PTGDR-1 and/or PTGDR-2 antagonists for preventing and/or treating systemic lupus erythematosus

Abstract

The present invention concerns a PTGDR-1 antagonist, a PTGDR-2 antagonist, a dual PTGDR-1/PTGDR-1 antagonist, or a combination of PTGDR-1 antagonist and PTGDR-2 antagonist, and pharmaceutical compositions containing them, for use for preventing and/or treating SLE.

Claims

1. A method for treating lupus nephritis in a patient in need thereof, wherein said method comprises administering said patient with a PTGDR-1 antagonist in an amount sufficient to limit an extent or reduce an occurrence of organ damage.

2. The method according to claim 1, wherein said antagonist is a small molecule antagonist.

3. The method according to claim 1, wherein said antagonist has formula (I) ##STR00020## or pharmaceutically acceptable salts thereof, wherein n is 0 or 1; m is 1, 2 or 3; R.sub.1 is H, C.sub.1-C.sub.3 alkyl, halogenated C.sub.1-C.sub.3 alkyl or cyclopropyl; R.sub.2 is 4-chlorophenyl or 2,4,6-trichlorophenyl.

4. The method according to claim 1, wherein said PTGDR-1 antagonist is laropiprant.

5. The method according to claim 1, wherein said PTGDR-1 antagonist is used in combination with at least a PTGDR-2 antagonist.

6. The method according to claim 5, wherein said PTGDR-2 antagonist has formula (VIII) ##STR00021## wherein R1 is H, fluorine, methyl, methoxy, benzyloxy, or hydroxyl, R2 is phenyl which is substituted by fluorine, chlorine, trifluoromethyl, methyl, ethyl, propyl, isopropyl, or methoxy, and Y is 0 or 1, or pharmaceutically acceptable salts thereof.

7. The method according to claim 6, wherein said PTGDR-2 antagonist is CAY10471 (TM30089).

8. The method according to claim 5, wherein said PTGDR-1 antagonist and PTGDR-2 antagonist are formulated in a single pharmaceutical composition.

9. The method according to claim 5, wherein said PTGDR-1 antagonist and PTGDR-2 antagonist are formulated in separate pharmaceutical compositions for simultaneous use, separate use, or use spread over time.

10. A method for treating lupus nephritis in a patient in need thereof, wherein said method comprises administering said patient with a pharmaceutical composition comprising a PTGDR-1 antagonist and a PTGDR-2 antagonist, or a dual PTGDR-1/PTGDR-2 antagonist in an amount sufficient to limit an extent or reduce an occurrence of organ damage.

11. A method for treating lupus nephritis in a patient in need thereof, wherein said method comprises administering said patient with a PTGDR-1 antagonist and a PTGDR-2 antagonist as a combined preparation for simultaneous use, separate use, or use spread over time.

12. A method for treating lupus nephritis in a patient in need thereof, wherein said method comprises administering said patient with a PTGDR-2 antagonist.

13. The method according to claim 1 wherein the PTGDR-1 antagonist prevents basophil homing to secondary lymphoid organs.

14. The method according to claim 1 wherein the PTGDR-1 antagonist prevents, limits the extent or reduces the increase in autoantibody titers and/or the occurrence of SLE flares and/or organ damages.

Description

FIGURES

(1) FIG. 1. Human blood basophil gating strategy and activation status in function of SLE disease activity.

(2) (a) Flow cytometric analysis of CD203c levels on blood basophils from subjects with inactive, mild or active SLE (n=60/40/82, respectively) compared to controls (CT, n=100). (b) Flow cytometric analysis of CD62L levels on blood basophils from subjects with inactive, mild or active SLE (n=43/33/66, respectively) compared to controls (CT, n=90). (c) Flow cytometric analysis of CD63 levels on blood basophils from subjects with inactive, mild or active SLE (n=14/6/12, respectively) compared to controls (CT, n=12). (a-c) Data are normalized to controls' mean and expressed in arbitrary units as means+s.e.m. Statistical analyses were by Mann-Whitney tests. NS: not significant, *: P<0.05, **: P<0.01, ***: P<0.001.

(3) FIG. 2. Basopenia and basophil activation status correlate with disease activity and are specific for lupus nephritis among other active renal diseases.

(4) (a) Blood basophils per μL as determined by flow cytometry from healthy controls (CT) and subjects with inactive, mild or active SLE (n=87/58/38/84, respectively) ad defined in the online methods. (b) Spearman correlation between blood basophil numbers and SLEDAI (r=−0.3629, p<0.0001) shown on a linear scale. (c) Proportion of blood HLA-DR.sup.+ basophils as determined by flow cytometry healthy controls (CT) and subjects with inactive, mild or active SLE (n=96/60/40/84, respectively). (d) Receiver-Operating Characteristic (ROC) analysis of the proportion of HLA-DR+ basophils in SLE patients (n=184) versus controls (n=97) (thick line, AUC=0.9091) and of dsDNA-specific IgG titers in SLE patients (n=123) versus controls (n=39) (dotted line, AUC=0.8384). (e) Blood basophils per μL as in (a) from subjects with the following active renal diseases: IgA-N: IgA nephropathy; HSP: Henoch-Schönlein purpura nephropathy; DN: Diabetic Nephropathy; MN: membranous nephropathy; OGN: Other glomerular nephropathies; NGKD: Non-Glomerular Kidney Diseases; aLN: active Lupus Nephritis; compared to healthy controls (n=40/20/39/22/42/46/81/87, respectively). (f) Proportion of blood HLA-DR+ basophils in patients with active renal diseases as in (e) (n=40/20/39/21/51/47/77, respectively) compared to controls (n=96). (a,c,e,f) Data are expressed as means+s.e.m. Statistical analyses were by Mann-Whitney tests. Comparison to healthy controls' median is shown above each bar and to the corresponding bars when indicated. NS: not significant, *: P<0.05, **: P<0.01, ***: P<0.001, ****: P<0.0001.

(5) FIG. 3. PGD.sub.2/PTGDRs and CXCL12/CXCR4 axes contribute to SLE patient specific basophil phenotype

(6) (a) Flow cytometric analysis of PTGDR-2 (CRTH2) levels on blood basophils from healthy controls (CT) and subjects with inactive, mild or active SLE (n=71/48/31/60, respectively). (b) 11β-prostaglandin F.sub.2α (11β-PGF2α) levels in plasma from controls and individuals with inactive, mild or active SLE (n=29/31/19/37, respectively) as measured by EIA. (c) Blood basophils per μl of blood in subjects with SLE classified on the basis of low (n=51) or high (n=34) 11β-PGF2α plasma levels (titer below or above CT titer mean+2 standard deviations, respectively). (d) Flow cytometric analysis of CXCR4 levels on blood basophils from CT and subjects with inactive, mild or active SLE (n=66/32/20/51, respectively). (e) CXCL12 levels in plasma from controls and individuals with inactive, mild or active SLE (n=63/43/29/59, respectively) as measured by ELISA. (f) Flow cytometric analysis of the levels of CD164 on blood basophils from CT and subjects with inactive, mild or active SLE (n=33/15/7/26, respectively). (a,d,f) Data are normalized to the mean of CT values and expressed in arbitrary units. (a-f), Statistical analyses were by Mann-Whitney tests. Comparison to healthy controls' median is shown above each bar and to the corresponding bars when indicated. Data are expressed as means+s.e.m. NS: not significant, *: P<0.05, **: P<0.01, ***: P<0.001, ****: P<0.0001.

