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WO2003094857A2 - Stimulation, par des recepteurs de type toll, qui inhibe la differentiation des osteoclastes - Google Patents

Stimulation, par des recepteurs de type toll, qui inhibe la differentiation des osteoclastes Download PDF

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WO2003094857A2
WO2003094857A2 PCT/US2003/014946 US0314946W WO03094857A2 WO 2003094857 A2 WO2003094857 A2 WO 2003094857A2 US 0314946 W US0314946 W US 0314946W WO 03094857 A2 WO03094857 A2 WO 03094857A2
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osteoclast
cell
tlr
osteoclast precursor
precursor cell
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WO2003094857A3 (fr
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Yongwon Choi
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University of Pennsylvania Penn
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    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N5/00—Undifferentiated human, animal or plant cells, e.g. cell lines; Tissues; Cultivation or maintenance thereof; Culture media therefor
    • C12N5/06—Animal cells or tissues; Human cells or tissues
    • C12N5/0602—Vertebrate cells
    • C12N5/0634—Cells from the blood or the immune system
    • C12N5/0643—Osteoclasts
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N2501/00—Active agents used in cell culture processes, e.g. differentation
    • C12N2501/05—Adjuvants
    • C12N2501/052—Lipopolysaccharides [LPS]
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N2501/00—Active agents used in cell culture processes, e.g. differentation
    • C12N2501/05—Adjuvants
    • C12N2501/056—Immunostimulating oligonucleotides, e.g. CpG
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N2501/00—Active agents used in cell culture processes, e.g. differentation
    • C12N2501/10—Growth factors
    • C12N2501/185—Osteoprotegerin; Osteoclast differentiation factor (ODF, RANKL)
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N2501/00—Active agents used in cell culture processes, e.g. differentation
    • C12N2501/20—Cytokines; Chemokines
    • C12N2501/22—Colony stimulating factors (G-CSF, GM-CSF)
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N2501/00—Active agents used in cell culture processes, e.g. differentation
    • C12N2501/90—Polysaccharides
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N2503/00—Use of cells in diagnostics
    • C12N2503/02—Drug screening

Definitions

  • Osteoclasts are multinucleated giant cells that reside in bone tissue and resorb calcified matrix (Vaananen et al., 2000, J Cell Sci 113 (Pt 3):377; Suda et al, 1999, Endocr Rev 20:345 ). Osteoclasts originate from hematopoietic precursor cells, which are cells that also give rise to macrophages or dendritic cells responsible for mediating immune responses (Kahn et al., 1975, Nature 258:325; Udagawa et al., 1990, Proc Natl Acad Sci U.S.A.
  • TNF-related activation-induced cytokine TRANCE, also called receptor activator of NF- KB ligand, osteoprotegerin ligand, or osteoclast differentiation factor
  • M-CSF macrophage colony stimulating factor
  • TLRs Toll-like receptors
  • Peptidoglycan (PGN) and lipoteichoic acid (LTA) act as ligands of TLR2 (Schwandner et al, 1999, J Biol Chem 274:17406; Aliprantis et al.,
  • Double-stranded RNA [ ⁇ oly(I:C) RNA], LPS, flagellin, and the CpG motif of unmethylated DNA (CpG DNA) act as ligands of TLR3, TLR4, TLR5, and TLR9, respectively (Alexopoulou et al., 2001, Nature 413:732; Poltorak et al., 1998, Science 282:2084; Hayashi et al, 2001, Nature 410:1099; Hemmi et al.,
  • TLR stimulation by diverse microbial products directly induces the maturation of dendritic cells, which is an essential step for subsequent adaptive immune responses (Kaisho, et al., 2001, Trends Immunol 22:78).
  • TLR stimulation by diverse microbial products directly induces the maturation of dendritic cells, which is an essential step for subsequent adaptive immune responses (Kaisho, et al., 2001, Trends Immunol 22:78).
  • . been suggested to be a potent stimulator of bone loss (Nair et al., 1996, Infect Immun 64:2371; Abu-Amer et al, 1997, J Clin Invest 100:1557; Sakuma et al., 2000, Infect Immun 68:6819; Ueda-N et al., 1998, Y4. J Periodontal Res. 33:509.
  • LPS has been shown to increase the number of osteoclast precursors via TNF- ⁇ action in vivo (Abu- Amer et al., 1997, J Clin Invest 100:1557; ).
  • Prostaglandins and the EP4 subtype receptor are implicated in osteoclast differentiation induced by LPS (Sakuma et al., 2000, Infect Immun 68:6819. ), suggesting an indirect role of LPS in osteoclast differentiation by promoting inflammatory responses accompanied by the production of prostaglandins and TNF- ⁇ .
  • LPS can enhance the survival, fusion, and activation of osteoclasts independent of IL-1, TNF- ⁇ , and TRANCE (Suda et al., 2002.
  • osteoclasts are derived from common precursor cells for macrophages and dendritic cells. Therefore an understanding of the role that osteoclasts may play in the immune response will aid in the development of therapies to treat patients suffering from bone loss associated with bacterial infection and bone loss resulting from other diseases.
  • the present invention provides methods and therapies for such treatments.
  • the invention includes a method of inhibiting differentiation of an osteoclast precursor cell.
  • the method comprises contacting the cell with at least one Toll-like receptor (TLR) ligand, wherein the TLR ligand stimulates at least one TLR on the osteoclast precursor cell, thereby inhibiting differentiation of the osteoclast precursor cell.
  • TLR Toll-like receptor
  • the contacting is performed in vivo in a mammal.
  • the mammal is a human.
  • the osteoclast precursor cell is selected from the group consisting of a bone marrow cell, a peripheral blood monocyte, and a cell of the . murine myeloid cell line RAW264.7.
  • the TLR ligand ⁇ U !SV from the group consisting of PGN, ⁇ oly(I:C) RNA, LPS, and CpG DNA.
  • the TLR is selected from the group consisting of TLR2, TLR3, TLR4 and TLR9.
  • the TLR ligand is LPS and the osteoclast precursor cell is contacted with the LPS at a concentration of 1 ng/ml of the LPS.
  • kits for inhibiting differentiation of an osteoclast precursor cell comprises at least one TLR ligand, wherein the TLR ligand stimulates at least one TLR on the osteoclast precursor cell, thereby inhibiting differentiation of the osteoclast precursor cell, the kit further comprising an applicator, and an instructional material for the use thereof.
  • the invention further includes a method of enhancing phagocytosis by an osteoclast precursor cell, the method comprising contacting the cell with at least one TLR ligand, wherein the TLR ligand stimulates at least one TLR on the osteoclast precursor cell and inhibits differentiation of the osteoclast precursor cell, thereby sustaining the phagocytic activity of the osteoclast precursor cell and enhancing phagocytosis by the cell.
  • the invention includes a method of enhancing phagocytosis by a population of osteoclast precursor cells.
  • the method comprises contacting the osteoclast precursor cells with at least one TLR ligand, wherein the TLR ligand stimulates at least one TLR on at least one osteoclast precursor cell in the population of osteoclast precursor cells and inhibits differentiation of the one osteoclast precursor cell, thereby sustaining the phagocytic activity of the osteoclast precursor cell and enhancing phagocytosis in the population of osteoclast precursor cells.
  • the invention also includes a method of maintaining an osteoclast precursor cell in a progenitor state in a mammal, the method comprising administering to a mammal at least one TLR ligand, wherein the TLR ligand stimulates at least one TLR on the osteoclast precursor cell, thereby maintaining the osteoclast precursor cell in the progenitor state in the mammal.
  • a method of identifying an osteoclast cell that is in a precursor state comprising the steps of: first contacting an osteoclast cell with at least one TLR ligand; then contacting the cell with M-CSF and TRANCE; and assaying the phagocytic activity of the cell; then assaying the unidentified cell for progenitor status; -- wherein when the cell is phagocytic, the cell is an osteoclast in a precurs6 ⁇ J ⁇ .3" •J 14y4b
  • the invention includes a kit for identifying a cell as an osteoclast precursor cell.
  • the kit comprises at least one TLR ligand, M-CSF, TRANCE; and a reagent that detects phagocytic activity.
  • the kit further comprises an applicator, and an instructional material for the use thereof.
  • the invention also includes a method of enhancing survival of a mature osteoclast, the method comprising contacting the mature osteoclast with at least one TLR ligand, thereby enhancing survival of the mature osteoclast.
  • kits for enhancing survival of a mature osteoclast comprising a TLR ligand, an applicator, and an instructional material for the use thereof.
  • a method of identifying a TLR that inhibits differentiation of an osteoclast precursor cell comprises contacting an osteoclast precursor cell with a test TLR ligand and comparing the level of inhibition of differentiation of the cell so contacted with an osteoclast precursor cell not contacted with the test TLR ligand, wherein inhibition of differentiation of the contacted cell when compared with differentiation of an osteoclast precursor cell not so contacted is an indication of that the test TLR ligand inhibits differentiation of the osteoclast precursor cell.
  • a method of treating a patient in need of a stable progenitor-stage osteoclast comprising contacting an osteoclast precursor cell with at least one TLR ligand wherein differentiation of the osteoclast precursor cell is inhibited, whereby a stable progenitor-stage osteoclast is formed, and administering the stable progenitor-stage osteoclast to a patient in need thereof.
  • a pharmaceutical composition comprising a stable progenitor-stage osteoclast and a pharmaceutically-acceptable carrier.
  • the invention includes a method of treating a patient in need of a stable phagocytic progenitor-stage osteoclast.
  • the method comprises administering to the patient at least one TLR-stimulating ligand, wherein the administration inhibits differentiation of an osteoclast precursor cell to provide the patient with a stable phagocytic progenitor-stage osteoclast.
  • the invention includes a method of identifying a TLR ligand that inhibits differentiation of an osteoclast precursor cell but does not stimulate production of tumor necrosis factor (TNF) alpha.
  • TNF tumor necrosis factor
  • Figure lb is a graph illustrating the percentage of osteoclast precursors and osteoclasts that incorporated the zymosan particles in each culture. Data are expressed as the mean ⁇ SD of four cultures.
  • FIG. 2 is an image depicting gene expression of TLRs in osteoclast precursors and osteoclasts.
  • Total RNA was prepared from osteoclast precursors and purified mature osteoclasts, and templates for PCR were synthesized with (+RT) or without (-RT) reverse transcriptase.
  • PCR products for mouse TLR1-TLR9 were separated by electrophoresis and visualized by ethidium bromide staining with ultraviolet light illumination.
  • Figure 3 a is a series of images depicting the induction of l ⁇ B phosphorylation by TLR ligands in osteoclast precursors.
  • Osteoclast precursors were cultured in 0.5% FCS-containing medium for 2 hours before treatment with TLR ligands. Cells were then treated with PGN (10 ⁇ g/ml), poly( C) RNA (100 ⁇ g/ml), LPS (1000 ng/ml), or CpG DNA (1 ⁇ M). After culturing for indicated times, cell lysates were harvested. Phospho-l ⁇ B ⁇ and a-actin was detected by western blotting. expression in osteoclast precursors.
