Login| Sign Up| Help| Contact|

Patent Searching and Data


Title:
USE OF LXR LIGANDS FOR THE MODULATION OF DENDRITIC CELLS (DCs)
Document Type and Number:
WIPO Patent Application WO/2006/077012
Kind Code:
A2
Abstract:
The present invention relates to the use of LXR in methods for identifying compounds which interfere with DC differentiation and/or maturation and to methods to identify LXR-mediated, DC-specific anti-inflammatory genes. Non-human mammalian animals may be used as in vivo model systems for identifying LXR binding compounds (in particular LXR agonists) inhibiting or preventing T cell activation, Th2-cytokine secretion, recruitment of inflammatory cells to the BAL fluid, and/or, peribronchial and/or perivascular infiltration of inflammatory cells. The present invention also discloses the use of an LXR agonist to prepare a medicament for the treatment of diseases or disorders wherein the inhibition or the prevention of DC differentiation and/or maturation, recruitment of inflammatory cells to the BAL fluid, Th2-cytokine secretion, and/or, peribronchial and/or perivascular infiltration of inflammatory cells is aimed at. The present invention finally relates to Dendritic Cell composition or DC precursor composition and to uses of these compositions to study the recruitment of inflammatory cells to the BAL fluid, Th2-cytokine secretion, and/or, peribronchial and/or perivascular infiltration of inflammatory cells in a model organism.

Inventors:
BELANGER CAROLE (FR)
DARTEIL RAPHAEL (FR)
HUM DEAN (FR)
Application Number:
PCT/EP2006/000043
Publication Date:
July 27, 2006
Filing Date:
January 05, 2006
Export Citation:
Click for automatic bibliography generation   Help
Assignee:
GENFIT S A (FR)
BELANGER CAROLE (FR)
DARTEIL RAPHAEL (FR)
HUM DEAN (FR)
International Classes:
G01N33/50; A61K31/00; A61P11/06
Domestic Patent References:
WO2004058819A22004-07-15
WO2003106435A12003-12-24
WO2003059884A12003-07-24
Foreign References:
US20040259948A12004-12-23
GB2381866A2003-05-14
Other References:
COLLINS ALAN R: "Pleiotropic vascular effects of PPARgamma ligands." DRUG NEWS & PERSPECTIVES. MAY 2003, vol. 16, no. 4, May 2003 (2003-05), pages 197-204, XP001206500 ISSN: 0214-0934
SHIAU A K ET AL: "ORPHAN NUCLEAR RECEPTORS: FROM NEW LIGAND DISCOVERY TECHNOLOGIES TO NOVEL SIGNALING PATHWAYS" CURRENT OPINION IN DRUG DISCOVERY AND DEVELOPMENT, CURRENT DRUGS, LONDON, GB, vol. 4, no. 5, September 2001 (2001-09), pages 575-590, XP009031659 ISSN: 1367-6733
CASTRILLO, A. ET AL: "Liver X receptor-dependent repression of matrix metalloproteinase-9 expression in macrophages." JOURNAL OF BIOLOGICAL CHEMISTRY, vol. 278, no. 12, 21 March 2003 (2003-03-21), pages 10443-10449, XP009049818 ISSN: 0021-9258
Attorney, Agent or Firm:
Becker, Philippe (25 rue Louis le Grand, Paris, FR)
Download PDF:
Claims:
CLAIMS
1. A method for identifying a compound which interferes with (inhibit or prevent) Dendritic Cell differentiation and/or maturation comprising the steps of: a) stimulating in vitro differentiation of Dendritic Cell precursors to Dendritic Cells and/or Dendritic Cell maturation, b) adding during, before or after step a), to said Dendritic Cells or Dendritic Cell precursors an LXR agonist stimulating said receptor, c) measuring the influence of said LXR agonist on the differentiation and/or maturation of said Dendritic Cell precursors or Dendritic Cells, d) optionally, repeating step a) to c) wherein, instead of LXR agonist, a reference compound known to inhibit said differentiation and/or maturation is added during, before or after step a), and measuring the influence of said reference compound on the differentiation and/or maturation of said Dendritic Cell precursors or Dendritic Cells and comparing these results with the results obtained in step c), e) identifying an LXR agonist interfering with (inhibiting or preventing) Dendritic Cell differentiation and/or maturation from the results of step c) and/or d), and, f) optionally, isolating and/or formulating the compound identified in step e).
2. The method according to claim 1 , wherein said Dendritic Cell or Dendritic Cell precursor is of myeloid, lymphoid or plasmacytoid origin.
3. The method according to claim 1 or 2, wherein said Dendritic Cell is directly isolated from (peripheral or cord) blood.
4. The method according to claim 1 or 2, wherein said Dendritic Cell precursor is a monocyte, a CD34+ hematopoietic progenitor cell or an IL3R plasmacytoid cell.
5. The method according to any of claims 1 to 4, wherein said Dendritic Cell differentiates from a monocyte.
6. The method according to any of claims 1, 2, 4 and 5, wherein said Dendritic Cell precursor is isolated from peripheral blood, cord blood, bone marrow, thymus or lymphoid tissues.
7. The method according to any of claims 1 to 6, wherein the stimulation of the differentiation of said Dendritic Cell precursors to Dendritic Cells or the maturation of said Dendritic Cells in step a) is achieved by treating with an agent chosen from the group consisting of allergens, inflammatory cytokines, CD40L, bacterial products, pathogens such as Escherichia coli, Candida, viruses or other agents.
8. The method according to any of claims 1 to 7, wherein said LXR agonist is identified using a binding assay and/or a signal transduction assay specific for said receptor.
9. The method according to any of claims 1 , 2 and 4 to 8, wherein said Dendritic Cell precursor differentiation to Dendritic Cells is measured through the analysis of cell surface expression of CD1a, CD11c, CD40, HLADR, transcription of CD40 and/or MMP9 and/or other differentiation marker.
10. The method according to any of claims 1 to 9, wherein the maturation of Dendritic Cells is measured through the analysis of the LPS (or other maturation agents) inducible expression of cell surface markers CD83, CD86, CD80, HLADR and/or through the analysis of the transcription of chemokines IP10, ELC, MCP1, RANTES, TARC, chemokine receptor CCR7 and/or other maturation agent.
11. The method according to claim 9 or 10, wherein said expression is analyzed via quantitative RTPCR analysis or by FACS analysis.
12. The method according to any of claims 1 to 11, wherein the Dendritic Cell precursor differentiation or DCs maturation is measured through the analysis of T cell immune response triggered by Dendritic Cells.
13. The method according to claim 12, wherein said T cell immune response is measured in a heterologous MLR assay thereby incubating Dendritic Cells with allogeneic T cells and measuring T cell proliferation.
14. The method according to any of claims 1 to 13, wherein the LXR agonist of step b) or the reference compound of step d) is T0901317 or GW3965, or a functional equivalent, or a combination thereof.
15. The method according to any of claims 1 to 14, wherein said method is a High Throughput Screening method.
16. A method to identify LXRmediated, Dendritic Cell specific, antiinflammatory target genes, comprising the steps of: a) stimulating in vitro the differentiation of Dendritic cell precursors to Dendritic Cells and/or the Dendritic Cell maturation, b) adding during, before or after step a) to said Dendritic Cells or Dendritic Cell precursors an LXR agonist stimulating said receptor, c) analyzing the influence (positive or negative) of said LXR agonist on the expression of secondary genes (target genes), by comparing the expression of the same genes in Dendritic Cell precursors or Dendritic Cells which were not treated by the LXR agonist, d) optionally repeating steps a) to c) wherein instead of LXR agonist, a reference compound known to induce said differentiation and/or maturation is added during, before or after step a), e) optionally, comparing the results of steps c) and d), f) identifying an LXR mediated target gene, wherein the expression of said target gene is down or upregulated upon the treatment using the LXR agonist, g) optionally, isolating a nucleic acid representing at least part of said target gene, and, h) optionally further identifying compounds having an activity on the expression of the target gene identified in step f) or on the activity of the protein encoded by said target gene.
17. A method according to claim 16, wherein said Dendritic Cell or Dendritic Cell precursor is of myeloid, lymphoid or plasmacytoid origin.
18. The method according to claim 16 or 17, wherein said Dendritic Cell is directly isolated from (peripheral or cord) blood.
19. A method according to claim 16 or 17, wherein said Dendritic Cell precursor is a monocyte, a CD34+ hematopoietic progenitor cells or an IL3R plasmacytoid cell.
20. The method according to any of claims 16 to 19, wherein said Dendritic Cell differentiates from a monocyte.
21. A method according to any of claims 16, 17, 19 and 20, wherein said Dendritic Cell precursor is isolated from peripheral blood, cord blood, bone marrow, thymus or lymphoid tissues.
22. A method according to any of claims 16 to 21, wherein the stimulation of the differentiation of said Dendritic Cell precursors to Dendritic Cells or the Dendritic Cell maturation in step a) is achieved by treating with an agent chosen from the group consisting of allergens, inflammatory cytokines, CD40L, bacterial products, pathogens such as Escherichia coli, Candida, viruses or other agent.
23. The method according to any of claims 16, 17, 19 to 22, wherein said Dendritic Cell precursor differentiation to Dendritic Cells is measured through the analysis of cell surface expression of CD1a, CD11c, CD40, HLADR, transcription of CD40 and/or MMP9 and/or other differentiation marker.
24. The method according to any of claims 16 to 23, wherein the maturation of Dendritic Cells is measured through the analysis of the LPS (or other maturation agents) inducible expression of cell surface markers CD83, CD86, CD80, HLADR and/or through the analysis of the transcription of chemokines IP10, ELC, MCP1 , RANTES, TARC, chemokine receptor CCR7 and/or other maturation agent.
25. The method according to claim 23 or 24, wherein said expression is analyzed via quantitative RTPCR analysis or by FACS analysis.
26. The method according to any of claims 16 to 25, wherein the Dendritic Cell precursor differentiation or DCs maturation is measured though the analysis of T cell immune response triggered by Dendritic Cells.
27. The method according to claim 26, wherein said T cell immune response is measured in a heterologous MLR assay thereby incubating Dendritic Cells with allogeneic T cells and measuring T cell proliferation.
28. A method according to any of claims 16 to 27, wherein said LXR agonist is identified using a binding assay and/or a signal transduction assay specific for said receptor.
29. A method according to any of claims 16 to 28, wherein said LXR agonist of step b) or said refereneecompound of step d) is T0901317 or GW3965, or a functional equivalent, or a combination thereof.
30. A method according to any of claims 16 to 29, wherein said influence of step c) is analysed through the analysis of the expression of a large battery of genes via quantitative RTPCR analysis.
31. A method according to any of claims 16 to 30, wherein said method is a High Throughput Screening method.
32. Use of a nonhuman mammalian animal as an in vivo model for identifying compounds inhibiting Th2cytokine secretion, recruitment of inflammatory cells to the BAL fluid, and/or, peribronchial and/or perivascular infiltration of inflammatory cells, comprising the steps of: a) sensitizing said animal by injecting OVA emulsified in alum thereby generating OVAspecific T cells in a nonhuman mammalian animal, b) incubating said animal for several days, c) challenging said animal with OVA aerosols, d) injecting an LXR agonist before each aerosol challenge in said animal, e) analyzing the inflammatory cells content of the BAL fluid of said animal, f) analyzing Th2cytokine secretion by T cells of said animal, g) measuring the level of Th2cytokine secretion in the BAL fluid, h) analyzing peribronchial and/or perivascular inflammatory cell infiltration in lung biopsies of said animal, i) identifying compounds as LXR agonists inhibiting Th2cytokine secretion, recruitment of inflammatory cells to the BAL fluid, and/or, peribronchial and/or perivascular infiltration of inflammatory cells, and, j) optionally, isolating and/or formulating the compound identified in step i).
33. Use of a nonhuman mammalian animal as an in vivo model for identifying compounds preventing Th2cytokine secretion, recruitment of inflammatory cells to the BAL fluid, and/or, peribronchial and/or perivascular infiltration of inflammatory cells, comprising the steps of: a) injecting an LXR agonist in a nonhuman mammalian animal, b) sensitizing said animal by injecting ovalbumine (OVA) emulsified in alum thereby generating OVAspecific T cells in said animal, c) incubating said animal for several days, d) challenging said animal with OVA aerosols, e) optionally, injecting an LXR agonist before each aerosol challenge in said animal, f) analyzing the inflammatory cells content of the BAL fluid of said animal, g) analyzing Th2cytokine secretion by T cells of said animal, h) measuring the level of Th2cytokine secretion in the BAL fluid, i) analyzing peribronchial and/or perivascular inflammatory cell infiltration in lung biopsies of said animal, j) identifying compounds as LXR agonists preventing Th2cytokine secretion, recruitment of inflammatory cells to the BAL fluid and/or peribronchial and/or perivascular infiltration of inflammatory cells, and, k) optionally, isolating and/or formulating the compound identified in step j).
34. Use of a nonhuman mammalian animal as an in vivo model for identifying compounds inhibiting T cell proliferation and/or Th2cytokine release through the analysis of the primary immune response, comprising the steps of: a) isolating OVAspecific T cells from the TCR transgenic mouse DO11.10, b) labeling the OVAspecific T cells obtained in step a) with CFSE, c) treating Dendritic Cells in vitro with an LXR agonist, d) pulsing Dendritic Cells of step c) with OVA, e) injecting the CFSElabelled T cells obtained in step b) in a nonhuman mammalian animal, f) analyzing T cell proliferation and/or Th2cytokine secretion by ex vivo OVA restimulated T cells, g) comparing T cell proliferation and/or Th2cytokine production analyzed in step f) with T cell proliferation and/or Th2cytokine production in an animal treated as described in the previous steps but wherein the Dendritic Cells were not treated with an LXR agonist, h) optionally comparing T cell proliferation and/or Th2cytokine production analyzed in step f) with T cell proliferation and/or Th2cytokine production in an animal treated as described in the previous steps but wherein the Dendritic Cells were treated with a reference compound, i) identifying compounds as LXR agonists inhibiting T cell proliferation and/or Th2 cytokine release, and, j) optionally, isolating and/or formulating the compound identified in step i).
35. Use of a nonhuman mammalian animal as an in vivo model for identifying compounds inhibiting the recruitment of inflammatory cells to the BAL fluid, Th2 cytokine secretion, and/or, peribronchial and/or perivascular infiltration of inflammatory cells through the analysis of the secondary immune response, comprising the steps of: a) treating Dendritic Cells with an LXR agonist, b) pulsing the Dendritic Cells of step a) with OVA, c) injecting the LXRtreated Dendritic Cells pulsed with OVA obtained in step b) in said non human mammalian animal, d) incubating said animal for several days, e) challenging said animal with OVA aerosols, f) analyzing inflammatory cell content of the BAL fluid of said animal, g) optionally extracting draining lymph nodes from said animal, h) optionally restimulating lymph nodes of step g) with OVA, i) optionally analyzing Th2cytokine secretion produced by the cells of step h), j) optionally measuring Th2cytokine secretion in the BAL fluid, k) optionally analyzing peribronchial and/or perivascular inflammatory cell infiltration in lung biopsies of said animal, I) comparing the cellular content of step f), Th2cytokine secretion optionally measured in step i) or j), and/or, peribronchial and/or perivascular inflammatory cell infiltration optionally measured in step k) with the cellular content, Th2cytokine secretion, and/or, peribronchial and/or perivascular inflammatory cell infiltration obtained from an animal treated as described in the previous steps but wherein the Dendritic Cells were not stimulated with the LXR agonist, m) optionally comparing the cellular content of step f), Th2cytokine secretion optionally measured in step i) or j), and/or, peribronchial and/or perivascular inflammatory cell infiltration optionally measured in step k) with the cellular content, Th2cytokine secretion, and/or, peribronchial and/or perivascular inflammatory cell infiltration obtained from an animal treated as described in the previous steps but wherein the Dendritic Cells were stimulated with a reference compound, n) identifying the LXR agonist as a compound inhibiting recruitment of inflammatory cells to the BAL fluid, Th2cytokine secretion, and/or, peribronchial and/or perivascular infiltration of inflammatory cells, if said LXR agonist reduces said cellular content, reduces Th2cytokine production as determined in step i) or j) and/or reduces peribronchial and/or perivascular inflammatory cell infiltration of step k) and, o) optionally isolating and/or formulating the compound identified in step n).
36. Use according to any of claims 32 to 35, wherein said animal is a mouse or a rat.
37. Use according to any of claims 34 to 36, wherein said Dendritic Cell is of myeloid, lymphoid or plasmacytoid origin.
38. Use according to any of claims 34 to 37, wherein said Dendritic Cell is directly isolated from (peripheral or cord) blood.
39. Use according to any of claims 32 to 38, wherein said LXR agonist or said reference compound is T0901317 or GW3965, or a functional equivalent, or a combination thereof.
40. Use according to any of claims 32 to 39, wherein said animal is used as a model for identifying compounds interfering with (inhibiting or preventing) asthma, more particularly allergyinduced asthma.
41. Use of an LXR agonist to interfere in vitro with (inhibit or prevent) Dendritic Cell precursor differentiation and/or Dendritic Cell maturation.
42. Use of an LXR agonist identified according to a method of claim 1 to 31 or identified by means of the use of a nonhuman mammalian animal according to any of claims 32 to 40 for the preparation of a medicament for the treatment of a disease associated with the recruitment of inflammatory cells to the BAL fluid.
43. Use of an LXR agonist identified according to a method of claim 1 to 31 or identified by means of the use of a nonhuman mammalian animal according to any of claims 32 to 40 for the preparation of a medicament for the treatment of a disease associated with increased Th2cytokine release.
44. Use of an LXR agonist identified according to a method of claim 1 to 31 or identified by means of the use of a nonhuman mammalian animal according to any of claims 32 to 40 for the preparation of a medicament for the treatment of a disease associated with peribronchial and/or perivascular infiltration of inflammatory cells.
45. An isolated Dendritic Cell composition or an isolated Dendritic Cell precursor composition comprising Dendritic Cells or Dendritic Cell precursors thereof which have been treated in vitro with an LXR agonist interfering with (inhibiting or preventing) Dendritic Cell precursor differentiation and/or Dendritic Cell maturation.
46. The Dendritic Cell composition or the Dendritic Cell precursor composition according to claim 45, wherein the stimulation of the differentiation of said Dendritic Cell precursors to Dendritic Cells or the Dendritic Cell maturation is achieved by treating with an agent chosen from the group consisting of allergens, inflammatory cytokines, CD40L, bacterial products, pathogens such as Escherichia coli, Candida, viruses or other agent.
47. The Dendritic Cell composition or Dendritic Cell precursor composition according to claim 45 or 46, wherein Dendritic Cell or Dendritic Cell precursor is of myeloid, lymphoid or plasmacytoid origin.
48. The Dendritic Cell composition according to any of claims 45 to 47, wherein said Dendritic Cell is directly isolated from (peripheral or cord) blood.
49. The Dendritic Cell precursor composition according to claim 45 or 46, wherein said Dendritic Cell precursor is a monocyte, a CD34+ hematopoietic progenitor cell or an IL 3R plasmacytoid cell.
50. The Dendritic Cell composition or Dendritic Cell precursor composition according to any of claims 45 to 49, wherein said Dendritic Cell differentiates from a monocyte.
51. The Dendritic Cell precursor composition according to any of claims 45, 46, 47, 49 and 50, wherein said Dendritic Cell precursor is isolated from peripheral blood, cord blood, bone marrow, thymus, or lymphoid tissues.
52. The Dendritic Cell composition or Dendritic Cell precursor composition according to any of claims 45 to 51 , wherein said LXR agonist is T0901317 or GW3965, or a functional equivalent, or a combination thereof.
53. Use of a Dendritic Cell composition or a Dendritic Cell precursor composition according to any of claims 45 to 52 to study the recruitment of inflammatory cells to the BAL fluid in a model organism.
54. Use of a Dendritic Cell composition or a Dendritic Cell precursor composition according to any of claims 45 to 52 to study the Th2cytokine release in the BAL fluid and/or by lymph node cells in a model organism.
55. Use of a Dendritic Cell composition or a Dendritic Cell precursor composition according to any of claims 45 to 52 to study the peribronchial and/or perivascular infiltration of inflammatory cells in a model organism.
56. Use of a Dendritic Cell composition or a Dendritic Cell precursor composition according to any of claims 45 to 52 to study asthma, more preferably allergyinduced asthma.
57. Use according to any of claims 42 to 44, wherein said disease is asthma, more preferably allergyinduced asthma.
58. Use according to any of claims 41 to 44, wherein said LXR agonist is T0901317 or GW3965, or a functional equivalent, or a combination thereof.
Description:
Use of LXR ligands for the modulation of Dendritic Cells (DCs)

