP pyroptosomes are separated from the reaction mixtures by centrifugation of the 96-well plate at 3000 rpm for 5 min in an Eppendorff table top centrifuge with 96-well plate rotor adaptors. The supernatants are then removed by aspiration and the wells are then washed with a buffer (CHAPS lysis buffer). After the washing step, the pyroptosomes are re-suspended in the CHAPS lyses buffer and then the fluorescence in each well is measured using a standard fluorescence microplate reader. If the library of small

11058275.3 95 molecules contains inhibitors of pyroptosome formation, those wells containing the inhibitory compounds will have very low or no fluorescence signal.

[00404] As a proof of principle, the effect of potassium chloride on pyroptosome formation using the 96-well plate format was tested. Potassium chloride was added to separate wells to a final concentration of 100, 200 or 300 mM, and then equal amounts of THP-I lysates containing ASC-GFP were added to each well. The plate was then incubated at 37⁰C for 30 min and then processed as above. As shown in Fig. 21B, the amount of pyroptosome formation decreased dramatically in the presence of increasing amounts of potassium chloride as compared to the control. Therefore, we believe that using this assay, we have a go od chance of identifying small molecules that inhibit ASC oligomerization. This assay can also be performed in a 96-well plate with a filter paper bottom. Plates for this filter-based HTS are available from Whatman® (Cat. # 7700-3306) (see http://www.whatman.com/products/?pageID=7.30.27.187). The assembled pyroptosomes can then be collected at the bottom of each well by applying vacuum to the whole plate. This would simplify and shorten the procedure by eliminating the centrifugation and aspiration steps. Once small molecules with pyroptosome inhibitory activity are identified, these molecules for their effect on pyroptosome formation induced by LPS or other proinflammatory stimuli in THP-I cells can be characterize. If these compounds can inhibit pyroptosome formation in vivo , and have no cell toxicity, we will further evaluate their potential anti -inflammatory activity in mouse models of inflammation.

[00405] The references cited herein and throughout the specification are incorporated herein by reference in their entirty.

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ASC coding sequence (SEQ. ID. No. 1) (Genbank Accession No.: BAA87339)

GAGGGCGCGATCCTGGCGTCCCCCGACGGCCTGGGGCCCCAATCCAGAGGCCTGGG

TGGGAGGGGACCAAGGGTGTAGTAAGGAAGCGCCTTTTGCTGGAGGGCAACGGAC

CGGGGCGGGGAGTCGGGAGACCAGAGTGGGAGGAAGGCGGGGAGTCCAGGTTCCG

CCCCGGAGCCGACTTCCTCCTGGTCGGCGGCTGCAGCGGGGTGAGCGGCGGCAGCG

GCCGGGGATCCTGGAGCCATGGGGCGCGCGCGCGACGCCATCCTGGATGCGCTGGA

11058275.3 106 GAACCTGACCGCCGAGGAGCTCAAGAAGTTCAAGCTGAAGCTGCTGTCGGTGCCGC

TGCGCGAGGGCTACGGGCGCATCCCGCGGGGCGCGCTGCTGTCCATGGACGCCTTG

GACCTCACCGACAAGCTGGTCAGCTTCTACCTGGAGACCTACGGCGCCGAGCTCAC

CGCTAACGTGCTGCGCGACATGGGCCTGCAGGAGATGGCCGGGCAGCTGCAGGCGG

CCACGCACCAGGGCTCTGGAGCCGCGCCAGCTGGGATCCAGGCCCCTCCTCAGTCG

GCAGCCAAGCCAGGCCTGCACTTTATAGACCAGCACCGGGCTGCGCTTATCGCGAG

GGTCACAAACGTTGAGTGGCTGCTGGATGCTCTGTACGGGAAGGTCCTGACGGATG

AGCAGTACCAGGCAGTGCGGGCCGAGCCCACCAACCCAAGCAAGTGCGGAAGCTC

TTCAGTTTCACACCAGCCTGGAACTGGACCTGCAAGGACTTGCTCCTCCAGGCCCTA

AGGGAGTCCCAGTCCTACCTGGTGGAGGACCTGGAGCGGAGCTGAGGCTCCTTCCC

AGCAACACTCCGGTCAGCCCCTGGCAATCCCACCAAATCATCCTGAATCTGATCTTT

TATACACAATATACGAAAAGCCAGCTTGAAAAAAAAAAA

GFP coding sequence (SEQ. ID. No. 2) (Genbank Accession No. E17099)

ATGAGTAAAGGAGAAGAACTTTTCACTGGAGTTGTCCCAATTCTTGTTGAATTAGAT

GGTGATGTTAATGGGCACAAATTTTCTGTCAGTGGAGAGGGTGAAGGTGATGCAAC

ATACGGAAAACTTACCCTTAAATTTATTTGCACTACTGGAAAACTACCTGTTCCATG

GCCAACACTTGTCACTACTTTCTCTTATGGTGTTCAATGCTTTTCAAGATACCCAGAT

CATATGAAACGGCATGACTTTTTCAAGAGTGCCATGCCCGAAGGTTATGTACAGGA

AAGAACTATATTTTTCAAAGATGACGGGAACTACAAGACACGTGCTGAAGTCAAGT

TTGAAGGTGATACCCTTGTTAATAGAATCGAGTTAAAAGGTATTGATTTTAAAGAA

GATGGAAACATTCTTGGACACAAATTGGAATACAACTATAACTCACACAATGTATA

CATCATGGCAGACAAACAAAAGAATGGAATCAAAGTTAACTTCAAAATTAGACACA

ACATTGAAGATGGAAGCGTTCAACTAGCAGACCATTATCAACAAAATACTCCAATT

GGCGATGGCCCTGTCCTTTTACCAGACAACCATTACCTGTCCACACAATCTGCCCTT

TCGAAAGATCCCAACGAAAAGAGAGACCACATGGTCCTTCTTGAGTTTGTAACAGC

TGCTGGGATTACACATGGCATGGATGAACTATACAAATAA

11058275.3 107

Claims

What is claimed:

1. A method of isolating and detecting ASC pyroptosomes in a sample, the method comprising centrifuging a sample to form a pellet and detecting the presence of ASC protein in said pellet.