  • Osteoclast precursors were treated with PGN (10 ⁇ g /ml), poly(I:C) RNA (100 ⁇ g /ml), LPS (1000 ng/ml), or CpG DNA (1 ⁇ M). After culturing for the indicated times, total RNA was isolated from the cells, and the expression of TNF- ⁇ mRNA was analyzed by northern blotting.
  • Figure 4a is a series of graphs illustrating the effects of TLR ligands on osteoclast differentiation and phagocytic function.
  • Osteoclast precursors derived from bone marrow cells of C57BL/6- (closed circles) or C3H/HeJ (open squares) mice were cultured with medium containing increasing concentrations of PGN, poly( C) RNA, LPS , and CpG DNA in the presence of TRANCE (300 ng/ml) and M- CSF (30 ng/ml) for 3 days. Then, cells were cultured with FITC conjugated zymosan particles for 1 hour.
  • FIG. 4b is a series of images depicting the effects of TLR ligands on osteoclast differentiation and phagocytic function.
  • Cells were treated with 1 ⁇ g/ml PGN, 1 ⁇ g/ml poly(I:C) RNA, 10 ng/ml LPS, and 1 ⁇ M CpG DNA, as set forth in Figure 4a. Note that there are no TRAP-positive cells in the bright field, as illustrated in the upper panels in Figure 4b. The upper right panel is the control sample which did not receive TLR-ligand treatment.
  • Figure 4c is a series of graphs illustrating human peripheral blood monocytes that were cultured with medium containing increasing concentrations of PGN, poly(LC) RNA, LPS, or CpG DNA in the presence of TRANCE and M-CSF for 4 days. After this time, cells were fixed, and stained for TRAP.
  • Figure 4d is a pair of graphs illustrating mouse osteoclast precursors and human blood monocytes that were cultured with medium containing increasing concentrations of EL- la for 3- and 4 days, respectively. After this time, cells were fixed, and stained for TRAP.
  • TRAP-positive multinucleated-cells (TRAP(+) MNCs) 1 ⁇ 0 ⁇ having more than 3 nuclei were counted as osteoclasts. All the data are GX ⁇ lJ, 'JS ) y4b the mean ⁇ SD of four cultures.
  • FIG 5 is a series of graphs illustrating the effects of TLR ligands on osteoclast differentiation from the RAW264.7 cell line.
  • RAW264.7 cells were cultured with medium containing increasing concentrations of PGN, poly (I: C) RNA, LPS, or CpG DNA with TRANCE for 4 days. Then, cells were fixed, and stained for TRAP.
  • TRAP-positive rnultinucleated cells (TRAP(+,) MNCs) having more than 3 nuclei were counted as osteoclasts. Data are expressed as the mean ⁇ SB of four cultures.
  • Figure 6a is a series of graphs illustrating the effects of TLR ligands on the survival of mature osteoclasts.
  • Osteoclasts were generated from osteoclast precursors derived from C57BL/6 (closed circles) or C3H/HeJ (open squares) mice by the treatment with TRANCE and M-CSF. Then, mature osteoclasts were purified dissociation solution and tapping. The initial number of mature osteoclasts immediately after purification (0 hours) was 185 ⁇ 12 cells/well. Purified mature osteoclasts were cultured in medium containing increasing concentrations of PUN, poly(LC) RNA, LPS, or CpG DNA for 12 hour. Then, cells were fixed and stained for TRAP.
  • TRAP-positive rnultinucleated cells (TRAP(+) MNCs) having more than 3 nuclei were counted as osteoclasts (A) LPS treatment did not enhance the viability of mature osteoclasts from TLR4-deficient C3H HeJ mice (A). Data are expressed as the mean ⁇ SD of four cultures.
  • Figure 7A is a graph depicting the level of TNF- ⁇ present in the culture media of a series of bone marrow-derived macrophage cultures, as quantified by ELISA.
  • CpG DNAs 1-10 and CpG standard DNA were added to separate cultures, which were then incubated for 48 hours at 37 °C, at which time the ELISA assay was conducted.
  • Figure 7B is a graph depicting the level of TNF- ⁇ present in the culture media of a series of bone marrow-derived macrophage cultures, as quantified by ELISA.
  • CpG DNAs 11, 12, and 17-20 and CpG standard DNA were added to . separate cultures, which were then incubated for 48 hours at 37 °C, at wm iK ⁇ i A: uA 4 "
  • Figure 8A is a graph depicting the absorbance at 405 nm of a series of mouse osteoclast precursor cultures.
  • CpG DNAs 1-10 and CpG standard DNA were added to separate cultures, which were cultured for 3 days at 37 °C, at which time the number of osteoclasts was quantified.
  • Figure 8B is a graph depicting the TRAP activity of a series of mouse osteoclast precursor cultures. CpG DNAs 11, 12, and 17-20 and CpG standard DNA were added to separate cultures, which were cultured for 3 days at 37 °C, at which time the TRAP activity was quantified.
  • Figure 9A is a graph depicting the absorbance at 405 nm of a series of mouse osteoclast precursor cultures. CpG DNAs 1-10 and CpG standard DNA were added to separate cultures, which were cultured for 3 days at 37 °C, at which time the number of osteoclasts was quantified.
  • Figure 9B is a graph depicting the TRAP activity of a series of mouse osteoclast precursor cultures. CpG DNAs 11, 12, and 17-20 and CpG standard DNA were added to separate cultures, which were cultured for 3 days at 37 °C, at which time the TRAP activity was quantified.
  • Figure 10 is a graph depicting the TRAP activity of a series of mouse osteoclast precursor cultures.
  • CpG DNAs 1-10 and 17-20 and CpG standard DNA were added to separate cultures, which were cultured for 4 days at 37 °C, at which time the TRAP activity was quantified.
  • the present invention relates to the discovery that some of the microbial products interacting with TLRs modulate osteoclast differentiation, thus contributing to the pathology of bone diseases such as periodontitis, osteomyelitis, and bacterial arthritis caused by microbial infection.
  • gene expression patterns of TLRs and their potential function have been analyzed and demonstrate that murine osteoclast precursors express all known TLRs.
  • Another feature of the invention is the discovery that TLR stimulation on osteoclast precursors inhibits osteoclastogenesis as induced by M-GSF and TRANCE. This discovery is in contrast to the positive effect ol on immune cell maturation.
  • another feature of the present invention involves altering the differentiation of a common precursor cell such as an osteoclast precursor cell such as an osteoclast precursor cell and therefore the behavior of these cell with respect to the immune response, by way of TLR stimulation.
  • a compound is used to inhibit osteoclast differentiation while having little or no effect on immune stimulation.
  • osteoclast TLR stimulation prolongs and supports the phagocytic activity of such cells, and accordingly assists in eliminating infective agents.
  • inhibition of the differentiation of an osteoclast precursor cell results in a cell in a precursor state that has enhanced phagocytic activity. The presence of this cell prevents the pathogenic effects of microbial invasion on bone by assisting in the clearance of the bacteria responsible for the infection.
  • An osteoclast precursor cell treated by a method of the present invention when administered to a patient, is also useful for regulating the balance between bone resorption and immune response.
  • a TLR-contacted osteoclast precursor cell of the invention By administering to a patient with a microbial infection of the bone a TLR-contacted osteoclast precursor cell of the invention, the patient benefits from the enhanced phagocytic effect of the non- differentiated osteoclast precursor cell without experienceing excess bone loss.
  • a stabilized phagocytic osteoclast precursor of the present invention has the benefit of contributing to the preservation of bone matrix when administered to a mammal as detailed above.
  • the non-differentiated osteoclast precursor cells should not possess the bone-resorbing activity of a differentiated, mature osteoclast.
  • a stabilized phagocytic osteoclast precursor of the present invention administered to a mammal to assist in the clearance of a bone infection should not destroy bone by way of bone resorption.
  • Amplification refers to any means by which a polynucleotide sequence is copied and thus expanded into a larger number of polynucleotide molecules, e.g., by reverse transcription, polymerase chain reaction, and ligase chain reaction.
  • Antisense refers particularly to the nucleic acid sequence of the non- coding strand of a double stranded DNA molecule encoding a protein, or to a sequence which is substantially homologous to the non-coding strand.
  • an antisense sequence is complementary to the sequence of a double stranded DNA molecule encoding a protein. It is not necessary that the antisense sequence be complementary solely to the coding portion of the coding strand of the DNA molecule.
  • the antisense sequence may be complementary to regulatory sequences specified on the coding strand of a DNA molecule encoding a protein, which regulatory sequences control expression of the coding sequences.
  • antibody refers to an immunoglobulin molecule which is able to specifically bind to a specific epitope on an antigen.
  • Antibodies can be_ intact immunoglobulins derived from natural sources or from recombinant sources and can be immunoreactive portions of intact immunoglobulins.
  • Antibodies are typically tetramers of immunoglobulin molecules.
  • the antibodies in the present invention may exist in a variety of forms including, for example, polyclonal antibodies, monoclonal antibodies, Fv, Fab and F(ab) 2 , as well as single chain antibodies and humanized antibodies (Harlow et al., 1999, Using Antibodies: A Laboratory Manual, Cold Spring Harbor Laboratory Press, NY; Harlow et al., 1989, Antibodies: A Laboratory Manual, Cold Spring Harbor, New York; Houston et al., 1988, Proc. Natl. Acad. Sci. USA 85:5879-5883; Bird et al., 1988, Science 242:423- 426).
  • apper as the term is used herein, is meant any device including, but not limited to, a hypodermic syringe, a pipette, and the like, for administering, for example, an inhibitor of osteoclast differentiation of the invention to a mammal.
  • "ammo acids” are represented by the full KbiLl xx ' S"?** by the three letter code corresponding thereto, or by the one-letter code corresponding thereto, as indicated in the following table:
  • Biological sample means a sample obtained from a mammal that can be used to assess, for example, the state of differentiation of an osteoclast. Such a sample includes, but is not limited to, a bone marrow cell or a peripheral monocyte.
  • a biological sample may be used in connection with diagnostic methods of the present invention, as well as with assays for the properties and biological activities of osteoclasts.
  • “Cleavage” is used herein to refer to the disassociation of a c uc bond between two amino acids in a polypeptide, thereby separating the polypeptide comprising the two amino acids into at least two fragments.
  • “Complementary” as used herein refers to the broad concept of subunit sequence complementarity between two nucleic acids, e.g., two DNA molecules. When a nucleotide position in both of the molecules is occupied by nucleotides normally capable of base pairing with each other, then the nucleic acids are considered to be complementary to each other at this position. Thus, two nucleic acids are complementary to each other when a substantial number (at least 50%) of corresponding positions in each of the molecules are occupied by nucleotides which normally base pair with each other (e.g., A:T and G:C nucleotide pairs). As defined herein, an antisense sequence is complementary to the sequence of a double stranded DNA molecule encoding a protein.