LXR-ligands, in particular LXR agonists, may be used to modulate in vitro and in vivo Dendritic Cell (DC) differentiation and/or maturation, and/or, asthmatic responses. Said responses may include the recruitment of inflammatory cells to the BAL (Broncho Alveolar Lavage) fluid, Th2-cytokine secretion, and/or, peribronchial and/or perivascular infiltration of inflammatory cells.

Technical field

The present invention relates to the use of LXR in methods for identifying compounds, in particular LXR agonists, which interfere (inhibit or prevent) with DC differentiation and/or maturation.

The present invention also relates to methods to identify LXR-mediated, DC-specific anti-inflammatory genes.

In addition, according to the present invention, non-human mammalian animals may be used as in vivo model systems for identifying LXR binding compounds (in particular LXR agonists) inhibiting or preventing Th2-cytokine secretion, recruitment of inflammatory cells to the BAL fluid, and/or, peribronchial and/or perivascular infiltration of inflammatory cells.

The present invention also relates to the use of a non-human mammalian animals as an in vivo model for identifying compounds inhibiting T cell proliferation and/or Th2-cytokine release through the analysis of primary immune response or for identifying compounds inhibiting the recruitment of inflammatory cells to the BAL fluid, Th2-cytokine secretion, and/or, peribronchial and/or perivascular infiltration of inflammatory cells through the analysis of secondary immune response.

The present invention discloses the use of an LXR agonist to prepare a medicament for the treatment of diseases or disorders wherein the inhibition or the prevention of DC differentiation and/or maturation is aimed at or for the treatment of diseases associated with the recruitment of inflammatory cells to the BAL fluid, with Th2-cytokine secretion, and/or, with peribronchial and/or perivascular infiltration of inflammatory cells. Said medicament may preferably be used to treat asthma, in particular allergy-induced asthma.

The present invention also relates to Dendritic Cell compositions or DC precursor compositions and to uses of these compositions to study the recruitment of inflammatory cells to the BAL fluid, Th2-cytokine secretion, and/or, peribronchial and/or perivascular infiltration of inflammatory cells in a model organism. In particular said model organism may be used to study asthma.

Background art

A major aim of the present invention is to find new agents to treat airway inflammation and/or asthma, in particular, allergy-induced asthma. The hallmarks of allergic asthma are infiltration of eosinophils into the bronchial wall and lumen, elevated serum IgE levels, mucus production in the airway and airway hyperresponsiveness. These processes involve a wide range of inflammatory cells (such as eosinophils, mast cells, T cells, CD4+ T cells, macrophages, and Dendritic Cells) and inflammatory mediators and enzymes (such as cysteinyl-leukotrienes, histamine, prostaglandins, kinins, IL-4, IL-5, IL-13, adenosine A1 receptor, mast cell tryptase and phosphodiesterase).

Current therapies for allergic asthma are aimed at relieving bronchoconstriction (beta agonists) or inflammation. Not all compounds with antiinflammatory or immunosuppressive activity are effective to treat airway inflammation and/or asthma. For instance the anti-inflammatory compounds nedocromil, cromolyn and theophylline are not as effective as inhaled corticosteroids to reduce the asthmatic response. On the other hand, strong immunosuppressive drugs such as cyclosporin are not used to treat asthma because the patient would be to sensitive to bacterial or virus infection. In addition, anti-inflammatory drugs that reduce a Th 1 -type of immune response (for instance in Th 1 -mediated autoimmune disease) will not be the best compounds to treat a Th2-mediated disease like asthma.

Corticosteroids are commonly used as anti-inflammatory agents in asthma. However, the long-term use of corticosteroids can produce serious side effects such as glaucoma, cataracts, osteoporosis, growth retardation in children and adolescents and decreased functioning of the adrenal gland.

These important side effects caused by corticosteroids stress the importance of further improving said therapy by finding new agents and new target molecules specific for said diseases. New asthma targets include leukotriene antagonists (however, not as effective as corticosteroids), monoclonal antibodies that specifically recognize IgE, cytokine or cytokine receptor modulators such as soluble IL- 4 receptor, chemokine or chemokine receptor modulators, inhibitors of PDE4, adenosine receptors, adhesion molecules, Th2-specific transcription factors and genes involved in airway remodeling.

LXRs, also named Liver X Receptors, were first identified as orphan receptors of the nuclear receptor superfamily. Two LXR proteins (α and β) are known to exist in mammals. The expression of LXRα is restricted, with highest levels in the liver (hence, the name Liver X Receptor) and lower but significant levels in kidney, intestine, spleen and adrenals. LXRβ expression is ubiquitous and has been found in

nearly every tissue examined. Later on, LXR was shown to be activated by a specific class of naturally occurring, oxidized derivatives of cholesterol, including 22(R)- hydroxylcholesterol, 24(S)-hydroxycholesterol and 24(S),25(S)-epoxycholesterol. These oxysterols are concentrated in tissues where cholesterol metabolism and LXR expression are high, such as liver, brain and placenta.

The compounds T0901317 (Schultz et al. (2000) Genes&Dev. Nov 15; 14(22): 2831-8) and GW3965 (Collins et al. (2002) J.Med.Chem. May 9; 45(10):1963- 6) were shown to be LXR selective agonists.

Further in vitro and in vivo evidences illustrated that LXR function is an essential regulatory component of cholesterol homeostasis. It was suggested that LXR agonists may be used as protective agents against atherosclerosis due to their coordinated effects on cholesterol synthesis, dietary cholesterol absorption, reverse cholesterol transport and bile acid synthesis and excretion.

Recently, WO 03/059884, WO 03/106435, Fowler et al. (2003), Joseph et al. (2003) and Castrillo et al. (2003) indicated a role of LXR ligands in keratinocyte- and macrophage-inflammatory responses.

WO 03/059884 relates to heterocyclic compounds, compositions and methods for modulating the activity of nuclear receptors, including LXR. In said application an extensive listing is given for the use of said LXR binding compounds (for instance on p.16, 1.7-21). One of said uses is the treatment of inflammation. However, said application only provides a hypothetical example of an in vivo study to test the effect of said compounds on plasma cholesterol and triglycerides levels (example 74, p.345- 346). In said application, no examples were given, nor were suggestions made, whether agonists or antagonists of said nuclear receptors are needed to treat inflammation.

WO 03/106435 relates to new compounds modulating LXR function suggesting their role as anti-sclerotic and anti-inflammatory agent. Also in this application no focus is set on a specific use of said compounds. A broad list is given of diseases which may be treated using the compounds of the invention such as aterosclerosis (and atherosclerosis), lipid-related diseases, inflammatory diseases, cardiovascular diseases, renal diseases, diabetes, cancer and Alhzheimer's disease (for instance on p.8, 1.24 to p.9, 1.1). The term "inflammatory diseases" is broadly defined as "diseases mediated by inflammatory cytokines" (I.32-33 of said paragraph). To test the antiinflammatory activity of the compounds of said invention, an irritant contact dermatitis model and an allergic contact dermatitis model has been hypothetically suggested (test example 4, p.431-432). However, no suggestion has been made whether LXR agonists or LXR antagonists exert this anti-inflammatory effect.

Further, LXR activators were found to stimulate epidermal differentiation and

development, improve permeability barrier homeostasis and inhibit epidermal proliferation. Keratinocytes generate the primary cytokines TNF-α and IL-1 in response to a variety of forms of cutaneous injury.

Fowler et al. (2003) (J. Invest. Dermatol. 120:246-255) demonstrated that compounds that activate LXR (such as 22(R)-hydroxycholesterol, 25- hydroxycholesterol or the non-sterol activator GW3965) reduce inflammation in animal models of irritant and allergic contact dermatitis by a receptor-mediated process (via both LXRα and LXRβ).

Joseph et al. (2003) (Nature Med. 9:213-219) demonstrated that LXRs and their ligands are negative regulators of macrophage inflammatory gene expression (iNOS,

COX-2, IL-6, IL-1β, G-CSF, MCP-1 , MCP-3, MIP-1β, IP-10 and MMP-9). They showed that LXR agonists reduce inflammation in a model of contact dermatitis and inhibit inflammatory gene expression in aortas of atherosclerotic mice. They identified LXR as lipid dependent regulators of inflammatory gene expression that may serve to link lipid metabolism and immune function in macrophages.

Matrix MetalloProteinases (MMPs) are enzymes that degrade extracellular matrix components during normal and pathogenic tissue remodelling. Inappropriate expression of MMPs contributes to the development of vascular pathology, including atherosclerosis. Castrillo et al. (2003) (J.Biol.Chem. 278:10443-10449) demonstrated that LXR agonists GW3965 or T0901317 specifically inhibit MMP-9 expression in macrophages at least in part through antagonism of the NFKB signalling pathway. They suggested the impact of both LXRs, LXRα and LXRβ, on macrophage inflammatory responses.

Summary of the invention

The present invention relies on the unexpected finding that LXRs are present on Dendritic Cells (DCs) and exert an unexpected function in said cells. According to the present invention, said receptors and the DCs carrying said receptors may be used to develop methods, assays and kits to screen compounds, in particular LXR binding compounds, which interfere with (inhibit or prevents) Dendritic Cell differentiation and/or maturation.

The present invention also points to the importance of the identification of LXR- mediated, DC specific anti-inflammatory target genes. Methods for said modulation and identification are described below. One of the aims of the present invention is to find new compounds which may be used to prepare medicaments for the treatment of diseases or disorders wherein a decrease of DCs activity/functionality, and/or, a decrease of recruitment of

inflammatory cells to the BAL fluid, Th2-cytokine secretion, and/or, peribronchial and/or perivascular infiltration of inflammatory cells is aspired.

In addition, the decrease in DC activity or functionality may be the result from a decrease of the differentiation and/or maturation of DC-precursors into fully mature and functional DCs.

In the present invention, the disease, which is in particular aimed at to be treated, is airway inflammation and/or asthma, i.e. allergy-induced asthma.

According to the present invention, non-human mammalian animals may be used as in vivo model systems for identifying LXR binding compounds (in particular LXR agonists) inhibiting or preventing Th2-cytokine secretion, recruitment of inflammatory cells to the BAL fluid, and/or, peribronchial and/or perivascular infiltration of inflammatory cells.

