US20160354437A1 - Compositions and methods for modulation of immune response - Google Patents

Compositions and methods for modulation of immune response Download PDF

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US20160354437A1
US20160354437A1 US15/117,629 US201515117629A US2016354437A1 US 20160354437 A1 US20160354437 A1 US 20160354437A1 US 201515117629 A US201515117629 A US 201515117629A US 2016354437 A1 US2016354437 A1 US 2016354437A1
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pop3
pop1
pop2
asc
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Christian Stehlik
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Northwestern University
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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides
    • A61K38/16—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • A61K38/17—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • A61K38/1703—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates
    • A61K38/1709—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates from mammals

Definitions

  • POP1, POP2, and/or POP3 are inhibited to enhance an immune response (e.g., to treat or prevent infection), or POP1, POP2, and/or POP3 are administered or activated to reduce an immune response (e.g., to treat or prevent autoimmune or inflammatory disease).
  • the innate immune system is essential as a first line of defense to rapidly detect invading pathogens and to elicit a proper immune response for clearing infections and initiating wound healing.
  • Infections are sensed by germline encoded pattern recognition receptors (PRRs) present in different compartments of immune and non-immune cells, and include Toll-like receptors (TLRs), RIG-1-like receptors (RLRs), AIM2-like receptors (ALRs) and Nod-like receptors (NLRRs).
  • PRRs germline encoded pattern recognition receptors
  • TLRs Toll-like receptors
  • RLRs RIG-1-like receptors
  • AIM2-like receptors AIM2-like receptors
  • NLRRs Nod-like receptors
  • PRRs are not limited to specifically recognize conserved molecules on pathogens referred to as pathogen associated molecular patterns (PAMPs), but also sense host-derived damage-associated molecular patterns (DAMPs).
  • PAMPs pathogen associated molecular patterns
  • DAMPs host-derived damage-associated molecular patterns
  • the NLR family consists of 22 intracellular cytosolic PRRs with a tripartite domain architecture, composed of a C-terminalleucine rich region (LRR), a central nucleotide binding NACHT domain, and an N-terminal effector domain crucial for downstream signaling.
  • LRR C-terminalleucine rich region
  • NACHT domain central nucleotide binding NACHT domain
  • N-terminal effector domain crucial for downstream signaling.
  • the NLR effector domain is either a transactivation domain (NLRAs), a baculovirus inhibitor of apoptosis repeat (BIR) (NLRBs), an unknown domain (NLRX), a caspase recruitment domain (CARD) (NLRCs), or a PYRIN domain (PYD) in the largest NLR subfatnily (NLRPs). While pathogen activation of some NLRCs is linked to signalosome activation, others can activate caspase-1. Similarly, some of the characterized NLRPs also detect PAMPs and DAMPs in the cytosol and respond with the formation and activation of caspase-I-activating inflammasomes in macrophages.
  • NLRAs transactivation domain
  • BIR baculovirus inhibitor of apoptosis repeat
  • NLRX unknown domain
  • CARD caspase recruitment domain
  • PYD PYRIN domain
  • the apoptotic speck-like protein containing a CARD (ASC, PYCARD, TMSI) is the essential adaptor for bridging NLRPs with caspase-1, and macrophages deficient in ASC are impaired in caspase-1 activation and maturation of IL-1 ⁇ and IL-18.
  • Inflammasomes are protein scaffolds linking PAMP and DAMP recognition by NLRP members to the activation of caspase-I-dependent processing and release of the inflammatory cytokines interleukin (IL)-I ⁇ and IL-18.
  • ALRs including AIM2 and IF116 activate inflammasomes or type I interferon, respectively. They sense cytosolic DNA in autoimmune disease to perpetuate disease, as well as DNA from bacteria and viruses during infection.
  • POP1, POP2, and/or POP3 are inhibited to enhance an immune response (e.g., to treat or prevent infection), or POP1, POP2, and/or POP3 are administered or activated to reduce an immune response (e.g., to treat or prevent autoimmune or inflammatory disease).
  • POP1-, POP2-, and/or POP3-based inflammasome are administered to: (a) inhibit inflammasome activity, (b) prevent IL-1 ⁇ IL-18, and/or type I interferon release, (c) interfere with caspase-1 activation, (d) to prevent self-perpetuation of inflammasome responses, and/or (e) to block excessive production of cytokines (e.g., in inflammatory disease).
  • POP1, POP2, and/or POP3 are neutralized and/or inhibited (e.g., by administration of an inhibitor) to: (a) enhance immune response, (b) to boost adjuvant activity, and/or (c) for more efficiently clearing infections.
  • compositions comprising, inflammasome-inhibitory peptides, polypeptides, and protein, and methods of treating autoimmune and/or chronic inflammatory diseases and conditions therewith.
  • polypeptides, peptides, and peptidomimetecs are provided that exhibit the inflammasome-inhibitory activity of POP1, POP2, or POP3 or an enhancement thereof, as well as methods of use thereof.
  • compositions comprising a peptide or polypeptide having less than 100% sequence identity with SEQ ID NO: 60 (full length POP1), encompassing a portion with at least 50% sequence identity (e.g., >50%, >60%, >70%, >75%, >80%, >85%, >90%, >95%) with SEQ ID NO: 60 (full length POP1), and exhibiting inflammasome-inhibitory activity.
  • a polypeptide or peptide has 100% sequence identity with all or a portion of POP1.
  • the peptide or polypeptide comprises a portion with at least 80% sequence similarity (e.g., >80%, >90%, >95%) with POP1.
  • the peptide or polypeptide has less than 100% sequence identity, but more than 50% sequence identity (e.g., >50%, >60%, >70%, >75%, >80%, >85%, >90%, >95%) with POP1. In some embodiments, the peptide or polypeptide has at least 80% sequence similarity with POP1.
  • compositions comprising a peptide or polypeptide having less than 100% sequence identity with SEQ ID NO: 61 (full length POP2), encompassing a portion with at least 50% sequence identity (e.g., >50%, >60%, >70%, >75%, >80%, >85%, >90%, >95%) with SEQ ID NO: 61 (full length POP2), and exhibiting inflammasome-inhibitory activity.
  • a polypeptide or peptide has 100% sequence identity with all or a portion of POP1.
  • the peptide or polypeptide comprises a portion with at least 80% sequence similarity (e.g., >80%, >90%, >95%) with POP2.
  • the peptide or polypeptide has less than 100% sequence identity, but more than 50% sequence identity (e.g., >50%, >60%, >70%, >75%, >80%, >85%, >90%, >95%) with POP2. In some embodiments, the peptide or polypeptide has at least 80% sequence similarity with POP2.
  • the composition comprises a peptide or polypeptide with less than 100% but more than 50% sequence identity (e.g., ⁇ 100%, but >50%, >60%, >70%, >75%, >80%, >85%, >90%, >95%) with SEQ ID NO: 62 (full length POP3).
  • the peptide or polypeptide has at least 80% sequence similarity (e.g., >80%, >85%, >90%, >95%) with POP3.
  • the peptide or polypeptide has less than 100% but more than 50% sequence identity (e.g., ⁇ 100%, but >50%, >60%, >70%, >75%, >80%, >85%, >90%, >95%) with POP3.
  • the peptide or polypeptide has at least 80% sequence similarity (e.g., >80%, >85%, >90%, >95%, 100%) with POP3.
  • a peptide or polypeptide has a portion with 100% sequence identity with POP3.
  • the peptide or polypeptide has less than 100% sequence identity, but more than 50% sequence identity (e.g., ⁇ 100%, but >50%, >60%, >70%, >75%, >80%, >85%, >90%, >95%) with POP3. In some embodiments, the peptide has at least 80% sequence similarity (e.g., >80%, >85%, >90%, >95%, 100%) with POP3.
  • a peptide is provided that is 10-50 amino acids in length (e.g., 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, and any ranges therein).
  • a synthetic peptide or polypeptide comprises at least 1 mutation (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, and any ranges therein) from the wild-type or a natural POP1, POP2, or POP3 sequence over the length of the peptide.
  • a synthetic peptide comprises at least 1 non-conservative mutation (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, and any ranges therein) from the wild-type or a natural POP1, POP2, or POP3 sequence over the length of the peptide.
  • a peptide comprises at least 1 conservative mutation (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, and any ranges therein) from the wild-type or a natural POP1, POP2, or POP3 sequence over the length of the peptide.
  • peptides have less than 100% but greater than 50% (e.g., 55%, 60%, 70%, 80%, 90%, 95%, and any ranges therein) sequence identity to a portion of POP1, POP2, or POP3 that is at least 5 amino acids in length (5, 10, 15, 20, 25, 30, 35, 40, 45, 50, and any ranges therein).
  • compositions comprising peptides and/or polypeptides that exhibit enhanced inflammasome-inhibitory activity relative to POP1, POP2, or POP3.
  • peptides and/or polypeptides exhibit >10% increased, >20% increased, >30% increased, >40% increased, >50% increased, >60% increased, >70% increased, >80% increased, >90% increased, >2-fold, >3-fold, >4-fold, >5-fold, >6-fold, >8 fold, >10-fold, or >20-fold inflammasome-inhibitory activity relative to POP1, POP2, or POP3.
  • enhanced inflammasome-inhibitory activity is exhibited in one or more assays descried herein (e.g., see Examples).
  • compositions e.g., small molecules, peptide, polypeptide, antibodies, nucleic acids, etc.
  • Such compositions may increase cellular levels of POP1, POP2, and/or POP3, interact with POP1, POP2, and/or POP3 to increase activity, colocalize POP1, POP2, and/or POP3 with inflammasomes, etc.
  • compositions comprising: (a) an POP1, POP2, or POP3 peptide or polypeptide described herein (e.g., in the preceding paragraphs); and (b) a physiologically acceptable buffer or carrier.
  • pharmaceutical preparations further comprise an additional therapeutic agent (e.g., for the treatment of: inflammation, pain, autoimmunity, etc.).
  • fusion peptides or polypeptides comprising: (a) a POP1-, POP2-, or POP3-peptide or polypeptide described herein (e.g., in the preceding paragraphs), and (b) a functional peptide or polypeptide segment.
  • the functional peptide or polypeptide segment comprises a signaling moiety, therapeutic moiety, localization moiety (e.g., cellular import signal, nuclear localization signal, etc.), detectable moiety (e.g., fluorescent moiety, contrast agent), or isolation/purification moiety (e.g., streptavidin, His 6 , etc.).
  • nucleic acid vectors e.g., plasmid, bacmid, viral vector (e.g., AAV) comprising polynucleotides encoding a POP1-, POP2-, or POP3-peptide or polypeptide described herein (e.g., in the preceding paragraphs).
  • vectors further comprise a promoter and/or one or more expression elements (e.g., transcription enhancer, translational start site, internal ribosome entry site, etc.).
  • methods comprising administering a polynucleotide or vector described herein to a subject or sample (e.g., for the treatment of autoimmunity or inflammation).
  • provided herein are methods of treating autoimmunity or inflammation or a related condition or disease comprising administering a POP1-, POP2-, or POP3-peptide or polypeptide described herein (e.g., in the preceding paragraphs) to a subject suffering from autoimmunity or inflammation or said related condition or disease.
  • kits for preventing autoimmunity or inflammation or a related condition or disease comprising administering a POP1-, POP2-, or POP3-peptide or polypeptide described herein (e.g., in the preceding paragraphs) to a subject at risk (e.g., family history, genetic predisposition, lifestyle, age, gender, etc.) of autoimmunity or inflammation or said related condition or disease.
  • a subject at risk e.g., family history, genetic predisposition, lifestyle, age, gender, etc.
  • compositions comprising one or more agents (e.g., nucleic acid, small molecule, peptide, polypeptide, antibody, aptamer, etc.) that inhibit the inflammasome-inhibitory activity of POP1, POP2, or POP3.
  • agents e.g., nucleic acid, small molecule, peptide, polypeptide, antibody, aptamer, etc.
  • inhibited inflammasome-inhibitory activity is exhibited in one or more assays descried herein (e.g., see Examples).
  • compositions comprising: (a) a POP1, POP2, or POP3 inhibitor; and (b) a physiologically acceptable buffer or carrier.
  • pharmaceutical preparations further comprise an additional therapeutic agent (e.g., for the treatment of: inflammartion, pain, autoimmunity, etc.).
  • fusion peptides or polypeptides comprising: (a) a POP1-, POP2-, or POP3-inhibitor, and (b) a functional peptide or polypeptide segment.
  • the functional peptide or polypeptide segment comprises a signaling moiety, therapeutic moiety, localization moiety (e.g., cellular import signal, nuclear localization signal, etc.), detectable moiety (e.g., fluorescent moiety, contrast agent), or isolation/purification moiety (e.g., streptavidin, His 6 , etc.).
  • nucleic acid vectors e.g., plasmid, bacmid, viral vector (e.g., AAV) comprising polynucleotides encoding a POP1-, POP2-, or POP3-inhibitor.
  • vectors further comprise a promoter and/or one or more expression elements (e.g., transcription enhancer, translational start site, internal ribosome entry site, etc.).
  • methods are provided comprising administering a polynucleotide or vector described herein to a subject or sample (e.g., for the treatment of infection).
  • provided herein are methods of treating infection of a wound or a condition or disease that is treated by an enhanced immune response comprising administering a POP1-, POP2-, or POP3-inhibitor to a subject.
  • methods of preventing infection or a related condition or disease comprising administering a POP1- or POP3-inhibitor to a subject at risk (e.g., geographic location, lifestyle, age, etc.) of infection.
  • FIGS. 1 a - f POP3 is a novel type-I interferon-inducible member of the POP family.
  • FIGS. 2 a - b POP3 is a gene located between IF116 and IFIX.
