COMPOSITIONS AND METHODS FOR MODULATION OF IMMUNE RESPONSE
Provided herein are compositions and methods for modulation of immune response via PYRIN domain-only proteins POP1 and/or POP3. In particular, POP1 and/or POP3 are inhibited to enhance an immune response (e.g., to treat or prevent infection), or POP1 and/or POP3 are administered or activated to reduce an immune response (e.g., to treat or prevent autoimmune or inflammatory disease).
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The present application claims the benefit of U.S. Provisional Patent Application Ser. No. 61/939,499, filed Feb. 13, 2014, which is incorporated by reference in its entirety.
FIELDProvided herein are compositions and methods for modulation of immune response via PYRIN domain-only proteins POP1, POP2, and/or POP3. In particular, 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).
BACKGROUNDThe 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). Activation of PRRs promotes an inflammatory host response through up-regulation of inflammatory mediators, including cytokines and chemokines to directly eliminate pathogens and to mount a long-lasting adaptive immune response. 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). 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. 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. 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-1β 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)-Iβ 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.
SUMMARYProvided herein are compositions and methods for modulation of immune response via PYRIN domain-only proteins POP1, POP2, and/or POP3. In particular, 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). In some embodiments, POP1-, POP2-, and/or POP3-based inflammasome (e.g., NLRP3 inflammasomes) inhibitors (e.g., POP1-, POP2-, or POP3-based peptide or peptidomimetics), are administered to: (a) inhibit inflammasome activity, (b) prevent IL-1β 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). In some embodiments, 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.
Provided herein are compositions comprising, inflammasome-inhibitory peptides, polypeptides, and protein, and methods of treating autoimmune and/or chronic inflammatory diseases and conditions therewith. In particular, 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.
Provided herein are 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. In some embodiments, a polypeptide or peptide has 100% sequence identity with all or a portion of POP1. In some embodiments, the peptide or polypeptide comprises a portion with at least 80% sequence similarity (e.g., >80%, >90%, >95%) with POP1. In some embodiments, 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.
Provided herein are 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. In some embodiments, a polypeptide or peptide has 100% sequence identity with all or a portion of POP1. In some embodiments, the peptide or polypeptide comprises a portion with at least 80% sequence similarity (e.g., >80%, >90%, >95%) with POP2. In some embodiments, 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.
In some embodiments, 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). In some embodiments, the peptide or polypeptide has at least 80% sequence similarity (e.g., >80%, >85%, >90%, >95%) with POP3. In some embodiments, 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. In some embodiments, the peptide or polypeptide has at least 80% sequence similarity (e.g., >80%, >85%, >90%, >95%, 100%) with POP3. In some embodiments, a peptide or polypeptide has a portion with 100% sequence identity with POP3. In some embodiments, 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.
In some embodiments, 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). In some embodiments, 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. In some embodiments, 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. In some embodiments, 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.
In some embodiments, 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).
In some embodiments, compositions are provided comprising peptides and/or polypeptides that exhibit enhanced inflammasome-inhibitory activity relative to POP1, POP2, or POP3. In some embodiments, 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. In some embodiments, enhanced inflammasome-inhibitory activity is exhibited in one or more assays descried herein (e.g., see Examples).
In some embodiments, compositions (e.g., small molecules, peptide, polypeptide, antibodies, nucleic acids, etc.) are provided (e.g., administered to a subject or cell) that enhance the inflammasome inhibitory activity of POP1, POP2, and/or POP3. 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.
In some embodiments, provided herein are pharmaceutical preparations 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. In some embodiments, pharmaceutical preparations further comprise an additional therapeutic agent (e.g., for the treatment of: inflammation, pain, autoimmunity, etc.).
In some embodiments, provided herein are 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. In some embodiments, 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, His6, etc.).
In some embodiments, provided herein are polynucleotides encoding a POP1-, POP2-, or POP3-peptide or polypeptide described herein (e.g., in the preceding paragraphs). In some embodiments, provided herein are 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). In some embodiments, vectors further comprise a promoter and/or one or more expression elements (e.g., transcription enhancer, translational start site, internal ribosome entry site, etc.). In some embodiments, methods are provided comprising administering a polynucleotide or vector described herein to a subject or sample (e.g., for the treatment of autoimmunity or inflammation).
In some embodiments, 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.
In some embodiments, provided herein are methods of 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.
In some embodiments, compositions are provided 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. In some embodiments, inhibited inflammasome-inhibitory activity is exhibited in one or more assays descried herein (e.g., see Examples).
In some embodiments, provided herein are pharmaceutical preparations comprising: (a) a POP1, POP2, or POP3 inhibitor; and (b) a physiologically acceptable buffer or carrier. In some embodiments, pharmaceutical preparations further comprise an additional therapeutic agent (e.g., for the treatment of: inflammartion, pain, autoimmunity, etc.).
In some embodiments, provided herein are fusion peptides or polypeptides comprising: (a) a POP1-, POP2-, or POP3-inhibitor, and (b) a functional peptide or polypeptide segment. In some embodiments, 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, His6, etc.).