The present invention also relates to the use of a non-human mammalian animals as an in vivo model for identifying compounds inhibiting T cell proliferation and/or Th2-cytokine release through the analysis of primary immune response or for identifying compounds inhibiting the recruitment of inflammatory cells to the BAL fluid, Th2-cytokine secretion, and/or, peribronchial and/or perivascular infiltration of inflammatory cells through the analysis of secondary immune response.

The present invention discloses the use of an LXR agonist to prepare a medicament for the treatment of diseases or disorders wherein the inhibition or the prevention of DC differentiation and/or maturation is aimed at and for the treatment of diseases associated with the recruitment of inflammatory cells to the BAL fluid, with

Th2-cytokine secretion and/or with peribronchial and/or perivascular infiltration of inflammatory cells. Said medicament may be used preferably to treat asthma, in particular allergy-induced asthma.

The present invention also relates to Dendritic Cell compositions or DC precursor compositions wherein said DCs or precursors thereof have been treated in vitro with an LXR ligand and to uses of these compositions to study the recruitment of inflammatory cells to the BAL fluid, Th2-cytokine secretion, and/or, peribronchial and/or perivascular infiltration of inflammatory cells, preferably to study asthma.

The present invention also relates to methods to modulate DCs in vitro.

Dendritic Cells are highly specialized antigen-presenting cells of the immune system. In particular, Dendritic Cells are the only antigen-presenting cells that can activate naϊve T cell and are therefore critical for the initiation of primary immune responses. Moreover, several studies have reported increased numbers of Dendritic Cells in the airways of allergic asthmatic subjects, suggesting that Dendritic Cells play a key role in asthma pathogenesis.

With the term "Th2-cytokine" is meant a cytokine produced typical for the Th2 response; examples of Th2-type cytokines are IL-4, IL-5, IL-6, IL-9, IL10 and IL-13. Predominant cytokines produced in a Th1-type response are IFN-γ, TNF-α, IL-2 or lymphotoxin (LT). As pointed out above, the prior art indicates a role of LXR ligands in lipid metabolism and in keratinocyte and macrophage-inflammatory responses. However, as keratinocytes, macrophages and Dendritic Cells show discrete expression profiles; said cells are clearly different. The fact that macrophages and Dendritic Cells are distinct is confirmed by the fact that the present inventors found that the LXR target genes are not exactly the same in both cell types. For instance, the present inventors found that cell-surface expression of CD 1a, which is not expressed in macrophages but is specific to Dendritic Cells, is highly reduced by LXR ligand. Moreover, the present invention shows that the genes CCR7 and ELC are regulated by LXR ligands. Such finding was not reported in studies with macrophages. The regulation of CCR7 and ELC in DCs indicates that LXR ligand can interfere with DC migration to lymphoid organs (see below). Therefore, based on the prior art a skilled person would not derive that LXR is present on DCs and would be able to interfere with DC functions.

Furthermore, prior to the present invention, there existed some evidence, suggested in WO 99/24400, that the decrease in LXR activation would be in favour of asthma treatment. Indeed, said patent application relates to novel derivatives of probucol for inhibiting oxidation and its subsequent damage that result or accompany respiratory disorders (such as asthma) (for instance on p.30, 1.1-24). According to said document, the probucol ester of the invention inhibits oxysterol synthesis (for instance on p.7, 1.26-29). From this prior art, a skilled person in the art would thus derive that inhibiting oxysterol synthesis (thus decreasing the activity of the LXR) may positively influence the treatment of asthma. Said application thus leads a skilled person in the art away from the present invention which uses an LXR-agonist (stimulating the LXR) for the treatment of asthma as disclosed herein.

Detailed description of the invention

In a first aspect, the present invention relates to a method for identifying a compound which interferes with (inhibits or prevents) Dendritic Cell (DC) differentiation and/or maturation comprising the steps of: . a) stimulating in vitro differentiation of Dendritic Cell precursors to Dendritic Cells and/or Dendritic Cell maturation, b) adding during, before or after step a), to said Dendritic Cells or Dendritic Cell precursors an LXR agonist stimulating said receptor,

c) measuring the influence of said LXR agonist on the differentiation and/or maturation of said Dendritic Cell precursors or Dendritic Cells, d) optionally, repeating step a) to c) wherein, instead of LXR agonist, a reference compound known to inhibit said differentiation and/or maturation is added during, before or after step a), and measuring the influence of said reference compound on the differentiation and/or maturation of said Dendritic Cell precursors or Dendritic Cells and comparing these results with the results obtained in step c), e) identifying an LXR agonist interfering with (inhibiting or preventing) Dendritic Cell differentiation and/or maturation from the results of step c) and/or d), and, f) optionally, isolating and/or formulating the compound identified in step e).

With the term "LXR", also named "Liver X Receptor" or "Lipid X Receptor", is meant the receptor as represented by the GenBank Accession No. NM_005693 (human LXRα) or by GenBank Accession No. NM_007121 (human LXRβ) . However, according to the present invention, said LXR may have an origin other than human such as the receptors represented by NM_013839 (murine LXRα), NM_009473 (murine LXRβ), NM_031627 (rat LXRα), NM_031626 (rat LXRβ) and NM_204542 (chicken LXRα). In addition, said receptor may be a variant thereof, carrying similar binding and/or signaling properties compared to the wild type receptor.

The cells used in the method of the present invention carrying said receptors may thus be of different origin such as human, monkey, mouse, rat, hamster, etc.

Dendritic Cell precursors (such as monocytes, CD34+ hematopoietic progenitor cells or IL-3R plasmacytoid cells - see below) can be differentiated in vitro into immature Dendritic Cells, which may be further matured by exposure to inflammatory mediators (the type of inflammatory agents that can be used will be further discussed in details). It is known that each of said stages in which said cells may reside is complex and comprises of different sub-stages. For Dendritic Cells, all of these sub-stages have not been identified yet. However, it is now well established that immature Dendritic Cells express high levels of CD 1a (key marker of Dendritic Cells) and have a high capacity to capture and process antigens while mature Dendritic Cells show a reduced expression of CD1a, have a reduced phagocytic activity, but express high levels of co- stimulatory molecules such as CD80 and CD86 and have a high capacity to stimulate T cells. Thus, through measuring the presence, the absence or the precise level of certain cell surface molecules or intracellular marker molecules, one may have an indication at which stage a Dendritic Cell approximately resides. The compound, which may interfere with the Dendritic Cell differentiation and/or maturation through the LXR, may be of any origin. For instance, said compound may be a small organic molecule from libraries of chemical compounds or collections of such molecules. Alternatively, said compound may be present in samples or extracts

from natural sources, e.g., plant, fungal or bacterial extracts or even in human tissue samples.

According to the present invention, all LXR agonists may interfere with DC differentiation and maturation; however the degree of interference (0% to 100%) depends on the property of the agonist. Indeed, according to the present invention, the activity of the LXR agonist may be divided in two major activities: a transactivation and a transrepression activity. Both activities may be full, partial or negligible.

Transactivation activity means the possibility to activate promoters bearing LXR response elements (LXREs), via the binding of liganded-LXR and coactivators at said elements.

Transrepression activity means the possibility to inhibit or interfere with the transactivation function of certain transcription factors (known examples are interference with NF-κB or AP-1 transcription factors). Preferably, LXR agonists of the present invention carry a dominant transrepression activity over their transactivation activity. According to the present invention, an agonist with a higher transrepression activity compared to its transactivation activity will probably result in a better interference of DC differentiation and/or maturation.

Importantly, an LXR ligand with weaker transactivation capacity will not lead to the undesirable effect linked to transactivation of SREBP-Ic which promotes hypertriglyceridemia [Repa (2000) Genes&Dev. 14:2819-2830; Yoshikawa (2001) MoI. Cell. Biol. 21:2991-3000].

According to the present invention, said LXR agonist carry preferably dominant (i.e. equal or more than 70%) transrepression activity (99, 95, 90, 85, 80, 75 or 70%), more preferably full (100%) transrepression activity, and no or negligible/minor (i.e. equal or less than 30%) transactivation activity (0, 1 , 5, 10, 20, 25 or 30%). However, the LXR agonist may also carry 70% to 30% of trans-repression activity (70, 65, 60, 55, 50, 45, 40, 35 or 30%) and 30% to 70% of transactivation activity (30, 35, 40, 45, 50, 55, 60, 65 or 70%).

The present invention also points to the fact that the choice of an LXR agonist, to achieve transactivation or transrepression, may not only be defined by the property of the ligand itself, but it may also be influenced by the environment created in the cell.

Preferably, the identification of a compound with improved properties compared to T0901317 or GW3965 is aimed at in the present invention. Therefore, T0901317, GW3965 or a functional equivalent (i.e. a molecule that can bind specifically to the Ligand Binding Domain (LBD) of LXR and transactivates or transrepresses specific target genes) or a combination thereof may be used as reference compound used in step d) of the above-described method.

The present inventors have found that the LXR agonists T0901317 and GW3965 share similar properties in terms of interfering with DC function in vitro and in trans-activating promoters containing LXR response elements.

From the prior art, many compounds have been identified which were found to bind LXR and modulate its activity. Examples of such agents are described in for instance, but are not limited to WO 00/66611 , WO 03/076418, WO 2004/009091, WO

03/060078, WO 03/106435, WO 03/059884 and US 6,645,955. All agents may easily be tested in a method according to the present invention.

The present invention points to the fact that the dominance of the transrepression activity of the LXR is only preferable, but not essential. Therefore, T0901317 or GW3965 (or a functional equivalent, or a combination thereof) may also be the LXR agonist of step b) of the above mentioned method.

Thus, preferably, the LXR agonist of step b) or the reference compound of step d) according to the present invention is T0901317 or GW3965 or a functional equivalent or a combination thereof.

The method described above may thus be considered as a general screening method to identify and possibly isolate and/or formulate interesting LXR agonists carrying DC modulatory properties. The development of specific screening assays to look for dominant trans-repression LXR agents is discussed below. According to the present invention, Dendritic Cells or Dendritic Cell precursors of which the differentiation and/or maturation is studied may be of myeloid, lymphoid or plasmacytoid origin.

Early in hematopoiesis, a pluripotent stem cell differentiates along one or two pathways, giving rise to either a lymphoid stem cell or a myeloid stem cell. The types and amounts of growth factors present in the microenvironment in which a particular stem cell resides control its differentiation. Both types of lymphoid and myeloid progenitor cells may generate Dendritric Cells (respectively of lymphoid or myeloid origin). The lymphoid progenitor or precursor, which is the terminology used for mice, is referred to as a plasmacytoid progenitor or precursor in human. According to the present invention, differentiated DCs can be directly isolated from blood using BDCA-1 or BDCA-3 (myeloid specific) or BDCA-2 and BDCA-4 (plasmacytoid specific) antibodies [Dzionek (2000) J.Immunol. 165:6037-6046]. Therefore, Dendritic Cells used in the method of the present invention may be directly isolated from (peripheral or cord) blood. Human DCs can differentiate in vitro from CD34+ hematopoietic progenitor cells of the cord blood, the bone marrow, peripheral blood [Caux (1992) Nature 360:258-61; Szabolcs (1995) J. Immunol. 154:5851-5861; Strunk (1996) Blood 87:1292-1302] or the thymus, from isolated monocytes [Palucka (1998) J.Immunol. 160:4587-4595]

(preferred method that will be described below) or from IL-3R plasmacytoid cells from the blood or lymphoid tissues [Banchereau (2000) J.Exp.Med. 192:39F-44). Therefore, the Dendritic Cell precursor used in the method of the present invention may be isolated from peripheral blood, cord blood, bone marrow, thymus or lymphoid tissues. The organs of the lymph system throughout the body, including the bone marrow, thymus, lymph nodes, spleen, tonsils, Peyer's patches and the lymphocyte aggregates on mucosal surfaces. As pointed out above, the Dendritic Cell precursor used in the method of the present invention may be a monocyte, a CD34+ hematopoietic progenitor cell or an IL-3 plasmacytoid cell. Preferably, said DCs differentiate from monocytes.

Murine myeloid DCs may commonly be obtained from bone marrow progenitor cells. Methods to isolate Dendritic Cells or Dendritic Cell precursors are known by a skilled person in the art. Differentiated murine bone marrow-derived DCs may be further treated with ovalbumine (OVA) before intratracheal injection in mice (see below). When using human DCs, said OVA treatment is not essential.

Monocyte-derived DCs are most commonly used to assess DC phenotype and function. A general protocol includes a first isolation of Peripheral Blood Mononuclear Cells (PBMCs) on Ficoll Gradient followed by a purification of monocytes by adherence [Bender (1996) J.lmmunol.Meth. 196:121-35] or by using CD14-antibody coated beads (Miltenyi Biotec) [Pickl (1996) J.Immunol. 157:3850-3859]. The purified monocytes are then cultured in presence of GM-CSF and IL-4 for a period of 5 to 7 days to generate immature Dendritic Cells.

In the method of the present invention, the stimulation of immature Dendritic Cells and the stimulation of the differentiation of Dendritic Cell precursors to Dendritic Cells in step a) may be achieved by treating with allergens (ex. der p 1 , der p 2, bet Via, ovalbumine, etc.), inflammatory cytokines (ex. TNFα, IL-1β, etc.), CD40L (also called CD154, is a co-stimulatory membrane protein belonging to the TNF superfamily), bacterial products (ex. LPS or LipoPolySaccharide), pathogens such as Escherichia coli, Candida, viruses (ex. Influenza, measles and dengue viruses, HIV, etc.) or other agents known in the art. The term "der p 1 or der p 2" means the Dermatophagoides pteronyssinus allergen. Said different agents may be purified or not.

The present invention further explains that said Dendritic Cell precursor differentiation to Dendritic Cells may be measured through the analysis of cell surface expression of CD1a, CD11c, CD40, HLA-DR, transcription of CD40 and/or MMP9 and/or other differentiation markers known in the art.

Maturation of Dendritic Cells may be measured through the analysis of LPS- inducible (or other maturation agents described above) expression of cell surface markers CD83, CD86, CD80, HLA-DR and/or through the analysis of the transcription

of chemokines IP-10, ELC (EBV-induced molecule 1 Ligand Chemokine), TARC (Thymus and Activation Regulated Chemokine), MCP-1 (Monocyte Chemoattractant Protein-1 ), RANTES (Regulated upon Activation, Normally T-Expressed and presumably Secreted), chemokine receptor CCR7 and/or other maturation markers known in the art.

The expression of said specific marker genes for DC maturation or DC precursor differentiation may be analyzed via quantitative RT-PCR analysis or by FACS (Fluorescence-Activated Cell Sorting) analysis.

The expression of internal control genes, such as GADPH, may also be studied in parallel to correct for experimental deviations. Changes of at least 10 %, including 15%, 20% and more, preferably 30%, in expression of said gene(s) in the presence of said agent compared to the expression level in said DCs in the absence of said agent, or compared to said expression level in said DCs in the presence of a reference compound are interpreted as an indication for the interference of said agent on DC precursor differentiation and/or DC maturation.

Alternatively, DC precursor differentiation or DC maturation may be measured through any method known in the art for the analysis of T cell immune response triggered by Dendritic cells.

