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Prostaglandin D2 amplifies lupus disease through basophil accumulation in lymphoid organs - PubMed

  • ️Mon Jan 01 2018

Prostaglandin D2 amplifies lupus disease through basophil accumulation in lymphoid organs

Christophe Pellefigues et al. Nat Commun. 2018.

Abstract

In systemic lupus erythematosus (SLE), autoantibody production can lead to kidney damage and failure, known as lupus nephritis. Basophils amplify the synthesis of autoantibodies by accumulating in secondary lymphoid organs. Here, we show a role for prostaglandin D2 (PGD2) in the pathophysiology of SLE. Patients with SLE have increased expression of PGD2 receptors (PTGDR) on blood basophils and increased concentration of PGD2 metabolites in plasma. Through an autocrine mechanism dependent on both PTGDRs, PGD2 induces the externalization of CXCR4 on basophils, both in humans and mice, driving accumulation in secondary lymphoid organs. Although PGD2 can accelerate basophil-dependent disease, antagonizing PTGDRs in mice reduces lupus-like disease in spontaneous and induced mouse models. Our study identifies the PGD2/PTGDR axis as a ready-to-use therapeutic modality in SLE.

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Conflict of interest statement

The authors declare no competing financial interests.

Figures

Fig. 1
Fig. 1

PGD2-PTGDR axis is associated with basopenia in SLE. a Basophils per µL of blood from CT and inactive, mild or active SLE patients (n = 116/55/42/103, respectively) as determined by FACS as described in Supplementary Fig. 1. b Representative FACS analysis of PTGDR-2 expression on blood basophils from a healthy control (CT), a patient with active SLE and isotype control (IC) staining. c PTGDR-2 levels on blood basophils from patients as in (a) (n = 101/49/34/78, respectively). d Representative FACS histograms of PrimeFlowTM RNA Assay showing PTGDR-1 RNA transcript expression by basophils from CT and active SLE individual compared to fluorescence minus one (FMO). e, f PTGDR-1 (e) and PTGDR-2 (f) RNA transcript expressions in basophils from CT (n = 6) and active SLE patients (n = 7) (ratio MFI/FMO) as in (d). g 11β-prostaglandin F2α (11β-PGF2α) levels in plasma from patients as in (a) (n = 44/33/28/66, respectively). h Basophils per µL of blood in patients with SLE grouped by low (n = 84) or high (n = 39) 11β-PGF2α plasma levels (high = levels above CT mean + 2 standard deviations). a, c, e–h Data are presented as median and interquartile ranges with whiskers representing 5–95 percentiles and the mean presented as a “+” symbol. Statistical analyses were by Mann−Whitney tests. e, f Data are presented as mean ± s.e.m. Statistical analyses were by unpaired Student t tests. a, c, e–h NS: not significant, *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001. Comparison to control group is shown above each bar and to the corresponding bars when indicated. A.U. arbitrary units

Fig. 2
Fig. 2

CXCL12-CXCR4 axis is associated with basopenia in SLE. a Representative FACS analysis of CXCR4 levels on blood basophils as in Fig. 1b. b CXCR4 levels on blood basophils as in Fig. 1a (n = 95/32/24/ 70, respectively). c Representative images of CXCR4 (green) and FcεRIα (red) expressions by CT or active SLE patient basophil captured by imaging flow cytometry (scale bar = 5 µm). d Total CXCR4 expression and e CXCR4 externalization score in basophils from CT (n = 3) and active SLE patients (n = 4) as in (b) (20 basophils per individual). f CXCR4 RNA transcript expressions in basophils as in Fig. 1d. g CXCL12 levels in plasma from patients as in Fig. 1a (n = 78/45/38/ 87, respectively). h Basophils per µL of blood in patients with active SLE grouped by low (n = 59) or high (n = 22) CXCL12 plasma levels as defined in Fig. 1h. b, d, e, g, h Data are presented as median and interquartile ranges with whiskers representing 5–95 percentiles and the mean presented as a “+” symbol. Statistical analyses were by Mann−Whitney tests. f Data are presented as mean ± s.e.m. Statistical analyses were by unpaired Student t tests. b, d−h NS: not significant, *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001. Comparison to control group is shown above each bar and to the corresponding bars when indicated. A.U. arbitrary units