In some embodiments, provided herein are polynucleotides encoding a POP1-, POP2-, or POP3-inhibitory peptiode or polypeptide described herein (e.g., in the preceding paragraphs). In some embodiments, provided herein are nucleic acid vectors (e.g., plasmid, bacmid, viral vector (e.g., AAV) comprising polynucleotides encoding a POP1-, POP2-, or POP3-inhibitor. In some embodiments, vectors further comprise a promoter and/or one or more expression elements (e.g., transcription enhancer, translational start site, internal ribosome entry site, etc.). In some embodiments, methods are provided comprising administering a polynucleotide or vector described herein to a subject or sample (e.g., for the treatment of infection).
In some embodiments, 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. In some embodiments, provided herein are 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.
As used herein, the term “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 1β (IL-1β) 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.
As used herein, the term “autoimmune disease” refers generally to diseases which are characterized as having a component of self-recognition. Examples of 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. For example, 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. For example, 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.
As used herein, the term “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.
As used herein, the term “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. For example, 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.
As used herein, the term “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. For example, a “synthetic POP1” (“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.).
The term “peptide mimetic” or “peptidomimetic” 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. Examples of peptidomimitics include chemically modified peptides, peptoids (side chains are appended to the nitrogen atom of the peptide backbone, rather than to the α-carbons), β-peptides (amino group bonded to the β carbon rather than the α carbon), etc.
As used herein, the term “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. The term “sequence similarity” refers to the degree with which two polymer sequences (e.g., peptide, polypeptide, nucleic acid, etc.) have similar polymer sequences. For example, 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). The “percent sequence identity” (or “percent sequence similarity”) 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. For example, if 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. As another example, if 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. For the purpose of calculating “percent sequence identity” (or “percent sequence similarity”) herein, any gaps in aligned sequences are treated as mismatches at that position.
As used herein, 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.). As used herein, the term “patient” typically refers to a subject that is being treated for a disease or condition.
As used herein, the term “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.
As used herein, the terms “administration” and “administering” refer 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. Exemplary 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.
As used herein, the terms “co-administration” and “co-administering” refer 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. Those of skill in the art understand that the formulations and/or routes of administration of the various agents or therapies used may vary. 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. Thus, 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.
As used herein, the term “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.
As used herein, the term “pharmaceutical 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.
The terms “pharmaceutically acceptable” or “pharmacologically acceptable,” as used herein, refer to compositions that do not substantially produce adverse reactions, e.g., toxic, allergic, or immunological reactions, when administered to a subject.
As used herein, 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. For examples of carriers, 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.
DETAILED DESCRIPTIONProvided herein are compositions and methods for modulation of immune response via PYRIN domain-only proteins POP1, POP2, and/or POP3. In particular, 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).
Although 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. However, the regulation of inflammasomes has been poorly understood. Experiments conducted during development of embodiments described herein indicate that 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. 2007; 75:1484-1492; Stehlik et al. Biochem J. 2003; 373:101-113; herein incorporated by reference in their entireties), POP3 interacts with the PYD of ALRs and thereby prevents recruitment of ASC. Although, it was found that recombinant POP3 interacted with NLRP3 in vitro, 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. Thus, POP3 evolved as a specific ALR inflammasome regulator.
Experiments further revealed that the human HIN-200 cluster is more complex than previously described and differs from mice. However, 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). However, p202 is barely detectable in C57BL/6 mice, but is highly expressed in BALB/c and NZB mouse strains. This is contemplated to be an important influence as to why POP3 expression in C57BL/6 mice was able to drastically alter the immune response upon MCMV infection using ‘humanized’ C57BL/6 mice. Two proteins encoding only a PYD are predicted in mice within the HIN-200 cluster, which both lack a human ortholog. Pydc3 (Ifi208) is predicted to encode only a PYD, but is significantly larger than POPs and might encode a HIN-200 domain, according to expressed sequence tags (Ludlow et al. Exp Cell Res. 2005; 308:1-17; Cridland J A, et al. BMC Evol Biol. 2012; 12:140; herein incorporated by reference in their entireties). Two predicted Pydc4 alternative transcripts encode only a PYD, but the longest transcript encodes a 586 aa protein, which is 95.2% identical to PYDC3. However, neither gene shows similarity with POP3 and no expression data or functional data are available. Thus, Aim2b, a predicted Aim2 splice form in mice, might most closely resemble the POP3 function in mice. Similar to the human HIN-200 locus, 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.
The type I IFN-responsiveness further distinguishes POP3 from other POP family members. Thus, POP3 represents one of the type I IFN-inducible proteins that antagonizes IFN-γ in macrophages and inflammasome activation (Guarda G, et al.