(7) FIG. 4. Associations between plasma 11β-PGF2α & CXCL12 titers, basophil CXCR4 & CD164 expression levels and lupus specific basopenia

(8) (a) Spearman correlation between blood basophil number per mL of blood and 11β-PGF2α plasma levels (r=−0.2585, P=0.0169, n=85) shown on a logarithmic scale. (b) Spearman correlation between blood basophil number per mL of blood and relative CXCR4 levels on basophils (as defined in FIG. 2d) (r=−0.4692, P<0.0001, n=101) shown on a logarithmic scale. (c) Blood basophils per μl of blood in subjects with active SLE classified on the basis of low or high CXCL12 plasma levels (titer below or above control titer mean+2 standard deviations, respectively). Data are expressed as means+s.e.m. Statistical analysis was by Mann-Whitney test. *: P<0.05. (d) Spearman correlation between blood basophil number per mL of blood and relative CD164 levels on basophils (as defined in FIG. 2f) (r=−0.4165, P=0.0029, n=49) shown on a logarithmic scale.

(9) FIG. 5. CXCR4-mediated basophil migration ex vivo and in vivo is enhanced by PGD.sub.2

(10) (a) Migration assays of human blood basophils from healthy controls (CT, n=6) and SLE patients (n=6) towards a CXCL12 gradient. (b) Migration assays of human blood basophils towards IL-3, CCL3, CCL5, CXCL2 and PGD.sub.2 gradients from healthy controls (Controls, n=8/4/3/4/7, respectively) and from SLE patients (SLE, n=6/3/6/3/5, respectively). (c) Relative dsDNA-specific IgE levels in plasma from inactive, mild or active SLE individuals (n=41/29/51, respectively) normalized to the control values mean (n=38) as measured by ELISA. (a-c) Statistical analyses were by Mann-Whitney tests compared to CT. (d) CXCR4 expression levels on blood basophils after 18 hours of incubation without (−) or with (+) PGD.sub.2, mouse anti-human IgE or IL-3 was assessed by flow cytometry. (e) Migration assays of human blood basophils stimulated as in (d) towards a CXCL12 gradient. (d,e) Statistical analyses were by paired Student t test. (f) Expression level variation of the indicated basophil markers between peritoneal and blood basophils from patients undergoing peritoneal dialysis and being treated for non-sterile peritonitis (n=6) were assessed by flow cytometry. Statistical analysis was by one sample t test compared to a 0 theoretical value. (a,b,e) Corrected migration as described in the methods. (a-f) Data are expressed as means±s.e.m. NS: not significant, #: P=0.06, *: P<0.05, **: P<0.01, ***: P<0.001, ****: P<0.0001.

(11) FIG. 6. Suboptimal IgE-mediated basophil activation leads to increased PTGDR-2 expression on human basophils.

(12) PTGDR-2 (CRTH2) expression levels (as defined in online methods) on blood basophils from healthy donors after 18 hours of incubation without (−) or with (+) PGD.sub.2, mouse anti-human IgE or IL-3. Statistical analyses were by paired Student t test.

(13) FIG. 7. PGD.sub.2 is sufficient to enhance the CXCL12-dependent basophil homing to SLOs occurring in lupus-prone mice

(14) (a) Ex vivo migration of basophils from whole WT or Lyn.sup.−/− splenocytes to CXCL12. (b) CXCR4 expression levels on basophils from the indicated compartments in aged WT (n=16) and Lyn.sup.−/− (n=14) animals. Data are normalized to the mean CXCR4 expression level of WT blood basophils. Statistical analyses placed directly above each bar compared the value for one given compartment to the blood compartment of the corresponding genotype. Statistical analyses between both genotypes for each compartment are also indicated. (c) Basophil number per peritoneum in young WT mice 24 hours after intraperitoneal (ip) injection of PBS or CXCL12 and compared to steady state (−) values (n=13/15/5, respectively). (d) CXCR4 expression levels on basophils from mesenteric lymph nodes (mLN) of young Lyn.sup.−/− mice 24 hours after PBS or PGD.sub.2 ip injection normalized to PBS injected mice values' mean. (e) Basophil number in mLN of young Lyn.sup.−/− mice 24 hours after ip injection of the indicated compound(s). (f) CXCR4 expression levels on spleen basophils from 15 weeks old WT mice incubated ex vivo for 24 hours with the indicated compound and normalized to the control values' mean. (a-f) Data are expressed as means±s.e.m. Basophils number and CXCR4 expression were assessed by flow cytometry. Statistical analyses were by unpaired t test with Welch's correction (a-e) and by paired Student t test (f). NS: not significant, #: P=0.058, *: P<0.05, **: P<0.01, ***: P<0.001, ****: P<0.0001.

(15) FIG. 8. CXCL12 or PGD.sub.2 ip injection in mice induce a CXCR4-dependent basophil accumulation in SLOs and peritoneum.

(16) (a) Proportion of basophil (×103) among living CD45+ cells in mesenteric lymph node (mLN) of young WT mice 24 hours after intraperitoneal (ip) injection of PBS (n=13) or CXCL12 (n=15) and compared to steady state (−) values (n=5). (b) Basophil number in spleen of young Lyn.sup.−/− mice 24 hours after ip injection of the indicated compound(s). (c) Basophil number in peritoneum of Lyn.sup.−/− mice 24 hours after ip injection of the indicated compound(s). Data are expressed as means±s.e.m. Statistical analyses were by unpaired Student t test with Welch's correction. NS: not significant, *: P<0.05, **: P<0.01, ***: P<0.001.

(17) FIG. 9: cAMP and PTGDRs specific agonist effects on CXCR4 expression by mouse spleen basophils ex vivo.

(18) (a-b) Relative CXCR4 expression levels on basophils (defined as CD19.sup.−TCRβ.sup.−CD3.sup.−CD49b.sub.+FcεRIα.sup.+CD123.sup.+)CD45.sup.lo and T cells (defined as CD45.sup.+CD3.sup.+TCRβ.sup.+ cells) in splenocytes incubated 4 hours without (−) or with the indicated concentration of N6,2′-O-dibutyryl-adenosine 3′:5′-cyclic monophosphate (db-cAMP) or 1 μM PGD.sub.2 as determined by flow cytometry. Statistical analyses were by paired Student t tests. NS: not significant, *: P<0.05, **: P<0.01, ***: P<0.001. (c) Relative CXCR4 expression levels on spleen basophils (defined as in (a)) incubated 4 hours without (0) or with the indicated concentration (nM) of the indicated compound(s). DK-PGD.sub.2: 13,14-dihydro-15-keto-PGD.sub.2 (PTGDR-2 specific agonist); BW245c: 3-(3-Cyclohexyl-3-hydroxypropyl)-2,5-dioxo-(R*,S*)-(±)-4-imidazolineheptanioc acid (PTGDR-1 specific agonist) as determined by flow cytometry. Statistical analysis was by two-ways ANOVA followed by a Tukey's multiple comparisons test. (a-c) Data are normalized to control value mean (per group, n=4 to 8). Data are expressed as mean+s.e.m. All experiments were realized with splenocytes from 8-12 weeks old WT mice.