Fig. 3
Fig. 3

CXCR4-dependent basophil migration is enabled by both PTGDR. a, b Migration assays of human blood basophils from CT (n = 6) and active SLE patients (n = 6) towards CXCL12 (a) and towards IL-3, CCL3, CCL5, CXCL2, and PGD2 (b) from healthy controls (Controls, n = 8/4/3/4/7, respectively) and from SLE patients (SLE, n = 6/3/6/3/5, respectively). c, d FACS analysis of CXCR4 expression levels (c) and migration towards CXCL12 (d) of purified CT human blood basophils after 18 h of incubation without (−) or with (+) 1 µM PGD2, anti-human IgE antibodies or IL-3. e, f, g CXCR4 expression levels were assessed as in (c) on purified human blood basophils after 4 h of incubation with 1 µM of the indicated compounds (except for ddAde: 50 µM) (PTGDR-1 agonist: BW245c, antagonist: Laropiprant; PTGDR-2 agonist: DK-PGD2, antagonist: CAY10471). Data are normalized to the mean value of the vehicle condition. For each condition, 3−12 independent experiments were conducted. h, i CXCR4 expression levels on mouse CD45+CD3+TCRβ+ T cells (h) and basophils (i) in splenocytes incubated 4 h without (−) or with the indicated concentration of db-cAMP or 1 µM PGD2 as determined by flow cytometry as described in Supplementary Fig. 1. j Fold increase in cAMP concentration in purified human basophils stimulated for 15 min with 1 µM of the indicated compound (except for ddAde (50 µM) and Forskolin (10 µM)) normalized to the unstimulated condition (pool of six independent experiments). a Data are expressed as medians and interquartile ranges. b, e–j Data are presented as mean ± s.e.m. a–j Statistical analyses were by Mann−Whitney tests (a, b), paired Student t test (c–i) or by Tukey’s multiple comparisons test (j). a–j NS: not significant, #P = 0.06, *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001. Comparison to control group is shown above each bar and to the corresponding bars when indicated. A.U. arbitrary units

Fig. 4
Fig. 4

PGD2 induces CXCR4-dependent basophil homing to SLO in lupus-prone mice. a Ex vivo migration of basophils from whole splenocytes to CXCL12 from young WT (n = 4) or Lyn–/– (n = 4) mice. b CXCR4 expression on basophils in aged WT (n = 16) and Lyn–/– (n = 14) animals (BM: bone marrow, LN: lymph nodes). Data are normalized to WT blood basophils mean value. Statistical analyses placed directly above each bar compared the value to the blood group for each genotype. c, d CXCL12 (c) and 11β-PGF2α (d) titers in serum from aged WT (n = 5) and Lyn–/– (n = 4) animals. e CXCL12 titers in protein extracts from indicated compartments in Lyn–/– mice as in (c). f CXCR4 expression on basophils from mLN of young Lyn–/– mice 24 h after PGD2 i.p. injection normalized to vehicle’s mean. g Basophil counts in the indicated compartments at steady state, 4 and 24 h after PGD2 i.p. injection in young Lyn–/– mice. h, j Basophil number in peritoneum (h) and mLN (j) of young Lyn–/– mice 24 h after i.p. injection of the indicated compound(s). i Representative contour plots showing mLN basophil proportion among living CD45+ cells (%) from mice as in (j). Basophil number and CXCR4 expression were assessed by flow cytometry as described in Supplementary Fig. 4a–c. a–j Data are presented as mean ± s.e.m. Statistical analyses were by unpaired Student t test. NS: not significant, *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001. Comparison to control group is shown above each bar and to the corresponding bars when indicated. A.U. arbitrary units

Fig. 5
Fig. 5

PGD2 injections induce basophil-dependent disease acceleration in young Lyn–/– mice. a, b Proportion of basophils among living CD45+ cells in lymph nodes (LN) (a) (cervical, brachial, and inguinal) and in spleen (b) from young Mcpt8DTR (WT) or Lyn–/– Mcpt8DTR (Lyn–/–) mice injected over 10 days with PBS or PGD2 along or not with diphtheria toxin-mediated basophil depletion (DT) as described in the Methods. From left to right, n = 3/4/4/4/4/5. c, d IA-IE expression on LN basophils (c) and CXCR4 expression on spleen basophils (d) in mice as in (a). NA not applicable. e, f Proportion of CD19+CD138+ cells among living CD45+ cells in spleen (e) and lymph nodes (f) in mice as in (a). g dsDNA-specific IgG levels in serum from mice as in (a). h Representative immunofluorescence staining for C3 and IgG deposits in kidneys from young Lyn–/– Mcpt8DTR mice treated as indicated (scale bar = 500 µm) and quantifications of these deposits in mice as in (a). i, j IL-4 and IL-1β levels in kidney protein extracts from mice as in (a). a–f Parameters were assessed by flow cytometry as described in Supplementary Fig. 4a–c and 8. g, i, j Parameters were assessed by ELISA. a–j Data are presented as mean ± s.e.m. Statistical analyses were by unpaired Student t tests. NS: not significant, #P = 0.06, §P < 0.1, *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001. Comparison to control group is shown above each bar and to the corresponding bars when indicated. A.U. arbitrary units