The ability of DCs to induce proliferation of allogeneic T cells in a primary MLR (Mixed Leukocyte Reaction) is commonly used for their functional evaluation [Steinman (2003) APMIS 11 1 :675-97]. It is now well established that terminally matured Dendritic Cells are the most potent stimulators of T cells. In the Mixed Leukocyte reaction assay, the polymorphic regions of donor MHC molecules are the stimulus that initiate the immune response. Alternatively, antigen-specific T cell response assays have also been reported using for example tetanus toxoid as a model antigen [Narendran (2002) Ann. N.Y.Acad. Sci. 958:170-174]. The present invention particularly suggests to measure said T cell immune response in a heterologous MLR (Mixed Leukocyte Reaction) assay thereby incubating Dendritic Cells from one donor with allogeneic naϊve CD4+ T cells isolated from a second donor and measuring T cell proliferation, preferably by BrdU (Bromodeoxyuridine) incorporation and alternatively by tritiated thymidine incorporation [Messele (2000) Clin.Diagn.Lab.lmmunol. 7: 687-692]. When in the method of the present invention, the T cell immune response is measured, this is preferentially performed in a heterologous MLR assay thereby incubating Dendritic Cells with allogeneic T cells and measuring T cell proliferation. The screening methods provided by the present invention may be amenable to high throughput assays. When using a High-Throughput Screening assay, a microarray system may be used. An agent that modulates the function of LXR is a molecule or a compound that increases or decreases LXR activity, including compounds that change

the binding of reference compounds and compounds that change LXR downstream signaling activities, i.e through the modulation of the interaction of LXR with secondary molecules. For example, the ability to reconstitute LXR/LXR-ligand binding either in vitro, on cultured cells or in vivo provides a tool for the identification of agents that disrupt said binding. Assays based on disruption of said binding can identify agents, such as small organic molecules, from libraries or collections of such molecules. Alternatively, such assays can identify agents in samples or extracts from natural sources, e.g., plant, fungal or bacterial extracts or even in human tissue samples. In one aspect, the extracts can be made from a library of chemical compounds, including, for example, T0901317 or GW3965, variants thereof, or other LXR ligands known in the prior art and derivatives or a combination thereof.

Standard physiological, pharmacological and biochemical procedures are available for testing and identifying compounds that can modulate the activity of nuclear receptors, including LXR. Such assays include, for example, biochemical assays such as binding assays, fluorescence polarization assays, FRET-based coactivator recruitment assays (for a general description see Glickman et al. (2002) J. Biomol. Screen. 7(1):3-10), as well as cell-based assays including co-transfection assays of LXR expression plasmids (with or without LXR coactivators) with luciferase reporter plasmids bearing LXR response elements (LXREs), or one-hybrid GAL4 assay to identify compounds that can activate a reporter plasmid containing GAL4 response element through binding of a GAL4-LXR (LBD or Ligand Binding Domain) chimera, and protein-protein interaction assays, such as the mammalian two-hybrid assay (Lehmann et al. (1997) J. Biol.Chem. 272:3137-3140).

High Throughput Screening systems are commercially available (see, e.g. Zymark Corp., Hopkinton, MA; Air Technical Industries, Mentor, OH; Beckman Instruments Inc., Fullerton, CA; Precision Systems Inc., Natick, MA) which enable these assays to be run in a high throughput mode. These systems automate the entire procedure, including all sample and reagent pipetting, liquid dispensing timed incubations and final readings of the microplate in detector(s) appropriate for the assay. These configurable systems provide high throughput and rapid start up as well as a high degree of flexibility and customization. The manufacturers of such systems provide detailed protocols for various high throughput systems. Thus, for example, Zymark Corp. provides technical bulletins describing screening systems for detecting the modulation of gene transcription, ligand binding, and the like. Assays that do not require washing or liquid separation steps are preferred for such High Throughput Screening systems and include biochemical assays such as fluorescence polarization assays (see for example, Owicki (2000) J. Biomol. Screen. 5(5):297), Scintillation Proximity Assays (SPA) (see, for example, Carpenter et al.

(2002) Meth.Mol. Biol. 190:31-49) and Fluorescence Resonance Energy Transfer (FRET) or time resolved FRET based coactivator recruitment assays (Mukherjee et al. 2002. J.Steroid Biochem.Mol.Biol. 81(3): 217-25 ; Zhou et al. (1998) Mol.Endocrinol. 12:1594-604). Such assays are generally performed using either the full length receptor or isolated Ligand Binding Domain (LBD) (to facilitate the purification procedure and the proteins can be tagged for example with histidine residues or GST (Glutathione-S-transferase)). In the case of LXR, said Ligand Binding Domains may comprise amino acids 264 to 447 (LXRq) or 278 to 461 (LXRβ) of the full length sequence. If a fluorescently labeled ligand is available, fluorescence polarization assays provide a way of detecting binding of compounds to the nuclear receptor of interest by measuring changes in fluorescence polarization that occurs as a result of the displacement of a trace amount of the label ligand by the compound. Additionally this approach can also be used to monitor the ligand dependent association of a fluorescently labeled coactivator peptide to the nuclear receptor of interest to detect ligand binding to the nuclear receptor of interest.

As found for other non-steroid hormone receptors, LXR functions as a heterodimer with the Retinoid X Receptor (RXR) to regulate gene expression [Willy (1997) Genes&Dev. 11:289-298; Lehmann (1997) J. Biol.Chem. 272:3137-3140]. The LXR/RXR heterodimer can be activated by ligands of both RXR and LXR, either separately or in synergy [Janowski (1996) Nature 383:728-31]. It is worth noting that LXRα was also shown to function as a monomer on cAMP-responsive transcriptional regulation of a number of genes, including renin and c-myc [Tamura (2000) PNAS 97:8513-8518; Anderson (2003) J.Biol.Chem. 278: 15252-15260]. A number of screening assays to identify compounds that interact with LXRα, LXRβ, or the heterodimeric forms have been described in the literature and some of these assays will be discussed below.

The binding of a potential ligand to the LBD domain of LXR can be measured in a Scintillation Proximity Assay (SPA) by assessing the degree to which a compound can compete off a radiolabeled ligand with known affinity for the receptor. Such assay was used to determine that naturally occuring oxysterols can bind directly to LXR at concentrations that occur in vivo [Janowski (1999) PNAS 96:266-271]. In this approach, the radioactivity emitted by a radiolabeled compound generates an optical signal when it is brought into close proximity to a scintillant such as a Ysi-copper containing bead, to which the nuclear receptor is bound. If the radiolabeled compound is displaced from the nuclear receptor, the amount of light emitted from the nuclear receptor bound scintillant decreases and this can be readily detected using standard

microplate liquid scintillation plate readers such as, for example, a Wallac MicroBeta reader.

The ability of compounds to bind to LXRα or LXRβ can also be measured by Fluorescence Resonance Energy Transfer (FRET) or time resolved FRET [Zhou (1998) MoI. Endocrinol. 12:1594-604]. Of particular interest will be to determine if a specific coactivator is recruited by LXR in transrepression versus transactivation mechanisms. Interestingly, it was recently reported that the two synthetic LXR ligands T0901317 and GW3965 showed some differences in coactivator recruitment [Miao (2004) J. Lipid Res. 45:1410-1417]. Since the present invention aims for the identification of LXR ligands with predominant transrepression activity, it will be of particular importance to select a FRET assay with coactivators that have more affinity with LXR ligands with good transrepression activity (ex. GW3965). Several LXR coactivators have been described such as SRC-1 , RIP140, TRAP220, SRC-3/ACTR [Kaneko (2003) J.Biol.Chem. 278:36091-36098], Asc-2 [Kim (2003) Mol.Cell.Biol. 23:3583-92], PGC-1 [Oberkofler (2004) Biochem.J. 381:357-63], GRIP-1 [Svensson (2003) EMBO J. 22:4625-4633]. LXR was also shown to interact with the co-repressors N-COR and SMRT [Hu (2003) Mol.Endocrinol. 17:1019-1026; Wagner (2003) Mol.Cell.Biol. 23:5780-5789]. Moreover, the LXR-corepressor interaction is isoform-specific, wherein LXRα has a very strong interaction while LXRβ only shows weak interaction [Hu (2003) MoI. Endocrinol. 17:1019-1026].

This assay can be exploited to measure the ligand dependent interaction of a secondary molecule with LXR in order to characterize the agonist or antagonist activity of compounds. For example the assay in this case involves the use a recombinant Glutathione-S-transferase (GST)-nuclear receptor Ligand Binding Domain (LBD) fusion protein and a synthetic biotinylated peptide sequenced derived from the receptor interacting domain of a secondary molecule. For example GST-LBD is labeled with a europium chelate (donor) via a europium-tagged anti-GST antibody, and the secondary molecule is labeled with allophycocyanin via a streptavidin-biotin linkage. The fluorescently labeled GST-LXR (LBD) and fluorescently labeled coactivator are mixed together and allowed to equilibrate for at least 1 hour prior to addition to either variable or constant concentrations of the sample for which the affinity is to be determined. After equilibration, the time-resolved fluorescent signal is quantitated using a fluorescent plate reader. The affinity of the compound can then be estimated from a plot of fluorescence versus concentration of compound added. In the presence of an agonist for LXR, the peptide is recruited to the GST-LBD bringing europium and allophycocyanin into close proximity to enable energy transfer from the europium chelate to the allophycocyanin. Upon excitation of the complex with light at 340 nm excitation energy absorbed by the europium chelate is transmitted to

the allophycocyanin moiety resulting in emission at 665 nm. If the europium chelate is not brought in to close proximity to the allophycocyanin moiety, there is little or no energy transfer and excitation of the europium chelate results in emission at 615 nm. Thus the intensity of light emitted at 665 nm gives an indication of the strength of the protein-protein interaction. The activity of a nuclear receptor antagonist can be measured by determining the ability of a compound to competitively inhibit (ie., IC50) the activity of an agonist for the nuclear receptor.

In addition, a variety of cell-based assay methodologies may be successfully used in screening assays to identify and profile the specificity of compounds of the present invention. These approaches include the co-transfection assay, translocation assays, complementation assays and the use of gene activation technologies to overexpress endogenous LXR.

Four basic variants of the co-transfection assay strategy exist, co-transfection assays using full-length LXR, co-transfection assays using chimeric LXR comprising the Ligand Binding Domain (LBD) of the nuclear receptor of interest fused to a heterologous DNA Binding Domain (DBD), assays based around the use of the mammalian two hybrid assay system and assays based around the use of the mammalian one hybrid assay system.

The basic co-transfection assay is based on the co-transfection into a selected cell type of an LXR expression plasmid together with a reporter plasmid comprising a marker gene whose expression is under the control of DNA sequence that is capable of interacting with LXR. It is now well established that LXR/RXR heterodimer binds to response elements termed LXREs, that are characterized by two direct hexameric repeats separated by four nucleotides (DR4 element) [Willy (1995) Genes&Dev. 9:1033-1045].

Treatment of the transfected cells with an agonist for LXR increases the transcriptional activity of that receptor which is reflected by an increase in expression of the reporter gene, which may be measured by a variety of standard procedures. As LXR functions as a heterodimer with RXR 1 the co-transfection assay can include the use of expression plasmids for both LXR and RXR, or functional binding domains thereof. Co-transfection assays require access to the full length LXR and suitable response elements that provide sufficient screening sensitivity and specificity to LXR. Genes encoding the following full-length proteins, which are suitable for use in the co- transfection studies and profiling the compounds described herein, include human RXRα (GenBank Accession No. NM_002957), human RXRβ (GenBank Accession No. XM_042579), human RXRy (GenBank Accession No. XM_053680), human LXRα (GenBank Accession No. NM_005693), human LXRβ (GenBank Accession No. NM_007121). Reporter plasmids may be constructed using standard molecular

biological techniques by placing nucleic acids (cDNA) encoding for the reporter gene downstream from a suitable minimal promoter. For example luciferase reporter plasmids may be constructed by placing cDNA encoding firefly luciferase immediately down stream from the herpes virus thymidine kinase promoter (located at nucleotides residues-105 to +51 of the thymidine kinase nucleotide sequence) which is linked in turn to the LXR response elements. Numerous methods of co-transfecting the expression and reporter plasmids are known to those of skill in the art and may be used for the co-transfection assay to introduce the plasmids into a suitable cell type. Ideally, the selected cell type will not endogenously express nuclear receptors that interact with the response elements used in the reporter plasmid. This will not be possible for LXRβ, which is ubiquitously expressed in all cell types. In contrast, LXRα is expressed in a more restricted manner. The presence of LXR coactivators, which play a key role in transactivation processes, is also taken into account for the selection of a cell line. Numerous reporter gene systems are known in the art and include, for example, alkaline phosphatase (see Berger J. et al. (1988) Gene 66:1-10; Kain, S. R. (1997) Methods. MoI. Biol 63:49-60), β-galactosidase (See, U. S. Patent No. 5,070, 012, issued Dec, 3,1991 to Nolan et al., and Bronstein I. et al., (1989) J.Chemilum.Biolum. 4:99-111), chloramphenicol acetyltransferase (See Gorman et al. (1982) Mol.Cell. Biol. 2:1044-51), β-glucuronidase, peroxidase, β-lactamase (U. S. Patent Nos. 5,741, 657 and 5,955, 604), catalytic antibodies, luciferases (U. S. Patents 5,221, 623; 5,683, 888; 5,674, 713; 5,650, 289; 5,843, 746) and naturally fluorescent proteins (Tsien, R. Y. (1998) Annu.Rev.Biochem. 67:509-44).

The use of chimeras comprising the Ligand Binding Domain (LBD) of LXR to a heterologous DNA Binding Domain (DBD) expands the versatility of cell-based assays by directing activation of LXR to defined DNA binding elements recognized by defined DNA Binding Domain (see WO 95/18380). This assay expands the utility of cell-based co-transfection assays in cases where the biological response or screening window using the native DNA Binding Domain is not satisfactory. In general the methodology is similar to that used with the basic co-transfection assay, except that a chimeric construct is used in place of the full length LXR. As with the full length LXR, treatment of the transfected cells with an LXR agonist increases the transcriptional activity of the heterologous DNA Binding Domain which is reflected by an increase in expression of the reporter gene as described above. For example, for such chimeric constructs, the DNA Binding Domains from defined nuclear receptors, or from yeast or bacterially derived transcriptional regulators such as members of the GAL 4 and Lex A /Umud super families are used.

A third cell-based assay of utility for screening LXR agonists is a mammalian two-hybrid assay that measures the ability of the LXR to interact with a cofactor in the presence of a ligand. The basic approach is to create three plasmid constructs that enable the interaction of the nuclear receptor with the interacting protein to be coupled to a transcriptional readout within a living cell. The first construct is an expression plasmid encoding a fusion protein comprising the interacting protein, or a portion of that protein containing the interacting domain, fused to a GAL4 DNA Binding Domain. The second expression plasmid comprises DNA encoding the LXR fused to a strong transcription activation domain such as VP16, and the third construct comprises the reporter plasmid comprising a reporter gene with a minimal promoter and GAL4 upstream activating sequences. Once all three plasmids are introduced into a cell, the GAL4 DNA Dinding Domain encoded in the first construct allows for specific binding of the fusion protein to GAL4 sites upstream of a minimal promoter. However because the GAL4 DNA Binding Domain typically has no strong transcriptional activation properties in isolation, expression of the reporter gene occurs only at a low level. In the presence of a ligand, the LXR-VP16 fusion protein can bind to the GAL4-interacting protein fusion protein bringing the strong transcriptional activator VP16 in close proximity to the GAL4 binding sites and minimal promoter region of the reporter gene. This interaction significantly enhances the transcription of the reporter gene, which can be measured for various reporter genes as described above. Transcription of the reporter gene is thus driven by the interaction of the interacting protein and nuclear receptor of interest in a ligand dependent fashion.