  • cDNA showing the open reading frame of POP3 (Genbank accession number: KF562078 (SEQ ID NO: 26))
  • FIGS. 3 a - d POP3 shows characteristic features of PYDs present in HIN-200 proteins.
  • Amino acid sequence of POP3 (SEQ ID NO: 32). The PYD is shaded grey. The predicted ⁇ -helices are marked with blue lines (bottom), while the corresponding ⁇ -helices of AIM2, as determined by crystal structure 1, are marked with a line (top).
  • FIGS. 4 a - k POP3 interacts with ALRs.
  • the POP3 antibody does not cross-react with other POP family members.
  • HEK293 cells were transfected with Myc-tagged POP1, POP2 and POP3 and immunoprobed with POP1, POP2 and POP3-specific antibodies.
  • the POP3 antibody does not cross-react with the related PYDs of AIM2 and IF116.
  • HEK293 cells were transfected with GFP or RFP-tagged POP3, AIM2-PYD and IFI16-PYD and immunoprobed with our POP3 antibody and with GFP and RFP antibodies as control. *denotes a cross-reactive protein.
  • FIGS. 5 a - k Silencing of POP3 in hM ⁇ enhances ALR-mediated IL-1 ⁇ and IL-18 release.
  • FIGS. 6 a - i Silencing of POP3 specifically affects the AIM2 inflammasome.
  • FIGS. 7 a - e Monocyte/macrophage-lineage-specific expression of POP3 in CD68-POP3 TG mice.
  • FIGS. 8 a - b A gating strategy used for immunophenotyping of peripheral blood and peritoneal lavage cells.
  • FIGS. 9 a - h POP3 expression in BMDM inhibits AIM2 and IF116 inflammasome-mediated cytokine release.
  • FIGS. 10 a - f POP3 function in mouse macrophages.
  • BMDM of UbiC-hCAR TG mice were immunoprobed for expression of hCAR ⁇ cyt using HEK293 cells transiently transfected with hCAR ⁇ cyt as a control.
  • WT (top panel) and UbiC-hCAR TG (bottom panel) BMDM were infected with increasing MOI of a GFP-expressing AdV and analyzed by fluorescence and phase contrast microscopy.
  • UbiChCAR TG BMDM were infected with low MOI of AdV expressing GFP or GFPPOP3 and transfected 48 h later with poly(dA:dT) or infected with MVA for 16 h and analyzed for secreted IL-1 ⁇ by ELISA.
  • FIG. 11 a - e POP3 interacts with AIM2 and IF116 in BMDM.
  • FIGS. 12 a - i CD68-POP3 TG mice are impaired in AIM2-dependent and viral DNA-induced host defense in vivo.
  • FIG. 13 A gating strategy for immunophenotyping of splenocytes. Splenocytes obtained 36 h after MCMV infection were gated according to established cell surface markers, as indicated.
  • FIGS. 14 a - b POP3 does not ameliorate MSU-induced peritonitis.
  • FIGS. 15 a - h Inducible expression of POP1 is reduced in inflammatory disease.
  • a Immunohistochemical staining of CD68 (red) and POP1 (brown) in human lung tissue.
  • e-h, POP1, HMGB1 and ASC transcripts were measured by Real-time PCR in (e) LPS-treated hM ⁇ ; (f) leukocytes from LPS infused human subjects; (g) 1 h anakinra pre-treated hM ⁇ , as indicated, before treatment with LPS for 24 h; (h) hM ⁇ treated with IL-1 ⁇ for 4 h.
  • FIGS. 16 a - d POP1 inhibits the NLRP3 inflammasome in human macrophages.
  • a HEK293 cells were transfected to express POP1 and ASCPYD as indicated, followed by immunoblot analysis with antibody to POP1 or c-Myc as indicated.
  • b CLUSTAL-W sequence alignment for POP1 and ASCPYD with dark shaded residues representing identical and light shaded residues representing conserved amino acids.
  • c POP1 transcripts were measured by Real-time PCR in LPS-treated THP-1 cells;
  • d THP-1 cells stably expressing Myc-POP1 were analysed by qPCR for POP1 expression.
  • Culture SN were analysed by ELISA for IL-1 ⁇ release in untreated cells (Ctrl) or in response to crude LPS or nigericin treatment in LPS-primed cells.
  • FIGS. 17 a - i POP1 inhibits the NLRP3 inflammasome in human macrophages.
  • THP-1 cells stably expressing GFP or GFP-POP1 were analysed by Real-time PCR for POP1 transcripts.
  • SN Culture supernatants (SN) from THP-1 cells stably expressing GFP or GFP-POP1 were analysed for IL-1 ⁇ release by ELISA (b) in untreated cells (Ctrl) or in response to nigericin or CPPD treatment or K+ depletion in LPS-primed cells; transfection of poly(dA:dT), flagellin, or MDP; or (c) LPS treatment, LPS transfection or incubation with LPS complexed with CTB.
  • d LPS primed THP-1 cells expressing GFP or GFP-POP1 were treated with nigericin and active caspase-1 determined by flow cytometry.
  • LPS primed THP-1 cells expressing GFP or GFP-POP1 were treated with nigericin or CPPD crystals and released LDH in culture supernatants was quantified.
  • THP-1 cells stably expressing shRNAs targeting POP1 or a scrambled Ctrl were analysed by Real-time PCR for POP1 transcripts and for IL-1 ⁇ release in culture SN in untreated cells (Ctrl) or in response to LPS.
  • Primary macrophages transfected with a scrambled Ctrl or POP1-specific siRNA were analysed for POP1 transcripts by Real-time PCR and culture SN for IL-1 ⁇ and IL-18 in response to LPS.
  • FIGS. 18 a - e POP1 inhibits nucleation of the NLRP3 inflammasome in human macrophages.
  • a Interaction of GST-POP1 with endogenous ASC in THP-1 total cell lysates (TCL) using GST as negative control and showing 10% TCL as input.
  • b Immunoprecipitation (IP) of proteins, with antibody to ASC, from HEK293 cells transfected to express NLRP3, ASC and POP1 as indicated, followed by immunoblot analysis alongside TCL.
  • IP Immunoprecipitation
  • IgG immunoglobulin G
  • IP of proteins, with antibody to HA from HEK293 cells transfected to express Myc-ASC, HA-ASCPYD and GFP-POP1 as indicated, followed by immunoblot analysis alongside TCL.
  • e Immunoblot of HA-ASCPYD and GFP-POP 1, as indicated, after protein cross linking; data are representative of two (a-c), three (d), and one (e) replicates.
  • FIGS. 19 a - c POP1 is specifically expressed in peripheral blood monocytes.
  • a, b (a) Peripheral blood cells and (b) BMDM from WT and CD68-POP1 (POP1) transgenic mice were analysed by Real-time PCR for POP1 expression.
  • c Gating strategy for peripheral blood cells isolated from mice.
  • FIG. 20 a - d POP1 is specifically expressed in tissue macrophages and conventional DCs.
  • a-d Analysis of POP1 expression by flow cytometry in (a) different monocyte populations in peripheral blood, (b) cell populations in bone marrow, (c) peritoneal cavity and (d) spleen isolated from WT and CD68-POP1 (POP1) transgenic mice. Data are representative of three (a-d) replicates.
  • FIGS. 21 a - c Gating strategy to define cell populations expressing transgenic POP1 in a, bone marrow, b, peritoneal cavity and c, spleen. Data are representative of three (a-c) replicates.
  • FIG. 22 a - f POP1 inhibits the NLRP3 inflammasome in mouse macrophages.
  • a Analysis of POP1 expression by flow cytometry in peripheral blood cell populations isolated from wild-type (WT) and CD68-POP1 transgenic (POP1) mice.
  • b Western Blot of POP1 expression in BMDM.
  • c Interaction of GST-POP1 with endogenous ASC in BMDM total cell lysates (TCL) using GST as negative control and showing 10% TCL as input.
  • d Immunoblot analysis of ASC polymerization in WT and POP1 BMDM left untreated or treated with LPS/ATP after cross linkage of pellets (P) and in TCL.
  • e Immunoblot analysis of caspase-1 and IL-1 ⁇ in culture supernatants (SN) of LPS-primed WT and POP1 BMDM treated with ATP, showing pro-caspase-1 expression in TCL for normalization.
  • f Flow Cytometric quantification of active caspase-1 in WT and POP1 BMDM in response to LPS/ATP; data are representative of three (a), two (c, d-f) and four (b) replicates.
  • FIG. 23 POP1 is specifically expressed in CD45+ cells in CD68-POP1 transgenic mice. Analysis of POP1 expression by flow cytometry in CD45- and CD45+ cell populations in liver isolated from wilt type (WT) and CD68-POP1 (POP1) transgenic mice. Data are representative of three replicates.
  • FIGS. 24 a - e POP1 inhibits IL-1 ⁇ release in mouse macrophages.
  • a-d Analysis of culture supernatants (SN) for IL-1 ⁇ , IL-18, IL-1 ⁇ and TNF- ⁇ by ELISA in (a, b) LPS primed and ATP treated (a) WT and CD68-POP1 transgenic (POP1) BMDM; (b) WT, POP1, ASC ⁇ / ⁇ and NLRP3 ⁇ / ⁇ BMDM; (c) LPS primed WT and POP1 BMDM cultured in K+ depleted medium; and (d) WT and POP1 PM treated with LPS/ATP or transfected with flagellin or poly(dA:dT).
  • LPS primed WT and POP1 BMDM were treated with nigericin or CPPD crystals and released LDH in culture supernatants was quantified.
  • FIGS. 25 a - c POP1 does not affect LPS mediated cell signalling and transcription of IL1b and IL18.
  • a CLUSTAL-W sequence alignment for human and mouse ASCPYD with dark shaded residues representing identical and light shaded residues representing conserved amino acids.
  • b Immunoblot analysis of total and phosphorylated (p-) I ⁇ B ⁇ , Jnk, p38 and p42/44 and ⁇ -tubulin in total cell lysates of wild-type (WT) and CD68-POP1 (POP1) BMDMs treated for the indicated times with LPS, analyzed with ‘pan-specific’ and phosphorylation-specific antibodies.
  • c Real-time PCR analysis of Il1b and Il18 transcripts in WT and POP1 BMDMs treated for 4 h with LPS.
  • FIGS. 26 a - i Monocyte/macrophage-specific expression of POP1 ameliorates LPS-induced peritonitis and CAPS.
  • WT wild-type
  • TG CD68-POP1 transgenic mice.
  • b c, Endotoxic shock was induced by i.p. injection of E.
  • f H&E staining of skin sections from above mice at day 8. Scale bar 100 ⁇ m (original) and 10 ⁇ m (magnification).
  • FIGS. 27 a - f Reduced LPS and ASC particle-induced neutrophil infiltration in CD68-POP1 transgenic mice.
  • a In vivo image of MPO activity in mice 3 h after i.p. injection of PBS or E. coli LPS (2.5 mg/kg body weight) in wild-type (WT) and CD68-POP1 transgenic mice. The range of the luminescence signal is from 1195 to 20677 photons/sec/cm2/sr.
  • b FACS purification of ASC-GFP particles from stable ASC-GFP expressing HEK293 cells and of ASC-GFP/RFP-POP1 particles from transiently transfected HEK293 cells.
  • c In vivo imaging as above in WT and CD68-POP1 mice 4 h after i.p. injection of PBS or 1 ⁇ 105 FACS-purified ASC-GFP particles. The range of the luminescence radiance is from 206 to 1080 photons/sec/cm2/sr.
  • d Coomassie staining of purified TAT-GFP and TAT-POP1.
  • f Mice were i.p.
  • FIGS. 28 a - h Expression of POP1 prevents ASC particle release and ameliorates ASC particle-induced inflammatory disease.
  • a, b LPS primed (a) THP-1 cells expressing GFP or GFP-POP1 were treated with nigericin and (b) WT and POP1 BMDM were treated with ATP and released ASC determined by immunoblot in total cell lysates (TCL) and culture supernatants (SN). c, as in (a), but SN were cross linked before analysis.
  • ASC-GFP particles were FACS purified and imaged by immunofluorescence microscopy, showing the characteristic filamentous structure. Scale bar is 2 ⁇ m.
  • Culture supernatants from THP-1 cells stably expressing GFP or GFP-POP1 were analysed for IL-1 ⁇ release by ELISA in LPS primed cells before (Ctrl) and after treatment with 1 ⁇ 103 FACS-purified ASC-GFP particles.
  • Mixed ASC-GFP/RFP-POP1 particles were FACS purified as above, showing identical structure. Scale bar is 2 ⁇ m.
  • Culture supernatants from THP-1 cells were analysed for IL-1 ⁇ release by ELISA in LPS primed cells before (Ctrl) and after treatment with 1 ⁇ 10 3 FACS-purified ASC-GFP and ASC-GFP/RFP-POP1 particles.
  • FIG. 29 Proposed function of POP1 as an IL-1 ⁇ -regulated NLRP3 inflammasome regulator.
  • NLRP3 activation causes inflammasome assembly and the release of pro-inflammatory mediators, including IL-1 ⁇ , which in turn triggers autocrine and paracrine signals that promote POP1 expression as a late response gene.
  • POP1 functions as an inhibitor for NLRP3 inflammasome assembly.
  • Pyroptosis also releases oligomeric ASC particles, which act as danger signals upon phagocytosis and trigger NLRP3-independent nucleation of soluble ASC and inflammasome assembly.
  • POP1 also prevents the release of ASC particles by preventing NLRP3 inflammasome nucleation.
  • FIGS. 30 a - b POP3 interacts with ASC.
  • A POP3-ASC-PYD interaction by GST pull down and
  • B co-immunoprecipitation in HEK293 cells.
  • FIGS. 31 a - b POP3 interacts with ALRs.
  • A GST-pull down of POP3 and the PYDs of ALRs
  • B co-immunoprecipitation in HEK293 cells between POP3 and myc-tagged ALRs (IF116 and AIM2), and self-inter-action of POP3 as control.