Immunity. 2011; 34:213-23; herein incorporated by reference in its entirety), and contributes to the anti-inflammatory and immunosuppressive functions of type I IFNs (González-Navajas J M, Lee J, David M, Raz E. Immunomodulatory functions of type I interferons. Nat Rev Immunol.; herein incorporated by reference in its entirety). Aim2−/− BMDM show elevated IFN-β 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. 2010; 11:395-402; herein incorporated by reference in their entireties). It was observed that POP3 silencing, which increases AIM2 signaling, also reduces IFN-β production, and accordingly, POP3 expression in hMΦ, THP-1 cells and BMDM promotes IFN-β production. Thus, besides promoting its own IFN-β-dependent production, experiments indicate that POP3 shifts the immune response from an inflammasome-dependent pro-inflammatory cytokine production to anti-inflammatory IFN-β production, thereby further blunting IL-1β and IL-18 signaling through upregulation of IL-1RA and IL-18BP (Kaser A, et al. Clin Exp Immunol. 2002; 129:332-8; Sciacca et al. J Neurovirol. 2000; 6 (Suppl 2):S33-7, herein incorporated by reference in their entireties). A similar mechanism has been proposed for LRRFIP2, which inhibits NLRP3 inflammasome activation by recruiting the pseudo caspase-1 substrate Flighthless-I (Jin J, et al. Nat Commun. 2013; 4:2075: herein incorporated by reference in its entirety), but also functions as a cytosolic DNA sensor, which promotes type I IFN production (Yang P, et al. Nat Immunol. 2010; 11:487-94; herein incorporated by reference in its entirety). The IFN-β-inducible expression pattern of POP3 as an early and late response gene was similarly observed in CD68-POP3 TG mice. Although, POP3 is lacking from mice, macrophage-specific TG expression revealed that POP3 is nevertheless functional in mice.
POP3 is protein exhibiting inflammasome inhibitory function. POP3 inhibits release of IL-1β 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-1β 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. Experiments were conducted during development of embodiments of the present invention that indicate
POP3 may be employed to specifically block IL-1β 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. Cell 156, 1207-22 (2014); Franklin et al. Nat Immunol 15, 727-37 (2014); herein incorporated by reference in their entireties). Thus, the PYD present in NLRP3 and ASC plays a crucial role in inflammasome assembly. Experiments conducted during development of embodiments described herein support such a model, where POP1 binds to the ASCPYD, which prevents ASCPYD interaction with active NLRP3PYD, and thus the crucial nucleation event for ASC oligomerization, as determined by non-reversible crosslinking. However, in spite of binding to the ASCPYD, POP1 did not prevent ASC self-polymerization. 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. Accordingly, ASC particle induced IL-1β release, which was prevented by POP1 expression. However, mixed particles composed of ASC and POP1 were inactive. Thus, POP1 expression not only prevents inflammasome assembly and cytokine release, but also the propagation of secondary inflammasome responses to bystander cells. Experiments conducted during development of embodiments described here in demonstrate slightly enhanced ASCPYD self-interaction in the presence of POP1, which indicated weak nucleation activity, similar to the NLRP3PYD, which can also nucleate ASCPYD polymerization, albeit significantly less efficient than full length, active NLRP36. Since the NLRP3PYD and POP1 interact through the same motif with the ASC PYD, a weak nucleation effect of POP1 under these in vitro conditions is possible and may also explains the previously observed positive effect of overexpressed POP1 on IL-1β release in inflammasome reconstitution assays (Stehlik et al. J. Immunol 171, 6154-63 (2003); herein incorporated by reference in its entirety). However, similar to the NLRP3PYD6, also POP1 does not have intrinsic prion activity, since POP1 does not form filaments when overexpressed on its own (Stehlik et al. Biochem. J 373, 101-113 (2003); herein incorporated by reference in its entirety).
Excessive and uncontrolled release of inflammasome mediators contribute to autoinflammatory and auto-immune disease (Strowig et al. Nature 481, 278-86 (2012); herein incorporated by reference in its entirety) and blocking in particular IL-1β has been proven beneficial in various inflammatory diseases in human and mice (Dinarello et al. Blood 117, 3720-32 (2011); herein incorporated by reference in its entirety). Furthermore, oligomeric ASC particles have been identified in CAPS and pulmonary disease patients (Franklin et al. Nat Immunol 15, 727-37 (2014); Baroja-Mazo et al. Nat Immunol 15, 738-48 (2014); herein incorporated by reference in their entireties). However, in spite of the tight regulation of this response, even a single point mutation in NLRP3 can drive excessive systemic inflammation (Hoffman et al. J Biol Chem 286, 10889-96 (2011); herein incorporated by reference in its entirety), indicating that also inflammasome regulatory mechanisms may be impaired. In agreement with this concept, POP1 expression was observed in lung tissue from healthy subjects, while CAPS and sepsis patients expressed significantly lower POP1 compared to healthy controls. This indicates that in addition to uncontrolled activation of NLRP3, also the subsequent recruitment of ASC, ASC oligomerization and consequently extracellular release of oligomeric ASC danger particles proceeds continuously, without a proper POP1-mediated shutdown mechanism.
Experiments conducted during development of embodiments described herein identified 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-1β and type I interferon. As such, 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.).
Experiments conducted during development of embodiments described herein identified POP2 as an inflammasome inhibitor. As such, 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.). Further, inhibition of POP2 activity us useful in the treatment or prevention of conditions (e.g., infection) in which an enhanced immune response is desired.