A fourth, and preferred, cell-based assay for screening LXR agonists is the mammalian one hybrid assay that measures the activity of LXR. This assay can be used to screen for agonists or antagonists. For this assay, an expression vector containing LBD (Ligand Binding Domain) of LXR fused to the DBD (DNA Binding Domain) of GAL4 may be generated. As a reporter plasmid, multiple copies of the GAL4 response element are inserted upstream of a minimal TK promoter linked to a reporter gene. These two constructs are transfected together in a relevant cell line expressing (known) LXR secondary molecules (co-activators or co-repressors described above). The measure of the luciferase activity (for example) is correlated with the amount of ligand added.

Any compound which is a candidate for activation of LXR may be tested by these methods. Generally, compounds are tested at several different concentrations to optimize the chances that activation of the receptor will be detected and recognized if present. As well, dose-response curves can be made to measure and compare the relative affinity of a given ligand and the maximal effect produced by each ligand.

Typically assays are performed in triplicate and vary within experimental error by less than 15%. Each experiment can be repeated three or more times with similar results.

Activity of the reporter gene can be conveniently normalized to the internal control and the data plotted as fold activation relative to untreated cells. A positive control compound (agonist) can be included alone with any aqueous or organic solvent

(DMSO (DiMethyl Sulfoxyde) being a preferred solvent) as high and low controls for the normalization of the assay data. Similarly, antagonist activity can be measured by determining the ability of a compound to competitively inhibit the activity of an agonist.

In the method of the present invention, said LXR agonist can be identified using a binding assay and/or a signal transduction assay specific for said receptor. A preferable example of a binding assay used in the method of the present invention is a one-hybrid GAL4 assay. Alternatively, or subsequently to the binding assay described above, a signal transduction assay specific for said receptor can be set up in order to identify the LXR agonist or to confirm if an LXR binding compound is an LXR agonist. According to the invention said signal transduction assay is preferentially secondary, following the above-proposed binding assay. An example of such a signal transduction assay may be a FRET assay.

Additionally, the LXR agonist, or compositions thereof, can be evaluated for their ability to increase or decrease the expression of target genes modulated by LXR in vivo (see below). This can be studied using Northern-blot, RT-PCR or oligonucleotide microarray analysis to measure RNA levels. Western-blot analysis can be used to measure expression of proteins encoded by LXR target genes. Genes that are known to be activated by LXR include ABCA1 [Costet (2000) J.Biol.Chem. 275:28240-28245], ABCG1 , ABCG5, ABCG-8 [Repa (2002) J.Biol.Chem. 277:18793- 18800], FAS [Joseph (2002) J.Biol.Chem. 277:11019-11025 ], ApoE [Laffitte (2001) PNAS 98:507-512 ], PLTP [Laffitte (2003) MoI. Cell. Biol. 23:2182-2191 ], CETP [Luo (2000) J.Clin. Invest. 105:513-520], CYP7A, PPARy [Seo (2004) Mol.Cell.Biol. 24:3430- 3444], Apo AIV [Liang (2004) Mol.Endocrinol. 18:2000-2010], Apo D [Hummasti (2004) J. Lipid Res. 45:616-625], SREBP-Ic, etc. In macrophages, LXR ligands were shown to inhibit the expression of inflammatory mediators such as inducible Nitric Oxide Synthases (NOS), CycloOXygenase 2 (COX-2), IL-6, and MMP9 in response to LPS (Lipo Poly Saccharide) stimulation [Joseph (2003) Nat.Med. 9:213-9 ; Castrillo (2003) J.Biol. Chem. 278:10443-10449].

The inventors of the present invention show that LXR ligands inhibit expression of CCR7, ELC, IP-10, CD83 and CD86.

According to the present invention, ECL and CCR7 have a crucial role in the migration of DCs to lymphoid organs, said genes are considered as important marker genes to measure the maturation of DCs.

A glucocorticoid receptor (GR) ligand with good trans-repressing activity has been recently reported [Coghlan (2003) Mol.Endocinol. 17:860-869]. This compound (AL-438) confers a full anti-inflammatory activity comparable to other steroids but has a reduced transactivation effect on genes involved on bone and glucose metabolism. Therefore, this compound may serve as a prototype to treat inflammatory diseases without having the detrimental side effects often associated with transactivation processes. Such compound with a dissociated transrepressing activity has not been described yet for LXR.

Changes of at least 15% or more, preferably 30%, in DC precursor differentiation and/or DCs maturation through the presence of said agent compared to the DC precursor differentiation and/or DCs maturation in the absence of said agent or compared to said DC precursor differentiation and/or DC maturation in the presence of a reference compound may identify an LXR agonist interfering with the DC precursor differentiation and/or DCs maturation through LXR. According to the present invention, changes in said differentiation and/or maturation may also go from 30% to 40, 50, 60, 70, 80, 90, up to 100%. The reference condition can be set as 100.

Many methods exist to isolate said agent from an extract or a (simple or complex) mixture of other compounds and may be performed without any difficulties by a skilled person in the art. In a second embodiment, the present invention elaborates on a method to identify LXR mediated, Dendritic Cell specific, anti-inflammatory target genes. For instance, according to the present invention, said method may comprise the steps of: a) stimulating in vitro the differentiation of Dendritic Cell precursor to Dendritic Cells and/or the Dendritic Cell maturation, b) adding during, before or after step a) to said Dendritic Cells or Dendritic Cell precursors an LXR agonist stimulating said receptor, c) analyzing the influence (positive or negative) of said LXR agonist on the expression of secondary genes (target genes), by comparing the expression of the same genes in Dendritic Cell precursors or Dendritic Cells which were not treated by the LXR agonist, d) optionally repeating steps a) to c) wherein, instead of LXR agonist, a reference compound known to induce said differentiation and/or maturation is added during, before or after step a), e) optionally, comparing the results of steps c) and d), f) identifying an LXR mediated target gene, wherein the expression of said target gene is down- or up-regulated upon the treatment using the LXR agonist, g) optionally, isolating a nucleic acid representing at least part of said target gene, and,

h) optionally further identifying compounds having an activity on the expression of the target gene identified in step f) or on the activity of the protein encoded by said target gene.

The effect of LXR ligands on cholesterol and fatty acid metabolism occurs via a transactivation mechanism on promoters bearing LXR response elements (LXREs). In contrast, the present invention suggests that the mechanism by which LXR ligand conferring anti-inflammatory activity occurs through a transrepression mechanism.

Therefore the present invention suggests that, for the treatment of diseases or disorders influenced by Dendritic Cell (DC) differentiation and/or maturation, the recruitment of inflammatory cells to the BAL fluid, Th2-cytokine secretion, and/or, peribronchial and/or perivascular infiltration of inflammatory cells, LXR ligands may be used preferably, that show better trans-repression than trans-activation capacities.

The present invention also relates to the treatment of airway inflammation and/or asthma. The ligands T0901317 and GW3965 are good trans-activators, however, a higher dose is needed for the trans-repression. Thus, a specific trans- repression screening assay needs to be developed to find molecules with a high trans- repressive activity. Once the target genes are identified whose expression is modulated by the activation of LXR, a further selection can be made for genes which are DC- specific and carry said trans-repression activity. Transcription regulator regions of said genes can be used as basis to develop specific screening assays (see above).

The present invention indicates that genes which are down-regulated by LXR include CD1a, CDHc 1 HLA-DR, ELC, CCR7, MMP-9, CD40 and CD80.

As previously mentioned, cell surface markers CD83, CD86, CD80, HLA-DR, chemokines ELC, IP-10, TARC, MCP-1, RANTES, chemokine receptor CCR7 (and other maturation agents known in the art) can be considered as possible primary marker genes to measure the maturation of DCs. Emphasis may be given to genes that have a key role on DC migration and antigen presentation function.

As mentioned for the previously method of the present invention, in the method to identify LXR mediated, DC specific, anti-inflammatory target genes, Dendritic Cell or Dendritic Cell precursor studied can be of myeloid, lymphoid or plasmacytoid origin.

As pointed out above, differentiated DCs can be directly isolated from blood, using BDCA-1 or BDCA-3 (myeloid specific) or BDCA-2 and BDCA-4 (plasmacytoid specific) antibodies [Dzionek (2000) J.Immunol. 165:6037-6046]. Therefore, Dendritic

Cells used in the methods of the present invention may be directly isolated from (peripheral or cord) blood.

Dendritic Cell precursors used can be a monocyte, a CD34+ hematopoietic progenitor cells of the cord blood, the adult bone marrow, peripheral blood or the thymus or an IL-3R plasmacytoid cells from blood or lymphoid tissues. Therefore,

Dendritic Cell precursor used in the method of the present invention may be a monocyte, a CD34+ hematopoietic progenitor or an IL-3R plasmacytoid cell and may be isolated from peripheral blood, cord blood, bone marrow, thymus or lymphoid tissues. Preferably, said Denditic Cell differentiates from a monocyte. In addition, the stimulation of DC precursors differentiation to Dendritic Cells or

DC maturation in step a) can be achieved by treating with an agent chosen from the group consisting of allergens (ex. der p 1 , der p 2, bet Via, ovalbumine, etc.), inflammatory cytokines (ex. TNFα, IL-1β, etc.), CD40L (also called CD154, is a co- stimulatory membrane protein belonging to the TNF superfamily), bacterial products (ex. LPS or Lipo Poly Saccharide), pathogens such as Escherichia coli, Candida, viruses (ex. Influenza, measles and dengue viruses, HIV, etc.) and other agents known in the art. Said different agents may be purified or not.

As mentioned for previous method, in this method the Dendritic Cell precursor differentiation to Dendritic Cells may be measured through the analysis of cell surface expression of CD1a, CD11c, CD40, HLA-DR, transcription of CD40 and/or MMP9 and/or other differentiation marker. Maturation of Dendritic Cells may be measured through the analysis of the LPS (or other maturation agents) inducible expression of cell surface markers CD83, CD86, CD80, HLA-DR and/or through the analysis of the transcription of chemokines IP-10, ELC, MCP-1 , RANTES, TARC, chemokine receptor CCR7 and/or other maturation agent. Both expressions may be analyzed via quantitative RT-PCR analysis or by FACS analysis. Alternatively, the function of Dendritic Cells may be measured though their capacity to trigger a T cell immune response. The Dendritic Cell precursor differentiation or DCs maturation can be measured though the analysis of T cell immune response triggered by Dendritic Cells. Said T cell immune response triggered by Dendritic Cells may be measured in a heterologous MLR assay thereby incubating Dendritic Cells with allogeneic T cells and measuring T cell proliferation.

According to the method of the present invention, said LXR agonist can be identified using a binding assay and/or a signal transduction assay specific for said receptor, said LXR agonist of step b) or said reference compound of step d) can be T0901317 or GW3965, or a functional equivalent, or a combination thereof and said influence of step c) can be analysed through the analysis of the expression of a large battery of genes via quantitative RT-PCR analysis.

In addition, said method may be a High Throughput Screening method. Said High Throughput Screening method can be set up using a microarray system.

Alternative approaches or suggestions, as indicated for the method for identifying an LXR agonist which interferes with (inhibits or prevents) DC precursor differentiation and/or DC maturation through LXR according to the present invention,

can be applied for the method to identify LXR mediated, Dendritic Cell specific, antiinflammatory target genes according to the present invention.

In a third embodiment, the present invention elaborates on the use of a non- human mammalian animal as an in vivo model for identifying compounds inhibiting Th2-cytokine secretion, recruitment of inflammatory cells such as macrophages, lymphocytes, neutrophils and/or eosinophils to the BAL fluid, and/or, peribronchial and/or perivascular infiltration of inflammatory cells comprising the steps of: a) sensitizing said animal by injecting OVA (Ovalbumine) emulsified in alum thereby generating OVA-specific T cells in a non-human mammalian animal, b) incubating said animal for several days, c) challenging said animal with OVA aerosols, d) injecting an LXR agonist (intranasally or other route of administration) before each aerosol challenge in said animal, e) analyzing the inflammatory cell (macrophage, lymphocyte, neutrophil, eosinophil, etc.) content of the BAL fluid of said animal, f) analyzing Th2-cytokine secretion by T cells of said animal, g) measuring the level of Th2-cytokine secretion in the BAL fluid, h) analyzing peribronchial and/or perivascular inflammatory cell infiltration in lung biopsies of said animal, i) identifying compounds as LXR agonists inhibiting Th2-cytokine secretion, recruitment of inflammatory cells to the BAL fluid, and/or, peribronchial and/or perivascular infiltration of inflammatory cells, and, j) optionally, isolating and/or formulating the compound identified in step i).

Preferably, OVA is emulsified in alum with 10 to 100 μg of OVA and a 2% alum suspension. The emulsion is given intraperitoneally and one or several sensitizations can be performed : for example, a first sensitization at day 0 and a second sensitization between day 4 and day 7.

According to the present invention, the OVA aerosol challenge may be performed two weeks after the final OVA-Alum injection. For instance, for the challenge, mice are exposed daily for 30 min for three consecutive days to nebulized

OVA (1% in PBS (Phosphate Buffered Saline)) in an exposure chamber connected to the outlet of an ultrasonic nebulizer that delivers an aerosol of particles. Twenty-four hours after the last aerosol exposure, mice may be sacrificed by intraperitoneal injection of pentobarbital sodium (60 mg/kg body weight) followed by exsanguination from the iliac vessels. The trachea is then surgically exposed and cannulated, and the bronchoalveolar lavage is performed as known by a skilled person in the art. The cells of the BAL fluid are then centrifuged, washed and counted on cytospin preparations.

The cellular composition of BAL fluid is an indicator of airway inflammation. In the OVA-

sensitized/OVA-exposed positive control group, there should be a significantly higher number of neutrophils, eosinophils, monocytes/macrophages and T lymphocytes compared to the negative control group OVA-sensitized/PBS aerosol.

As pointed out below, the development of asthma and airway inflammation is influenced by inflammatory cells, in particular by two major types of cells: eosinophilic and neutrophilic cells. Some types of said diseases or disorders are more influenced by eosinophil infiltration; others are more influenced by neutrophil infiltration, and others by a combination thereof. Several studies involving the analysis of BAL fluid from adult asthmatic subjects revealed an increased total cell number and an increased eosinophils and lymphocytes number, CD4+ T lymphocytes being the predominant T cell subtype. In contrast, other studies with infants or children with asthma showed a predominant increased of neutrophils in the BAL fluid [Le Bourgeois (2002) Chest 122:791-797]. Other types of said diseases or disorders may be influenced by other type of inflammatory cells. For example, it was found that CD8+ T lymphocytes predominate in subjects with Chronic Obstructive Pulmonary Disease (COPD) [Ho (2002) Chest 121:1421-1426]. Moreover, the presence of macrophages in the BAL fluid constitutes another marker of inflammation but is less characteristic for asthma.

Consequently, through the observation of the influence of the LXR agonist on the recruitment of inflammatory cells (such as neutrophils, lymphocytes, macrophages, etc.), in particular on the recruitment of eosinophils, a skilled person in the art may get a hint which diseases or disorders may be treated efficiently using a medicament comprising said specific LXR agonist.

Examples of Th2-cytokines which can be studied in the method of the present invention, are IL4, IL-5, IL-6, IL-9, 11-10 and IL-13. Mostly, the secretion of IL-4 and IL-5 is studied by a skilled person in the art to analyze the activation of the Th2 response. In the same step of the use of the present invention, the secretion of IFN-γ, TNF-a, IL-2 or lymphotoxin can be studied as marker to analyze the Th 1 -response.