  • FIG. 33 Knock-down of POP3 enhances IL-1 ⁇ in hM ⁇ .
  • hM ⁇ were transfected with siRNAs primed with ultrapure LPS (10 ng/ml) and infected with vaccinia virus.
  • IL-1 ⁇ in culture SN was analyzed 12 hrs post infection.
  • FIGS. 34 a - b POP3 is stabilized by MG132 IFN.
  • A,B Cells were infected with a POP3 expressing adenovirus and treated with MG132 or IFN and analyzed by immunoblot.
  • FIG. 35 hM ⁇ were infected with a GFP-POP3 AdV or ctrl, stained for endogenous IF116 and DNA and analyzed by microscopy.
  • FIG. 36 HEK293 cells were transfected, non-reversibly crosslinked and analyzed by immunoblot.
  • FIGS. 38 a - b Purification and delivery of recombinant TAT-POPs into human and mouse macrophages to impair inflammasome activity.
  • Purified recombinant TAT-POP1, TAT-POP2, TAT-POP3, and TAT-GFP was delivered into human THP-1 and mouse-J774A1 macrophages, followed by activation with LPS. Released mature IL-1 ⁇ was determined by ELISA.
  • Human THP-1 macrophages were incubated with TAT-GFP control or TAT-POP1 for 20 minutes, followed by treatment with LPS (300 ng/ml) for 16 hours to activate inflammasomes.
  • J774A1 cells were incubated with TATGFP control, TAT-POP1, TAT-POP2, or TAT-POP3 (black and grey bars represent a low and a higher TAT-peptide concentration, respectively) for 20 minutes, followed by treatment with LPS (300 ng/ml) for 16 hours to activate inflammasomes, followed by incubation with ATP (5 mM) for 30 minutes to induce release of mature IL-1 ⁇ and incubation in fresh medium for 3 hours (ATP is required for mouse macrophages to release processed IL-1 ⁇ ). Release of IL-1 ⁇ was assessed in culture supernatants by ELISA (BD Siosciences) and represented as fold induction compared to uninduced control cells. One representative experiment is shown.
  • inflammasome refers to a multiprotein complex comprising caspase 1, PYCARD, NALP and sometimes caspase 5 (a.k.a. caspase 11 or ICH-3). Inflammasomes are expressed in myeloid cells and are a component of the innate immune system. The exact composition of an inflammasome varies and depends on the activator which initiates inflammasome assembly. Inflammasomes promote the maturation of the inflammatory cytokines Interleukin 1 ⁇ (IL-1 ⁇ ) and Interleukin 18 (IL-18). Inflammasomes are responsible for activation of inflammatory processes, and have been shown to induce cell pyroptosis, a process of programmed cell death distinct from apoptosis.
  • IL-1 ⁇ Interleukin 1 ⁇
  • IL-18 Interleukin 18
  • autoimmune disease refers generally to diseases which are characterized as having a component of self-recognition.
  • autoimmune diseases include, but are not limited to, Autoimmune hepatitis, Multiple Sclerosis, Systemic Lupus Erythematosus, Myasthenia Gravis, Type I diabetes, Rheumatoid Arthritis, Psoriasis, Hashimoto's Thyroiditis, Grave's disease, Ankylosing Spondylitis Sjogrens Disease, CREST syndrome, Scleroderma, etc.
  • Most autoimmune diseases are also chronic inflammatory diseases. This is defined as a disease process associated with long-term (>6 months) activation of inflammatory cells (e.g., leukocytes).
  • the chronic inflammation leads to damage of patient organs or tissues.
  • Many other diseases are inflammatory disorders, but are not know to have an autoimmune basis.
  • Atherosclerosis Congestive Heart Failure, Crohn's disease, Colitis (e.g., Ulcerative Colitis), Polyarteritis nodosa , Whipple's Disease, Primary Sclerosing Cholangitis, etc.
  • the clinical manifestations of autoimmune and inflammatory diseases range from mild to severe. Mild disease encompasses symptoms that may be function-altering and/or comfort-altering, but are neither immediately organ-threatening nor life-threatening. Severe disease entails organ-threatening and/or life-threatening symptoms.
  • severe autoimmune disease is often associated with clinical manifestations such as nephritis, vasculitis, central nervous system disease, premature atherosclerosis or lung disease, or combinations thereof, which require aggressive treatment and may be associated with premature death.
  • peptide refers a short polymer of amino acids linked together by peptide bonds. In contrast to other amino acid polymers (e.g., proteins, polypeptides, etc.), peptides are of about 50 amino acids or less in length.
  • a peptide may comprise natural amino acids, non-natural amino acids, and/or modified amino acids.
  • a peptide may be a subsequence of naturally occurring protein or a non-natural sequence.
  • mutant peptide refers to a variant of a peptide having a distinct amino acid sequence from the most common variant occurring in nature, referred to as the “wild-type” sequence.
  • a mutant peptide may be a subsequence of a mutant protein or polypeptide (e.g., a subsequence of a naturally-occurring protein that isn't the most common sequence in nature), or may be a peptide that is not a subsequence of a naturally occurring protein or polypeptide.
  • a “mutant POP3 peptide” may be a subsequence of a mutant version of POP3 or may be distinct sequence not found in naturally-occurring POP3 proteins.
  • synthetic peptide refers to a peptide having a distinct amino acid sequence from those found in natural peptides and/or proteins.
  • a synthetic protein is not a subsequence of a naturally occurring protein, either the wild-type (i.e., most abundant) or mutant versions thereof.
  • sPOP1 peptide (“sPOP1 peptide”) is not a subsequence of naturally occurring POP1.
  • a “synthetic peptide,” as used herein, may be produced or synthesized by any suitable method (e.g., recombinant, chemical synthesis, enzymatic synthesis, etc.).
  • peptide mimetic refers to a peptide-like molecule that emulates a sequence derived from a protein or peptide.
  • a peptide mimetic or peptidomimetic can contain amino acids and/or non-amino acid components.
  • peptidomimitics include chemically modified peptides, peptoids (side chains are appended to the nitrogen atom of the peptide backbone, rather than to the ⁇ -carbons), ⁇ -peptides (amino group bonded to the ⁇ carbon rather than the ⁇ carbon), etc.
  • sequence identity refers to the degree to which two polymer sequences (e.g., peptide, polypeptide, nucleic acid, etc.) have the same sequential composition of monomer subunits.
  • sequence similarity refers to the degree with which two polymer sequences (e.g., peptide, polypeptide, nucleic acid, etc.) have similar polymer sequences.
  • similar amino acids are those that share the same biophysical characteristics and can be grouped into the families, e.g., acidic (e.g., aspartate, glutamate), basic (e.g., lysine, arginine, histidine), non-polar (e.g., alanine, valine, leucine, isoleucine, proline, phenylalanine, methionine, tryptophan) and uncharged polar (e.g., glycine, asparagine, glutamine, cysteine, serine, threonine, tyrosine).
  • acidic e.g., aspartate, glutamate
  • basic e.g., lysine, arginine, histidine
  • non-polar e.g., alanine, valine, leucine, isoleucine, proline, phenylalanine, methionine, tryptophan
  • uncharged polar e.g.
  • the “percent sequence identity” is calculated by: (1) comparing two optimally aligned sequences over a window of comparison (e.g., the length of the longer sequence, the length of the shorter sequence, a specified window, etc.), (2) determining the number of positions containing identical (or similar) monomers (e.g., same amino acids occurs in both sequences, similar amino acid occurs in both sequences) to yield the number of matched positions, (3) dividing the number of matched positions by the total number of positions in the comparison window (e.g., the length of the longer sequence, the length of the shorter sequence, a specified window), and (4) multiplying the result by 100 to yield the percent sequence identity or percent sequence similarity.
  • a window of comparison e.g., the length of the longer sequence, the length of the shorter sequence, a specified window, etc.
  • peptides A and B are both 20 amino acids in length and have identical amino acids at all but 1 position, then peptide A and peptide B have 95% sequence identity. If the amino acids at the non-identical position shared the same biophysical characteristics (e.g., both were acidic), then peptide A and peptide B would have 100% sequence similarity.
  • peptide C is 20 amino acids in length and peptide D is 15 amino acids in length, and 14 out of 15 amino acids in peptide D are identical to those of a portion of peptide C, then peptides C and D have 70% sequence identity, but peptide D has 93.3% sequence identity to an optimal comparison window of peptide C.
  • percent sequence identity or “percent sequence similarity” herein, any gaps in aligned sequences are treated as mismatches at that position.
  • the term “subject” broadly refers to any animal, including but not limited to, human and non-human animals (e.g., dogs, cats, cows, horses, sheep, poultry, fish, crustaceans, etc.).
  • the term “patient” typically refers to a subject that is being treated for a disease or condition.
  • an effective amount refers to the amount of a composition sufficient to effect beneficial or desired results.
  • An effective amount can be administered in one or more administrations, applications or dosages and is not intended to be limited to a particular formulation or administration route.
  • administering refers to the act of giving a drug, prodrug, or other agent, or therapeutic treatment to a subject or in vivo, in vitro, or ex vivo cells, tissues, and organs.
  • routes of administration to the human body can be through space under the arachnoid membrane of the brain or spinal cord (intrathecal), the eyes (ophthalmic), mouth (oral), skin (topical or transdermal), nose (nasal), lungs (inhalant), oral mucosa (buccal), ear, rectal, vaginal, by injection (e.g., intravenously, subcutaneously, intratumorally, intraperitoneally, etc.) and the like.
  • co-administration refers to the administration of at least two agent(s) or therapies to a subject. In some embodiments, the co-administration of two or more agents or therapies is concurrent. In other embodiments, a first agent/therapy is administered prior to a second agent/therapy.
  • a first agent/therapy is administered prior to a second agent/therapy.
  • the appropriate dosage for co-administration can be readily determined by one skilled in the art. In some embodiments, when agents or therapies are co-administered, the respective agents or therapies are administered at lower dosages than appropriate for their administration alone.
  • co-administration is especially desirable in embodiments where the co-administration of the agents or therapies lowers the requisite dosage of a potentially harmful (e.g., toxic) agent(s), and/or when co-administration of two or more agents results in sensitization of a subject to beneficial effects of one of the agents via co-administration of the other agent.
  • a potentially harmful agent e.g., toxic
  • treatment means an approach to obtaining a beneficial or intended clinical result.
  • the beneficial or intended clinical result may include alleviation of symptoms, a reduction in the severity of the disease, inhibiting a underlying cause of a disease or condition, steadying diseases in a non-advanced state, delaying the progress of a disease, and/or improvement or alleviation of disease conditions.
  • composition refers to the combination of an active agent with a carrier, inert or active, making the composition especially suitable for diagnostic or therapeutic use in vitro, in vivo or ex vivo.
  • compositions that do not substantially produce adverse reactions, e.g., toxic, allergic, or immunological reactions, when administered to a subject.
  • the term “pharmaceutically acceptable carrier” refers to any of the standard pharmaceutical carriers including, but not limited to, phosphate buffered saline solution, water, emulsions (e.g., such as an oil/water or water/oil emulsions), and various types of wetting agents, any and all solvents, dispersion media, coatings, sodium lauryl sulfate, isotonic and absorption delaying agents, disintigrants (e.g., potato starch or sodium starch glycolate), and the like.
  • the compositions also can include stabilizers and preservatives.
  • stabilizers and adjuvants see, e.g., Martin, Remington's Pharmaceutical Sciences, 15th Ed., Mack Publ. Co., Easton, Pa. (1975), incorporated herein by reference in its entirety.
  • POP1, POP2, and/or POP3 are inhibited to enhance an immune response (e.g., to treat or prevent infection), or POP1, POP2, and/or POP3 are administered or activated to reduce an immune response (e.g., to treat or prevent autoimmune or inflammatory disease).
  • inflammasome-produced cytokines are necessary for host defense and metabolic health, excessive and uncontrolled cytokine production contributes to pathological inflammation and autoinflammatory diseases. Hence, factors that promote a balanced inflammasome response are essential for maintaining homeostasis.
  • the regulation of inflammasomes has been poorly understood.
  • the type I IFN-inducible POP3 is one of the proteins that function to maintain a balanced inflammasome response in humans by specifically inhibiting ALR inflammasome assembly in response to immunogenic DNA. While other POPs directly interact with the inflammasome adaptor ASC (Dorfleutner A, et al. Infect Immun.
  • POP3 interacts with the PYD of ALRs and thereby prevents recruitment of ASC.
  • POP3 interacts with the PYD of ALRs and thereby prevents recruitment of ASC.
  • NLRP3 functional impairment of NLRP3-dependent inflammasome formation and activation was not observed in vitro and in vivo.
  • POP3 was not recruited to the endogenous ligand-induced NLRP3-ASC complex, but was recruited to MVA-induced endogenous AIM2, where it prevented ASC recruitment.
  • POP3 evolved as a specific ALR inflammasome regulator.
  • mice may employ an alternative mechanism for ALR inflammasome regulation through the DNA-binding HIN-200 family member p202, which lacks the PYD and is not encoded in humans, but may function as an antagonist for AIM2 in mice (Roberts T L, et al. Science. 2009; 323:1057-60; herein incorporated by reference in its entirety).
  • p202 is barely detectable in C57BL/6 mice, but is highly expressed in BALB/c and NZB mouse strains.
  • Aim2b a predicted Aim2 splice form in mice, might most closely resemble the POP3 function in mice.
  • the rat HIN-200 chromosomal region is also predicted to encode four HIN-200 proteins (Rhin2, Rhin3, Rhin4, Aim2) and the putative POP Rhin5, which is twice the size of POP3 and shares less than 14% sequence identity and also lacks any expression data. Contrary to mice, it is speculated that humans evolved POP3 to interfere with ALR inflammasome assembly.