In some embodiments, POP1, POP2, and/or POP3 are administered to a cell or subject. In some embodiments, POP1, POP2, and/or POP3 polypeptide, peptide, or peptidomimetic is administered. In some embodiments, a composition that enhances the activity of POP1 and/or POP3 is administered. In some embodiments, a composition that colocalizes POP1 and/or POP3 with inflammasomes is administered.
In some embodiments, a protein, polypeptide, or peptide is provided 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%). In some embodiments, a protein, polypeptide, or peptide is provided that 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)). In some embodiments, a protein, polypeptide, or peptide is provided that exhibits inflammasome inhibitory activity.
In some embodiments, a protein, polypeptide, or peptide is provided 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%). In some embodiments, a protein, polypeptide, or peptide is provided that 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)). In some embodiments, a protein, polypeptide, or peptide is provided that exhibits inflammasome inhibitory activity.
In some embodiments, a protein, polypeptide, or peptide is provided 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%). In some embodiments, a protein, polypeptide, or peptide is provided that 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)). In some embodiments, a protein, polypeptide, or peptide is provided that exhibits inflammasome inhibitory activity.
In some embodiments, exogenous POP1, POP2, POP3, fragments thereof, or peptides or polypeptides having 60-100% sequence identity thereto (e.g., and maintaining POP1, POP2, and/or POP3 activity) are administered to a cell or subject. In other embodiments, POP1, POP2 and/or POP3 activating agents (e.g., peptides, small molecules, etc.) are administered to a cell or subject. In some embodiments, administration of the foregoing results in inflammasome inhibitions, inhibition of cytokine production, inhibition of IL-1β release, inhibition of type I interferon release, blocking signaling from NLRs, etc.
In some embodiments, a composition is administered that inhibits inflammasome activity. In some embodiments, a composition is administered that inhibits inflammasome assembly.
Experiments conducted during development of embodiments described herein identified 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-1β and type I interferon. As such, 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). In some embodiments, one or more inhibitors (e.g., anti-POP1 antibody, anti-POP3 antibody, fragments thereof) of POP1 and/or POP3 (e.g., anti-POP1 antibody, anti-POP3 antibody, fragments thereof) are administered (e.g., to a cell, tissue, subject, etc.) for the treatment or prevention of infection (e.g., bacterial or viral).
Experiments conducted during development of embodiments described herein identified POP2 as an inflammasome inhibitor. As such, 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.). Further, inhibition of POP2 activity us useful in the treatment or prevention of conditions (e.g., infection) in which an enhanced immune response is desired.
In some embodiments, a POP1, POP2, and/or POP3 inhibitor is a small molecule, peptide, polypeptide, protein, antibody, nucleic acid, etc. In some embodiments, a POP1, POP2, and/or POP3 binding agent (e.g., antibody, antibody fragment, aptamer, etc.) is administered that neutralizes POP1, POP2, and/or POP3 activity. In some embodiments, such a binding agent recognizes an epitope displayed by POP1, POP2, and/or POP3. In some embodiments, binding agents (e.g., antibody, antibody fragment, aptamer, etc.) capable of binding and/or neutralizing POP1, POP2, and/or POP3 are provided. In some embodiments, an antibody is a humanized antibody, antibody fragment, multivalent antibody, monoclonal antibody, neutralizing antibody, or any suitable combination thereof.
In some embodiments, compositions comprise a POP1, POP2, and/or POP3 neutralizing antibody or antibody fragment. The term “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.
In some embodiments, a composition is administered that promotes inflammasome activity. In some embodiments, a composition is administered that promotes inflammasome assembly.
In some embodiments, methods are provided screening test agents (e.g., pharmaceuticals, drugs, peptides, antibodies, aptamers, or other test agents) for one or more 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.). In some embodiments, 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.
In some embodiments, screening assays for assessing cellular behavior or function are provided. For example, the response of cells, tissues, or subject to interventions (e.g., POP1/POP2/POP3 inhibition, POP1/POP2/POP3 administration, POP1/POP2/POP3 activation, etc.) may be monitored by assessing, for example, cellular functions using animal or cell culture models. 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.).
EXPERIMENTAL Example 1 The PYRIN Domain-Only Protein POP3 Inhibits AIM2-Like Receptor Inflammasomes and Regulates Responses to DNA Virus Infections MicepCD68-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 (hCARΔcyt)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.
Macrophage Isolation, Culture and TransfectionhMΦ 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 μg/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. Immunity. 2012; 36:464-76; herein incorporated by reference in its entirety). 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. hMΦ, THP-1 cells, BMDM and PM were treated for the indicated times with 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-β (1500 U/mL; Millipore), MG132 (10 μM; 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 μM).
Virus PreparationRecombinant 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 μm pore size filters. KSHV was subsequently concentrated by ultracentrifugation at 20,000 rpm for 90 min (SW28 rotor, 4° C.). Viral pellets were resuspended in EBM2 medium (Lonza), 0.45 μm 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.
PlasmidspCDNA3-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. No.: KF562078), 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.