  • antisense sequence be complementary solely to the coding portion of the coding strand of the DNA molecule.
  • the antisense sequence may be complementary to regulatory sequences specified on the coding strand of a DNA molecule encoding a protein, which regulatory sequences control expression of the coding sequences.
  • a “compound” as the term is used herein is any molecule that would be recognized as a potential “drug” to one of skill in the art.
  • a “coding region” of a gene consists of the nucleotide residues of the coding strand of the gene and the nucleotides of the non-coding strand of the gene which are homologous with or complementary to, respectively, the coding region of an mRNA molecule which is produced by transcription of the gene.
  • a "coding region" of an mRNA molecule also consists of the nucleotide residues of the mRNA molecule which are matched with an anticodon region of a transfer RNA molecule during translation of the mRNA molecule or which encode a stop codon.
  • the coding region may thus include nucleotide residues corresponding to amino acid residues which are not present in the mature protein encoded by the mRNA molecule (e.g. amino acid residues in a protein export signal sequence).
  • a "deleterious effect” as used herein refers to a negative effect on a living subject.
  • a toxin when administered to a living cell, may inhibit one or more cellular processes or pathways, resulting in a cell which is impaired in _ function and/or viability.
  • Such an effect of the toxin is referred to as a deitu i ⁇ u 1 V4b effect.
  • “Differentiation” as used herein refers to the maturation of a cell from the precursor, or “progenitor” stage to the mature cell stage.
  • the "effective concentration” of a polypeptide of the invention is the threshold availability of a biologically active polypeptide of interest, below which the biological function of the polypeptide is not observed in an organism.
  • the effective concentration of a cellular receptor is the point below which, for a particular reason (such as inhibition with a drug via binding of the drug molecule to the polypeptide), the overall number of cellular receptors contributing to the recognized biological function of that receptor is no longer sufficient to maintain what is recognized as "normal” or “typical” biological homeostasis.
  • Encoding refers to the inherent property of specific sequences of nucleotides in a polynucleotide, such as a gene, a cDNA, or an mRNA, to serve as templates for synthesis of other polymers and macromolecules in biological processes having either a defined sequence of nucleotides (i.e., rRNA, tRNA and mRNA) or a defined sequence of amino acids and the biological properties resulting therefrom.
  • a gene encodes a protein if transcription and translation of mRNA corresponding to that gene produces the protein in a cell or other biological system.
  • Both the coding strand the nucleotide sequence of which is identical to the mRNA sequence and is usually provided in sequence listings, and the non-coding strand, used as the template for transcription of a gene or cDNA, can be referred to as encoding the protein or other product of that gene or cDNA.
  • “Expression vector” refers to a vector comprising a recombinant polynucleotide comprising expression control sequences operatively linked to a nucleotide sequence to be expressed.
  • An expression vector comprises sufficient cis- acting elements for expression; other elements for expression can be supplied by the host cell or in an in vitro expression system.
  • Expression vectors include all those known in the art, such as cosmids, plasmids (e.g., naked or contained in liposomes) and viruses that incorporate the recombinant polynucleotide.
  • a first region of an oligonucleotide "flanks" a second region of the oligonucleotide if the two regions are adjacent one another or if the two regions are separated by no more than about 1000 nucleotide residues, and preferably no more than about 100 nucleotide residues.
  • fragment as applied to ordinarily be at least about 20 nucleotides in length, typically, at least about 50 nucleotides, more typically, from about 50 to about 100 nucleotides, preferably, at least about 100 to about 500 nucleotides, even more preferably, at least about 500 nucleotides to about 1000 nucleotides, yet even more preferably, at least about 1000 to about 1500, even more preferably, at least about 1500 nucleotides to about 2000 nucleotides, yet even more preferably, at least about 2000 to about 2500, even more preferably, at least about 2500 nucleotides to about 2600 nucleotides, yet even more preferably, at least about 2600 to about 2650, and most preferably, the nucleic acid fragment will be greater than about 2652 nucleotides in length.
  • “Homologous” as used herein refers to nucleotide sequence similarity between two regions of the same nucleic acid strand or between regions of two different nucleic acid strands. When a nucleotide residue position in both regions is occupied by the same nucleotide residue, then the regions are homologous at that position. A first region is homologous to a second region if at least one nucleotide residue position of each region is occupied by the same residue. Homology between two regions is expressed in terms of the proportion of nucleotide residue positions of the two regions that are occupied by the same nucleotide residue.
  • a region having the nucleotide sequence 5'-ATTGCC-3' and a region having the nucleotide sequence 5'-TATGGC-3' share 50% homology.
  • the first region comprises a first portion and the second region comprises a second portion, whereby, at least about 50%, and preferably at least about 75%, at least about 90%, or at least about 95% of the nucleotide residue positions of each of the portions are occupied by the same nucleotide residue. More preferably, all nucleotide residue positions of each of the portions are occupied by the same nucleotide residue.
  • an "instructional material” includes a publication, a recording, a diagram, or any other medium of expression which can be used to communicate the usefulness of the composition of the invention for its designated use.
  • the instructional material of the kit of the invention may, for example, be affixed to a container which contains the composition or be shipped together with a container which contains the composition. Alternatively, the instructional material may be shipped separately from the container with the intention that the instructional material and the composition be used cooperatively by the recipient.
  • isolated nucleic acid refers to a nucleic acid which has been separated from sequences which flank it in a naturally occurring state, e.g., a DNA fragment which has been removed from the sequences which are normally adjacent to the fragment, e.g., the sequences adjacent to the fragment in a genome in which it naturally occurs.
  • the term also applies to nucleic acids which have been substantially purified from other components which naturally accompany the nucleic acid, e.g., RNA or DNA or proteins, which naturally accompany it in the cell.
  • the term therefore includes, for example, a recombinant DNA which is incorporated into a vector, into an autonomously replicating plasmid or virus, or into the genomic DNA of a prokaryote or eukaryote, or which exists as a separate molecule (e.g, as a cDNA or a genomic or cDNA fragment produced by PCR or restriction enzyme digestion) independent of other sequences. It also includes a recombinant DNA which is part of a hybrid gene encoding additional polypeptide sequence.
  • ligand refers to a molecule that binds to another molecule. For example, insulin is a ligand for the insulin receptor.
  • maintaining refers to the act of preserving the current state of something.
  • maintaining a cell in an undifferentiated state refers to the act of preserving the cell in an undifferentiated state, i.e., preventing the cell from progressing to a differentiated, mature state.
  • mature osteoclast as used herein in reference to an osteoclast cell describes an osteoclast cell that has fully differentiated, i.e., a cell that is not in a precursor or progenitor state.
  • a “mature osteoclast” is a rnultinucleated cell that is non-phagocytic and has bone-resorption activity.
  • Near-occurring as applied to an object refers to the fact that the object can be found in nature. For example, a polypeptide or polynucleotide sequence that is present in an organism (including viruses) that can be isolated from a source in nature and which has not been intentionally modified by man is naturally-occurring.
  • A refers to adenosine
  • C refers to cytidine
  • G refers to guanosine
  • T refers to thymidine
  • U refers to uridine.
  • nucleotide sequence encoding an amino acid sequence includes all nucleotide sequences that are degenerate versions of each 14q_ l ⁇ other and that encode the same amino acid sequence. Nucleotide sequent mat 1 V b encode proteins and RNA may include introns.
  • two polynucleotides as "operably linked” is meant that a single-stranded or double-stranded nucleic acid moiety comprises the two polynucleotides arranged within the nucleic acid moiety in such a manner that at least one of the two polynucleotides is able to exert a physiological effect by which it is characterized upon the other.
  • a promoter operably linked to the coding region of a gene is able to promote transcription of the coding region.
  • a "polynucleotide” means a single strand or parallel and anti-parallel strands of a nucleic acid.
  • a polynucleotide may be either a single-stranded or a double-stranded nucleic acid.
  • nucleic acid typically refers to large polynucleotides.
  • oligonucleotide typically refers to short polynucleotides, generally no greater than about 50 nucleotides. It will be understood that when a nucleotide sequence is represented by a DNA sequence (i.e., A, T, G, C), this also includes an RNA sequence (i.e., A, U, G, C) in which "U" replaces "T.”
  • the left-hand end of a single-stranded polynucleotide sequence is the 5'- end; the left-hand direction of a double-stranded polynucleotide sequence is referred to as the 5 '-direction.
  • the direction of 5' to 3' addition of nucleotides to nascent RNA transcripts is referred to as the transcription direction.
  • the DNA strand having the same sequence as an mRNA is referred to as the "coding strand”; sequences on the DNA strand which are located 5' to a reference point on the DNA are referred to as “upstream sequences”; sequences on the DNA strand which are 3' to a reference point on the DNA are referred to as "downstream sequences.”
  • osteoclast is a rnultinucleated giant cell residing in bone tissue.
  • An osteoclast cell resorbs calcified bone matrix, and originates from a hematopoietic precursor cell that also give rise to macrophages or dendritic cells which mediate immune responses.
  • osteoclast precursor cell is a hematopoietic cell that has not differentiated, i.e., is in a "progenitor” state and possesses phagocytic activity.
  • An osteoclast is said to be in a "precursor state” or a “progenitor state” when the osteoclast is a hematopoietic cell that has not differentiated but would become an "osteoclast" as defined above.
  • Porture refers to the engulfing of a substance, such as but not limited to a bacterial cell, by another cell.
  • a "portion" of a polynucleotide means at least at least about twenty sequential nucleotide residues of the polynucleotide. It is understood that a portion of a polynucleotide may include every nucleotide residue of the polynucleotide.
  • Primer refers to a polynucleotide that is capable of specifically hybridizing to a designated polynucleotide template and providing a point of initiation for synthesis of a complementary polynucleotide. Such synthesis occurs when the polynucleotide primer is placed under conditions in which synthesis is induced, i.e., in the presence of nucleotides, a complementary polynucleotide template, and an agent for polymerization such as DNA polymerase.
  • a primer is typically single-stranded, but may be double-stranded. Primers are typically deoxyribonucleic acids, but a wide variety of synthetic and naturally occurring primers are useful for many applications.
  • a primer is complementary to the template to which it is designed to hybridize to serve as a site for the initiation of synthesis, but need not reflect the exact sequence of the template. In such a case, specific hybridization of the primer to the template depends on the stringency of the hybridization conditions. Primers can be labeled with, e.g., chromogenic, radioactive, or fluorescent moieties and used as detectable moieties.
  • Probe refers to a polynucleotide that is capable of specifically hybridizing to a designated sequence of another polynucleotide.
  • a probe specifically hybridizes to a target complementary polynucleotide, but need not reflect the exact complementary sequence of the template. In such a case, specific hybridization of the probe to the target depends on the stringency of the hybridization conditions.
  • Probes can be labeled with, e.g., chromogenic, radioactive, or fluorescent moieties and used as detectable moieties.