  • POP3 represents one of the type I IFN-inducible proteins that antagonizes IFN- ⁇ in macrophages and inflammasome activation (Guarda G, et al.
  • Aim2 ⁇ / ⁇ BMDM show elevated IFN- ⁇ production (Fernandes-Alnemri T, et al. Nat Immunol. 2010; 11:385-393; Rathinam V A, et al. The AIM2 inflammasome is essential for host defense against cytosolic bacteria and DNA viruses. Nat Immunol.
  • POP3 is protein exhibiting inflammasome inhibitory function. POP3 inhibits release of IL-1 ⁇ and type I interferon. POP3 interacts with the central inflammasome adaptor ASC and thereby blocks the signaling from Nod-like receptors (NLRs), and with two pattern recognition receptors of the AIM2-like receptor family, namely AIM2 and IF116. POP3 is a small 13 kDa protein and delivery of a recombinant protein into macrophages shows inhibitory activity. In some embodiments, provided herein are POP3 peptides, polypeptides, and/or peptidomimetics.
  • POP3 is a novel regulator for cytosolic pattern recognition receptors of the NLR and ALR family. Therefore it is significant for blocking excessive production of cytokines in inflammatory disease.
  • IL-1 ⁇ is a very potent cytokine, and excessive production of it is linked to many inflammatory and autoimmune diseases. Many autoimmune diseases also show a type I interferon signature and type I interferon is required for inflammasome activation.
  • POP3 may be employed to specifically block IL-1 ⁇ generation or to neutralize POP3 to boost immune responses for adjuvant activity or to clear infections. Since POP3 most potently interacts with ALRs, which sense cytosolic DNA, which is present during autoinnnune disease and bacterial and viral infections, both, neutralizing as well as mimicking POP3 provides useful therapies.
  • NLRP3 inflammasome regulatory proteins exist to maintain a proper level of activity and in particular, to limit its activity during the resolution phase of this response. Experiments were conducted during development of embodiments described herein to determine if POP1 is one of these proteins.
  • the NLRP3 inflammasome and its mediators are tightly regulated at multiple steps, ranging from transcription to posttranslational modification of individual components, yet the essential step is the assembly of the NLRP3 inflammasome platform.
  • Recent evidence supports a model where NLRP3PYD binding to ASCPYD induces an initial nucleation event of the ASCPYD and subsequent prion-like ASCPYD self-polymerization (Lu et al. Cell 156, 1193-206 (2014); Cai et al.
  • ASCPYD Upon overexpression of both proteins, the high expression levels of ASCPYD are sufficient to promote self-polymerization through the prion activity of the ASCPYD.
  • Each ASCPYD interacts with two other ASCPYD molecules within filaments (Lu et al. Cell 156, 1193-206 (2014); Vajjhala et al. J Biol Chem 287, 41732-41743 (2012); herein incorporated by reference in their entireties), and since the residues necessary for ASCPYD polymerization are conserved in POP1, it is contemplated that POP1 could replace ASCPYD within filaments, without providing the CARD for caspase-1 nucleation, polymerization and activation.
  • POP1 and POP3 as inflammasome inhibitors, blockers of signaling from Nod-like receptors (NLRs), and capable of blocking cytokine production (e.g., excessive cytokine production), for example production of IL-1 ⁇ and type I interferon.
  • NLRs Nod-like receptors
  • POP1 and/or POP3 are useful for the treatment or prevention of diseases and conditions in which overactivity of inflammasomes or excessive cytokine production are causative or symptomatic (e.g., autoimmune diseases, inflammatory diseases, etc.).
  • POP2 as an inflammasome inhibitor.
  • POP2 is useful for the treatment or prevention of diseases and conditions in which overactivity of inflammasomes or excessive cytokine production are causative or symptomatic (e.g., autoimmune diseases, inflammatory diseases, etc.).
  • inhibition of POP2 activity us useful in the treatment or prevention of conditions (e.g., infection) in which an enhanced immune response is desired.
  • POP1, POP2, and/or POP3 are administered to a cell or subject.
  • POP1, POP2, and/or POP3 polypeptide, peptide, or peptidomimetic is administered.
  • a composition that enhances the activity of POP1 and/or POP3 is administered.
  • a composition that colocalizes POP1 and/or POP3 with inflammasomes is administered.
  • a protein, polypeptide, or peptide that has at least 60% sequence identity with POP1 (e.g., 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 98%, 99%). In some embodiments, a protein, polypeptide, or peptide is provided that has less than 100% sequence identity with POP1. In some embodiments, a protein, polypeptide, or peptide is provided that has at least 60% sequence similarity with POP1 (e.g., 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 98%, 99%).
  • a protein, polypeptide, or peptide has at least 60% (e.g., 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 98%, 99%) with a portion of POP1 at least 8 amino acids in length (e.g., 8 amino acids, 9 amino acids, 10 amino acids, 11 amino acids, 12 amino acids, 13 amino acids, 14 amino acids, 15 amino acids, 16 amino acids, 17 amino acids, 18 amino acids, 19 amino acids, 20 amino acids, 25 amino acids, 30 amino acids, 35 amino acids, 40 amino acids, 45 amino acids, 50 amino acids, or more, or any ranges therein (e.g., 15-25 amino acids)).
  • a protein, polypeptide, or peptide is provided that exhibits inflammasome inhibitory activity.
  • a protein, polypeptide, or peptide that has at least 60% sequence identity with POP2 (e.g., 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 98%, 99%). In some embodiments, a protein, polypeptide, or peptide is provided that has less than 100% sequence identity with POP2. In some embodiments, a protein, polypeptide, or peptide is provided that has at least 60% sequence similarity with POP2 (e.g., 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 98%, 99%).
  • a protein, polypeptide, or peptide has at least 60% (e.g., 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 98%, 99%) with a portion of POP2 at least 8 amino acids in length (e.g., 8 amino acids, 9 amino acids, 10 amino acids, 11 amino acids, 12 amino acids, 13 amino acids, 14 amino acids, 15 amino acids, 16 amino acids, 17 amino acids, 18 amino acids, 19 amino acids, 20 amino acids, 25 amino acids, 30 amino acids, 35 amino acids, 40 amino acids, 45 amino acids, 50 amino acids, or more, or any ranges therein (e.g., 15-25 amino acids)).
  • a protein, polypeptide, or peptide is provided that exhibits inflammasome inhibitory activity.
  • a protein, polypeptide, or peptide that has at least 60% sequence identity with POP3 (e.g., 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 98%, 99%). In some embodiments, a protein, polypeptide, or peptide is provided that has less than 100% sequence identity with POP3. In some embodiments, a protein, polypeptide, or peptide is provided that has at least 60% sequence similarity with POP3 (e.g., 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 98%, 99%).
  • a protein, polypeptide, or peptide has at least 60% (e.g., 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 98%, 99%) with a portion of POP3 at least 8 amino acids in length (e.g., 8 amino acids, 9 amino acids, 10 amino acids, 11 amino acids, 12 amino acids, 13 amino acids, 14 amino acids, 15 amino acids, 16 amino acids, 17 amino acids, 18 amino acids, 19 amino acids, 20 amino acids, 25 amino acids, 30 amino acids, 35 amino acids, 40 amino acids, 45 amino acids, 50 amino acids, or more, or any ranges therein (e.g., 15-25 amino acids)).
  • a protein, polypeptide, or peptide is provided that exhibits inflammasome inhibitory activity.
  • exogenous POP1, POP2, POP3, fragments thereof, or peptides or polypeptides having 60-100% sequence identity thereto are administered to a cell or subject.
  • POP1, POP2 and/or POP3 activating agents are administered to a cell or subject.
  • administration of the foregoing results in inflammasome inhibitions, inhibition of cytokine production, inhibition of IL-1 ⁇ release, inhibition of type I interferon release, blocking signaling from NLRs, etc.
  • a composition is administered that inhibits inflammasome activity. In some embodiments, a composition is administered that inhibits inflammasome assembly.
  • POP1 and POP3 as inflammasome inhibitors, blockers of signaling from Nod-like receptors (NLRs), and capable of blocking cytokine production (e.g., excessive cytokine production), for example production of IL-1 ⁇ and type I interferon.
  • NLRs Nod-like receptors
  • inhibition of POP1 and/or POP3 is useful for the treatment or prevention of diseases and conditions in which inflammasomes and/or cytokine production provide treatment (e.g., bacterial or viral infection).
  • one or more inhibitors e.g., anti-POP1 antibody, anti-POP3 antibody, fragments thereof
  • POP1 and/or POP3 e.g., anti-POP1 antibody, anti-POP3 antibody, fragments thereof
  • POP1 and/or POP3 are administered (e.g., to a cell, tissue, subject, etc.) for the treatment or prevention of infection (e.g., bacterial or viral).
  • infection e.g., bacterial or viral
  • POP2 as an inflammasome inhibitor.
  • POP2 is useful for the treatment or prevention of diseases and conditions in which overactivity of inflammasomes or excessive cytokine production are causative or symptomatic (e.g., autoimmune diseases, inflammatory diseases, etc.).
  • inhibition of POP2 activity us useful in the treatment or prevention of conditions (e.g., infection) in which an enhanced immune response is desired.
  • a POP1, POP2, and/or POP3 inhibitor is a small molecule, peptide, polypeptide, protein, antibody, nucleic acid, etc.
  • a POP1, POP2, and/or POP3 binding agent e.g., antibody, antibody fragment, aptamer, etc.
  • a binding agent e.g., antibody, antibody fragment, aptamer, etc.
  • binding agents capable of binding and/or neutralizing POP1, POP2, and/or POP3 are provided.
  • an antibody is a humanized antibody, antibody fragment, multivalent antibody, monoclonal antibody, neutralizing antibody, or any suitable combination thereof.
  • compositions comprise a POP1, POP2, and/or POP3 neutralizing antibody or antibody fragment.
  • neutralizing antibody or “antibody that neutralizes” refers to an antibody that reduces at least one activity of a polypeptide comprising the epitope to which the antibody specifically binds. In certain embodiments, a neutralizing antibody reduces an activity in vitro and/or in vivo.
  • a composition is administered that promotes inflammasome activity. In some embodiments, a composition is administered that promotes inflammasome assembly.
  • test agents e.g., pharmaceuticals, drugs, peptides, antibodies, aptamers, or other test agents
  • activities described herein e.g., POP1, POP2, or POP3 inhibition; POP1, POP2, or POP3 activation; POP1, POP2, or POP3 localization; inflammasome inhibition; blocking NLR signaling; inhibiting cyctokine production; etc.
  • test agents e.g., pharmaceuticals, drugs, peptides, antibodies, aptamers, or other test agents identified using screening assays of the present invention find use in the treatment of autoimmune diseases, inflammatory diseases, infections, etc.
  • screening assays for assessing cellular behavior or function are provided.
  • the response of cells, tissues, or subject to interventions e.g., POP1/POP2/POP3 inhibition, POP1/POP2/POP3 administration, POP1/POP2/POP3 activation, etc.
  • Such assays find particular use for characterizing, identifying, validating, selecting, optimizing, or monitoring the effects of agents (e.g., small molecule-, peptide-, antibody-, nucleic acid-based drugs, etc.) that find use in the treatment of diseases and conditions described herein (e.g., autoimmune diseases, inflammatory diseases, infections, etc.).
  • agents e.g., small molecule-, peptide-, antibody-, nucleic acid-based drugs, etc.
  • the PYRIN Domain-Only Protein POP3 Inhibits AIM2-Like Receptor Inflammasomes and Regulates Responses to DNA Virus Infections
  • pCD68-POP3 was generated by replacing CAT in pCAT-Basic containing the human CD68 promoter and the macrophage-specific IVS-1 enhancer with POP3 and flanking the cassette with AatII restriction sites.
  • the AatII fragment was excised, purified and B6.TgN(CD68-POP3) TG mice were generated by pronuclear injection into C57BL/6 embryos. Two lines were initially analyzed and subsequently a single line was used for most experiments and genotyping was outsourced to Transnetyx.
  • B6.TgN(UbiC-hCAR) TG mice were generated by pronuclear injection of a BglII fragment from pUBI containing the ubiquitin C promoter/intron41 and the human coxsackie and adenovirus receptor (hCAR) with deleted cytoplasmic domain (hCAR ⁇ cyt)40.
  • Mice were housed in a specific pathogen-free animal facility and all experiments were performed on age and gender-matched 8-12 weeks old mice conducted according to procedures approved by the Northwestern University Committee on Use and Care of Animals.
  • hM ⁇ were isolated from healthy donor blood after obtaining informed consent under a protocol approved by Northwestern University Institutional Review Board by Ficoll-Hypaque centrifugation (Sigma) and countercurrent centrifugal elutriation in the presence of 10 ⁇ g/ml polymyxin B using a JE-6B rotor (Beckman Coulter), as described 51 and transfected in 24-well dishes (2.5 ⁇ 105 cells) with 120 nM siRNA duplexes (F2/virofect; Targeting Systems) and analyzed 72 hr posttransfection (POP3 stealth siRNA sense strand: 5′-CAUGGCAUUUCUGGGAAUGCAUGUU-3′ (SEQ ID NO: 1), POP3 siRNA#2 sense strand: 5′-GAGCAGGAAACGGUAUAUGUGGGA-3′ (SEQ ID NO: 2), and Ctrl stealth siRNA, Invitrogen) (Khare S, et al.
  • BMDM BMDM were flushed from femurs and tibia and differentiated in L929-conditioned medium (25%) in DMEM medium supplemented with 10% heat inactivated FCS (Invitrogen) and analyzed after 7 days. Resting or elicited peritoneal macrophages (PM) were isolated by peritoneal lavage before or 3-5 days after i.p. injection of 1 ml 4% aged thioglycollate medium. THP-1 cells were obtained from ATCC and were routinely tested for mycoplasma contamination. THP-1 cells were stably transduced with pLEX-based lentiviral particles.