(19) FIG. 10. Blockade of basophil accumulation in SLOs dampens lupus-like disease activity

(20) (a,b) Proportion of basophils (CD19.sup.−TCRβ.sup.−CD49b.sup.+FcεRIα.sup.+CD123.sup.+)CD45.sup.lo) among singlets living CD45.sup.+ cells in mesenteric (a) and other (cervical, brachial and inguinal) lymph nodes (b) from 10 to 12 weeks-old Lyn.sup.−/− mice injected over ten days with PBS (open circles) or PGD.sub.2 alone (open squares), and PGD.sub.2 injected and basophil depleted (MAR-1) (open diamonds) or not (IgG) (open triangles) as described in the methods. (c) IA-IE expression levels on LN basophils from mice as in (b). NA: not applicable. (d,e) Proportion of short lived plasma cells CD19.sup.+CD138.sup.+ among singlets living CD45.sup.+ cells in spleen (d) and lymph nodes (e) in the same mice as in (b). (f) Representative immunofluorescence staining for C3 and IgG deposits in kidneys from mice as indicated in (b) (scale bar=1 mm) and their corresponding quantifications in PBS (n=3), PGD.sub.2 (n=4), PGD.sub.2+control IgG (n=3) and PGD.sub.2+MAR-1 (n=3) injected mice. (g) Fold increase in urine albumin/creatinine ratio before and after PBS or PGD.sub.2 10 days-long treatment. (a-e) Basophil, plasma cell numbers and IA-IE expression levels were assessed by flow cytometry. (a-g) Data are expressed as means±s.e.m. Statistical analyses were by unpaired student t tests. #: P=0.0571, *: P<0.05, **: P<0.01, ***: P<0.001, ****: P<0.0001. One representative out of three independent experiments is shown.

(21) FIG. 11. Blockade of basophil accumulation in SLOs dampens lupus-like disease activity

(22) Quantifications of immunofluorescence staining for C3 and IgG deposits in kidneys from aged Lyn.sup.−/− mice treated (n=8) or not (vehicle, n=9) with PTGDR-1 and PTGDR-2 antagonists for ten days.

(23) FIG. 12. Targeting PTGDRs blocks CXCR4-mediated basophil accumulation in SLOs and reduces the lupus-like disease activity.

(24) (a-h) Comparisons between aged wild-type (WT) and Lyn.sup.−/− mice treated or not (vehicle) for 10 days with PTGDR-1 and PTGDR-2 antagonists as described in the online methods. (a,b,c) Flow cytometric analysis of basophils among living CD45.sup.+ cells in lymph nodes (cervical, axillar, inguinal and mesenteric) (a) and spleen (b). (c) CXCR4 expression levels on spleen basophils. (d) Proportion of short lived plasma cells CD19.sup.+CD138.sup.+ among living CD45.sup.+ cells in lymph nodes as in (a) was determined by flow cytometry. (e) Optical density (O.D.) values at 450 nm of dsDNA-specific IgG in plasma from the indicated mice as measured by ELISA (×10.sup.3). (f) Total IgE levels as measured by ELISA in plasma from the indicated mice. (g,h) IL-4 (g) and IL-1β (h) concentration in pg per mg of total kidney protein extract from the indicated mice as measured by ELISA. (a-h) WT vehicle, n=5; WT treated, n=4; Lyn.sup.−/− vehicle, n=8; Lyn.sup.−/− treated, n=8. Data are expressed as mean+s.e.m. Statistical analyses were by unpaired Student t tests. NS: not significant, *: P<0.05, **: P<0.01.

(25) FIG. 13. Treatment with PTGDRs antagonists reduces specifically basophil accumulation in SLOs leading to reduced short-lived plasma cell number and serum ANA titers.

(26) (a-d) Comparisons between aged wild-type (WT) and Lyn.sup.−/− mice treated or not (vehicle) with PTGDR-1 and PTGDR-2 antagonists for ten days. (a,b) Flow cytometric analysis of basophil proportion among CD45+ bone marrow (BM) cells (a) and blood leukocytes (b). (c) Flow cytometric analysis of short lived plasma cells CD19+CD138+ among living CD45+ cells. (d) Anti-nuclear antibodies (ANA) levels as measured by ELISA in plasma from the indicated mice. (a-d) WT vehicle, n=5; WT treated, n=4; Lyn.sup.−/− vehicle, n=8; Lyn.sup.−/− treated, n=8. Data are expressed as means+s.e.m. (a-c) Statistical analyses were by unpaired Student t test. (d) Statistical analysis was by Mann-Whitney test. NS: not significant, *: P<0.05, **: P<0.01.

(27) FIG. 14. Graphical abstract

(28) In systemic lupus erythematosus (SLE), a loss of self-tolerance induces the expansion of autoreactive (AR) T and B cells. Autoreactive plasma cells secrete autoreactive antibodies which will bind self-antigens of nuclear origin and complement factors to form circulating immune complexes (CIC). The deposition of these CIC or autoreactive antibodies in target organs is associated with local lesions, inflammation (and PGD.sub.2 production), and organ damages. Healthy basophils can get activated by the binding of CIC to Fc receptors (FIERI and FcγRs) to express more prostaglandin D2 (PGD.sub.2) receptors (PTGDRs) and activation markers such as CD203c. As chronic inflammation settles, so does the secretion of various inflammatory mediators in blood, including PGD.sub.2. PGD.sub.2 is sufficient to induce PGD.sub.2 production by circulating basophils themselves leading to an autocrine effect of PGD.sub.2. This leads to an increased surface expression of CXCR4 and enable basophil sensitivity to CXCL12 gradients. As a result, basophils are more eager to migrate to SLOs, which are known to secrete more CXCL12 during lupus pathogenesis. There, basophils support autoreactive T and B cells through their expression of activating molecules such as mBAFF, MHC-II or the secretion of various cytokines such as IL-4 and IL-6. Moreover, basophils can promote autoreactive antibody production and IgE class switching of B cells. As CIC and autoreactive IgE titers increase, so will targeted organ inflammation, PGD.sub.2 and CXCL12 titers and basophils homing to SLOs. It is assumed that basophils drive an amplification loop of the disease and blocking their recruitment to SLOs would prevent rise in autoantibody titers and consequent flares.

EXAMPLES

Example 1: Materials and Methods

(29) Mice.

(30) C57BL/6J wild-type (WT) mice were purchased from Charles River Laboratories (L'Arbresle, France) and Lyn.sup.−/− mice on a pure C57BL/6 background were bred in our animal facility. For lupus-like disease studies, mice were aged for a minimum of 40 weeks before treatment and analysis. For other ex vivo or in vivo analysis, young mice were between 8 and 12 weeks old, unless otherwise specified. Mice were maintained in specific pathogen-free conditions, used in accordance with French and European guidelines and approved by local ethical committee and by the Department of Research of the French government under the animal study proposal 02484.01.

(31) Patients.