The type of T cell response is often studied by the analysis of cytokines produced by lymph node T cell restimulated in vitro with specific antigens such as OVA. These studies are usually performed with draining lymph nodes such as mediastinal lymph node cell suspensions, which contains naϊve and effector T cell of CD4 and CD8 subtype.

The analysis of peribronchial and/or perivascular inflammatory cell infiltration in lung biopsies can be performed as mentioned: at 24 hours after the last aerosol, lung biopsies are fixed, paraffin embedded, cut into four-micrometer sections and stained with hematoxylin and eosin for histological analysis. A skilled person in the art can identify peribronchial and/or perivascular eosinophil-rich infiltrates and airway mucosal changes typical of goblet cell hyperplasia.

The LXR agonist used according to the present invention may be any compound which stimulates LXR activity. As mentioned above, said agonist may be

T0901317 or GW3965, or a functional equivalent or combination thereof, or any compound which was described or found to modulate LXR or a novel discovered LXR agonist. In addition, said LXR agonist, applied according to the present invention, may be a single compound or may be present in a composition. Said composition may be a pharmacological composition to be tested for DCs differentiation/maturation, for recruitment of inflammatory cells to the BAL fluid, for Th2-cytokine secretion, and/or, for peribronchial and/or perivascular infiltration of inflammatory cells. In addition, said composition can be tested to analyze its effect on airway inflammation and/or asthmatic responses. The above-described use of the present invention may be considered as secondary and/or tertiary screenings assays to evaluate LXR ligands for their efficiency to have an effect on the above-mentioned physiological processes. Said compound or composition may have the same properties as the compound or composition to be used in human or animal patients.

LXR agonists, to be tested for their DC differentiation/maturation modulating activity, for their modulating activity of recruitment of inflammatory cells to the BAL fluid, for their Th2-cytokine secretion modulating activity, and/or, for their modulating activity of peribronchial and/or perivascular infiltration of inflammatory cells, may be formulated and administered orally, or parentally through intravenous, subcutaneous, intramuscular, percutaneous, intranasal or intrarectal route or the like, or by inhalation. Preferably, said agonists are administered by nebulization or intranasally.

Dosage forms for oral administration include tablets, pills, powders, granules, liquids, suspensions, syrups, capsules, etc. The tablets may be formulated according to a conventional process by using additives consisting of an excipient such as lactose, starch, calcium carbonate, crystalline cellulose or silicic acid; a binder such as carboxymethylcellulose, methylcellulose, calcium phosphate or polyvinylpyrrolidone; a disintegrator such as sodium alginate, sodium bicarbonate, sodium laurylsulfate or stearic acid monoglyceride; a humectant such as glycerin; an absorbent such as kaolin or colloidal silica; a lubricant such as talc or granular boric acid, etc.

The pills, powders and granules may be prepared by conventional processes also using additives similar to those mentioned above.

Liquid preparations such as the liquids, suspensions and syrups can be formulated also according to conventional processes. As a carrier, for example, a glycerol ester such as tricaprylin, triacetin or an iodized poppy oil fatty acid ester; water; an alcohol such as ethanol; or an oily base such as liquid paraffin, coconut oil, soybean oil, sesame oil or corn oil is used.

The capsules are formulated by filling a powdery, granular or liquid pharmaceutical composition, or the like, in gelatin capsules, or the like.

Dosage forms for intravenous, subcutaneous and intramuscular administration include injections in the forms of sterilized aqueous solutions, non-aqueous solutions, etc. In an aqueous solution, for example, a physiological saline solution or the like is used as a solvent. In a non-aqueous solution, for example, propylene glycol, polyethylene glycol, a vegetable oil such as olive oil, an organic ester which is acceptable for injection such as ethyl oleate or an iodized poppy oil fatty acid ester, or the like is used as a solvent. To the pharmaceutical preparations for injection are optionally added an isotonizing agent, a preservative, a humectant agent, an emulsifier, a dispersant, a stabilizer, etc., and the preparation may be sterilized by applying an adequate treatment such as filtration through a bacterium-retaining filter, blending of a germicide or irradiation. Also, the preparation may be prepared as an aseptic solid preparation which is used by dissolving in sterilized water or a sterilized solvent for injection just prior to use.

Further, the LXR agonist may be used in the form of a clathrate compound prepared by using α, β or γ-cyclodextrin, a methylated cyclodextrin, or the like. The compound may be used also as an injection of lipoid form.

Dosage forms for percutaneous administration preparations include ointments, creams, lotions, solutions, etc.

Examples of the base of an ointment include a fatty acid such as castor oil, olive oil, sesame oil or safflower oil; lanolin; white, yellow or hydrophilic vaseline; wax; a higher alcohol such as oleyl alcohol, isostearyl alcohol, octyldodecanol or hexyldecanol; a glycol such as glycerin, diglycerin, ethylene glycol, propylene glycol, sorbitol or 1 ,3-butanediol; etc. Further, as a solubilizing agent for a compound of the present invention, ethanol, dimethyl sulfoxide polyethylene glycol, etc. may be compounded. Optionally, a preservative such as a paraoxybenzoic acid ester, sodium benzoate, salicylic acid, sorbic acid or boric acid; an antioxidant such as butylhydroxyanisole or dibutylhydroxytoluene; etc. may be added. Further, in order to stimulate percutaneous absorption in an ointment, an absorption promoter such as diisopropyl adipate, diethyl sebacate, ethyl caproate or ethyl laurate may be compounded. Also, for stabilization, the LXR agonist may be used in the form of a clathrate compound prepared by using α, β or γ-cyclodextrin, a methylated cyclodextrin, etc. An ointment can be prepared by a conventional process. In the creams, dosage forms of oil-in-water type are preferable with the aim of stabilizing the LXR agonist. Further, the above-mentioned fatty oil, higher alcohol, glycol, or the like may be used as the base of a cream, and diethylene glycol, propylene glycol, sorbitan mono fatty acid ester, polysorbate 80, sodium laurylsulfate,

or the like may be used as the emulsifier of a cream. Further, the above-mentioned preservative, antioxidant, or the like may be added, as necessary. Furthermore, as in the case of ointment, the LXR agonist can be used in the form of a clathrate compound prepared by using a cyclodextrin or a methylcyclodextrin. A cream can be prepared according to a conventional process.

Examples of the lotions include a suspension-type lotion, an emulsion-type lotion and a solution-type lotion. The suspension-type lotion may be prepared by using a suspending agent such as sodium alginate, traganth or sodium carboxymethylcellulose, and optionally by adding an antioxidant, a preservative, etc. The emulsion-type lotion may be prepared according to a conventional process by using an emulsifier such as sorbitan mono fatty acid ester, polysorbate 80 or sodium laurylsulfate. The LXR agonist can be dissolved in an alcohol such as ethanol, and optionally an antioxidant, a preservative, or the like is added.

Besides the above-mentioned dosage forms, pastas, poultices, aerosols, etc., may be cited. Pharmaceutical preparations having these dosage forms can be prepared according to conventional processes.

Pharmaceutical LXR agonist preparations for intranasal administration are supplied in the form of a liquid or powdery composition. As the base of the liquid preparation, water, saline, a phosphate buffer solution, an acetate buffer solution, or the like is used, and the liquid preparation may contain further a surfactant, an antioxidant, a stabilizer, a preservative and/or a thickener. As the base for the powdery preparation, a water-absorbent base is preferable. Examples of the water-absorbent base include polyacrylate salts such as sodium polyacrylate, potassium polyacrylate and ammonium polyacrylate; cellulose lower-alkyl ethers such as methylcellulose, hydroxyethylcellulose, hydroxypropyl-cellulose and sodium carboxymethylcellulose; and polyethylene glycol, polyvinyl pyrrolidone, amylose, pullulan, etc., which are easily soluble in water. Further, they include cellulose compounds such as crystalline cellulose, [alpha]-cellulose and cross-linked sodium carboxymethylcellulose; starch compounds such as hydroxypropyl starch, carboxymethyl starch, cross-linked starches, amylose, amylopectin and pectin; proteins such as gelatin, casein and sodium caseinate; gums such as gum arabic, tragacanth gum and glucomannan; and polyvinylpolypyrrolidone, cross-linked polyacrylic acid and salts thereof, cross-linked polyvinyl alcohols, etc., which are scarcely soluble in water. These compounds may be used alone or in mixtures of two or more thereof. The powdery preparation may be further compounded with an antioxidant, a coloring agent, a preservative, a disinfectant, an antiseptic, etc. These liquid and powdery preparations can be applied, for example, by using a spraying device, etc.

For intrarectal administration, ordinary suppositories such as gelatin soft capsule are used.

Further, for inhalation, a powdery or liquid composition prepared by using an

LXR agonist alone or in combination with, an adequate biocompatible vehicle can be administered to disease sites using an applicator such as a spraying device, a nebulizer or an atomizer. Alternatively, an active ingredient comprising an LXR agonist may be administered by using a pMDI (volumetric sprayer) in which a suspension or solution prepared by suspending or dissolving the active ingredient in a spraying agent for aerosol such as alternative flon is filled. Furthermore, the LXR agonist may be dissolved in an ethanol aqueous solution, the solution is filled in an adequate sprayer.

A pharmaceutically effective dose of the active LXR agonist tested depends on administration route, age and sex of the patient and the conditions of the disease, but it is ordinarily about 0.001-100 μg per day, preferably about 0.01-50 μg per day, and administration frequency is ordinarily 1-3 time per day. The LXR agonist is preferably prepared, so as to meet these conditions.

When the LXR agonists are administered as a composition, and not as a simple one-agent solution, as normally would be administered to a patient, a skilled person may also evaluate possible side-effects of said agents on the metabolic conditions in said model animals. The effect of an LXR agonist on the modulation of physiological processes such as airway eosinophilic inflammation and asthma is evaluated through the comparison of the in vivo changes related to recruitment of inflammatory cells such as macrophages, lymphocytes, neutrophils and/or eosinophils to the BAL fluid, related to Th2-cytokine secretion, and/or, related to peribronchial and/or perivascularinfiltration of inflammatory cells.

For example, inflammatory cells content, Th2-cytokine secretion, and/or, peribronchial and/or perivascular infiltration of inflammatory cells in a model wherein an LXR agonist is used are compared with inflammatory cells content, Th2-cytokine secretion, and/or, peribronchial and/or perivascular infiltration of inflammatory cells of model wherein the animal is treated identically with a reference compound or wherein the animal is untreated or wherein an animal received a carrier molecule or a vehicle. To measure the Th2 response, the secretion of IL-4, IL-5, IL-6, IL-9, IL-10 and IL-13 is studied. IFN-γ, TNF-α, IL-2 or lymphotoxiη secretion is mostly studied to test in parallel the effect of the LXR agonists on the Th1- response. The above-mentioned method describes the possibility to study the inhibition of recruitment of inflammatory cells such as macrophages, lymphocytes, neutrophils and/or eosinophils to the BAL fluid, the inhibition of Th2-cytokine secretion, and/or, the inhibition of peribronchial and/or perivascular infiltration of inflammatory cells.

According to the present invention, said model animals may also be used to study the prevention of said recruitment of inflammatory cells to the BAL fluid, the prevention of Th2-cytokine secretion, and/or, the prevention of peribronchial and/or perivascular infiltration of inflammatory cells. For instance, the present invention suggests the use of a non-human mammalian animal as an in vivo model for identifying compounds preventing Th2- cytokine secretion, recruitment of inflammatory cells such as macrophages, lymphocytes, neutrophils and/or eosinophils to the BAL fluid, and/or, peribronchial and/or perivascular infiltration of inflammatory cells, comprising the steps of: a) injecting an LXR agonist (intranasally or other route of administration) in a non human mammalian animal, b) sensitizing said animal by injecting ovalbumine (OVA) emulsified in alum thereby generating OVA-specific T cells in said animal, c) incubating said animal for several days, d) challenging said animal with OVA aerosols, e) optionally, injecting an LXR agonist (intranasally or other route of administration) before each aerosol challenge in said animal, f) analyzing the inflammatory cell (macrophage, lymphocyte, neutrophil, eosinophil, etc.) content of the BAL fluid of said animal, g) analyzing Th2-cytokine secretion by T cells of said animal, h) measuring the level of Th2-cytokine secretion in the BAL fluid, i) analyzing peribronchial and/or perivascular inflammatory cell infiltration in lung biopsies of said animal, j) identifying compounds as LXR agonists preventing Th2-cytokine secretion, recruitment of inflammatory cells such as macrophages, lymphocytes, neutrophils and/or eosinophils to the BAL fluid, and/or, peribronchial and/or perivascular infiltration of inflammatory cells, and, k) optionally, isolating and/or formulating the compound identified in step j).

For this use, the same comments apply as mentioned above for the previous use of non-human mammalian animals.

Alternatively, a non-human mammalian animal may be used as an in vivo model for identifying compounds inhibiting T cell proliferation and/or Th2-cytokine release through the analysis of the primary immune response. Th2-cytokines may for instance be released from lymph node cells. Said use may comprise the steps of: a) isolating OVA-specific T cells from the TCR (T Cell Receptor) transgenic mouse

DO11.10, b) labeling the OVA-specific T cells obtained in step a) with Carboxy Fluorescein diacetate Succinimidyl Ester (CFSE),

c) treating Dendritic Cells in vitro with an LXR agonist, d) pulsing Dendritic Cells of step c) with ovalbumine (OVA); CD40, MCH-classll, CD80 and/or CD86 markers may hereby be induced, e) injecting the CFSE-labelled T cells obtained in step b) in a non-human mammalian animal, preferably 2 days before injecting the Dendritic Cells obtained in step d), f) analyzing T cell proliferation and/or Th2-cytokine secretion by ex vivo OVA- restimulated T cells, g) comparing T cell proliferation and/or the Th2-cytokine production analyzed in step f) with T cell proliferation and/or Th2-cytokine production in an animal treated as described in the previous steps but wherein the Dendritic Cells were not treated with an LXR agonist, and, h) optionally, comparing T cell proliferation and/or Th2-cytokine production analyzed in step f) with T cell proliferation and/or Th2-cytokine production in an animal treated as described in the previous steps but wherein the Dendritic Cells were treated with a reference compound, i) identifying compounds as LXR agonists inhibiting T cell proliferation and/or Th2- cytokine release, and, j) optionally, isolating and/or formulating the compound identified in step i).

According to the present invention, OVA-specific T cells of step a) are isolated from spleen and lymph nodes of a TCR transgenic mouse (DO11.10) [Murphy (1990) Science 250:1720-1723] and are labeled with CFSE (for Carboxy Fluorescein diacetate Succinimidyl Ester) which passively diffuses into cells and becomes fluorescent after being cleaved by intracellular esterases. CFSE serves as a tracer for measuring T cell proliferation, as cell divisions correspond to sequential halving of CFSE fluorescence. In said use, Dendritic Cells are treated in vitro with an LXR agonist. As previously mentioned said Dendritic Cells may be of myeloid, lymphoid, or plasmacytoid origin, preferably myeloid origin, and may be obtained as mentioned before. The conditions at which said agonist is applied (concentrations and the compositions) may be easily derived for the skilled person in the art. For example, for the murine models of allergic asthma with adoptive transfer of DCs, the DCs will be treated in vitro with different doses of the LXR agonist (generally from 10-8M to 10-5M) and the non-toxic dose at which an effect is observed on DC precursor differentiation and on DC maturation will be selected for the in vivo studies.