  • E. coli LPS 600 ng/mL E. coli LPS (0111:B4, Sigma) or pre-treated with ultra-pure E. coli LPS (0111:B4; Invivogen) (100 ng/mL), MSU (400 ng/mL; Invivogen); mouse and human IFN- ⁇ (1500 U/mL; Millipore), MG132 (10 ⁇ M; Calbiochem), mIL-1Ra (100 ng/mL; R&D Systems) or recombinant IL-1Ra (anakinra, 10 mg/mL, Amgen).
  • Cells were transfected with poly(dA:dT) (2 ng/mL; Sigma), MDP (20 ug/mL; Invivogen), Salmonella thyphimurium flagellin (140 ng/mL; Invivogen) and Bacillus anthracis Lethal toxin and protective antigen (1 ug/mL; List Biological Laboratories) using Lipofectamine 2000 (Invitrogen). Where indicated, cells were pulsed for 20 min with ATP (5 mM; Sigma) or treated for 45 min with nigericin (5 ⁇ M).
  • Recombinant adenovirus was generated by cloning GFP or GFP-POP3 into pShuttle, recombination with pAdEasy in E. coli BJ5183 and purification from HEK293N cells on a caesium chloride gradient.
  • Lentiviral particles were generated in HEK293T- Lenti cells (Clontech) transfected with pLEX containing GFP or GFP-POP3 and the packaging plasmids pMD.2G and psPAX2 (Addgene plasmids 12259 and 12260).
  • Murine cytomegalovirus (MCMV, Smith strain, ATCC #VR-1399) was obtained from the American Type Culture Collection (ATCC) and propagated in mouse embryo fibroblast SG-1 cells (ATCC #CRL-1404) for cell-based experiments and passaged twice for 2 weeks each, in the salivary glands of 6-8 week-old BALB/c mice after i.p. injection of 1.5 ⁇ 105 PFU/mL MCMV.
  • mice were euthanized and salivary glands collected, homogenized in HBSS, clarified and the viral titer determined by plaque formation assay and a Taqman qPCR assay based on MCMV iE and glycoprotein B (Invitrogen), using a MCMV standard curve and stored in aliquots at ⁇ 80° C.
  • Non-infected clarified salivary gland homogenates were used for mock infection.
  • 2.5 ⁇ 105 macrophages were infected with 1 ⁇ 105 PFU/well in 24-well plates.
  • Vaccinia virus (MVA, modified Vaccinia virus Ankara, ATCC # VR-1508) was obtained from ATCC and amplified in hamster fibroblast BHK-21 cells (ATCC #CCL-10).
  • MVA titer was determined by a plaque-forming assay using BHK-21 cells. 2.5 ⁇ 105 macrophages were infected with 1 ⁇ 106 PFU/well in 24-well plates. Kaposi's Sarcoma-associated herpes virus (KSHV) lytic cycle was induced from BCBL-1 cells by supplementing media with TPA (20 ng/mL). KSHV-containing culture SN was collected after 96 h, clarified by centrifugation (330 ⁇ g for 5 min followed by centrifugation at 1540 ⁇ g for 30 min) and filtered through 0.45 ⁇ m pore size filters. KSHV was subsequently concentrated by ultracentrifugation at 20,000 rpm for 90 min (SW28 rotor, 4° C.).
  • KSHV Kaposi's Sarcoma-associated herpes virus
  • Viral pellets were resuspended in EBM2 medium (Lonza), 0.45 ⁇ m filtered, and titered on the endothelial cell line iHMVEC52.
  • KSHV was used to infect 2.5 ⁇ 105 macrophages at 1.2 ⁇ 105 IU/24-well.
  • pCDNA3-based expression constructs for ASC, POP1 and POP2 are described in, for example, Dorfleutner A, et al. Virus Genes. 2007; 35:685-694; Dorfleutner A, et al. Infect Immun 2007; 75:1484-1492; Stehlik et al. Biochem J. 2003; 373:101-113; Khare S, et al. Immunity. 2012; 36:464-76; Bryan et al. J Inflamm (Lond) 2010; 7:23; Bryan et al. J Immunol. 2009; 182:3173-82; herein incorporated by reference in their entireties).
  • POP3 Acc.
  • AIM2 AIM2-PYD, IF116, IFI16-PYD, IFIX, IFIX-PYD, MNDA, MNDA-PYD, were generated by standard PCR from cDNAs and expressed sequence tags (EST) (Open Biosystems) and cloned in pcDNA3, pLEX or pShuttle with N-terminal myc, HA, Flag, GFP or RFP tags.
  • EST expressed sequence tags
  • Rabbit polyclonal and mouse monoclonal POP3 antibodies were custom raised (KLH-conjugated-CGSPSSARSVSQSRL), rabbit polyclonal antibody to ASC (Chemicon clone 2E1-7 and custom), mouse monoclonal antibody to ASC (custom), mouse polyclonal antibody to caspase-1 (Santa Cruz Biotech clone M-20), mouse monoclonal antibody to hCAR (Santa Cruz Biotech clone Mab.E[mh1]), mouse monoclonal antibody to GFP (Santa Cruz Biotech clone B-2), mouse monoclonal antibody to dsRED (Santa Cruz Biotech clone F-9), mouse monoclonal antibody to myc (Roche and Santa Cruz Biotech clone 9E10), mouse monoclonal antibody to the N-terminus of IF116 (Santa Cruz Biotech clone 1G7), mouse monoclonal antibody to the C-terminus of IF116 (Ab
  • IP co-immunoprecipitations
  • Bound proteins were separated by SDS-PAGE, transferred to PVDF membranes and analyzed by immunoblotting with indicated antibodies and HRP-conjugated secondary antibodies, ECL detection (Pierce), and image acquisition (Ultralum). TCL (5%) were also analyzed where indicated.
  • Endogenous NLRP3 and AIM2 inflammasome complexes were similarly purified from ultrapure LPS-primed (16 hrs, 100 ng/mL) THP-1 cells following nigericin treatment (45 min, 5 ⁇ M) or MVA-infection (90 min), respectively.
  • POP3 was cloned into pGEX-4T1 and affinity purified as a GST fusion protein from E. coli BL21.
  • Proteins were either prepared by in vitro transcription/translation (TNT Quick Coupled Transcription/Translation, Promega), or TCL were prepared from IFN-3-treated (16 hrs) BMDM or THP-1 cells by lysis (50 mM Hepes pH 7.4, 120 mM NaCl, 10% Glycerol, 2 mM EDTA, 0.5% Triton X-100, supplemented with protease inhibitors) as a source of endogenous proteins, and cleared lysates were incubated with immobilized GST-POP3 or GST control for 16 hrs at 4° C., followed by extensive washing with lysis buffer and analysis as above.
  • TNT Quick Coupled Transcription/Translation Promega
  • BMDM For ASC cross-linking, 4 ⁇ 10 6 BMDM were seeded in 60 mm plates and subjected to cross-linking as described 55. Briefly, cells were transfected with 1 ⁇ g/ml poly (dA:dT) for 5 hrs, supernatants were removed, cells rinsed with ice-cold PBS and lysed (20 mM Hepes pH 7.4, 100 mM NaCl, 1% NP-40, 1 mM sodium orthovanadate, supplemented with protease inhibitors) and further lysed by shearing.
  • hM ⁇ were grown on cover slips and either IFN- ⁇ treated for 16 hrs or infected with GFP, GFP-POP3 expressing adenovirus, MVA for 2 hrs or KSHV for 8 hrs, fixed, permeabilized, and immunostained with AIM2 (Cell Signaling clone 8055), IF116 (Santa Cruz Biotech clone 1G7) and POP3 (custom raised) antibodies and secondary Alexa Fluor 546-conjugated antibodies and DAPI (Invitrogen) (Bryan et al. J Immunol. 2009; 182:3173-82; herein incorporated by reference in its entirety). Images were acquired by fluorescence microscopy on a Nikon TE2000E2-PFS with a 100 ⁇ oil objective and image deconvolution (Nikon Elements).
  • IL-1 ⁇ , IL-18, TN ⁇ , IL-6, IFN- ⁇ and IFN- ⁇ secretion was quantified from clarified culture SN obtained from hM ⁇ , BMDMs, PM and from mouse serum by ELISA (BD Biosciences, eBiosciences, Invitrogen). Samples were analyzed in triplicates and repeated at least three times, showing a representative result. Active caspase-1 p10 was detected by immunoblot in TCA-precipitated serum-free culture supernatants 4 hrs after treatment (Khare S, et al. Immunity. 2012; 36:464-76; herein incorporated by reference in its entirety).
  • mRNA expression of target genes was quantified by RT-PCR or in vivo by using gold nanoparticles conjugated to specific oligonucleotides duplexed with Cy5-labeled reporter strands, which are non-toxic and are endocytosed by live cells (SmartFlares; Millipore). Subsequent analysis was performed by flow cytometry in combination with linage specific markers. 7-12 weeks old mice received an i.p. injection of MCMV (105 PFU) for 6 hrs. Mice were euthanized and peritoneal cells were obtained by lavage.
  • MCMV 105 PFU
  • Blood was obtained by retro-orbital bleeding, collected in EDTA-containing tubes, and incubated for 16 hrs with control or POP3 specific SmartFlares (1:1000 dilution in HBSS). Subsequently, cells were blocked with Fc-Block (2.4G2, BD), stained with fluorochrome-conjugated antibodies (see below), fixed and depleted of red blood cells using BD FACS Lysing solution (BD Biosciences) and analyzed on a BD LSR II flow cytometer. Data were compensated and evaluated using FlowJo software (Tree Star, Ashland, Oreg., USA).
  • Leukocyte subsets were identified as following: CD4 T cells as CD4+(RM4-5, BD), CD8 T cells as CD8+(53-6.7, BD), B cells as B220+(RA3-6B2, BD), NK cells as NK1.1+(PK-136, BD), neutrophils as CD11b+(M1/70, eBioscience) and Ly6G+(1A8, BD), monocytes as CD11b+(M1/70, eBioscience) and Ly-6C+(AL-21, BD) and macrophages as CD11b+(M1/70, eBioscience) and F4/80+(BM8, eBioscience).
  • the POP3 TaqMan assay was custom designed: POP3-Fwd: 5′-AGCACGAGTAGCCAACTTGATT-3′ (SEQ ID NO: 3), POP3-Rev: 5′-GGTCTTCCTCACTGCAGACA-3′ (SEQ ID NO: 4) and POP3-FAM probe: 5′-CCATGCCAGCGTTTTA-3′ (SEQ ID NO: 5).
  • the RT-PCR primers for POP3 were: POP3-Fwd: 5′-ATGGAGAGTAAATATAAGGAG-3′ (SEQ ID NO: 6), POP3-Rev: 5′-TCAACATGCATTCCCAGAAAT-3′ (SEQ ID NO: 7).
  • RPMI 1640 with 10% FCS, 2 mM glutamine, 100 U penicillin/0.1 mg streptomycin/mL, 10 mM HEPES buffer, and 1 mM sodium pyruvate.
  • Splenocytes were counted (Countess cell counter; Invitrogen) and 3 ⁇ 106 splenocytes were directly stained for IFN- ⁇ expression, and an additional 3 ⁇ 106 splenocytes were first suspended in complete RPMI medium and stimulated for 4 hrs in the presence of leukocyte activation cocktail (2 ⁇ L/mL, BD Biosciences) before staining (Rathinam V A, et al. Nat Immunol. 2010; 11:395-402; herein incorporated by reference in its entirety).
  • leukocyte activation cocktail 2 ⁇ L/mL, BD Biosciences
  • Splenocytes were pre-incubated with mouse Fc block, and labeled with pre-titrated fluorescent antibodies to B220, CD4, CD8, CD11b, CD69, NK1.1, as described above and Ly49H (3D10, eBioscience).
  • Intracellular staining for IFN- ⁇ was accomplished using a BD Cytofix/Cytoperm Kit according to the manufacturer's specifications (BD Biosciences) and dead cells were excluded using Aqua live/dead staining (Invitrogen). At least 400,000 events per sample were acquired on a BD LSRII instrument and data were analyzed with FlowJo software (TreeStar, Inc).
  • mice 10-12 week-old age and gender matched WT and CD68-POP3 mice were randomly i.p. injected with either PBS (0.5 mL/mouse) or MSU crystals in PBS (10 mg in 0.5 mL PBS/mouse). 5 hrs after MSU injection, mice were i.p. administered the luminescent Xenolight Rediject Inflammation probe (200 mg/kg, PerkinElmer) (Gross S, et al. Nat Med. 2009; 15:455-61; herein incorporated by reference in its entirety). Images were exposed for 5 min (IVIS Spectrum, PerkinElmer) and luminescence quantified with Living Image (PerkinElmer).
  • mice were also euthanized 7 hrs after MSU injection and peritoneal cavities were flushed with 2 mL of ice-cold PBS/10% FBS, clarified by centrifugation, and analyzed for IL-1 ⁇ by ELISA.
  • POP3 is Expressed in Response to Type-I Interferons
  • POP3 A previously undescribed human POP family member, POP3, was identified (Genbank accession number: KF562078) ( FIG. 1 a ).
  • the POP3 cDNA revealed an open reading frame of 342 bp ( FIG. 2 a ) encoded from a single exon located within the IFN-inducible gene cluster between IF116 and pyrin and HIN domain family member 1 (PYHIN1) on chromosome 1q23, which also contains AIM2 and myeloid cell nuclear differentiation antigen (MNDA) ( FIG. 2 b ).