(32) Blood samples were collected from adult subjects enrolled in a prospective long term study of systemic lupus erythematosus (SLE) and chronic renal diseases. The study was approved by the Comite Regional de Protection des Personnes (CRPP, Paris, France) under the reference ID-RCB 2014-A00809-38. Diagnostics of inpatients were not known by the investigators at the time of sample processing and flow cytometry analysis. SLE samples were obtained from in- and outpatients and clinical data were harvested after approval by the Comission Nationale de l'Informatique et des Libertés (CNIL). All SLE subjects fulfilled the American College of Rheumatology classification criteria for SLE. SLE and healthy control (HC) donor characteristics are shown in Table 1 (below). Lupus activity was assessed by SELENA-SLEDAI (Safety of Estrogens in Lupus Erythematosus National Assessment—SLE Disease Activity Index) scores. Based on the SLEDAI score, lupus activity was classified as inactive (0-1), mild (2-4) and active (>4). All samples were collected in heparin blood collection tubes and processed within 4 hours. A written informed consent was obtained from all subjects. Active lupus nephritis subjects were defined by histologically active classes III or IV+/−V nephritis, in accordance with the ISN/RPS classification (Weening, J. J., et al., J. Am. Soc. Nephrol. 15, 241-250 (2004)).

(33) Antibodies and Flow Cytometry

(34) All antibodies were from commercial sources. Flow cytometry acquisition was done with a LSRII Fortessa using DIVA software (BD Biosciences). Blood sample processing procedure was as previously described (Charles, N. et al., Nat. Med. 16, 701-707 (2010)). All data relative to marker expression levels are expressed as the ratio between the geometric mean fluorescence intensity (Geo MFI) of the indicated marker on the cells of interest and the Geo MFI of the corresponding isotype control. Data were normalized or not as indicated in figure legends. Data analysis was realized with FlowJo v.X.0.7 (Treestar).

(35) Chemokines, Cytokines, 11β-PGF.sub.2α and Immunoglobulin Measurement Assays

(36) All commercial assays were performed according to the manufacturer instructions. 11β-Prostaglandin F.sub.2α enzyme immunoassay (EIA) kits were from Cayman Chemicals (Ann Arbor, Mich.). Mouse ANA enzyme linked immunosorbent assay (ELISA) kits were from ADI (San Antonio, Tex.). Human and mice CXCL12 ELISA kits were from R&D Systems (Minneapolis, Minn.). Assessment of cytokine content in the kidney was previously described (Charles, N. et al., Nat. Med. 16, 701-707 (2010)). Mouse IL4 and ID1β ELISA kits were from BioLegend (San Diego, Calif.). Mouse IgE Quantification ELISA kits were from Bethyl Laboratories (Montgomery, Tex.). Human and mouse anti-dsDNA IgG and IgE were quantified as previously described (Charles, N. et al., Nat. Med. 16, 701-707 (2010)). Absorbance was assessed by an Infinite 200 Pro plate reader (TECAN, Männedorf, Switzerland).

(37) Human Basophil Purification and Enrichment.

(38) Human basophils were purified to >95% by negative selection with the Human Basophils Enrichment kit (Stemcell Technologies, Grenoble, France) for culture, stimulation and chemotaxis experiments. In some chemotaxis experiments, human basophils were enriched to 3-5% by negative selection with the Human PE positive selection kit (Stemcell Technologies) by using a cocktail of PE-conjugated anti-CD3, CD19 and CD89 (BioLegend). These kits were used following manufacturer instructions.

(39) Imaging Flow Cytometry

(40) Basophils were enriched to 3-5% as described above and frozen at −80° C. in 90% FCS 10% dimethyl sulfoxide until enough samples were collected. Thawed cells were stained, fixed (IC fixation buffer, eBioscience) and permeabilized (Wash Perm Buffer, BioLegend) following the manufacturers' instructions. Anti-human CXCR4 or its isotype (BioLegend) were used for intracellular staining. DAPI was added prior to cytometry analysis. Basophils were gated as Singlets cells/Focus high/DAPI high/PE.sup.− CD123.sup.+ FcεRIα.sup.+ CD303.sup.−. CXCR4 expression was determined for each basophil as the ratio of the geometric mean of their CXCR4 intensity on the mean basophil CXCR4 FMO (Fluorescence Minus One) intensity. Internalization scores were determined using Fc□RI□ staining as a membrane marker and CXCR4 staining as the probe. For each sample, externalization score corresponds to [1—internalization score]. All analyses were performed using the ImageStream X Mark II imaging flow cytometer and the IDEAS v6 software (AMNIS).

(41) Basophil Culture and Stimulation

(42) Human basophils and mouse splenocytes were cultured in culture medium (RPMI 1640 with Glutamax and 20 mM HEPES, 1 mM Na-pyruvate, non-essential amino acids 1× (all from Life Technologies, Saint-Aubin, France), 100 μg/ml streptomycin and 100 U/ml penicillin (GE Healthcare, Vélizy, France) and 37.5 μM β-mercaptoethanol (Sigma-Aldrich, MO)) supplemented with 20% heat-inactivated fetal calf serum at 37° C. and 5% CO.sub.2. 18 hours-long stimulation prior to migration assays were done in culture medium at 1×10.sup.6 cells per mL by adding 1 nM of IL-3 (Peprotech), 1 μM of prostaglandin D2 (PGD.sub.2), 1 μM of the PTGDR-1 specific agonist BW245c, 1 μM of the PTGDR-2 specific agonist 13,14-dihydro-15-keto Prostaglandin D.sub.2 (DK-PGD.sub.2) (all from Cayman chemicals) or 5 ng/mL of anti-IgE (mouse anti-human IgE or rat anti-mouse IgE, both from Thermo Scientific). All cells were washed twice before any migration assay. Control conditions were always with the same vehicle concentration as stimulated conditions.

(43) For CXCR4 overexpression modulation by PTGDRs antagonists, PGD.sub.2 and H-PGDS inhibitor, purified basophils were resuspended in RPMI containing 0.1% BSA+/− the following compounds: vehicle (ethanol 0.1‰), 1 μM PTGDR-1 antagonist Laropiprant, 1 μM PTGDR-2 antagonist CAY10471, 1 or 10 μM PGD.sub.2, and 1 μM of Prostaglandin D Synthase (hematopoietic-type) Inhibitor I (catalog #16256) (all from Cayman Chemical). Cells were incubated for 4 hours at 37° C. and 5% CO.sub.2 and surface expression of the indicated markers were assessed by flow cytometry.