  • progenitor refers to an undifferentiated cell. More specifically, it refers to a cell in any metabolic state prior to differentiation. For example, a hematopoietic cell is a progenitor cell, as it has yet to differentiate into a mature cell.
  • Recombinant polynucleotide refers to a polynucleotide having sequences that are not naturally joined together.
  • An amplified or assembled n ⁇ 1 ⁇ 0 ⁇ recombinant polynucleotide may be included in a suitable vector, and the y?vL-d/*i y 6 be used to transform a suitable host cell.
  • a recombinant polynucleotide may serve a non-coding function (e.g., promoter, origin of replication, ribosome-binding site, etc.) as well.
  • a host cell that comprises a recombinant polynucleotide is referred to as a "recombinant host cell.”
  • a gene which is expressed in a recombinant host cell wherein the gene comprises a recombinant polynucleotide produces a "recombinant polypeptide.”
  • a "recombinant polypeptide” is one which is produced upon expression of a recombinant polynucleotide.
  • Polypeptide refers to a polymer composed of amino acid residues, related naturally occurring structural variants, and synthetic non-naturally occurring analogs thereof linked via peptide bonds, related naturally occurring structural variants, and synthetic non-naturally occurring analogs thereof. Synthetic polypeptides can be synthesized, for example, using an automated polypeptide synthesizer.
  • protein typically refers to large polypeptides.
  • peptide typically refers to short polypeptides.
  • polypeptide sequences the left-hand end of a polypeptide sequence is the amino-terminus; the right-hand end of a polypeptide sequence is the carboxyl-terminus.
  • promoter/regulatory sequence means a nucleic acid sequence which is required for expression of a gene product operably linked to the promoter/regulator sequence.
  • this sequence may be the core promoter sequence and in other instances, this sequence may also include an enhancer sequence and other regulatory elements which are required for expression of the gene product.
  • the promoter/regulatory sequence may, for example, be one which expresses the gene product in a tissue specific manner.
  • first molecule is said to "stimulate" a second molecule when the first molecule effect an event upon binding to or interacting with the second molecule.
  • “Survival” of a cell as used herein refers to the act of a cell remaining viable. When a cell is no longer “surviving,” it is no longer considered viable.
  • a “therapeutically effective amount” of a compound is t &SJ ⁇ I ⁇ 'i ⁇ 6 compound which is sufficient to provide a beneficial effect to the subject to which the compound is administered.
  • TLRs Tolllike receptors
  • the present invention provides a method of inhibiting differentiation of an osteoclast precursor cell, wherein an osteoclast precursor cell is contacted with a TLR ligand.
  • a TLR on the osteoclast precursor cell is stimulated and consequently, differentiation of the osteoclast cell is inhibited.
  • the cell exhibits enhanced phagocytic activity.
  • TLR ligands which serve to inhibit osteoclast differentiation are preferably ligands which do not stimulate the immune response in other ways, i.e., which do not stimulate production of TNF- ⁇ .
  • TLR ligand herein should be construed o mean any TLR ligand, but to preferably mean a TLR ligand which inhibits osteoclast differentiation without stimulating the immune response.
  • the cell may exhibit enhanced phagocytic activity which, for the purpose of the present invention is not considered to come under the umbrella of immune stimulation.
  • an osteoclast precursor cell is contacted with a TLR ligand in vivo.
  • an osteoclast precursor cell in contacted with a TLR ligand in vitro.
  • One aspect of this embodiment of the invention comprises administration of a contacted osteoclast precursor cell to a patient for therapeutic purposes, including, but not limited to enhancing the immune response of such a patient through increased phagocytic activity provided by a contacted osteoclast precursor cell administered to the patient.
  • the present invention also provides a method of maintaiffin ⁇ ri •J/1 y b osteoclast precursor cell in a progenitor state, wherein an osteoclast precursor cell is contacted with a TLR ligand.
  • a TLR on the osteoclast precursor cell is stimulated and consequently, differentiation of the osteoclast cell is inhibited, maintaining the osteoclast precursor cell in a progenitor state.
  • an osteoclast precursor cell is contacted with a TLR ligand in vitro.
  • an osteoclast precursor cell in contacted with a TLR ligand in vivo.
  • an osteoclast precursor cell is contacted in a subject, preferably a mammal.
  • mammals useful in the present invention include, but are not limited to, human, monkey, sheep, rat, and mouse.
  • the mammal is a human.
  • Other mammals useful in the present invention would be known to one of skill in the art.
  • an osteoclast precursor cell may be administered to a subject in the form of a pharmaceutical composition, described in detail elsewhere herein.
  • a TLR on an osteoclast precursor cell When a TLR on an osteoclast precursor cell is stimulated, the cell does not differentiate to become a mature osteoclast. Accordingly, such a cell administerd to a patient or stimulated within a patient retains phagocytic activity and can contributes to the local immune response in the patient.
  • a TLR-stimulated osteoclast may destroy by phagocytosis bacteria involved in bone infection at the site of the TLR-stimulated, non-differentiated osteoclast. The removal and destruction of bacteria involved in a bone infection assists in the immune response of an infected patient, and decreases the healing and recovery time of the patient.
  • a TLR-stimulated, non- differentiated osteoclast in a patient remains in a progenitor state. Without differentiating into a mature osteoclast, the TLR-stimulated osteoclast precursor does not possess bone-resorbing, i.e., "osteoclast" activity.
  • TLR-stimulation of an osteoclast precursor can stabilize the osteoclast precursor cell in a progenitor state, thus decreasing the loss of bone.
  • the inhibition of osteoclast differentiation by stimulation of one or more TLRs on an osteoclast in a patient, or by administration of an osteoclast z z stimulated by one or more TLRs, provides a mammal with beneficial activity while supporting the immune response.
  • such osteoclast-mediated bone-preserving activity is useful in a mammal suffering from a bacterial bone infection.
  • the inhibition of the differentiation of an osteoclast to form a mature osteoclast prevents the mature osteoclast-associated bone resorption associated with mature osteoclasts in a mammal, while at the same time increasing phagocytic activity for the clearance of a bacterial bone infection.
  • the present invention therefore fills a need in connection with the clearance of bacteria in bacterial bone infections, as an increase in the immune response is typically accompanied by an increased loss of bone.
  • an osteoclast precursor cell may be isolated from a mammal.
  • Mammals useful in the present invention include, but are not limited to, human, monkey, sheep, rat, and mouse.
  • the mammal is a human.
  • Methods of isolating, culturing, and manipulating such osteoclast precursor cells are well-known to one of skill in the art.
  • an osteoclast precursor cell is isolated from a population of bone marrow cells.
  • an osteoclast precursor cell is isolated from a population of peripheral blood monocytes.
  • an osteoclast precursor cell is isolated from a population of cells derived from the myeloid cell line RAW264.7.
  • mouse bone marrow cells are cultured in a minimal essential medium containing 10% fetal bovine serum with M-CSF (5 ng/ml) for 12 hours in 100-mm diameter dishes (1 x 10 7 cells/10 ml/dish) to separate adherent cells and non-adherent cells. Then, non-adherent cells are harvested and cultured with M-CSF (30 ng/ml) in 100-mm diameter dishes (5 x 10 6 cells/10 ml/dish).
  • osteoclast precursors prepared by other methods are useful in the present invention, and the present example should therefore not limit the methods by which these cells are prepared.
  • TLRs Toll-like receptors
  • immune cells such as macrophages and dendritic cells.
  • TLRs are also expressed on osteoclast precursor cells. More specifically, all of the presently-known murine TLRs - TLR1, TLR2, TLR3, TLR4, TLR5, TLR6, TLR7, TLR8, and TLR9 - are expressed on osteoclast precursor cells. Accordingly, it is a feature of the present invention to contact osteoclast precursor cells by way of one or more TLRs. Contact of the TLRs on an osteoclast precursor cell with a ligand is useful to inhibit differentiation of such an osteoclast precursor cell and maintain the osteoclast precursor in a progenitor state.
  • TLRs have been shown herein to be expressed on osteoclast precursor cells as described in detail in the Experimental Examples section below. Briefly, to determine which TLRs are expressed in an osteoclast precursor cell, RNA from the osteoclast precursor cell is analyzed using a reverse transcriptase polymerase chain reaction (RT-PCR) followed by southern blot hybridization to the RT-PCR products. Primers specific for each TLR under investigation - i.e., TLR1, TLR2, TLR3, TLR4, TLR5, TLR6, TLR7, TLR8, TLR9 - are used in connection with the analysis of TLR expression, as will be understood by one of skill in the art.
  • RT-PCR reverse transcriptase polymerase chain reaction
  • contacting an osteoclast precursor cell with a TLR ligand stimulates a single TLR, e.g., TLR3.
  • contacting an osteoclast precursor cell with a TLR ligand may stimulate more than one TLR, e.g., TLR3, TLR2, and TLR1 , simultaneously.
  • the TLR ligand may directly stimulate each stimulated TLR by, for example, direct binding to the TLR.
  • TLRs include such TLRs as are discovered in the future, since any osteoclast cell receptor which would be determined to be a TLR according to well-established biochemical criteria known in the art, would be understood by the routineer as being capable of inhibiting osteoclast differentiation when stimulated with the appropriate ligand, as disclosed throughout the instant specification and as amply demonstrated and exemplified herein.
  • the present invention is not limited in any way to the particular TLRs set forth herein; rather, the invention includes those TLRs known in the art or to be discovered in the future.
  • TLR lignads which inhibit osteoclast differentiation do not stimulate the immune response.
  • a TLR on an osteoclast precursor cell of the present invention ay ⁇ c stimulated by numerous ligands, as are known to one of skill in the art.
  • an osteoclast precursor cell is contacted with a ligand that is a microbial constituent.
  • microbial constituents include, by way of a non- limiting example, lipopolysaccharide (LPS).
  • LPS lipopolysaccharide
  • Additional TLR ligands useful in the present invention include peptidoglycan, lipoteichoic acid, flagellin, dsRNA (poly(LC) RNA), and the CpG motif of unmethylated DNA.
  • TLR ligands While the interaction of a number of TLR ligands with their cognate TLR receptor has been reported - i.e., peptidoglycan and lipoteichoic acid with TLR2, LPS with TLR3 - the present invention should be understood to encompass the stimulation of any osteoclast TLR receptor with any TLR ligand, wherein such stimulation results in the inhibition of osteoclast precursor cell differentiation. Accordingly, the present invention is not limited in any way to the particular TLR ligands set forth herein; rather, the invention includes those TLR ligands known in the art or to be discovered in the future.
  • TLR ligands to be discovered in the future can be identified and characterized by means of their interaction with and effect on their corresponding TLR, and should be considered within the scope of the present invention, as the present invention encompasses the novel demonstration that an osteoclast precursor cell can be prevented from differentiating by means of contacting the osteoclast precursor cell with multiple ligands having specificity for multiple TLRs.