  • PYHIN1 IFN-inducible gene cluster between IF116 and pyrin and HIN domain family member 1 (PYHIN1) on chromosome 1q23, which also contains AIM2 and myeloid cell nuclear differentiation antigen (MNDA)
  • MNDA myeloid cell nuclear differentiation antigen
  • POP3 encodes a single PYD of 113 aa with 5 ⁇ -helices, whereas the PYD of AIM2 consists of 6 ⁇ -helices.
  • the 3rd AIM2-PYD ⁇ -helix appears to be unstructured in POP3 ( FIG. 3 a ), pronounced to the structure observed for the PYD of NLRP1, which forms a flexible loop instead of ⁇ -helix 3 and is predicted to become stabilized upon PYD-PYD interaction 23.
  • POP3 showed high sequence similarity to the PYD of AIM2 ( FIG. 1 b , Table 1), and showed overall high similarity to the PYDs of HIN-200 family members ( FIG. 3 b ) (Jin et al. J Biol Chem. 2013; 288:13225-35; herein incorporated by reference in its entirety).
  • POP3 shared several of the characteristic sequence motifs within ⁇ -helices 1 and 2 of HIN-200 PYDs, but not those present within ⁇ -helices 5 and 6 ( FIG. 3 c ).
  • POP3 most likely originated from exon duplication of the AIM2-PYD, reminiscent to POP1, which is derived from the PYD of ASC (Stehlik C, Krajewska M, Welsh K, Krajewski S, Godzik A, Reed J C.
  • the PAAD/PYRIN-only protein POP1/ASC2 is a modulator of ASC-mediated NF- ⁇ B and pro-Caspase-1 regulation. Biochem J. 2003; 373:101-113; herein incorporated by reference in its entirety).
  • POP3 revealed low sequence homology with the PYDs of mouse and human ASC and NLRP3 ( FIG. 1 b , Table 1).
  • POP3 mRNA was expressed in monocytic cell lines and human primary macrophages (hM ⁇ ), but not in B and T cells ( FIG. 1 c ). Similar to ALRs, POP3 expression was upregulated in response to IFN- ⁇ in hM ⁇ , but the TLR4 agonist LPS did not induce POP3 expression ( FIG. 1 d ). Accordingly, POP3 was also detected by immunoblot in IFN-3-treated THP-1 macrophages ( FIG. 1 e ).
  • the POP3 expression pattern was unique, since neither POP1 nor POP2 were regulated by IFN- ⁇ , emphasizing a selective role of POP3 within the type I-IFN-mediated host response.
  • POP3 expression was upregulated as an early response gene within the first two hours, as well as a late response gene after 48 hours of IFN- ⁇ treatment, which was distinctive from AIM2, IF116, IFNB and the IFN-stimulated gene RSAD2 (also known as VIPERIN) ( FIG. 10 .
  • the IFN- ⁇ -inducible POP3 is a member of the POP family and shows similarity to the PYDs of HIN-200 proteins.
  • AIM2 modified DNA originating from latent Kaposi's Sarcoma-Associated Herpesvirus (KSHV) infection is recognized by IF116 within the nucleus in vitro (Kerur N, et al. Cell Host Microbe. 2011; 9:363-75; herein incorporated by reference in its entirety).
  • KSHV Kaposi's Sarcoma-Associated Herpesvirus
  • KSHV infection caused partial cytosolic redistribution of IF116 as quickly as 2 hours p.i. (data not shown), which was more prominent 8 hours p.i.
  • POP3 partial co-localization of IF116 with POP3
  • KSHV did not cause aggregation of IF116 in hM ⁇ at the titer used in our experiments.
  • GST-POP3 also purified endogenous AIM2 and IF116 ( FIG. 4 g ).
  • POP1 and POP2 did not bind to the inflammasome adaptor ASC.
  • weak binding of recombinant POP3 to NLRP3 we also observed in vitro ( FIG. 4 g ), in spite of the rather low degree of homology and the presence of the HIN-200 PYD-specific sequence motifs within POP3.
  • POP3 also caused a reduced interaction of ectopically expressed ASC and AIM2 in HEK293 cells by co-immunoprecipitation, indicating that POP3 is able to disrupt ALR inflammasome complex assembly by competing with ASC for the PYD binding site in AIM2 ( FIG. 4 j ). These data indicate that POP3 functions selectively as an ALR inflammasome inhibitor.
  • AIM2 inflammasome assembly causes the formation of ASC oligomers (Fernandes-Alnemri et al. Nature. 2009; 458:509-13; herein incorporated by reference in its entirety).
  • AIM2 and ASC in HEK293 cells caused the formation of ASC dimers and oligomers, but not transfection of ASC and POP3 or POP3, ASC or AIM2 alone.
  • POP3 AIM2-mediated ASC dimers and oligomers were significantly reduced ( FIG. 4 k ), indicating that POP3 can inhibit the PYD-dependent recruitment of ASC to AIM2.
  • POP3 is a previously undescribed IFN- ⁇ -inducible protein, which directly interacts with the ALRs AIM2 and IFI16 through PYD-PYD interaction to prevent inflammasome formation.
  • POP3 Inhibits ALR-Mediated IL-1 ⁇ and IL-18 Release
  • AIM2 and IF116 function as cytosolic and nuclear inflammasome-activating DNA-sensors, respectively (Fernandes-Alnemri T, et al. Nat Immunol. 2010; 11:385-393; Rathinam V A, et al. Nat Immunol. 2010; 11:395-402; Jones J W, et al. Proc Natl Acad Sci USA. 2010; 107:9771-6; Kerur N, et al. Cell Host Microbe. 2011; 9:363-75; herein incorporated by reference in their entireties).
  • POP3 following siRNA-mediated silencing in hM ⁇ , as determined by RT-PCR ( FIG.
  • IL-1 ⁇ release in response to cytosolic double strand (ds) DNA was significantly enhanced ( FIG. 5 a ).
  • Comparable results were also obtained with a second POP3-targeting siRNA ( FIG. 6 a ).
  • POP3 silencing did not affect IL-1 ⁇ release triggered upon activation of non-ALR inflammasomes, including inflammasome responses to LPS, the NLRP1 inflammasome in response to B. anthracis lethal toxin (LeTx) (Boyden et al. Nat Genet.
  • THP-1 cells are widely used to study inflammasome responses and elevated AIM2-dependent release of IL-1 ⁇ in POP3 silenced THP-1 cells was also observed in response to AIM2-, but not NLRP3-dependent stimuli ( 6 b ).
  • FIG. 5 d Increased MVA-induced IL-18 release upon silencing of POP3 was also observed ( FIG. 5 d ).
  • This effect of POP3 was specific for inflammasome-dependent cytokines, since the release of the inflammasome-independent cytokines TNF ( FIG. 5 e ) and IL-6 ( 6 c ) was not affected by POP3 silencing.
  • POP3 silencing also did not affect mRNA expression of ASC, AIM2 and IF116, as determined by real-time PCR ( FIG. 5 f ) or protein expression ( FIG. 6 d ).
  • THP-1 cells stably expressing GFP-POP3, but not GFP control showed significantly reduced release of IL-1 ⁇ ( FIG.
  • IFI16 functions as a sensor promoting IFN- ⁇ production in response to DNA virus infection
  • Weholzner L, et al. Nat Immunol. 2010; 11:997-1004; herein incorporated by reference in its entirety In agreement with the elevated IFN- ⁇ secretion in Aim2 ⁇ / ⁇ macrophages, which indicates that it may negatively regulate IFN- ⁇ production, it was observed that silencing of POP3 decreases IFN- ⁇ production in response to MVA infection of hM ⁇ ( FIG.
  • mice are lacking from mice (Stehlik & Dorfleutner. J Immunol. 2007; 179:7993-8; herein incorporated by reference in its entirety). Similar to the close chromosomal location of POP1 and ASC, POP3 is found next to AIM2 and IF116, and POP3 is also absent in mice, despite significant amplification of this gene cluster ( FIG. 7 a ).
  • transgenic (TG) mice were generated expressing POP3 from the human CD68 (hCD68) promoter in combination with the IVS-1 intron containing a macrophage-specific enhancer (Gough et al.
  • POP3 Inhibits ALR-Mediated Cytokine Release in BMDM
  • POP3 expression results in a significant decrease of IL-1 ⁇ release in response to poly(dA:dT) transfection and MVA or MCMV infections, but not in response to MDP, flagellin, LPS+ATP or monosodium urate crystals (MSU) in BMDM ( FIG. 9 a, b ) and in peritoneal macrophages ( FIG. 10 a ).
  • POP3 co-purified IF116 and AIM2, but not ASC from BMDM ( FIG. 11 a ), similar to what was observed in THP-1 cells ( FIG. 4 g ). As observed in human THP-1 cells, recombinant POP3 also weakly co-purified NLRP3 in BMDM in vitro ( FIG. 11 a ).
  • ASC oligomerization was analyzed in response to AIM2 inflammasome stimulation with poly(dA:dT) in WT and CD68-POP3 TG BMDM as a readout for AIM2 inflammasome formation (Fernandes-Alnemri et al. Nature. 2009; 458:509-13; herein incorporated by reference in its entirety).
  • Insoluble ASC monomers, dimers and oligomers were drastically decreased in the presence of POP3 ( FIG. 11 b ), supporting impaired AIM2 inflammasome formation in the presence of POP3.
  • Inflammasome formation is essential for caspase-1 activation, and although the protein amount of pro-caspase-1 was not altered in POP3 expressing BMDM, active caspase-1 p10 was significantly reduced in response to MVA and MCMV, but not in response to LPS+ATP in culture supernatants ( FIG. 11 c ), further emphasizing the functional specificity of POP3 for AIM2, but not NLRP3 inflammasome formation.
  • the POP3 effect was specific for caspase-1 and was not caused by modulating NF- ⁇ B activation, since the NF- ⁇ B-inducible cytokines TNF and IL-6 were equally secreted ( FIG. 9 e, f ).
  • POP3 Blunts ALR-Mediated Anti-Viral Host Defense In Vivo
  • Aim2 ⁇ / ⁇ mice are severely impaired in mounting an efficient host response to MCMV infection, due to a deficiency in the inflammasome-dependent systemic IL-18 release.
  • IL-18 acts in synergy with IL-12 to stimulate IFN- ⁇ production by splenic NK cells, which is crucial for the early anti-viral response against DNA viruses, including MCMV.
  • WT and CD68-POP3 TG mice were challenged with MCMV, and, similar to Aim2 deficiency, serum IL-18 and IFN- ⁇ concentrations were also strongly decreased in CD68-POP3 expressing mice at 36 hours post i.p. infection. However, TNF serum concentrations were not affected ( FIG. 12 a ).
  • CD68-POP3 TG mice displayed a similar spleen weight as WT mice after MCMV infection, but showed slightly reduced splenocyte numbers ( FIG. 12 b ).
  • IL-18 is required for Ly49H+NK cell expansion (Andrews et al. Nat Immunol. 2003; 4:175-81; herein incorporated by reference in its entirety).
  • Reduced IL-18 concentration in CD68-POP3 TG mice was observed in experiments conducted during development of embodiments described herein ( FIG. 12 a ).
  • CD68-POP3 TG mice displayed a decreased number of NK1.1+Ly49H+ cells, whereas NK1.1+Ly49H ⁇ cells were increased. However, comparable numbers of T and B cells were found ( FIG. 12 c and FIG. 13 ).
  • CD68-POP3 TG mice had significantly less IFN- ⁇ producing splenic NK cells ex vivo at 36 hours post infection ( FIG. 12 d, e ), reminiscent of Aim2 ⁇ / ⁇ mice 6.
  • This response was specific to MCMV and not due to an intrinsic defect of splenic NK cells from CD68-POP3 TG mice to produce IFN- ⁇ , since activation of WT and POP3 splenic NK cells with the phorbol ester PMA and ionomycin ex vivo, produced comparable numbers of IFN- ⁇ +NK cells ( ⁇ 95%) ( FIG. 12 e ).
  • CD68-POP3 TG mice displayed elevated serum IFN- ⁇ concentration at early (11 hours), but not at later time points (36 hours) post MCMV infection ( FIG. 12 f ). These results indicate that the deficient IFN- ⁇ response is due to impaired systemic IL-18 observed in CD68-POP3 TG mice upon MCMV infection. Significant increase in the splenic MCMV titer in CD68-POP3 TG mice was observable ( FIG. 12 g ). The 2-fold increase was comparable to the 2-fold increase observed in Asc ⁇ / ⁇ mice in a previously published experiment (Rathinam V A, et al. Nat Immunol. 2010; 11:395-402; herein incorporated by reference in its entirety).
  • the PYRIN Domain-Only Protein POP1 Inhibits Inflammasome Assembly and Ameliorates Inflammatory Disease
  • pCD68-POP1 was generated by replacing CAT in pCAT-Basic containing the human CD68 promoter and the macrophage-specific IVS-1 enhancer (Khare et al. Nat Immunol 15, 343-353 (2014); Iqbal et al. Blood blood-2014-04-568691 (2014); herein incorporated by reference in their entireties) with GFP-POP1 and flanking the cassette with AatII restriction sites.
  • the AatII fragment was excised, purified and B6.TgN(CD68-POP1) TG mice were generated by pronuclear injection into C57BL/6 embryos by the Northwestern University Transgenic and Targeted Mutagenesis Facility.
  • mice C57BL/6 wild type (WT) and Lysozyme M-Cre knock-in mice (CreL) were obtained from the Jackson Laboratories and NLRP3 ⁇ / ⁇ , ASC ⁇ / ⁇ and floxed NLRP3 A350V knock-in mice were described earlier (Mariathasan et al. Nature 430, 213-8 (2004); Mariathasan et al. Nature 440, 228-232 (2006); Brydges et al Immunity 30, 875-87 (2009); herein incorporated by reference in their entireties). Mice were housed in a specific pathogen-free animal facility and all experiments were performed on age and gender-matched, randomly assigned 8-14 week old mice conducted according to procedures approved by the Northwestern University Committee on Use and Care of Animals.