(44) Basophil Migration and Apoptosis Assays

(45) Migration assays were performed in culture medium supplemented with 0.1% bovine serum albumin (BSA, Sigma-Aldrich) in Transwell 5 μm polycarbonate permeable support 6.5 mm inserts (Corning, N.Y., N.Y.) for 3 hours at 37° C. and 5% CO.sub.2 with 1×10.sup.5 purified basophils or 2×10.sup.5 enriched basophils at 1×10.sup.6 cells per mL. Purified or enriched basophils from the upper and bottom chambers were counted at the end of the assay. Basophil content and phenotype was determined by flow cytometry by analyzing more than 100 basophils. Purification or enrichment of human basophils didn't show any difference in the measured migration for all tested chemokines. Migration was defined as the ratio between the number of basophils in the bottom chamber and the number of basophils in the upper plus the bottom chambers. Spontaneous migration was defined as the migration observed without any chemokine in the bottom chamber. Corrected migration was defined as the difference between specific and spontaneous migration. For migration assays the following concentrations (known to be optimal) were used for each compound: Human IL-3: 300 pM (Peprotech), human CCL3, CCL5, and CXCL12: 50 nM; CXCL2 (all from BioLegend) and PGD.sub.2 (Cayman chemicals): 100 nM. Migration to human CCL3, CCL5, CXCL2, and PGD.sub.2 was done in the presence of IL-3 at 300 pM in both chambers. Migration with IL-3 represents chemokinetism: IL-3 was added in both chambers to the same concentration and compared to the spontaneous migration observed without IL-3. Effects of 24 hours incubation with IL-3 or PGD2 (as described above) on basophil apoptosis were estimated by using the FITC Annexin V Apoptosis Detection Kit from BD Biosciences and used accordingly to manufacturer's instructions.

(46) In Vivo Experiments

(47) For CXCL12-induced basophil in vivo migration assays, 200 μL of PBS containing 100 ng of murine CXCL12 or PBS alone were injected intraperitoneally (ip) in 8-12 weeks old WT mice. For PGD.sub.2-induced basophil in vivo migration assays, 100 μL of PBS (with 2 μL of ethanol) alone, or PBS±20 nmoles of PGD.sub.2±200 μg of AMD3100 (all from Cayman Chemicals) were injected ip in 8-12 weeks old Lyn.sup.−/− mice. In all cases, 24 hours later, mice were euthanized and peritoneal lavage, blood, mesenteric lymph nodes and spleen were collected and prepared for FACS analysis as previously described (Charles, N. et al., Nat. Med. 16, 701-707 (2010)). For acceleration of disease development by PGD.sub.2 injections, 12 weeks old Lyn.sup.−/− mice were injected ip with 20 nmoles of PGD.sub.2 or vehicle in PBS, every two days for 10 days, for a total of 6 injections. Mice were analyzed the day following the last injection. For treatment with PTGDRs antagonists, aged WT and Lyn.sup.−/− mice were treated by oral doses of 5 mg/kg of Laropiprant and CAY10471 (Cayman chemicals) or equivalent dose of ethanol (vehicle) in tap water twice a day for ten days. Then, mice were euthanized and blood, plasma, spleen, bone marrow, kidneys and lymph nodes (cervical, brachial, inguinal and mesenteric) were analyzed as previously described (Charles, N. et al., Nat. Med. 16, 701-707 (2010)). The treatment didn't affect weight and cell numbers in the different organs. Cell viability was assessed by the utilization of Ghost Dye Violet 510 (Tonbo, San Diego, Calif.).

(48) Analysis of Glomerular Deposition of IgG and C3, and Kidney Function.

(49) Kidney preparation for immunofluorescence analysis of C3 and IgG deposits was as previously described (Charles, N et al. Nat. Med., 2010, 16, 701-707, 2159). Quantification of C3 and IgG deposits was realized by using ImageJ software (v1.49p, NIH, USA). A minimum of 20 glomeruli was quantified per kidney. For assessment of kidney function the albumin/creatinine ratio (ACR) was determined. Urine was collected from each mouse before and after treatment. The albumin concentration was measured with a mouse albumin ELISA (Bethyl laboratories, Montgomery, Tex.). A creatinine assay (R&D systems, Minneapolis, Minn.) was used to determine urine creatinine concentrations. Results are expressed as a fold increase corresponding to the ratio of the ACR after/before treatment.

(50) Statistical Analysis.

(51) Distribution was assessed with D'Agostino-Pearson omnibus normality test or Kolmogorov-Smirnov test, depending on sample size, to perform appropriate analyses. When more than 2 groups were compared, one-way analysis of variance (ANOVA) tests were conducted before the indicated post-tests when significance (p<0.05) was reached. All tests run were two-tailed. Statistics were performed with GraphPad Prism V5 and V6 (GraphPad) and with STATA 12 (Statacorp) softwares.

Example 2: Results

(52) Specific SLE and Lupus Nephritis Basophil Phenotype

(53) On a cohort of individuals with SLE (n=188, Table 1), we first validated that SLE subject basophils had an activated phenotype as shown by increased CD203c (a basophil activation marker) and CD62L (L-selectin, involved in leukocyte rolling) expressions as compared to healthy control (HC) ones (n=98, FIG. 1a-b, Table 1) (Charles, N. et al., Nat. Med. 16, 701-707 (2010)).

(54) However, SLE basophils did not display a degranulated phenotype (as measured by their CD63 expression level, FIG. 1c). Basopenia appeared to be a good marker of disease correlating with SLE disease activity index (SLEDAI, American College of Rheumatology Ad Hoc Committee on Systemic Lupus Erythematosus Response, C. The American College of Rheumatology response criteria for systemic lupus erythematosus clinical trials: measures of overall disease activity. Arthritis Rheum. 50, 3418-3426 (2004)) (Spearman r coefficient=−0.3629, P<0.0001) (FIG. 2a,b), whereas proportion of HLA-DR positive basophils was better (Receiver Operating Characteristic (ROC) Area Under Curve (AUC)=0.9091) than anti-dsDNA IgG (ROC AUC=0.8384) to discriminate SLE subjects from healthy control (HC) (ROC AUC comparison by DeLong method (DeLong, E. R. et al., Biometrics 44, 837-845 (1988)): P=0.03) (FIG. 2c,d). Moreover, basopenia and high proportion of HLA-DR+ basophils were specific markers for active lupus nephritis when compared to other active renal diseases (FIG. 2e,f). Of note, these SLE-specific basophil parameters were independent of SLE patient treatments at the time of blood harvesting and independent of gender (data not shown). Altogether, these data validated that activated basophils, peripheral basopenia and high proportion of HLA-DR+ basophils are hallmarks of active SLE individuals. Moreover, our data strongly suggest that lupus environment drives a basophil sub-optimal activation (without a detectable degranulation response) and a basophil redistribution to SLOs.