Fig. 6
Fig. 6

PTGDR blockade dampens basophil accumulation in SLO in a lupus environment. a, e Representative contour plots of basophils among living CD45+ cells in LN and spleen from aged Lyn–/– mice (a) and pristane-injected WT mice (e) treated or not (vehicle) with both PTGDR-1 and PTGDR-2 antagonists for 10 days. Proportions are shown on the plots. b−d Comparisons between aged wild-type (WT) treated (n = 4) or not (n = 5) with both PTGDR antagonists and aged Lyn–/– mice treated or not (n = 12) with Laropiprant (n = 5), CAY10471 (n = 4), or both antagonists (n = 9). b, c Proportion of basophils among living CD45+ cells in LN (b) and spleen (c). d CXCR4 expression on spleen basophils in mice as in (c). f–h Comparisons between aged WT mice 24 weeks after injection of either PBS or pristane and treated or not with both PTGDR antagonists (from left to right: n = 12/4/9/4). f, g Proportion of basophils among living CD45+ cells in LN (f) and spleen (g). h CXCR4 expression on spleen basophils in mice as in (f). b–d, f–h Data were determined by flow cytometry as described in Supplementary Fig. 4a–c and represent two pooled independent experiments for each model. Data are presented as mean ± s.e.m. Statistical analyses were by unpaired Student t tests. NS: not significant, *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001. Comparison to control group is shown above each bar and to the corresponding bars when indicated. A.U. arbitrary units

Fig. 7
Fig. 7

Blockade of basophil accumulation in SLO dampens lupus-like disease activity. a, b Proportion of CD19+CD138+ short-lived plasma cells among living CD45+ cells in lymph nodes (a) and in spleen (b) from mice as described in Fig. 6b, determined by flow cytometry as described in Supplementary Fig. 8. c, d dsDNA-specific IgG levels (optical density (O.D.) at 450 nm×102) (c) and total IgE levels (d) in blood from mice as in (a) as measured by ELISA. e, f IL-4 (e) and IL-1β (f) concentrations of total kidney protein extracts from mice as in (a), measured by ELISA. g−i Representative immunofluorescence staining for C3 and IgG deposits in kidneys from aged Lyn–/– mice treated or not (vehicle) with both PTGDR antagonists (scale bar = 500 µm) (g) and their quantifications in mice as in (a) (h, i). a–g Data represent two pooled independent experiments. Data are presented as mean ± s.e.m. Statistical analyses were by unpaired Student t tests. NS: not significant, #P = 0.06, *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001. Comparison to control group is shown above each bar and to the corresponding bars when indicated. A.U. arbitrary units

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References

    1. Davidson A. What is damaging the kidney in lupus nephritis? Nat. Rev. Rheumatol. 2016;12:143–153. doi: 10.1038/nrrheum.2015.159. - DOI - PMC - PubMed
    1. Kandala NB, et al. Belimumab: a technological advance for systemic lupus erythematosus patients? Report of a systematic review and meta-analysis. BMJ Open. 2013;3:e002852. doi: 10.1136/bmjopen-2013-002852. - DOI - PMC - PubMed
    1. Voehringer D. Protective and pathological roles of mast cells and basophils. Nat. Rev. Immunol. 2013;13:362–375. doi: 10.1038/nri3427. - DOI - PubMed
    1. Charles N, Hardwick D, Daugas E, Illei GG, Rivera J. Basophils and the T helper 2 environment can promote the development of lupus nephritis. Nat. Med. 2010;16:701–707. doi: 10.1038/nm.2159. - DOI - PMC - PubMed
    1. Otsuka A, et al. Basophils are required for the induction of Th2 immunity to haptens and peptide antigens. Nat. Commun. 2013;4:1739. doi: 10.1038/ncomms2740. - DOI - PMC - PubMed

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