  • PGN prevents an osteoclast precursor cell from differentiating by specific interaction with TLR2
  • LPS prevents an osteoclast precursor cell from differentiating by specific interaction with TLR4.
  • Figures 7, 8, 9 and 10 describe elsewhere herein, there is described disclosure which differentiates the effects of a TLR ligand and an osteoclast differentiation from immune stimulation
  • a range of TLR ligand concentrations may be used to contact a TLR on an osteoclast precursor cell according to the methods of the present invention.
  • an osteoclast precursor cell is contacted with a TLR ligand at a concentration between 1 pg/ml and 1 g/ml. More preferably, the concentration of TLR ligand is between 10 pg/ml and 1 mg/ml. Even more preferably, the concentration of TLR ligand used is between 100 pg/ml and 1 ⁇ g/ml.
  • an osteoclast precursor cell is contacted wn ⁇ Lu concentration of 1 ng/ml.
  • the concentration of TLR ligand used to contact an osteoclast precursor cell will vary, depending upon many factors, including, but not limited to, pharmacological potency of the ligand, size of the ligand, and inclusion of any additional substances conferring a synergistic effect in combination with the ligand. Further, the concentration of TLR ligand used to contact an osteoclast precursor cell may depend upon agents or adjuvants combined with the TLR ligand, such as those used with pharmaceutical compositions, as described elsewhere herein in detail. Further, in an aspect of the present invention, more than one type of
  • TLR ligand may be used concomitantly to contact an osteoclast precursor cell using any method of the present invention.
  • both LPS and peptidoglycan may be used in conjunction to contact an osteoclast precursor cell using a method of the present invention. It will be understood by one of skill in the art, based on the knowledge of TLR ligands and their cognate receptors, that certain combinations of TLR ligands may produce an additive or synergistic effect on an osteoclast precursor cell when administered concomitantly according to a method of the present invention.
  • stimulation of an osteoclast precursor cell using a method of the present invention can have therapeutic applications.
  • enhancing the phagocytic activity in a patient has the benefit of enhancing the immune response in the patient.
  • the administration of a TLR- stimulated, non-differentiated phagocytic osteoclast to a patient with a bacterial bone infection can provide the patient with an enhanced defense against the bone infection.
  • the phagocytic activity of the administered osteoclast can aid in the local isolation and clearance of the bacteria responsible for the bone infection.
  • Bacterial bone infections stimulate a strong immune response.
  • bone infections can induce a strong immune response on the part of the host due to microbial products such as peptidoglycan, lipteichoic acid, and lipopolysaccharide.
  • Host responses to such infection include the upregulation of TNF- ⁇ production by macrophage and dendritic cell activation.
  • the resulting TNF- ⁇ contributes to the local immune response against the bacterial infection, but TNF- ⁇ also enhances the differentiation of osteoclasts, as well as the survival ot ⁇ c - * /1 V4b mature osteoclasts.
  • the infected patient can experience increased bone loss during the immune response. For this reason, a TLR ligand that inhibits osteoclast differentiation while having little or no effect on TNF- ⁇ production is prefered.
  • a TLR-stimulated, non-differentiated phagocytic osteoclast can have the added benefit to the patient of minimizing bone loss associated with enhancing the immune response. Because a non-differentiated (i.e., "immature") osteoclast does not possess the bone-resorbing activity of a differentiated, mature osteoclast, a patient to which a TLR-stimulated, non-differentiated phagocytic osteoclast is administered will not experience further destruction of bone, but can experience an increased rate of clearance of infection.
  • the present invention features a method of enhancing phagocytic activity of an osteoclast precursor cell, wherein an osteoclast precursor cell is contacted with a TLR ligand.
  • a TLR on the osteoclast precursor cell is stimulated and consequently, differentiation of the osteoclast cell is inhibited.
  • the osteoclast precursor cell In the undifferentiated or "precursor" state, the osteoclast precursor cell possesses phagocytic activity, whereas in the differentiated or "mature” state, the osteoclast does not possess or exhibit phagocytic activity.
  • the phagocytic activity of the osteoclast precursor is supported and sustained for a length of time longer than had the osteoclast precursor cell been permitted to differentiate into a mature osteoclast.
  • phagocytic activity is enhanced in an isolated osteoclast precursor cell by contacting the osteoclast precursor cell with a TLR ligand.
  • Yet another embodiment of the present invention features a method of enhancing phagocytosis in a mammal, wherein an osteoclast precursor cell in a mammal is contacted with a TLR ligand.
  • a TLR on the osteoclast precursor cell is stimulated and consequently, differentiation of the osteoclast cell is inhibited.
  • the osteoclast precursor cell In the undifferentiated or "precursor" state, the osteoclast precursor cell possesses phagocytic activity, and therefore, stabilization of the precursor state of tnc ⁇ tsuc ⁇ i tu supports and sustains the phagocytic activity of the osteoclast precursor in the mammal.
  • the osteoclast precursor cell In the undifferentiated or "precursor" state, the osteoclast precursor cell does not possesses bone-resorbing activity, and therefore, stabilization of the precursor state of the osteoclast supports and sustains a cell that does not have the ability to destroy bone.
  • a method of enhancing phagocytosis enhances the immune response in a mammal without contributing to bone loss during the immune response.
  • a TLR on the osteoclast precursor cell is stimulated and consequently, differentiation of the osteoclast cell is inhibited.
  • the osteoclast precursor cell In the undifferentiated or "precursor” state, the osteoclast precursor cell possesses phagocytic activity, and therefore, stabilization of the precursor state of the osteoclast supports and sustains the phagocytic activity of the osteoclast precursor in the mammal.
  • the osteoclast precursor cell does not possesses bone-resorbing activity, and therefore, stabilization of the precursor state of the osteoclast supports and sustains a cell that does not have the ability to destroy bone.
  • the present invention also features a method of identifying an osteoclast cell as an osteoclast precursor cell, wherein an osteoclast cell is first contacted with at least one TLR ligand, then contacted with M-CSF and TRANCE, then assayed for phagocytic activity. Subsequently, the osteoclast cell is positively identified as an osteoclast precursor cell by demonstrating phagocytic activity of the cell. Identification of an osteoclast cell by a method of the present invention is useful in the determination of the differentiated state of an osteoclast cell prior to the administration of such a cell to a patient.
  • the identification of an osteoclast cell as an osteoclast precursor cell begins by contacting an osteoclast cell with a TLR ligand, which ligand prevents the cell from differentiating, as described in detail above. Any TLR ligand as described elsewhere herein may be used to contact an osteoclast cell in this embodiment of the invention.
  • an osteoclast cell is contacted with 1 ng/ml LPS in the presence 01 JUU ng/ml TRANCE and 30 ng/ml M-CSF and incubated at 37 °C for three days, after which time the resulting cells are cultured with fluorescein isothiocyanate (FIT C)- conjugated zymosan particles for one hour. After culturing, cells are washed with phosphate-buffered saline, fixed, and stained for tartrate-resistant acid phosphatase (TRAP), and cells staining positive for TRAP presents evidence of phagocytic activity, thereby identifying the cells as osteoclast precursor cells.
  • FIT C fluorescein isothiocyanate
  • one embodiment of the present invention features a method of enhancing survival of a mature osteoclast, wherein a mature osteoclast is contacted with a TLR ligand.
  • a mature osteoclast is contacted with a TLR ligand.
  • the survival of the mature osteoclast is enhanced in comparison to a second mature osteoclast, where the second mature osteoclast is not contacted with the TLR ligand.
  • any TLR and any TLR ligand may be used, as such TLRs and TLR ligands have been described in detail above in connection with the more general disclosure of the use of TLRs and TLR ligands to inhibit differentiation of an osteoclast precursor cell.
  • any source of an osteoclast as described above in connection with the more general disclosure of the use of TLRs and TLR ligands to inhibit differentiation of an osteoclast precursor cell is equally applicable as a source for methods of the invention in which a mature osteoclast is contacted with a TLR ligand to enhance the survival of the mature osteoclast.
  • a mature osteoclast is contacted with peptidoglycan to enhance the survival of the osteoclast.
  • a mature osteoclast having a TLR2 receptor is contacted withußschreib peptidoglycan to enhance the survival of the osteoclast.
  • a mature osteoclast is contacted with LPS to enhance the survival of the osteoclast.
  • a mature osteoclast having a TLR4 receptor is contacted with LPS to enhance the survival of the osteoclast.
  • a mature osteoclast is cultured for twelve hours in a medium containing 10 ng/ml LPS, which is a TLR4 ligand.
  • the LPS -containing culture contains viable, mature osteoclasts, indicating survival of mature osteoclasts in the presence of the TLR4 ligand, LPS.
  • an aspect of the invention includes methods of treating such diseases by, among other things, administering a TLR-stimulating ligand that can preferentially inhibit the loss of bone matrix.
  • the present invention also encompasses methods of treating a patient in need of a stable phagocytic progenitor stage osteoclast.
  • a phagocytic progenitor stage osteoclast is useful as a part of a local immune response in a patient, where such an osteoclast may provide phagocytic activity to a patient, without increasing bone- resorption in the patient.
  • Stabilization of a phagocytic progenitor stage osteoclast in the progenitor state results when the osteoclast is prevented from fully differentiating into a mature osteoclast.
  • contacting a TLR on an osteoclast precursor cell with a TLR ligand inhibits precursor cell and stabilizes the cell in the progenitor state required to support phagocytic activity.
  • a method for treating a patient in need of a stable phagocytic progenitor stage osteoclast in which a phagocytic progenitor stage osteoclast is contacted with a TLR ligand for the purpose of inhibiting differentiation of the progenitor stage osteoclast.
  • a TLR ligand for the purpose of inhibiting differentiation of the progenitor stage osteoclast.
  • the progenitor stage osteoclast In the undifferentiated or "precursor" state, the progenitor stage osteoclast possesses phagocytic activity, and therefore, stabilization of the progenitor state of the osteoclast supports and sustains the phagocytic activity of the progenitor stage osteoclast.
  • the TLR lignad is chosen so as to inhibit differentiation of the osteoclast which having little or no effect on TNF- ⁇ production.
  • a stabilized progenitor stage osteoclast prepared as described elsewhere herein can be administered to a patient in need thereof using techniques known to one of skill in the art.
  • administration to a patient of a stable phagocytic progenitor stage osteoclast contributes to a local immune response in a patient, at the site of administration, thereby treating a patient in need of a stable phagocytic progenitor stage osteoclast.
  • a further aspect of this embodiment of the invention is that the patient will not experience an increase in bone resorption in conjunction with the administration of a stable phagocytic progenitor stage osteoclast, as the administered osteoclast does not possess bone-resorbing activity.
  • a method for treating a patient in need of a stable phagocytic progenitor stage osteoclast in which a TLR-stimulating ligand is provided to a patient in need thereof.
  • a TLR on the progenitor stage osteoclast is stimulated and consequently, differentiation of the progenitor stage osteoclast is inhibited.