Subsequently, Dendritic Cells pulsed with OVA are used according to the present invention to induce an asthmatic response in an in vivo asthma model. Said pulsed DCs are preferably injected intratracheally in said animal. Furthermore, after said injection said animal is incubated preferably 10 days to allow induction of immunological responses. Moreover, said Dendritic Cells of step c) are pulsed with

ovalbumine (OVA), preferably at a concentration of 10 ug of OVA as a 2% alum suspension. When pulsed, several maturation markers such as CD40, MHC-class II, CD80 and/or CD86 are induced.

Th2-cytokine production by ex vivo OVA-restimulated lymph node T cells may be studied as mentioned before. The proliferation of T cells is measured by using CFSE as a cell tracer (as described above).

According to the present invention, a non-human mammalian animal may also be used as an in vivo model for identifying compounds inhibiting the recruitment of inflammatory cells such as macrophages, lymphocytes, neutrophils and/or eosinophils to the BAL fluid, Th2-cytokine secretion, and/or, peribronchial and/or perivascular infiltration of inflammatory cells through the analysis of the secondary immune response. Said use may comprise the steps of: a) treating Dendritic Cells with an LXR agonist, b) pulsing the Dendritic Cells of step a) with ovalbumine (OVA), c) injecting the LXR-treated Dendritic Cells pulsed with OVA obtained in step b) in said non human mammalian animal, d) incubating said animal for several days, e) challenging said animal with OVA aerosols, f) analyzing inflammatory cell (macrophage, lymphocyte, neutrophil, eosinophil, etc.) content of the BAL fluid of said animal, g) optionally, extracting draining lymph nodes from said animal, h) optionally, restimulating lymph nodes of step g) with OVA, i) optionally, analyzing Th2-cytokine secretion produced by the cells of step h), j) optionally, measuring Th2-cytokine secretion in the BAL fluid, k) optionally, analyzing peribronchial and/or perivascular inflammatory cell infiltration in lung biopsies of said animal,

I) comparing the cellular content of step f), the Th2-cytokine secretion optionally measured in step i) or j) and/or peribronchial and/or perivascular inflammatory cell infiltration optionally measured in step k) with the cellular content, the Th2-cytokine secretion and/or peribronchial and/or perivascular inflammatory cell infiltration obtained from an animal treated as described in the previous steps but wherein the Dendritic Cells were not stimulated with the LXR agonist, m) optionally, comparing the cellular content of step f), Th2-cytokine secretion optionally measured in step i) or j) and/or peribronchial and/or perivascular inflammatory cell infiltration optionally measured in step k) with the cellular content, Th2-cytokine secretion, and/or, peribronchial and/or perivascular inflammatory cell infiltration obtained from an animal treated as described in the previous steps but wherein the Dendritic Cells were stimulated with a reference compound,

n) identifying the LXR agonist as a compound inhibiting recruitment of inflammatory cells to the BAL fluid, Th2-cytokine secretion, and/or, peribronchial and/or perivascular infiltration of inflammatory cells, if said LXR agonist reduces said cellular content, reduces Th2-cytokine production as determined in step i) or j) and/or reduces peribronchial and/or perivascular inflammatory cell infiltration of step k), and, o) optionally, isolating and/or formulating the compound identified in step n).

Dendritic Cells pulsed with OVA are used according to the present invention to induce an asthmatic response in an in vivo asthma model. The present application shows that if DCs are previously treated with an LXR ligand, this asthmatic response could be reduced. The murine model with adoptive transfer of pulsed-DCs may thus be used to evaluate LXR ligands in secondary or tertiary screening assays.

Said pulsed DCs are preferably injected intratracheally in said animal. Furthermore, after said injection said animal is incubated preferably 10 days to allow induction of immunological responses. Draining lymph nodes, such as mediastinal lymph nodes are removed, homogenized and resuspended in culture media using standard methods of the art. The ex vivo production of cytokines by the collected T cells is measured after restimulation with 10μg/ml of OVA for 4 days as described in [Hammad (2004) Am.JPathol. 164:263-271]. The cytokine production studied relates to the Th2-cytokines IL-4, IL-5, IL-6, IL-

9, IL-10 and IL-13 produced by T cells. IFN-γ, TNF-α, IL-2 or lymphotoxin concentrations may be studied as reference for the Th 1 -response.

According to the present invention, said non-human mammalian animal may, for instance, be a mouse or a rat. As pointed out before, said Dendritic Cell may be of myeloid, lymphoid or plasmacytoid origin. Furthermore, said Dendritic Cell may be directly isolated from (peripheral or cord) blood. In addition, said LXR agonist or said reference compound may be T0901317 or GW3965, or a functional equivalent, or a combination thereof. In addition, said animal may be used as a model for identifying compounds interfering with (inhibiting or preventing) asthma, more particularly allergy-induced asthma.

Fourth, the present invention elaborates on the in vitro use of an LXR agonist to interfere with (inhibit or prevent) Dendritic Cell precursor differentiation and/or DC maturation. As pointed out before, all LXR agonists may be candidates to exert said effect. Consequently, LXR agonist may be used for the preparation of a medicament for the treatment of a disease associated with DC differentiation and/or maturation.

However, it may be more preferable to use agonists carrying an optimal ratio of their transrepression over their transactivation activity (see above).

Consequently, according to a fifth embodiment of the invention, LXR agonist identified according to a method of the present invention or identified by means of the use of a non-human mammalian animal according to the present invention, may be used to prepare a medicament for the treatment of a disease associated with recruitment of inflammatory cells to the BAL fluid. In addition, according to the present invention, said agonist may be used to prepare a medicament for the treatment of a disease associated with increased Th2-cytokine release. Finally, said agonist may be used to prepare a medicament for the treatment of a disease associated with peribronchial and/or perivascular infiltration of inflammatory cells. Further, for the treatment of airway eosinophilic inflammation and/or asthma as listed in the present application, LXR agonists can be administered alone or in combination with conventional medicines.

Effectiveness to treat specific diseases or disorders aimed in the present invention by the application of LXR agonists has been demonstrated by experiments using T0901317 in mouse models for allergy-induced asthma. These compounds have their effect through the modulation of Th2-cytokine secretion and through the modulation of recruitment of inflammatory cells to the BAL fluid. However, said effects are not limited to said compound but also apply to other LXR agonists.

An LXR agonist may be combined with other agents also carrying activity on airway inflammation, or carrying activity on other cellular processes. For instance said cellular processes may be linked to the dysregulation of other inflammatory responses or may be related to non-inflammatory responses. Examples of said non-inflammatory responses may be the treatment of cholesterol mediated processes (diabetes, cholestasis, atherosclerosis, or related diseases). For instance, vitamin D3 and DEX interfere strongly with DC function and produce a distinct DC phenotype [Xing (2002) Biochem.Biophys.Res.Commun. 297:645-52]. Therefore, LXR ligands, glucocorticoids (i.e. dexamethasone and analogues thereof), anti-histamines, Platelet Activating Factor (PAF) antagonists, leukotriene antagonists, 5-lipoxygenase inhibitors, COX(2) inhibitors, anticholinergic agents, methyl xanthines or β-adrenergic agents may be combined in a preparation to treat the diseases according to the present invention.

Alternatively, the present invention suggests that some LXR agonists will have dominant activity in DC modulation (i.e. in DC precursors differentiation and/or in DCs maturation), and other may have dominant activity in modulating cholesterol metabolism. Therefore, the present inventors suggest that it is possible to perform a combined treatment of DC-related and cholesterol-related diseases through the use of specific LXR-agonists. Dosage information for these agents is well known.

A sixth embodiment of the present invention relates to an isolated Dendritic Cell composition or an isolated Dendritic Cell precursor composition comprising DCs or DC

precursors thereof which have been treated in vitro with an LXR agonist. Said agonist may interfere with , (inhibiting or preventing) DC precursor differentiation and/or with DCs maturation. Said cells may subsequently be sensitized in vitro with the maturation agents described above. For instance, in the method of the present invention, the stimulation of the differentiation of Dendritic Cell precursors to Dendritic Cells or Dendritic Cell maturation may be achieved by treating with allergens (ex. der p 1, der p 2, bet Via, ovalbumine, etc.), inflammatory cytokines (ex. tNFα, IL-1β, etc.), CD40L (also called CD154, is a co-stimulatory membrane protein belonging to the TNF superfamily), bacterial products (ex. LPS or LipoPolySaccharide), pathogens such as Escherichia coli, Candida, viruses (ex. Influenza, measles and dengue viruses, HIV, etc.) or other agents known in the art. Said different agents may be purified or not.

As explained above, said Dendritic Cell or Dendritic Cell precursor may be of myeloid, lymphoid or plasmacytoid origin. According to the present invention, differentiated DCs can be directly isolated from blood, using BDCA-1 or BDCA-3 (myeloid specific) or BDCA-2 and BDCA-4 (plasmacytoid specific) antibodies. Therefore, Dendritic Cells used in the method of the present invention may be directly isolated from (peripheral or cord) blood.

Said Dendritic Cell precursor may be a monocyte, a CD34+ hematopoietic progenitor cell of the cord blood, the adult bone marrow, the peripheral blood or the thymus or an IL-3R plasmacytoid cell from the blood or lymphoid tissues. The Dendritic Cell precursor may thus be isolated from peripheral blood, cord blood, bone marrow, thymus or other lymphoid tissues and may be a monocyte, a CD34+ hematopoietic progenitor or an IL-3R plasmacytoid cell. Preferably said Dendritic Cell differentiates from a monocyte. The LXR agonist used to treat the Dendritic Cell composition or the Dendritic Cell precursor composition according to the present invention may be T0901317 or GW3965 or a functional equivalent or a combination thereof.

The present invention further elaborates on the use of a Dendritic Cell composition or a Dendritic Cell precursor composition according to the present invention -to. study the_recruitment of inflammatory cells to the BAL fluid in a model organism. The present invention also elaborates on the use of a Dendritic Cell composition or a Dendritic Cell precursor composition according to the present invention to study the release of Th2-cytokine in the BAL fluid and by lymph node cells in a model organism. Finally, the present invention also elaborates on the use of a Dendritic Cell composition or a Dendritic Cell precursor composition according to the present invention to study the peribronchial and/or perivascular infiltration of inflammatory cells in a model organism. Said models may be specific for a disease associated with said physiological changes.

According to the present invention, the LXR agonist identified according to a method of the present invention or identified by means of the use of a non-human mammalian animal according to the present invention, may be used to prepare a medicament for the treatment of asthma, preferably allergy-induced asthma. The present invention also relates to the use of a Dendritic Cell composition or a Dendritic Cell precursor composition according to the present invention to study asthma, preferably allergy-induced asthma. Many types of asthma exist: a schematic representation of the different kinds of asthma is given in Figure 1. As discussed above, the development of asthma and airway inflammation is influenced by inflammatory cells, in particular by two major types of cells : eosinophilic and neutrophilic cells. Some types are more influenced by eosinophilic infiltration; others more influenced by neutrophilic infiltration. Of all diseases depicted, the allergen- triggered disease (allergy-induced asthma) is the most influenced by eosinophylic migration. As the secondary effect of LXR ligands was found in the present invention to inhibit said eosinophilic migration, it becomes obvious that especially allergen-induced asthma may be treated using said LXR agonists. That LXR agonists are efficient in allergy induced asthma models is confirmed by the experimental findings described in the present application. However, diseases from the group consisting of Eosinophilic Intrinsic Asthma, Eosinophilic Bronchitis, Occupational Asthma, COPD, Bronchiectasis may also be treated using LXR ligands as the LXR ligand can also repress the effect of a Th1 stimuli such as LPS. The present invention also suggests that the disease chosen from the group consisting of Viral Wheeze/PV Cough, Neutrophilic Asthma and Bronchitis may be treated.

According to the present invention, the LXR agonist T0901317 or GW3965 or a functional equivalent or a combination thereof may be used to interfere in vitro with (inhibit or prevent) DC differentiation and/or maturation or to study a disease associated with the recruitment of inflammatory cells to the BAL fluid, with the release of Th2-cytokine in the BAL fluid and/or by lymph node cells, and/or, with peribronchial and/or perivascular infiltration of inflammatory cells. The LXR agonist, T0901317 or GW3965 or a functional equivalent or a combination thereof, may be also used according to any uses or methods of the present invention.

Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. The content of all above cited prior art is incorporated by reference. Exemplary methods and materials are described below, although methods and materials similar or equivalent to those described herein can be used in the practice or testing of the present invention. In case of conflict, the present specification, including definitions, will control. The materials, methods, and examples are illustrative only and

not intend to be limiting. Other features and advantages of the invention will be apparent from the following examples and drawings.

FIGURES

Figure 1 Asthma and airway inflammation, (taken from AJ. Wardlaw et al. (2002)

Clin.Science 103:201-211)

Shown is a schematic representation of the complex and indirect relationship between airway and asthma. Many conditions are characterized by airway inflammation in which either Th2/eosinophilic or Th1/neutrophilic responses predominate. Although there is a bias towards eosinophilic inflammation being associated with asthma, this is far from absolute. The reason why in some cases inflammation leads to bronchoconstriction and AHR (Airway HyperResponsiveness - an asthma-like phenotype with wheeze and bronchodilator-responsive shortness of breath), whereas in others it leads only to symptoms of bronchitis (cough and sputum production), is at the crux of what causes asthma. Although the term asthma-like or asthma describes variable airflow obstruction and AHR, it is believed these are the most specific hallmarks of the disease, it should be remembered that patients with asthma usually also have symptoms of bronchitis. Mwt, molecular weight; Eos. Branch., eosinophilic bronchitis; Occ. Asthma, occupational asthma; PV, post viral; BHR, bronchial hyper-responsiveness; COPD , chronic obstructive pulmonary disease.

Figure 2 Expression of LXRα and LXRβ in myeloid and plasmacytoid DCs. mRNA expression of LXR α and LXRβ was analyzed by RT-PCR. Monocyte-derived DCs were stimulated with 100 ng/ml of LPS for 24 hours and plasmacytoid DCs were stimulated with 3 μM of CpG ODN 2006 (δ'-TGCTGCTTTTGTGCTTTTGTGCTT) and IL-3 (10ng/ml) for 24 hours.

Figure 3 In vitro effects of LXR agonists on DC modulation: Interference of LXR ligands T0901317 and GW3965 with the differentiation of monocytes into Dendritic Cells. LXR ligands GW3965 (A and B) and T0901317 (C and D) were added at day 3 of differentiation and monocyte-derived DCs were characterized at day 6 for cell-surface expression of CD1a by FACS (Fluorescence-Activated Cell Sorting) analysis and for CD40 transcriptional activity by RT-PCR.

Figure 4 In vitro effects of LXR agonists on DC modulation : Interference of LXR ligands T0901317 and GW3965 with LPS-inducible transcription of ELC and CCR7 in Dendritic Cells.

Monocyte-derived DCs were preincubated for 2 hours with LXR ligand GW3965 (A and B) or T0901317 (C and D) and then stimulated with LPS (Lipo Poly Saccharide) for an additional 16 hours. The transcriptional activity of the chemokine ELC (Ebv-induced molecule 1 Ligand Chemokine) and its receptor CCR7 was analysed by quantitative RT-PCR.

Figure 5 In vitro effects of LXR agonists on DC modulation : Interference of LXR ligand T0901317 with the capacity of DCs to stimulate the proliferation of allogeneic T cells. A heterologous MLR (Mixed Leukocyte Reaction) assay was performed with increasing quantities of DCs (treated or not with T0901317) incubated with naϊve CD4+ T cells for 7 days. T cell proliferation was measured by BrdU (Bromodeoxyuridine) incorporation.