Immunoblot Analysis, Immunoprecipitation and ImmunohistochemistryRabbit 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 (Abeam clone ab104409), rabbit polyclonal antibody to the C-terminus of AIM2 (Cell Signaling Technology clone 8055), rabbit polyclonal antibodies to IκBα (clone 44D4)/p-IκBα (clone 14D4), JNK (clone 9252)/p-JNK (9251), p38 (clone 9212)/p-p38 (clone 12F8), p42/44 (clone 9102)/p-p42/44 (9101), IRF3 (clone D83B9), p-IRF3 (clone 4D4G) (all Cell Signaling Technology) and mouse monoclonal antibody to β-tubulin (Santa Cruz Biotech clone TU-02), mouse monoclonal antibody to GST (Santa Cruz Biotech clone B-14) and mouse monoclonal antibody to NLRP3 (Adipogen clone Cryo-2) were used for immunoblot. For co-immunoprecipitations (IP), HEK293 cells were transfected with GFP-POP3, HA-ASC, RFP-AIM2 or empty plasmid in 100 mm dishes (Lipofectamine 2000, Invitrogen). Cells were lysed (50 mM Hepes pH 7.4, 150 mM NaCl, 10% Glycerol, 2 mM EDTA, 0.5% Triton X-100, supplemented with protease inhibitors) 36 hrs post transfection. Cleared lysates were subjected to IP by incubating with immobilized antibodies as indicated for 16 hrs at 4° C., followed by extensive washing with lysis buffer. 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 μM) or MVA-infection (90 min), respectively. For GST pull down experiments, 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. For ASC cross-linking, 4×106 BMDM were seeded in 60 mm plates and subjected to cross-linking as described 55. Briefly, cells were transfected with 1 μg/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. Cleared lysates were stored for immunoblot analysis and the insoluble pellets were resuspended in 500 μl PBS, supplemented with 2 mM disuccinimydyl suberate (DSS, Pierce) and incubated with rotation at room temperature for 30 min. Samples were centrifuged at 5,000 rpm for 10 min at 4° C. and the cross-linked pellets were resuspended in 50 μl Laemmli sample buffer and analyzed by immunoblot. Human lung tissue was embedded in paraffin, cut into 3 μm sections, mounted, deparaffinized and immunostained with mouse monoclonal CD68 (Dako) and rabbit polyclonal POP3 and peroxidase (HRP)/DAB+ and alkaline phosphatase (AP)/Fast Red enzyme/chromogen combinations (Dako) and specific isotype controls (Dako) and haemotoxylin counterstaining of nuclei.
Immunofluorescence MicroscopyhMΦ were grown on cover slips and either IFN-β 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).
Cytokine and Caspase-1 MeasurementIL-1β, IL-18, TNα, IL-6, IFN-β and IFN-γ secretion was quantified from clarified culture SN obtained from hMΦ, 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 Analysis
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. 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). Doublets and debris were excluded and leukocytes were identified using the pan-hematopoietic marker CD45 (30-F11, BD). 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). Monocytes were further subdivided into Ly-6Chi classical or inflammatory and Ly-6Clo/med non-classical or resident monocytes. Total RNA was isolated from hMΦ, BMDM or mouse blood using Trizol (Invitrogen) or the mouse RiboPure-blood RNA isolation kit (Invitrogen), treated with DNase I, reverse transcribed with GoScript (Promega) and analyzed by TaqMan Real-time gene expression system using predesigned FAM labeled primer/probes on an ABI 7300 Real time PCR machine (Applied Biosystems) and displayed as relative expression compared to GAPDH or β-actin. 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).
In Vivo Virus Infection and Intracellular IFN-γ Staining8-10 week-old age and gender matched wild type (WT) and CD68-POP3 mice were randomly infected with 1×105 to 1×106 PFU by i.p. injection of MCMV or mock salivary gland homogenates and euthanized after 36 hrs. Spleens were digested with collagenase type D (Roche) (1 mg/mL) and DNase I (Roche) (0.1 mg/mL) in HBSS at 37° C. for 15 min, passed through 40 μm nylon cell strainers (BD Biosciences), after which red cells were lysed using 1×BD Pharm Lyse buffer (BD Biosciences), and washed with complete RPMI medium (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-γ 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 μL/mL, BD Biosciences) before staining (Rathinam V A, et al. Nat Immunol. 2010; 11:395-402; herein incorporated by reference in its entirety). 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-γ (XMG1.2, eBioscience) 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).
MSU-Induced Peritonitis10-12 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-1β by ELISA.