  • the progenitor stage osteoclast In the undifferentiated or "precursor" state, the progenitor stage osteoclast possesses phagocytic activity, and therefore, stabilization of the progenitor state of the osteoclast supports and sustains the phagocytic activity of the progenitor stage osteoclast in the patient.
  • enhancing phagocytosis of an osteoclast precursor cell contributes to the immune response in a patient, thereby treating a patient in need of a stable phagocytic progenitor stage osteoclast.
  • a further aspect of this embodiment of the invention provides that the patient will not experience an increase in bone resorption in conjunction with the administration of a stable phagocytic progenitor stage osteoclast, as the administered osteoclast does not possess bone-resorbing activity.
  • TLR-stimulating ligand administered to a patient may be effected by one of many means known to one of ordinary skill in the art of drug delivery. Formulations, dosages, and methods of administration of such a TLR- stimulating ligand are discussed in detail hereinbelow.
  • a TLR on an osteoclast precursor cell useful in a method of the present invention may be stimulated by numerous ligands.
  • an osteoclast precursor cell is contacted with a microbial constituent.
  • the present invention is not limited in any way to the particular TLR ligands set forth herein; rather, the invention includes those TLR ligands known in the art or to be discovered in the future, for reasons that have been described in detail above.
  • an aspect of the present invention provides a method for identifying a TLR ligand that has the property of inhibiting the differentiation of an osteoclast precursor cell.
  • an osteoclast precursor cell is first contacted with a test TLR ligand, and assessed for its ability to differentiate when compared with an osteoclast cell that was not contacted with the test compound.
  • Inhibition of differentiation in the presence of the test TLR ligand compared with differentiation in its absence is an indication that the test IJUJ ⁇ ug ⁇ u ⁇ inhibits differentiation.
  • One measurement of inhibition of differentiation, as discribed herein, is whether the cell exhibits increased phagocytic activity. Detection of phagocytic activity is an indication of the undifferentiated precursor state of the osteoclast, and therefore, detection of such activity is an indication that the test TLR ligand has positively inhibited differentiation of the osteoclast precursor cell.
  • an osteoclast cell is first contacted with a test TLR ligand, then contacted with M-CSF and TRANCE, then assayed for phagocytic activity. Subsequently, the osteoclast cell is positively identified as an osteoclast precursor cell by demonstrating phagocytic activity of the cell. Identification of an osteoclast precursor cell by a method of the present invention is useful in the determination of the differentiated state of an osteoclast cell for the purpose of identifying a test compound that has the property of inhibiting the differentiation of an osteoclast precursor cell.
  • the immunostimulation, and the inhibition of the differentiation of an osteoclast precursor cell obtained upon stimulation of an osteoclast precursor cell TLR can be separable and distinct.
  • Immunostimulation of an osteoclast precursor cell can be measured by upregulation of TNF- ⁇ production, as described elsewhere herein. While it has been described herein that stimulation of an osteoclast precursor cell TLR can inhibit differentiation of the osteoclast precursor cell, and that the same TLR stimulation can enhance the phagocytic activity of the osteoclast precursor cell, which is also a measure of immunostimulation of an osteoclast precursor cell, it is also shown that immunostimulation and inhibition of differentiation of an osteoclast precursor cell can be separable and distinct.
  • an osteoclast precursor cell is contacted with a TLR ligand.
  • the osteoclast is examined for immunostimulation as measured by TNF- ⁇ production and for inhibition of differentiation, measured as described herein.
  • an contacted with CpG5. After culturing the osteoclast precursor cell, the cell is assayed for TNF- ⁇ upregulation as an indication of the level of immunostimulation.
  • the cell is then assayed for phagocytic activity as a measure of the inhibition of differentiation.
  • the measured values for immunostimulation and phagocytic activity of the osteoclast precursor cell are then compared with those values obtained performing the same analysis with the control DNA sequence set forth in SEQ ID NO:l, CpG, which exhibits both an immunostimulatory effect and a differentiation- inhibitory effect when used to contact an osteoclast precursor cell.
  • a difference in the ratio of the immunostimulatory effect to the differentiation-inhibitory effect for an osteoclast precursor cell contacted with CpG5 as to when an osteoclast precursor cell is contacted with CpG is an indication that CpG5 has a different effect on immunostimulation of an osteoclast precursor cell than CpG5 has on the differentiation-inhibitory activity of the same cell.
  • the present invention further encompasses various kits relating to inhibition of differentiation of an osteoclast precursor cell.
  • kits relating to inhibition of differentiation of an osteoclast precursor cell are an effective tool in combating infection associated with bone.
  • a kit of the present invention for the inhibition of differentiation of an osteoclast precursor cell comprises at least one TLR ligand, such as PGN, poly(I:C) RNA, LOS, and CpG DNA, and an instructional material detailing the use of the kit as described in the methods provided herein.
  • the TLR ligand or ligands of the kit should stimulate at least one of TLR1, TLR2, TLR3, TLR4, TLR5, TLR6, TLR7, TLR8 and TLR9 on an osteoclast precursor cell.
  • a kit for the inhibition of differentiation of an osteoclast precursor cell further comprises reagents and/or materials necessary for the inhibition of differentiation of an osteoclast precursor cell in a mammal.
  • Such reagents and/or . .I ⁇ materials include, but are not limited to devices for delivery of a TLR li i ⁇ / ! ⁇ ! ⁇ •J/1 V , mammal.
  • kits which will be supplied in the instructional material, comprise the methods described herein.
  • the kit in combination with the instructional material describing its method of use may be used to the benefit of the mammal.
  • kits for identifying an osteoclast precursor cell comprising at least one TLR ligand and further comprises a reagent useful for detecting phagocytic activity of an osteoclast precursor cell, M-CSF, TRANCE, and an instructional material detailing the use of the kit.
  • the TLR ligand of the kit should stimulate at least one of TLR1, TLR2, TLR3, TLR4, TLR5, TLR6, TLR7, TLR8 and TLR9 on an osteoclast precursor cell.
  • kits for enhancing survival of a mature osteoclast comprising at least one TLR ligand, such as PGN, poly(I:C) RNA, LOS, and CpG DNA, and an instructional material detailing the use of the kit.
  • the TLR ligand of the kit should stimulate at least one of TLR1, TLR2, TLR3, TLR4, TLR5, TLR6, TLR7, TLR8 and TLR9 on an osteoclast precursor cell.
  • the present invention encompasses the use of pharmaceutical compositions of a stable phagocytic progenitor-stage osteoclast to practice methods of the invention, the compositions comprising a stable phagocytic progenitor-stage osteoclast and a pharmaceutically-acceptable carrier.
  • the invention also encompasses the use pharmaceutical compositions of at least one appropriate TLR stimulator to practice the methods of the invention, the compositions comprising at least one appropriate TLR stimulator and a pharmaceutically-acceptable carrier.
  • the term "pharmaceutically-acceptable carrier” means a chemical composition with which an appropriate stable phagocytic progenitor-stage osteoclast or at least one appropriate TLR stimulator may be combined and which, following the combination, can be used to administer the appropriate pharmaceutical composition to a mammal.
  • compositions useful for practicing the invention may be administered to deliver a dose of between 1 ng/kg/day and 100 mg/kg/day.
  • Pharmaceutical compositions that are useful in the methouo il ll! J/1 V4 , invention may be administered systemically in oral solid formulations, aerosol, topical or other similar formulations.
  • such pharmaceutical compositions may contain pharmaceutically-acceptable carriers and other ingredients known to enhance and facilitate drug administration.
  • compositions such as nanoparticles, liposomes, resealed erythrocytes, and immunologically based systems may also be used to administer an appropriate table phagocytic progenitor-stage osteoclast or TLR stimulator according to the methods of the invention.
  • Compounds which are identified using any of the methods described herein may be formulated and administered to a mammal for treatment of the diseases disclosed herein are now described.
  • the invention encompasses the preparation and use of pharmaceutical compositions comprising a compound useful for treatment of the diseases disclosed herein as an active ingredient.
  • a pharmaceutical composition may consist of the active ingredient alone, in a form suitable for administration to a subject, or the pharmaceutical composition may comprise the active ingredient and one or more pharmaceutically acceptable carriers, one or more additional ingredients, or some combination of these.
  • the active ingredient may be present in the pharmaceutical composition in the form of a physiologically acceptable ester or salt, such as in combination with a physiologically acceptable cation or anion, as is well known in the art.
  • the term "pharmaceutically acceptable carrier” means a chemical composition with which the active ingredient may be combined and which, following the combination, can be used to administer the active ingredient to a subject.
  • physiologically acceptable ester or salt means an ester or salt form of the active ingredient which is compatible with any other ingredients of the pharmaceutical composition, which is not deleterious to the subject to which the composition is to be administered.
  • compositions described herein may be prepared by any method known or hereafter developed in the art of pharmacology.
  • preparatory methods include the step of bringing the active ingredient into association with a carrier or one or more other accessory ingredients, and then, if necessary or desirable, shaping or packaging the iuuii i muj a desired single- or multi-dose unit.
  • compositions are principally directed to pharmaceutical compositions which are suitable for ethical administration to humans, it will be understood by the skilled artisan that such compositions are generally suitable for administration to animals of all sorts. Modification of pharmaceutical compositions suitable for administration to humans in order to render the compositions suitable for administration to various animals is well understood, and the ordinarily skilled veterinary pharmacologist can design and perform such modification with merely ordinary, if any, experimentation. Subjects to which administration of the pharmaceutical compositions of the invention is contemplated include, but are not limited to, humans and other primates, and other mammals.
  • compositions that are useful in the methods of the invention may be prepared, packaged, or sold in formulations suitable for oral, parenteral, pulmonary, intranasal, buccal, or another route of administration.
  • Other contemplated formulations include projected nanoparticles, liposomal preparations, resealed erythrocytes containing the active ingredient, and immunologic ally-based formulations.
  • the relative amounts of the active ingredient, the pharmaceutically acceptable carrier, and any additional ingredients in a pharmaceutical composition of the invention will vary, depending upon the identity, size, and condition of the subject treated and further depending upon the route by which the composition is to be administered.
  • the composition may comprise between 0.1% and 100% (w/w) active ingredient.
  • a pharmaceutical composition of the invention may further comprise one or more additional pharmaceutically active agents.
  • additional agents include anti-emetics and scavengers such as cyanide and cyanate scavengers.
  • Controlled- or sustained-release formulations of a pharmaceutical composition of the invention may be made using conventional technology.
  • an "oily" liquid is one which comprises a carbon- containing liquid molecule and which exhibits a less polar character than water.
  • Liquid formulations of a pharmaceutical composition of which are suitable for oral administration may be prepared, packaged, and sold either in liquid form or in the form of a dry product intended for reconstitution with water or another suitable vehicle prior to use.
  • parenteral administration of a pharmaceutical composition includes any route of administration characterized by physical breaching of a tissue of a subject and administration of the pharmaceutical composition through the breach in the tissue.