(55) TABLE-US-00001 TABLE 1 SLE Patients and control characteristics Lupus patients Inactive Mild Active Healthy Variables All SLE (SLEDAI ≤ 1) (1 ≤ SLEDAI ≤ 4) (SLEDAI > 4) controls Demographic characteristics n 188 61 41 86 110 Age, mean ± SD, years 37.8 ± 12.3 43.4 ± 14.1 36.1 ± 10.2 34.7 ± 10.6 34.5 ± 14.9 Female Gender, n (%) 167 (89) 51 (84) 36 (88) 80 (93) 51 (47) Lupus characteristics Disease duration, mean ± SD, years 10.5 ± 8.2  11.7 ± 9.0  12.1 ± 7.5  9.1 ± 8.0 — Anti-dsDNA Ab positive, n (%) 104 (55) 10 (18) 27 (64) 67 (79) — History of lupus nephritis, n (%) 144 (77) 36 (59) 29 (66) 79 (92) — SLEDAI Mean ± SD 6.6 ± 7.6 0.0 ± 0.2 2.9 ± 1.0 13.0 ± 6.8  — Median (range) 4 (0-43) 0 (0-1) 2 (2-4) 12 (5-43) — Treatment characteristics Current prednisone dose (mg/day) Mean ± SD 23.6 ± 80.8 4.1 ± 3.9 7.2 ± 8.8  44.8 ± 115.4 — 15 mg/day or higher, n (%) 38 (20) 0 (0) 4 (9) 34 (40) — Concurrent immunosuppressive therapy (n, %) hydroxychloroquine, n (%) 159 (84) 54 (88) 38 (93) 67 (78) — mycophenolate mofetil, n (%) 50 (27) 15 (24) 16 (36) 19 (22) — cyclophosphamide, n (%) 3 (2) 0 (0) 0 (0) 3 (3) — azathioprine, n (%) 26 (14) 8 (13) 7 (17) 11 (13) — SLEDAI: Systemic Lupus Erythematosus Disease Activity Index.

(56) PGD.sub.2/PTGDRs and CXCR4/CXCL12 Axes in Basophils from SLE Subjects

(57) To decipher basophil activation and redistribution to SLOs during lupus pathogenesis, we analyzed on basophils from SLE subjects, versus HC, the expression levels of receptors for chemotactic molecules known to be dysregulated in individuals with lupus or chronic inflammatory diseases (Pellefigues, C. & Charles, N., Curr. Opin. Immunol. 25, 704-711 (2013)). Most of the screened receptor expressions were not significantly different from the ones observed on HC basophils (Table 2). Of note, Thymic Stromal Lymphopoietin Receptor (TSLP-R), IL-33 receptor (T1/ST2), C—C motif ligand receptor (CCR) 4, CCR6 and CCR7 could not be detected on basophils (Table 2).

(58) However, PTGDR-2 expression was increased on basophils from SLE individuals (Table 2, FIG. 3a) as did its ligand titers in their plasma (118-PGF.sub.2a levels, the main plasmatic PGD.sub.2 metabolite, are presented) (FIG. 3b). An inverse correlation between 11β-PGF.sub.2α titers and blood basophil counts in subjects with SLE was found (Spearman r=−0.2585, P=0.0169) (data not shown). Moreover, high levels of 11β-PGF.sub.2α were associated with increased basopenia in SLE subjects (FIG. 3c). Together, these data strongly suggest that PGD.sub.2 and its receptors are associated with basophil activation and extravasation during lupus.

(59) CXCR4 expression was increased on basophils from all SLE individuals, but active SLE patients showed an even more marked increase (Table 2, FIG. 3d). CXCL12 plasma titers followed the same pattern of increase as its receptor did on basophils (FIG. 3e). Basophil CXCR4 expression levels were negatively correlated with blood basophil count in SLE subjects (Spearman r=−0.4692, P<0.0001) (FIG. 4b). Moreover, in active SLE subjects, high CXCL12 titers were associated with a more pronounced basopenia (FIG. 4c). Endolyn (CD164) is a transmembrane syalomucin enhancing sensitivity to CXCL12 when associated to CXCR4 and is also known as a human basophil activation marker. CD164 levels on basophils from active SLE subjects followed the same expression pattern as CXCR4 and were correlated with basopenia (Spearman r=−0.4165, P=0.0029), suggesting an increased sensitivity of SLE basophils to CXCL12 in vivo (FIG. 3f and FIG. 4d). Basophils are known to express CXCR4 mostly intracellularly. Analyses by imaging flow cytometry showed that SLE patient basophils had an increased CXCR4 content and that it was more externalized than in HC basophils (data not shown).

(60) TABLE-US-00002 TABLE 2 Basophil surface marker expression level relative to SLE disease activity Normalized expression levels (to CT mean) on basophils from: Chemokine Mean (±Se, n, p Mann Whitney test vs CT) or cytokine Healthy Inactive SLE Mild SLE Active SLE receptor volonteers patients patients patients analyzed CD# Ligand(s) (controls, CT) (SLEDAI ≤ 1) (1 < SLEDAI ≤ 4) (SLEDAI > 4) CCR1 CD191 CCL3, 5, 1 1.013 1.199 1.11  7, 23 (±0.14, (±0.17, (±0.13, (±0.18, 13, 1) 15, 0.78) 8, 0.40) 19, 1) CCR2 CD192 CCL2, 7, 1 1.04  1.03  1.04  8, 13, 16 (±0.11, (±0.15, (±0.25, (±0.2, 30, 1) 19, 0.89) 14, 0.57) 20, 0.58) CCR3 CD193 Eotaxin 1  0.8529  0.9268  0.9925 (CCL11, (±0.09, (±0.10, (±0.11, (±0.13, 24, 26) 39, 1) 10, 0.4642) 13, 0.9663) 20, 0.9681) CCR4 CD194 CCL2, 4, ND ND ND ND 5, 17, 22 CCR5 CD195 CCL5, 3, 1  0.7726 1.377 1.031 4, 3L1 (±0.14, (±0.09, (±0.42, (±0.19, 6, 1) 5, 0.43) 3, 0.38) 7, 0.94) CCR6 CD196 CCL20 ND ND ND ND CCR7 CD197 CCL19, 21 ND ND ND ND CXCR1 CD181 IL-8 1 1.104 1.197 1.187 (±0.12, (±0.21, (±0.17, (±0.27, 25, 1) 6, 0.63) 6, 0.28) 13, 0.90) CXCR2 CD182 IL-8, 1 1.181  0.9324 1.053 CXCL1, 2, (±0.04, (±0.12, (±0.08, (±0.12, 3, 5 53, 1) 27, 0.85) 15, 0.21) 37, 0.22) CXCR4 CD184 CXCL12 1  .sup. 1.317 ** .sup. 1.279 *  .sup. 2.622 *** (±0.04, (±0.09, (±0.11, (±0.42, 66, 1) 32, 0.0068) 20, 0.0385) 51, <0.001) PTGDR-2 CD294 PGD.sub.2 1 1.404 .sup. 1.329 * .sup. 1.328 * (CRTH2) (±0.06, (±0.11, (±0.13, (±0.11, 71, 1) 48, 0.0091) ** 31, 0.0446) 60, 0.0493) Endolyn CD164 CXCR4 1 .sup. 1.281 * 1.158   .sup. 1.873 **** (±0.05, (±0.11, (±0.11, (±0.18, 33, 1) 15, 0.0207) 7, 0.0943) 26, <0.0001) TSLP-R — TSLP ND ND ND ND IL33-R T1/ST2 IL33 ND ND ND ND Abbreviations used: SE: Standard Error; ND: Not Detected; CD: Cluster of Differentiation; CT: controls; CCL: C-C motif ligand; CCR: C-C motif ligand receptor; CXCL: C-X-C motif ligand; CXCR: C-X-C motif ligand receptor; CRTH2: Chemoattractant Receptor-homologous molecule expressed on Th2 cells (DP2, PTGDR-2); PGD.sub.2: Prostaglandin D.sub.2; PTGDR: PGD2 receptor; TSLP: Thymic stromal lymphopoietin (-R: receptor); Statistical analyses were by Mann-Whitney tests. * P < 0.05, ** P < 0.01, *** P < 0.001, **** P < 0.0001.