Figure 6 One-hybrid GAL4-LXR assay.

Representative diagram of a one-hybrid GAL4 assay using a reporter plasmid containing 5 tandem repeats of GAL4 response elements (Gal4RE(x5)) and an expression plasmid encoding a GAL4(DBD)-LXR(LBD) chimera.

Figure 7 FRET assay.

Representative diagram of a FRET (Fluorescence Resonance Energy Transfer) assay using a GST-LXR(LBD) fusion protein, a biotinylated LXXLL peptide derived from a known coactivator, a GST-APC conjugated antibody and a RPE (R-PhycoErythrin)- conjugated streptavidin which constitutes the donor molecule.

Figure 8 In vivo asthma model for identifying compounds interfering with T cell proliferation and/or Th2-cytokine release by lymph node cells through the analysis of the primary immune response. The intratracheal injection of OVA-pulsed DCs treated with the LXR ligand impairs the primary immune response by interfering with cytokine secretion of lymph node T cell.

Figure 9 Schematic representation of the murine model of allergic asthma with adoptive transfer of OVA-pulsed DCs.

Figure 10 In vivo asthma model for identifying compounds interfering with (inhibiting) recruitment of inflammatory cells to the BAL fluid, with Th2-cytokine secretion, and/or,

with peribronchial and/or perivascular infiltration of inflammatory cells through the analysis of the secondary immune response. Cellular composition of the BAL fluid.

Figure 11 In vivo asthma model for identifying compounds interfering with (inhibiting) recruitment of inflammatory cells to the BAL fluid, with Th2-cytokine secretion, and/or, with peribronchial and/or perivascular infiltration of inflammatory cells through the analysis of the secondary immune response. Analysis of the cytokine secretion by lymph node T cell.

EXAMPLES

Example 1 : Expression of LXRα and LXRβ in myeloid and plasmacytoid DCs. mRNA expression of LXRα and LXRβ was analyzed by RT-PCR using the following oligonucleotides: 5'-TTCCTCCTGACTCTGCGGTG (sense oligo) and 5'- TCCTGGCTTCCTCTCTGAGG (antisense oligo) for LXRα and 5'- AGCAGCAGCAGGAGTCACAGTC (sense oligo) and 5'-

CTTGAGCCGCTGTTAGCTGGAC (antisense oligo) for LXRβ. Monocyte-derived DCs were stimulated with 100 ng/ml of LPS for 24 hours and plasmacytoid DCs were stimulated with 3 μM of CpG ODN 2006 (δ'-TGCTGCTTTTGTGCTTTTGTGCTT) and IL-3 (10ng/ml) for 24 hours.

The present example demonstrates that both LXRα and LXRβ are expressed in myeloid and plasmacytoid DCs (mDC and pDC - see Figure 2). Interestingly, LXRα is down-regulated in plasmatocytoid DCs activated with CpG oligos. In contrast, LXRβ is up-regulated, suggesting that these two LXRs may have different roles in DCs. However, according to the present invention, in the primary screening assay for selecting LXR agonists, the selection of compounds binding to both receptor subtypes is aimed at.

Example 2: In vitro effect of LXR agonists on DC modulation.

The synthetic LXR ligands, T0901317 and GW3965, were tested on the phenotype and maturation function of DCs.

The LXR ligands were first tested on the normal differentiation of monocytes into DCs by measuring the acquisition of the DC phenotype. For these experiments, GW3965 or T0901317 were added at day 3 of differentiation and DC differentiation markers were examined at day 6 (the cytokines GM-CSF and IL-4 were present throughout the differentiation process).

It was found that LXR ligands T0901317 or GW3965 interfered with cell-surface expression of CD1a, CD11c, CD40 and HLA-DR and with the transcription of CD40 and MMP9. A dose response analysis of CD1a and CD40 is shown in Figure 3. These results showed that LXR ligands interfere with the differentiation of DCs and skewed DC toward the acquisition of an unusual phenotype.

The effect of LXR ligands on LPS-inducible maturation of DCs was then investigated. For these experiments, monocyte-derived DCs obtained at day 6 of differentiation were pre-treated with LXR ligands GW3965 or T0901317 for 2 hours and then stimulated with LPS for 16 hours. The maturation markers ELC, CCR7, IP-10 were down regulated by T091317, as well as co-stimulatory molecules CD80 and CD86 and MHC Il expression (the results for ELC and CCR7 are shown in Figure 4). The effect of LXR ligands on reducing the expression of chemokine ELC and its receptor CCR7, which are key genes involved in DC motility, strongly suggest that LXR ligands inhibit the migration of DCs to secondary lymphoid organs and therefore may interfere with the initiation of the immune response triggered by DCs. Moreover, the reduction of MHC class Il expression and co-stimulatory molecules such as CD80 and CD86 indicate that the antigen presentation function will be altered by LXR ligands.

Indeed, it was found that monocyte-derived DCs treated with the LXR ligand have a reduced ability to stimulate the proliferation of naϊve CD4+ T cell in a Mixed Leukocyte Reaction (MLR) assay. For this assay, increasing quantities of LPS-matured DCs (treated or not with LXR ligand) were incubated with a fixed quantity of CD4+ naϊve T cell derived from another blood donor. After incubation for 5 days, BrdU was added for another 24 hours, and BrdU incorporation in T cells was measured by ELISA with anti-BrdU antibodies coupled to a chemiluminescent moiety. The results of the MLR assay are shown in Figure 5.

The transactivation of known LXR target genes such as ABCA1 , ApoE, and PLTP was also observed, suggesting that LXR ligands are fully functional in DCs and thus play a role in cholesterol and lipoprotein metabolism of DCs.

In summary, the effect of the LXR ligand T0901317 was tested on the expression of known differentiation and maturation markers and on the antigen presentation function of DCs. The maturation markers ELC, CCR7, IP-10, CD80, CD83, CD86, MHC class Il were down regulated by T091317, suggesting that an LXR agonist could reduce the migration of DCs in the secondary lymphoid organs and reduce the potential of DCs to initiate an immune response. To get a better insight into the overall effect of the LXR ligand, in vivo studies were initiated using several murine models of allergic asthma.

Example 3: High Throughput Screen (HTS) for LXR agonists: one-hybrid GAL-4.

One strategy to identify compounds which interact with the Ligand Binding Domain (LBD) of LXR is the development of a cell-based GAL-4 screening assay. The concept of said assay is depicted in Figure 6. This assay also allows the development of HTS screening assays to screen for LXR ligands, in particular LXR agonists. Alternatively, a cell-based GAL4 assay may serve as a primary screening of pharmacological compounds; followed by a secondary screening, such as a FRET assay (see example 4). Another potential secondary screening may include the use of endogenous LXR target genes (see below).

For cell-based GAL-4 screening assay, an expression vector containing the DNA Binding Domain (DBD) of GAL4 is fused to the Ligand Binding Domain (LBD) of LXR. In addition, a reporter plasmid comprising multiple copies of the GAL4 response element upstream of a minimal TK promoter is made. Both constructs are then transfected together in a relevant cell line which expresses known LXR co-activators or co-repressors. Examples of said co-activators are SRC-1, ASC2, PGC-1 , RIP140, TRAP220, SRC-3/ACTR and GRIP-1 and said co-repressors are NCOR1 and SMRT/NCOR2. One day after the transfection, the nuclear ligand T091317 or the pharmacological products are added and the luciferase activity is measured 24 hours later. This assay can serve to screen for T091317 analogs or other molecules that can fit the ligand binding pocket and mimick the effect of T091317. High level of induction with T0901317 or GW3965 was obtained in COS-7 and HepG2 cells but other cell lines may also be tested.

Example 4: High Throughput Screen for LXR agonists : FRET.

The concept of FRET assay is depicted in Figure 7. This assay can be used to screen for agonists or antagonists. First a GST (Glutathion S-Transferase) fusion construct containing the LBD domain of LXR is produced in bacteria. For the FRET assay, the GST-LXR chimeric protein is incubated with a biotinilated peptide derived from a co-activator or co-repressor of LXR, a GST-APC conjugated antibody which serves as an acceptor molecule, and a RPE (R-PhycoErythrin)-conjugated streptavidin which constitutes the donor molecule. A positive interaction of the co-activator or co- repressor peptide with the LBD brings in proximity the donor molecule (RPE-SA) with the acceptor molecule (GSP-APC antibody). Following the incubation, the RPE chromatophore excites at 495 nm and if the acceptor molecule is within the range of 80 A, the energy is transferred to the APC chromatophore and the emission is measured at 670 nm. The most relevant co-activators or co-repressors (described above for the GAL4 assay) are tested in this assay.

Example 5: High throughput screen for LXR agonists: endogenous LXR targets.

Several LXR target genes involved in cholesterol and fatty acid metabolism have been identified so far. Examples are ABCA1 , ABCG5, ABCG8, cholesterol ester transfer protein, cyp7A1, acetyl-coenzyme A, fatty acid synthetase, fatty acid binding protein, apolipoprotein E, SREBP-Ic, P450, stearoyl-CoA desaturase 1 and insig-2. However, the endogenous LXR target genes involved in inflammation and immunity are less known. Recently, Kim et al. (2003) (MoI. Cell. Biol. 23:3583-92) showed, by transcription profiling of LXR ligand treated mice, that IKKb is an inducible target gene of LXR.

In order to identify LXR target genes involved in the reduction of the inflammatory process, assays using T0901317-treated DCs (with or without an inflammatory stimuli) are performed. Said inflammatory stimuli are chosen from the group consisting of der p1 , LPS or CD40L. The identification of (a) LXR target gene(s) in DCs allows the development of assays with a DC-specific LXR target gene. Microarrays of DCs treated with LXR ligand will help to identify DC-specific LXR target gene.

Using RT-PCR analysis, some genes, such as ABCA1 , ApoE, and PLTP were found to be induced by the LXR ligand T0901317. It is believed that these genes are transactivated through the binding of LXR to response elements found in their promoter region. In contrast, the LXR ligand interfered with the LPS-inducible expression of ELC, CCR7 and MMP9. Since many genes involved in inflammatory processes are regulated by NFKb, it was suggested that LXR may interfere through antagonism of NFKB signaling pathway [Castillo (2003) J.Biol.Chem. 278:10443-10449]. Other signaling pathways such as the IL-4 inducible STAT6 pathway, the PMA-inducible GATA-3 pathway, and the TGF-β signaling pathway may be tested to determine the influence of LXR ligands.

Several trans-repressive type of screen already exist for the glucocorticoid receptor (GR) [Li (2003) J.Biol.Chem. 278:41779-41788; Coghlan (2003) Mol.Endocinol. 17:860-869; Vanden Berghe (1999) MoI. Pharmacol. 56:797-806], a skilled person in the art may thus easily derive all necessary details to set up such an assay from said reference (Li et al. (2003) J.Biol.Chem). For example, one of the assay tested the ability of GR ligands to transrepress the TNFα/IL-1β induced E-selectin promoter activity [Coghlan (2003) Mol.Endocinol. 17:860-869]. This transrepression assay was performed by co-transfection of a reporter plasmid containing the promoter region of E-selectin together with a GR expression plasmid. Following transfection, cells were treated with TNFα and IL-1β in the absence or presence of compound. In parallel, a transactivation assay was performed using a reporter plasmid containing a promoter bearing GR response elements such as the tyrosine aminotransferase promoter. These assays led to the identification of a compound (AL-438) that confers a

better transrepression activity than the well characterized prednisolone and a weaker transactivation capacity than prednisolone.

Example 6: In vivo asthma model for identifying compounds interfering or preventing Th2-cytokine secretion, recruitment of inflammatory cells to the BAL fluid, and/or, peribronchial and/or perivascular infiltration of inflammatory cells.

This model is used to study the effect of the LXR ligand on existing eosinophilia. For this model, mice are sensitized by an injection of ovalbumine (OVA) emulsified in alum. The effect of the LXR ligand is tested by injecting the ligand intranasally, at the time of sensitization. OVA aerosol challenges are given for three consecutive days between day 10 to day 12, and LXR ligand is given intranasally at least four hours before each challenge. However, the time at which the LXR ligand is given is not limited to said period. The asthmatic response is analyzed 24 hours after the last challenge and includes the histological analysis of peribronchial and/or perivascular infiltration of inflammatory cells in lung biopsies, BAL fluid analysis (cellular content and cytokine secretion) and airway hyperactivity .

Alternatively, this model can also be used to test if LXR ligands can prevent asthma in which case the injection of the LXR ligand is performed before the sensitization with OVA.

Example 7: In vivo asthma model for identifying compounds interfering with T cell proliferation and/or Th2-cytokine release by lymph node cells through the analysis of the primary immune response.

In order to investigate if the LXR ligand interferes with the primary immune response, a model was used wherein the proliferation of OVA-specific T cell labeled with CFSE (CarboxyFluorescein diacetate Succinimidyl Ester) was analyzed following the intratracheal injection of bone-marrow derived DCs, treated or not with the LXR ligand T0901317. It was shown that T0901317-treated DCs have a reduced ability to initiate a primary immune response which was observed by a reduction in the secretion of IL-4, IL-5 and IL-10 by lymph node T cell (Figure 8).

For these experiments, a TCR transgenic model in which CFSE-labeled T cells were injected 2 days before sensitization was used.

Example 8: In vivo asthma model for identifying compounds interfering with (inhibiting) recruitment of inflammatory cells to the BAL fluid, with Th2-cytokine secretion, and/or, with peribronchial and/or perivascular infiltration of inflammatory cells through the analysis of the secondary immune response.

The nuclear receptor LXR has been evaluated in vitro and was shown to interfere with Dendritic Cell phenotype and function (see above). In this example the in vivo inhibitory effect of the LXR ligand on the asthmatic response using a murine model of asthma with adoptive transfer of DCs is studied. Using a murine model of allergic asthma with adoptive transfer of OVA-pulsed

DCs, it was shown that the LXR ligand interfered with the immune response associated with asthma. The experimental approach followed is schematically represented in Figure 9. For these experiments, bone marrow-derived DCs were treated with the LXR ligand 1 hour before the pulse with OVA. As seen in human monocyte-derived DCs, The phenotypic characterization of pulsed DCs showed that the inducible expression of CD40, MHCII, CD80 and CD86 by OVA was also altered by the presence of the LXR ligand (data not shown). The OVA-pulsed DCs were then injected intratracheal^ and 10 days later, the mice received 3 consecutive OVA aerosol challenges. At day 13, the BAL fluid was analyzed for cellular content and the thoracic lymph nodes were extracted, restimulated with OVA for 4 days, and analyzed for cytokine secretion. The analysis of the BAL fluid showed that the treatment of DCs with the LXR ligand had an effect on the secondary immune response which is reflected by a reduction in the number of macrophages, lymphocytes and eosinophils (Figure 10). As well, the level of the Th2-cytokines, IL-4, IL-5 produced by T cell were reduced (Figure 11). As a conclusion, the present invention indicates that LXR-treated DC, i.e. using

T0901317 inhibits airway eosinophila in BAL fluid and inhibits secretion of Th2- cytokines such as IL-4, IL-5 and IL-10. The present invention provides thus evidence for the role of LXR agonists in airway inflammation.

It is to be understood that while the invention has been described in conjunction with the detailed description thereof, the foregoing description is intended to illustrate and not limit the scope of the invention, which is defined by the scope of the appended claims. Other aspects, advantages, and modifications are within the scope of the following claims.