  • Parenteral administration thus includes, but is not limited to, administration of a pharmaceutical composition by injection of the composition, by application of the composition through a surgical incision, by application of the composition through a tissue-penetrating non-surgical wound, and the like.
  • parenteral administration is contemplated to include, but is not limited to, subcutaneous, intraperitoneal, intramuscular, intrasternal injection, and kidney dialytic infusion techniques.
  • Formulations of a pharmaceutical composition suitable for parenteral administration comprise the active ingredient combined with a pharmaceutically acceptable carrier, such as sterile water or sterile isotonic saline. Such formulations may be prepared, packaged, or sold in a form suitable for bolus administration or for continuous administration.
  • Injectable formulations may be prepared, packaged, or sold in unit dosage form, such as in ampules or in multi-dose containers containing a preservative.
  • Formulations for parenteral administration include, but are not limited to, suspensions, solutions, emulsions in oily or aqueous vehicles, pastes, and implantable sustained-release or biodegradable formulations. Such formulations may further comprise one or more additional ingredients including, but not limited to, suspending, stabilizing, or dispersing agents.
  • the active ingredient is provided in dry (i.e. powder or granular) form for reconstitution with a suitable vehicle (e.g. sterile pyrogen-free water) prior to parenteral administration of the reconstituted composition.
  • a suitable vehicle e.g. sterile pyrogen-free water
  • compositions may be prepared, packaged, or sold in the form of a sterile injectable aqueous or oily suspension or solution.
  • This suspension or solution may be formulated according to the known art, and may comprise, in addition to the active ingredient, additional ingredients such as the dispersing agents, wetting agents, or suspending agents described herein.
  • Such sterile injectable formulations may be prepared using a non-toxic parenterally-acceptable diluent or solvent, such as water or 1,3-butane diol, for example.
  • diluents and solvents include, but are not limited to, Ringer's solution, isotonic sodium chloride solution, and fixed oils such as synthetic mono- or di-glycerides.
  • compositions for sustained release or implantation may comprise pharmaceutically acceptable polymeric or hydrophobic materials such as an emulsion, an ion exchange resin, a sparingly soluble polymer, or a sparingly soluble salt.
  • dosages of the compound of the invention which may be administered to an animal range in amount from 1 microgram to about 100 grams per kilogram of body weight of the animal. While the precise dosage administered will vary depending upon any number of factors, including but not limited to, the type of animal and type of disease state being treated, the age of the animal and the route of administration.
  • the dosage of the compound will vary from about 1 mg to about 10 g per kilogram of body weight of the animal. More preferably, the dosage will vary from about 10 mg to about 1 g per kilogram of body weight of the animal.
  • the compound may be administered to an animal as frequently as several times daily, or it may be administered less frequently, such as once a day, once a week, once every two weeks, once a month, or even lees frequently, such as once every several months or even once a year or less.
  • the frequency of the dose will be readily apparent to the skilled artisan and will depend upon any number of factors, such as, but not limited to, the type and severity of the disease being treated, the type and age of the animal, etc.
  • mice Three- to four-week old C57BL/6 and C3H/HeJ male mice were obtained from The Jackson Laboratory (Bar Harbor, ME) and housed in a specific pathogen-free facility at the University of Pennsylvania. All procedures were performed according to the University of Pennsylvania Institutional Animal Care and Use Committee Guidelines.
  • a soluble form of recombinant TRANCE was purified from insect cells as described previously (Wong et al.,1997. J Exp Med 186:2075).
  • Recombinant human M-CSF was obtained from Genetics Institute (Cambridge, MA).
  • LPS from Escherichia coli (O55:B5), PGN from Staphylocaccus aureus, and Poly(I:C) RNA were purchased from Sigma-Aldrich Co. (St. Louis, MO), Fluka (Sigma-Aldrich Group, St. Louis, MO), and Amersham Biotech (Piscataway, NJ), respectively.
  • Phosphothioate stabilized GpCv DNA (TCCATGACGTTCCTGATGCT; SEQ ID NO: 1) was synthesized in the core facility of the University of Pennsylvania.
  • Osteoclast precursors were prepared essentially as described previously (Kobayashi et al., 2000, J. Exp Med 191:275; Takeshita et al, 2000, J. Bone Miner Res 15:1477).
  • mouse bone marrow cells were cultured in a- minimal essential medium ( ⁇ -MEM) (Life Technologies, Inc., Gaithersburg, MD) containing 10% fetal bovine serum (FBS) with M-CSF (5 ng/ml) for 12 hours in 100- mm diameter dishes (Corning, Inc., NY, NY; 1 x 10 7 cells/10 ml/dish) to separate adherent cells and non-adherent cells.
  • ⁇ -MEM minimal essential medium
  • FBS fetal bovine serum
  • M-CSF 5 ng/ml
  • osteoclast precursors were cultured with TRANCE (300 ng/ml) and M-CSF (30 ng/ml) for 3 days in 96-well culture plates (Corning, NY, NY; 2 x 10 4 cells/0.2 ml/well) or in 60-mm diameter dishes (Corning; 5 x 10 5 cells/5 ml/dish).
  • TRANCE 300 ng/ml
  • M-CSF 30 ng/ml
  • Fluorescein-conjugated zymosan A (S. cerevisiae) Bio Particle ® (Molecular Probes, Eugene, OR) was added to osteoclast precursor and osteoclast cultures in 96-well culture plates (20 ⁇ g/0.2 ml/well). After 1 hour of culturing, cells were washed with PBS to remove the particles that were not incorporated by the cells. Cells were fixed and stained for tartrate-resistant acid phosphatase (TRAP), which is strongly expressed in osteoclasts (Suda et al., 1997, Methods Enzymol 282:223). Zymosan particles incorporated by the cells were visualized by ultraviolet light illumination under microscopic examination.
  • TRIP tartrate-resistant acid phosphatase
  • RT-PCR Polymerase chain reaction amplification of reverse-transcribed RNA
  • RNA from osteoclast precursors and purified mature osteoclasts in culture dishes 60 mm-diameter was prepared using Trizol solution (Life
  • First- strand cDNA was synthesized from total RNA using Superscript II (Life Technologies, Gaithersburg, MD): and was subjected to PCR amplification with Taq polymerase (Sigma, St. Louis, MO). Primers for-mouse TLRs used in these studies are following:
  • TLR1 5'-CTTCAGACTTCTGACATCCTCTCA-3' (forward, nucleotides; 355-378) (SEQ ID NO:2) and 5 ' -TTC A AGC ACAC ACTTGATGTTAGA-3 ' (reverse, nucleotides 855-878) (SEQ ID NO:3)
  • osteoclast precursors were cultured for indicated periods in the presence of PGN (10 ⁇ g/ml), poly(l:C) RNA (100 ⁇ g/ml), LPS (1 ⁇ g/ml), or CpG DNA (1 ⁇ M). Cells were then washed with ice-cold PBS and then lysed in sample buffer (62.5 mM Tris-HCl, pH 6.8, 2% SDS, 10% glycerol, 50 mM dithiothreitol, 0.1% breomophenol blue).
  • Cell lysates (20 ⁇ g of protein) were resolved by 10% SDS-polyacrylamide gel electrophoresis and transferred onto PVDF membranes (Millipore, Bedford, MA) and probed with anti-phos ⁇ ho-I ⁇ B ⁇ antibodies (1 ⁇ g/ml) (New England Biolabs Inc, Beverly, MA) or anti- ⁇ -actin (1 ⁇ g/ml) (Oncogene, San Diego, CA) antibodies.
  • osteoclast precursors or purified osteoclasts in 60-mm diameter dishes were cultured with PGN (10 ⁇ g/ml), poly(I:C) RNA (100 ⁇ g/ml), LPS (1000 ng/ml), and CpG DNA (1 ⁇ M) for the indicated periods, and then were subjected to total RNA isolation using Trizol (Life Technologies).
  • cDNA probes encoding mouse TNF- ⁇ were labeled with 32 P using a cDNA labeling kit (Amersham, Arlington Heights, IL).
  • Osteoclast precursors were generated from mouse bone marrow cells by M-CSF treatment. There were no TRAP-positive or multinucleated cells in osteoclast precursor preparations (Fig. 1A). After 3 days of culturing with TRANCE and M-CSF, both mono- and multi-nuclear TRAP-positive cells appeared (Fig. 1 A). When osteoclast precursors were cultured on dentine slices with TRANCE and M- CSF, resorption pits were formed on the dentine slices, indicating that TRAP-positive multinucleated cells are mature osteoclasts.
  • TLR2 and TLR4 were prominently expressed (Fig. 2).
  • TLRs expressed on osteoclast precursors respond to various microbial products, they were stimulated with PGN, poly(I:C) RNA, LPS, or CpG DNA, the ligands for TLR2, TLR3, TLR4 or TLR9, respectively.
  • PGN poly(I:C) RNA
  • LPS poly(I:C) RNA
  • CpG DNA CpG DNA
  • all of the TLR ligands efficiently induced NF-KB activation, which is a hallmark of TLR stimulation (Fig. 3 A).
  • TLR stimulation in osteoclast precursor cells by these ligands upregulated expression of TNF- ⁇ (Fig. 3B).
  • TLR stimulation of osteoclast precursors leads to cellular responses similar to those observed in macrophages.
  • osteoclast precursors prepared from C2H/HeJ mice which contain a mutation in the gene encoding TLR4, differentiated to osteoclasts even in the presence of LPS.
  • all the other TLR ligands tested (PGN, dsRNA, and CpG DNA) strongly inhibited osteoclast differentiation (Fig. 4A).
  • Cells treated with TLR ligands in the presence of TRANCE and M-CSF for 3 days still incorporated zymosan particles within 1 hour (Fig. 4B), demonstrating that they maintained their phagocytic capacities rather than differentiating into non-phagocytic mature osteoclasts (Fig. 1).
  • TLR ligands When human peripheral blood monocytes were used as osteoclast precursors, TLR ligands also , ⁇ strongly inhibited osteoclast differentiation induced by M-CSF and 4C). Therefore, these results indicate that TLR stimulation by microbial products elicits strong negative signals for osteoclast differentiation from monocytic osteoclast precursor cells.
  • the signaling pathway induced by TLRs shares many biochemical components with that of the IL-1 receptor (BL-1R) (e.g, MyD88, IRAK, and TRAF6), raising the questions of whether IL-l ⁇ can inhibit osteoclast differentiation.
  • BL-1R IL-1 receptor
  • EL-l ⁇ is a potent stimulator of NF- ⁇ B and also upregulates production of TNF- ⁇ in bone marrow-derived osteoclast precursors
  • EL-1R stimulation did not antagonize osteoclast differentiation (Fig. 4D).
  • TLR stimulation inhibits osteoclast differentiation induced by TRANCE
  • the murine myeloid RAW264.7 cell line was treated with TRANCE in the presence or absence of various TLR ligands (Fig. 5).