  • CC-RE-Lot 2 NIH Clinical Center Reference endotoxin
  • Peripheral blood-derived hM ⁇ were isolated from healthy donor blood after obtaining informed consent under a protocol approved by Northwestern University Institutional Review Board by Ficoll-Hypaque centrifugation (Sigma) and countercurrent centrifugal elutriation in the presence of 10 ⁇ g/ml polymyxin B using a JE-6B rotor (Beckman Coulter), as described (Khare et al. Nat Immunol 15, 343-353 (2014); herein incorporated by reference in its entirety).
  • Bone marrow cells flushed from femurs were differentiated into BMDM with GM-CSF either in recombinant form (100 ng/ml; Peprotech), or conditioned medium from L929 (25%) in DMEM medium supplemented with 10% heat inactivated FCS (Invitrogen), 5% horse serum (Gibco) and analyzed after 7 days.
  • Peritoneal macrophages (PM) were obtained by peritoneal lavage.
  • HEK293 and THP-1 cells were obtained from ATCC and were routinely tested for mycoplasma contamination by PCR.
  • hM ⁇ were transfected in 24-well dishes (3.3 ⁇ 10 5 cells) with 100 nM siRNA duplexes (F2/virofect; Targeting Systems) and analyzed 72 hr posttransfection (POP1 siRNA sense strand: 5′-ccuccuacuacgaggacuatt-3′ and Ctrl siRNA F, Santa Cruz, Qiagen), as described (Khare et al. Nat Immunol 15, 343-353 (2014); herein incorporated by reference in its entirety). Transfection efficiency was confirmed by qPCR. THP-1 cells were stably transduced with pLEX or pLKO-based lentiviral particles using magnetic beads (ExpressMag, Sigma) and selected with Puromycin.
  • Recombinant lentivirus was produced in HEK293- lenti cells (Clontech) by Xfect-based transfection (Clontech) with pLKO or pLEX and the packaging plasmids pMD.2G and psPAX2 (Addgene plasmids 12259 and 12260), followed by concentration of virus-containing conditioned medium (Lenti-X Concentrator, Clontech).
  • POP1 shRNA#1 5′-ccggtcctactacgaggactacgcactcgagtgcgtagtcctcgtagtaggattttttg-3′ (TRC N0000423651; SEQ ID NO: 8); POP1 shRNA#3: 5′-ccggacaagctggtcgcctcctactctcgagagtaggaggcgaccagcttgtttttttg-3′ (TRC N0000436892; SEQ ID NO: 9) and a non-targeting scrambled control shRNA (Sigma).
  • E. coli LPS 600 ng/mL E. coli LPS (0111:B4, Sigma) or pre-treated with ultra-pure E. coli LPS (0111:B4; Invivogen) (100 ng/mL), recombinant IL-1 ⁇ (10 ng/ml, Millipore), recombinant IL-1Ra (anakinra, 10 mg/mL, Amgen), MDP (20 ug/mL; Invivogen), CPPD (125 ug/ml; Invivogen), Cholera Toxin B subnit (CTB) (10 ug/ml; List Biological Laboratories) for 16 h.
  • CTB Cholera Toxin B subnit
  • Cells were transfected with poly(dA:dT) (2 ng/mL; Sigma), Salmonella thyphimurium flagellin (140 ng/mL; Invivogen), ultra-pure E. coli LPS (0111:B4; Invivogen) (1000 ng/mL) using Lipofectamine 2000 (Invitrogen). Where indicated, cells were pulsed for 20 min with ATP (5 mM; Sigma) or treated for 45 min with nigericin (5 ⁇ M). In caspase-1 inhibition studies, the caspase-1 inhibitor zYVAD-fmk was added to cells 30 min prior stimulation with LPS.
  • LPS-primed cells were incubated for 3 h in K + free medium containing 0.8 mM MgCl 2 , 1.5 mM CaCl 2 , 10 mM HEPES, 5 mM Glucose, 140 mM NaCl, pH 7.2.
  • mice 8-12 weeks old female WT and CD68-POP1 TG mice had their abdomen shaved under anaesthesia, and were randomly selected for i.p. injection with PBS or LPS (2.5 mg/kg, E. coli 0111:B4, Sigma). After 3 h, mice were i.p. injected with XenoLight Rediject Inflammation probe (200 mg/kg, PerkinElmer) (Gross et al. Nat Med 15, 455-61 (2009); herein incorporated by reference in its entirety) and in vivo bioluminescence was captured by imaging (IVIS Spectrum, PerkinElmer) 10 min post injection with a 5 min exposure on anesthetized mice. Images were quantified with Living Image software (PerkinElmer).
  • Endotoxic shock was induced by i.p. injection of a lethal dose of 20 mg/kg LPS ( E. coli 0111:B4) and mice were monitored 4 times daily for survival. Body temperature was measured with an animal rectal probe. Blood was collected 3 h post LPS injection by mandibular bleed, and serum cytokine levels were quantified by ELISA.
  • Floxed NLRP3 A350V mice (Brydges et al. Immunity 30, 875-87 (2009); Brydges et al. J Clin Invest 123, 4695-4705 (2013); herein incorporated by reference in their entireties) were crossed with Lysozyme M-Cre recombinase (CreL) and CD68-POP1 TG mice and male and female offsprings analysed for body weight and survival. Histological analysis was performed at day 8 after birth.
  • HEK293 cells were infected with an ASC-GFP-expressing lentivirus as described above.
  • Total cell lysates were prepared by hypotonic lysis (20 mM HEPES-KOH, pH 7.5, 5 mM MgCl 2 , 0.5 mM EGTA, 0.1% CHAPS, supplemented with protease inhibitor) and aggregation of ASC-GFP was induced by incubation of cell lysates at 37° C. for 30 min as previously described (Fernandes-Alnemri et al. Methods Enzymol 442, 251-270; herein incorporated by reference in its entirety).
  • ASC-GFP particles were sorted by flow cytometry and polymerization of ASC-specks was confirmed by fluorescent microscopy.
  • HEK293 cells transiently transfected with ASC-GFP and RFR-POP1 were used for isolation of ASC/POP1 particles.
  • LPS-primed THP-1 cells were treated with 2 ⁇ 10 3 particles for 16 h.
  • mice 14 weeks old male WT and CD68-POP1 TG mice had their abdomen shaved under anaesthesia, and were randomly selected for i.p. injection with PBS or FACS-purified ASC-GFP particles (1 ⁇ 10 5 particles/mouse).
  • mice were i.p. injected with XenoLight Rediject Inflammation probe (200 mg/kg, PerkinElmer) (Gross et al. Nat Med 15, 455-61 (2009); herein incorporated by reference in its entirety) and in vivo bioluminescence was captured by imaging (IVIS Spectrum, PerkinElmer) 10 min post injection with a 5 min exposure on anesthetized mice 15 . Images were quantified with Living Image software (PerkinElmer). Peritoneal lavage fluids were collected and assayed for IL-1 ⁇ by ELISA.
  • Rabbit polyclonal and mouse monoclonal POP1 antibodies were custom raised, rabbit polyclonal antibody to ASC (Santa Cruz Biotech), mouse monoclonal antibody to ASC (custom), mouse polyclonal antibody to caspase-1 (Santa Cruz Biotech clone M-20), mouse monoclonal antibody to GFP (Santa Cruz Biotech clone B-2), mouse monoclonal antibody to myc (Roche and Santa Cruz Biotech clone 9E10), mouse monoclonal antibody to HA (Santa Cruz Biotech clone F-7), rabbit polyclonal antibody to IL-1 ⁇ (Santa Cruz Biotech), rabbit polyclonal antibodies to I ⁇ B ⁇ (clone 44D4)/p-I ⁇ B ⁇ (clone 14D4), JNK (clone 9252)/p-JNK (9251), p38 (clone 9212)/p-p38 (clone 12F8), p42/44 (clone 9102)/p-p
  • IP Co-Immunoprecipitations
  • HEK293 cells were transfected with GFP, GFP-POP1, HA-ASC, MYC-NLRP3, HA-ASC PYD , MYC-ASC PYD or empty plasmid in 100 mm dishes using Lipofectamine 2000 (Invitrogen).
  • POP1 was expressed from pGEX-4T1 and affinity purified as a GST fusion protein from E. coli BL21 (Stehlik et al. Biochem. J 373, 101-113 (2003); herein incorporated by reference in its entirety). Protein lysates were prepared from LPS-treated (16 h) BMDM or THP-1 cells by lysis (50 mM Hepes pH 7.4, 120 mM NaCl, 10% Glycerol, 2 mM EDTA, 0.5% Triton X-100, supplemented with protease inhibitors), and cleared lysates were incubated with immobilized GST-POP1 or GST control for 16 h at 4° C., followed by extensive washing with lysis buffer and analysis as above.
  • BMDM 4 ⁇ 10 6 BMDM were seeded in 60 mm plates and subjected to cross-linking.
  • Cells were either left untreated or treated with LPS (4 h) and pulsed with ATP (20 min), culture SN were removed, cells rinsed with ice-cold PBS and lysed (20 mM Hepes pH 7.4, 100 mM NaCl, 1% NP-40, 1 mM sodium orthovanadate, supplemented with protease inhibitors) and further lysed by shearing.
  • Human lung tissue was embedded in paraffin, cut into 3 ⁇ m sections, mounted, deparaffinized and immunostained with mouse monoclonal CD68 (Dako) and rabbit polyclonal POP1 (custom raised) and peroxidase (HRP)/DAB + and alkaline phosphatase (AP)/Fast Red enzyme/chromogen combinations (Dako) and specific isotype controls (Dako) and hematoxylin counterstaining of nuclei.
  • Mouse tissues were dissected, fixed in 10% formalin, embedded in paraffin, sectioned and stained with hematoxylin and eosin (H&E) at the Northwestern University Mouse Histology and Phenotyping Laboratory.
  • ELISA IL-1 ⁇ , IL-1 ⁇ , IL-18 and TNF ⁇ secretion was quantified from clarified culture SN obtained from hM ⁇ , THP-1 cells, BMDMs, PM and from mouse serum by ELISA (BD Biosciences, eBiosciences, Invitrogen). Samples were analyzed in triplicates and repeated at least three times, showing a representative result.
  • Blood was collected into EDTA-containing tubes either via facial vein bleed (from live animals) or via cardiac puncture (from euthanized animals). Whole blood was stained with fluorochrome-conjugated antibodies and erythrocytes were then lysed using BD FACS lysing solution (BD Biosciences). Cells from the peritoneal cavity were harvested after lavage of the peritoneal cavity with 10 ml of ice-cold MACS buffer. Bone marrow was harvested from femurs and tibias. Spleen and lungs were digested using mixture of Collagenase D and DNase I (Roche) in HBSS at 37° C. for 30 min and filtered through 40 ⁇ m nylon mesh.
  • BD FACS lysing solution BD Biosciences
  • Erythrocytes were lysed using BD Pharm Lyse (BD Biosciences) and cells were counted using Countess automated cell counter (Invitrogen). Dead cells were discriminated using trypan blue. Cells were stained with live/dead Aqua (Invitrogen) or eFluor 506 (eBioscience) viability dyes, incubated with FcBlock (BD Bioscience) and stained with fluorochrome-conjugated antibodies. Data were acquired on a BD LSR II flow cytometer (BD Biosciences). Compensation and analysis of the flow cytometry data were performed using FlowJo software (TreeStar). “Fluorescence minus one” controls were used when necessary to set up gates.
  • 6xHIS-POP1 and a 6xHIS-GFP cDNAs in pET28a were fused with the HIV TAT sequence (YGRKKRRQRRR (SEQ ID NO: 10)) by standard PCR, produced under native conditions in E.
  • mice 12 weeks old male WT mice had their abdomen shaved under anaesthesia, were randomly selected for i.p. injection with TAT-GFP or TAT-POP1 (40 ⁇ g/kg) for 30 min prior LPS i.p. injection (2.5 mg/kg, E. coli 0111:B4, Sigma), and were quantified for MPO activity in vivo 1 h later, as described above.
  • NLRP3 inflammasome activity is required for homeostasis in several tissues, including the lung and contributes to the pathology of lung inflammation (De Nardo et al. Am J Pathol 184, 42-54 (2014); herein incorporated by reference in its entirety).
  • POP1 expression was found in human lung tissue, particularly in CD68 + alveolar macrophages (M ⁇ ) by immunohistochemistry ( FIG. 15 a ), using a custom raised antibody that neither cross reacts with other POPs (Khare et al. Nat Immunol 15, 343-353 (2014); herein incorporated by reference in its entirety), nor with the highly similar ASC PYD ( FIG. 16 a, b ).
  • Cryopyrinopathies or Cryopyrin-associated periodic syndromes; CAPS
  • CAPS Cryopyrin-associated periodic syndromes
  • the late response expression of POP1 potentially enables inflammasome functions in early host defense and may provide a mechanism to counter excessive release of late mediators that perpetuate systemic inflammation.
  • This LPS-inducible expression of POP1 was also observed in leukocytes isolated from human subjects following LPS infusion in vivo (Calvano et al. Nature 437, 1032-7 (2005); herein incorporated by reference in its entirety) ( FIG. 15 f ).
  • LPS is a potent activator of IL1B transcription through I ⁇ B ⁇ -regulated NF- ⁇ B activation (Scheibel et al.
  • POP1 Inhibits Inflammasome-Mediated Cytokine Release in Human Macrophages
  • THP-1 GFP-POP1 human monocytic THP-1 cell line stably expressing POP1 (THP-1 GFP-POP1 ) was generated, which was confirmed by qPCR ( FIG. 17 a ).