(61) CXCL12 is described as one of the most overexpressed gene during peritoneal dialysis and is actively secreted together with PGD.sub.2 during peritonitis. To study human basophil migration in vivo, we analyzed them both in blood and in peritoneal dialysis fluid from patients being treated for a non-sterile peritonitis. CXCR4 expression was dramatically increased on human basophils recruited to the inflamed peritoneum as compared to their blood counterparts (data not shown). This basophil recruitment was associated with a peripheral basopenia (data not shown), as previously shown in active chronic idiopathic urticarial (Jain, S. Dermatology research and practice 2014, 674709), strongly suggesting that human CXCR4+ basophils can migrate in vivo to CXCL12- and PGD.sub.2-secreting inflamed tissues, and that peripheral basopenia reflects this active basophil recruitment.

(62) Altogether these data identified both PGD.sub.2/PTGDRs and CXCL12/CXCR4 axes as basophil activation pathways during SLE flares and which may account for their associated basopenia. Therefore, these axes may contribute to the described basophil accumulation in SLOs in individuals with active lupus.

(63) PGD.sub.2/PTGDRs Axis Enhances CXCR4-Dependent Basophil Migration During Lupus

(64) In order to evaluate the functional consequences of the above findings, ex vivo migration assays of purified basophils from HC and active SLE subjects were performed. Strikingly, SLE basophils were attracted to CXCL12 gradients while HC basophils did not (FIG. 5a), reflecting their differences in CXCR4 and CD164 expression (FIG. 3d,f). However, no difference was detected with other common basophil chemo-attractant compounds, including PGD.sub.2 (FIG. 5b). Since PGD.sub.2 titers were associated with basopenia in SLE (FIG. 3c) and since autoreactive IgE are prevalent in active SLE subjects (FIG. 5c and Dema, B., et al. PLoS One 9, e90424 (2014)), we next investigated if these factors could potentiate basophil migration towards CXCL12. Standard culture of purified human basophils is known to induce intracellular CXCR4 externalization, a process inhibited in the presence of IL-3. Priming purified basophils during 18 hours with 1 μM PGD.sub.2 enhanced their CXCR4 expression and their migration towards CXCL12 (FIG. 5d,e) and induced PTGDR-2 internalization (data not shown), without inducing their apoptosis (data not shown). This PGD.sub.2 priming induced a slight increase in the high affinity IgE receptor alpha chain (FcεRIα) expression on basophil surface, which may increase their sensitivity to IgE-dependent stimulation (data not shown, Sub-optimal anti-IgE stimulation (MacGlashan, D., Jr., Clin. Exp. Allergy 40, 1365-1377 (2010)) (i) tended to increase CXCR4 expression on basophils (FIG. 5d) which may influence their migration towards CXCL12 although statistical significance was not reached (FIG. 5e), (ii) increased PTGDR-2 expression levels on basophils (FIG. 6a) and (iii) did not induce basophil degranulation (data not shown). None of other tested compounds (CCL3, CXCL2 and CCL5), known to have an effect on basophils and to be dysregulated during SLE, induced an increased CXCR4 externalization ex vivo as PGD.sub.2 did (data not shown).

(65) CXCL12 is described as one of the most overexpressed gene during peritoneal dialysis and is actively secreted together with PGD.sub.2 during peritonitis. To study human basophils migration in vivo, we analyzed them both in blood and in peritoneal dialysis fluid from patients being treated for a non-sterile peritonitis. CXCR4 was found to be dramatically increased on human basophils recruited to the inflamed peritoneum as compared to their blood counterparts (FIG. 5e). This basophil recruitment was associated with a peripheral basopenia (supplementary FIG. 5b), as previously shown in active chronic idiopathic urticaria. This demonstrated that human CXCR4+ basophils can migrate in vivo to CXCL12- and PGD.sub.2-secreting inflamed tissues.

(66) We next studied the mechanism by which PGD.sub.2 induced CXCR4 externalization by human basophils. Both PTGDR-1 and PTGDR-2 were cooperatively involved since antagonism of one, the other or both receptor(s) led to block CXCR4 externalization (data not shown). Moreover, blocking PTGDRs led to a decreased spontaneous CXCR4 externalization. This suggested that either ex vivo culture and/or PGD.sub.2-mediated stimulation of human basophils led them to produce PGD.sub.2 to have an autocrine effect as eosinophils do. To confirm this hypothesis, we used a specific H-PGDS inhibitor which resulted in the same inhibition of spontaneous CXCR4 externalization than the one induced by the PTGDR antagonists, and to a decreased PTGDR-2 internalization (data not shown). Together with the fact that the H-PGDS inhibitor effect was overcome only by a ten-fold higher PGD.sub.2 concentration (data not shown), we here confirmed our above hypothesis that PGD.sub.2 led to CXCR4 externalization partially by stimulating PGD.sub.2 production by basophils themselves.

(67) Altogether, these data strongly suggest that the PGD.sub.2/PTGDRs axis directly influences the CXCL12 sensitivity of SLE patient basophils by increasing both their CXCR4 expression and externalization, and that lupus environment (including autoreactive IgE, CXCL12 and PGD.sub.2) facilitates this cross-talk resulting in basophil extravasation and peripheral basopenia. Thus, PGD.sub.2 might be required to allow CXCR4-dependent basophil migration to SLOs in SLE individuals. Indeed, both PGD.sub.2/PTGDRs and CXCL12/CXCR4 axes were associated with basopenia and disease activity in lupus subjects (FIG. 3).

(68) CXCR4/CXCL12 and PGD.sub.2/PTGDRs Axes in Lyn.sup.−/− Lupus-Prone Mice

(69) We previously showed that aged Lyn.sup.−/− mice develop a basophil-dependent T.sub.H2 bias contributing to an IgE-, IL-4- and basophil-dependent lupus-like nephritis (Charles, N. et al., Nat. Med. 16, 701-707 (2010); Charles, N., et al., Immunity 30, 533-543 (2009)). In this lupus-like disease model, basophils accumulate in SLOs leading to an amplification loop of the disease (Charles, N., et al., Immunity 30, 533-543 (2009)).

(70) We next assessed whether this mouse model was involving both CXCL12/CXCR4 and PGD.sub.2/PTGDRs axes as SLE subjects did. In ex vivo migration assays, Lyn.sup.−/− spleen basophils migrated towards CXCL12 whereas their WT counterparts did not (FIG. 7a) mimicking the observed differences between SLE subjects and HC (FIG. 5a). CXCR4 expression levels were increased on Lyn.sup.−/− basophils from blood, bone marrow (BM) and SLOs as compared to their WT counterparts in aged animals (FIG. 7b). Moreover, CXCR4 expression was increased on WT and Lyn.sup.−/− basophils from SLOs as compared to their blood counterparts suggesting an involvement of CXCR4 in their accumulation in these organs (FIG. 7b). CXCL12 intraperitoneal (ip) injection in WT mice induced in vivo basophil migration to the injection site (FIG. 7c) and to the draining mesenteric lymph nodes (mLN) (FIG. 8a).