  • the RAW264.7 cell line differentiates into mature osteoclasts in response to TRANCE stimulation alone (27).
  • TLR ligands PGN, LPS, or CpG DNA are added to the culture, TRANCE-induced differentiation of osteoclasts from the RAW264.7 cell line was strongly inhibited.
  • the inhibitory effect of poly (I:C) RNA on TRANCE-induced osteoclastogenesis of the RAW264.7 cell line was not as potent as on-bone-marrow derived precursors.
  • TLR2 and TLR4 were found in mature osteoclasts (Fig. 2), the effects of TLR ligands on the survival of mature osteoclasts was also examined.
  • mature osteoclasts were highly purified and cultured in the absence or presence of TLR ligands (Fig 6A, B). In the absence of any stimuli, most multinucleated, mature osteoclasts died within 12 hours. However, PGN and LPS induced the survival of 14q4 ⁇ mature osteoclasts in a dose-dependent manner (Fig. 6A).
  • TLR stimulation has been shown herein to effect both immunostimulatory response as well as inhibition of osteoclast precursor differentiation
  • TLR stimulation can also preferentially effect one or the other of immunostimulatory response or inhibition of osteoclast precursor differentiation.
  • CpG standard DNA (as set forth above), and CpG DNAs 1-12 and 17- 20 were added to bone marrow-derived macrophage cultures, one for each of the CpG DNAs and for the CpG DNA standard.
  • the cultures were incubated for 48 hours at 37 °C, at which time the level of TNF- ⁇ in the culture media was quantified by ELISA (Figs. 7A & 7B).
  • CpG standard DNA (as set forth above), and CpG DNAs 1- 12 and 17-20 were added to mouse osteoclast precursor cultures, again, with one culture for each of the CpG DNAs and for the CpG DNA standard.
  • CpG DNAs were added to the individual osteoclast precursor cultures simultaneously with TRANCE (300 ng/ml) and M-CSF (30 ng/ml). Cells were cultured for 3 days, at which time TRAP activity and the number of osteoclasts were quantified (Figs. 8 A, 8B, 9 A &9B).
  • CpG standard DNA (as set forth above), and CpG DNAs 1-12 and 17-20 were added to human monocyte cultures, again, with one culture for each of the CpG DNAs and for the CpG DNA standard.
  • CpG DNAs were added to the individual osteoclast precursor cultures simultaneously with TRANCE (300 ng/ml) and M-CSF (30 ng/ml). Cells were cultured for 4 days, at which time TRAP activity and the number of osteoclasts were quantified (Fig. 10). ⁇ ⁇ n ⁇ / ⁇ ⁇ o ⁇
  • results show, for example, that the TLR9 ligand stimulator of TNF- ⁇ production. Because TNF- ⁇ production is an indicator of immunostimulatory effect of osteoclast precursor cells, this result demonstrates that CpG-5 effects only weak immunostimulatory activity from osteoclast precursor cells upon TLR stimulation. However, CpG-5 exhibits a measurably stronger inhibition of osteoclast differentiation in comparison to the other CpG oligos tested, demonstrating that the immunostimulatory and- the osteoclast differentiation inhibitory effects obtained by way of stimulation of a TLR on an osteoclast precursor cell are separable and can be distinct.
  • osteoclast precursors also expressed TLRs. Stimulation via TLRs induced the activation of NF- B, and upregulated the expression levels of TNF- ⁇ mRNA.
  • TLR ligands are potent inhibitors of osteoclast differentiation induced by M-CSF and TRANCE from bone marrow osteoclast precursors or from human peripheral blood monocytes. TRANCE induced differentiation of the RAW264.7 cell line to osteoclasts can be also inhibited by TLR ligands, indicating that TRL ligands inhibit the osteoclastogenic pathway induced by TRANCE.
  • IFN- ⁇ , IL-4, and GM-CSF are produced by macrophages and known to inhibit osteoclast differentiation (Bendixen et al., 2001, Proc Natl Acad Sci USA 98:2443;Fox et al., 2000, Biochem Biophys Res Commun 276:868; Miyamoto, et al., 2001, Blood 98:2544).
  • TLR ligands neutralizing antibodies to IFN- ⁇ , IL-4, and GM-CSF with TLR ligands added to osteoclast cell cultures do not affect the negative effect of TLR ligands on osteoclast differentiation, demonstrating that TLR ligands inhibit osteoclast differentiation independent of IFN- y, IL-4, and GM-CSF.
  • the cytoplasmic tails of TLRs are extremely similar to the cytoplasmic domain of the IL-1R called the Toll/IL-IR homology (TIR) domain (Kaisho, et al., 2001, Trends Immunol 22:78; Akira et al., 2000, J Endotoxin Res 6:383).
  • the TIR domain forms a complex with myeloid differentiation factor 88 (MyD88-), DL-1 receptor-associated kinase (IRAK), and tumor necrosis factor receptor-associated factor (TRAF 6), and signaling induces phosphorylation and degradation of I ⁇ B to activate NF- ⁇ B (Kaisho, et al., 2001, Trends Immunol 22:78; Akira et al., 2000, J Endotoxin Res 6:383; Muzio et al, 2000, Microbes Infect 2:251; Wong et al., 1999, Mol Cell 4:1041).
  • TLR ligands different from TLR ligands, the present invention demonstrates that IL-l ⁇ does not inhibit osteoclast differentiation- induced by TRANCE. Therefore, signaling molecules unique to the TLR stimulation pathway are likely to play roles in the inhibition of osteoclast differentiation, the mechanism of which may be determined by future studies.
  • microbial products inhibit osteoclast differentiation via TLRs.
  • This particular aspect of the present invention is counter-intuitive and unexpected, since bacterial infection can cause inflammatory bone diseases such as periodontitis, osteomyelitis, and bacterial arthritis (Nair et al., 1996, Infect Immun 64:2371). Bone mineral density is reduced in such diseases because of excessive bone resorption by osteoclasts.
  • LPS has been suggested to be a potent stimulator of -bone loss by causing the increase in the number of osteoclasts in mice (Nair et al., 1996, Infect Immun 64:2371; Abu- Amer et al., 1997, J Clin Invest 100:1557; Sakuma et al., 2000, Infect Immun 68:6819; Ueda-N et al., 1998, Y4. J Periodontal Res. 33:509).
  • LPS can also directly enhance the survival of mature osteoclasts (Suda et al., 2002. J Cell Physiol 190:101), suggesting that TL 46 stimulation provides anti-apoptotic signals in mature osteoclasts.
  • the present invention demonstrates that mature osteoclasts express TLR4 for LPS, that mature osteoclasts express TLR2, and that PGN (the ligand for TLR2) enhances the survival of mature osteoclasts (Fig. 2 and Fig. 6).
  • osteoclast precursors are much more sensitive to TLR stimulation.
  • a complete inhibition of osteoclast differentiation can be achieved by 1 ng/ml of LPS, at which concentration LPS has no effect on the survival of mature osteoclasts (Fig. 4A and Fig. 6A).
  • TLR stimulation is a potent negative regulator of osteoclastogenesis and the fact that excessive bone resorption by osteoclasts is associated with bacterial infection. It has been shown recently that alveolar bone destruction in periodontitis caused by infection of Gram-negative bacteria is mediated by enhanced osteoclastogenesis due to T cell immunity (Teng et al., 2000, J Gun Invest 106:R59). CD4 + T cell responses to bacterial infection and TRANCE expressed on activated CD44 + T cells are required for increased number of osteoclasts and their enhanced activities leading to bone destruction.
  • LPS is a potent inducer of dendritic cell maturation, which is required for successful priming of antigen-specific T cells.
  • TLR ligands can induce the production of various proinflammatory cytokines such as TNF- ⁇ , EL-1, or IL-12 by activating macrophages or dendritic cells (Kaisho, et al., 2001, Trends Immunol 22:78; Aderem, A., 2001, Grit Care Med 29:516; Underhill et al, 1999, Proc Natl Acad Sci).
  • the present invention . . , , . .
  • osteoclast p ⁇ t ⁇ PJSLi' i 4 2 4b produce proinflammatory cytokines such as TNF- ⁇ in response to various TLR ligands.
  • TLR stimulation inhibited osteoclast differentiation
  • osteoclast precursors treated with TLR ligands still retained high levels of mechanism for the clearance of bacterial infection. Therefore, the net outcome of TLR stimulation of osteoclast precursors is likely to enhance immune responses for bacterial clearance.
  • This enhancement of immune responses can be achieved by promoting cytokine production from precursor cells and by inhibiting their differentiation into nonphagocytic, non-immune cells such as mature osteoclasts. Since these cells can differentiate into mature osteoclasts if TLR ligands are removed, it appears that, after microbial infection is cleared, the presence of residual activated T cells can lead to the differentiation of phagocytic precursors into mature, bone-resorbing osteoclasts. In addition to promoting inflammatory responses, TNF- ⁇ can also enhance the differentiation and survival of osteoclasts and upregulate M-CSF gene expression in osteoblast/stromal cells (Kobayashi et al., 2000, J. Exp Med 191:275; Lam et al, 2000, J.
  • osteoclast precursors are extremely responsive to microbial constituents via TLRs. Interaction of these microbial products with TLRs on osteoclast precursors appears to favor the role of osteoclast precursors as part of the pro-inflammatory system by inhibiting their differentiation into mature osteoclasts and by promoting the production of inflammatory cytokines. TLRs are thus likely to regulate the balance of immune responses and bone metabolism during acute attacks of vertebrate hosts by various microbes.

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Abstract

Selon la présente invention, il s'avère que la stimulation effectuée par des récepteurs de type Toll sur des précurseurs d'ostéoclastes inhibe la différentiation des ostéoclastes. De plus, la stimulation par ces récepteurs de type Toll sur les précurseurs d'ostéoclastes soutient l'activité phagocytique des précurseurs des ostéoclastes, contribuant ainsi à la réponse immune.
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CN110882381A (zh) * 2019-12-18 2020-03-17 中国人民解放军陆军军医大学第一附属医院 重组IFN-λ1蛋白在制备防治炎症性骨丢失药物中的应用

Non-Patent Citations (4)

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ITOH ET AL.: 'Lipopolysaccharide promotes the survival of osteoclast via Toll-like receptor 4, but cytokine production of osteoclasts in response to lipopolysaccharide is different from that of macrophages' THE JOURNAL OF IMMUNOLOGY vol. 170, 2003, pages 3688 - 3695, XP002970753 *
KIKUCHI ET AL.: 'Gene expression of osteoclast differentiation factor is induced by lipopolysaccharide in mouse osteoblasts via Toll-like receptors' THE JOURNAL OF IMMUNOLOGY vol. 166, 2001, pages 3574 - 3579, XP001120607 *
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* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN110882381A (zh) * 2019-12-18 2020-03-17 中国人民解放军陆军军医大学第一附属医院 重组IFN-λ1蛋白在制备防治炎症性骨丢失药物中的应用

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