POP3 is Expressed in Response to Type-I InterferonsA previously undescribed human POP family member, POP3, was identified (Genbank accession number: KF562078) (
Since PYDs usually exhibit homotypic interactions and POP3 contained several HIN-200 PYD-specific sequence motifs and displayed high homology to the PYD of AIM2, experiments were conducted during development of embodiments described herein to determine if POP3 was able to bind to the PYD of HIN-200 proteins. GST-POP3, but not GST control, bound to the PYD of AIM2 and IF116. However, GST-POP3 showed no significant interaction with the PYDs of MNDA and PYHIN1 (
This observation was further supported by co-localization studies. AIM2 and IF116 have been shown to co-localize with ASC, albeit at different sites and in response to different stimuli (Ludlow et al. Exp Cell Res. 2005; 308:1-17; Fernandes-Alnemri et al. Nature. 2009; 458:509-13; Kerur N, et al. Cell Host Microbe. 2011; 9:363-75; herein incorporated by reference in their entireties). While DNA from Modified Vaccinia virus Ankara (MVA) and murine cytomegalovirus (MCMV) is sensed by AIM2 in the cytosol11-14, 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). AIM2 was predominantly localized to cytosolic punctate structures, and this pattern was not altered in response to GFP adenovirus (AdV) infection (
Accordingly, GST-POP3 also purified endogenous AIM2 and IF116 (
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). Only co-transfection of 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. However, in the presence of POP3, AIM2-mediated ASC dimers and oligomers were significantly reduced (
In response to DNA virus infection, 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). In the absence of POP3, following siRNA-mediated silencing in hMΦ, as determined by RT-PCR (
POP1 and POP2 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 (
Next, the effect of POP3 expression on AIM2 and other inflammasomes was analyzed. Consistent with the increased inflammasome response observed in the absence of POP3 in hMΦ, POP3 expression resulted in a significant decrease of IL-1β 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 (
The PYDs of human and mouse ALRs are well conserved. POP3 co-purified IF116 and AIM2, but not ASC from BMDM (
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-γ 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-γ concentrations were also strongly decreased in CD68-POP3 expressing mice at 36 hours post i.p. infection. However, TNF serum concentrations were not affected (
A functional specificity of POP3 for ALRs is further supported by the observation that WT and CD68-POP3 TG mice did not show significant differences in their response to MSU crystal-challenge in vivo. Severity of MSU-induced peritonitis was comparable in both genotypes, showing similar peritoneal IL-β concentrations 7 hours after MSU challenge (
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. C57BL/6 wild type (WT) and Lysozyme M-Cre knock-in mice (CreL) were obtained from the Jackson Laboratories and NLRP3−/−, ASC−/− and floxed NLRP3A350V 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.
Human SubjectsAvailable datasets of septic and CAPS patients in the NCBI Gene Expression Omnibus (GEO) database were searched for the inclusion of POP1 and were analysed for expression of POP1 and ASC with GEO2R. GEO accession number GDS3085 contains expression data of leukocytes of critically ill adult patients not diagnosed with sepsis (n=17), diagnosed with Gram negative, Gram positive or mixed sepsis (n=55) that were analysed using CompuGen Human OligoLibrary V1.0 (Tang et al. Crit Care Med 36, 1125-8 (2008); herein incorporated by reference in its entirety). GEO accession number GSE13904 contains expression data obtained from whole blood drawn from pediatric healthy controls (n=18) and patients diagnosed with systemic inflammatory response syndrome (SIRS), sepsis or septic shock (n=209) that were analysed with the Affymetrix Human Genome U133 Plus 2.0 Array (Wong et al. Crit Care Med 37, 1558-66 (2009); herein incorporated by reference in its entirety). GEO accession number GSE40561 contains expression data obtained from whole blood drawn from CINCA (n=2) and MWS (n=5) that were analysed using Illumina human HT-12 beadchips (Boisson et al. Nat Immunol 13, 1178-86 (2012); herein incorporated by reference in its entirety). GEO accession number GSE3284 contains expression data of leukocytes isolated at the indicated times from human healthy male and female subjects (n=8) after intravenous administration of NIH Clinical Center Reference endotoxin (CC-RE-Lot 2) (2 ng/kg body weight) over a 5 min period that were analyzed with Affymetrix Hu133A and Hu133B oligonucleotide array (Calvano et al. Nature 437, 1032-7 (2005); herein incorporated by reference in its entirety).
Macrophage Isolation, Culture and TransfectionPeripheral blood-derived hMΦ 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 μg/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. hMΦ were transfected in 24-well dishes (3.3×105 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). hMΦ, THP-1 cells, BMDM and PM were treated for the indicated times with 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-1β (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. 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 μM). 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 MgCl2, 1.5 mM CaCl2, 10 mM HEPES, 5 mM Glucose, 140 mM NaCl, pH 7.2.
LPS-Induced Peritonitis8-12 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.
CryopyrinopathyFloxed NLRP3A350V 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.
ASC-GFP Particle PurificationHEK293 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 MgCl2, 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 2×103 particles for 16 h.
ASC-Particle-Induced Peritonitis14 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 (1×105 particles/mouse). After 4 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 mice15. Images were quantified with Living Image software (PerkinElmer). Peritoneal lavage fluids were collected and assayed for IL-1β by ELISA.
PlasmidspcDNA3 and pGEX-based expression constructs for ASC, POP1, NLRP3, ASCPYD and NLRP3PYD were described earlier (Stehlik et al. J. Immunol 171, 6154-63 (2003); Khare et al. Nat Immunol 15, 343-353 (2014); Stehlik et al. Biochem. J 373, 101-113 (2003); herein incorporated by reference in their entireties).