(71) In vivo i.p. injection of PGD.sub.2 increased CXCR4 expression on mLN Lyn basophils (FIG. 7d), as it did ex vivo on human basophils (FIG. 5d), and drove their accumulation in SLOs and peritoneum (FIG. 7e and FIG. 8b,c). Moreover, in vivo ip injection of PGD.sub.2 in Lyn.sup.−/− mice led to a significant, but transient, peripheral basopenia when compared to steady state conditions (data not shown) as observed in peritoneal dialysis patients (data not shown). This PGD.sub.2-induced basophil recruitment was strictly dependent on the CXCL12/CXCR4 axis since co-injection with AMD3100, a specific antagonist of CXCR4, completely abolished basophil recruitment both in SLOs and peritoneum (FIG. 7e and FIG. 8b,c). In mice, PGD.sub.2-induced CXCR4 up-regulation was as well mediated by both PTGDR-1 and PTGDR-2 (CRTH2), as shown by the effects of their specific agonists (BW245c and DK-PGD.sub.2, respectively) on spleen WT basophils ex vivo (FIG. 7f). Both agonists induced a significant but much lower increase in CXCR4 expression on other WT splenocytes including T and B cells (data not shown).

(72) PTGDR-1 is known to induce cyclic adenosine monophosphate (cAMP) production upon engagement by PGD.sub.2. We next analyzed the effect of a membrane permeable cAMP (N6,2′-O-dibutyryl-adenosine 3′:5′-cyclic monophosphate (db-cAMP)) on CXCR4 externalization by mouse WT spleen basophils ex vivo. Basophils externalized CXCR4 upon db-cAMP exposure (FIG. 9a) with a 100 fold higher sensitivity than T cells to this compound (FIG. 9b). PGD.sub.2 was unable to induce enough cAMP through PTGDR-1 to lead to CXCR4 externalization by T cells in these settings, unlike what was observed on basophils (FIG. 9a,b). Dose response experiments of each PTGDR specific agonists in the presence or not of H-PGDS inhibitor suggested again that both PTGDRs were able to cooperatively induce CXCR4 externalization on basophils through PTGDR-2-induced PGD.sub.2 synthesis acting in an autocrine way on PTGDR-1-mediated cAMP production (FIG. 9c).

(73) These results strongly suggest that in Lyn.sup.−/− old mice, as in SLE subjects, PGD.sub.2 enhances in vivo CXCL12-dependent basophil accumulation in inflamed tissues and SLOs by modulating their CXCR4 expression levels through the activation of both PTGDR-1 and PTGDR-2.

(74) PGD.sub.2 Chronic Exposure Accelerates Lupus-Like Disease Development in a Basophil-Dependent Manner

(75) Therefore, a more chronic exposure to PGD.sub.2 in lupus-prone mice before they start developing the disease should lead to a chronic accumulation of basophils in SLOs, an increased number of autoreactive plasma cells and to an acceleration of disease development. To verify this hypothesis, we repeatedly injected ip PGD.sub.2 to young (12 weeks-old) Lyn mice every two days over ten days. As expected, basophils increased their CXCR4 expression levels (data not shown) and accumulated systemically in SLOs (FIG. 10a,b) where they were activated as shown by their increased IA-IE expression (FIG. 10c). This was associated with an increased proportion of CD19+CD138.sup.+ plasma cells in SLOs (FIG. 10d,e), resulting in an increased deposition of immune complexes in the kidney as shown by C3 and IgG deposition quantification (FIG. 10f). Consequently, nearly all the PGD.sub.2 injected Lyn.sup.−/− mice had increased albuminuria unlike their PBS-injected counterparts at the end of the protocol (FIG. 10g). Other immune cell types analyzed didn't show any significant increase in their CXCR4 surface expression (data not shown). Importantly, this PGD.sub.2-induced lupus-like disease acceleration was dependent on basophils since antibody-mediated (MAR-1) basophil depletion during the whole protocol led to a complete rescue of the PGD.sub.2 effects on disease development (FIG. 10b-e). These results confirmed that PGD.sub.2, by enabling CXCR4-dependent basophil accumulation in SLOs, contributes to lupus-like disease and to autoantibody-mediated kidney damage.

(76) Targeting the PGD.sub.2 Axis Reduces CXCR4-Mediated Basophil Accumulation in SLOs and Dampens Lupus-Like Disease

(77) Targeting the CXCL12/CXCR4 axis in murine lupus has already been described and showed some efficacy on disease activity (Balabanian, K., et al., J. Immunol. 170, 3392-3400 (2003), Wang, A., et al., J. Immunol. 182, 4448-4458 (2009)). However, CXCR4 antagonism (with AMD3100), initially developed as an anti-HIV drug, is known to induce a release of hematopoietic stem cells and interfere with homeostatic functions (Devi, S. et al. J. Exp. Med. 210, 2321-2336, (2013), Hummel, S. et al., Curr. Opin. Hematol. 21, 29-36 (2014)). Preventing the CXCR4 up-regulation on basophils from lupus-prone Lyn.sup.−/− mice by blocking the PGD.sub.2/PTGDRs axis seemed a safer approach to disable the basophil-dependent amplification loop of autoantibody production in SLOs.

(78) Then, we treated aged Lyn and WT mice by oral gavage with both specific antagonists of PTGDR-1 and PTGDR-2, Laropiprant and CAY10471, respectively, at a dose of 5 mg/kg each, twice daily for ten days. This treatment led to a dramatic reduction in basophil numbers in SLOs of Lyn.sup.−/− animals (FIG. 12a,b) associated with a decreased CXCR4 expression on spleen basophils (FIG. 12c). Of note, BM and blood basophil proportions were not affected by the treatment (FIG. 13a,b). These results validated the approach consisting of disabling basophil accumulation in SLOs by targeting the PTGDRs.

(79) As expected, this reduction in basophil accumulation was associated with a significant decrease of CD19.sup.+CD138.sup.+ short-lived plasma cell numbers (FIG. 12d and FIG. 13c). Strikingly, proportions of all other immune cell populations analyzed (B cells, neutrophils, Ly6C.sup.+ monocytes, and Ly6C.sup.− monocytes) remained unaffected by the treatment as did their CXCR4 expression levels (data not shown). PTGDRs blockade in Lyn.sup.−/− animals, by disabling basophil accumulation in SLOs, decreased their autoantibody titers (FIG. 12e and FIG. 13d) and their T.sub.H2 bias as measured by total IgE plasma concentrations (FIG. 12f). Consequently, this treatment allowed as well a significant decrease in the kidney content of C3 and IgG deposits (FIG. 11) and the pro-inflammatory cytokines IL-4 and IL-1β (FIG. 12g,h). Therefore, a short-term treatment with PTGDRs antagonists allowed an efficient dampening in the disease activity observed in our lupus-prone animals.

(80) Altogether, these results suggest that aiming the PGD.sub.2/PTGDRs axis could be a valuable new therapeutic approach in SLE. Indeed, PTGDRs blockade, by breaking the CXCL12-dependent basophil homing to SLOs, might turn-off the basophil-dependent amplification loop of autoantibody production, efficiently preventing flares and subsequent organ damage in SLE (FIG. 14).