2. The method according to claim 1, wherein the centrifugal force is no more than 5000 x G.

3. The method according to claim 1, wherein the sample is a lysate of macrophages.

4. The method according to claim 1, wherein the presence of an ASC protein is detected by immunochemistry.

5. A method of diagnosing inflammation in an individual, the method comprising detecting an ASC pyroptosome in a sample from an individual, wherein a detectable presence of an ASC pyroptosome indicates that said individual is suffering from inflammation.

6. The method of claim 5, wherein the sample from said individual is a macrophage.

7. The method of claim 5, wherein the inflammation is caused by an inflammatory disease or a pathogen infection.

8. The method of any of claims 5-7, wherein the presence of an ASC pyroptosome in an individual is detected by a method according to claim 1.

9. The method according to claim 8, wherein the sample is a lysate of macrophages.

10. The method according to claim 8, wherein the centrifugal force is no more than 5000 x G.

11. The method according to claim 8, wherein the presence of an ASC protein is detected by immunochemistry.

12. A method of detecting the presence of a microbial pathogen, the method comprising the steps of:

(a) contacting a reporter cell with a test sample suspected of containing a microbial pathogen;

(b) detecting ASC pyroptosome; and

(c) determining that the test sample contains a microbial pathogen, wherein an ASC pyroptosome is detected.

11058275.3 108

13. A method for screening and identification of a compound that inhibits inflammation, the method comprising the steps of:

(a) contacting a compound to be screened with a reporter cell in the presence of a proinflammatory stimulus;

(b) detecting ASC pyroptosome; and

(c) selecting the compound wherein ASC pyroptosome in the presence of the compound is not detectable or an amount of ASC pyroptosome is reduced compared to a reference amount.

14. A method for screening for a compound that inhibits the interaction of pyrin with PSTPIPl and/or activation of pyrin by PSTPIPl, the method comprising the steps of:

(a) contacting a compound to be screened with a reporter cell expressing pyrin, PSTPIPl, and ASC protein;

(b) detecting an amount of ASC pyroptosome; and

15. The method of any of claims 12-14, wherein the presence of an ASC pyroptosome in said reporter cell is detected by a method according to claim 1.

16. The method according to claim 15, wherein the centrifugal force is no more than 5000 g.

17. The method according to claim 15, wherein the presence of an ASC protein is detected by immunochemistry.

18. The method of any of claims 12-14, wherein said reporter cell is a macrophage.

19. The method of any of claims 12-14, wherein said reporter cell stably expresses ASC tagged with green fluorescent protein.

20. The method of any of claims 12, 13, 14, 18, 19, wherein the presence of an ASC pyroptosome in said reporter is detected by fluorescence.

21. The method of any of claims 12, 13, 14, 18, 19, wherein the presence of an ASC pyroptosome in said reporter is detected by immunocytochemistry.

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22. A method for screening and identification of a compound that inhibits inflammation, the method comprising the steps:

(a) contacting a compound to be screened with a cell lysate;

(b) detecting an amount of ASC pyroptosome;

(c) comparing the amount of ASC pyroptosome with a reference amount; and

(d) selecting the compound wherein no detectable ASC pyroptosome is formed in the presence of the compound or there is a reduced amount of ASC pyroptosome formed in the presence of the compound compared to the reference amount.

23. The method of claim 22, wherein a presence of an ASC pyroptosome in said cell lysate is detected by a method according to claim 1.

24. The method according to claim 23, wherein the centrifugal force is no more than 5000 x G.

25. The method of claim 23, wherein an ASC pyroptosome is detected by immunochemisty.

26. The method of claim 22, wherein the cell lysate is from a macrophage that is stably expressing an ASC-GFP fusion protein.

27. The method of claim 22, wherein the cell lysate is from a non-macrophage that is stably expressing an ASC-GFP fusion protein.

28. The method of claim 26 or 27, wherein the cell lysate is SlOO lysate.

29. The method of claim 26 or 27, wherein the cell lysate is crude lysate.

30. The method of any of claims 22-29, wherein the presence of an ASC pyroptosome in said reporter is detected by fluorescence.

31. A method of determining the effectiveness of an anti-inflammatory treatment in a subject, the method comprising:

(a) obtaining a sample from a subject at a first time point;

(b) obtaining a sample from the subject at a second time point, the second time point being after the administration of an anti-inflammatory treatment;

(c) detecting and/or analyzing the ASC pyroptosome; and

11058275.3 110 (d) comparing the ASC pyroptosome in each sample, wherein a decrease in the ASC pyroptosome in the second time point sample in comparison with the first time point sample is an indication that the anti-inflammatory treatment is effective.

32. The method of claim 31, wherein the ASC pyroptosome is detected according to claim 1.

33. The method of claims 31 or 32, wherein the sample is a sample of macrophages.

34. A method of treatment of inflammation in a subject, the method comprising administering an effective amount of a BBox-peptide and a pharmaceutically acceptable carrier.

35. The method of claim 34, wherein the inflammation is due to an auto-inflammation disease.

36. The use of a BBox-peptide for the manufacture of a medicament for the treatment of inflammation.

37. The use of claim 36, wherein the inflammation is due to an auto-inflammation disease.

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