Antibody-Based DetectionRabbit 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-1β (Santa Cruz Biotech), rabbit polyclonal antibodies to IκBα (clone 44D4)/p-IκBα (clone 14D4), JNK (clone 9252)/p-JNK (9251), p38 (clone 9212)/p-p38 (clone 12F8), p42/44 (clone 9102)/p-p42/44 (9101) (all Cell Signaling Technology) and mouse monoclonal antibody to β-tubulin (Santa Cruz Biotech clone TU-02), mouse monoclonal antibody to GST (Santa Cruz Biotech clone B-14), mouse monoclonal antibody to β-actin (Sigma clone AC-74) and mouse monoclonal antibody to NLRP3 (Adipogen clone Cryo-2) were used for immunoblot.
Co-Immunoprecipitations (IP):
HEK293 cells were transfected with GFP, GFP-POP1, HA-ASC, MYC-NLRP3, HA-ASCPYD, MYC-ASCPYD or empty plasmid in 100 mm dishes using Lipofectamine 2000 (Invitrogen). 24 h post transfection, cells were lysed under hypotonic conditions (20 mM HEPES pH 7.4, 10 mM KCl, 1 mM EDTA, supplemented with protease inhibitors) using a 23½ G syringe needle, cleared and adjusted to 50 mM Hepes pH 7.4, 150 mM NaCl, 10% Glycerol, 2 mM EDTA, 0.5% Triton X-100, supplemented with protease inhibitors, and subjected to IP by incubating with sepharose immobilized antibodies as indicated for 16 h at 4° C., followed by extensive washing with lysis buffer. Bound proteins were separated as above. TCL (5-10%) were also analyzed where indicated. Endogenous NLRP3 inflammasome complexes were similarly purified from ultrapure LPS-primed (16 h, 100 ng/mL) THP-1 cells following nigericin treatment (45 min, 5 μM).
GST Pull Down:
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.
ASC Cross-Linking:
4×106 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. Cleared lysates were stored for immunoblot analysis and the insoluble pellets were resuspended in 500 μl PBS, supplemented with 2 mM disuccinimydyl suberate (DSS, Pierce) and incubated with rotation at room temperature for 30 min. Samples were centrifuged at 5,000 rpm for 10 min at 4° C. and the cross-linked pellets were resuspended in 50 μl Laemmli sample buffer and analyzed by immunoblot.
Immunohistochemistrv:
Human lung tissue was embedded in paraffin, cut into 3 μm 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-1α, IL-1β, IL-18 and TNFα secretion was quantified from clarified culture SN obtained from hMΦ, 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.
Flow Cytometry:
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 μm nylon mesh. 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.
Active Caspase-1:
Cells were primed with LPS for 4 h, followed by a 20 min pulse with ATP. After 1 h incubation in fresh medium, active caspase-1 p10 was detected in 10% TCA precipitated culture supernatants (SN). Total cell lysate (TCL) and concentrated culture SN pellets were resuspended in Laemmli buffer. Samples 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). Alternatively, cells were incubated with biotin-conjugated YVAD-CMK (Anaspec) for 30 min at 37° C. with 5% CO2 before LPS priming (40 min) and ATP stimulation (20 min) Alexa Fluor 647 conjugate streptavidin (Molecular Probes) was used to quantify active caspase-1 by flow cytometry.
Lactate Dehydrogenase (LDH) ReleaseCleared culture supernatants were analyzed for LDH release as a measurement for pyroptosis by colorimetric enzyme activity assay accordingly to the manufacturer's instructions (Clontech LDH cytotoxicity detection assay).
Cell Penetrating Recombinant Proteins6xHIS-POP1 and a 6xHIS-GFP cDNAs in pET28a (Invitrogen) were fused with the HIV TAT sequence (YGRKKRRQRRR (SEQ ID NO: 10)) by standard PCR, produced under native conditions in E. coli BL21 after induction with 2 mM IPTG for 4 h and affinity purified on Talon columns (Invitrogen) in the presence of 10 mM imidazole (pH 6.5), eluted with 1 M imidazole and further purified by anion exchange chromatography (pH6.5, Macro-Prep High S, Biorad) to eliminate endotoxin, which was verified by the LAL assay, desalting (Econo-Pac columns 10DG, Biorad) and protein concentration (Nanosep 3k, Pall Life Sciences). C57BL/6 mice were i.p. injected with 50 μg TAT-GFP, followed by peritoneal lavage after 1 h and FACS analysis with the indicated lineage markers for uptake control. 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 μg/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.
Quantitative Real-Time PCRTotal RNA was isolated from hMΦ, BMDM or THP-1 cells using Trizol reagent (Invitrogen) and from mouse peripheral blood using the mouse RiboPure-blood RNA isolation kit (Invitrogen), treated with DNase I, reverse transcribed with GoScript (Promega) and analyzed by TaqMan Real-time gene expression system using predesigned FAM labeled primer/probes on an ABI 7300 Real time PCR machine (Applied Biosystems) and displayed as relative expression compared to β-actin.