  • THP-1 GFP-POP1 revealed diminished secretion of mature IL-1 ⁇ in response to NLRP3 activation with nigericin, CPPD or K + depletion, and in response to activation of the AIM2, NLRC4 and NLRP1 inflammasomes with poly(dA:dT) transfection, flagellin transfection or with MDP treatment in LPS-primed cells, respectively ( FIG. 17 b ).
  • POP1 blocked IL-1 ⁇ release by the non-canonical inflammasome in response to cytosolic LPS upon LPS treatment, cytosolic LPS upon LPS transfection or cytosolic delivery of LPS with cholera toxin subunit B (CTB) ( FIG. 17 c ). Comparable results were obtained for myc-tagged POP1 ( FIG. 16 d ). Accordingly, THP-1 GFP-POP1 cells also showed markedly blunted NLRP3-mediated caspase-1 activity in LPS-primed and nigericin-treated cells ( FIG. 17 d ), and consequently reduced LDH release ( FIG. 17 e ).
  • TLR-mediated NF- ⁇ B priming is necessary for NLRP3 inflammasome activation (Bauernfeind et al. J Immunol 183, 787-91 (2009); Juliana et al. J Biol Chem 287, 36617-22 (2012); Schroder et al. Immunobiology 217, 1325-9 (2012); herein incorporated by reference in their entireties), but, in contrast to over-expression in epithelial cell lines, stable POP1 expression in THP-1 cells did neither affect phosphorylation of I ⁇ B ⁇ ( FIG. 17 h ) nor transcription of IL1B ( FIG. 17 i ) in response to LPS, thus implicating POP1 in directly regulating the NLRP3 inflammasome in M ⁇ .
  • POP1 specifically bound to endogenous ASC, but not to the PYD-containing PRRs NLRP3 and AIM2 in LPS-primed THP-1 cells ( FIG. 18 a ), emphasizing the selectivity of these interactions. POP1 also indirectly co-purified pro-caspase-1, and since POP1 interacts with the ASC PYD , which did not affect the CARD-mediated binding of ASC to caspase-1 ( FIG. 18 a ). Binding of ASC to NLRP3 induces ASC PYD nucleation, which provides the oligomeric platform essential for caspase-1 activation (Lu et al.
  • mice Since all POPs, including POP1, are lacking from mice (Stehlik et al J Immunol 179, 7993-8 (2007); Khare et al. Nat Immunol 15, 343-353 (2014); herein incorporated by reference in their entireties), GFP-POP1 transgenic (TG) mice were generated and POP1 expression to M ⁇ was restricted using the hCD68/IVS-1 promoter/enhancer (Iqbal et al. Blood blood-2014-04-568691 (2014); Khare et al. Nat Immunol 15, 343-353 (2014); Gough et al. Immunology 103, 351-61 (2001); herein incorporated by reference in their entireties), based on the expression observed in CD68 + M ⁇ ( FIG. 15 a ).
  • FIG. 19 a qPCR analysis of whole blood cell RNA revealed expression of POP1 specifically in CD68-POP1 TG, but not in wild-type (WT) mice ( FIG. 19 a ).
  • POP1 expression from the CD68/IVS-1 promoter in BMDM POP1 was LPS-inducible ( FIG. 19 b ), thus recapitulating the inducible expression found in hM ⁇ ( FIG. 15 e, f ).
  • Flow cytometry analysis of peripheral blood demonstrated POP1 expression selectively in monocytes ( FIG. 22 a , FIG. 17 c ), with equal expression in classical Ly6C hi CD43 ⁇ , intermediate Ly6C int CD43 + and non-classical Ly6C lo CD43 + monocytes ( FIG. 21 a ).
  • POP1 was also expressed in the myeloid-(MP), M ⁇ and DC-(MDP) and common DC precursor (CDP) in bone marrow ( FIG. 20 b , 21 a ), large peritoneal M ⁇ (LPM), small peritoneal M ⁇ (SPM) and peritoneal DC ( FIG. 18 c , 22 b ), as well as in splenic red pulp M ⁇ (RPM), monocytes and CD11b + DCs, but not plasmacytoid DC (pDC) ( FIG. 18 d , 23 c ). Expression was also detected in BMDM by immunoblot ( FIG. 22 b ).
  • Monocyte/M ⁇ -specific POP1 expression was also observed in other tissues, with no detectable expression in CD45 ⁇ cells ( FIG. 23 ).
  • Mouse and human ASC are highly homologous within the PYD ( FIG. 25 a ), and as observed for THP-1 cells, POP1 also interacted with mouse ASC, but not NLRP3 or AIM2 in BMDM ( FIG. 22 c ).
  • oligomerization of ASC requires nucleation by NLRP3 and the subsequent ASC polymerization can be captured by non-reversible cross-linking and functions as a readout for inflammasome activation (Fernandes-Alnemri et al.
  • BMDM POP1 lacked active caspase-1 p10 and mature IL-1 ⁇ in culture supernatants of LPS/ATP treated cells to a similar extent as the caspase-1 inhibitor zYVAD-fmk ( FIG. 22 e ), and revealed reduced active caspase-1 as quantified by flow cytometry in intact cells ( FIG. 22 f ).
  • POP1 Prevents Inflammasome-Dependent Cytokine Release in Mouse Macrophages
  • BMDM POP1 LPS/ATP treated BMDM POP1 also displayed significantly reduced levels of IL-1 ⁇ , IL-1 ⁇ and IL-18 in culture supernatants by ELISA. However, secretion of TNF ⁇ , which occurs independently of caspase-1, was not affected ( FIG. 24 a ). Significantly, reduced IL-1 ⁇ release in BMDM POP1 was comparable to BMDM ASC ⁇ / ⁇ and BMDM NLRP3 ⁇ / ⁇ ( FIG. 24 b ). K + efflux is the unifying mechanism of NLRP3 activation (Mu ⁇ oz-Planillo et al Immunity 38, 1142-1153 (2013); herein incorporated by reference in its entirety).
  • BMDM POP1 culturing BMDM POP1 in K + -free medium showed impaired IL-1 ⁇ release compared to BMDM WT ( FIG. 24 c ).
  • peritoneal macrophages PM POP1
  • PM POP1 peritoneal macrophages
  • BMDM POP1 also showed reduced LDH release, and thus pyroptosis, when compared to BMDM WT in response to NLRP3 activation ( FIG.
  • Caspase-11 is responsible for LPS- and Gram negative bacteria-induced lethal shock, but ASC and NLRP3 are both necessary for amplifying this lethal response to LPS in vivo (Kayagaki et al. Nature 479, 117-21 (2011); herein incorporated by reference in its entirety). Accordingly, Asc ⁇ / ⁇ and Nlrp3 ⁇ / ⁇ mice are protected from LPS-induced lethality in response to moderate LPS doses (Kayagaki et al. Nature 479, 117-21 (2011); Mariathasan et al. Nature 440, 228-232 (2006); Mariathasan et al. Nature 430, 213-8 (2004); herein incorporated by reference in their entireties).
  • CD68-POP1 TG mice experienced significantly less hypothermia ( FIG. 26 b ) and were significantly more protected from a lethal LPS dose ( FIG. 26 c ). Compared to 100% lethality in WT mice, only 30% of CD68-POP1 TG mice died within 96 hours, which is similar to ASC ⁇ / ⁇ mice (Mariathasan et al. Nature 430, 213-8 (2004); herein incorporated by reference in its entirety). Consistent with reduced neutrophil infiltration and increased survival, serum IL-1 ⁇ and IL-18 levels were also reduced, but TNF ⁇ levels remained unchanged ( FIG. 26 d ).
  • CAPS can be recapitulated in mice by knocking-in CAPS-associated NLRP3 mutations (Brydges et al. Immunity 30, 875-87 (2009); Meng et al. Immunity 30, 860-74 (2009); Brydges et al. J Clin Invest 123, 4695-4705 (2013); herein incorporated by reference in their entireties).
  • a mouse model for Muckle Wells Syndrome (MWS) was employed, where floxed NLRP3 A350V , corresponding to human NLRP3 A352V , is expressed exclusively in myeloid cells in the presence of lysozyme M-Cre (CreL) (Brydges et al.
  • NLRP3 A350V/+ CreL mice develop systemic inflammation affecting multiple organs, display characteristic skin inflammation and die within two weeks of birth, a phenotype caused by excessive IL-1 ⁇ and IL-18 release and pyroptosis.
  • NLRP3 A350V/+ CreL mice had inflammatory skin abscesses and lesions shortly after birth, which developed into scaling erythema, but NLRP3 A350V/+ CreL CD68-POP1 mice did not display this phenotype ( FIG. 26 e ).
  • Extracellular ASC particles are phagocytized by macrophages and directly nucleate soluble ASC to activate caspase-1 in an ASC-dependent, but NLRP3-independent process (Franklin et al. Nat Immunol 15, 727-37 (2014); herein incorporated by reference in its entirety).
  • FACS-purified ASC-GFP particles FIG. 28 d , FIG. 27 b
  • IL-1 ⁇ release in LPS primed THP-1 GFP cells but not in THP-1 GFP-POP1 cells ( FIG. 28 d )
  • POP1 incorporates into newly polymerized ASC PYD filaments, as suggested above ( FIG. 18 d, e ).
  • ASC CARD density is reduced to a level that is insufficient to nucleate caspase-1 and caspase-1 activation is prevented 6 .
  • mixed ASC-GFP/RFP-POP1 particles were generated ( FIG. 28 e , FIG. 27 b ), which, contrary to ASC-GFP particles, failed to cause IL-1 ⁇ release in THP-1 cells ( FIG. 28 e ).
  • Injection (i.p.) of ASC-GFP particles into WT mice resulted in neutrophil recruitment ( FIG. 28 f , FIG. 27 c ) and IL-1 ⁇ release ( FIG.
  • FIG. 30 shows that POP3 can interact with itself and ASC (with the PYRIN domain[PYD] of ASC) in vitro (left) and in vivo (right).
  • FIG. 31 shows that POP3 can interact with the PYDs of the ALRs AIM2 and IF116 in vitro (left) and in vivo (right).
  • FIG. 32 shows that POP3 can inhibit mutant NLRP3-mediated inflammasome activation in the inflammasome reconstitution system.
  • FIG. 33 shows that knock-down of POP3 in primary human macrophages enhances vaccinia virus-mediated inflammasome activation and subsequent IL-1 ⁇ release, which is inhibited by knock-down of AIM2 and ASC, which are both involved in sensing vaccinia virus infection.
  • FIG. 34 shows that POP3 is regulated through protein stability and is degraded by the proteasome, which is blocked by MG132, a proteasome inhibitor. Also interferon causes stability of POP3 through its posttranslational modification.
  • FIG. 35 shows that POP3 expression recruits IF116 from the nucleus to the cytosol, where both proteins co-localize, which supports that both proteins can interact.
  • FIG. 36 shows that POP3 expression prevents the interaction between AIM2 and the inflammasome adaptor ASC.
  • AIM2 and ASC form oligomeric complexes in the absence of POP3.
  • FIG. 37 shows that POP3 expression blocks DNA (dsVACV70mer)-induced and IF116 mediated activation of interferon response elements.
  • FIG. 38 shows that delivery of recombinant POPs fused to the cell permeable TAT peptide into macrophages can block inflammasome activity.

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WO2018133139A1 (fr) 2017-01-21 2018-07-26 宁波知明生物科技有限公司 Application de paeéniflorin-6'-o-benzène sulfonate en médecine pour le traitement du syndrome de sjögren
WO2020223595A1 (fr) * 2019-05-01 2020-11-05 H. Lee Moffitt Cancer Center And Research Institute, Inc. Modification guidée par la structure d'inhibiteurs de l'inflammasome nlrp3 à base de peptides pour syndromes myélodysplasiques (smd)
JP2021514183A (ja) * 2018-02-08 2021-06-10 スタイプ セラピューティクス エーピーエス 改変免疫調節ペプチド
US20210238228A1 (en) * 2018-08-16 2021-08-05 H. Lee Moffitt Cancer Center And Research Institute, Inc. Stapled peptides as pyrin-domain targeted nlrp3 inflammasome inhibitors

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US11725035B2 (en) * 2016-08-09 2023-08-15 Stipe Therapeutics Aps Methods of treating a disorder associated with with insufficient stimulator of interferon genes (STING) activity
GB201815045D0 (en) * 2018-09-14 2018-10-31 Univ Ulster Bispecific antibody targeting IL-1R1 and NLPR3

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US7300749B2 (en) * 2000-02-17 2007-11-27 Millennium Pharmaceuticals, Inc. Molecules of the pyrin domain protein family and uses thereof
US20040002593A1 (en) * 2002-04-04 2004-01-01 Reed John C. PAAD domain-containing polypeptides, encoding nucleic acids, and methods of use
WO2006110591A2 (fr) * 2005-04-07 2006-10-19 University Of South Florida Polypeptides inhibiteurs du nfkb de pop2, acides nucleiques et methodes d'utilisation associees
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US20160354437A1 (en) 2014-02-13 2016-12-08 Northwestern University Compositions and methods for modulation of immune response
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WO2018133139A1 (fr) 2017-01-21 2018-07-26 宁波知明生物科技有限公司 Application de paeéniflorin-6'-o-benzène sulfonate en médecine pour le traitement du syndrome de sjögren
JP2021514183A (ja) * 2018-02-08 2021-06-10 スタイプ セラピューティクス エーピーエス 改変免疫調節ペプチド
US20210238228A1 (en) * 2018-08-16 2021-08-05 H. Lee Moffitt Cancer Center And Research Institute, Inc. Stapled peptides as pyrin-domain targeted nlrp3 inflammasome inhibitors
WO2020223595A1 (fr) * 2019-05-01 2020-11-05 H. Lee Moffitt Cancer Center And Research Institute, Inc. Modification guidée par la structure d'inhibiteurs de l'inflammasome nlrp3 à base de peptides pour syndromes myélodysplasiques (smd)

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