IL-1β-Induced Expression of POP1 is Reduced in Inflammatory Disease PatientsNLRP3 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 (MΦ) by immunohistochemistry (
To investigate the role of POP1 in NLRP3 inflammasome activation a human monocytic THP-1 cell line stably expressing POP1 (THP-1GFP-POP1) was generated, which was confirmed by qPCR (
POP1 Blocks NLRP3PYD-Mediated ASCPYD Nucleation in Human Macrophages
Recruitment of ASC to upstream sensors is essential for inflammasome activation. POP1 specifically bound to endogenous ASC, but not to the PYD-containing PRRs NLRP3 and AIM2 in LPS-primed THP-1 cells (
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 MΦ 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+MΦ (
LPS/ATP treated BMDMPOP1 also displayed significantly reduced levels of IL-1β, IL-1α and IL-18 in culture supernatants by ELISA. However, secretion of TNFα, which occurs independently of caspase-1, was not affected (
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). Experiments were conducted to determine whether POP1 also modulates NLRP3 activity in vivo. As NLRP3 inflammasome-released IL-1β is essential for neutrophil recruitment during sterile inflammation (McDonald et al. Science 330, 362-366 (2010); herein incorporated by reference in its entirety), WT and CD68-POP1 TG mice we injected i.p. with a low dose of LPS and determined neutrophil infiltration 3 hours after LPS challenge by quantifying myeloperoxidase (MPO) activity in vivo. Contrary to PBS, injection of LPS recruited a substantial number of neutrophils into the peritoneal cavity, which was completely abolished in CD68-POP1 TG mice (
Polymerized ASC particles have been detected in the serum of active CAPS patients (Baroja-Mazo et al. Nat Immunol 15, 738-48 (2014); herein incorporated by reference in its entirety), which are released from macrophages through inflammasome-dependent pyroptosis and act as danger signals on neighbouring cells (Franklin et al. Nat Immunol 15, 727-37 (2014); Baroja-Mazo et al. Nat Immunol 15, 738-48 (2014); herein incorporated by reference in their entireties). POP1 prevented ASC nucleation (
All publications and patents provided herein incorporated by reference in their entireties. Various modifications and variations of the described compositions and methods of the invention will be apparent to those skilled in the art without departing from the scope and spirit of the invention. Although the invention has been described in connection with specific preferred embodiments, it should be understood that the invention as claimed should not be unduly limited to such specific embodiments. Indeed, various modifications of the described modes for carrying out the invention that are obvious to those skilled in the relevant fields are intended to be within the scope of the present invention.
Claims
1. A method of treating or preventing a disease or condition associated with inflammation comprising administering a composition that increases the activity of POP1, POP2, and/or POP3.
2. The method of claim 1, wherein the composition comprises a POP1, POP2, and/or POP3 polypeptide, peptide, or peptidomimitic.
3. The method of claim 2, wherein the polypeptide or peptide comprises 100% sequence identity with all or a portion of POP1, POP2, or POP3.
4. The method of claim 2, wherein the polypeptide or peptide comprises less than 100% sequence identity, but greater than 70% sequence identity with all or a portion of POP1, POP2, or POP3.
5. The method of claim 1, wherein the composition comprises an agent that enhances the inflammasome inhibitory activity of POP1, POP2, and/or POP3.
6. The method of claim 5, wherein the composition is a small molecule, peptide, polypeptide, protein, or antibody.
7. The method of claim 1, wherein the disease or condition is an autoimmune disease.
8. The method of claim 1, wherein the disease or condition is an inflammatory disease.
9. The method of claim 1, wherein the composition is co-administered with one or more additional treatments for the disease or condition
10. A method of treating or preventing infection comprising administering a composition that inhibits the activity of POP1, POP2, and/or POP3.
11. The method of claim 10, wherein the composition is a small molecule, peptide, polypeptide, protein, or antibody.
12. The POP1, POP2, or POP3 protein, polypeptide, or peptide that comprises less than 100% sequence identity, but greater than 70% sequence identity with all or a portion of POP1, POP2, or POP3.
13. A POP1, POP2, or POP3 protein, polypeptide, or peptide for use as a medicament.
14. The POP1, POP2, or POP3 protein, polypeptide, or peptide of claim 13, comprising less than 100% sequence identity, but greater than 70% sequence identity with all or a portion of POP1 or POP3.
15. The POP1, POP2, or POP3 protein, polypeptide, or peptide of claim 13 for use in the treatment or prevention of an autoimmune or inflammatory disease or condition.
16. A POP1, POP2, or POP3 protein, polypeptide, or peptide for use in the treatment or prevention of an autoimmune or inflammatory disease or condition.
17. A POP1, POP2, or POP3 inhibitor.
13. A POP1, POP2, or POP3 protein, polypeptide, or peptide for use as a medicament.
14. The POP1, POP2, or POP3 protein, polypeptide, or peptide of claim 13, comprising less than 100% sequence identity, but greater than 70% sequence identity with all or a portion of POP1, POP2, or POP3.
15. The POP1, POP2, or POP3 protein, polypeptide, or peptide of claim 13 for use in the treatment or prevention of an autoimmune or inflammatory disease or condition.
16. A POP1, POP2, or POP3 protein, polypeptide, or peptide for use in the treatment or prevention of an autoimmune or inflammatory disease or condition.
Type: Application
Filed: Feb 13, 2015
Publication Date: Dec 8, 2016
Applicant: Northwestern University (Evanston, IL)
Inventor: Christian Stehlik (Evanston, IL)
Application Number: 15/117,629